U.S. patent application number 10/011202 was filed with the patent office on 2002-07-18 for batch devices for the reduction of compounds from biological compositions containing cells and methods of use.
Invention is credited to Hei, Derek J..
Application Number | 20020094568 10/011202 |
Document ID | / |
Family ID | 56289848 |
Filed Date | 2002-07-18 |
United States Patent
Application |
20020094568 |
Kind Code |
A1 |
Hei, Derek J. |
July 18, 2002 |
Batch devices for the reduction of compounds from biological
compositions containing cells and methods of use
Abstract
Methods and devices are provided for reducing the concentration
of low molecular weight compounds in a biological composition
containing cells while substantially maintaining a desired
biological activity of the biological composition. The device
comprises highly porous adsorbent particles, and the adsorbent
particles are immobilized by an inert matrix.
Inventors: |
Hei, Derek J.; (Concord,
CA) |
Correspondence
Address: |
Charles D. Holland
Morrison & Foerster LLP
755 Page Mill Road
Palo Alto
CA
74304-1018
US
|
Family ID: |
56289848 |
Appl. No.: |
10/011202 |
Filed: |
December 7, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10011202 |
Dec 7, 2001 |
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09112400 |
Jul 8, 1998 |
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09112400 |
Jul 8, 1998 |
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09003113 |
Jan 6, 1998 |
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Current U.S.
Class: |
435/308.1 ;
210/656 |
Current CPC
Class: |
B01D 15/00 20130101;
C07D 405/12 20130101; A61M 1/0218 20140204; B01J 20/20 20130101;
B01J 20/28004 20130101; C07F 9/64 20130101; A01N 1/0205 20130101;
C07D 493/04 20130101; B01J 20/28057 20130101; A61M 1/3679 20130101;
A61L 2/00 20130101; A61M 1/0209 20130101; A01N 1/0242 20130101;
A61L 2/0082 20130101; B01J 20/28019 20130101; B01J 20/28026
20130101; B01J 20/28078 20130101; A01N 1/0278 20130101; C07D 219/10
20130101; B01J 20/28028 20130101; A01N 1/0236 20130101 |
Class at
Publication: |
435/308.1 ;
210/656 |
International
Class: |
C12M 003/00; C12M
001/00; B01D 015/08 |
Claims
1. A device for reducing the concentration of a low molecular
weight compound in a biological composition containing cells,
wherein the device comprises an inert matrix containing highly
adsorbent particles, and wherein the diameter of the adsorbent
particles ranges from about 100 .mu.m to about 1500 .mu.m, and
wherein the cells in the biological composition treated with the
device substantially maintain their desired biological activity,
and wherein the device is for use in a batch process.
2. A device according to claim 1 further comprising a container
that is compatible with the biological composition.
3. A device according to claim 1, wherein the adsorbent particles
have a surface area greater than about 750 m.sup.2/g.
4. A device according to claim 3, wherein the adsorbent particles
have a surface area between about 1000 m.sup.2/g and 3000
m.sup.2/g.
5. A device according to claim 1, wherein the adsorbent particles
are polyaromatic adsorbent particles.
6. A device according to claim 5, wherein the polyaromatic
adsorbent particles possess superior wetting properties.
7. A device according to claim 5, wherein the polyaromatic
adsorbent particles comprise a hypercrosslinked polystyrene
network.
8. A device according to claim 1, wherein the adsorbent particles
are activated carbon particles.
9. A device according to claim 8, wherein the activated carbon
particles are derived from a synthetic source.
10. A device according to claim 9, wherein the low molecular weight
compound is a quencher.
11. A device according to claim 9, wherein the activated carbon
particles are roughly spherical and are of a diameter ranging from
about 300 .mu.m to about 900 .mu.m.
12. A device selected from devices according to claim 1, 5 or 8,
wherein the inert matrix is a synthetic polymer fiber.
13. A device according to claim 12, wherein the synthetic polymeric
fiber comprises a polymer core with a high melting temperature
surrounded by a sheath with a lower melting temperature.
14. A device according to claim 12, wherein the inert matrix is a
particulate network.
15. A device according to claim 14, wherein the particulate network
comprises polyethylene particles.
16. A device according to claim 1, wherein the adsorbent particles
are immobilized in the matrix.
17. A device according to claim 1, wherein the low molecular weight
compound is a nucleic acid targeting agent or a
photosensitizer.
18. A device according to claim 1, wherein the low molecular weight
compound is an acridine derivative, a psoralen derivative or a
dye.
19. A device according to claim 1, wherein the low molecular weight
compound is a biological response modifier.
20. A device according to claim 19, wherein the biological response
modifier is activated complement.
21. A device according to claim 1, wherein the low molecular weight
compound is a quencher.
22. A device according to claim 1, wherein the low molecular weight
compound is a polyamine derivative.
23. A device according to claim 1, wherein the desired biological
activity maintained by the cells is suitability for infusion.
24. A device according to claim 1, wherein the biological
composition comprises platelets.
25. A device according to claim 24, wherein treatment of the
biological composition with a device over a period of about 5 days
results in less than about a 10% loss in platelet count.
26. A device according to claim 25, wherein the device further
comprises a container that is compatible with the biological
composition, and wherein the inert matrix is a synthetic polymer
fiber comprising a polymer core with a high melting point
surrounded by a sheath with a lower melting temperature, and
wherein the adsorbent particles are polyaromatic adsorbents
comprising a hypercrosslinked polystyrene network, and wherein the
surface area of the particles is greater than about 750 m.sup.2/g,
and wherein the adsorbent particles are immobilized in the
matrix.
27. A device according to claim 26, wherein the desired biological
activity maintained by the cells is suitability for infusion.
28. A device according to claim 27, wherein the biological
composition is treated with the device at a temperature of about
22.degree. C. for between 0.5 hour and 7 days.
29. A device according to claim 26, wherein the low molecular
weight compound is an acridine derivative, a psoralen derivative or
a dye.
30. A device according to claim 26, wherein the low molecular
weight compound is a biological response modifier.
31. A device according to claim 30, wherein the biological response
modifier is activated complement.
32. A device according to claim 1, wherein the biological
composition comprises red blood cells.
33. A device according to claim 32, wherein treatment of the
biological composition with the device over a period of up to 35
days results in less than about 1% hemolysis.
34. A device according to claim 33, wherein the device further
comprises a container that is compatible with the biological
composition, and wherein the inert matrix is a synthetic polymer
fiber comprising a polymer core with a high melting point
surrounded by a sheath with a lower melting temperature, and
wherein the adsorbent particles are activated carbon particles
derived from a synthetic source, and wherein the diameter of the
particles ranges from about 300 .mu.m to about 900 .mu.m, and
wherein the surface area of the particles is greater than about 750
m.sup.2/g, and wherein the adsorbent particles are immobilized in
the matrix.
35. A device according to claim 34, wherein the desired biological
activity maintained by the cells is suitability for infusion.
36. A device according to claim 34, wherein the low molecular
weight compound is an acridine derivative, a psoralen derivative or
a dye.
37. A device according to claim 34, wherein the low molecular
weight compound is a biological response modifier.
38. A device according to claim 37, wherein the biological response
modifier is activated complement.
39. A method for reducing the concentration of a low molecular
weight compound in a biological composition containing cells
comprising treating the biological composition with a device of
claim 1 or 34 for between 0.5 hour and 5 weeks, wherein the cells
in the biological composition treated with the device substantially
maintain their desired biological activity.
40. A method according to claim 39, wherein the cells in the
biological composition treated with the device substantially
maintain their desired biological activity after being treated with
the device for between 1.0 hour and 5 weeks.
41. A method according to claim 39, wherein the low molecular
weight compound is an acridine derivative.
42. A method according to claim 39, wherein the low molecular
weight compound is a psoralen derivative.
43. A method according to claim 39, wherein the low molecular
weight compound is a dye.
44. A method according to claim 39, wherein the low molecular
weight compound is a biological response modifier.
45. A method according to claim 44, wherein the biological response
modifier is activated complement.
46. A method according to claim 39, wherein the biological
composition is first treated with the device at a temperature of
about 22.degree. C. for between about 0.5 and about 36 hours.
47. A method according to claim 39, wherein the biological
composition is first treated with the device at a temperature of
about 22.degree. C. for between about 0.5 and about 24 hours.
48. A method according to claim 39, wherein the biological
composition is first treated with the device at a temperature of
about 22.degree. C. for between about 0.5 and about 12 hours.
49. A method according to claim 47, wherein the blood product i s
subsequently treated with the device at a temperature of about
4.degree. C. for up to 5 weeks.
50. A biological composition containing cells, wherein the
biological composition is suitable for infusion, and wherein the
biological composition is produced by treating a biological
composition containing cells with a device according to claim 1 or
34 for a period between about 0.5 hour and 5 weeks.
51. A device for 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 adsorbent particles, wherein the adsorbent
particles are immobilized by an inert matrix.
52. A method for reducing the concentration of a low molecular
weight compound in a biological composition containing cells
comprising treating the biological composition with a device
according to claim 26 for between about 0. 5 hours and about 7
days, wherein the cells in the biological composition treated with
the device substantially maintain their desired biological
activity.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part application of
copending application Ser. No. 09/003,113, filed Jan. 6, 1998,
which is incorporated by reference in its entirety.
TECHNICAL FIELD
[0002] The present invention relates to methods and devices for the
reduction of compounds in biological compositions. The compounds
have a molecular weight ranging from about 100 g/mol to about
30,000 g/mol.
BACKGROUND ART
[0003] 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. Bertolini et al., Vox Sang 62:82
(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. Bock et al.,
Transfusion 31:333 (1991) (Sepacell PL-5A, Asahi, Tokyo, Japan);
J.D. Sweeney et al., Transfusion 35:131 (1995) (Leukotrap PL, Miles
Inc., Covina, Calif.); and M. van Marwijk et al., Transfusion 30:34
(1990) (Cellselect, NPBI, Emmer-Compascuum, The Netherlands;
Immugard Ig-500, Terumo, 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.
[0004] 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. Ishihara et al. J Biomed. Mat.
Res. 28:1347 (1994). Such polymers, however, are not designed for
the adsorption of low molecular weight compounds.
[0005] Various dialysis means are able to remove low molecular
weight compounds from plasma and whole blood. For example, dialysis
can successfully remove low 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.
[0006] 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.
[0007] 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.
[0008] 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.
DISCLOSURE OF THE INVENTION
[0009] 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.
[0010] 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.
[0011] The present invention provides a device for reducing the
concentration of low molecular weight compounds in a biological
composition containing cells such that the cells are suitable for
their intended use.
[0012] In one embodiment, the device comprises an inert matrix
containing highly adsorbent particles having a diameter ranging
from about 100 .mu.m to about 1500 .mu.m. The cells in the
biological composition treated with the device substantially
maintain their desired biological activity. The device is for use
in a batch process.
[0013] In another embodiment, the device further comprises a
container that is compatible with the biological composition.
[0014] In another embodiment, the adsorbent particles have a
surface area greater than about 750 m.sup.2/g. In another
embodiment, the adsorbent particles have a surface area between
about 1000 m.sup.2/g and 3000 m.sup.2/g.
[0015] In another embodiment, the adsorbent particles are
polyaromatic adsorbent particles.
[0016] In another embodiment, the polyaromatic adsorbent particles
possess superior wetting properties.
[0017] In another embodiment, the polyaromatic adsorbent particles
comprise a hypercrosslinked polystyrene network.
[0018] In another embodiment, the adsorbent particles are activated
carbon particles.
[0019] In another embodiment, the activated carbon particles are
derived from a synthetic source.
[0020] In another embodiment, the low molecular weight compound is
a quencher.
[0021] In another embodiment, the activated carbon particles are
roughly spherical and are of a diameter ranging from about 300
.mu.m to about 900 pm.
[0022] In another embodiment, the inert matrix is a synthetic
polymer fiber.
[0023] In another embodiment, the synthetic polymeric fiber
comprises a polymer core with a high melting temperature surrounded
by a sheath with a lower melting temperature.
[0024] In another embodiment, the inert matrix is a particulate
network.
[0025] In another embodiment, the particulate network comprises
polyethylene particles.
[0026] In another embodiment, the adsorbent particles are
immobilized in the matrix.
[0027] In another embodiment, the low molecular weight compound is
a nucleic acid targeting agent or a photosensitizer.
[0028] In another embodiment, the low molecular weight compound is
an acridine derivative, a psoralen derivative or a dye.
[0029] In another embodiment, the low molecular weight compound is
a biological response modifier.
[0030] In another embodiment, the biological response modifier is
activated complement.
[0031] In another embodiment, the low molecular weight compound is
a quencher.
[0032] In another embodiment, the low molecular weight compound is
a polyamine derivative.
[0033] In another embodiment, the desired biological activity
maintained by the cells is suitability for infusion.
[0034] In another embodiment, the biological composition comprises
platelets.
[0035] In another embodiment, treatment of the biological
composition with a device over a period of about 5 days results in
less than about a 10% loss in platelet count.
[0036] In another embodiment, the device comprises an inert matrix
containing highly adsorbent particles having a diameter ranging
from about 100 .mu.m to about 1500 .mu.m. The cells in the
biological composition treated with the device substantially
maintain their desired biological activity. The device is for use
in a batch process. The biological composition comprises platelets.
Treatment of the biological composition with the device over a
period of about 5 days results in less than about a 10% loss in
platelet count. The device further comprises a container that is
compatible with the biological composition, and wherein the inert
matrix is a synthetic polymer fiber comprising a polymer core with
a high melting point surrounded by a sheath with a lower melting
temperature, and wherein the adsorbent particles are polyaromatic
adsorbents comprising a hypercrosslinked polystyrene network, and
wherein the surface area of the particles is greater than about 750
m.sup.2/g, and wherein the adsorbent particles are immobilized in
the matrix. An example of desired biological activity maintained by
the cells includes, without limitation, suitability for infusion.
Examples of low molecular weight compounds reduced by the device
include, without limitation, an acridine derivative, a psoralen
derivative, a dye and a biological response modifier such as
activated complement. A device of this embodiment, as an example,
may be used to treat the biological composition at a temperature of
22.degree. C. for between about 0.5 hour and about 7 days.
[0037] In another embodiment, the biological composition comprises
red blood cells.
[0038] In another embodiment, treatment of the biological
composition with the device over a period of up to 35 days results
in less than about 1% hemolysis.
[0039] In another embodiment, the device comprises an inert matrix
containing highly adsorbent particles. The cells in the biological
composition treated with the device substantially maintain their
desired biological activity. The device is for use in a batch
process. The biological composition comprises red blood cells.
Treatment of the biological composition with the device over a
period of up to 35 days results in less than about 1% hemolysis.
The device further comprises a container that is compatible with
the biological composition, and wherein the inert matrix is a
synthetic polymer fiber comprising a polymer core with a high
melting point surrounded by a sheath with a lower melting
temperature, and wherein the adsorbent particles are activated
carbon particles derived from a synthetic source, and wherein the
diameter of the particles ranges from about 300 .mu.m to about 900
.mu.m, and wherein the surface area of the particles is greater
than about 750 m.sup.2/g, and wherein the adsorbent particles are
immobilized in the matrix. An example of desired biological
activity maintained by the cells includes, without limitation,
suitability for infusion. Examples of low molecular weight
compounds reduced by the device include, without limitation, an
acridine derivative, a psoralen derivative, a dye, a biological
response modifier such as activated complement, and a quencher such
as glutathione.
[0040] The present invention also provides a method for reducing
the concentration of a low molecular weight compound in a
biological composition containing cells, wherein the cells in the
biological composition treated with a device substantially maintain
their desired biological activity after treatment for between 0.5
hour and 5 weeks.
[0041] In one embodiment, the device comprises an inert matrix
containing highly adsorbent particles having a diameter ranging
from about 100 .mu.m to about 1500 .mu.m. The cells in the
biological composition treated with the device substantially
maintain their desired biological activity. The device is for use
in a batch process.
[0042] In another embodiment, the device comprises an inert matrix
containing highly adsorbent particles having a diameter ranging
from about 100 .mu.m to about 1500 .mu.m. The cells in the
biological composition treated with the device substantially
maintain their desired biological activity. The device is for use
in a batch process. The biological composition comprises platelets.
Treatment of the biological composition with the device over a
period of about 5 days results in less than about a 10% loss in
platelet count. The device further comprises a container that is
compatible with the biological composition, and wherein the inert
matrix is a synthetic polymer fiber comprising a polymer core with
a high melting point surrounded by a sheath with a lower melting
temperature, and wherein the adsorbent particles are polyaromatic
adsorbents comprising a hypercrosslinked polystyrene network, and
wherein the surface area of the particles is greater than about 750
m.sup.2/g, and wherein the adsorbent particles are immobilized in
the matrix.
[0043] In another embodiment, the device comprises an inert matrix
containing highly adsorbent particles. The cells in the biological
composition treated with the device substantially maintain their
desired biological activity. The device is for use in a batch
process. The biological composition comprises red blood cells.
Treatment of the biological composition with the device over a
period of up to 35 days results in less than about 1% hemolysis.
The device further comprises a container that is compatible with
the biological composition, and wherein the inert matrix is a
synthetic polymer fiber comprising a polymer core with a high
melting point surrounded by a sheath with a lower melting
temperature, and wherein the adsorbent particles are activated
carbon particles derived from a synthetic source, and wherein the
diameter of the particles ranges from about 300 .mu.m to about 900
.mu.m, and wherein the surface area of the particles is greater
than about 750 m.sup.2/g, and wherein the adsorbent particles are
immobilized in the matrix.
[0044] In another embodiment, the cells in the biological
composition treated with the device substantially maintain their
desired biological activity after being treated with the device for
between about 1.0 hour and 5 weeks.
[0045] In another embodiment, the low molecular weight compound is
an acridine derivative, a psoralen derivative or a dye.
[0046] In another embodiment, the low molecular weight compound is
a biological response modifier.
[0047] In another embodiment, the biological response modifier is
activated complement.
[0048] In another embodiment, the biological composition is treated
with the device at a temperature of about 22.degree. C. for between
about 0.5 and about 36 hours.
[0049] In another embodiment, the biological composition is treated
with the device at a temperature of about 22.degree. C. for between
about 0.5 and about 24 hours.
[0050] In another embodiment, the biological composition is treated
with the device at a temperature of about 22.degree. C. for between
about 0.5 and about 12 hours.
[0051] In another embodiment, the biological composition is treated
with the device at a temperature of about 22.degree. C. for between
about 0.5 and about 24 hours and is subsequently treated with the
device at a temperature of about 4.degree. C. for up to 5
weeks.
[0052] The present invention also provides a composition containing
cells, wherein the biological composition is suitable for infusion.
The biological composition is produced by treating a biological
composition containing cells with a device for a period between
about 0.5 hour and 5 weeks.
[0053] In one embodiment, the device comprises an inert matrix
containing highly adsorbent particles having a diameter ranging
from about 100 .mu.m to about 1500 .mu.m. The cells in the
biological composition treated with the device substantially
maintain their desired biological activity. The device is for use
in a batch process.
[0054] In another embodiment, the device comprises an inert matrix
containing highly adsorbent particles having a diameter ranging
from about 100 .mu.m to about 1500 .mu.m. The cells in the
biological composition treated with the device substantially
maintain their desired biological activity. The device is for use
in a batch process. The biological composition comprises platelets.
Treatment of the biological composition with the device over a
period of about 5 days results in less than about a 10% loss in
platelet count. The device further comprises a container that is
compatible with the biological composition, and wherein the inert
matrix is a synthetic polymer fiber comprising a polymer core with
a high melting point surrounded by a sheath with a lower melting
temperature, and wherein the adsorbent particles are polyaromatic
adsorbents comprising a hypercrosslinked polystyrene network, and
wherein the surface area of the particles is greater than about 750
m.sup.2/g, and wherein the adsorbent particles are immobilized in
the matrix.
[0055] In another embodiment, the device comprises an inert matrix
containing highly adsorbent particles. The cells in the biological
composition treated with the device substantially maintain their
desired biological activity. The device is for use in a batch
process. The biological composition comprises red blood cells.
Treatment of the biological composition with the device over a
period of up to 35 days results in less than about 1% hemolysis.
The device further comprises a container that is compatible with
the biological composition, and wherein the inert matrix is a
synthetic polymer fiber comprising a polymer core with a high
melting point surrounded by a sheath with a lower melting
temperature, and wherein the adsorbent particles are activated
carbon particles derived from a synthetic source, and wherein the
diameter of the particles ranges from about 300 .mu.m to about 900
.mu.m,and wherein the surface area of the particles is greater than
about 750 m.sup.2/g, and wherein the adsorbent particles are
immobilized in the matrix.
[0056] The present invention also provides a device for reducing
the concentration of small organic compounds in a blood product
while substantially maintaining a desired biological activity of
the blood product.
[0057] In one embodiment, the device comprises highly porous
adsorbent particles, wherein the adsorbent particles are
immobilized by an inert matrix.
BRIEF DESCRIPTION OF THE DRAWINGS
[0058] 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.
[0059] FIG. 2 schematically represents a portion of one embodiment
of the immobilized adsorbent media of the present invention.
[0060] 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.
[0061] FIG. 4 diagrammatically represents a cross-sectional view of
one embodiment of immobilized adsorbent media in which the
adsorbent beads are immobilized within the fibers of the
immobilized adsorbent media and the heat seals that encompass
samples of fiberized resin.
[0062] FIG. 5 is a graph showing a comparison of adsorption
kinetics for removal of aminopsoralens from platelets with
Dowex.RTM. XUS-43493 and Amberlite.RTM. XAD-16 HP loose adsorbent
beads and immobilized adsorbent media containing Amberlite.RTM.
XAD-16.
[0063] FIG. 6 is a graph showing a comparison of adsorption
kinetics for removal of aminopsoralens from platelets with
immobilized adsorbent media containing Amberlite.RTM. 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.
[0064] 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.RTM. XUS-43493 beads.
[0065] 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.RTM. XAD-16 and Dowex.RTM.
XUS-43493 for aminopsoralens.
[0066] 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.RTM. XAD-16 (bottom) and Dowex.RTM. 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.
[0067] FIG. 10 is a graph showing a comparison of adsorption of
aminopsoralens over a 3-hour period from plasma using
Amberlite.RTM. XAD-16 wet in several different solutions.
[0068] FIG. 11 is a graph showing a comparison of the kinetics of
adsorption of methylene blue over a 2-hour period from plasma.
[0069] FIG. 12 depicts the chemical structures of acridine,
acridine orange, 9-amino acridine, and
5-[(.beta.-carboxyethyl)amino]acridine.
[0070] FIG. 13 is a graph showing plots the data for adenine
capacity (y-axis) and 5-[(.beta.-carboxyethyl)amino]acridine
capacity (x-axis) for various resins.
[0071] FIGS. 14A and 14B is a graph showing a comparison of the
adsorption kinetics for removal of
5-[(.beta.-carboxyethyl)amino]acridine with Dowex.RTM. XUS-43493
and Purolite.RTM. MN-200 and Amberlite.RTM. XAD-16 HP.
[0072] FIG. 15 is a graph showing a comparison of the adsorption
kinetics for removal of 9-amino acridine and acridine orange with
Dowex.RTM. XUS-43493.
[0073] FIG. 16 is an illustration of a batch configuration for the
immobilized adsorption device (IAD).
[0074] FIG. 17 is a graph showing a comparison of the adsorption
isotherms for various Ambersorbs as compared to Purolite
MN-200.
[0075] FIG. 18 is a graph showing a comparison of the levels of
5-[(.beta.-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 G277 IAD (500 g/m.sup.2) over 4 weeks of
storage at 4.degree. C.
[0076] FIG. 19 is a graph showing the effect of enclosure material
on adsorption kinetics for 5-[(.beta.-carboxyethyl)amino]acridine
in PRBCs.
[0077] 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.
[0078] FIG. 21 is a graph showing a comparison of percent hemolysis
for the non-immobilized and immobilized adsorbent particle Pica
G-277 activated carbon.
[0079] FIG. 22 is a graph showing kinetics for removal
4'-(4-amino-2-oxa) butyl-4,5',8 trimethylpsoralen from platelet
concentrates.
[0080] 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
[0081] 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.
[0082] 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.
[0083] Definitions
[0084] 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 FIG. 12).
[0085] 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. 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.
[0086] The term "aromatic," "aromatic compounds," and the like
refer broadly to compounds with rings of atoms having delocalized
electrons. The monocyclic compound benzene (C.sub.6H.sub.6) 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).
[0087] 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).
[0088] 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 recipient. In a preferred embodiment,
biocompatible housings are blood bags, including a platelet storage
container or red blood cell storage container.
[0089] 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.
[0090] 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.
[0091] The term "blood bag" refers to a form of blood product
container.
[0092] 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.
[0093] 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 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.
[0094] 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).
[0095] 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.
[0096] 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 azocannine 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 of the present invention may be practiced in
conjunction with any dye that is a cyclic compound.
[0097] 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.
[0098] 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.
[0099] 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.
[0100] The phrase "high temperature activation process" refers to a
high temperature process that typically results in changes in
surface area, porosity and surface chemistry of the treated
material due to pyrolysis and/or oxidation of the starting
material.
[0101] 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.
[0102] 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.
[0103] 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.
[0104] The term "macropores" generally means that the diameter of
the pores is greater than about 500 .ANG.. The term micropores
refers to pores with diameters less than about 20 .ANG.. The term
mesopores refers to pores with diameters greater than about 20
.ANG.. and less than about 500 .ANG..
[0105] The term "macroporous" is used to describe a porous
structure having a substantial number of pores with diameters
greater than about 500 .ANG..
[0106] 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.
[0107] 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 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.
[0108] 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.
[0109] 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.
[0110] 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.
[0111] The term "polystyrene network" refers broadly to polymers
containing styrene (C.sub.6H.sub.5CH.dbd.CH.sub.2) monomers; the
polymers may be linear, consisting of a single covalent alkane
chain with phenyl substituents, or cross-linked, generally with m-
or p-phenylene residues or other bifunctional or hypercrosslinked
structure, to form a two-dimensional polymer backbone or 3D
network.
[0112] The term "psoralen removal means" refers to a substance or
device that is able to remove greater than about 80% 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.
[0113] 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 of
about 99%.
[0114] 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%.
[0115] 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.
[0116] 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.
[0117] 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.
[0118] 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 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. Pat. Nos. 3,975,481,
4,110,391, 4,460,530, 4,880,843 and 4,925,880, incorporated by
reference herein. 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.
[0119] 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 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 "wetting
agent" is distinguishable from a "stabilizing agent" in that the
former is believed to reopen adsorbent pores of those 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 will. A general
discussion of wetting and wetting agents is set forth in U.S. Pat.
No. 5,501,795 to Pall et al., hereby incorporated by reference.
[0120] 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 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 more preferably 10% after 5 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
Klein H. G. , ed. Standards for Blood Banks and Transfusion
Services, 17.sup.th Ed., Bethesda, Md.: American Association of
Blood Banks, 1996, incorporated by reference herein.
[0121] 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
maintains cell viability or a suitable coagulation factor
level.
[0122] 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.
[0123] 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 compounds (e.g.
inactivation compounds, response modifiers) such that infusion
provides intended function without detrimental side effects.
[0124] 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).
[0125] The term "4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl psoralen"
is alternatively referred to as "S-59."
[0126] The term "N-(9-acridinyl)-.beta.-alanine" is alternatively
referred to as "5-[(.beta.-carboxyethyl)amino]acridine." It is
further alternatively referred to as "S-300."
[0127] The term "XUS-43493" is alternatively referred to as
"Optipore 493."
[0128] Adsorbent Particles
[0129] 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.
[0130] 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 .mu.m in diameter and less than about 1500 .mu.m in
diameter; preferably, the particles are between about 200 .mu.m and
about 1300 .mu.m in diameter; more preferably, the particles are
between about 300 .mu.m and about 900 .mu.m in diameter.
[0131] A high surface area is characteristic of the particles.
Preferably, the particles have a surface area between about 750
m.sup.2/g and about 3000 m.sup.2/g. More preferably, the particles
have a surface area between about 1000 m.sup.2/g and about 3000
m.sup.2/g.
[0132] 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.
[0133] 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.RTM., which is available from PICA USA Inc.
(Columbus, Ohio), Norite ROX 0.8, which is available from Norit
Americas, Inc. (Atlanta, Ga.), Ambersorb.RTM. 572, which is
available from Rohm & Haas (Philadelphia, Pa.), and G-277.RTM.,
which is available from PICA (Columbus, Ohio).
[0134] In another preferred embodiment, the particles can be
hydrophobic resins. Nonlimiting examples of hydrophobic resins
include the following polyaromatic adsorbents: Amberlite.RTM.
adsorbents (e.g., Amberlite.RTM. XAD-2, XAD-4, and XAD-16),
available from Rohm and Haas (Philadelphia, Pa.); Amberchromo
adsorbents available from Toso Haas (TosoHass, Montgomeryville,
Pa.); Diaion.RTM.//Sepabeads.RTM. Adsorbents (e.g., Diaion.RTM.
HP20), available from Mitsubishi Chemical America, Inc. (White
Plains, N.Y.); Hypersol-Macronet.RTM. Sorbent Resins (e.g,
Hypersol-Macronet.RTM. Sorbent Resins MN-200, MN-150 and MN-400)
available from Purolite (Bala Cynwyd, Pa.); and Dowex.RTM.
Adsorbents (e.g, Dowex.RTM. XUS-40323, XUS-43493, and XUS-40285),
available from Dow Chemical Company (Midland, Mich.).
[0135] Preferred particles are hydrophobic resins which are
polyaromatic adsorbents comprising a hypercrosslinked polystyrene
network, such as Dowex.RTM. XUS-43493 (known commercially as
Optipore.RTM. L493 or V493) and Purolite MN-200.
[0136] Hypercrosslinked polystyrene networks, such as Dowex.RTM.
XUS-43493 and Purolite MN-200 are non-ionic macroporous and
macroreticular resins. The non-ionic macroreticular and macroporous
Dowex.RTM. XUS-43493 has a high affinity for 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 (ie. 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.
[0137] Hypercrosslinked polystyrene networks, such as Dowex.RTM.
XUS-43493 and Purolite MN-200 are preferably in the form of
spherical particles with a diameter range of about 200 .mu.m to
about 1300 .mu.m. Adsorbent particles, including for example,
Dowex.RTM. XUS-43493, preferably have extremely high internal
surface areas and relatively small pores (e.g. average diameter 46
.ANG.). The internal surface area of the particle can be from about
300 to about 1100 m.sup.2/g; preferably about 900 to about 1100
m.sup.2/g; most preferably about 1100 m.sup.2/g. The majority of
the pores of the particle can be greater than 25 .ANG.and less than
800 .ANG.; preferably from about 25 .ANG.to about 150 .ANG.; most
preferably from about 25 .ANG.to about 50 .ANG.. 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. Puroliteo has many similar characteristics to Dowex.RTM.
XUS-43493, such as high affinity for psoralens and superior wetting
properties, and is also a preferred adsorbent particle.
[0138] 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 levels of extractable
residual monomer, crosslinkers and other organic extractables.
[0139] 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 copolymerization 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.RTM.
XAD-4 is produced by such a procedure.
[0140] In contrast to the conventional networks described above,
the preferred adsorbents of the present invention (e.g., Dowex.RTM.
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.
[0141] 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 Polymers 13:27-42 (1990); Tsyurupa et al.,
Reactive Polymers 25:69-78 (1995), hereby incorporated by
reference.
[0142] Several cross-linking agents have been successfully employed
to produce the bridges between polystyrene chains, including
p-xylene dichloride (XDC), monochlorodimethyl ether (MCDE),
1,4-bis-chloromethyldiphenyl (CMDP),
4,4'-bis-(chloromethyl)biphenyl (CMB), dimethylformal (DMF),
p,p'-bis-chloromethyl-1,4-diphenylbutane (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. Pat.
No. 3,729,457, hereby incorporated by reference.
[0143] 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 (i.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.
[0144] Some of the structurally-related characteristics of the
above-described adsorbent particles are summarized in Table A.
1TABLE A Mean Surface Mean Pore Mesh Resin Chemical Nature Area
(m.sup.2/g) Diam. (.ANG.) Size (.mu.m) Amberlite .RTM. Adsorbents -
Robin and Haas XAD-2 polyaromatic 300 90 20-60 XAD-4 polyaromatic
725 40 20-60 XAD-7 polymethacrylate 450 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 .RTM. Adsorbents - Toso Haas CG-71m
polymethacrylate 450-550 200-300 50-100 CG-71c polymethacrylate
450-550 200-300 80-160 CG-161m polyaromatic 800-950 110-175 50-100
CG-161c polyaromatic 800-950 110-175 80-160 Dialon .RTM.//Sepabeads
.RTM. Adsorbents - Mitsubishi Chemical HP20 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 polymethacrylate 500 200-800 25-50 HP20SS
polyaromatic 500 300-600 75-150 SP20MS polyaromatic 500 300-600
50-100 Dowex .RTM. Adsorbents - Dow Chemical Company XUS-40285
functionalized 800 25 20-50 XUS-40323 polyaromatic 650 100 16-50
XUS-43493 polyaromatic 1100 46 20-50
[0145] Processing the Adsorbent Particles
[0146] 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.
[0147] 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 adsorbent is certified
sterile (USP XXI), pyrogen-free (LAL), and free of detectable
extractables (DVB and total organics).
[0148] 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.RTM. 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.
[0149] One advantage of the cleaned/processed adsorbent is an
extremely low level of particles with diameters less than 30 .mu.m.
Preliminary testing on adsorbents (Dowex.RTM. XUS-43493 and
Amberlite.RTM. 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 .mu.m) were essentially absent.
[0150] The Use of Wetting Agents and Stabilizing Agents with
Adsorbent Resins
[0151] Methods may be used for preventing drying and loss of
adsorption capacity of particles, such as Amberlite.RTM. which lose
some of their adsorption capacity under certain conditions (e.g.,
drying).
[0152] 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
.gamma.-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.
[0153] 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.RTM.
XUS-43493 and Purolite.RTM. MN-200) generally do not require a
wetting agent because the crosslinks prevent the pores from
collapsing. Unlike Amberlite.RTM. XAD-16, these macroreticular
adsorbents retain a very high proportion of their initial activity
when they are dried.
[0154] In a third method, loss of adsorption capacity upon drying
may be prevented by hydrating Amberlite.RTM. XAD-16 and related
adsorbents (e.g., Amberlite.RTM. XAD-4) in the presence of a
non-volatile wetting agent. For example, when using Amberlite.RTM.
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.
[0155] 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.
[0156] 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, Ill.). Adsorbent beads
like Amberlite.RTM. XAD-16 may be stabilized in ethanol and
glycerol.
[0157] 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(OCH.sub.2CH.sub.2).sub.nOH, 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).
[0158] Inert Matricesfor Particle Immobilization
[0159] 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.
[0160] Most preferably, the synthetic fibers are polyester fibers
(Air Quality Filtration (AQF), a divisior 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.
[0161] 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).
[0162] Exemplary natural polymer fibers include cellulose fibers
derived from a variety of sources, such as jute, kozu, krafi and
manila hemp. Networks of synthetic or natural polymer fibers have
been used to make filters as described in U.S. Pat. Nos. 4,559,145
and 5,639,376, which are herein incorporated by reference.
[0163] 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 polyolefins, such as polyethylene.
[0164] Polymeric fibers such as those described above may be
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.
[0165] Immobilization of Particles
[0166] 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 fiber network with adsorbent particles immobilized
therein.
[0167] 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.
[0168] Technology for immobilization of adsorbent beads on a fiber
network to construct air filters has been described in U.S. Pat.
No. 5,486,410 and U.S. Pat. No. 5,605,746, incorporated by
reference herein. As depicted in FIG. 1, the polymer fibers 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. Pat. No. 5,190,657 to Heagle et al., hereby
incorporated by reference. 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.degree. C..times.1 min.)
causing 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 FIG. 2. As depicted in FIG. 3 and
FIG. 4 (not to scale), generally speaking, the fiber networks
contain three layers; two outer layers 607 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 FIG.
3 and FIG. 4, heat seals 608 may be made in the resulting fiberized
resin at predetermined intervals; for example, heat seals can be
made in the fiberized 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 blood 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 FIG.
4, the adsorbent beads 606 are not secured to the fibers
themselves, but 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.
[0169] 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.
[0170] 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), which are herein
incorporated by reference. The particles can be immobilized using a
wet-laid process, as described in U.S. Pat. Nos. 4,559,145 and
4,309,247, which are herein incorporated by reference. The
particles can be immobilized using a melt-blown process, as
described in U.S. Pat. No. 5,616,254, which is herein incorporated
by reference. Where a wet-laid process is used to construct a
matrix from natural polymer fibers, the inert matrix preferably
includes a binding agent to bond the adsorbent particles to the
fibers. Nonlimiting examples of binding agents include melamine,
polyamines and polyamides. The matrix typically contains 1% or less
of such binding agents.
[0171] 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.
[0172] 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
fibers 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.
[0173] Preferably, the resulting adsorption medium comprises known
amounts of adsorbent per area. The adsorbent per area is from about
I 00 g/m.sup.2 to about 500 g/m.sup.2, preferably from about 250
g/m.sup.2 to about 350 g/m.sup.2. 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).
[0174] 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 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 g/m.sup.2, more preferably about 50-80% adsorbent at a
loading of about 250-350 g/m.sup.2.
[0175] Coating the Adsorbent Particles
[0176] The surface hemocompatibility 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, N.Y.) and
cellulose-based polymers, erg, ethyl cellulose, which may be
obtained from Dow Chemical (Midland, Mich.). 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 hemocompatibility and reduce the risk
of small particle generation due to mechanical breakdown.
[0177] The 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.
[0178] The coating can be applied in a number of different methods,
including radio frequency glow discharge polymerization, as
described in U.S. Pat. No. 5,455,040, which is hereby incorporated
by reference and the Wurster coating process (performed by
International Processing Corp. (Winchester, Ky.).
[0179] 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.RTM. XUS-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.
[0180] 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.
[0181] 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.
[0182] 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%).
[0183] 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.
[0184] Devices
[0185] Devices are provided for reduction of compounds from
biological compositions containing cells. The device is a batch
device. An example of a batch device is shown in FIG. 16. Batch
devices are known in the literature and are described, for example,
in PCT publication WO 96/40857, incorporated by reference
herein.
[0186] 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 the adsorbent medium and the bag is
agitated for a specified period of time.
[0187] For example, in one embodiment, an adsorption medium, e.g.,
immobilized Dowex.RTM. XUS-43493, is placed inside a blood product
container (e.g., a PL 146 Plastic container (BaxterHealthcare Corp.
(Deerfield, Ill.) kept on either a platelet shaker (Helmer
Laboratories (Novesvill, Ind.)) or rotator (Helmer Laboratories
(Novesvill, Ind.)) 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.degree. C. to about
22.degree. C.
[0188] Methods can be used to reduce the presence of adsorbent
particles that may come loose from the adsorbent medium.
[0189] 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.RTM. 200, 800,
1200 hydrophilic polyethylene sulfonate (PES) membranes (Gelman
Sciences (Ann Arbor, Mich.)); Durapore.RTM. hydrophilic modified
polyvinylidene difluoride (PVDF) (Mantee America Corp. (San Diego,
Calif.)) and hydrophilic modified polysulfone membranes with
integrated hydrophobic vents, e.g. Gemini membranes, (Millipore
(Marlborough, Mass.)); and membranes comprising polycarbonate with
a polyvinylidene coating (Poretics (Livermore, Calif.)). The
containers can be sterilized after addition of the adsorption
medium.
[0190] Preferred Embodiment for Biological Compositions Containing
Cells
[0191] 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.
[0192] Biological response modifiers like the anaphalatoxin C3a and
the terminal membrane attack complex SC5b-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.
[0193] 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 SC5b-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.
[0194] 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
m.sup.2/g.
[0195] Particularly preferred particles for this embodiment are
polyaromatic adsorbents comprising a hypercrosslinked polystyrene
network, such as Dowex.RTM. XUS-43493 or Purolite MN-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.
[0196] Exemplary compounds that are reduced or controlled by the
devices, materials and methods of this embodiment are psoralens,
psoralen derivatives, isopsoralens, psoralen photoproducts,
acridines, acridine derivatives, methylene blue, plastic
extractables, biological response modifiers, quenchers and
polyamine derivatives.
[0197] 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., less than 10%
loss). One method contemplated by the present invention allows
extended storage by improving the hemocompatibility of the
adsorbent surface.
[0198] 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 5 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 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.
[0199] 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.
[0200] 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 m.sup.2/g.
[0201] 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.
[0202] In one embodiment the particles used in devices for 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 ROX 0.8, which
is available from Norit Americas Inc. (Atlanta, Ga.); and G-277,
which is available from Pica U.S.A. (Columbus, Ohio).
[0203] 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 .mu.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.
[0204] 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.RTM. adsorbents (e.g., Amberlite.RTM. XAD-2, XAD-4, and
XAD-16), available from Rohm and Haas (Philadelphia, Pa.);
Amberchrom.RTM. adsorbents available from Toso Haas (Toso Haas,
Montgomeryville, Pa.); and Diaion.RTM.//Sepabeads.RTM. Adsorbents
(e.g., Diaion.RTM. HP20), available from Mitsubishi Chemical
America, Inc. (White Plains, N.Y.). In a particularly preferred
embodiment the particles are Hypersol-Macronet.RTM. Sorbent Resins
(e.g., Hypersol-Macronet.RTM. Sorbent Resins MN-200, MN-1 50 and
MN-400) available from Purolite (Bala Cynwyd, Pa.) or Dowex.RTM.
Adsorbents (e.g., Dowex.RTM. XUS-43493, and XUS-40285), available
from Dow Chemical Company (Midland, Mich.).
[0205] 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 Unitika (Osaka, Japan) and
Hoechst Trevira (Augsberg, Germany).
[0206] 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.
[0207] It is preferable that the device contact the red blood
composition at about 4.degree. C. or about 22.degree. 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 5
contacted with the device at about 22.degree. C. for no more than
about 36 hours. In another embodiment, the red blood cell
composition is contacted with the device at about 22.degree. C. for
no more than about 24 hours. In another embodiment, the red blood
composition is contacted with the device at about 22.degree. C. for
no more than about 12 hours. In another embodiment, the red blood
composition is contacted with the device at about 22.degree. C. for
no more than 6 hours.
[0208] 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.degree. C. for a time ranging from about
0.5 to about 24 hours and then stored at about 4.degree. C. for up
to about 5 weeks. In another embodiment, the device is contacted
with the red blood composition at about 22.degree. C. for a time
ranging from about 0.5 to about 12 hours and then stored at about
4.degree. C. for up to about 5 weeks. In another embodiment, the
device is contacted with the red blood composition at about
22.degree. C. for a time ranging from about 0.5 to about 6 hours
and then stored at about 4.degree. C. for up to about 5 weeks.
[0209] The use of an adsorption medium comprising adsorbent
particles 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.
[0210] 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-Coming Diagnostics, Medfield, Mass.). 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.
[0211] 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
5-[(.beta.-carbethyoxyethyl)amino]-acridine and glutathione in a
red blood cell sample. Standard HPLC assays can be used to
determine concentrations of 5-[(.beta.-carboxyethyl)amino]acridine
and glutathione in red blood cells contacted with the device. Assay
mobile phases are 10 mM H.sub.3PO.sub.4 in HPLC water and 10 mM
H.sub.3PO.sub.4 in acetonitrile. Zorbax SB-CN and YMC ODSAM-303
columns are available from MacMod Analytical, Inc. (Chadds Ford,
Pa.) and YMC, Inc. (Wilmingtion, N.C.).
[0212] 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.
[0213] Applications
[0214] 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 photoactivation 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. Pat. Nos.
5,459,030 and 5,559,250, incorporated by reference herein, 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.
[0215] In one embodiment the present invention contemplates a
method of inactivating pathogens in solution, wherein the method
comprises: a) providing, in any order: i) 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
for 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
adsorbent particles, wherein the adsorbent particles are
immobilized by an inert matrix.
[0216] In addition to the pathogen inactivating compound, reactive
degradation products thereof can be reduced from the material such
as a blood product, for example prior to transfusion.
[0217] 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
component(s) of interest being collected and retained and the other
components being returned to the donor. The donor receives
replacement fluids during the reinfusion process to help compensate
for the volume and pressure loss caused by component removal.
Apherersis systems are described in PCT publication W096/40857,
hereby incorporated by reference.
[0218] Low Molecular Weight Compounds
[0219] 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 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.
[0220] Small Organic Compounds
[0221] A diverse set of small organic compounds can be adsorbed by
the device of the present invention. The molecules can be cyclic or
acyclic. In one embodiment the compounds are preferably, cyclic
compounds such as psoralens, acridines or dyes. In another
embodiment the compounds are thiols.
[0222] 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.
[0223] 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: 5'-bromomethyl-4,4',
8-trimethylpsoralen, 4'-bromomethyl-4,5',8-trimethylpsoralen,
4'-(4-amino-2-aza)butyl-4,5',8-t- rimethylpsoralen,
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'-(5-amino-2-aza)pentyl-4,5', 8-trimethylpsoralen,
4'-(6-amino-2-aza)hexyl-4,5',8-trimethylpsoralen,
4'-(7-amino-2,5-oxa)hep- tyl-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-trimethylpsorale- n, 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-2,6,1 1-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-trimethylpsor- alen. Preferably, the
psoralen is 4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl- psoralen.
[0224] Acridines
[0225] Nonlimiting examples of acridines include acridine orange,
acriflavine, quinacrine, N1, N1-bis
(2-hydroxyethyl)-N4-(6-chloro-2-metho-
xy-9-acridinyl)-1,4-pentanediamine,
9-(3-hydroxypropyl)aminoacridine, N-(9-acridinyl)glycine,
S-(9-acridinyl)-glutathione. In a preferred embodiment the acridine
is N-(9-acridinyl)-p-alanine, alternatively, named
5-[(.beta.-carboxyethyl)amino]acridine.
[0226] Dyes
[0227] Nonlimiting examples of dyes include phenothiazines such as
methylene blue, neutral red, toluidine blue, crystal violet and
azure A, phenothiazones such as methylene violet Bernthsen,
phthalocyanines such as aluminum 1,8,15, 22-tetraphenoxy-29H,3 1
H-phthalocyanine chloride and silica analogues, and hypericin.
Preferably, the dye is methylene blue or toluidine blue. More
preferably, the dye is methylene blue.
[0228] The term "thiazine dyes" includes dyes that contain a sulfur
atom in one or more rings. The most common thiazine dye is
inethylene blue [3,7-Bis(dimethylamino)-phenothiazin-5-ium
chloride). Other thiazine dyes include, but are not limited to,
azure A, azure C and thionine, as described e.g. in U.S. Pat. No.
5,571,666 to Schinazi.
[0229] 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, Mich.), and as described e.g. in
U.S. Pat. No. 5,571,666 to Schinazi, hereby incorporated by
reference.
[0230] Quenchers
[0231] 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 Number 282173000600,
filed Jan. 6, 1998, the disclosure of which is incorporated herein.
In this 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.
[0232] 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.
[0233] Plastic Extractables
[0234] The concentration of a group of low molecular weight
compounds that are extractables from plastic storage containers and
tubing used to handle biological 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). 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.
[0235] 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.
[0236] 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.
[0237] Biological Response Modifiers (BRMs)
[0238] 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 of Immunology, 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, C5a); cytokines such
as IL-1, IL-6 and IL-8; and chemokines such as RANTES and MIP.
[0239] The accumulation of BRMs in a blood product during storage
can adversely affect 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).
[0240] Polyamine Derivatives
[0241] 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.
[0242] Polyethylene Glycols
[0243] 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.
[0244] Examples of compounds that may be removed by the device of
the present invention include linear or branched polyethylene
glycols attached to activating moeities which may include cyanuryl
chloride, succinimidyl esters, oxycarbonyl imidazole derivatives,
nitrophenyl carbonate derivatives, glycidyl ether derivatives, and
aldehydes.
[0245] 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
[0246] 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.
[0247] In the experimental disclosure which follows, the following
abbreviations apply: eq (equivalents); M (Molar); .mu.M
(micromolar); N (Normal); mol (moles); mmol (millimoles); .mu.mol
(micromoles); nmol (nanomoles); g (grams); mg (milligrams); .mu.g
(micrograms); Kg (kilograms); L (liters); mL (milliliters);
.mu.L(microliters); cm (centimeters); mm (millimeters); .mu.m
(micrometers); nm (nanometers); min. (minutes); s and sec.
(seconds); J (Joules, also watt second); .degree. 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,
Wis.); Asahi (Asahi Medical Co., Ltd., Tokyo, Japan); Baker (J.T.
Baker, Inc., Phillipsburg, N.J.); Barnstead (Barnstead/Thermolyne
Corp., Dubuque, Iowa); Becton Dickinson (Becton Dickinson
Microbiology Systems; Cockeysville, Md.); Bio-Rad (Bio-Rad
Laboratories, Hercules, Calif.); Cerus (Cerus Corporation; Concord,
Calif.); Chrono-Log (Chrono-Log Corp.; Havertown, Pa.);
Ciba-Corning (Ciba-Coming Diagnostics Corp.; Oberlin, Ohio);
Consolidated Plastics (Consolidated Plastics Co., Twinsburg, Ohio);
Dow (Dow Chemical Co.; Midland, Mich.); Eppendorf (Eppendorf North
America Inc., Madison, WI); Gelman (Gelman Sciences, Ann Arbor,
Mich.); Grace Davison (W.R. Grace & Co., Baltimore, Md.);
Helmer (Helmer Labs, Noblesville, Ind.); Hoechst Celanese (Hoechst
Celanese Corp., Charlotte, N.C.); International Processing Corp.
(Winchester, Ky.); Millipore (Milford, Mass.); NIS (Nicolet, a
Thermo Spectra Co., San Diego, Calif.); Poretics (Livermore,
Calif.); Purolite (Bala Cynwyd, Pa.); Rohm and Haas (Chauny,
Fiance); Quidel (San Diego, Calif.); Saati (Stamford, Conn.);
Scientific Polymer Products (Ontario, N.Y.); Sigma (Sigma Chemical
Company, St. Louis, Mo.); Spectrum (Spectrum Chemical Mfg. Corp.,
Gardenia, Calif.); Sterigenics (Corona, Calif.); Tetko, Inc.
(Depew, N.Y.); TosoHaas (TosoHass, Montgomeryville, Pa.); Wallac
(Wallac Inc., Gaithersburg, Md.); West Vaco (Luke, W. Va.); YMC
(YMC Inc., Wilmington, N.C.); 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); ft.sup.3/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 (Luria
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{fourth root} (Mohm); PAS III (platelet additive
solution III); dH.sub.2O (distilled water); IAD (immobilization
adsorption device); SCD (sterile connection [connect] device).
[0248] One of the examples below refers to HEPES buffer. This
buffer contains 8.0 g of 137 mM NaCl, 0.2 g of 2.7 mM KCl, 0.203 g
of 1 mM MgCl.sub.2(6H.sub.20), 1.0 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.RTM. XAD-16
[0249] 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.RTM. XAD-16 was compared with devices
containing free (i.e., not immobilized) Amberlite.RTM. XAD-16 HP
and dowex.RTM. XUS-43493.
Preparation of Fiberized Resin and Adsorbent Beads
[0250] Immobilized adsorbent media containing Amberlite.RTM. 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.RTM.
XAD-16 at a loading of 130 g/m.sup.2 (i.e., each square meter of
fiber contained 130 g of adsorbent beads).
[0251] The fiberized resin was cut into squares (14 cm.times.14
cm), and the resulting sections contained approximately 2.5 g of
dry Amberlite.RTM. XAD-16. The Amberlite.RTM. 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.RTM. XAD-16 and other adsorbents are also
effective and are contemplated by the present invention. It should
be noted that fiberized resin containing other types of beads
(e.g., bridged or hypercrosslinked resins like Dowex.RTM.
XUS-43493) do not require a wetting step for effective psoralen
removal.
[0252] Amberlite.RTM. 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.RTM. 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.RTM. 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.times.7 cm square; 30 .mu.m openings) were then filled with 2.5
g (dry weight) of either the loose Amberlite.RTM. XAD-16 HP or
dowex.RTM. XUS-43493 beads.
[0253] The fiberized resin and adsorbent-containing pouches were
sterilized by autoclaving on "wet" cycle for 45 minutes at 121 CC.
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.
[0254] Contacting Fiberized Resin And Adsorbent Beads With
Psoralen-Containing Platelet Concentrate (PC)
[0255] 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 .mu.M
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/cm.sup.2 of UVA. Following illumination, the treated PCs were
transferred into the PL 2410 Plastic containers containing either
fiberized resin with immobilized Amberlite.RTM. XAD-16, loose
Amberlite.RTM. XAD-16 HP or loose dowex.RTM. XUS-43493, or into an
empty PL 2410 Plastic container as a control. The PL 2410 Plastic
containers (Baxter) were then placed on a Helmer platelet incubator
at 22.degree. C. and agitated at approximately 70
cycles/minute.
[0256] 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 KH.sub.2PO.sub.4, pH=3.5) containing
trimethylpsoralen (TMP) as the internal standard. Proteins and
other macromolecules were precipitated by incubating the samples at
4.degree. C. for 30 minutes. The samples were then centrifuged and
the supernatant was filtered (0.2 .mu.meter) and analyzed on a C-1
8 reversed phase column (YMC ODS-AM 4.6 mm .times.250 mm) by
running a linear gradient from 65% solvent A (25 mM
KH.sub.2PO.sub.4, pH =3.5), 35% B (methanol) to 80% B in 20
minutes.
[0257] 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 pH/Blood 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
[0258] 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 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.times.14 cm). As the data in FIG. 5
indicate, the kinetics of 4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl
psoralen adsorption are very comparable 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.
[0259] 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.times.14 cm fiberized XAD-16 (130 g resin/cm.sup.2) wet
in 30% ethanol; and iii) 2.5 g XUS-43493 (<5% 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.
[0260] As indicated in Table 1, the day-5 pH and pO.sup.2 values
were slightly elevated relative to the day 5 control for samples
containing non-immobilized beads (XAD-16 HP and XUS-43493). The
experiment with the fiberized resin had pH and PO.sub.2 values
which were more comparable to the control. Platelet counts
indicated a 9-22% platelet loss following 5 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 5) and performed better in
all in vitro assays when compared to device containing
non-immobilized XAD-16 or XUS-43493 adsorbent particles.
2TABLE 1 Platelet Hypo- Count Hypotonic (10.sup.11/30 Shape shock
GMP-140 Sample pH pO.sub.2 pCO.sub.2 0 mL) change Aggreg response
Morph (%) Control 6.98 56 31 4.40 .+-. Day 0 0.18 Control 6.89 80
27 4.21 .+-. 0.58 .+-. 83 .+-. 3 0.24 .+-. 297 60.6 Day 5 0.16 0.03
0.02 XAD-16 6.98 110 22 3.65 .+-. 0.38 .+-. 70 .+-. 6 0.29 .+-. 278
53.8 HP 0.09 0.09 0.09 (-13.3%) AD 6.90 72 28 3.82 .+-. 0.56 .+-.
80 .+-. 4 0.27 .+-. 292 55.8 AD-16 0.15 0.12 0.04 (-9.3%) XUS-43493
6.98 115 20 3.27 .+-. 0.37 .+-. 51 .+-. 2 0.64 .+-. 271 67.0 0.05
0.28 0.10 -22.3%)
[0261] Though an understanding of the mechanism underlying the
higher pH and pO.sub.2 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
pO.sub.2 values which were more comparable to the control than the
XUS-43493 or XAD-16 beads.
[0262] 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 XUS-43493 involves transfer of the platelets from
this device after 8 hours of exposure; this procedure results in
<5% loss in platelets.
[0263] 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/pO.sub.2, shape change, aggregation, morphology and GMP-140.
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
[0264] 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.
[0265] 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 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
[0266] 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.RTM. XAD-16 and for fiberized resin comprising
immobilized activated charcoal.
Preparation of Fiberized Resin
[0267] Hoechst Celanese prepared fiberized resin containing
Amberlite.RTM. XAD-16 HP (Rohm and Haas). The fiberized resin
containing the Aniberlite.RTM. 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/m.sup.2 (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.RTM.
XAD-16. The composition of the fibers for each fiberized resin was
the same.
[0268] The fiberized resin was cut into squares (14 cm.times.14
cm); the resulting sections contained approximately 2.5 g of dry
Amberlite.RTM. XAD-16. Next, the fiberized resin was sterilized by
autoclaving on "wet" cycle for 45 minutes at 121.degree. 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.
Contacting Fiberized Resin with Psoralen-Containing PC
[0269] 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 150 .mu.M
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 J/cm.sup.2 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.degree. C. and agitated at approximately 70 cycles/minute.
[0270] 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 KH.sub.2PO.sub.4, pH=3.5)
containing trimethylpsoralen (TMP) as the internal standard.
Proteins and other macromolecules were precipitated by incubating
the samples at 4.degree. C. for 30 minutes. The samples were then
centrifuged and the supernatant was filtered (0.2 .mu.meter) and
analyzed on a C-18 reversed phase column (YMC ODS-AM 4.6
mm.times.250 mm) by running a linear gradient from 65% solvent A
(25 mM KH.sub.2PO.sub.4, pH=3.5), 35% B (methanol) to 80% in 20
minutes.
[0271] 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 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
[0272] 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
(.ltoreq.0.5 .mu.M residual
4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl psoralen after 4
hours).
[0273] Platelet yield and in vitro platelet function for each of
the fiberized resin were also evaluated and the data are summarized
in Table 2.
3TABLE 2 Platelet Count (10.sup.11/300 Shape Aggrega- Sample pH
pO.sub.2 pCO.sub.2 mL) Change tion Control 7.07 59 23 3.34 .+-.
0.13 1.17 .+-. 90 .+-. 5 Day 0 0.07 Control 6.88 112 21 3.09 .+-.
0.22 0.52 .+-. 72 .+-. 8 Day 5 0.02 Fiberized Resin 6.93 117 20
2.55 .+-. 0.16 0.39 .+-. 69 .+-. 6 with (-17%) 0.13 XAD-16
Fiberized Resin 7.07 100 20 2.83 .+-. 0.04 0.74 .+-. 61 .+-. 0 with
(-8%) 0.05 AQF-500-B Fiberized Resin 6.99 107 20 2.82 .+-. 0.16
0.46 .+-. 65 .+-. 2 with (-9%) 0.06 AQF-375-B
[0274] Referring 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
pO2, 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.
[0275] The charcoal-based fiberized resin provided good results in
both the shape change and aggregation assays. Although the shape
change result for the AQF-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
[0276] 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.RTM. XUS-43493
and fiberized resin containing Amberlite.RTM. XAD-16 coated with
pHEMA.
Preparation of pHEMA-Coated Adsorbent Beads and Fiberized Resin
[0277] Dowex.RTM. XUS-43493 (commercially known as Optipore.RTM.
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 50 mg/ml.
[0278] 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.degree. C., and an air flow rate of approximately
200 ft.sup.3/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.
[0279] A device containing non-immobilized dry (uncoated)
Dowex.RTM. XUS-43493 (2.5 g) and pHEMA-coated Dowex.RTM. XUS-43493
(3.0 g or 5.0 g) were prepared by placing the desired mass of
adsorbent into a square 30 .mu.m polyester mesh pouch (7 cm.times.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 containers were sterilized by
either E-beam (NIS) or gamma irradiation (SteriGenics) to 2.5 MRad;
as previously alluded to, E-beam sterilization is generally
preferred.
[0280] Hoechst Celanese prepared fiberized resin containing
Amberlite.RTM. XAD-16 according to the method described in Example
1. The fiberized resin was cut into squares (14 cm.times.14 cm);
the resulting sections contained approximately 2.5 g of dry
Amberlite.RTM. XAD-16. The Amberlite.RTM. 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.degree. 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
[0281] 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 .mu.M
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/cm.sup.2 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.degree. C. and agitated at approximately 70
cycles/minute.
[0282] 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 KH.sub.2PO.sub.4, pH=3.5)
containing trimethylpsoralen (TMP) as the internal standard.
Proteins and other macromolecules were precipitated by incubating
the samples at 40.degree. C. for 30 minutes. The samples were then
centrifuged and the supernatant was filtered (0.2 pmeter) and
analyzed on a C-18 reversed phase column (YMC ODS-AM 4.6 mm
.times.250 mm) by running a linear gradient from 65% solvent A (25
mM KH.sub.2PO.sub.4, pH =3.5), 35% B (methanol) to 80% in 20
minutes.
[0283] 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
[0284] 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/65% synthetic media (PAS III) with pHEMA-coated and
uncoated Dowex.RTM. XUS-43493 beads. Specifically,
4'-(4-amino-2-oxa)butyl-4,5',8-- trimethyl psoralen removal with
3.0 g Dowex.RTM. XUS-43493 coated with 3.7% (w/w) 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-tri-
methyl psoralen removal with 2.5 g (dry) uncoated Dowex.RTM.
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
resistance to diffusion of 4'-(4-amino-2-oxa)butyl-4,5',8-tri-
methyl psoralen to the interior of the adsorbent particles.
[0285] The adsorption kinetics of
4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl psoralen removal were also
determined with 5.0 g dowex.RTM. 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
[0286] 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.
4 TABLE 3 Platelet Yield, Polymer Coating Adsorbent Day Coating
Level (%) Mass (g) 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
[0287] 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
[0288] 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 pum polyester
mesh pouch (7 cm.times.7 cm); the control comprised treated PC
stored in a PL 2410 Plastic container alone. The results are
summarized in Table 4.
5TABLE 4 Platelet GM-140 Count Shape Activation Sample pH pO.sub.2
(10.sup.11/300 L) Change Aggregation HSR Morphology (%) Control
7.03 67 4.94 .+-. -- -- -- -- -- Day 0 0.24 Control 6.87 84 4.58
.+-. 0.96 .+-. 76 .+-. 2 0.35 .+-. 295 70.0 Day 5 0.08 0.05 0.03
(-0%) Uncoated 6.95 128 3.49 .+-. 0.38 .+-. 44 .+-. 0 0.47 .+-. 260
58.3 XUS-43493 0.08 0.00 0.12 (-23.8%) 3.7% 6.93 108 4.19 .+-. 0.59
.+-. 59 .+-. 5 0.32 .+-. 240 58.5 pHEMA 0.09 0.03 0.04 XUS-43493
(-8.5%) Gamma 3.7% 6.88 92 4.35 .+-. 0.71 .+-. 69 .+-. 9 0.40 .+-.
264 54.8 pHEMA 0.11 0.06 0.04 XUS-43493 (-5.0%) E-beam
[0289] Referring to Table 4, the day-5 pH values appear to be very
stable compared to the control. The pO.sub.2 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.
[0290] 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 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.
[0291] Another hemocompatibility study was performed comparing a
device containing non-immobilized uncoated XUS-43493 (2.5 g beads;
30 .mu.m polyester mesh pouch; 7 cm.times.7 cm), uncoated fiberized
resin (14 cm.times.14 cm) containing Amberlite.RTM. XAD-16, and
fiberized resin containing pHEMA-coated Amberlite.RTM. XAD-16. The
favorable effect of pHEMA on the fiberized resin is indicated by
the results presented in Table 5
6TABLE 5 Platelet GM-140 Count Shape Activation Sample pH pO.sub.2
(10.sup.11/300 L) Change Aggregation HSR Morphology (%) Control
7.11 50 3.01 .+-. 0.10 -- -- -- -- -- Day 0 Control 6.91 122 2.85
.+-. 0.16 0.60 .+-. 78 .+-. 4 0.31 .+-. 302 72.5 Day 5 (-0%) 0.09
0.01 Uncoated 7.00 151 2.11 .+-. 0.07 0.35 .+-. 55 .+-. 4 0.57 .+-.
267 68.7 XUS- (-26.0%) 0.03 0.03 43493 Uncoated 6.94 126 2.23 .+-.
0.13 0.52 .+-. 82 .+-. 4 0.45 .+-. 297 65.3 Fiberized (-21.8%) 0.03
0.01 Resin with XAD-16 pHEMA- 6.95 120 2.44 .+-. 0.11 0.59 .+-. 86
.+-. 5 0.46 .+-. 305 63.0 Fiberized (-14.4%) 0.01 0.02 Resin with
XAD-16
[0292] As indicated by the data regarding in vitro platelet
function and platelet yield in Table 5, coating with pHEMA brought
the pO.sub.2 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
[0293] 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.RTM. XAD-16 and Dowex.RTM. XUS-43493 adsorbent
beads were used in the experiments of this example.
Methodology
[0294] Amberlite.RTM. XAD-16 HP (Rohm & Haas (Philadelphia,
Pa.)) and Dowex.RTM. XUS-43493 (Supelco, Bellefonte, Pa.) were
dried to <5% water in a 80.degree. 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.degree. C. oven; drying the adsorbent
at temperatures >120.degree. 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.
[0295] 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 subjected to any pre-treatment,
while the optimally wet samples of adsorbent were prepared by
wetting the adsorbent with 30% ethanol/70% dH.sub.2O. The
optimally-wet adsorbent was rinsed with dH.sub.2O to remove
residual ethanol. The adsorbent was prepared just prior to the
adsorption study to assure that drying did not occur.
[0296] Each of the adsorption studies was performed using 100%
human plasma containing 150 .mu.M
4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl psoralen spiked with
.sup.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
.sup.3H-4'-(4-amino-2-oxa- )butyl-4,5',8-trimethyl psoralen were
determined. Samples (200 .mu.L) 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.RTM. XAD-16 and Dowex.RTM.
XUS-43493 Treated with Glycerol
[0297] 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.RTM. XAD-16 and Dowex.RTM.
XUS-43493. Adsorbent samples were wet in the ethanol/glycerol
solutions for 15 minutes prior to drying for 48 hours at 80.degree.
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.
[0298] 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 for maintaining high
adsorbent capacity after drying. Control samples that were wet in
50% ethanol/50% dH.sub.2O (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.
[0299] 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).
[0300] 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 5.0 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.RTM. XAD-1 6 And dowex.RTM.
XUS-43493 Treated With Glycerol Or PEG
[0301] 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.RTM.
XAD-16 (bottom) and Dowex.RTM. 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.
[0302] As indicated by the data in FIG. 9 and predictable based on
the its "macronet" structure, the capacity of dowex.RTM. XUS-43493
was not affected by drying ("No Tx" sample). Conversely, the
Amberlite.RTM. 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.RTM. XAD-16 Treated with Glycerol
or PEG
[0303] 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-t- rimethyl psoralen over a 3-hour
period from 100% plasma using Amberlite.RTM. XAD-16 wet in several
different solutions. Specifically, the data in FIG. 10 represents
XAD-16 i) 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% ethanol/50% dH.sub.2O (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.RTM. XAD-16 samples that were wet in 50%
glycerol/50% ethanol or 50% 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.
[0304] The data presented in this example indicate that treating
Amberlite.RTM. 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
[0305] This example is directed at the ability of a variety of
different polymeric adsorbent materials to remove methylene blue
from fresh frozen plasma.
[0306] 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.RTM. XAD-16 HP (Rohm and Haas), MN-200
(Purolite), and Dowex.RTM. 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.
[0307] Fiberized resin containing XAD-16 was prepared as generally
described in Example 1. Briefly, a 2 cm.times.7 cm (i.e., 14
cm.sup.2) strip of fiberized resin containing 130 g/m.sup.2 XAD-16
was first wet in 70% ethanol and then rinsed exhaustively in
distilled water.
[0308] 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 .mu.M. 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.
[0309] A 30 mL sample of the 100% plasma containing 10 .mu.M
methylene blue was added to each vial. The vials were placed on a
rotator at room temperature. Samples (200 .mu.L) 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 KE.sub.2PO.sub.4,
pH=3.5). Proteins and other macromolecules were precipitated by
incubating the samples at 4.degree. C. for 30 minutes. Samples were
centrifuged and the supernatant was filtered (0.2 .mu.m) and
analyzed on a C-18 reversed phase column (YMC ODS-AM, 4.6
mm.times.250 mm) by running a linear gradient from 65% solvent A
(25 mM KH.sub.2PO.sub.4, pH=3.5), 35% B (Methanol) to 80% B in 20
minutes. The limit of detection for the HPLC assay was
approximately 0.5 .mu.M methylene blue.
[0310] 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 XUS-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 cm.sup.2 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.
[0311] 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
[0312] 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-[(.beta.-carboxyethyl)amino]acridine, from PRBCs.
[0313] The chemical structures of several acridines are depicted in
FIG. 12. As indicated in FIG. 12, 9-amino acridine and
5-[(.beta.-carboxyethyl- )amino]acridine are aminoacridines.
Resin Selectivity
[0314] Equilibrium adsorption of compound
5-[(.beta.-carboxyethyl)amino]ac- ridine was studied with several
types of resins. The polymeric adsorbent resins evaluated were
Amberlite.RTM. XAD-2, XAD-4, XAD-7, and XAD-16 HP (Rohm and Haas);
Purolite.RTM. MN-150, MN-170, MN-200, MN-300, MN-400, MN-500, and
MN-600; and Dowex.RTM. XUS-43493 and XUS-40285 (Dow Chemical Co.).
In addition, several Amberlite.RTM. anion exchange resins (IRA-958,
IRA-900, IRA-35, IRA-410 and IRA-120; Rohm and Haas) and an
Amberlite.RTM. weak cation exchange resin (DP-1; Rohm and Haas)
were tested. Moreover, several charcoals were evaluated, including
Hemosorba.RTM. AC (Asahi), PICA G277 and Norit A Supra (both
commercially available from American Norit). Finally, Porapak.RTM.
RDx (Waters), a styrene vinyl pyrrolidone copolymer which has
affinity for nitro aromatic compounds, was also tested.
[0315] Initially, an equilibrium adsorption study was performed
with samples of each resin to evaluate capacity for
5-[(.beta.-carboxyethyl)am- ino]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.RTM. (Baxter) containing 100 ,M of
5-[(.beta.-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.RTM. 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 than 100
.mu.M 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-[(.beta.-carboxyethyl)amino]acridine and adenine by HPLC.
[0316] For the HPLC procedure, each sample was diluted 2-fold with
sample diluent (50% methanol, 25 mM KH.sub.2PO.sub.4, pH=3.5), and
proteins and other macromolecules were precipitated by incubating
the samples at 4.degree. 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
.times.250 mm) by running a linear gradient from 75% solvent A (25
mM KH.sub.2PO.sub.4, pH =3.5), 25% B (methanol) to 80% B in 20
minutes. For 5-[( .beta.-carboxyethyl)aminojacr- idine removal,
estimated capacities (.mu.mole/g) at C.sub.f=1 .mu.M were
determined from single adsorption measurements with C.sub.o=100
.mu.M; the results are set forth in the second column of Table 6
(ND=not detectable). For adenine removal, estimated capacities
(mmole/g) at C.sub.f=1 mM were estimated from single adsorption
measurements with C.sub.o=1.5 mM. The results are set forth in the
third column of Table 6 (ND=not detectable).
7 TABLE 6 Estimated 5-[(13- carboxyethyl)amino]acri- Adenine
Capacity dine Capacity (.mu.mole/g) (mmole/g) Resin at C.sub.f = 1
.mu.M at C.sub.f = 1 mM 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-150 1.6 0.24 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 0277 5.0 ND Norit A Supra ND ND Porapak RDx
0.1 0.01 IRA-410-D 0.0 0.00
[0317] While the use of any resin capable of adsorbing acridine
compounds is contemplated, preferred resins selectively adsorb
5-[(.beta.-carboxyethyl)amino]acridine over adenine and exhibit low
hemolysis. FIG. 13 plots the data for adenine capacity (y-axis) and
5-[(.beta.-carboxyethyl)amino]acridine capacity (x-axis) for
various resins. As indicated by the data in Table 6 and FIG. 13,
the Dowex.RTM. XUS-43493 and Purolite.RTM. MN-200 resins had the
highest 5-[(.beta.-carboxyethyl)amino]acridine capacity; moreover,
when both high 5-[(.beta.-carboxyethyl)amino]acridine capacity and
low adenine capacity were considered, the Amberlite.RTM. XAD-16 HP
performed well.
[0318] The results from the in vitro experiments described in this
example suggest that Dowex.RTM. XUS-43493 and the related resin
Purolite.RTM. MN-200 are preferred resins for the removal of
acridine compounds from PRBCs.
Example 7
Removal of Acridine Compounds from Packed Red Blood Cells With
Styrene-divinylbenzene Adsorbents
[0319] 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.RTM. solution.
[0320] A. Experimental Procedures
[0321] For the experiments of this example, stock solutions (10 mM)
of 5-[(.beta.-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-[(.beta.-carboxyethyl)amino]acridine was added to a
solution containing 25% plasma/75% saline to achieve a final
concentration of 100 .mu.M, while the acridine orange and the
9-amino acridine compounds were added to solutions containing 25%
plasma/75% Adsolo (Baxter) Red Cell Preservation Solution to
achieve a final acridine concentration of 100 .mu.M.
[0322] The adsorbents utilized were Amberlite.RTM. XAD-16 HP (Rohln
and Haas); Purolite.RTM. MN-200, and Dowex.RTM. 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.RTM.
solution containing 100 .mu.M acridine was added to each tube
containing adsorbent. The tubes were then placed on a rotator at
room temperature, and 500 .mu.L samples of solution were removed at
various times and stored for later analysis.
[0323] Samples were analyzed by HPLC for levels of residual
acridine. Each sample was diluted 2-fold with sample diluent (50%
methanol, 25 mM KH.sub.2PO.sub.4, pH=3.5), and proteins and other
macromolecules were precipitated by incubating the samples at
4.degree. C. for 30 minutes. The samples were then centrifuged and
the supernatant was filtered (0.2 .mu.meter) and analyzed on a C-18
reversed phase column (YMC ODS-AM 4.6 mm.times.250 mm) by running a
linear gradient from 75% solvent A (25 mM KH.sub.2PO.sub.4,
pH=3.5), 25% B (methanol) to 80% B in 20 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
5-[(.beta.-carboxyethyl)amino]acridine.
[0324] B. Adsorption Kinetics
[0325] The kinetics of adsorption of
5-[(.beta.-carboxyethyl)amino]acridin- e over a 3-hour period from
25% plasma/75% saline were compared for Dowex.RTM. XUS-43493,
Purolite.RTM. MN-200, and Amberlite.RTM. XAD-16 HP. FIG. 14A and
FIG. 14B both represent residual 5-[(.beta.-carboxyethyl)ami-
no]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, 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
5-[(.beta.-carboxyethyl)amino]acridine.
[0326] FIG. 15 compares the adsorption kinetics for removal of
9-amino acridine and acridine orange from 25% plasma/75% Adsol.RTM.
with Dowex.RTM. XUS-43493. Referring to FIG. 15, 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. By comparison, the capacity of the dowex.RTM. XUS-43493 for
acridine orange was lower, which may be related to the presence of
two tertiary amino groups on acridine orange.
Example 8
Preparation of PRBCs Treated with a
5-[(.beta.-carboxyethyl)amino]acridine Derivative and
Glutathione
[0327] 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 PL146 (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-[(.beta.-carboxyethyl)amino]acridine derivative for a final
concentration of 300 82 M. The PRBC mixture was agitated for 1
minute using a wrist action shaker (manufacturer). The
5-[(.beta.-carboxyethyl)a- mino]acridine derivative and glutathione
treated PRBCs were allowed to incubate at room temperature
overnight to allow break down of the
5-[(.beta.-carboxyethyl)amino]acridine derivative into
5-[(.beta.-carboxyethyl)amino]acridine.
Example 9
HPLC Assay for 5-[(62 -carboxyethyl)amino]acridine in PRBC and PRBC
Supernatant
[0328] The sample (100 .mu.L) was diluted with 100 .mu.L of saline
and the resulting mixture was vortexed. To the solution was added
300 .mu.L of 20 mM H.sub.3PO.sub.4 in CH3CN, and the mixture was
vortexed for 15 sec. The sample was centrifuged at 13,200 rpm for 5
minutes. The supernatant (200 .mu.L) was diluted into 800 .mu.L of
cold 0.1 M HCl and vortexed. The sample was filtered into an
autosampler vial using a 0.2 .mu.m Gelman Acrodisc filter. The HPLC
conditions were as follows: (Manufacturer and part no. for column
and general column.) Column=Zorbax SB-CN, 4.6 mm.times.150 mm, 3.5
.mu.m particles; guard column=(4.6 mm .times.. 12.5 mm, 5 .mu.m
particles (Mac Mod Analytical, Inc. (Chadds Ford, Pa.)); the mobile
phase for A was 10 mM H.sub.3PO.sub.4 in HPLC water; the mobile
phase for C was 10 mM H.sub.3PO.sub.4 in acetonitrile; temperature
was 20.degree. C.; sample volume was 100 .mu.L; gradient conditions
were as follows:
8 Flow Rate Time A (%) C (%) (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
[0329] the DAD settings were as follows:
9 Detection Detection Reference Reference Signal Wavelength
Bandwidth Wavelength Bandwidth A 410 5 580 20 B 260 5 580 20
Example 10
HPLC Assay for Glutathione in PRBC and PRBC Supernatant
[0330] 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.times.4.6 mm, 5.mu.m particle; guard
column=Brownlee, 15.times.3.2 mm, 7 .mu.m particle; the mobile
phase for A was 10 mM H.sub.3PO.sub.4 in HPLC water; the mobile
phase for C was 10 mM H.sub.3PO.sub.4 in acetonitrile; the
temperature was 15.degree. C.; the sample volume was 75 .mu.L; the
gradient conditions were as follows:
10 Flow Rate Time A (%) C (%) (mL/min) 0.00 95.0 5.0 0.5 6.00 95.0
5.0 0.5 8.00 10.0 90.0 1.0 9.00 95.0 5.0 1.0 20.00 STOP STOP
STOP
[0331] the DAD settings were as follows:
11 Detection Detection Reference Reference Signal Wavelength
Bandwidth Wavelength Bandwidth A 205 10 600 100
Example 11
Method of screening Adsorbents
[0332] 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.
12 Solution C Solution D 3.2 mM Adenine 53.2 mM Na citrate 2
H.sub.2O 85 mM mannitol 5.4 mM NaH.sub.2PO.sub.4 2 H.sub.2O 100 mM
glucose 38 mM Na.sub.2HPO.sub.4 2 H.sub.2O 6.2 mM HCl
[0333] The plasma-Erythrosol solution was spiked with
5-[(.beta.-carboxyethyl)amino]acridine to a final concentration of
300 .mu.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
tared 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 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-[(.beta.-carboxyethyl)-amino]acrid- ine. 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.
13TABLE 7 Adsorbent Screen--Removal of
5-[(.beta.-carboxyethyl)amino]acridine from 25% Plasma/75%
Erythrosol. ("LOD" is limit of detection.) Final Corrected Wetting
Acridine Capacity Estimated Adsorbent Chemistry Mass (g) Required?
C.sub.r (.mu.M) (.mu.mole/g) K (L/g) Purolite MN-150 Weak base, PS-
0.108 No 1.4 13.5 9.7 DVB Purolite MN-170 Weak base, PS- 0.105 No
0.4 14.0 38.4 DVB Purolite MN-200 Nonfunctional, 0.077 No 0.9 19.1
21.5 PS-DVB Purolite MN-300 Weak Base, PS- 0.111 No 1.0 13.2 13.4
DVB Purolite MN-400 Strong base, PS- 0.102 No 1.1 14.3 13.0 DVB
Purolite MN-500 Strong acid, PS- 0.098 No 0.6 15.0 24.2 DVB
Purolite MN-600 Weak acid, PS- 0.112 No 0.8 13.1 16.7 DVB Dowex
Optipore L- Nonfunctional 0.189 No 0.3 7.8 22.5 493 PS-DVB 46 A,
1100 m.sup.2/g Dowex Optipore L- Weak base, PS- 0.245 No 0.5 6.0
11.2 285 DVB 25 A, 800 m.sup.2/g Amberlite XAD-2 Nonfunctional
0.190 Yes 117.5 4.7 0.0 PS-DVB 90 A, 300 m.sup.2/g Amberlite XAD-4
Nonfunctional 0.118 Yes 1.6 12.4 7.7 PS-DVB 40 A, 725 m.sup.2/g
Amberlite XAD-7 Polyacrylic ester 0.097 Yes 117.4 9.1 0.1 90 A, 450
m.sup.2/g Amberlite XAD-16 Nonfunct. 0.101 Yes 44.1 12.4 0.3
polystyrene 100 A, 800 m.sup.2/g Hemosorba AC Activated 0.149 No
LOD >9.8 >196.8 charcoal pHEMA-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. 0.067 No 239.1 4.1 0.0 macroreticular PS, Cl-form
Amberlite IRA 35 Weak base 0.076 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
Cl-form Amberlite IRA 958 Quat. Am. 0.084 No 304.9 -0.6 0.0
macroreticular Polystyrene, Cl- form Amberlite DP-1 Carboxylic
0.066 No 292.8 0.1 0.0 macroreticular PS Amberlite IR-120 Sulfonic
acid gel 0.079 No 1.4 18.6 13.1 PS H-form Diaion HPA 75 Quat. alkyl
amine 0.091 No 97.2 10.8 0.1 highly porous PS 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 Cl-form Amberlite
IRA-958 Quat. Am. 0.139 No 296.3 -0.1 0.0 macroreticular PS Cl-form
Ambersorb 563 Synthetic AC, 550 0.228 No 0.4 6.4 17.5 m.sup.2/g,
high hydrophobicity Ambersorb 572 Synthetic AC, 0.267 No LOD
>5.5 >55.0 1100 m.sup.2/g, low hydrophobicity Ambersorb 575
Synthetic AC, 800 0.258 No LOD >5.7 >56.9 m.sup.2/g, mid
hydrophobicity PICA G277 AC Activated charcoal 0.283 No LOD >5.2
>51.9 PICA NC506 AC Activated charcoal 0.266 No LOD >5.5
>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 charcoal 0.268 No LOD >5.5
>54.8 Norti B Supra Activated charcoal 0.258 No LOD >5.7
>56.9 Norit Supra E Activated charcoal 0.266 No 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 0.233 No LOD >6.3 >63.1
Norit CN1 Activated charcoal 0.261 No LOD >5.6 >56.4 Norit
G60 Activated charcoal 0.230 No LOD >6.4 >64.0 Norit ROX,O,8
Activated charcoal 0.246 No LOD >6.0 >59.8 Norti Darco
Activated charcoal 0.219 No LOD >6.7 >67.3 (20 .times. 50)
PICAtif Medicinal Activated charcoal 0.129 No LOD >11.4
>113.8 Davison Silica Unmodified silica 0.245 No 77.0 4.4 0.1
Grade 15 Davison Silica Unmodified silica 0.255 No 140.1 3.0 0.0
Grade 636 BioRad AG501- Mixed bed ion 0.083 No 119.4 10.5 0.1 X8(D)
exchange Amberlite 200 Sulfonic acid 0.080 No 46.5 15.5 0.3
macroret. PS, Na- form MP-3 (C-18) SPE Sulfonated C-18 0.239 Yes
1.7 6.1 3.7 media resin Alltech C-18 SPE C-18 modified 0.234 Yes
64.4 4.9 0.1 silica BioRad t-Butyl C-4 modified 0.213 Yes 163.9 3.1
0.0 HIC polymethacrylate Baker C-18 SPE C-18 modified 0.263 Yes
94.9 3.8 0.0 silica Waters Sep Pak C- C-18 modified 0.190 Yes 81.3
5.6 0.1 18 silica Baker C-4 SPE C-4 modified 0.232 Yes 178.3 2.5
0.0 silica Waters Bondapak C-8 modified 0.213 Yes 156.9 3.2 0.0 C-8
silica Waters Bondapak C-4 modified 0.228 Yes 251.0 0.9 0.0 C-4
silica Amberchrom cg- Polystyrene 0.192 Yes 79.3 5.6 0.1 161 xcd
150 A, 900 m.sup.2/g Amberchrom cg- Polystyrene 0.176 Yes 243.7 1.4
0.0 1000 sd 1000 A, 250 m.sup.2/g Amberchrom cg- Polystyrene 0.135
Yes 172.1 4.5 0.0 300 md 300 A, 700 m.sup.2/g Amberchrom cg-71
Polymethacrylate 0.189 Yes 156.4 3.6 0.0 md 250 A, 500 m.sup.2/g
Waters Porapak PS-vinyl 0.125 Yes 307.8 -0.6 0.0 RDx pyrrolidone
porous adsorbent CUNO Delipid Resin-modified 0.663 No 245.8 0.4 0.0
Media cellulose CUNO Weak base 0.361 No 280.8 0.2 0.0 DEAE media
modified cellulose Sigma DE Diatomaceous 0.179 No 317.6 -0.7 0.0
earth Diaion SP-850 Polystyrene 0.236 Yes 1.4 6.2 4.4 38 A, 1000
m.sup.2/g Diaion SP-207 Brominated PS 0.191 No 211.1 2.2 0.0 105 A,
650 m.sup.2/g Diaion HP-2MG Polymethacrylate 0.258 Yes 182.6 2.2
0.0 170 A, 500 m.sup.2/g Diaion HP-20 Polystyrene 0.175 Yes 107.4
5.3 0.0 260 A, 500 m.sup.2/g Amberlite 1180 Polystyrene-DVB 0.094
Yes 9.9 15.1 1.5 300 A, 600 m.sup.2/g Amberilte 1600
Polystyrene-DVB 0.208 Yes 73.0 5.3 0.1 Amberlite XAD-
Polystyrene-DVB 0.172 Yes 61.0 6.8 0.1 2000 42 A, 580 m.sup.2/g
Amberlite XAD- Polystyrene-DVB 0.203 Yes 173.0 3.0 0.0 2010 280 A,
660 m.sup.2/g Dowex XUS- Polystyrene-DVB 0.213 Yes 102.6 4.5 0.0
40323 100 A, 650 m.sup.2/g Whatman DE-52 Weak base 0.284 No 177.5
2.0 0.0 modified cellulose Whatman CM-32 Weak acid 0.294 No 268.7
0.4 0.0 modified cellulose Whatman QA-52 Strong base 0.264 No 285.2
0.2 0.0 modified cellulose Whatman SE-53 Strong acid 0.238 No 294.3
0.0 0.0 modified cellulose Pharmacia Q Seph Strong base 0.111 No
267.8 1.2 0.0 FF modified agarose Pharmacia S Seph Strong acid
0.112 No 267.0 1.2 0.0 FF modified agarose Toyopearl QAE- Wead base
0.118 No 266.8 1.2 0.0 550 C modified agarose Toyopearl Butyl
Hydrophobic (C- 0.112 Yes 232.8 2.7 0.0 650-M 4) modified
methacrylate Toyopearl SP- Strong acid 0.111 No 230.2 2.9 0.0 550C
modified methacrylate Toyopearl CM- Weak acid 0.118 No 249.0 1.9
0.0 650M modified methacrylate Toyopearl Weak base 0.112 No 260.4
1.5 0.0 DEAE-650M modified methacrylate Toyopearl Super Q Strong
base 0.111 No 263.2 1.4 0.0 650C modified methacrylate
[0334]
14TABLE 8 Adsorbent Screen--Removal of Glutathione from 25%
Plasma/75% Erythrosol Estimated GSH Estimated Capacity at HPLC Area
Final GSH Capacity at C.sub.r = 30 .mu.M Adsorbent (mAU*sec) Conc.
(.mu.M) C.sub.r (.mu.mole/g) (.mu.mole/g) Purolite 336.1 2375 28.9
0.4 MN-150 Purolite 386.7 2732 12.8 0.1 MN-170 Purolite 445.5 3147
-9.6 -0.1 MN-200 Purolite 356.7 2520 21.7 0.3 MN-300 Purolite 297.0
2099 44.1 0.6 MN-400 Purolite 392.8 2775 11.5 0.1 MN-500 Purolite
392.4 2773 10.1 0.1 MN-600 Dowex 428.5 3027 -0.7 0.0 XUS-43493
Dowex 251.6 1778 24.9 0.4 XUS-40285 Amberlite 420.8 2973 0.7 0.0
XAD-2 Amberlite 449.0 3172 -7.3 -0.1 XAD-4 Amberlite 448.0 3165
-8.5 -0.1 XAD-7 Amberlite 412.2 2912 4.4 0.0 XAD-16 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 441.1
3116 -8.6 -0.1 IRA 900 Amberlite 434.4 3069 -4.5 0.0 IRA 35
Amberlite 413.0 2918 4.8 0.0 IRA 410 D Amberlite 446.2 3152 -9.1
-0.1 IRA 958 Amberlite 433.3 3061 -4.6 0.0 DP-1 Amberlite 388.6
2745 16.2 0.2 IRA-120 Diaion HPA 75 447.3 3160 -8.8 -0.1 Duolite
S-761 312.9 2211 42.6 0.6 MP-3 428.0 3024 -0.5 0.0 Alltech 411.3
2906 2.0 0.0 C-18 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 >56.1 >167.9 Ambersorb 575 0.0 LOD >58.1 >173.8
PICA 0.0 LOD >53.0 >158.4 G277 AC PICA 0.0 LOD >56.4
>168.7 NC506 AC PICAtif 0.0 LOD >53.6 >160.4 Med. AC West
VACO 0.0 LOD >65.7 >196.5 CX-S Norit A Supra 0.0 LOD >55.9
>167.2 Norti B Supra 0.0 LOD >58.1 >173.7 Norit Supra E
0.0 LOD >56.3 >168.5 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 CN1 0.0 LOD >57.5 >172.1 Norit G60
0.0 LOD >65.3 >195.4 Norit ROX,O,8 0.0 LOD >61.0 >182.4
Norti Darco 0.0 LOD >68.6 >205.3 (20 .times. 50) PICAtif 0.0
LOD >116.2 >347.4 Medicinal 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 BioRad 445.6 3148 -8.9 -0.1
AG501-X8(D) Amberlite 200 458.1 3237 -14.8 -0.1 BioRad t-Butyl
484.0 3419 -9.8 -0.1 HIC Baker C-18 427.7 3022 -0.4 0.0 SPE Waters
Sep 422.2 2983 0.4 0.0 Pak C-18 Baker C-4 SPE 429.8 3036 -0.8 0.0
Waters 405.5 2865 3.2 0.0 Bondapak C-8 Waters 390.8 2761 5.2 0.1
Bondapak C-4 Amberchrom 414.8 2931 1.8 0.0 cg-161 xcd Amberchrom
373.5 2639 10.2 0.1 cg-1000 sd Amberchrom 396.2 2799 7.4 0.1 cg-300
md Amberchrom 396.4 2800 5.3 0.1 cg-71 md Waters 449.9 3179 -7.1
-0.1 Porapak RDx CUNO Delipid 452.5 3197 -1.5 0.0 Media CUNO DEAE
213.7 1510 20.6 0.4 media Sigma 473.9 3348 -9.7 -0.1 Diatomaceous
Earth Diaion SP-850 424.1 2996 0.1 0.0 Diaion SP-207 471.6 3332
-8.7 -0.1 Diaion 429.7 3036 -0.7 0.0 HP-2MG 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 407.4 2879 3.5 0.0 XAD-2000 Amberlite 423.0 2988
0.3 0.0 XAD-2010 Dowex 372.7 2633 8.6 0.1 XUS-40323 Whatman 384.7
2718 5.0 0.1 DE-52 Whatman 514.8 3637 -10.8 -0.1 CM-32 Whatman
398.1 2813 3.5 0.0 QA-52 Whatman 458.0 3236 -4.9 0.0 SE-53
Pharmacia Q 377.6 2668 15.0 0.2 Seph FF Pharmacia S 390.5 2759 10.8
0.1 Seph FF Toyopearl 395.1 2792 8.8 0.1 QAE-550 C Toyopearl 372.9
2635 16.2 0.2 Butyl 650-M Toyopearl 404.2 2856 6.5 0.1 SP-550C
Toyopearl 394.0 2784 9.2 0.1 CM-650M Toyopearl 392.3 2772 10.2 0.1
DEAE-650M Toyopearl 384.6 2717 12.8 0.1 Super Q 650C
Example 12
Adsorption Capacities for Adsorbents
[0335] Adsorption isotherm experiments were carried out to
determine the adsorptive capacities (.mu.mole
5-[(.beta.-carboxyethyl)-amino]acridine/g adsorbent) for various
types of adsorbents. FIG. 17 shows adsortion isotherms obtained for
several Ambersorbs as compare to the adsorption isotherm for
Purolite MN-200. Adsorption studies were performed in 25%
plasma/75% Erythrosol solutions containing 0.3-3 mM
5-[(.beta.-carboxyethyl)amino]acridine and 0.6-10 mM GSH. Samples
were agitated for 24 hours at room temperature. Calculations using
the adsorption capacity form Table 7 (22 .mu.mole/g) determined
that aproximately 4 g of Purolite MN-200 would be required to
reduce the level of 5-[(.beta.-carboxyethyl)-amino]acridine in a
300 mL unit of PRBC from 300 .mu.M to a final level of 1 .mu.M.
Less than 1 g of Ambersorb 572 (130 .mu.mole/g) would be required
to achieve comparable removal. Asimilar calculion 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
500 .mu.M in the 150 mL of supernatant (50% HCT).
Example 13
Long Term Removal of Breakdown Products
[0336] This experiment examined
5-[(.beta.-carboxyethyl)-amino]acridine and GSH levels in PRBCs
from which a fiberized PICA G-277 activated carbon device (AQF,
7.3g, 500 m.sup.2/g) was removed after 24 hours of exposure. This
study was conducted in parallel with studies where the PRBCs had
continued device exposure. FIG. 18 shows that the concentrations of
5-[(.beta.-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.
[0337] The concentration of 5-[(.beta.-carboxyethyl)-amino]acridine
was reduced to 5 .mu.M in initially shaken PRBCs after 35 days of
storage in the presence of a removal device (MN-200). This
indicates that 5-[(.beta.-carboxyethyl)-amino]acridine removal does
occur in static storage conditions at 4.degree. C.
Example 14
Effect of Enclosure Material (membrane, woven, non-woven) on an
IAD
[0338] 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 G277 activated carbon media (AQF 500
g/m.sup.2) was surrounded by a heat-sealed membrane, woven
polyester, or non-woven polyester material. PRBC units (300 mL)
were dosed with 300 .mu.M of a degradable
5-[(.beta.-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-[(.beta.-carboxyethyl)-a- mino]acridine was monitored over 24
hours. FIG. 19 shows 5-[(.beta.-carboxyethyl)-amino]acridine levels
in the supernatant of 300 mL PRBC units exposed to IADs consisting
of fiberized Pica G277 activated carbon (500 g/m2) and enclosed by
a membrane, woven, or non-woven material. PRBCs (Erythrosol,
glucose, 62% HCT) were dosed with 300 .mu.M of a degradable
5-[(.beta.-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.
[0339] These studies indicate that the Tetko woven enclosure shows
the fastest removal kinetics for
5-[(.beta.-carboxyethyl)-amino]acridine over 24 hours. Final levels
achieved for all enclosure materials after 2 weeks were similar,
with 5-[(.beta.-carboxyethyl)-amino]acridine concentrations
decreasing to approximately 2 .mu.M after 1 day, but rising back to
10 .mu.M near day 8.
Example 15
Effect of the Compound Adsorption Device on Red Blood Cell
Function
[0340] Indicators of red blood cell function were monitored over
the course of 5-[(.beta.-carboxyethyl)-amino]acridine and GSH
removal experiments for various device configurations. Parameters
measured included percentage lysis, ATP and K.sup.+ 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 G277 devices. Levels of K.sup.+ in PRBCs
were found to increase with time. The temperature at which removal
occurred did not influence K.sup.+ 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.sup.+ 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 0.1 and 1% after 24 hours. As shown in Table 10 and Table
11 and graphed in FIGS. 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.degree. 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-im-mobilized
adsorbent. Similar observations have been observed for other IADs
as compared to the same non-immobilized adsorbent.
15TABLE 9 ATP Concentration (.mu.mol/dL) in PRBCs over time Time
Control (no IAD) MN-200 IAD PICA G277 IAD 0 hr 76 78 79 4 hr 80 79
82 8 hr 82 82 81 24 hr 83 83 86 8 days 73 70 78 22 days 50 42 50 35
days 25 23 28
[0341]
16TABLE 10 Percent Lysis in PRBCs (56% HCT) exposed to different
IADs over time Time Control (no IAD) MN-200 IAD PICA G277 IAD 0 hr
0.07 0.09 0.13 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
[0342]
17TABLE 11 Percent lysis in PRBCs (55% HCT) exposed to different
loose particle adsorbents enclosed in woven mesh. Amber- Darco
Control Purolite Hemosorba sorb AC Pica Time no IAD MN-200 CH-350
572 20 .times. 50 G277 0 hr 0.10 0.07 0.08 0.07 0.06 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
0.11 0.39 1.06 0.16 0.13 0.92 Day 14 0.12 0.48 1.11 0.22 0.13 0.40
Day 21 0.15 0.59 1.24 0.29 0.17 0.57 Day 28 0.22 0.60 1.52 0.40
0.26 0.74 Day 35 0.30 0.78 1.85 0.55 0.41 1.05 Day 42 0.53 1.08
2.72 0.86 0.79 1.60
[0343] 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% lysis. Those
adsorbents were significantly better at minimizing hemolysis of red
blood cells than the Purolite MN-200, Hemosorba CH-350 and Pica
G277 adsorbents.
[0344] Table 12 shows a comparative study where supernatants from
red blood cell samples containing glutathione and
5-[(.beta.-carboxyethyl)ami- no]acridine were 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
5-[(.beta.-carboxyethyl)-amino]acridine and glutathione. Both
natural activated carbons (Norit and PICA) and synthetic activated
carbons (Ambersorb) proved to be effective at compound
reduction.
18TABLE 12 Properties of Several Different Adsorbents Acridiene GSH
Cap{circumflex over ( )} Cap{circumflex over ( )} (.mu.mole/g)
(.mu.mole/g) Adsorbent Manufacturer Description.sup.@ C.sub.r = 500
.mu.M C.sub.r = 1 .mu.M MN-200 Purolite Hypercrosslinked 0.0*
17.sup.+ macroreticular PS-DVB, 200-1200 .mu.m particles 1100
m.sup.2/g Optipore L-493 Dow Chemical Hypercrosslinked 0.0* 22*
macroreticular PS-DVB, 300-840 .mu.m particles 1100 m.sup.2/g, 46
.ANG. avg. pore diameter Duolite GT-73 Rohm & Haas Macroporous
adsorbent 1.7* 10* with thiol functional groups Norit A Supra Norit
Americas, Steam lignite AC, >2790* 560.sup.+ Inc. 2000
m.sup.2/g, 97% < 150 .mu.m particles Picatiff Med. PICA Powdered
AC from >2670* >53* coconut husk, 2000 m.sup.2/g, 8-35 .mu.m
particles Norit ROX 0.8 Norit Steam-activate peat AC, >3040*
>60* extruded 900 m.sup.2/g, 840-1000 .mu.m cylinders Ambersorb
572 Rohm & Haas Synthetic AC from >2800* 134.sup.+
sulfonated 1100 m.sup.2/g, 300-840 .mu.m particles Microporous (ca.
50% pores < 20.ANG.) G-277 PICA Granular activated >2640*
>52* carbon from coconut husk, .sup.@PS-DVB =
polystyrene-divinyl benzene, AC = activated carbon {circumflex over
( )} Values listed as ">" were single measurements with residual
levels below the assay LOD *Estimated from single-point adsorption
studies in 25% plasma, 75% Erythrosol. .sup.+Estimated from
multi-point adsorption isotherms in 25% plasma, 75% Erythrosol.
Example 16
[0345] 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,
N.C.). The performance of this adsorbent media in a batch removal
device for platelets was evaluated.
[0346] Platelet Preparation
[0347] Single donor apheresis platelet units containing
3.5-4.5.times.10.sup.11 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
concentration of 150 .mu.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/cm.sup.2 UV-A and transferred
into the appropriate removal device for the study. All experiments
included a control platelet unit which was photochemically treated
(150 .mu.M psoralen, 3.0 J/cm.sup.2 UVA) but was not contacted with
a removal device.
[0348] Device Preparation
[0349] 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 .mu.m polyester mesh
(Tetko). Mesh pouches containing the media were autoclaved
(121.degree. 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
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.
[0350] Adsorption Kinetics
[0351] 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.degree. 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.degree. 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 KH.sub.2PO.sub.4 buffer.
[0352] In vitro Platelet Function
[0353] 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.
[0354] Following 5 or 7 days of storage, the platelet count and pH
were determined for each platelet unit. The pH and pO2/pCO2 was
measured using a CIBA-Coming model 238 Blood Gas Analyzer. The
platelet count of each sample was determined using a Baker
9118+Hematology analyzer.
[0355] 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.
[0356] 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.
[0357] 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 VS whole blood
aggregometer.
[0358] 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).
[0359] Platelet activation as indicated by expression of p-selectin
(Granular Membrane Protein, GMP-140) was measured. CD62 antibody
was used for the test sample, mouse control IgG1 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.
[0360] Adsorption Kinetics
[0361] The impact of incorporating the adsorbent beads into a fiber
matrix was investigated. Platelet units containing
4.0.times.10.sup.11 platelets in 300 mL of 35% plasma, 65% PAS III
were treated with 150 .mu.M 4'-(4-amino-2-oxa)butyl-4,5',
8-trimethyl psoralen+3.0 J/cm.sup.2 UVA. IADs were prepared from
Hoechst-L493 with an adsorbent loading of 450 g/m.sup.2. 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 psoralen by HPLC.
FIG. 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 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/m.sup.2.
[0362] 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. The 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 .mu.M residual
4'-(4-amino-2-oxa)butyl-4,5',8-trime- thyl psoralen in under 4
hours of contact.
[0363] 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/m.sup.2) 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/m.sup.2 indicated that
adsorption kinetics were not affected by incorporation into a fiber
matrix at a low level of loading.
[0364] Platelet Yield and In Vitro Platelet Function
[0365] 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.
[0366] 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 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.degree. 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.
19TABLE 13A (Day 5) Comparison of Hoechst-L493 Fiberized Media to
Standard Removal Device Platelet Yield and In Vitro Function
following 5-Day Storage Sample Platelet Count Yield
(.times.10.sup.11/300 mL) (%) pH pCO.sub.2 pO.sub.2 Control (+ PCT
- RD) 3.50 .+-. 0.11 100 6.91 18 120 5.0 g Hoechst/L-493 3.41 .+-.
0.06 97 .+-. 4 6.95 21 95 Transfer at 24 hr 5.0 g Hoechst/L-493
3.33 .+-. 0.08 95 .+-. 4 6.93 24 87 No Transfer 2.5 g Dowex
Optipore 2.60 .+-. 0.07 74 .+-. 3 7.04 13 146 L-493 Baxter Lot
FX1032 D96F20042R No Transfer Shape Aggre- Change HSR gation
Morphology MP-140 Control (+ PCT - 0.83 .+-. 0.31 .+-. 69 .+-. 4
273 67.2 IAD 0.26 0.08 5.0 g Hoechst/L-493 0.90 .+-. 0.43 .+-. 83
.+-. 0 268 61.5 Transfer at 24 hr 0.02 0.04 5.0 g Hoechst/L-493
0.83 .+-. 0.40 .+-. 80 .+-. 2 280 58.9 No Transfer 0.12 0.01 2.5 g
Dowex 0.24 .+-. 0.38 .+-. 47 .+-. 3 240 71.3 Optipore L-493 0.04
0.02 Baxter Lot FX1032 D96F20042R No Transfer
[0367]
20TABLE 13B (Day 7) Comparison of Hoechst-L493 Fiberized Media to
Standard Removal Device Platelet Yield and In Vitro Function
following 7-Day Storage Sample Platelet Count Yield
(.times.10.sup.11/300 mL) (%) pH pCO.sub.2 pO.sub.2 Control (+ PCT
- 3.45 .+-. 0.06 100 6.97 13 126 IAD) 5.0 g Hoechst/L- 3.28 .+-.
0.22 95 .+-. 7 6.90 18 105 493 Transfer at 24 hr 5.0 g Hoechst/L-
3.11 .+-. 0.15 90 .+-. 5 6.88 21 100 493 No Transferred 2.5 g Dowex
2.29 .+-. 0.07 66 .+-. 2 7.04 9 161 Optipore L-493 Baxter Lot
FX1032 D96F20042R No Transfer Shape Aggre- Change HSR gation
Morphology GMP-140 Control (+ PCT - 0.54 .+-. 0.25 .+-. 48 .+-. 0
284 80.1 IAD) 0.05 0.03 5.0 g Hoechst/L- 0.76 .+-. 0.64 .+-. 86
.+-. 11 267 72.7 493 0.02 0.16 Transfer at 24 hr 5.0 g Hoechst/L-
0.67 .+-. 0.29 .+-. 79 .+-. 7 280 66.1 493 0.00 0.04 No Transfer
2.5 g Dowex 0.31 .+-. 0.28 .+-. 29 .+-. 11 261 77.1 Optipore L-493
0.09 0.06 Baxter Lot FX1032 D96F20042R No Transfer
[0368] 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.
[0369] 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.
21TABLE 14A (Day 5) Evaluation of Gamma Sterilized Hoechst-L493
Fiberized Media Platelet Yield and In Vitro Function following
5-Day Storage (No Transfer) Sample Platelet Count Yield
(.times.10.sup.11/300 mL) (%) pH pCO.sub.2 pO.sub.2 Control (+ PCT
- 3.96 .+-. 0.19 100 6.89 25 88 IAD) 5.0 g Hoechst/L-493 3.68 .+-.
0.16 93 .+-. 6 6.89 27 70 7.5 g Hoechst/L-493 3.44 .+-. 0.11 87
.+-. 5 6.87 26 84 2.5 g Dowex 2.85 .+-. 0.13 72 .+-. 5 7.04 13 145
Optipore L-493 Baxter Lot FX1032 D96F20042R Shape Morph- Change SR
ggregation ology GMP-140 Control (+ PCT - 0.56 .+-. .26 .+-. 0.04
73 .+-. 1 289 65.1 IAD) 0.12 5.0 g Hoechst/ 0.45 .+-. .40 .+-. 0.00
76 .+-. 2 278 54.5 L-493 0.04 7.5 g Hoechst/ 0.48 .+-. .25 .+-.
0.01 73 .+-. 2 290 55.4 L-493 0.06 2.5 g Dowex 0.04 .+-. .30 .+-.
0.01 38 .+-. 3 232 62.8 Optipore L-493 0.06 Baxter Lot FX1032
D96F20042R
[0370]
22TABLE 14B (Day 7) Evaluation of Gamma Sterilized Hoechst-L493
Fiberized Media Platelet Yield and In Vitro Function following
7-Day Storage (No Transfer) Platelet Count Yield Sample
(.times.l0.sup.11/300 mL) (%) pH pCO.sub.2 pO.sub.2 Control(+ 3.88
.+-. 0.18 100 6.99 18 93 PCT-IAD) 5.0 g Hoechst 3.67 .+-. 0.08 95
.+-. 5 6.92 22 81 /L-493 7.5 g 3.48 .+-. 0.07 90 .+-. 4 6.91 20 91
Hoechst/L-493 2.5 g Dowex 2.65 .+-. 0.22 68 .+-. 6 7.04 9 159
Optipore L- 493 Baxter Lot FX1032 D96F20042R Aggreg- Shape atio
Morph- Sample Change HSR n ology Control 0.53 .+-. 0.12 0.23 .+-.
0.02 65 .+-. 5 260 5.0 g Hoechst/L-493 0.54 .+-. 0.00 0.30 .+-.
0.01 83 .+-. 8 266 7.5 g Hoechst/L-493 0.53 .+-. 0.09 0.29 .+-.
0.01 81 .+-. 13 280 2.5 g Dowex Optipore 0.23 .+-. 0.04 0.25 .+-.
0.03 33 .+-. 9 228 L-493 Baxter Lot FX1032 D96F20042R
[0371] 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 5.0 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.
[0372] 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 .mu.M 4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl psoralen +3.0
J/cm.sup.2 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/TVA) 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.
Example 17
Comparison of AQF Fiberized Beads vs. Free Beads in PRBCs
[0373] This study compared the removal of S-300
(N-(9-acridinyl)-.beta.-al- anine) and glutathione from PRBC using
AQF fiberized vs. free beads of Ambersorb 572.
[0374] 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 .mu.M. This was
mixed manually and allowed to incubate at room temperature for 4
hours.
[0375] 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/m.sup.2) 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.degree. 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.sup.+ concentration, and pH (see table 16).
[0376] 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 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.sup.+ levels
are much higher after 24 hours for the free beads, indicating
substantial loss or red blood cell function.
23TABLE 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 supematant
(glutathione) after treatment. Ambersorb 572 beads in AQF Ambersorb
572 fiberized media free beads Time Exp. 1 Exp. 2 Exp. 1 Exp. 2
Control Residual 0 33.5 33.7 31.1 32.1 32.9 S-300 2 hr 4.3 6.5 5.5
6.9 42.6 .mu.M 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 2.1 113.8 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
17.1 17.4 174.7 5 weeks 16.7 19 12.5 15.4 171.2 Residual 0 6.55
6.59 6.40 6.50 6.40 gluta- 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 6 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 0.35 0.49 6.40
[0377]
24TABLE 16 % Hemolysis, K+, ATP, and pH comparison of PRBC treated
with Ambersorb 572 as beads in a fiberized media vs. free beads.
Ambersorb 572 beads in AQF fiberized Ambersorb 572 media free beads
Time Exp. 1 Exp. 2 Exp. 1 Exp. 2 Control % 0 0.13 0.14 0.13 0.11
0.15 Hemolysis 2 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 weeks 0.45 0.39 9.94 15.55 0.25
5 weeks 0.47 0.36 10.19 19.30 0.28
[0378]
25 Ambersorb 572 beads in AQF fiberized Ambersorb 572 media free
beads Time Exp. 1 Exp. 2 Exp. 1 Exp. 2 Control K.sup.+ 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 .mu.mol/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 24.18 23.40 pH 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
[0379] This study looked at the effect of the mode of agitation
used during IAD treatment on removal of S-300
(N-(9-acridinyl)-.beta.-alanine) and glutathione and on percentage
hematocrit, percentage hemolysis, ATP concentration, K.sup.+
concentration, and pH.
[0380] 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/m.sup.2) 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 15
treated as follows:
26 Test Agitation Type 4.degree. C. Storage Article during first 24
hours at room temperature Condition 1 platelet shaker (72
cycles/min) static 2 platelet shaker (72 cycles/min) orbital shaker
intermittent 3 orbital shaker (72 cycles/min) static 4 platelet
rotator (6 cycles/min) static 5 Nutator (3-D rotation, 24
cycles/min) static 6 static (no agitation) static 7(control)
platelet shaker (no media or enclosure) static
[0381] 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 of hemolysis than the
other methods. Table 17 shows the S-300 and glutathione levels.
Table 18 shows the red cell function.
27TABLE 17 S-300 and glutathione levels in units treated with
various agitiation modes with AQF fiberized Ambersorb 572. Test
article 1 2 3 4 5 6 7 RT agitation mode PS PS OS PR N static
Control 4.degree. C. condition static OS static static static
static static Resi- 0 34.7 33.8 34.3 34.2 34.7 34.8 33.9 dual 2 hr
4.4 4.7 5.0 4.0 3.6 32.5 43.9 S-300 4 hr 2.9 2.5 2.2 2.7 2.1 35.9
57.4 .mu.M 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
weeks 16.3 8.6 17.7 17.1 18.0 22.6 160.6 5 weeks 13.7 4.0 14 12.9
14.4 14.4 166.8 Resi- 0 5.97 6.02 5.98 5.96 5.92 5.92 6.93 dual 2
hr 0.52 0.71 0.84 0.65 0.55 4.39 5.99 gluta- 4 hr 0.74 0.82 0.73
0.67 0.64 4.33 6.93 thione 6 hr 0.59 0.67 0.59 0.6 0.55 3.79 6.41
mM 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 1.15 5.03 PS =
platelet shaker, OS = orbital shaker, PR. = platelet rotator, N =
Nutator
[0382]
28TABLE 18 % hemolysis, K.sup.+, ATP, and pH results with various
agitation modes using fiberized AQF Ambersorb 572. 7 Test article 1
2 3 4 5 6 Con- RT agitation mode PS PS OS PR N static trol
4.degree. C. condition static OS static static static static static
% 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 0.43 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 weeks 0.76 0.53 0.33 1.18 1.26 0.20 0.19 5 weeks 1.18 0.75 0.40
1.18 1.34 0.34 0.24 K.sup.+ 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 weeks 18.82 19.08 18.54 19.32 19.22 18.74 20.40
5 weeks 31.65 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
.mu.mol/dL 1 week 62.40 62.01 65.72 63.18 62.60 66.11 66.50 2 weeks
50.31 48.36 51.29 50.12 49.14 53.43 51.29 5 weeks 23.40 22.43 23.99
23.79 24.18 26.13 23.99
Example 19
Removal of Activated Complement by fiberized Ambersorb 572 treated
PRBCs
[0383] This study looked at the formation of complement fragments
C3a and SC5b-9 in PRBCs after treatment with an S-300 derivative
and glutathione followed by removal using AQF fiberized media.
[0384] 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:
29 Test Article (PRBC unit #) Description 1 no IAD 2 no IAD-ice
incubated 3 4.8 g Ambersorb IAD (400 g/m.sup.2) (no enclosure) 4 7
sheets cellulose acetate membrane (47 mm dia.)
[0385] 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.degree. C. Unit 2 was stored at 4.degree. C.
continuously.
[0386] 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.times.g for 15 minutes
and 450 .mu.L of each supernatant was mixed with 50 .mu.L of cold
200 mM EDTA and vortexed. These were frozen rapidly on dry ice and
stored at -70 .degree. C.
[0387] Enzyme immunoassays (Quidel) were used to detect the
formation of complement fragments C3a and SC5b-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.
[0388] 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
420 C. The sample treated with the IAD showed lower levels of C3a
and SC5b-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.
30TABLE 19 Complement fragment C3a and SC5b-9 levels with various
treatments. 2 3 4 Test article 1 no IAD AQF no cellulose Treatment
no IAD 4.degree. C. pouch acetate C3a 0 411 354 391 388 ng/mL 24 hr
609 383 -1 701 5 days 665 404 26 N/A SC5b-9 0 38 37 18 25 ng/mL 24
hr 120 52 22 87 5 days 226 N/A 34 N/A
[0389]
31TABLE 20 S-300 concentration (PRBC), glutathione concentration
(supernatant), and % hemolysis with various treatments. 2 3 4 Test
article 1 no IAD AQF no cellulose Treatment no IAD 4.degree. C.
pouch acetate Residual 0 30.55 30.18 29.38 29.6 S-300 24 hr 121.39
61.93 2.51 118.39 .mu.M 5 days 142.36 136.33 12.35 153.58 gluta- 5
days 7.15 7.26 0.72 7.07 thione 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
[0390] 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.
[0391] The media that was studied in this example is comprised of
Purolite MN-200 adsorbent particles (200-1200 .mu.m) immobilized in
ultra high molecular weight polyethylene (UHMWPE) particulate
matrix. Representative media is manufactured by Porex (Fairbum,
Ga.). Disks of immobilized adsorbent media were formed by mixing
approximately 50% (w/w) Purolite MN-200 (200-1200 .mu.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, Calif.).
[0392] The fiber matrix IAD was comprised of Purolite MN-200
immobilized in a non-woven polyester matrix at loading of 300 g
adsorbent/m.sup.2. Approximately 2.5 g (adsorbent mass) of
immobilized Purolite MN-200 was placed in a pouch constructed from
30 .mu.m woven polyester material (Tetko, DePew, N.Y.). 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, Calif.).
[0393] Units of ABO-matched platelet concentrates comprised of
platelets (3-5.times.10.sup.11 cells) suspended in approximately
300 mL of 35% autologous plasma, 65% platelet additive solution
were obtained from Sacramento Blood Bank (Sacramento, Calif.). 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 psoralen). 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, Ind.) at room
temperature (22.degree. C.) for the duration of the experiment.
[0394] 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 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.
[0395] The kinetics for psoralen adsorption are shown in FIG. 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).
[0396] 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 drastically reduced platelet loss while the
kinetics of psoralen removal are still very rapid.
[0397] 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.
32TABLE 21 Comparison of Day 5 Platelet Yield and In Vitro Platelet
Function for Fiber Matrix and Particulate Matrix IADs Platelet
Count Sample (.times.10.sup.11/300 mL) % Yield pH pCO.sub.2
PO.sub.2 Control 2.89 .+-. 0.06 100 6.95 22 116 AQF Fiber 2.71 .+-.
0.11 94 .+-. 4 6.96 23 112 Matrix IAD (300 g/m.sup.2 MN-200) Porex
Particulate 2.61 .+-. 0.10 90 .+-. 4 6.90 22 123 Matrix IAD (50%
MN-200)
[0398]
33 Shape Morph- Sample Change HSR Aggregation ology GMP-140 Control
1.03 .+-. 0.33 0.71 .+-. 44 .+-. 3 308 67.0 0.28 AQF Fiber 0.96
.+-. 0.14 0.53 .+-. 60 .+-. 0 311 63.9 Matrix IAD 0.06 (300
g/m.sup.2 MN-200) Porex 1.00 .+-. 0.05 0.42 .+-. 64 .+-. 1 304 66.2
Particulate 0.01 Matrix IAD (50% MN- 200)
[0399] 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.
[0400] 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 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.
* * * * *