Note: Descriptions are shown in the official language in which they were submitted.
ACTIVATION OF BIOCOMPATIBLE POLYMERS
WITH sIoLoGIcALs WHOSE BINDING
COMPLEMENTS ARE PATHOLOGICAL EFFECTORS
TECHNICAL FIELD
This invention relates to biocompatible polymers
having immobilized reactive biologicals thereon which bind
selected specific pathological effectors or specific
groups of pathological effectors associated with diseased
body fluids.
BACKGROUND OF THE INVENTION
The course of many disease states is often reflected
by elevated levels of specific blood proteins and other
molecules. This phenomenon is typically utilized as a
diagnostic tool to define the pathology and to follow the
course of clinical treatment. In many instances, these
specific blood components are directly or indirectly
responsible for the primary and secondary manifestations
of the disease proc~ss. "Autoimmune" diseases can be
described as diseases characterized by circulating
antibodies to endogenous substrates and tissue proteins
required by the body for normal growth and maintenance.
"Neoplastic" diseases are typically characterized by
uncontrolled growth of an undifferentiated transformed
cell line which evades or compromises the body's natural
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defense mechanisms by producing immunosuppressant blocking
factors, surface antigen masking components and/or growth
regulator constituents. Specific compartmentalization of
these pathological effectors onto a biocompatible
substrate is consistent with the restoration of "normal"
body function by removal of the pathological effectors of
the disease process.
The basic function of the organs, cells and
molecules that comprise the immune system is to recognize
and to eliminate from the body foreign substances. ~hese
foreign substances are eliminated by reaction between the
foreign substance and antibodies which are formed in
response to the substance. In general, this function is
performed eficiently and without detriment to the host.
However, in certain instances, disturbances can occur
which can lead to pathogenic disorders such as, for
example, an uncontrolled response (allergic disorders) or
an abnormal response (autoimmune disease). The
pathogenesis of both of these disorders is related
directly or indirectly to the production of antibodies
with cross reactivities to either environmental antigens
~allergens) or self-antigens.
An autoimmune disease is a pathological condition
arising when a host responds immunologically by produetion
of antibodies with reactivity to a self-antigen. Auto-
immunity ean affeet almost every part of the body, and
generally involves a reaetion between a self-antigen and
an immunoglobulin (Ig~l or IgG) antibody. Representative
autoimmune diseases can involve the thyroid, kidney,
pancreas, neurons, gastric mucosa, adrenals, skin, red
eells and synovial membranes as well as thyroglobulin,
insulin, deoxyribonueleie acids and immunoglobulins.
For some types of autoimmune and neoplastie
diseases, non-speeifie immunosuppressant treatments, sueh
as whole body X-irradiation or the administration of
eytotoxie drugs, have been used with limited sueeess. The
disadvantages of sueh treatment inelude the toxieity of
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the agents used, and the increased incidence of various
cancers, especially lymphomas and reticulum cell sarcomas,
following such therapy. In addition, the use of non-
specific agents for chronic cellular suppression greatly
increases the susceptibility of the patient to serious
infection from environmental fungi, bacterla and viruses
which under ordinary circumstances would not cause
problems. The invention disclosed herein is specific in
that it removes only the pathological effector or those
groups of pathological effectors which are related to and
responsible for the manifestations of a particular
disease.
In viewing the prior art, one finds that most
recently there have been generally two approaches to
therapeutic treatments for autoimmune and/or neoplastic
diseases. The first of these is to introduce a material
into the patient which causes a specific type of
immunological tolerance to be produced. This suppression
of antibody response would then effect a tolerance to the
offending antigen. A typical example of this type of
approach is U.S. 4,222,907 issued to Katz on September 16,
1981. In this reference, the diseased patient is given a
therapeutic treatment which consists of introducing
conjugates of an antigen linked to a D-glutamic acid:
D-lysine copolymer.
The second approach has been the extracorporeal
route. The procedures generally involve the removal of
hole blood, separation of cellular and soluble blood
substances, substitution or treatment of blood plasma and
recombination-infusion of the treated whole blood. The
first example of this approach would be plasma substitu-
tion or exchange with salt, sugar and/or protein solutions
and is described by McCullough et al, "Therapeutic Plasma
Exchange," Lab. ~led. 12(12), p. 745 (1981). Plasma
exchange is a rather crude technique that requires a large
volume of replacement solution. A second example of this
approach involves physical and/or biochemical modlfication
7~
of the plasma portion of whole blood. Typical of the
state of the art of this therapeutic treatment are, for
example, the Terman et al article "Extracorporeal Immuno-
adsorption: Initial Experience in Human Systemic Lupus
Erythematosus,`' The Lancet, October 20, 1979, pages
824-826. This article describes a hemodialysis type
system utilizing two mechanical filters with a DN~
collodian charcoal filter be.ween said two mechanical
filters. Typical of this state of the art, however, the
adsorbant column is only semispecific for immune
components because the charcoal substrate will
nonspecifically delete many vital low molecular weight
constituents from the treated plasma. A second
application of this approach can be illustrated by the
Terman et al article "Specific Removal of Circulated
Antigen by Means of Immunoadsorption," FEBS Letters, Vol.
61, No. 1, January, 197~, pages 59-62. This re f erence
teaches the specific removal of radiolabeled antigen by
antibody treated cellulosic membranes. The author~
however, demonstrates that control membranes have a
significant capacity to non-specifically adsorb proteins.
A third application of this approach is illustrated
by the Bansal et al article "Ex vivo Removal of 5erum IgG
in a Patient With Colon Carcinoma," Cancer, ~2(1), pp.
1-18 (1978). This report teaches the semi-specific
adsorption of immunoglobulin by ex vivo treatment of
plasma with formalin and heat-killed Staphylococcus
aureas. The biological activity of certain strains of S.
aureas is attributed to a molecule prPsent on the cell
wall, called Protein A, which interacts and binds with the
Fc portion of mammalian IgG. This treatment, because it
interacts with the Fc moiety, does not discriminate
between normal and pathological IgG components and experi-
ments have shown the possibility of significant side
ef~ects.
A fourth application of this approach can be
illustrated by the Malchesky et al article "On-line
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Separation of Macromolecu]es by Membrane Filtration With
Cryogelation," Artif. Organs ~:205, 19~0. This
publication teaches the semi-specific removal of
cryoglobulin substances from plasma by the combination o~
filtration and cold treatment chambersO The incidence and
composition of cryoglobular precipitates are not
necessarily consistent with or indicative of many
autoimmune or neoplastic diseases.
Another problem associated with the current state of
the art is that without systems using mechanical filtra-
tion, the specific pathological effectors desired to be
removed have not been removed in large enough amounts to
do much good for the diseased patient in that the columns
do not specifically absorb substantially only the desired
specific pathological effectors.
It has now been found that high specificity of
pathological effector removal can be effectuated by
treatment of blood and/or plasma in an economical manner
using the present invention.
SU~l~IARY OF THE I~IVENTION
Broadly stated, this invention relates to a
biospecific polymer having immobilized reactive
biologicals, said biologicals having high specific
activity for bindlng complements which are pathological
effectors comprising a biocompatible polymer support, with
or without a spacer attached to said biocompatible polymer
support having a physical size which forces said spacer to
extend from the surface of said biocompatible polymer
support, and a biological or biologicals immobilized on
the biocompatible polymer support or the spacer, via
chemical bonding, and characterized in that said
biological or biologicals retain their reactivity for
binding specific pathological effectors or specific groups
of pathological effectors.
This invention also relates to a regimen for the
therapeutic treatment of autoimmune diseases comprising
::~Z~4~
passing a diseased patient's blood, plasma or other body
fluid over a biospecific polymer having immobilized
reactive biologicals, thereby removing the desired patho-
logical effectors from said patient's blood or plasma and
then returning said blood to said patient.
Further, this invention, broadly stated, relates to
a method of producing these biospecific polymers having
immobilized reactive biologicals which have high specific
activity for binding complements which are pathological
effectors.
Also relating to this invention is a method of
producing biospecific polymers on a mechanical support to
provide excellent mechanical integrity.
These and other ob~ects of the present invention are
disclosed and described in the detailed description below
and in the appended claims.
DETAILED_DESCRIPTION
I. BIOCOMPATIBLE POLYMER ~UPPORT
The biocompatible polymer supports useful in the
present invention are materials which tend not to cause
adverse effects when in contact with body fluids such as,
for example, plasma or whole blood, while at the same time
maintaining a reactive but immobilized biological oriented
such that the biological is extended out from the surface
of said polymer support. The materials which are suitable
are those which may be cast into films and other physical
forms, while at the same time being susceptible to having
said biologicals chemically bound to them without damaging
either themselves or the biologicals bound thereto. The
types of materials generally contemplated to be suitable
are those known in the art as hydrogels and may be either
copolymers or homopolymers.
~ lodified cellulose and cellulosic derivatives,
particularly cellulose acetate, have also found utility as
biocompatible supports useful in the present invention.
By modified cellulosic derivatives what is meant is that
the cellulosic polymer is surface modified by covalently
linking pendant blocompatible surface groups to the
cellulosic substrate polymer rendering it more
- biocompatible. Such surface groups are well known and
need not be described here, however, for purposes of the
present invention, albumin has shown particular utility as
a modifying group. Methods of attaching such groups are
described hereinbelow.
Homopolymers may also be used as suitable
biocompatible polymer supports in the present invention.
It is to be understood, however, that when homopolymers
are discussed, they include materials which can also be
identified as slightly cross-linked homopolymers. That
is, they contain a relatively small amount of a second
component either intrinsic in the production of the
monomer or added purposely to insure enough cross-linking
so as to protect the homopolymer from slowly dissolving
away in an aqueous media, such as blood. An example of
this type of homopolymer which is often slightly cross-
linked is hydroxyethyl methacrylate (HEMA).
Referring to the hydrogels, suitable polymers may
either be regular homopolymers containing substantially no
other material in their matrices, or they may be
copolymers prepared from two or more monomers such as
styrene and vinyl acetate, for example. In certain
instances, this type of tailoring of the copolymers with
various monomers may enhance the desira~le properties of
the biocompatible polymer support material. Examples of
suitable monomers which may be copolymerized, include, for
example, hydroxyethyl methacrylate and glycidyl
methacrylate.
Also useful are terpolymers which are a subclass of
copolymers containing three monomers which are
polymerized. An example of a suitable terpolymer is
glycidyl methacrylate/N-vinyl pyrrolidone/hydroxyethyl
methacrylate ~GMA/NVP/HEMA).
In addition to the specific copolymers and homo-
polymers listed above, copolymers, prepared with or
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without various additional monomers, and homopolymers
suitable in the present invention may be polymerized from
the following monomers: hydroxyalkyl acrylates and
hydroxyalkyl methacrylates, for example, hydroxyethyl
acrylate, hydroxypropyl acrylate, and hydroxybutyl
methacrylate; epoxy acrylates and epoxy methacrylates,
such as, for example, glycidyl methacrylate; amino alkyl
acrylates and amino alkyl methacrylates; N-vinyl
compounds, such as, for example, N-vinyl pyrrolidone,
N-vinyl carbazole, N-vinyl acetamide, and N-vinyl
succinimide; amino styrenes; polyvinyl alcohols and
polyvinyl amines, which must be made from suitable
polymeric precursors; polyacrylamide and various
substituted polyacrylamides; vinyl pyridine; vinyl
sulfonate and polyvinyl sulfate; vinylene carbonate; vinyl
acetic acid, and vinyl crotonic acid; allyl amine and
allyl alcohol; vinyl glycidyl ether and allyl glycidyl
ether. Processes and procedures for creating copolymers
andtor homopolymers from the above monomers are well-known
and understood in that particular art. These parameters
are not critical to the instant invention with the caveat
that the final copolymer and/or homopolymer is nontoxic
for animal, including human, use.
The method used to cast these materials into a form
suitable for use in the present invention is not of
critical importance. One presently preferred method is
spin casting and is exemplified in Examples 2, 3 and 4.
II. BIOLOGICALS
In the context of the present invention, biological
and/or biologicals may be defined as a chemical compound
~hich possesses an ability to covalently bond to the
biocompatible polymer support or spacer (defined herein-
below), while at the same time retaining an activity to
bind a desired pathological-causing constituent. It is to
be understood that, in addition, the biological or
biologicals employed must be of such size that they
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covalently bond to the surface of the polymer support and
are not small enough to penetrate the porous matrix of the
polymer support and be chemically bonded therefore inside
or in the interior of the support material. In this
light, a spacer may be employed to insure that the
reactive si~e of the biological, which remains and is
susceptible to bonding with the desired pathological
constituent, can in fact be presented to this constituen~,
i.eO, that it is held outward away -Erom the support so as
to come into contact with the body fluid flowing over the
support. It is obvious from the above that, of course,
the reactivity for binding the desired pathological
constituent is, in fact, retained after immobilization of
the biological or biologicals onto the biocompatible
polymer support. Examples of materials which may be used
as biologicals include, for example: acetylcholine
receptor proteins, histocompatibility antigens,
ribonucleic acids, basement membrane proteins,
immunoglobulin classes and subclasses, myeloma protein
receptors, complement co~ponents, myelin proteins, and
various hormones, vitamins and their receptor components.
Particular examples are, for example, attaching insulin to
a biocompatible polymer support to remove anti-insulin
antibody which is associated with the autoimmune disease
insulin resistance; attaching anti-Clq and/or Clq to a
biocompatible polymer support to remove immune complexes
which are associated with connective tissue and prolifera-
tive diseases such as, for example, rheumatoid arthritis
and carcinoma.
Any generally known method of chemical attachment
will suffice for attaching the biologicals to the bio-
compatible polymer support, with the caveat that the
biological still has at least one active site for the
particular autoimmune disease-associated component.
Generally, the methods of chemical attachment used fall
into three classes or routes of attachment. These three
routes are, 1) spontaneous attachment, 2) chemical
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activation of terminal functional groups, and 3) coupling
reagent attachment. Spontaneous covalent attachment of
biologicals to polymer support surface proceeds via
chemically reactive groups extending from the polymer
support. Thus, for exampl~, reactive groups such as
aldehyde and epoxy extending from the polymer support
readily couple biologicals containing available hydroxyl,
amino or thiol groups. Also, for example, free aldehyde
groups on the polymer support couple via acetal linkages
with hydroxyl-containing biologicals and via imide
linkayes with amino-containing molecules. Additionally,
for example, free oxime groups couple via al~ylamine,
ether and thioether lin};ages with biologicals containing
amine, hydroxyl and thio groups respectively. For
purposes of convenience all said attachments and couplings
are defined herein as immobilizations. More extensive
discussions of these reactions may be round, for example,
in "Chemical Procedures for Enzyme Immobilization of
Porous Cellulose Beads," Chen, L. F. et al, Biotechnology
and Bioengineering, Vol. XIX , pp. 1463-1473 (1977) and
"Epoxy Activated Sepharose," 6B, Pharmacia Fine Chemicals,
Affinity Chromatography, pp. 27-32 (1979).
Chemical activation of terminal functional groups
may be accomplished by activating polymer surface func-
tional groups by chemical modification of their terminal
components. This method can be exemplified by ~he oxida-
tion of terminal epoxy functions with periodic acid to
form active aldehyde groups. This method is further
exemplified, for example, in "Immobilization of Amyloglu-
cosidose on Poly [(Glycidyl ~ethacrylate) Co ~Ethylene
Dimethacrylate)] Carrier and Its Derivatives," Svec, F. et
al, Biotechnology and Bioengineering, Vol. XX~ pp. 1319-
13~8 (197~). The immobilization of the biologicals
proceeds as described hereinabove. Condensation reactions
may be accomplished between free carboxyl and amine groups
via carbodiimide activation of the carboxy groups as is
described, for example, in "New Approaches to Non-
Thrombogenic Materials," Hoffman et al, Coagulation -
Current Research and Clini_al Applications, Academic
Press, N.Y. (1973). Briefly the immobilization of the
biologicals is effected by carbodiimide activation by
either the polymer or biological carboxyl groups and
condensation with a free amine to form a stable peptide
bond. The final orientation of the biological is
generally a factor as to whether an amine or a carboxyl
containing polymer be utilized.
Coupling reagent attachment can be accomplished
using a variety of coupling agents to form covalent
bridges between polymers and biologicals. Here free
hydroxyl and/or amine containing polymers and biologicals
are covalently coupled by reagents such as, for example,
cyanogen bromide, diisocyanates, dialdehydes and trichloro
-s~triazine. More exhaustive discussion of this technique
may be found for example, in the Chen et al article cited
hereinabove.
The preferred method of immobilizing a reactive
biological onto a biocompatible polymer substrate in a
given case generally is dictated by the molecular loca-
tions of the reac-tive binding moiety of the biological and
the functional groups on the biological and polymer
substrate which can be covalently combined. For example,
it is presently preferred in the case o~ polymer
substrates containing terminal hydroxy functions to
activate by treatment with an alkaline solution o~
cyanogen bromide (10 to 20% w/v~. Typically the reaction
mixture is maintained at room temperature (20 to 25C)
~or about 30 minutes. The p~l of the solution is
mainLained in a range of about 10 to 12, by the ~.ddition
of alkaline material, e.g., KOH or NaOH. The polymer is
extensively washed with physiological saline (0.9 gm%) and
incubated with solutions of a puri-fied biological
dissolved in a slightly alkaline buffer solution for 12 to
16 hours at 2 to 8C. The polymer is extensively rinsed
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with physiological saline to remove unbound or
nonspecifically bound biological components.
Biologicals are immobilized on glycidyl containing
polymers via ether, thioether or alkylamine bonds.
Epoxy-activated polymer substrates are rinsed and swollen
with aqueous neutral buffer solutions at room temperature.
Purified biologicals, dissolved borate~ carbonate or
phosphate buffer solutions are incubated with the glycidyl
polymer substrate for 12 to 20 hours at 4 to 30C.
Excess and nonspecifically bound biologicals are removed
by rinsing the polymer with saline, acetic acid (0.2 to
l.OM) and phosphate-buffered (pH = 7.2 ~ 0.2) saline
solutions. Activation of amine and carboxyl containing
polymer matrices are effected by treatment with purified
biologicals dissolved in sliyhtly acidic (pH 4.5 to 6.5)
buffer solutions of a water soluble carbodiimide. Bio-
logicals are covalently coupled to polymer support
substrates by incubation of polymer support, biological
and carbodiimide reactants for 12 to 16 hours at 2~ to
8C. ~he polymer-biological conjugates are washed alter-
nately in acid then alkaline rinses until the rinse
solutions are clear of biological and carbodiimide
reactants.
In order to determine the specific binding charac-
teristics of the polymer i~nobili2ed biologicals,
physiological serum solutions of complementary
biomolecules were treated with activated membranes. The
amounts of biomolecule were measured radiochemically.
Significant reduction of specific biomolecules resulted
following brief exposures to the biologically modified
polymer substrates.
III. SPACERS
In the present invention, a spacer may be defined as
a molecule or compound which is capable of attachment to
the surface of a biospecific polymer support, is large
enough to extend from the surface of said support and is
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capable of immobilizing a biological and/or biologicals.
The spacer insures that the active site of the biological
is held outward away from the support so as to contact the
body fluid more efficiently. It is obvious from the above
that, of course, the reactivity for binding with the
desired dlsease complex is, in fact, retained after
immobilization of the biological or biologicals onto the
spacer and therefore onto the biocompatible polymer
support.
The spacers are derived from organic mol~cules
having at least two reactive functional groups generally
situated at opposing ends of the molecule. Such groups
serve as attachment vehicles capable of coupling the
spacer to the polymer support and to the biological. The
reactive functional groups on the spacer may be the same
or different with the caveat that they react with func-
tional groups along the surface of the polymer support and
the functional groups extending from the biological
forming covalent bonds. Any known method for carrying out
such coupling reactions will suffice~ For example, the
me~hods described hereinabove outlining coupling routes
for attaching a biological directly onto a polymer support
may be used.
Suitable examples of spacers which may be used in
the present invention, where the reactive -Eunctional
groups are the same, include, ~or example, 1,6-diamino-
hexane, glutaraldehyde, 1,4-cyclohexane-dicarboxylic acid,
ethylenediamine tetraacetic acid, triethylene glycol,
1,4-butanediol diglycidyl ether, methylene-p-phenyl
diisocyanate and succinic anhydride. Examples of spacers
in which the reactive functional groups are not the same
include, for example, 6-aminocaproic acid, p-nitrobenzoyl
chloride, 1,2-epoxy-3-(p-nitrophenoxy) propane,
aminopropyltriethoxy-silane and homocysteine thiolactone.
Polypeptides and proteins may also be used as
spacers in the present invention. Albumin, a low affinity
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protein, for example, has been successfully employe~ as a
spacer. In addition, albumin and other natural proteins
serve to render the polymer support more blocompatible.
Finally, it is understood that certain materials may
act simultaneously as a spacer and as the activator in the
reaction used to combine the spacer and the biocompatible
support. Examples of these kinds of compounds, include,
for example, gluteraldehyde and 1,4-butanediol diglycidyl
ether.
IV. SUPPORT MEMBER
Most, if not all, of the suitable biocompatible
polymer supports have very low mechanical stability. Most
of these materials are, in fact, gels or gel-like as
opposed to materials which have high mechanical stability,
such as, for example, sheets of polypropylene. Thus, in
most embodiments utilizing the present invention, a
support member which is mechanically stable is necessary.
This support member allows large surace areas to be
utilized to insure rapid and medically, as well as commer-
cially, acceptable levels of immune disease-associated
component removal. The support member, besides being
mechanically stable, should also be inexpensive and must
be sterilizable so as to be made compatible for use in a
system wherein the blood of a diseased patient is to be
treated by the present invention. Examples o~ materials
whieh are suitable for the present invention as support
members include, for example, filter paper, polyester
fiber, polycarbonates, retieulated polyurethanes, NORYL '
a polyphenylene oxide polymer manufaetured by the General
Eleetrie Company, microporous polyolefins sueh as
polypropylene, and eotton cloth.
Many methods of attaching the activated or
biocompatible polymer support having biologicals
chemically attached may be utilized. Thus, for example,
methods such as spin coating, horizontal casting, vacuum
impregnating, dip coating, dip coating with later
crosslinking, and solution copolymerization may be used.
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Specific examples of these methods may be found in the
examples hereinbelow.
V. ~HERAPEUTIC REGIMEN
Broadly stated, the presen~ly contemplated
therapeutic r~gimen of the present invention is for the
therapeutic treatment of autoimmune and other diseases
comprising exposing a diseased patient's blood or plasma
having a biospecific polymer having immobilized reactive
biologicals, thereby removing the specific pathological
effectors from said patient's blood or plasma and then
returning said blood to patient. This therapeutic treat-
ment may or may not necessitate the use of blood
separation techniques. Thus the treatment is contemplated
to be carried out in a manner similar to a dialysis
treatment with the advantage that total blood separation
may not be needed and that there is very little if any
physical damaging of normal blood components.
It is also possible, of course, to utilize the
present invention and the process of the present invention
in the treatment of plasma. The plasma may be obtained
from whole blood by any of the currently known and
practiced methods. Thus, for example plasma may be
separated from a patient's blood by known methods, then
treated by the present invention and then recombined with
the other blood components and returned to the patient
using currently known procedures. In addition plasma
which is being used in known medical treatments may
utilize the present invention to treat said plasma be~ore
being administered to a patient requiring plasma from a
blood bank for example. Obviously whole blood from a
blood bank may also be treated by and benefit from the
present invention.
It is also to be understood that the current
invention may also be used with other body fluids to
effect removal of pathological effectors.
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- 16 ~
Because of the advantages of the present invention
mentioned above as well as others which will be clear to a
person skilled in this art many types of disease states
are contemplated to respond to the present invention used
in a therapeutic regimen. Broadly stated six groups of
disease states could be advantageously treated. These slx
disease categories are disorders of immune components,
druq excesses, toxin exposure, imbalances of body
substances, infections, and neoplastic states. Many
diseases are currently treated using plasmapheresis and
cytopheresis where the desired result is removal of a
specific substance. The present invention and the process
of the invention would apply to these diseases currently -
treated by plasmapheresis and cytopheresis.
Examples of immune complex diseases which can be
treated are, for example, any disease states involving
antibody, antigen, antibody-antigen, antigen-antigen and
antibody-antibody interactions, cell surface complexes,
cytoplasmic complexes, etc.
Examples of drug overdoses which can be treated are,
for example, overdoses of iron, dioxin, aspirin, TYLENOL~
methotrexate and other tricyclics.
Examples of poisons and toxins for which the present
invention is suitable are, for example, lead; aluminum,
mushrooms (Anatoxin~ and organic phosphates.
Body substances when present in excess can lead to
disease. Examples of these which can be eliminated using
the present invention include, for exam?le, cholesterol,
uric acid, immunoglobulins, sickle cells, uremic toxins,
bilirubin, porphyrin, cortisol and prostaglandins.
Some examples of infectious agents which may be
treated are, for example, viral disorders such as
cytomegalovirus; protozoan disorders such as malaria,
trypanosomes and leishmanias; bacterial infections such as
strepotococci; fungus infections such as tinea versicolor;
mycoplasma such as pleuro-pneumonia-like organisms;
rickettsia diseases such as typhus and spotted fevers;
- 17 -
spirochetes such as syphilis and chlamydia~agents in the
psittacosis lympho-granuloa-trachoma disease group.
Neoplasms which are treatable using the present
invention include, for example, the lymphomas, sarcomas,
carcinomas and leukemias. These may be removed by
specific removal of a cell line; inhibitors, initiators of
the disease and combinations thereof.
Further examples of disease states ~7hich may be
treated using the present invention include, for example,
the following:
Infections such as; Post streptococcal
glomerulonephritis, Subacute bacterial endocarditis,
Secondary syphilis, Pneumococcal sepsis, Lepromatous
leprosy, Ventricular shunt infection, Infectious
mononucleosis, Typhoid fever, Subacute sclerosing
encephalitis, Landry-Guillain-Barre syndrome, Hepatitis B
infection, Quartan malaria, Schistosomiasis, and
Trypanosomiasis.
Neoplasmas such as; Hepatoma, Lymphoma and Hodgkins
disease, Acute leukemia, Hypernephroma, Carcinoma of the
colon, Bronchogenic carcinoma, and Burkitts lymphoma.
Connective Tissue Disorders such as; Periarteritis
nodosa, Chronic glomerulonephritis, Acute or subacute
thyroiditis, Vinyl chloride poisoning, Chronic liver
disease, Mixed cryoglobulinemias, Berger 7 5 disease or IgA
nephropathy, ~apidly progressive glomerulonephritis, and
Sickle ce~l anemia.
Flematologic Diseases such as, Thrombic
thrombocytopenic purpura, Autoimmune hemolytic anemia,
Idiopathic thrombocytopenic purpura, Idiopathic
neutropenia, Cold hemagglutinin disease, Paroxysmal cold
hemoglobinuria, Circulating anticoagulants, Acquired
hemophilia, the leukemias, the lymphomas, Erythroblastosis
fetalis, Pernicious anemia, and Rh diseases.
Neurologic Diseases such as; Acute demyelinating
encephalitis, Multiple Sclerosis, Landry's paralysis,
'74.~'~
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Guillain-Barre syndrome, Peripheral neuritis, and
Myasthenla gravis.
Collagen Diseases such as; Raynaud's, Lupus
Erythematosus, Polyarteritis nodosa, Scleroderma,
Dermatomyositis, Sjogren's syndrome, Rheumatoid arthritis,
Rheumatic fever, and Erythema nodosa.
Endocrine Diseases such as, for example; Cushing's
syndrome & disease, Thyroiditis, Thyrotoxicosis, Addison's
disease, and Aspermatogenesis.
Gastrointestinal Diseases such as; Portal cirrhosis,
Acute hepatitis, Chronic active hepatitis, Lupoid
hepatitis, Biliary cirrhosis, ~lcerative colitis, Regional
enteritis, and Pancreatitis.
Mi.scellaneous Diseases such as, for example;
Hypercholesterolemia, Glomerulonephritis, Basement
membrane disease, Psychogenic states - drugs, Postaortic
valve prosthesis - hemolytic anemia, Exfoliative
dermatitis, Id reaction, Psoriasis, Behcet's syndrome,
Carcinoma, Subacute bacterial endocarditis, Hypertension,
Asthma, Hereditary angioneurotic edema, Meningococcemia,
Crohn disease, Hepatic encephalopathy and Raynaud disease.
Further, Diseases characterized by Antibodies to
Nuclear Antigens, Cytoplasmic Antigens, Cell Surface
Antigens, and Subclasses may be treated by the present
invention. Suitable examples include, for example;
Antibodies to Native-DNA (aouble stranded~ or single and
double stranded, Antibodies to SS DNA, Antibodies to
Deoxyribonucleoprotein, Antibodies to Histone, Antibodies
to Sm, Antibodies to RNP, Antibodies to Sc 1-1 -
Scleroderma, Antibodies to SS-A - Sjogren syndrome,
Sicca complex, Antibodi.es to RAP - Rheumatoid Arthritis,
Sjogren syndrome, Antibodies to PM-1
Polymyositis-dermatomyositis, and Antibodies to nucleolar-
Systemic sclerosis, Sjogren syndrome.
Also, Antibodies Associated With Specific Autoimmune
Disorders such as; Antibodies to smooth muscle - Chronic
~lepatitis, Antibodies to acetylcholine receptors
1 9
Myasthenia gravis, Antibodies to basement membrane at the
dermal-epidermal junction ~ Bullous pemphigoid, Antibodies
to the mucopolysaccharide protein complex or intracellular
cement substance - Pemphigus, Antibodies to
immunoglobulins - Rheumatoid arthritis, Antibodies to
glomerular basement membrane ~ Glomerulonephritis,
Goodpasture's syndrome, Idiopathic primary hemasiderosis,
Antibodies to erythrocytes - Autoimmune hemolytic
anemia, Antibodies to the thyroid - Hashimoto's,
Antibodies to intrinsic factor - Pernicious anemia,
Antibodies to platelets - Idiopathic thrombocytopenic
purpura, Alloimmunization, Antibodies to mitochondria -
Primary biliary cirrhosis, Antibodies to salivary duct
cells - Sjogren's syndrome, Antibodies to the adrenal -
Idiopathic adrenal atropathy, Antibodies to thyroid
microsomal - Grave's Disease, Antibodies to thyroglobulin
- Addison's Disease, and Antibodies to islet cells -
Diabetes Mellitus.
Paraproteinemias such as, for example; Multiple
myeloma, Macroglobulinemia, Cryoglobulinemia, and Light
chain disease,
Hyperlipidemia such as; Primary biliary cirrhosis
and Familial Hypercholesterolemia.
Endocrinopathies such as; Grave disease and Diabetes
mellitus.
Alloimmunization such as, Hemolytic disease of the
newborn and Renal homograft rejection.
Also, suitable for treatment using the present
invention include, for example, Post Transfusion Purpura
and Autoantibody Diseases such as, Goodpasture's syndrome,
yasthenia gravis, Pemphigus vulgaris, Hematological
disease, Idiopathic (autoimmune) thrombocytopenic purpura,
~utoimmune hemolytic anemia, Inhibitor to factor VIII and
Polyradiculopathy/Guillain-Barre Syndrome.
Immune Complex Diseases may also be treated and
include, for example; Systemic Lupus Erythematosus,
- 20 -
Polyarteritis nodosa, Cutaneous vasculitis, Rheumatoid
arthritis, Glomerulonephritis, and Dermatomyositis.
While not subscribing to any one particular theory
over another a review of the probable progression of
autoimmune pathology suggests that the pathological
sequence is very likely initiated by a free antigen
challenge, followed by antibody evolution and complexing
and finali~ed by antibody excess and complement fixation
of formed complexes. Thus, for proper selection of the
biospecific polymer formulation and provision for proper
efficacy would require preliminary diagnostic procedures
to determine the predominant form of the autoimmune
effector. An illustrative example of this is described
below for the treatment of rheumatoid disease. Briefly,
rheumatoid disease can be characterized as following the
progression from a) free RF antigen (atypical Ig)
(rheumatic condition), b) free RF antibody evolution and
RF complexing and finally c) antibody excess and
complement activated RF complex fixation. Thus treatment
of rheumatoid disease in its early development could be
determined by detection of atypical immunoglobulins by
monoclonal rheumatoid factor (m-RF) antibodies. Treatment
at this stage would be best effected by m-RF activated
biospecific polymers to remove the offending antigen and
thus preventing the evolution of endogenous RF (e-RF)
antibodies. Diagnostic evidence of e-RF would indicate
the utilization of biospecific polymers having both m-RF
and aggregated gamma globulin active biologicals (RF
antigen). Alternatively, two biospecific polymers in
series, each having one type of active biological could be
utilized. In either case this combination of m-RF and
aggregated gamma globulin would adsorb both the offending
antigen and antibody molecules to sequester the disease
progression. In the case where significant levels of RF
antigen- antibody complex is detected, biospecific
polymers containing Clq and/or collagen effector molecules
.
- 21 -
would be indicated. Finally, if the disease process has
progressed to the stage of complement fixation of formed
immune complexes an effective biospecific polymer would
contain one or more anti-complement antibodies such as,
for example, anti-Clq, anti-C3 or anti-C4. Again the
hiologicals, if more than one is desirable, can be
immobilized on a single biocompatible support or each can
be on a separate support and connected in series in
relation to the blood or plasma flow.
As has been proposed above, effective use of the
present invention is realized by thorough definition of
the dynamics and stage of the immune response for
effective disease management.
Today, plasmapheresis and cytophoresis are the
treatments for disease by removal of noxious substances or
cells from the blood. It is currently believed that any
disease treated by plasmapheresis and/or cytopheresis,
where the desired result is the removal of a specific
substance, can be advantageously treated with the product
and process of the present invention.
More specifically, a presently contemplated
therapeutic regimen for whole blood may be illustrated as
follows:
a~ a vascular access is provided which will allow
for;
b) a blood flow of from about 30 ml/min. to about
200 ml/min.,
c) an anticoagulant is administered to the blood;
and
d) a pumping means is provided;
e) the blood is passed in contact with the
present invention;
f) depending on the anticoagulant used,
additional medication may be needed or desired to
neutralize the anticoagulatory effect on said treated
blood;
g) the treated blood is returned to the patient.
- 22 -
The time frame presently contemplated for the above
regimen is approximately from about 2 hours to about
4 hours~ It is realized, of course, that depending upon
the situation such time frame may be either shortened or
lengthened.
A presently contemplated therapeutic regimen for
plasma may be illustrated as follows:
a) a vascular access is provided which will allow
for;
b) a blood flow of from about 30 ml/min. to about
200 ml/min.,
c) an anticoagulant is administered to the blood;
and
d) a pumping means is provided;
e) a plasma-formed blood component separation
means is provided;
f) the plasma is passed in contact with the
present invention;
g) filtration through a 0.2 micron filter to
remove any microemboli, bacteria and/or fungi,
h) the treated plasma and the formed blood
components are recombined;
i) depending on the anticoagulant used,
addltional medication may be needed or desired to
neutralize the anticoagulating effect on said treated
blood;
j) the treated blood is returned to the patient.
The vascular access may be provided using well known
techniques and procedures in the medical arts. Thus, for
exa~ple, an indwelling large bore cannula may be used
intravenously or arterially. Examples of suitable veins
and arteries include the antecubital vein, subclavian vein
and brachial or radial arteries. It is further understood
that an arterial venous shunt or fistulae (AV shunt) may
also be used. In this case the heart is the pumping
means. If an AV shunt or fistulae is not used the
preferred pumping means during venous access is a
t
- ~3 ~
roller-peristalic pump capable of providing a flow rate of
from about 30 ml/min to about 200 ml/min.
Suitable anticoagulants useful in the process of the
present invention include, for example, acid citrate
dextrose (approximately 1 ml to every 8 ml of whole
blood), heparin, heparin/acid citrate dextrose mixtures
(e.g. 1250 IU heparin in 125 ml acid citrate dextrose/L),
and prostaglandin. It is ~o be appreciated that in using
anticoagulants such as heparin and prostaglandin it is
generally understood that a counteracting medication
should be administered to the treated blood or plasma
before returning or giving said blood or plasma to a
patient.
Further, in the case of treating plasma, it is
understood that any conventional methods of removing the
formed blood components may be used. Suitable examples of
methods of separating plasma from formed blood components
include, plasmapheresis, centrifugal cell separation, and
cell sedimentation in a plasma bag. Where possible both
continuous separation and intermittent (batch) separation
are suitable - the aforementioned methods of separation
are independent of the present invention and its use.
Finally, the form of the present invention is,
generally, not critical. Thus the present invention may
utilize a biocompatible support containing the biological
in the form of sheets, hollow fibers, cylindrical fibers,
reticular networks, cylindrical or rectangular channels,
beads and combinations thereof for example. The use of a
fluidized bed may also be advantageous in some cases.
EXAMPLE 1
This example describes one method of casting the
biocompatible polymer support and a method of chemically
attaching a biological directly to the polymer supportO
This example also is used to describe the use of a system
having no mechanical support associated with it.
- 24 -
ABSORPTION OF ANTI-INSULIN ~NTIBODIES USING
INSULIN ACTIVATED POLY-HYDROXYETHYL
METHACRYLATE (p-HEMA) MEMBRANE
A. Polymer castlng. Solutions of monomer were
prepared by combining 15.0 g 2-hydroxyethyl methacrylate
(Polysciences Inc., Warrington, PA), 15.0 g ethylene
glycol (Fisher Scientific, Pittsburg, PA), 0.08 g sodium
bisulfite (Fisher) and 0.036 g ammonium persulfate
(Fisher). The solution was stirred for 15 minutes at room
temperature. Approximately 5 ml of solution was placed on
a glass plate ~5" l x 5" w x 3/8"t) in the center of a
polyethylene spacer (10 mil thick) cut to form a gasket
with a 4" x 4" window. A second glass plate was placed
over the gasket and solution, clamped in place and the
entire assembly incubated at 60C overnight. The clamps
were removed and the glass plates were pried slightly
apart and transferred to a deionized water bath for at
least 24 hours. The swollen polymer membrane was
carefully removed from the glass plates and was rinsed-
hydrated for at least three days in fresh exchanges of
deionized water (500 ml per day).
B. Polymer activation. Membrane discs (5 mm
-
diameter were cut from the polymer sheet for activation
and analysis. A 10-20 gm % cyanogen bromide (Eastman
Kodak Co., Rochester, NY) solution was prepared by
dissolving 1.69 g of finely divided BrCN crystals in 10 ml
of 0.2 ~ Na2CO3 (pH 11.1) with continuous stirring at 4C.
The pH of the solution was maintained above 11 by the
dropwise addition of 5N NaOH until the crystals were
dissolved and the pEi was stabilized. Four membrane discs
were placed in a small sieve and rinsed with approximately
5 ml 0.1 N HCl and incubated for 15 minutes in the
cyanogen bromide solution. The discs were each rinsed at
least two more times with 5 ml portions of 0.1 N HC1 and
incubated overnight in 5.0 ml U-100 regular ILETIN
insulin injection solution (Eli Lilly, Indianapolis, Ind.)
~ `
L'742~
- 25 -
which had been adjusted to a pH of 8.7 by the addition of
1 N NaOH. The memhrane discs were rinsed with 5 ml 0.5 M
NaClj 0.lM Na2CO3 solution and 3 times in 5 ml aliquots of
phophate (0.05M) buffered saline (0.9 gm %) solution
(pH=7.~).
C. ~valuation of membrane adsorption of
anti-insulin antibody. A double antibody competitive
~._ . . _
binding radioim~unoassay was performed by incubating 560
pg (picogram) I125 labeled porcine insulin (New England
Nuciear, Boston, MA) and serial dilutions (980 to 15 pg)
of non labeled porcine insulin (Cambridge Nuclear,
Billerica, MA) or the p-HEMA membrane discs with 280 pg o~
guinea pig anti-porcine insulin antibody (New England
Nuclear) in 0.5 ml of phosphate ~0.05 M) buffered (pH 7.4)
saline (0.9 gm ~) containing 1 gm % Bovine Serum Albumin
(Sigma Chemical Co., St. Louis, MO) for two hours at room
temperature. The p-HE~ discs were removed from each test
solution. A 0.1 ml aliquot of goat anti-guinea pig gamma
globulin was added to each test tube. The test solutions
were mixed and incubated ~or an additional two hours at
room temperature. A 1.0 ml aliquot of cold t2-4C)
phosphate buffered saline (pH 7.4) was added to each tube.
Each test solution was mixed and centrifuged for 15
minutes at 4C at 7500G and the supernatant decanted into
20 ml scintillation vials.- The supernatant was gelled
with 5.0 ml Aquasol liquid scintillation fluid (New
England Nuclear) and counted in an Isocap 300 Counter
(Searle Analytic Inc., DesPlaines, I~) for 4.0 minutes.
Insulin treated membrane discs adsorbed lllpg anti-insulin
antibody from solution or 283Pg per sq. cm. surface area.
EXAMPLE 2
-
This example describes how an unsupported
biospecific membrane may be produced. It also describes
how 6-aminocaproic acid (having a six carbon chain) may be
used as a spacer for attaching insulin to the
~i
`c.~
- 26 -
biocompatible polymer support used to remove insulin
antibody and adsorption of anti-:insulin antibodies using
the insulin activated poly-hydroxyethyl methacrylate
(p-HEMA) membrane.
A. Polymer casting. Solutions of monomer were
prepared by combining 15.0 g 2-hydroxyethyl methacrylate
~Polysciences Inc., Warrington, PA), 15.0 g ethylene
glycol (Fisher Scientific, Pittsburg, PA), 0.08 g sodium
bisulfite (Fisher) and 0.036 g ammonium persulfate
(Fisher). The solution was stirred for 15 minutes at room
temperature. Approximately 5 ml of solution was placed on
a glass plate (5" l x 5" w x 3/8"t) in the center of a
polyethylene spacer (10 mil thick) cut to form a gasket
with a 4" x 4" window. A second glass plate was placed
over the gasket and solution, clamped in place and the
entire assembly incubated at 60C overnight. The clamps
were removed and the glass plates were pried slightly
apart and transferred to a deionized water bath for at
least 24 hours. The swollen polymer membrane was
carefully removed from the glass plates and was rinsed
hydrated for at least three days in fresh exchanges of
deionized water (500 ml per day).
s. Polymer activation. Membrane discs were
prepared as previously described in Example 1. A 10-20 gm
~ cyanogen bromide (Eastman Kodak Co., Rochester, NY)
solution was prepared by dissolving 1.69 g of finely
divided BrCN crystals in 10 ml of 0.2 M Na2CO3 (pH 11.1~
with continuous stirring at 4~C. The pH of the solution
was maintained above 11 by the dropwisè addition of 5N
NaOH until the crystals were dissolved and the pH was
stabili7ed. Four membrane discs were placed in a small
sieve and rinsed with approximately 5 ml 0.1 N HCl and
incubated for 15 minutes in the cyanogen bromide solution.
The discs were each rinsed at least two more times with 5
ml portions of 0.1 N HCl and incubated overnight in 10 ml
of a 10 gm % 6-aminocaproic acid solution (w/v) (Sigma
Chemical Co.) prepared in 0.1 M Na2CO3, 0.5 M NaCl buffer
~z~
~ 27 ~
solution, pH 8.6. Membrane discs were rinsed with 5 ml
0.1 M Na2C03, 0.5 M NaCl buffer and three 5 ml aliquots of
phosphate (0.05 M) buffered (pH=7~4) saline (0.9 gm %)
solution. The membrane discs were removed from the rinse
solution, activated by incubation in 10 ml of a 10% (w/v)
1-cyclohexal-3-(2 morpholinoethyl) carbodiimide (Sigma
Chemical Co.) solution prepared in 0.1 M (2~N-morpholino]
ethanesulfonic acid) (MES) buffer (pH 6.0~ for thirty
minutes at room temperature and each disc rinsed in 5 ml
of cold (4C) phosphate buffered saline solution.
Duplicate membrane discs were incubated overnight in 5.0
ml of either U-100 regular ILETIN insulin injection
solution or pork insulin regular ILETIN solutions (Eli
Lilly, Indianapolis, IN) at 4Co Membrane discs were
removed from the protein solutions and rinsed three times
in 5 ml of phosphate buffered saline solution.
C. Evaluation of membrane adsorption of
anti-insulin antibody. A double antibody competitive
binding radioimmunoassay was performed by incubating 560
pg I labeled porcine insulin (New England Nuclear,
Boston, MA) and serial dilutions (980 to 15 pg3 of non
labeled porcine insulin (Cambridge Nuclear, Billerica, MA)
or p-HEMA membrane discs with 280 pg of guinea pig
anti-porcine insulin antibody (New England Nuclear) in 0.5
ml phosphate (0.05 M~ buffered IpH 7.4) saline (0.9 gm %)
containing 1 gm % bovine serum albumin (Sigma Chemical
Co., St. Louis, M~) for two hours at room temperature.
The p-HE~ discs were removed from each test solution. A
0.1 ml aliquot of goat anti-guinea pig gamma ~lobulin was
added to each test tube. The test solutions were mixed
and incubated for an additional two hours at room
temperature. A 1.0 ml aliquot of cold (2-4C) phosphate
buffered saline (pH 7.4) was added to each tube. Each
test solution was mixed and centrifu~ed for 15 minutes at
4C at 7500 G and the supernatant decanted into 20 ml
scintillation vials. The supernatant was gelled with 5.0
ml Aquasol liquid scintillation fluid (New England
Nuclear) and counted in an Isocap 300 Counter (Searle
~'7g~2~
~ 28 -
Analytic Inc., DesPlaines, IL) for 4.0 minutes. Insulin
treated membrane discs adsorbed 271 pg anti-insulin
antibody from solution or 690 pg per sq. cm~ surface area.
EXAMPLE 3
This example describes a method of casting the
biocompatible polymer supports, both with and without
mechanical support, via spin casting. This example also
describes a second way of chemically binding the
biological to the biocompatible polymer support.
ADSORPTION OF ANTI-HUMAN IMMUNOGLOBULIN GlIgG)
ANTIBODIES (RHEUMATOID TYPE "FACTORS"~ USING
I~MUNOGLOBULIN ACTIVATED POLY-HYDROXYETHYL
METHACRYLATE-CO-GLYCIDYL METHACRYLATE (p-HE~L) MEMBRANES:
I. Polymer castin~. The following example
describes the production of both supported and unsupported
polymer membranes by spin casting techniques.
A. SPin Casting Device
The spin casting device consists of a closed
aluminum drum with 1/4 in. thick walls. The inside
dimensions of the drum are 4 in. in diameter and 5 in. in
length. The drum is connected to a motor (Fisher
Dyna-Mix; Fisher Scientific Co., Pittsburg, PA) which
spins the drum, and the drum rpm is measured with a st~obe
phototachometer (Model 1891M Power Instrument Inc.,
Skokie, IL). A heat blower gun (Fisher Scientific Co.)
hPats ~he spinning drum; thermocouples measure the
internal drum temperature and the temperature of the air
flowing over the drum. The drum is purged with nitrogen
before and during the polymerization.
B. Supported Membrane Production
Whatman Grade 50 hardened filter paper (Fisher
Scientific, Pittsburg, PA) was used as a support backing
to provide mechanical strength for these spin castings.
The paper was cut into rectangular sheets (4-15/16 x
- 29 -
12-7/16 in.) and then soaked in ethylene glycol ~EG)
(Fisher Scientific Co., Cat. No. E-177) for 30 min. at
room temperature. The excess glycol was drained from the
paper; after draining, the paper contained 2-4 g of EG.
The conditioned paper was curled in the form of a cylinder
and placed inside of the spin casting drum. The outside
edge of the paper was pressed against the drum wall to
expel any air between the wall and the paper. When the
paper is in place, it is preferable but not necessary that
the ends of the paper are butted up against each other;
there can be some overlap. The paper backing was checked
for entrapped air pockets; if any existed, they were
removed with a rubber policeman.
For polymerizations which produce a very adhesive
polymer, the spin casting cylinder can first be lined with
a sheet of silicon release paper by placing the nontreated
side of the paper against the cylinder. The conditioned
Whatman filter can then be placed against the release
paper carefully so as not to entrap air.
C. Polymerization Formulations
The following are representative polymerization
formulations currently being used. In each case, the
initiator was stirred with the reactive monomer(s) at room
temperature for 30 minutes or until the initiator
dissolved.
.~Z~7~
- 30 ~
GMA-HEMA (50/50) Copolymer
. .
6.25 g 2-Hydroxyethyl Methanoxylate (~IEMA)
6.25 g Glycidyl Methacrylate (GMA)
12~5 g Ethylene Glycol (EG)
0.02 g 2,2' Azobis(2-amidinopropane)
Hydrochloride [~BAP)
5~-NVP-HEMA (50/40/10~ Copolymer
6.25 g GMA
1.25 g NVP
5.00 g HEMA
12.5 g EG*
0.02 g ABAP
* The ethylene glycol weight includes 2-4 g of EG on
the Whatman paper.
D. Spin Casting Procedure
-
While the initiator was dissolving in the
monomer(s), the drum was loaded into the spin casting
assembly. The drum was spun at 1400 rpm at room
temperature and purged with nitrogen for 15 minutes after
which the initiator-monomer solution t25.0 ml) was
injected into the drum with a hypodermic syringe having a
flexible Teflon tip. The nitrogen purge was resumed and
the drum speed increased to 2,900 rpm.
The fan (ca. 35 ft3/min.l and heater on the heat gun
were started, and the drum was heated at 70-75C for 90
minutes. The heat was then shut off, but the fan was left
on to cool the drum until the internal drum temperature
dropped to about 30C. The cool drum was removed from the
spin casting apparatus and filled with deionized water.
After soaking for an hour, the casting was removed from
the drum.
II. Polymer activation. Membrane discs were
prepared as previously described in Example 1. Fourteen
individual discs were each incubated in 1.0 ml of 1.0 M
- 31 -
hexane diamine (Eastman Kodak, Rochester, N.Y.) solution
for 72 hours at 4C. The discs were removed from the
hexane diamine solution and washed three times with 2 ml
phosphate buffered saline solution. A 4.0 gm % human
gamma globulin (HGG) (Sigma Chemical Co.) solution was
prepared by dissolving 4.0 gm HGG in 100 ml 0.1 M MES
buffer (pH 6.0) solution with gentle stirring at room
temperature. After the protein was completely dissolved,
serial dilutions were made by successive transers of 1.0
ml protein solution to 9.0 ml MES solution to yield
protein concentrations of 4 mg/ml, 400 ug/ml, 40 ug/ml and
4 ug/ml of buffer. Two individual polymer discs were each
incubated in 0.5 ml of the protein solutions and 0.5 ml of
a 0.25 M 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide
~Sigma Chemical Co.) solution prepared in MES buffer for
72 hours at ~C. Each membrane disc was removed from the
protein solution and rinsed 3 times with 2 ml of cold
(4C) phosphate buffered saline.
III. Evaluation of membrane adsorption of anti-IgG
antibody from physiological solutions. A radioimmunoassay
was per~ormed by incubating individual membrane discs with
10 ng I goat anti-human Ig~ (New England Nuclear) in
1.0 ml PBS which contained 1.0 gm % Human Serum Albumin
(Sigma Chemical Co.) for two hours at room temperature.
The radiotracer solution was removed and each membrane was
rinsed three times with Z.O ml PBS solution. The
membranes were incubated in the last rinse solution
overnight at 4~. Individual membranes were removed from
the rinse solutions and counted in an Innotron Hydragamma
counter (Scientific Products) for one minute each. Counts
per minutP were converted to disintegrations per minute
~DPM) by division with the detector efficiency. The
amount of adsorbed antibody was approximated by dividing
the average DPM by the radiotracer specific activity. The
following results were obtained:
- 3~ -
HGG TreatmentAnti IgG Adsorbed
(mg/ml~(py per sq. cm.)*
20.0 2453
2.0 1919
- 0.2 1271
0.02 664
0.002 **
* Picograms of radiotracer material per square
centimeter of membrane.
** Background activity.
EXAMPLE 4
This example shows the use of amino caproic acid as
a spacer for gamma-globulin.
Adsorption of anti-human Immunoglobulin G (Ig G)
antibodies (Rheumatoid type "Factors") using
immunoglobulin activated poly-hydroxyethyl
methacrylate co-glycidyl methacrylate (p-HEGL) membranes.
A. Polymer casting. Spin Cast p-HEGL membranes
were prepared as described in Example 3.
B. Polymer_ derivatization and activation.
Membrane discs were prepared and ~reated as described in
Example 3 except that 1.0 M 6-amino caproic acid (Sigma
Chemical Co.) was substituted for hexane diamine as a
derivitization and spacer agent.
C. Evaluation of membrane adsorption of anti-IgG
antibody from physiological solutions. A radioimmunoassay
was performed as described in Example 3 and the following
results were obtained:
- 33 -
HGG Treatmentanti IgG adsorbed
(mg/ml)(pg per s~. cm.)*
20.0 2395
2.0 1828
0.2 1310
0.02 732
0.002 158
* Picograms of radiotracer material per square
centimeter of membrane.
EXAMPLE 5
This example shows the use of albumin (67,000 MW) as
a spacer for folate. The folate is used to remove folic
acid binding protein.
Adsorption of Folic Acid Binding Proteins (FABP) hy
Folate-Albumin activated poly hydroxyethyl methacrylate
(p-HE~) membranes:
A. Polymer Casting. Filter paper supported
p-HEMA polymer memb~anes were spin cast as described in
Example 2 utilizing the following polymer formulation;
15.0 g 2-Hydroxyethyl Methacrylate
15.0 g Ethylene Glycol
0.08 g Sodium Metabisulfite
O.036 g Ammonium Persul~ate
B. Polymer derivatization and activation. A
~olic acid Bovine Serum Albumin complex was prepared by
carbodiimide condensation of folate carboxyl groups with
albumin terminal amine groups. To achieve this 200 mg
folic acid ISigma Chemical Co.) was dissolved in 8 ml 0.1
N NaOH and 400 mg 1-cyclohexyl-3(2-morpholinoethyl)-
carbodiimide metho-p-toluenesulfonate (Sigma Chemical Co.)
was dissolved in 2.0 ml 0.1 M MES buffer (pH 6.0) and 1.0
gm Bovine Serum Albumin (BSA) was dissolved in 40.0 ml 0.1
- 34 ~
M MES buffer. The solutions were combined, mixed and
incubated for 72 hours at 4C. The unreacted folate and
carbodiimide was removed from solution by trea-tlng 20 ml
of the mixture with 20 ml of a BSA (2.5 am ~) - charc3al
~1.25 gm %) suspension for thirty minutes at 4C. The
suspension was centrifuged for 15 min. at 3400 G at 4C
decanted and filtered through a 0.22 micron filter.
Membrane discs were prepared and treated with
cyanogen bromide solution as previously described in
Example 1. After the discs were rinsed in cold saline
solution, sets o eight discs were added to and incubated
in 20 ml of either physiological saline, 160 mg ~ BSA or
the folate albumin complex solution previously prepared.
The discs were incubated for 72 hours at 4C. Each
membrane set was removed from solution, blotted dry and
placed in 20 ml saline solution at 4C to rinse for at
least 24 hours. Duplicate membranes were treated with 8
ml of 1% gluteraldehyde solution for 1 minute and rinsed
overnight in 20 ml of phosphate buffered saline buffer.
C Evaluation of membrane adsorption of folic
.
acid binding protein (FABP~ from physiological solution.
A competitive protein binding radioassay was performed by
incubating 370 pg 3H-pteroylglutamic Acid ~PGA) (Amersham
Corp., Arlington Heights, IL) and standard dilutions (4S
to 348 pg) o~ non-radioactive PGA (Sigma Chemical Co.~ or
p-HE~lA membrane discs with 234 pg binding activity of FABP
(Kamen, B.A. and Caston, J.D., "Direct Radiochemical Assay
for Serum Folate: Competition between 3H-Folic Acid and
5-Methyl-tetrahydrofolic Acid for a Folate Binder", ~.
Lab. Clin. Med., 83, 164, 1974) in 1.0 ml of C.05 M
phosphate buffer (pH 7.6) which contained 20 ul of folate
free normal human serum and 5 mg sodium ascorbate (Sigma
Chemical Co.). The radioassay tubes were mixed, incubated
for 30 minutes at room temperature and 10 minutes at 4C.
Individual membrane discs were removed from the test
solutions and 0.5 ml of a cold (4C) BSA (2.5 gm ~) -
charcoal (1.25 gm %) suspension was added to each tube.
- 35 -
All test solutions were incubated for 10 minutes at 4C
and centrifuged at 2000 G for 15 minutes at 4C. The
supernatants were decanted into 20 ml scintillation vials.
Twelve (12.0) ml liquid scintillation fluid (Fisher
Scientific, Pittsburg, PA) was added to each vial.
Samples were counted in an Isocap 300 Counter ISearle
Analytic Inc.) for 2 minutes each. The following results
were obtained:
Membrane TreatmentFABP Adsorbed
(pg/sq. cm.)
Saline 67
Cyanogen Bromide 54
Bovine Serum Albumin 39
Folate BSA Complex 758
The above-described examples serve to illustrate the
present invention without restricting it in any way. It
will be obvious to those in the art that various changes
and modifications may be made without departing from the
spirit and scope of the present invention.
