Title of the Invention
METHOD FOR IMMUNE SWITCHING
Background of the Invention
1. Field of the Invention
This invention relates to the encapsulation of an immune modulator.
The pore size of the capsule acts as an immune switch. Encapsulation technology system and its targeted therapeutic applications are detailed.
2. The Prior Art:
Encapsulation technology is the packaging , carrier and delivery
system for cells and drugs. The technology immunoprotects cells and acts as
an extend-release-matrix for drugs. This approach is used to develop cure and treatment for many diseases. Several polymers have been used but
alginate is particularly favored for sensitive applications such as islet cell
encapsulation. Alginates are commercially available. Use of these heterogeneous and impure commercial preparations have lead to bioincompatible, hemoincompatible and inflammatory reactions with fibrosis,
thrombosis, abscess formations, infections, immunorejections and graft failures. This led to bottleneck in technology and prevented it from advancement in larger animal models from 1970 to 1990s. Endotoxin content of commercial alginate preparations was 100,000 E.UJg or greater.
In recent years, purified alginate and other polymers have been
introduced commercially. Prokop A. and Wang T. G. have studied and
reviewed the subject in "Purification of Polymers Used for Fabrication of an Immunoisolation Barrier" published in 'Annals of NY academy of Medicine',
1997, Vol.831: 223-231. Several methods for cell encapsulations have been
described in recent years using purified alginate. U. S. Patent No. 5,459,054 to Skjak- Braek et al describes the method of encapsulating cells with
purified alginate. The material has been used successfully to
microencapsulate islet cells to cure type 1 diabetes. U. S. Patent 5,879,709 to Soon-Shiong et aland US. Patent No.5, 573, 934 to Hubell et al details the
method to further improve biocompatibility of alginate by additional coating
with PEG and its applications. US. Patent No. 5,874,099 to Dionne et al
describes applications of purified alginate material in cell transplant
technology in general. Leblond F. A. et al evaluated biocompatibility, hemocompatibility and immune tolerance of alginate in "Studies on Smaller
(~ 315 micron ) Microcapsules: IV. Feasibility and Safety of Intrahepatic Implantation of Small Alginate Poly-L -Lysine Microcapsules". The findings
were published in "Cell Transplantation", Vol. 8, pp. 327-337, 1999. The removal of endotoxin appears to be the critical factor in above improvements of clinical results.
Endotoxins or Lipopolysaccharides(LPS) activate innate immune responses through CD 14 dependent and CD 14 independent pathways. CD 14
dependent pathway act through Human Toll-like Receptors(HTLRs), while CD 14 independent pathway act through complement mediated pathways.
Both pathways coordinate and translate mechanical and chemical signals
through dendritic cells to generate specific adaptive immune responses. Dendritic cells thus essentially act as a transducer. Further, translation of innate immune responses to adaptive immune responses also occur through breakdown products of C3 complement such as C3b and C3d. Recent
advances in endotoxin research suggest a human being is 1,000 times more
sensitive to the effects of endotoxin than small animal models. Fetal bovine
serum is frequently used in encapsulated technology for cells. It contains
lipopolysaccharide binding protein and sensitizes the effects of endotoxins.
Bacterial and viral contaminants also act as endotoxin sensitizers. Above factors can also increase endotoxin sensitivity by 1,000 fold or more. The
combined effects in human beings therefore could be 1,000 to 1,000,000 fold increased sensitivity to endotoxin. This leads to activation of innate and adaptive immune responses and both causes or contributes to immune rejections, bioincompatibility and graft failure. Outside the capsule it leads
to fibrosis and choking of oxygen and nutritional supply, while inside the capsule death of cells occur by apoptotic and nonapoptotic mechanisms.
Complement inhibitors that affect complement pathways at various junctures such as at Cl, C3, C5a, C5b-C9 and Factor D have been used with success to control and inhibit endotoxin mediated effects. In experimental
models, both control of endotoxin mediated effects and improved survival have been demonstrated.
U. S. Patent No. 4,265,908 to Conrow et al describes the
complement inhibitory effects of sulfonic salts and its various formulations for therapeutic uses. Hydrogel formulation of sulfonic salts and its polymer
with agarose have been described by Iwata H. et al in "Strategy for
Developing Microbeads Applicable to Isletxenotransplantation into a
Spontaneous Diabetic NOD Mouse" in the J. of Biomedical Materials
Research, 28(10), 1201-07, 1994. Sulfonic polymer, have been successfully
incorporated to improve biocompatibility in hollow fibers of polysulfone and
biosulfane used for hemodialysis. Sulfonic polymer component has additionally heparin-like properties. Labarre D. J. reviews this property in Ηeparin-like Polymer Surfaces: Control of Coagulation and Complement
Activation by Insoluble Functionalized Polymers" in The Int. J. of Artificial Organs 13:10, 651-657, 1990. Pascual M. et al details the "Specific
Interactions of Polystyrene Biomaterials with Factor D of Human Complement" in Biomaterials, 14, 9, 665-670, 1993. Kilpatrick J. M. et al details "Control of the Alternate Complement Pathway: Inhibition of Factor
D", Chapter 13, pp. 203-225, in controlling the complement system for novel
drug development, edited by Mazarakis H. and Swart S. J. published by IBC,
1997. Rustagi P. K. et al details "Development of Novel Broad Spectrum Serine Protease Inhibitors for use as Anticoagulants", Chapter 15, pp.307- 320, in "Anticoagulant, Antithrombotic and Thrombolytic Therapeutics II",
IBC series, 1998. US. Patent No. 5,660,825 to Sims P. et al describes the
composition of polypetide that has complement inhibitory property and its
therapeutic applications. U. S. Patent No. 5,679,345 to Sanfilippo et al
describes a method for preventing complement dependent rejection of organs
or tissue transplants using antibodies that prevent the formation of C5b-C9, membrane attack complex.
As shown by these prior art examples, bioincompatibility,
hemoincompatibility and immune rejections are major problems in medicine and surgery including implantation and transplantation biology. Further, it
is obvious that the rate-limiting step in the clinical application of foreign materials is the discovery of chemicals and compounds that have inhibitory
effects on bioincompatible and immune reactions. LPS and complement mediated effects are best described in relation to microbial infections. It has been recently hypothesized that "the simple antigen migration-localization
principle should further our understanding of the events that occur with or
without therapeutic intervention in a variety of infectious, neoplastic, and
autoimmune diseases or after transplantation, and may offer improved rationales for prevention and treatment". (Starzl T.E. and Zinkernagel R.M.,
"Review Article: Antigen Localization and Migration in Immunity and
Tolerance", The New England Journal of Medicine, Vol. 339, No. 26, pp. 1905- 1913).
Summary of the Invention
It is an object of the present invention to improve biocompatibility,
hemocompatibility and immune tolerance of polymers such as alginate by reducing its endotoxin content to a minimal level.
It is a further object of the present invention to combine ultrapure alginate with immune modulator for therapeutic purposes.
It is another object of the present invention to control the action and duration of immune modulators by combining ultrapurified alginate and sodium polystyrene sulfonate in a hydrogel formulation.
It is an additional object of the present invention to regulate the pore
size of the hydrogel to less than 100 kD to selectively permit inhibition of immune system for targeted therapeutic applications in implantations and transplantation biology.
It is a further object of the present invention to relax the pore size of
the hydrogel to more than 200 kD to permit selective activation of the
immune response for targeted therapeutic applications in certain infections, cancers and vaccine developments.
It is an additional object of the present invention to characterize
drug-device components of the encapsulation technology to develop encapsulation technology system and its targeted therapeutic applications.
It is a further object of the invention to develop an encapsulation
technology system that obviates prior art problems and meet or exceed minimal safety criteria of endotoxin content specified by the FDA for drug-
devices.
I achieve the above objects of my invention by first developing an
Encapsulated Cell Device as detailed in my U.S. Patent No. 5,976,780.
Ultrapurified alginate and sulfonic polymer are combined and cellular material is processed to remove sensitizing factors. This strategy helps prevent the harmful effects of endotoxin.
When 2% alginate with 5% polystyrene sodium are reacted with Ca
100 mM/L, instant hydrogel formulation occurs. Both exchange Na and bind
with Ca. This leads to the encapsulation of polystyrene sulfonate in
ultrapurified alginate gel. The pore size of the gel obtained is 50-100 kD.
Polystyrene sulfonate is a 50 kD polymer but it does not leach out on repeated washing with water or saline and it forms strong, stable bonds with
alginate. Factor D, is a 25 kD protein. It readily permeates the gel and avidly binds with polystyrene sulfonate to provide complement inhibition. In the
prior art, agarose gel was combined with polystyrene sulfonate. The hydrogel
has pores of 10 kD. This would protect NOD mice from immune rejection simply because of its lower molecular cut off as detailed in my U. S. Patent
No. 5,976,780. Complement inhibition could not account for such effects
because all complement proteins have molecular weight above 25 kD. Sodium polystyrene sulfonate thus was excluded to perform its complement
inhibitory effects. When combined with agarose, to prevent leaching,
additional coatings with polybrene and carboxy methylcellulose were required. In sensitive applications such as cell transplant, the procedure of agarose with polystyrene sulfonate requires repeated exposure to thermal trauma. When used intravenously, polystyrene sulfonate acts as a
suspension-solution and tends to precipitate out causing unreliable dosing,
has a tendency to clump and, due to more avid binding with Factor H, it may activate the alternate complement system. These problems have been
obviated when ultrapurified alginate is used in combination with polystyrene sulfonate.
Factor D circulates in the blood in the cone, of 1-2 meg/ ml but is a powerful complement activator. Thus, for example, cobra venom factor will not be able to convert C3 to C3c in the absence of Factor D. However, addition of even 1% of normal concentration of Factor D in -vitro is capable of
restoring C3 to C3c conversion. By contrast, bonded Factor D with sulfonic polymer in 100 times greater concentration fails to restore C3 conversion.
This indicates total loss of Factor D activity following its binding with
sulfonic polymer. The pore size of 50-100 kD prevents binding of Factor H which is a complement inhibitor. Factor H, as a complement inhibitor, accentuates the degradation of C3 convertase, binds with breakdown
products of C3 convertase and prevents costimulation of B cells and work with Decay Accelerating Factor (DAF) and Factor I to rapidly inactivate C3
convertase. Its binding with polystyrene sulfonate and its inactivation of complement inhibitory property, is therefore not desirable. Selective binding
of Factor D and selective exclusion of Factor H may help potentiate complement inhibition in a 50-100 kD pore size gel. The strategy helps improve reliability of sulfonic polymer as a complement inhibitor. By preemptive blockage of the complement system, transducer function
of dendritic cells is not activated. This way, complement inhibitors also
act as an inhibitor of adaptive immune responses.
Encapsulation of sodium polystyrene sodium in 200 kD pore size alginate will have opposite effects viz. activation of complement system by more avid binding with Factor H. This in turn will lead to activation of
transducer function of dendritic cells. Thus, complement activation also
contributes to activation of adaptive immune responses. The relaxation of pore size can readily be obtained by using appropriate dilution of alginate
and CaCl concentration and by shortening the reaction time. The activation
of immune response is beneficial in vaccine development as an adjuvant to speed immune responses. Thus, it can be used as military or civilian
preparedness against bioterrorism. Certain bacterial infections, viral
infections, parasitic infections and cancers evade immune system and thrive by binding with Factor H or by secreting Factor H-like substances.
Streptococcus pyogenes, Neisseria gonorrhoeae, HIV, Echincoccus granulosus and Yersinia entercolitica are such examples of infections that evade
immune responses by binding to Factor H. Cancers, such as of cervical, bladder and renal cells, evade immune response by secreting Factor H-like
substances. Use of Factor H antibodies has been shown to bring efficient
killing of resistant strains of N. Gonorrhea and HIV in in-vitro experiments. It is speculated that encapsulation of sodium polystyrene sulfonate in 200 kD
pore size polymer will allow direct binding of Factor H and deplete its
availability for pathogens and tumors. Thus, it will transiently activate the complement system. Concomitant administration of antibiotics, antivirals or antitumor agents will therefore be more effective in eradicating such
pathogens or tumors.
Sodium polystyrene sulfonate, when administered intravenously in
experimental animals, in doses of 10 mg/ kg or more, completely inhibits
complement pathways. Further as Factor D inhibitor, it has broad spectrum inhibitory effects on serine protease of chymotrypsin family. This property
has protected death of mice from endotoxinemia. Ultrapurified alginate and
polystyrene sulfonate act synergistically to inhibit proinflammatory cytokines and complement system. By depriving survival stimuli, such as endotoxin,
C3b, C3d and cytokines, antiapoptotic proteins of the monocytes and Bel family are not activated. The strategy breaks the critical linkage, i.e.
breakdown products of C3 convertase and activation of monocyte/
dendrocytes to initiate adaptive immunity. This helps prevent activation of adaptive immunity as well. Dendritic cells are not activated and therefore do
not express second signal. This leads to arrest in T cell mediated adaptive responses. Lymphocytes and monocytes die " by neglect or passive death".
Cytokines and complement mediated signals are also needed to upregulate adhesion receptors and for cellular activation and aggregation. Failure to
provide such signal, leads to nonthrombotic endothelial surface and noninflammatory state. Different hydrogel formulations can readily be
prepared using commercially available devices for microencapsulation and hollow fibers for macroencapsulation. Such devices permit targeted
therapeutic applications in-vitro, in-vivo or ex-vivo. In-vivo applications could be both extravascular or intravascular.
Brief Description of the Drawings
In the accompanying drawings to which reference is made in the
instant specification which is to be read in conjunction therewith, like
reference numerals are used to indicate the parts in the various views:
FIG. 1 shows endotoxin or LPS effects on afferent and efferent arm of immune responses.
FIG. 2 compares the endotoxin content of a purified alginate with
ultrapurified alginate.
FIG. 3 is the immune switch according to the invention.
Description of the Preferred Embodiment
As shown in FIG. 1, endotoxin causes activation and amplification of
effector or efferent arms of immune responses through its binding with LBP and complement. The items in the boxes 10, 12, 14, 16 and 18 show the activation of cellular pathways leading to activation of cytokines, costimuli
and adhesion receptors. The end result is bioincompatibility, hemoincompatibility and immune rejections. In a worst case scenario, septic shock and death may occur. Removal of endotoxin to its minimal level thus
prevents the activation of items 10, 12, 14, 16 and 18. Blockage of cellular pathways 32, 34 ,36, and 38 occurs and contributes to biocompatibility, hemocompatibility and immune tolerance as shown 44.
Endotoxins contribute to activation of adaptive immunity through C3 breakdown products and monocyte/dendritic cell activation 10 and activation of dendritic cells leads to expression of costimulli, such as B7-1 and B7-2.
Costimulation of B 7-1 and B7-2 are the critical events that lead to migration
of dendritic cells to secondary lymphoid tissues and activate adaptive immunity. For the activation of CD 4+ T mediated humoral immune responses, CD + 8 T cell mediated immune responses and B cell mediated
antibody responses, at least two signals are needed. In the absence of second signal or B7 costimulli, T and B cells fail to cause immune responses and lead
to anergy. Prevention of dendritic cell maturation therefore is the critical
step to induce immune tolerance 42 to foreign materials including cell and
organ transplantations. This occurs by blockage 32 of dendritic cells and its maturation.
Majority of immunosuppressives such as cyclosporine, tacrolimus (FK
506), Azathioprine, Mycophenolatemofetil(CellCept), MuromonaCD3 (OKT3;Orthobiotech), Interleukin-2 receptor antagonist
(Basiliximab(Simulect, Novartis) and Daclizumab(Zenapax, Hoffmann-La-
Roche) and antibodies to T cell or its costimulli used for induction protocol are targeted to inhibit T cell mediated effector or efferent immune responses
40 to cause immunosuppression after the expression of costimulli or after the activation of immune system or maturation of dendritic cell. Removal of
endotoxin, dampening of inflammatory cytokines and complement inhibition provide preemptive strategy to prevent activation of immune responses and
lead toward immune tolerance in cell and organ transplant. Presence of
memory clone of T and B cells may still activate the immune system. However, in the absence of costimuli the response tends to be milder. Hydrogel nature of the formulation allows one to combine any immuno-
suppressives to further strengthen immuno-suppressive effect 40 to cause immunosuppression. Complement inhibitor as a hydrogel formulation can be
used as a drug additionally to inhibit complement mediated events including
whole body inflammatory responses and septic shock. Hydrogel formulation permits encapsulation of any cell to further protect from immunorejections.
By enlarging pores of the hydrogel to 200 kD, the encapsulated drug
acts as an immune activator that has a range of applications in infections, cancers and vaccine developments. As shown in FIG. 3, by controlling the
pore size the immune switch function is regulated. Since any drug can be
encapsulated, the immune switch function and its potency can be controlled for target specific applications. The micro hydrogel 60 according to the invention contains polystrene sulfonate 62 Factor D and Factor H inhibitor
64. Hydrogel 60 includes pores 50 which, on the one hand, may have a pore
size 66 having a nominal molecular weight cutoff between 50kd and lOOkd. Such pore size would exclude Factor H, as illustrated by arrow 68, and would allow selective entry of Factor D, as illustrated by arrow 70. This results in
immune inhibition. Alternatively, pores 50 may be of a pore size 72 having a
nominal molecular weight cutoff greater than 200kD. Pore size 72 would allow preferential entry of Factor H due to more avid and rapid binding of
Factor H as compared to Factor D, as illustrated by arrow 74. Arrow 76 indicates delayed entry of Factor D.
ENCAPSULATION TECHNOLOGY SYSTEM AND ITS TARGETED
THERAPEUTIC APPLICATIONS
Recently, FDA has defined encapsulation technology as 'Therapeutic Drug-Device'. Drug components may be cellular or noncellular and will vary
depending upon the targeted therapeutic application. The invention herein provides a device component which may be a capsule, syringe, microcapsule,
macrocapsule or ex -vivo instrument. It has the same variability as a drug
component. A matrix component i.e. alginate, however, is the common component and its function is to provide biocompatibility,
hemocompatibility and immunetolerance. Addition of gelling agent, such as CaCl, imparts the immunoisolatory and extended-release functions to the
matrix. Thus standardization of the two common components, viz. matrix
and gelling agent, in the form of a kit permits the development of targeted therapeutic applications using different devices and cellular or noncellular
therapeutic agents. Encapsulation of an immune modulator broadens the range of targeted therapeutic applications in implantations, transplantations,
certain infections, cancers and vaccines. Since immune modulator is a sulfonic polymer, addition of sulfonic group improves basic biocompatibility,
hemocompatibility and immunetolerance of the encapsulated device. Use of
ultrapurified alginate of high 'G' monomer composition, removal of divalent
toxins and presence of sulfonic group contribute to increased mechanical strength and salt bridge stability over a wide pH range. In other words, encapsulation of an immune modulator such as sulfonic polymer results in
the development of an encapsulation technology system that overcomes prior
art problems and provides a wide range of targeted therapeutic applications. Described below are methods to formulate encapsulation devices of different
shapes and sizes that allow targeted therapeutic applications.
1. Drug Formulation: Alginate is the most common polymer used for the encapsulating cellular and noncellular materials. Below, basic alginate
fomulation is described that meets or exceeds FDA acceptance criteria for minimal endotoxin content for encapsulated devices. Such formulation is standardized for its chemical properties as detailed in my U.S. Patent No.
5,976,780.
Step 1. Obtain UP alginate from sources as powder.
EX. UP MVG ( S. NO. Property Product Code: Ultrapurified 28023316).
Endotoxin content 700 E.UJg dry weight.
Step 2. Prepare 2% solution of defined quantity in 0.9% NaCl solution.
Endotoxin content in 2% 100 ml solution will be 1400 E.UJdl or 14 EU/ml.
Assuming 0.9% NaCl is endotoxin free.
Comment: Alginate powder dissolves poorly and tends to clump. Magnetic
shaker and vigorous shaking are required to adequately dissolve alginate powder. It may take up to 24 hours to completely dissolve alginate powder in the solution.
Step 3. Add appropriate preservative or antibiotics to prevent bacterial and fungal contamination and improve shelf life.
Step 4. Adjust pH to 7.4. Sterile filter solution through 0.45 micron filter.
Step 5. Add dry or wetted PROSEP*-Rem Tox to above solution. Add 1 gm for every 10 ml solution.
Step 6: Gently mix the suspension for 3 hrs. The mixing can be carried out by placing the container on a roller-mixer. Do not use magnetic stirrer at this stage. This can cause break-up of glass beads.
Step 7. Remove the superpatent by allowing the PROSEP* Rem Tox to settle
and decant the solution. Alternately a glass sintered funnel can be used to separate the beads from the solution. For 2% 100 ml solution, endotoxin content will be reduced from 1400 EU/dl to 14 EU/dl.
Step 8. Supply alginate solution is 2% ready-to-use sterile solution in vials of 50 ml
size. Each vial will have endotoxin content of 7 E.U.
Above ready-to-use polymer solution should be further characterized for
mirobial growth and shelf life. Appropriate preservatives may be added. Prior to use, alginate solution is diluted to 1% for cellular or noncellular applications by
using 0.9% NaCl. Further pH adjustments are not necessary. For most cellular and noncellular applications, requirements of alginate are 5 ml to 100ml. 1% ml
alginate solution contains 7 E.U. of endotoxin. FDA requirement of endotoxin content limit is 5 E.U./kg i.e. 350 EU/70 kg or 20 EU/device. Above procedure meets
and exceeds such requirements. In addition to meeting FDA criteria of minimal
endotoxin content, hyrogel formulations should be mechanically stable and should not expand or contract or crack or alter its pore size following implantation. Chemical composition of UP Alginate as defined in U.S. Patent 5,976,780 is free of
divalent toxins and rich in 'G' polymer content. Isotonic gel in 0.9% NaCl, will lead to mechanically stable hydrogel and will maintain its defined pore size. Addition of sulfonic group further increases salt bridge stability over wider pH range.
Above solution now can readily be used for encapsulating cellular as well as
noncellular material. Additionally, the solution can be used to develop target
specific therapeutic applications. Thus, for microencapsulation,
macroencapsulation or ex-vivo blood contacting applications, commercial devices such as INNOVA microencapsulator or A/G technology hollow fibers and cartridges can readily be used. Use of adaptable nozzle permit one to obtain preselected size
and provide protocol flexibility. Further sonification or fragmentation of
microcapsule lead to formation of ultramicrocapsules. Less than 5 micron size
particles can safely be administered due to its hemocompatibility. The mean size of capillaries is 8 micron and the mean size of RBC is 5 micron. Therefore, particles less than 5 micron will not lead to vascular obstruction. Encapsulation of sulfonic
polymer in 50-100 kDa pore size will allow prevention of ischemia-reperfusion
injuries. Porus nature of the gel will allow oxygen diffusion to readily take place