CA2099376A1 - Stabilization of proteins by cationic biopolymers - Google Patents
Stabilization of proteins by cationic biopolymersInfo
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- CA2099376A1 CA2099376A1 CA002099376A CA2099376A CA2099376A1 CA 2099376 A1 CA2099376 A1 CA 2099376A1 CA 002099376 A CA002099376 A CA 002099376A CA 2099376 A CA2099376 A CA 2099376A CA 2099376 A1 CA2099376 A1 CA 2099376A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1629—Organic macromolecular compounds
- A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
- A61K9/1647—Polyesters, e.g. poly(lactide-co-glycolide)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/593—Polyesters, e.g. PLGA or polylactide-co-glycolide
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1629—Organic macromolecular compounds
- A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1629—Organic macromolecular compounds
- A61K9/1658—Proteins, e.g. albumin, gelatin
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Medicinal Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Medicinal Preparation (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
A method is described for the incorporation of proteins in the form of specific noncovalent complexes with polycationic reagents, into sustained release systems, where the polycation stabilizes the protein against inactivation while it resides in the delivery device, and retards release of the protein from the delivery device. Alternatively, the polycation-protein complex itself serves as a depot for release of the protein active agent, rather than a polymeric matrix. The end result is the release of the active agent with retention of biological activity, with a high cumulative field, over a sustained period of time.
Description
WO 92t1 1844 PCr/USsl/09771 ~f ~9~76 STABlLIZATlON OF PROTEIMS BY CATIONIC BIOPOLYMERS
Background of the InYention This inven~ion is in the field of delivery systems forpharmaceutical agents and is especially rela~ed to methods for the stabili~ation of proteins using cationic polymers.
Sustained release devices have been developed over the past several years based on a broad range of technologies, directed to the delivery of a wide selection of pharmaceutical agents. The physical formats for such devices include use of microparticles, slabs or sirnilar macroscopic systems designed for implantation, gels and emulsions, and other preparations conceived to preserve the active agent in the delivery system for an extended period of time.
The mechanism of release-from matrix-type sustained release devices is generally understood to occur by hindered diffusion of the active agent through the carrier matrix, or by erosion of the matl~x over tune resulting in the liberation of the incorporated active agent. These processes are not mutually exclusive, and both mechanisms may be simultaneously active in the case of a given system.
In recent years sustained release devices have been used for the delivery of protein pharmaceutical agents, primaril;y as a result of the availability of recombinant proteins which have been developed for therapeutic applications in a wide variety of pathological conditions.
~evelopment of such systems creates greater challenges to overcome than in the ca~se of low molecular weight drugs and pharmaceutically active substances, since prote~ns inherently have only marginal conformational stability, and can frequently be susceptible to conditions or process~s which result in inaclivaiion or desla~ration. ;n contrast to the degradation or deterioration of low molecular weight pharmaceuticals, the struc~ral alterations in proteins leading to inactivation need not involve changes ~n the covalent saucture of the protein, but can be entirely the consequence :
WO 92/11844 pcr/uss1/o977l ..
Background of the InYention This inven~ion is in the field of delivery systems forpharmaceutical agents and is especially rela~ed to methods for the stabili~ation of proteins using cationic polymers.
Sustained release devices have been developed over the past several years based on a broad range of technologies, directed to the delivery of a wide selection of pharmaceutical agents. The physical formats for such devices include use of microparticles, slabs or sirnilar macroscopic systems designed for implantation, gels and emulsions, and other preparations conceived to preserve the active agent in the delivery system for an extended period of time.
The mechanism of release-from matrix-type sustained release devices is generally understood to occur by hindered diffusion of the active agent through the carrier matrix, or by erosion of the matl~x over tune resulting in the liberation of the incorporated active agent. These processes are not mutually exclusive, and both mechanisms may be simultaneously active in the case of a given system.
In recent years sustained release devices have been used for the delivery of protein pharmaceutical agents, primaril;y as a result of the availability of recombinant proteins which have been developed for therapeutic applications in a wide variety of pathological conditions.
~evelopment of such systems creates greater challenges to overcome than in the ca~se of low molecular weight drugs and pharmaceutically active substances, since prote~ns inherently have only marginal conformational stability, and can frequently be susceptible to conditions or process~s which result in inaclivaiion or desla~ration. ;n contrast to the degradation or deterioration of low molecular weight pharmaceuticals, the struc~ral alterations in proteins leading to inactivation need not involve changes ~n the covalent saucture of the protein, but can be entirely the consequence :
WO 92/11844 pcr/uss1/o977l ..
3 7 ~
of disruption of an ex~ensive system of noncovalent interactions which are responsible for the preservation of the nativ~ three dimensional structure of the protein. This is the basis for the greater lability of proteins.
Certain features of sustained release devices e xacerbate the poten~l for the inactivation of protein active agents. These include the fact that large amounts of solid protein are introduced into the delivery system (either as pure preparations or n~ixed with additives and excipients), and that the physical attributes of the delivery systems themselves may present interfaces which promote denaturation. Hydrating the solid protein under physiological conditions in vivo results in formation of a protein gel or a highly concentrated solution of the protein. Under these circumstances it is quite possible for the protein to become aggregated or denatured due to interacttons with neighboring molecules or upon exposure to the interface with the delivery system.
In order to overcome these potential problems, prote~lls h~ve been formulated with excipients intended to stabili~e the protein in the milieu of the pharmaceutical product. It has long been known that a variety of low molecular weight compounds have the effect of preserving the activity of proteins and enzymes in solution. These include simple salts, as described by P. H. von Hippel and K.-Y. Wong, "Neutral Salts: the Generality of Their Effects on the Stability of Macromolecular Conformations", Science 145, 577-580 (1964), buffer salts and polyhydroxylated compounds such as glycerol, mannitol, sucrose and polyethylene glycols, K. Gekko and S. N.
Timasheff, "Mechanism of Protein Stabilization by Glycerol: Preferen~al Hydra~on in Glycerol-Water Mixtures", Biochemistrv 20, 4667~676 ~1981); K. Geldco and T. Morikawa, "Pleferential Hydration of Bovine Serum Albumin in Polyhydric Alcohol-Water Mix~res", J. Biochem. 90, 39-50 (1981~; and J. C. Lee and L. L. Y. Lee, "Preferential Solvent Interactions lietween Proteins and Polyethylene Glycols", J. Biol. Chem.
WO 92/l 1844 Pcr/uss1/o977 '5 --3--2~937~
256, 625-631 (1981). Certain biocompa~ble polymers have also been applied ~or this purpose, such as various polysaccharides and synthetic polymers includmg polyvinylpyrrolidone, for example. Even benign detergents such as polyoxyethylene sorbitan monooleate (Tween 80TM) have been included to preserve bioactivity in pharmaceutical formulations. Use of these materials has been irnplemented over many years, for example, with soluble preparations of vaccines and insulin, long before recombinant protein pharrnaceutical agents became available.
Except for the detergents, the mechanism by which these substances exert their stabilizing effect has become evident in recent years as a result of thorough investigation. It has been shown that stabilization occurs as a result of a general thersnodynamic phenomenon prevalent in these ternary systems, wherein the cosolute (for example, the polyol) is preferentially excluded from the domain of the protein, and the protein is preferentially hydrated. As a rcsult, the protein is stabilized by enhancement of the hydrophobic interactions which are generally thought to confer stability on the native tertiary structure of the protein, as compared with the protein in the absence of the cosolute.
Use of these excipients may be associated with certain disadvantages. For example, the thermodynamic effects require high concentrations of the cosolute in order to be effective. Under certain conditions, high concentrations of polysaccharides may even lead to phase separation of the protein. Alternatively, low molecular weight excipients have high solubilities and high diffusion coefficients, so that they are depleted from the delivery device considerably more rapidly than the active agent. The beneficial effects of the excipient are therefore transient, OCCDg only in the ini~al stages of the dura~ion of ~e release of ~e protein. This condi~on leaves the protein pharmaceu~dcal still wi~in the .
wo 92/l 184q PCI/US91/09771 ~i9937~
sustained release device, prone to inactivation due to interrnolecular aggregahon and interaction with ~e surface of the device.
It is therefore an object of the present invention to enhance the amount of release and stability of proteins incorporated into polymeric matrices for controlled drug delivery.
It is a further object of the present invention to provide a method and compositions that can be used with a variety of compounds to enhance stability, with minimum effort and expense.
I~ is another object of the present invention to provide a method and compositions that can be used as biodegra~able, biocompatible depots for controlled drug delivery.
Summary of the Invention A method is described for the incorporation of biologically active agen~s, especially protein ph~maceutical agen~, in ~ fonn of specific noncovalent complexes with polycationic reagents, into sustained release systems, where the polycation stabilizes the protein against inactivation while it resides in the delivery device, and retards release of the protein due to the added effects of dissociation of the complex according to the law of mass action. The end result is the release of the active agent with retention of biological activity, with a high cumulative yield, over a sustained period of time.
In a second embodiment of this method and compositions, the polycation-protein complex itself serves as a depot for release of the prote-in active agent, rather than a polymeric ma~c. ~ the most prefe~ed embodirnent, the complexing polyelectrolyte is both biocompatible and biodegradable.
', , " ~ , ..
~ . .; .
wo 92/11844 PCr/US91/09771 -5- ~9~76 Examples are provided demonstrating complex forma~on (for example, between erythropoiehn and chitosan) and enhanced stabili~y and release from polymeric devices of proteins (such as Factor Vl~.
Brief Description oP the Drawil}gs Figure 1 is the percent ery~ropoietin (EPO) released from poly(DL-Iactide-co-glycolide) (50.50) microspheres in 50 mM sodium phosphate pH 7.3 at 37C, for EPO:chitosan ratios, expressed as percents of total solids, of 12:7, 6:10 and 17:0 over time (days).
Figure 2 is a graph of the cumulative units of Factor vm activity released per mg of poly(lactic acid) microspheres, containing either poly(arginine) (2 mg/ml) complexed with Factor VIII at 30% loading, over time (days) or Factor vm in NaCl-CaCl2-glycine buffer.
Figure 3 is a graph of the percent cumulative release over time (days) for bov~ne serum albumin (BSA):sucrose (5:5) ~ight squa~es);
BSA:protamine (5:5) (~iangles); and BSA (dark squares), all at 10% by weight loading.
Detailed Description of the Invention The majority of the prior art processes and phenomena relating to stabilit~ and release of compounds from polymeric matrices is based on general physical chemical principles, except for the process of erosion of sustained release systems, which involves actual che nical degradation of the matr~x. The method and compositions described herein, in contrast, are based on a reversible chemical ~nteraction between the compound to be released and a stabilizing compound.
In the preferred embodiment, the bido~cally ac~ve agent is a protein or peptide (including nahlral, recombinant, synthetic, high and low molecular weight proteins or peptides). It could also be a nucleic acid, a ' wo 92/11844 PCr/US91/09771 --6- 2 ~ ~ ~ 3 7 6 polysaccharide, a carbohydrate or derivatives thereof, a low molecular weight organic molecule or pharmacological agent. Complex formation between proteins and biological polycations can be used for proteins whose isoelectric point (pI) is acidic or neutral, as well as any protein having acidic side chains clustered together on the surface of the protein when it is in its native, active confo~nation. Proteins with acidic or neutral pI
values have a preponderance of acidic over basic side chains in ~heir structures. These are the groups which are available for interaction with the polycation, primarily by electrostatic interactions. The polycation has the capability of binding several molecules of protein per molecule of polycation. If the pxotein is also polyvalent in binding sites for the polycation, the complex will likely aggregate or precipitate, in analogy ~o the antigen-antibody precipitin reaction. If the protein is monovalent for the polycation the complex will remain soluble, presumably as a complex compAsed of many prot~ mole~:ules bound to each polyca~ion molecule.
The complexed protein is stabilized relative to the case of the absence of the polycation, both in a~ueous solution or suspension, and when incoIporated into sustained release devices.
The polycation must be biocompatible and, preferably, biodegradable. A variety of polycations can be used. Simple polyamino acids such as poly~ysine) or poly(arginine) are useful materials. Their molecular weights should be 4,000 daltons or greater, preferably about 50,000 or greater. Protamine is another useful polycation. Chitosan is useful primar~y for acidic proteins, since it precipitates at pH values greater than about 6.5. Other biological polycations are also applicable for the purposes of this inven~on.
The weight ra~o of protein to polycation can be in the range l:1000 (when the protein has a very high biological ac~vity per un~t weight, so that the overall dosing requirement is low) to 20:1 (in the wo 92/11844 pcr/ussl/09771 1<'~.~
of disruption of an ex~ensive system of noncovalent interactions which are responsible for the preservation of the nativ~ three dimensional structure of the protein. This is the basis for the greater lability of proteins.
Certain features of sustained release devices e xacerbate the poten~l for the inactivation of protein active agents. These include the fact that large amounts of solid protein are introduced into the delivery system (either as pure preparations or n~ixed with additives and excipients), and that the physical attributes of the delivery systems themselves may present interfaces which promote denaturation. Hydrating the solid protein under physiological conditions in vivo results in formation of a protein gel or a highly concentrated solution of the protein. Under these circumstances it is quite possible for the protein to become aggregated or denatured due to interacttons with neighboring molecules or upon exposure to the interface with the delivery system.
In order to overcome these potential problems, prote~lls h~ve been formulated with excipients intended to stabili~e the protein in the milieu of the pharmaceutical product. It has long been known that a variety of low molecular weight compounds have the effect of preserving the activity of proteins and enzymes in solution. These include simple salts, as described by P. H. von Hippel and K.-Y. Wong, "Neutral Salts: the Generality of Their Effects on the Stability of Macromolecular Conformations", Science 145, 577-580 (1964), buffer salts and polyhydroxylated compounds such as glycerol, mannitol, sucrose and polyethylene glycols, K. Gekko and S. N.
Timasheff, "Mechanism of Protein Stabilization by Glycerol: Preferen~al Hydra~on in Glycerol-Water Mixtures", Biochemistrv 20, 4667~676 ~1981); K. Geldco and T. Morikawa, "Pleferential Hydration of Bovine Serum Albumin in Polyhydric Alcohol-Water Mix~res", J. Biochem. 90, 39-50 (1981~; and J. C. Lee and L. L. Y. Lee, "Preferential Solvent Interactions lietween Proteins and Polyethylene Glycols", J. Biol. Chem.
WO 92/l 1844 Pcr/uss1/o977 '5 --3--2~937~
256, 625-631 (1981). Certain biocompa~ble polymers have also been applied ~or this purpose, such as various polysaccharides and synthetic polymers includmg polyvinylpyrrolidone, for example. Even benign detergents such as polyoxyethylene sorbitan monooleate (Tween 80TM) have been included to preserve bioactivity in pharmaceutical formulations. Use of these materials has been irnplemented over many years, for example, with soluble preparations of vaccines and insulin, long before recombinant protein pharrnaceutical agents became available.
Except for the detergents, the mechanism by which these substances exert their stabilizing effect has become evident in recent years as a result of thorough investigation. It has been shown that stabilization occurs as a result of a general thersnodynamic phenomenon prevalent in these ternary systems, wherein the cosolute (for example, the polyol) is preferentially excluded from the domain of the protein, and the protein is preferentially hydrated. As a rcsult, the protein is stabilized by enhancement of the hydrophobic interactions which are generally thought to confer stability on the native tertiary structure of the protein, as compared with the protein in the absence of the cosolute.
Use of these excipients may be associated with certain disadvantages. For example, the thermodynamic effects require high concentrations of the cosolute in order to be effective. Under certain conditions, high concentrations of polysaccharides may even lead to phase separation of the protein. Alternatively, low molecular weight excipients have high solubilities and high diffusion coefficients, so that they are depleted from the delivery device considerably more rapidly than the active agent. The beneficial effects of the excipient are therefore transient, OCCDg only in the ini~al stages of the dura~ion of ~e release of ~e protein. This condi~on leaves the protein pharmaceu~dcal still wi~in the .
wo 92/l 184q PCI/US91/09771 ~i9937~
sustained release device, prone to inactivation due to interrnolecular aggregahon and interaction with ~e surface of the device.
It is therefore an object of the present invention to enhance the amount of release and stability of proteins incorporated into polymeric matrices for controlled drug delivery.
It is a further object of the present invention to provide a method and compositions that can be used with a variety of compounds to enhance stability, with minimum effort and expense.
I~ is another object of the present invention to provide a method and compositions that can be used as biodegra~able, biocompatible depots for controlled drug delivery.
Summary of the Invention A method is described for the incorporation of biologically active agen~s, especially protein ph~maceutical agen~, in ~ fonn of specific noncovalent complexes with polycationic reagents, into sustained release systems, where the polycation stabilizes the protein against inactivation while it resides in the delivery device, and retards release of the protein due to the added effects of dissociation of the complex according to the law of mass action. The end result is the release of the active agent with retention of biological activity, with a high cumulative yield, over a sustained period of time.
In a second embodiment of this method and compositions, the polycation-protein complex itself serves as a depot for release of the prote-in active agent, rather than a polymeric ma~c. ~ the most prefe~ed embodirnent, the complexing polyelectrolyte is both biocompatible and biodegradable.
', , " ~ , ..
~ . .; .
wo 92/11844 PCr/US91/09771 -5- ~9~76 Examples are provided demonstrating complex forma~on (for example, between erythropoiehn and chitosan) and enhanced stabili~y and release from polymeric devices of proteins (such as Factor Vl~.
Brief Description oP the Drawil}gs Figure 1 is the percent ery~ropoietin (EPO) released from poly(DL-Iactide-co-glycolide) (50.50) microspheres in 50 mM sodium phosphate pH 7.3 at 37C, for EPO:chitosan ratios, expressed as percents of total solids, of 12:7, 6:10 and 17:0 over time (days).
Figure 2 is a graph of the cumulative units of Factor vm activity released per mg of poly(lactic acid) microspheres, containing either poly(arginine) (2 mg/ml) complexed with Factor VIII at 30% loading, over time (days) or Factor vm in NaCl-CaCl2-glycine buffer.
Figure 3 is a graph of the percent cumulative release over time (days) for bov~ne serum albumin (BSA):sucrose (5:5) ~ight squa~es);
BSA:protamine (5:5) (~iangles); and BSA (dark squares), all at 10% by weight loading.
Detailed Description of the Invention The majority of the prior art processes and phenomena relating to stabilit~ and release of compounds from polymeric matrices is based on general physical chemical principles, except for the process of erosion of sustained release systems, which involves actual che nical degradation of the matr~x. The method and compositions described herein, in contrast, are based on a reversible chemical ~nteraction between the compound to be released and a stabilizing compound.
In the preferred embodiment, the bido~cally ac~ve agent is a protein or peptide (including nahlral, recombinant, synthetic, high and low molecular weight proteins or peptides). It could also be a nucleic acid, a ' wo 92/11844 PCr/US91/09771 --6- 2 ~ ~ ~ 3 7 6 polysaccharide, a carbohydrate or derivatives thereof, a low molecular weight organic molecule or pharmacological agent. Complex formation between proteins and biological polycations can be used for proteins whose isoelectric point (pI) is acidic or neutral, as well as any protein having acidic side chains clustered together on the surface of the protein when it is in its native, active confo~nation. Proteins with acidic or neutral pI
values have a preponderance of acidic over basic side chains in ~heir structures. These are the groups which are available for interaction with the polycation, primarily by electrostatic interactions. The polycation has the capability of binding several molecules of protein per molecule of polycation. If the pxotein is also polyvalent in binding sites for the polycation, the complex will likely aggregate or precipitate, in analogy ~o the antigen-antibody precipitin reaction. If the protein is monovalent for the polycation the complex will remain soluble, presumably as a complex compAsed of many prot~ mole~:ules bound to each polyca~ion molecule.
The complexed protein is stabilized relative to the case of the absence of the polycation, both in a~ueous solution or suspension, and when incoIporated into sustained release devices.
The polycation must be biocompatible and, preferably, biodegradable. A variety of polycations can be used. Simple polyamino acids such as poly~ysine) or poly(arginine) are useful materials. Their molecular weights should be 4,000 daltons or greater, preferably about 50,000 or greater. Protamine is another useful polycation. Chitosan is useful primar~y for acidic proteins, since it precipitates at pH values greater than about 6.5. Other biological polycations are also applicable for the purposes of this inven~on.
The weight ra~o of protein to polycation can be in the range l:1000 (when the protein has a very high biological ac~vity per un~t weight, so that the overall dosing requirement is low) to 20:1 (in the wo 92/11844 pcr/ussl/09771 1<'~.~
209~37~
converse situation). The preferred range for the weight ratio will be l:100 to 10:1. The pH at which the complex is fonned will affect the process.
The overall state of charge of the protein will be a ~nction of pH, since proteins are polyampholytes. The pH must be one at which the protein retains full biological activity, which is a property unique to each protein.
The pH may also affect the charge on the polycation in certain cases, or, as with chitosan, actually affect its solubility. Of course, once in~oduced in vivo, release devices incorporating these complexes will experience pH
values approxima~ng physiological pH.
The fabrication of sustained release systems containing protein-polycation complexes differs little from the processes currently used for incorporating protein formulations. Liquid formulations can be employed in the manufacture of sustained release microspheres in conventianal solvent evaporation procedures. Solid formulations, typically prepared as lyophilized solids from the li~uid, can also be used. In particul~, solid preparations of protein-polycation formulations can be micronized, i.e., fragmented to produce particles in the size ~nge from less ~an I
micrometer to about 5 micrometers, using the procedures outlined by Gombotz, et al., in U.S. Serial No. 07/345,684 filed May 1, 1989, the teachings of which are .incorporated herein, summarized as follows.
The biologically active molecule is first dissolved in a solvent that can be lyophilized to forrn a solution having a concentration ranging from approxirnately 0.1 to 25% (w/v). The solvent rnay be pure water or can be buffered to a parLicular pH or ionic strength. The solvent may also be organic. The solution may contain the biologically active molecule alone, n~ix~res of two or more Iypes of biologically active molecules alone, mixtures of biologically active molecules and stabilizers, or any combination thereof. ~ order to reduce the particle size of these preparations to the greatest extent, the composition should be suspended in . . , -W0 92/11844 PCr/US91/09771 ~ .
2~37~
a medium ~n which not only the solvent but also the buffer s:alts are vola~le under conditions of lyophilization. Examples of buffers removed by lyophilization include ammonium bicarbonate and other vola~le ammonium salts.
The soluhon is then atomized into a low temperature liquified gas using any one of several devices, such as ultrasonic nozzles, pressure nozzles, pneumatic nozzles and rotary nozzles. The liquified gas can be liquid argon (-185.6C~, liquid nitrogen (-195.8C), liquid oxygen (-182.9C) or any other gas that results in the immediate freezing of the atomized particles into frozen particles. Oxygen is not preferred for proteins since it is explosive and may also cause oxidation of the proteirl.
The liquified gas is removed by evaporation at a temperature at which the solvent remains frozen, leaving behind frozen particles. The frozen solvent is removed from the particles by Iyophilization to yield porous par~c!es. These particles can ~ary in dia;ncter dcpcnding on the technique used for their aton~zation, but generally range from approximately 10 to 50 micrometers.
These protein particles can be incorporated into biodegradable polymer microspheres using the processes taught by Gombotz, et al., U.S.
Serial No. 07/346,143 filed May 1, 1989, the teachings of which are incorporated herein, or other more conventional techniques. Polymers that can be used to forrn the microspheres include bioerodible polymers such as poly(lactic acid), poly~actic-co-glycolic acid), poly(caprolactone), polycarbonates, polyan~ides, polyanhydrides, polyamino acids, polyortho esters, polyacetals, polycyanoacrylates and degradable polyurethanes,and non-erodible polymers such as polyacrylates, ethylene-viny} ace~te and other acyl substituted cellulose acetates and derivatives thereof, non-erodible polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl '' ' .: .' ' ' . :, ' wo 92/l ~844 P~r/uss1tos77l ~: g ~99376 fluoride, poly(v~yl imidazole), chlorosulphonated polyolefins, and polyethylene oxide.
The method of Gombotz, et al., is summarLzed as follows.
Polymer and agent to be encapsulated in solution or dispersion are atomi~ed using an ultrasonic device into a liquified gas which overlays a bed of frozen non-solvent. The microspheres are immediately frozen by the Iiquified gas. The solvent is slowly removed from these spheres as they thaw and sink onto and then into very cold non-solvent which extracts the solvent as it and the spheres thaw, leaving microspheres conta~ning the encapsulated agent. The liquified gas can be liquid argon (-185.6C), liquid nitrogen (-195.8C), liquid oxygen (-182.9C) or any other gas that results in the imrnediate freezing of the atomized par~cles into ~rozen spheres.
The product microspheres have been shown to exhibit sustained release in ~irr~ and in ~vo uith a broad variety of proteins and e~zym~s.
The loadings of the active formulation of the protein-polycation complex in such sustained release systems can be from S to 50% (w/w), preferably in the range 10~0%.
Release of the protein active agent from microspheres containing protein-polycation complexes can occur according to one of seve~al mechanisms. First, dissociation of the protein from the complex would occur only in situ in the domain of the sustained delivery system. The free protein diffuses out of the device, while the polycation relT~ns behind. The polgcation presumably is still bound in a network of the protein-polycation complex (in the case of proteins tt-at are polyvalent for ~e polycation), or bound to other protein molecules (1n the case of pro~eins that are rnonovalent for ~e polycation). In either case, it is likely ~at the diffusion coefflcient of the polycaticn molecule is much lower than that of the free pro~ein, so that it remains within the device. Second, ~e . .
:
Wo 92/11844 PCI/US91/09771 -1~
~i399~76 protein-polycation complex, to the extent that it is soluble, diffi~ses out of the sustained de~ivery device into the release sink. Ilt then undergoes dissociation to release the protein active agent into the medium. Third, free ~i.e., uncomplexed) molecules of protein and po]Lycation leave the sustained release device independently and possibly s~imultaneously. They remain uncomplexed to the extent permi~ed by the law of mass ac~on. In reality, it is l~sely that a combination of these effects is operative.
It has been discovered that proteins can form complexes with biological polycations in Yitro; in many cases turbidity or formation of a precipitate actuallLy occurs. This observation has led to the use of such complexes as depots or reservoirs for stabilization of the protein active agent and for incorporation into sustained release systems. In this embodiment of this method and compositions, the polycation-protein complex itself serves as a depot for release of the protein active agent, rather than a polymeric maerix.
The requirements for a polycation-protein complex to serve as a reservoir for the sustained release of the protein as the ac~àve agent in a pharmaceutical formulation can be surnmarized as follows. First, the assoc~ation constant for the formation of the complex should be relatively high, a proper~y which may be achieved by virtue of cooperativity in the process of forming the complex. A consequence of having a high association constant is that the concentration of free protein will remain relatively low. Under such conditions, when the release mechanism is govemed by diffllsion, the rate of release can be dirninished because the flux is p~roportional to ~e concentration gradient established between the inner and outer phases. With a low concentration of protein es~ablished in ~e inner phase, the rate of diffusion will be low. Second, the concentration of polycation should be relatively low, so that the ac~ve agent is the prevalent component by weight in the formuhtion, if so .
.
.. ,- , .~ ' ~
WO 92/~ 1844 PCr/US~l/0977l desired. This is readily achievable because the high association constant ensures that most or all of ~e polycation par~cipates in complex formation.
Third, the molecular weight of the polycation should be relatively high, so that its diffusion coefficient will be low. In this way the active agent will be preferentially depleted firom the ma~ix or depot prior to the polycation.
The present inven~on will be further understood by the following non-limiting examples.
Example 1: Formation of a complex between bovine serwn albumin and chitosan.
1 g of chito~san was dissolved in 100 ml of 1 æ acetic acid. The pH of the resulting solution was 3Ø The solu~on was titrated with sodium hydroxide to pH of 6.0, avoiding precipitation and gel formation by ~u~ c.' ito~n. This is termed neu~ralized chitosan.
12.0 mg of bovine serum albumin (BSA) was dissolved in 1.0 ml 5 mM ammonium bicarbonate. 20 microliter aliquots of neutralized chitosan were added to the BSA, as well as to a buffer blank. A thick cloudy precipitate formed with the BSA, which was more profound and extensive than that observed with buffer alone. The latter is ascribed to pH-induced precipitation of chitosan. Centrifugation was used to determine whether precipitation occurred in the liquid supernatant with successive additions of chitosan. Generation of incremental turbidity ended at about the point where 200 microliters of the chitosan solution had been added to the BSA, coIIesponding to 2.0 mg chitosan.
The equivalence point was reached at a weight ratio of BSA:chitosan of about 5:1.
.
wo 92/1~844 PCr/US91/09771 f Example 2: Fo~nation of a complex between bo~ne hemoglobin and chitosan.
10.2 mg of bovine hemoglobin (EIb) was dissolved in 1.0 ml deionized water. Up ~o 40 microliters of neutralized chitosan was added in portions. With the first additions a dark agglome~ate formed, corresponding to partial depletion of color from the solution. Further addition of chitosan did not lead to a quantitative precipitation of ~e Hb.
Example 3: Preparation of and in vitro ~elease from PLGA
microspheres containing the erythropoietin~hitosan complex.
Chitosan acetate at pH S WélS used to dissolve recombmant human erythropoietin (EPO) with varying ratios of chitosan:EPO. These formulations were micron~zed according to the method set forth in Gombotz et al. (U. S. Serial No. 07/345,684) and incorporated into copoly~l),L-lactide, glycolide) (50:50, Boe}lringer-Ingelhe~m R~3 503) using the procedures of Gombotz et al. (U. S. Serial No. 07/345,143).
The final loading ratios, in weight percentages of the final microsphere preparation, were 6% EPO: 10.3% chitosan, 12% EPO: 6.8% chitosan, and 17% EPO alone.
These microspheres were subjected to in v~tro release stu~ies at 37C, using the follo~1ving release buffer: 50 n~ sodium phosphate, 0.9%
MaCI, 2% (w/v) ovalbun~n, pH 7.2. The release results are shown in Figure 1. It is evident that, as compared to the abænce of chitosan, incorpoIation of the polycation profoundly reduces the burst effect upon ~e release of EPO from the microspheres.
Example 4: Prepar~tion and in vi~o release of P~ A microspheres containing the Factor Vm-poly(arginine) complex.
Human recombinant Factor vm was recons~tuted to 200 units/ml in 0.2 M NaCl, 0.55 M glycine, 0.005 M CaCl2, 12 mg/ml hurnan serum WO 92/11844 PCr/US91/09771 , .
2~37~
albumin. To this solution was added polyarginine [(Arg)81 at 2 mg/ml.
The solution was subjected to a change in composition of the bu~fer to O.l M proline, 2.5 mM CaCl2, pH 7.35 by passing the reconstib~ted mixture through a SephadexR G-25 column equilibrated with ~e proline - CaC12 solution. The product was then micronized according to the proccdure of Gombotz, et al., in U.S. Serial No. 07/345,684, and incorporated ~nto microspheres comprised of poly~actic acid) as the carrier matrix, at a loading of the for nulated Factor vm preparation of 30% (w/w) using the procedure described by Gombotz, et al. in U.S. Serial No. 07/345,143.
This preparation is referred to as "poly(arginine)" in Figure 2.
A similar microsphere preparation was made using human recombinant Factor vm reconstituted to lO0 unitstml in 0.1 M NaCl, 0.275 M glycine, 0.0025 M CaC12, 6 mg/ml human serum album~n. This preparation was similarly incorporated into PLA microspheres at 30%
loading. This prepa~a~on is re~erred to as "NaCl-Glyc~ne" ~n };igure 2.
The two rnicrosphere preparations were subjected to in vi~ro release experiments at 37C, by immersLng approximately lO mg of microspheres in l.0 rnl aliquots of a release buffer consisting of O.l M
NaCl, O.l M glycine, lO mM HEPES, 2.5 mM CaCl2, 2 mg/lT~l hurnan serum albumin, pH 7.2 m a l.5 ml microfuge tube, and agitated gently.
Fresh aliquots of release medium were applied for each time point. The activity was assayed using the CoatestR kit for Factor VIII produced by Kabi Vitrum and distributed by Helena Laboratories, Inc. The color resulting from release of p-nitrophenolate from a synthetic substrate, as deternnned in microtiter plate format using a plate reader, and expressed as the cumulative percent of inçolporated acti~ity released per mg of microspheres, is given for the two preparations in Figure 2. It is evident ~at Factor VIII formuL~ted wi~ (Arg)n has led to markedly enhanced and sustained release l~ne~cs compared to omission of (Arg)n.
wo 92/11844 PCT/US91/09771 ~; ' `
~' ~3~7 ~
Example 5: Bo~ine Serum Albumin-Protamine Complex Release from Copoiy(lactid~glycolide) Microspheres.
A globulin-free preparation of bovine semm albumin (BSA) obtained from Sigma Chemical Co. was mixed 1:1 (w:w) with sucrose or WIth protan~ine sulfate. The resulting solu~ons, as well as a solution of BSA alone, were micronized according to Gombotz, et al., as described in U.S. Serial No. 07/345,684. The protein-excipient formulations were incorporated into microspheres of copoly(DL-lactide,glycolide) (50:50) following the procedures of Gombotz, et al., U.S. Serial No. 07/345,143, with total loadings of 10% by weight. These microspheres were placed in 20 rnM sodium phosphate, 0.15 M sodium chloride, 1.5 rnM sodium azide, pH 7.S, at 37 C to measure in vitro release.
The cumulative release over 68 days is shown in Figure 3. The surge in release that occurs between about days 20 and 28 is ascribed to dcgradatioil Or the polymeT matrix, expos~ng fresh reservoirs Or prv~ein for release to the medium. The results show that incorporation of protamine sulfate gives enhanced release characteristics as compared to the incorporation of an equa} arnount of sucrose. The extent of release in the first hour, termed the burst, is diminished, and the steady, near-zero-order release of protein is sustained for a longer duration. For BSA
without added excipients, the burst release is the lowest of the three cases shown, but the degradation phase releases a large fraction of the protein over a relatively short period of time; further release continues for the remainder of the time period considered. Of the three preparations shown, the incorporation of protan~ine sulfate leads to the most monotonic release of rotein after ~e burst.
. . . . .
,
converse situation). The preferred range for the weight ratio will be l:100 to 10:1. The pH at which the complex is fonned will affect the process.
The overall state of charge of the protein will be a ~nction of pH, since proteins are polyampholytes. The pH must be one at which the protein retains full biological activity, which is a property unique to each protein.
The pH may also affect the charge on the polycation in certain cases, or, as with chitosan, actually affect its solubility. Of course, once in~oduced in vivo, release devices incorporating these complexes will experience pH
values approxima~ng physiological pH.
The fabrication of sustained release systems containing protein-polycation complexes differs little from the processes currently used for incorporating protein formulations. Liquid formulations can be employed in the manufacture of sustained release microspheres in conventianal solvent evaporation procedures. Solid formulations, typically prepared as lyophilized solids from the li~uid, can also be used. In particul~, solid preparations of protein-polycation formulations can be micronized, i.e., fragmented to produce particles in the size ~nge from less ~an I
micrometer to about 5 micrometers, using the procedures outlined by Gombotz, et al., in U.S. Serial No. 07/345,684 filed May 1, 1989, the teachings of which are .incorporated herein, summarized as follows.
The biologically active molecule is first dissolved in a solvent that can be lyophilized to forrn a solution having a concentration ranging from approxirnately 0.1 to 25% (w/v). The solvent rnay be pure water or can be buffered to a parLicular pH or ionic strength. The solvent may also be organic. The solution may contain the biologically active molecule alone, n~ix~res of two or more Iypes of biologically active molecules alone, mixtures of biologically active molecules and stabilizers, or any combination thereof. ~ order to reduce the particle size of these preparations to the greatest extent, the composition should be suspended in . . , -W0 92/11844 PCr/US91/09771 ~ .
2~37~
a medium ~n which not only the solvent but also the buffer s:alts are vola~le under conditions of lyophilization. Examples of buffers removed by lyophilization include ammonium bicarbonate and other vola~le ammonium salts.
The soluhon is then atomized into a low temperature liquified gas using any one of several devices, such as ultrasonic nozzles, pressure nozzles, pneumatic nozzles and rotary nozzles. The liquified gas can be liquid argon (-185.6C~, liquid nitrogen (-195.8C), liquid oxygen (-182.9C) or any other gas that results in the immediate freezing of the atomized particles into frozen particles. Oxygen is not preferred for proteins since it is explosive and may also cause oxidation of the proteirl.
The liquified gas is removed by evaporation at a temperature at which the solvent remains frozen, leaving behind frozen particles. The frozen solvent is removed from the particles by Iyophilization to yield porous par~c!es. These particles can ~ary in dia;ncter dcpcnding on the technique used for their aton~zation, but generally range from approximately 10 to 50 micrometers.
These protein particles can be incorporated into biodegradable polymer microspheres using the processes taught by Gombotz, et al., U.S.
Serial No. 07/346,143 filed May 1, 1989, the teachings of which are incorporated herein, or other more conventional techniques. Polymers that can be used to forrn the microspheres include bioerodible polymers such as poly(lactic acid), poly~actic-co-glycolic acid), poly(caprolactone), polycarbonates, polyan~ides, polyanhydrides, polyamino acids, polyortho esters, polyacetals, polycyanoacrylates and degradable polyurethanes,and non-erodible polymers such as polyacrylates, ethylene-viny} ace~te and other acyl substituted cellulose acetates and derivatives thereof, non-erodible polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl '' ' .: .' ' ' . :, ' wo 92/l ~844 P~r/uss1tos77l ~: g ~99376 fluoride, poly(v~yl imidazole), chlorosulphonated polyolefins, and polyethylene oxide.
The method of Gombotz, et al., is summarLzed as follows.
Polymer and agent to be encapsulated in solution or dispersion are atomi~ed using an ultrasonic device into a liquified gas which overlays a bed of frozen non-solvent. The microspheres are immediately frozen by the Iiquified gas. The solvent is slowly removed from these spheres as they thaw and sink onto and then into very cold non-solvent which extracts the solvent as it and the spheres thaw, leaving microspheres conta~ning the encapsulated agent. The liquified gas can be liquid argon (-185.6C), liquid nitrogen (-195.8C), liquid oxygen (-182.9C) or any other gas that results in the imrnediate freezing of the atomized par~cles into ~rozen spheres.
The product microspheres have been shown to exhibit sustained release in ~irr~ and in ~vo uith a broad variety of proteins and e~zym~s.
The loadings of the active formulation of the protein-polycation complex in such sustained release systems can be from S to 50% (w/w), preferably in the range 10~0%.
Release of the protein active agent from microspheres containing protein-polycation complexes can occur according to one of seve~al mechanisms. First, dissociation of the protein from the complex would occur only in situ in the domain of the sustained delivery system. The free protein diffuses out of the device, while the polycation relT~ns behind. The polgcation presumably is still bound in a network of the protein-polycation complex (in the case of proteins tt-at are polyvalent for ~e polycation), or bound to other protein molecules (1n the case of pro~eins that are rnonovalent for ~e polycation). In either case, it is likely ~at the diffusion coefflcient of the polycaticn molecule is much lower than that of the free pro~ein, so that it remains within the device. Second, ~e . .
:
Wo 92/11844 PCI/US91/09771 -1~
~i399~76 protein-polycation complex, to the extent that it is soluble, diffi~ses out of the sustained de~ivery device into the release sink. Ilt then undergoes dissociation to release the protein active agent into the medium. Third, free ~i.e., uncomplexed) molecules of protein and po]Lycation leave the sustained release device independently and possibly s~imultaneously. They remain uncomplexed to the extent permi~ed by the law of mass ac~on. In reality, it is l~sely that a combination of these effects is operative.
It has been discovered that proteins can form complexes with biological polycations in Yitro; in many cases turbidity or formation of a precipitate actuallLy occurs. This observation has led to the use of such complexes as depots or reservoirs for stabilization of the protein active agent and for incorporation into sustained release systems. In this embodiment of this method and compositions, the polycation-protein complex itself serves as a depot for release of the protein active agent, rather than a polymeric maerix.
The requirements for a polycation-protein complex to serve as a reservoir for the sustained release of the protein as the ac~àve agent in a pharmaceutical formulation can be surnmarized as follows. First, the assoc~ation constant for the formation of the complex should be relatively high, a proper~y which may be achieved by virtue of cooperativity in the process of forming the complex. A consequence of having a high association constant is that the concentration of free protein will remain relatively low. Under such conditions, when the release mechanism is govemed by diffllsion, the rate of release can be dirninished because the flux is p~roportional to ~e concentration gradient established between the inner and outer phases. With a low concentration of protein es~ablished in ~e inner phase, the rate of diffusion will be low. Second, the concentration of polycation should be relatively low, so that the ac~ve agent is the prevalent component by weight in the formuhtion, if so .
.
.. ,- , .~ ' ~
WO 92/~ 1844 PCr/US~l/0977l desired. This is readily achievable because the high association constant ensures that most or all of ~e polycation par~cipates in complex formation.
Third, the molecular weight of the polycation should be relatively high, so that its diffusion coefficient will be low. In this way the active agent will be preferentially depleted firom the ma~ix or depot prior to the polycation.
The present inven~on will be further understood by the following non-limiting examples.
Example 1: Formation of a complex between bovine serwn albumin and chitosan.
1 g of chito~san was dissolved in 100 ml of 1 æ acetic acid. The pH of the resulting solution was 3Ø The solu~on was titrated with sodium hydroxide to pH of 6.0, avoiding precipitation and gel formation by ~u~ c.' ito~n. This is termed neu~ralized chitosan.
12.0 mg of bovine serum albumin (BSA) was dissolved in 1.0 ml 5 mM ammonium bicarbonate. 20 microliter aliquots of neutralized chitosan were added to the BSA, as well as to a buffer blank. A thick cloudy precipitate formed with the BSA, which was more profound and extensive than that observed with buffer alone. The latter is ascribed to pH-induced precipitation of chitosan. Centrifugation was used to determine whether precipitation occurred in the liquid supernatant with successive additions of chitosan. Generation of incremental turbidity ended at about the point where 200 microliters of the chitosan solution had been added to the BSA, coIIesponding to 2.0 mg chitosan.
The equivalence point was reached at a weight ratio of BSA:chitosan of about 5:1.
.
wo 92/1~844 PCr/US91/09771 f Example 2: Fo~nation of a complex between bo~ne hemoglobin and chitosan.
10.2 mg of bovine hemoglobin (EIb) was dissolved in 1.0 ml deionized water. Up ~o 40 microliters of neutralized chitosan was added in portions. With the first additions a dark agglome~ate formed, corresponding to partial depletion of color from the solution. Further addition of chitosan did not lead to a quantitative precipitation of ~e Hb.
Example 3: Preparation of and in vitro ~elease from PLGA
microspheres containing the erythropoietin~hitosan complex.
Chitosan acetate at pH S WélS used to dissolve recombmant human erythropoietin (EPO) with varying ratios of chitosan:EPO. These formulations were micron~zed according to the method set forth in Gombotz et al. (U. S. Serial No. 07/345,684) and incorporated into copoly~l),L-lactide, glycolide) (50:50, Boe}lringer-Ingelhe~m R~3 503) using the procedures of Gombotz et al. (U. S. Serial No. 07/345,143).
The final loading ratios, in weight percentages of the final microsphere preparation, were 6% EPO: 10.3% chitosan, 12% EPO: 6.8% chitosan, and 17% EPO alone.
These microspheres were subjected to in v~tro release stu~ies at 37C, using the follo~1ving release buffer: 50 n~ sodium phosphate, 0.9%
MaCI, 2% (w/v) ovalbun~n, pH 7.2. The release results are shown in Figure 1. It is evident that, as compared to the abænce of chitosan, incorpoIation of the polycation profoundly reduces the burst effect upon ~e release of EPO from the microspheres.
Example 4: Prepar~tion and in vi~o release of P~ A microspheres containing the Factor Vm-poly(arginine) complex.
Human recombinant Factor vm was recons~tuted to 200 units/ml in 0.2 M NaCl, 0.55 M glycine, 0.005 M CaCl2, 12 mg/ml hurnan serum WO 92/11844 PCr/US91/09771 , .
2~37~
albumin. To this solution was added polyarginine [(Arg)81 at 2 mg/ml.
The solution was subjected to a change in composition of the bu~fer to O.l M proline, 2.5 mM CaCl2, pH 7.35 by passing the reconstib~ted mixture through a SephadexR G-25 column equilibrated with ~e proline - CaC12 solution. The product was then micronized according to the proccdure of Gombotz, et al., in U.S. Serial No. 07/345,684, and incorporated ~nto microspheres comprised of poly~actic acid) as the carrier matrix, at a loading of the for nulated Factor vm preparation of 30% (w/w) using the procedure described by Gombotz, et al. in U.S. Serial No. 07/345,143.
This preparation is referred to as "poly(arginine)" in Figure 2.
A similar microsphere preparation was made using human recombinant Factor vm reconstituted to lO0 unitstml in 0.1 M NaCl, 0.275 M glycine, 0.0025 M CaC12, 6 mg/ml human serum album~n. This preparation was similarly incorporated into PLA microspheres at 30%
loading. This prepa~a~on is re~erred to as "NaCl-Glyc~ne" ~n };igure 2.
The two rnicrosphere preparations were subjected to in vi~ro release experiments at 37C, by immersLng approximately lO mg of microspheres in l.0 rnl aliquots of a release buffer consisting of O.l M
NaCl, O.l M glycine, lO mM HEPES, 2.5 mM CaCl2, 2 mg/lT~l hurnan serum albumin, pH 7.2 m a l.5 ml microfuge tube, and agitated gently.
Fresh aliquots of release medium were applied for each time point. The activity was assayed using the CoatestR kit for Factor VIII produced by Kabi Vitrum and distributed by Helena Laboratories, Inc. The color resulting from release of p-nitrophenolate from a synthetic substrate, as deternnned in microtiter plate format using a plate reader, and expressed as the cumulative percent of inçolporated acti~ity released per mg of microspheres, is given for the two preparations in Figure 2. It is evident ~at Factor VIII formuL~ted wi~ (Arg)n has led to markedly enhanced and sustained release l~ne~cs compared to omission of (Arg)n.
wo 92/11844 PCT/US91/09771 ~; ' `
~' ~3~7 ~
Example 5: Bo~ine Serum Albumin-Protamine Complex Release from Copoiy(lactid~glycolide) Microspheres.
A globulin-free preparation of bovine semm albumin (BSA) obtained from Sigma Chemical Co. was mixed 1:1 (w:w) with sucrose or WIth protan~ine sulfate. The resulting solu~ons, as well as a solution of BSA alone, were micronized according to Gombotz, et al., as described in U.S. Serial No. 07/345,684. The protein-excipient formulations were incorporated into microspheres of copoly(DL-lactide,glycolide) (50:50) following the procedures of Gombotz, et al., U.S. Serial No. 07/345,143, with total loadings of 10% by weight. These microspheres were placed in 20 rnM sodium phosphate, 0.15 M sodium chloride, 1.5 rnM sodium azide, pH 7.S, at 37 C to measure in vitro release.
The cumulative release over 68 days is shown in Figure 3. The surge in release that occurs between about days 20 and 28 is ascribed to dcgradatioil Or the polymeT matrix, expos~ng fresh reservoirs Or prv~ein for release to the medium. The results show that incorporation of protamine sulfate gives enhanced release characteristics as compared to the incorporation of an equa} arnount of sucrose. The extent of release in the first hour, termed the burst, is diminished, and the steady, near-zero-order release of protein is sustained for a longer duration. For BSA
without added excipients, the burst release is the lowest of the three cases shown, but the degradation phase releases a large fraction of the protein over a relatively short period of time; further release continues for the remainder of the time period considered. Of the three preparations shown, the incorporation of protan~ine sulfate leads to the most monotonic release of rotein after ~e burst.
. . . . .
,
Claims (24)
1. A stabilized composition for controlled release of a biologically active protein comprising a biocompatible polymeric matrix incorporating a complex of a protein or peptide and a biocompatible polycation.
2. The composition of claim 1 wherein the polycation is complexed with the protein in a ratio between approximately 1:1000 protein:polycation by weight and 20:1 protein:polycation.
3. The composition of claim l wherein the polycation is complexed with the protein in a ratio between approximately 1:100 protein:polycation by weight and 10:1 protein:polycation.
4. The composition of claim 1 wherein the protein has a pI of less than 8.
5. The composition of claim 1 wherein the polymer forming the matrix is selected from the group consisting of biocompatible synthetic and natural polymers.
6. The composition of claim 5 wherein the polymer is selected from the group consisting of poly(lactic acid), poly(lactic-co-glycolic acid), poly(caprolactone), polycarbonates, polyamides, polyanhydrides, polyamino acids, polyortho esters, polyacetals, polycyanoacrylates and degradable polyurethanes, and non-erodible polymers such as polyacrylates, ethylene-vinyl acetate and other acyl substituted cellulose acetates and derivatives thereof, non-erodible polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinyl imidazole), chlorosulphonated polyolefins, and polyethylene oxide.
7. The composition of claim 1 wherein the polycation is selected from the group of polyamino acids, basic proteins and catonic polysaccharides.
8. The composition of claim 1 wherein the polycation has a molecular weight of 4,000 daltons or greater.
9. The composition of claim 8 wherein the polycation has a molecular weight of about 50,000 or greater.
10. The composition of claim 1 wherein the matrix is the supramolecular aggregate formed by the polycation in complexed with the biologically active agent.
11. The composition of claim 10 wherein the protein is polyvalent in its interaction with the polycation.
12. A method for stabilizing a biologically active protein in a controlled release device comprising forming a complex of a protein and biocompatible polycation and incorporating the complex into a biocompatible polymeric matrix.
13. The method of claim 12 wherein the polycation is complexed with the protein in a ratio between approximately 1:1000 protein:polycation by weight and 20:1 protein:polycation.
14. The method of claim 12 wherein the polycation is complexed with the protein in a ratio between approximately 1:100 protein:polycation by weight and 10:1 protein:polycation.
15. The method of claim 12 wherein the protein has a pI of less than 8.
16. The method of claim 12 wherein the polymer forming the matrix is selected from the group consisting of synthetic and natural polymers.
17. The method of claim 16 wherein the polymer is selected from the group consisting of poly(lactic acid), poly(lactic-co-glycolic acid), poly(caprolactone), polycarbonates, polyamides, polyanhydrides, polyamino acids, polyortho esters, polyacetals, polycyanoacrylates and degradable polyurethanes,and non-erodible polymers such as polyacrylates, ethylene-vinyl acetate and other acyl substituted cellulose acetates and derivatives thereof, non-erodible polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinyl imidazole), chlorosulphonated polyolefins, and polyethylene oxide.
18. The method of claim 12 wherein the polycation is selected from the group of polyamino acids, basic proteins, and cationic polysaccharides.
19. The method of claim 12 wherein the polycation has a molecular weight of 4,000 daltons or greater.
20. The method of claim 19 wherein the polycation has a molecular weight of about 50,000 or greater.
21. The method of claim 12 wherein the polymeric matrix is formed by the polycation in combination with the protein.
22. The method of claim 16 wherein the protein is a protein polyvalent in its interaction with the polycation.
23. The method of claim 12 further comprising providing an amount of polycation in combination with protein effective to alter the release of the biologically active agent from the polymeric matrix.
24. The method of claim 12 further comprising providing an amount of polycation in combination with protein effective to maintain the activity of the protein as compared to the activity of the protein in the polymeric matrix in the absence of the polycation.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US63732591A | 1991-01-03 | 1991-01-03 | |
US637,325 | 1991-01-03 |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2099376A1 true CA2099376A1 (en) | 1992-07-04 |
Family
ID=24555459
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002099376A Abandoned CA2099376A1 (en) | 1991-01-03 | 1991-12-31 | Stabilization of proteins by cationic biopolymers |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0565618A4 (en) |
JP (1) | JPH07503700A (en) |
AU (1) | AU653771B2 (en) |
CA (1) | CA2099376A1 (en) |
WO (1) | WO1992011844A1 (en) |
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-
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- 1991-12-31 CA CA002099376A patent/CA2099376A1/en not_active Abandoned
- 1991-12-31 EP EP19920903702 patent/EP0565618A4/en not_active Withdrawn
- 1991-12-31 JP JP4503421A patent/JPH07503700A/en active Pending
- 1991-12-31 AU AU91652/91A patent/AU653771B2/en not_active Ceased
- 1991-12-31 WO PCT/US1991/009771 patent/WO1992011844A1/en not_active Application Discontinuation
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GB202007428D0 (en) | 2020-05-19 | 2020-07-01 | Fabricnano Ltd | Polynucleotide synthesis |
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GB202110595D0 (en) | 2021-07-22 | 2021-09-08 | Fabricnano Ltd | Functionalised nucleic acid structure |
Also Published As
Publication number | Publication date |
---|---|
AU9165291A (en) | 1992-08-17 |
EP0565618A1 (en) | 1993-10-20 |
EP0565618A4 (en) | 1994-06-29 |
WO1992011844A1 (en) | 1992-07-23 |
AU653771B2 (en) | 1994-10-13 |
JPH07503700A (en) | 1995-04-20 |
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