| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | January 5, 1999 |
| PATENT TITLE |
Polyphosphazene polyelectrolyte immunoadjuvants |
| PATENT ABSTRACT | A polyphosphazene polyelectrolyte immunoadjuvant having improved water solubility, a vaccine composition of the immunoadjuvant and an antigen or immunogen and methods of their use in producing or enhancing an immunoprotective response in a host are disclosed |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | June 7, 1995 |
| PATENT REFERENCES CITED |
Cohen et al., "Ionically Cross-Linkable Polyphosphazene: A Novel Polymer for Microencapsulation ", J. Amer. Chem. Soc. 112,7832-7833. Andrianov et al., "Controlled release using ionotropic polyphosphazene hydrogels"J. Contr. Rel. 27, 69-77. Aldrich Chemical Catalog, p. 731. |
| PATENT CLAIMS |
We claim: 1. A composition for inducing an immunogenic response in a mammal, comprising: an antigen and a polyphosphazene polyelectrolyte that has immunoadjuvant activity and said polyphosphazene polyelectrolyte is a copolymer having the formula: ##STR6## wherein each A is independently selected from X or Y in which (1) X is ##STR7## in which R.sub.1 is --O-- or --OR.sub.5 -- where R.sub.5 is a C.sub.1 -C.sub.5 alkylene moiety, R.sub.2 is at least one carboxylic acid moiety which is attached to the ring directly or through an alkylene and R.sub.3 is hydrogen or an alkoxy which is also attached to the ring; (2) Y is (OCH.sub.2 CH.sub.2).sub.n OR.sub.6 in which n is 1 to Z wherein Z is a number which provides an average molecular weight of 750 and R.sub.6 is a C.sub.1 to C.sub.10 hydrocarbon; (3) X and Y are in a range of ratios of from about 1.5:1 to about 11.5:1; and (4) m is 3 to 100,000. 2. The composition of claim 1 wherein the polymer contains at least 10 percent or more A groups that are not susceptible to hydrolysis under the conditions of use. 3. The composition of claim 1 wherein the polyphosphazene polyelectrolyte is cross-linked with a multivalent cation. 4. The composition of claim 3 wherein the multivalent cation is selected from the group consisting of calcium, copper, aluminum, magnesium, strontium, barium, tin, zinc and iron. 5. The composition of claim 1 wherein the antigen is selected from the group consisting of protein, peptide and glycoprotein. 6. The composition of claim 1 wherein the polyphosphazene polyelectrolyte is covalently conjugated with an antigen. 7. The composition of claim 1 wherein X is carboxylatophenoxy and Y is selected from the group consisting of: --OC.sub.2 H.sub.4 OCH.sub.3 and --OC.sub.2 H.sub.4 C.sub.2 H.sub.4 OCH.sub.3 8. A method of inducing an immune response in a mammal comprising the steps of administering to the mammal antigen and a polyphosphazene polyelectrolyte that has immunoadjuvant activity and said polyphosphazene polyelectrolyte is a copolymer having the formula: ##STR8## wherein each A is independently selected from X or Y in which (1) X is ##STR9## in which R.sub.1 is --O-- or --OR.sub.5 -- where R.sub.5 is a C.sub.1 -C.sub.3 alkylene moiety, R.sub.2 is at least one carboxylic acid moiety which is attached to the ring directly or through an alkylene and R.sub.3 is hydrogen or an alkoxy which is also attached to the ring; (2) Y is (OCH.sub.2 CH.sub.2).sub.n OR.sub.6 in which n is 1 to Z wherein Z is a number which provides an average molecular weight of 750 and R.sub.6 is a C.sub.1 to C.sub.10 hydrocarbon; (3) X and Y are in a range of ratios of from about 1.5:1 to about 11.5:1; and (4) in is 3 to 100,000; thereby inducing an immune response in said mammal. 9. The method of claim 8 wherein the antigen is conjugated with the polyphosphazene polyelectrolyte. 10. The method of claim 8 wherein the antigen and polyphosphazene are administered separately to proximate sites. 11. The method of claim 8 wherein the antigen and polyphosphazene are first combined and the combination is administered to the mammal. 12. The method of claim 8 wherein the method of administration is parenteral. 13. The method of claim 8 wherein the method of administration is oral. 14. A polyphosphazene polyelectrolyte adjuvant which has immunoadjuvant activity and is a copolymer having the formula: ##STR10## wherein each A is independently selected from X or Y in which (1) X is ##STR11## in which R.sub.1 is --O-- or --OR.sub.5 -- where R.sub.5 is a C.sub.1 -C.sub.3 alkylene moiety, R.sub.2 is at least one carbolic acid moiety which is attached to the ring dry or through an alkylene and R.sub.3 is hydrogen or an alkoxy which is also attached to the ring; (2) Y is (OCH.sub.2 CH.sub.2).sub.n OR.sub.6 in which n is 1 to Z wherein Z is a number which provides an average molecular weight of 750 and R.sub.6 is a C.sub.1 to C.sub.10 hydrocarbon; (3) X and Y are in a range of ratios of from about 1.5:1 to about 11.5:1; and (4) m is 3 to 100,000. 15. The polyphosphazene polyelectrolyte of claim 14 wherein the polymer contains at least 10 percent or more A groups that are not susceptible to hydrolysis under the conditions of use. 16. The polyphosphazene polyelectrolyte of claim 14 wherein the polyphosphazene polyelectrolyte is cross-linked with a multivalent cation. 17. The polyphosphazene polyelectrolyte of claim 16 wherein the multivalent cation is selected from the group consisting of calcium, copper, aluminum, magnesium, strontium, barium, tin, zinc and iron. 18. A composition comprising the polyphosphazene polyelectrolyte of claim 14 and an antigen selected from the group consisting of protein, peptide, and glycoprotein. 19. The polyphosphazene polyelectrolyte of claim 14 wherein the polyphosphazene polyelectrolyte is covalently conjugated with an antigen. 20. The polyphosphazene polyelectrolyte of claim 14 wherein X is a -carboxylatophenoxy. 21. A composition for inducing an immunogenic response in an animal, comprising: an antigen and a polyphosphazene polyelectrolyte that has immunoadjuvant activity and is a copolymer having the formula: ##STR12## wherein each A is independently selected from X or Y in which (1) X is ##STR13## in which R.sub.1 is --O-- or --OR.sub.5 -- where R.sub.5 is a C.sub.1 -C.sub.3 alkylene moiety, R.sub.2 is at least one carboxylic acid moiety which is attached to he ring directly or trough an alkylene and R.sub.3 is hydrogen or an alkoxy which is also attached to the ring; (2) Y is selected from the group consisting of --OC.sub.2 H.sub.4 OCH.sub.3 and --OC.sub.2 H.sub.4 OC.sub.2 H.sub.4 OCH.sub.3 ; (3) X and Y are present in a range of ratios of from about 1.5:1 to about 11.5:1; and (4) m is 3 to 100,000. 22. The composition of claim 3 wherein the multivalent cation is selected from the group consisting of poly(amino acid), poly(ethyleneimine), poly(vinylamine) and polysaccharides. 23. The composition of claim 1 wherein the antigen is selected from the group consisting of a polysaccharide, a glycolipid, a nucleic acid and an antigen derived from a cell, bacteria, virus particle or a portion of any of them. 24. A composition comprising the polyphosphazene polyelectrolyte adjuvant of claim 14 and an antigen selected from the group consisting of a glycolipid, a polysaccharide, a nucleic acid and an antigen derived from a cell, bacteria, virus particle or a portion of any of them. 25. The composition of claim 1 wherein the antigen is a nucleic acid. 26. The composition of claim 1 wherein the antigen is derived from a cell, bacteria, virus particle or a portion of any of them. 27. The polyphosphazene polyelectrolyte of claim 16 wherein the multivalent cation is selected from the group consisting of poly(amino acid), poly(ethyleneimine), poly(vinylamine) and polysaccharides. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
This application relates to the field of polymers for biomedical applications, and in particular describes polymers that are useful as immunoadjuvants. A wide variety of antigens stimulate the production of antibodies in animals and confer protection against subsequent infection. However, some antigens are unable to stimulate an effective immune response. The immunogenicity of a relatively weak antigen is often enhanced by the simultaneous administration of the antigen with an adjuvant, a substance that is not immunogenic when administered alone, but will induce a state of mucosal and/or systemic immunity when combined with the antigen. It has been traditionally thought that adjuvants, such as mineral oil emulsions or aluminum hydroxide, form an antigen depot at the site of injection that slowly releases antigen. Recent studies by Allison and Byars, in: "Vaccines: New Approaches to Immunological Problems:, R. W. Ellis, ed., p. 431, Butterworth-Heinemann, Oxford (1992) indicate that adjuvants enhance the immune response by stimulating specific and sometimes very narrow branches of the immune response by the release of cytokines. Unfortunately, many immunoadjuvants, such as Freund's Complete Adjuvant, are toxic and are therefore only useful for animal research purposes, not human vaccinations. Freund's Complete Adjuvant contains a suspension of heat-killed Mycobacterium tuberculosis in mineral oil containing a surfactant and causes granulomatous lesions in animals at the site of immunization. Freund's adjuvant may also cause the recipient of a vaccine to test positive for tuberculosis. Some synthetic polyelectrolytes have been found to provide immunostimulation when combined with an antigen. For example, the adjuvant activity of polyacrylic acid (PAA), copolymers of acrylic acid and N-vinylpyrrolidone (CP-AAVPD), poly-2-methyl-5-vinyl pyridine (PMVP), poly-4-vinylN-ethylpyridinium bromide (PVP-R.sub.2) and similar compounds, when conjugated to an antigen, has been studied by Petrov et al., Jhurnal Vses. Khim. Ob-va im. D. I. Mendeleeva, 33: 22-42 (1988). The immunomodulatory effect of polyelectrolyte complexes containing many of these same polyelectrolytes has also been more recently reviewed by Petrov et al., Sov. Med. Rev. D. Immunol., 4: 1-113 (1992). However, the toxicity and biodegradability of these polymers has not been studied and may prevent use of these polymers as adjuvants for use in humans. A non-toxic adjuvant or carrier having the ability to stimulate an immune response to non-antigenic or weakly antigenic molecules would fill a long-sought need in the development and administration of vaccines. The present invention provides an adjuvant that can be safely administered to humans and animals with minimal toxicity, is biodegradable and which has the additional advantage of being highly water soluble. The present invention also provides a rapid and efficient method of synthesizing a polymer, such as polyphosphazene, for use as an adjuvant. Thus, in one aspect, the present invention provides a polyphosphazene polyelectrolyte wherein at least a portion of said polyphosphazene polyelectrolyte has the formula: ##STR1## wherein each A is independently selected from X or Y in which (1) X is ##STR2## in which R.sub.1 is --N(R.sub.4)--, --O-- or --OR.sub.5 -- where R.sub.4 is hydrogen or a C.sub.1 -C.sub.4 alkyl(preferably hydrogen) and R.sub.5 is a C.sub.1 -C.sub.3 alkylene(preferably methylene) moiety, R.sub.2 is at least one carboxylic acid moiety which can be attached at any ring position directly or through an alkylene, preferably a methylene and R.sub.3 is hydrogen or another substituent moiety such as an alkoxy, for example a methoxy moiety; (2) Y is selected from the group consisting of (a) (OCH.sub.2 CH.sub.2).sub.n OR.sub.6 in which n is 1 to 120 and R.sub.6 is a C.sub.1 to C.sub.10 hydrocarbon, e.g. an aliphatic saturated or unsaturated hydrocarbon(preferably a C.sub.1 to C.sub.3 aliphatic hydrocarbon) or an aromatic hydrocarbon(including alkyl substituted aromatic hydrocarbons); and (b) (B) OR.sub.7 in which B is a polyoxypropylene/polyoxyethylene block copolymer and R.sub.7 is a C.sub.1 to C.sub.10 hydrocarbon, an aliphatic saturated or unsaturated hydrocarbon or an aromatic hydrocarbon, including alkyl substituted aromatic hydrocarbons; (3) X and Y are in a range of ratios of 1:40 to 40:1; and (4) m is 3 to 100,000. The phosphazene is a polyelectrolyte that is biodegradable and exhibits minimal toxicity when administered to animals, such as humans. It is ideal for use as an immunoadjuvant. Two examples of polyphosphazenes that are particularly preferred as immunoadjuvants are poly›p-(carboxylatophenoxy) (methoxyethoxy) phosphazene! and poly›p-(carboxylatophenoxy) (methoxyethoxyethoxy) phosphazene!. When cross-linked with a multivalention, the polymer becomes less soluble, resulting in slower release of the polymer from the site of administration. In another aspect the invention provides a composition comprising the polyphosphazene polyelectrolyte of the invention in combination with an antigen or immunogen in the form of a vaccine. In another aspect the invention provides a method of inducing or enhancing an immunoprotective response to an immunological challenge in a host which comprises administering to said host an immunogen capable of sensitizing said host to said immunological challenge and a polyphosphazene polyelectrolyte immunoadjuvant of the invention. The immunogen and adjuvant can be administered separately or as a composition. A vaccine composition is prepared by either mixing or conjugating the polymer adjuvant with an antigen prior to administration. Alternatively, the polymer and antigen can be administered separately to the same site. The term "pharmaceutically acceptable cation" refers to an organic or inorganic moiety that carries a positive charge and that can be administered as a counteraction in a phosphazene polyelectrolyte. The polymeric adjuvant of the invention is a polyphosphazene that is soluble in water at physiological pH. Polyphosphazenes are polymers with backbones consisting of alternating phosphorus and nitrogen, separated by alternating single and double bonds. Each phosphorous atom is covalently bonded to two pendant groups ("A"). The repeat unit in the polyphosphazenes of this invention has the following formula: ##STR3## wherein each A is independently selected from X or Y as described above. In general, when the polyphosphazene has more than one type of pendant group, the groups will vary randomly throughout the polymer, and the polyphosphazene is thus a random copolymer. Phosphorous can be bound to two like groups, or two different groups. Polyphosphazenes with two or more types of pendant groups can be produced by reacting poly(dichlorophosphazene) with the desired nucleophile or nucleophiles in a desired ratio. The resulting ratio of pendant groups in the polyphosphazene will be determined by a number of factors, including the ratio of starting materials used to produce the polymer, the temperature at which the nucleophilic substitution reaction is carried out, and the solvent system used. While it is very difficult to determine the exact substitution pattern of the groups in the resulting polymer, the ratio of groups in the polymer can be easily determined by one skilled in the art. A preferred phosphazene polyelectrolyte immunoadjuvant contains pendant groups that include carboxylic acid moieties. While the acidic groups are usually on nonhydrolyzable pendant groups, they can alternatively, or in combination, also be positioned on hydrolyzable groups. Most preferably the polymer is a polyphosphazene that includes pendant groups that include carboxylic acid moieties on pendant groups that do not hydrolyze under the conditions of use. In one preferred embodiment, X is selected from the group consisting of: ##STR4## In another aspect the invention provides a polyphosphazene having the formula: ##STR5## wherein m is 3 to 100,000 and each B is Ortho--O--C.sub.6 H.sub.4 COOH This polyphosphazene is also suitable for use as an immunoadjuvant in accordance with the invention. The phosphazene polyelectrolytes of the invention contain ionized or ionizable pendant groups that render the polyphosphazene anionic, cationic or amphiphilic. The ionic groups can be in the form of a salt, or, alternatively, an acid or base that is or can be at least partially dissociated. Any pharmaceutically acceptable monovalent cation can be used as counterion of the salt, including but not limited to sodium, potassium, and ammonium. The phosphazene polyelectrolytes can also contain non-ionic side groups. The phosphazene polyelectrolyte can be biodegradable or nonbiodegradable under the conditions of use. The ionized or ionizable pendant groups are preferably not susceptible to hydrolysis under the conditions of use. The phosphazene polyelectrolyte is preferably biodegradable to prevent eventual deposition and accumulation of polymer molecules at distant sites in the body, such as the spleen. The term biodegradable, as used herein, means a polymer that degrades within a period that is acceptable in the desired application, typically less than about five years and most preferably less than about one year, once exposed to a physiological solution of pH 6-8 at a temperature of approximately 25.degree. C.-37.degree. C. Crosslinked polyphosphazenes for use as immunoadjuvants can be prepared by combining a phosphazene polyelectrolyte with a metal multivalent cation such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, or cadmium. Polyphosphazenes, including phosphazene polyelectrolytes, can be prepared by a macromolecular nucleophilic substitution reaction of poly(dichloro phosphazene) with a wide range of chemical reagents or mixture of reagents in accordance with methods known to those skilled in the art. Preferably, the phosphazene polyelectrolytes are made by reacting the poly(dichloro phosphazene) with an appropriate nucleophile or nucleophiles that displace chlorine. Desired proportions of hydrolyzable to non-hydrolyzable side chains in the polymer can be obtained by adjusting the quantity of the corresponding nucleophiles that are reacted with poly(dichlorophosphazene) and the reaction conditions as necessary. Preferred polyphosphazenes for immunoadjuvant activity have a molecular weight of over 1,000. In another embodiment, the polyphosphazene polymer adjuvant of the invention is employed in a composition to induce an immunogenic response, in particular, a protective response when the composition is parenterally or orally administered to a patient. The antigen with which the adjuvants of the invention are used can be derived from a cell, bacteria or virus particle or portion thereof. The antigen can be a protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid or combination thereof which elicits an immunogenic response in an animal, for example, a mammal, bird, or fish. The immunogenic response can be humoral or cell mediated. In the event the material to which the immunogenic response is to be directed is poorly antigenic, it may be conjugated to a carrier such as albumin or to a hapten, using standard covalent binding techniques, for example, with one of the several commercially available reagent kits. In one embodiment, the polymer is used to deliver nucleic acid which encodes antigen to a mucosal surface where the nucleic acid is expressed. In such a case, the polymer is in the form of a hydrogel (crosslinked) and is in particulate form. Examples of preferred antigens include viral proteins such as influenza proteins, human immunodeficiency virus (HIV) proteins and hepatitis B proteins, and bacterial proteins and lipopolysaccharides such as gram negative bacterial cell walls and Neisseria gonorrhea proteins. An immunogenic composition, or vaccine, is prepared by combining the polymer adjuvant with an antigen. Approximately 0.5-0.0001 parts of antigen is added to one part polymer, preferably by stirring a solution of polymer and antigen until a solution or suspension is obtained, preferably for 10 minutes or more at 25.degree. C. The polymer is preferably combined with the antigen using a method dispersing the antigen uniformly throughout the adjuvant. Methods for liquefying the polymer include dissolving the polymer in an aqueous-based solvent, preferably having a pH range of between 7.1 and 7.7, and melting the polymer. The latter is useful only when the antigen is stable at the polymer melting temperature. The antigen is then mixed with the polymer. The polymer and the antigen, in solid form, for example, when the antigen is lyophilized, can also be physically mixed together, for example, by compression molding. The polymer can also be used to encapsulate the antigen, for example, using the method of U.S. Pat. No. 5,149,543 to Cohen, et al., the teachings of which are incorporate herein, or by spray drying a solution of polymer and antigen. Alternatively, microspheres containing the antigen and adjuvant can be prepared by simply mixing the components in an aqueous solution, and then coagulating the polymer together with the substance by mechanical forces to form a microparticle. The microparticle can be stabilized, if necessary or desired, using electrolytes, pH changes, organic solvents, heat or frost to form polymer matrices encapsulating biological material. The polymer of the present invention is soluble in physiologically buffered saline (PBS). The polymer can also be covalently conjugated with the antigen to create a water-soluble conjugate in accordance with methods well-known to those skilled in the art, usually by covalent linkage between an amino or carboxyl group on the antigen and one of the ionizable side groups on the polymer. In an alternative preferred embodiment, the polymer is cross-linked with a multivalent ion, preferably using an aqueous solution containing multivalent ions of the opposite charge to those of the charged side groups of the polyphosphazene, such as multivalent cations if the polymer has acidic side groups or multivalent anions if the polymer has basic side groups. Preferably, the polymers are cross-linked by di and trivalent metal ions such as calcium, copper, aluminum, magnesium, strontium, barium, tin, zinc, and iron, organic cations such as poly(amino acid)s, or other polymers such as poly(ethyleneimine), poly(vinylamine) and polysaccharides. It will be understood by those skilled in the art that the immunogenic vaccine composition can contain other physiologically acceptable ingredients such as water, saline or a mineral oil such as Drakeol.TM., Markol.TM., and squalene, to form an emulsion. The immunogenic composition can be administered as a vaccine by any method known to those skilled in the art that elicits an immune response, including parenterally, orally, or by transmembrane or transmucosal administration. Preferably, the vaccine is administered parenterally (intravenously, intramuscularly, subcutaneously, intraperitoneally, etc.), and preferably subcutaneously. Nonlimiting examples of routes of delivery to mucosal surfaces are intranasal (or generally, the nasal associated lymphoid tissue), respiratory, vaginal and rectal. The dosage is determined by the antigen loading and by standard techniques for determining dosage and schedules for administration for each antigen, based on titer of antibody elicited by the polymer-antigen administration, as demonstrated by the following examples. Although in the preferred embodiment the polymer antigen mixture is administered simultaneously, in an alternative embodiment, the polymer and antigen are administered separately to the same or nearby site. The polymer serves to attract cells of the immune system to the site, where they process the antigen. |
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