US20060193820A1 - Immunostimulating polyphosphazene compounds - Google Patents

Immunostimulating polyphosphazene compounds Download PDF

Info

Publication number
US20060193820A1
US20060193820A1 US11/355,737 US35573706A US2006193820A1 US 20060193820 A1 US20060193820 A1 US 20060193820A1 US 35573706 A US35573706 A US 35573706A US 2006193820 A1 US2006193820 A1 US 2006193820A1
Authority
US
United States
Prior art keywords
polyphosphazene polymer
antigen
polymer
compound
cooh
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US11/355,737
Other languages
English (en)
Inventor
Alexander Andrianov
Alexander Marin
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Parallel Solutions Inc
Original Assignee
Parallel Solutions Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Parallel Solutions Inc filed Critical Parallel Solutions Inc
Priority to US11/355,737 priority Critical patent/US20060193820A1/en
Assigned to PARALLEL SOLUTIONS, INC. reassignment PARALLEL SOLUTIONS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ANDRIANOV, ALEXANDER K., MARIN, ALEXANDER
Publication of US20060193820A1 publication Critical patent/US20060193820A1/en
Priority to US12/806,941 priority patent/US20100322891A1/en
Priority to US13/743,824 priority patent/US20130136758A1/en
Priority to US14/323,083 priority patent/US20140314707A1/en
Priority to US14/710,798 priority patent/US20150238600A1/en
Priority to US14/977,848 priority patent/US9687546B2/en
Priority to US15/610,747 priority patent/US20170258900A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/80—Polymers containing hetero atoms not provided for in groups A61K31/755 - A61K31/795
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • A61K39/292—Serum hepatitis virus, hepatitis B virus, e.g. Australia antigen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G79/00—Macromolecular compounds obtained by reactions forming a linkage containing atoms other than silicon, sulfur, nitrogen, oxygen, and carbon with or without the latter elements in the main chain of the macromolecule
    • C08G79/02—Macromolecular compounds obtained by reactions forming a linkage containing atoms other than silicon, sulfur, nitrogen, oxygen, and carbon with or without the latter elements in the main chain of the macromolecule a linkage containing phosphorus
    • C08G79/025—Polyphosphazenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/35—Allergens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • FIG. 1 represents serum IgG titers after immunization of mice with HBsAg formulated with Polymer 1 and Polymer 2.
  • HBsAg, HBsAg formulated with PCPP, and HBsAg formulated with Alum were used as controls (5 BALB/c mice per group; HBsAg: 1 ⁇ g/mouse; polymers: 50 ⁇ g/mouse; single dose intramuscular injection; 16 week data).
  • FIG. 2 represents serum IgG titers after immunization of mice with X-31 influenza formulated with Polymer 1 and Polymer 2.
  • X-31, X-31 formulated with PCPP, and X-31 formulated with Alum were used as controls (5 BALB/c mice per group; X-31: 5 ⁇ g/mouse; polymers: 50 ⁇ g/mouse; single dose intramuscular injection; 16 week data).
  • This application relates to polyphosphazene polymers having immunomodulating activity and the biomedical use of such polyphosphazene polymers in conjunction with an antigen or an immunogen.
  • antigens stimulate the production of antibodies in animals thereby conferring protection against infection. However, some antigens are unable to stimulate an effective immune response treatment.
  • 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 which will induce a state of systemic, humoral, and/or mucosal immunity when combined with the antigen.
  • adjuvants such as mineral oil emulsions or aluminum hydroxide, form an antigen depot at the site of injection that slowly releases antigen.
  • 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 it 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.
  • PAA polyacrylic acid
  • CP-AAVPD copolymers of acrylic acid and N-vinylpyrrolidone
  • PMVP poly-2-methyl-5-vinyl pyridine
  • PVP-R 2 poly-4-vinylN-ethylpyridinium bromide
  • V. A. Kabanov From Synthetic Polyelectrolytes to Polymer—Subunit Vaccines, Pure Appl. Chem., 2004, 76, 9, 1659-1677).
  • these polymers require covalent attachment of antigen to the polymer in order to elicit effective immune responses, which can present manufacturing and regulatory challenges.
  • toxicity and biodegradability of the majority of these polymers have not been studied and may prevent use of these polymers as adjuvants for applications in humans.
  • PCPP Poly[di(carboxylatophenoxy)phosphazene]
  • PCPP Polyphosphazene polymer
  • L. G. Payne, S. A. Jenkins, A. L. Woods, E. M. Grund, W. E. Geribo, J. R. Loebelenz, A. K. Andrianov, B. E. Roberts, Poly[di(carboxylatophenoxy)phosphazene] (PCPP) is a potent immunoadjuvant for an influenza vaccine; Vaccine 16, 92-98 (1998)).
  • more potent immunoadjuvants than PCPP are needed.
  • 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 polyphosphazenes that may be used as adjuvants and are superior in their immunoadjuvant activity to PCPP.
  • Polyphosphazenes are polymers with backbones including alternating phosphorus and nitrogen atoms, separated by alternating single and double bonds. Each phosphorous atom is covalently bonded to two pendant groups (“A”).
  • the repeat unit of a polyphosphazene has the following formula:
  • each “A” may be the same, or different, and wherein the unit is repeated “n” times.
  • the polymer When the polyphosphazene has only one type of pendant group or side group repeatedly attached to its backbone the polymer is said to be a homopolymer. When the polyphosphazene has more than one type of pendant group and the groups vary randomly throughout the polymer, the polyphosphazene is a random copolymer. Phosphorous can be bound to two like groups, or to two different groups.
  • Polyphosphazenes can be produced by reacting macromolecular precursor—poly(dichlorophosphazene) with the desired nucleophiles, such as alcohols, amines, -or thiols.
  • Polyphosphazenes with two or more types of pendant groups can be produced by reacting poly(dichlorophosphazene) with two or more types of nucleophiles in a desired ratio. Nucleophiles can be added to the reaction mixture simultaneously or in sequential order.
  • 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 order of addition, 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.
  • the polymers of the present invention may be produced by initially producing a reactive macromolecular precursor—poly(dichlorophosphazene).
  • the pendant groups are then substituted onto the polymer backbone by reaction between the reactive chlorine atoms on the backbone and the appropriate organic compound.
  • an organic compound containing hydroxyl group and ester group may be reacted with the reactive chlorine atoms on the polymer backbone.
  • One or a mixture of organic compounds can be used to result in a homopolymer or mixed substituent copolymers correspondingly.
  • Hydroxyl group of the organic compound can be activated with sodium, sodium hydride, or sodium hydroxide by procedures known in the art and then reacted with chlorine atoms attached to the polyphosphazene backbone.
  • ester functionalities of the pendant groups may be hydrolyzed to yield carboxylic acid functionalities. All ester functionalities can be hydrolyzed to achieve full conversion into the acid groups, or, if desired, the reaction can be stopped before the completion resulting in a mixed substituted copolymer containing both acid and ester functionalities. Polymer then can be dissolved in aqueous solutions at the desired concentration. Acid groups can be also converted into salt form, such as sodium or potassium, if required to improve solubility or to achieve desired polymer conformation and physico-chemical characteristics.
  • the present invention provides a polyphosphazene polymer that contains repeating units of the following formula: wherein in each monomeric unit of the polymer, each R is the same or different, and wherein in at least a portion of the monomeric units of the polymer one or more of the R groups is “W.”
  • W is: wherein at least one of C, C′, or C′′ includes —COOH; each of d, f, f′, f′, are from 0 to 3; and
  • the polyphosphazene polymer as hereinabove described and as hereinbelow described has an overall molecular weight of 5,000 g./mol. to 10,000,000 g./mol. It is a further aspect of the instant invention that the polyphosphazene polymer contains a minimum number of monomer units having the hereinabove-described and hereinbelow-described immunostimulating carboxylic acid containing groups.
  • the polyphosphazene polymer contains at least 10 of the above-described carboxylic acid containing monomeric units.
  • R groups may be one or more of a wide variety of substituent groups.
  • substituent groups there may be mentioned: aliphatic; aryl; aralkyl; alkaryl; carboxylic acid; heteroaromatic; carbohydrates, including glucose; heteroalkyl; halogen; (aliphatic)amino-including alkylamino-; heteroaralkyl; di(aliphatic)amino-including dialkylamino-, arylamino-, diarylamino-, alkylarylamino-; -oxyaryl including but not limited to -oxyphenylCO 2 H, -oxyphenylSO 3 H, -oxyphenylhydroxyl and -oxyphenylPO 3 H; -oxyaliphatic including -oxyalkyl, -oxy(aliphatic)CO 2 H, -oxy(aliphatic)SO 3 H, -oxy(aliphatic)(aliphatic)O 3 H, -oxy(aliphatic)
  • the present invention provides a polyphosphazene polymer that contains repeating units of the following formula: wherein in each monomeric unit of said polymer, each R is the same or different, and wherein in at least a portion of the monomeric units of the polymer one or more of the R groups is: and wherein d is from 0 to 15.
  • the polyphosphazene polymer as hereinabove described and as hereinbelow described has an overall molecular weight of 5,000 g./mol. to 10,000,000 g./mol. It is a further aspect of the instant invention that the polyphosphazene polymer contains a minimum number of monomer units having the hereinabove-described and hereinbelow-described immunostimulating carboxylic acid containing groups.
  • the polyphosphazene polymer contains at least 10 of the above-described carboxylic acid containing monomeric units.
  • R groups may be one or more of a wide variety of substituent groups.
  • substituent groups there may be mentioned: aliphatic; aryl; aralkyl; alkaryl; carboxylic acid; heteroaromatic; carbohydrates, including glucose; heteroalkyl; halogen; (aliphatic)amino-including alkylamino-; heteroaralkyl; di(aliphatic)amino-including dialkylamino-, arylamino-, diarylamino-, alkylarylamino-; -oxyaryl including but not limited to -oxyphenylCO 2 H, -oxyphenylSO 3 H, -oxyphenylhydroxyl and -oxyphenylPO 3 H; -oxyaliphatic including -oxyalkyl, -oxy(aliphatic)CO 2 H, -oxy(aliphatic)SO 3 H, -oxy(aliphatic)(aliphatic)O 3 H, -oxy(aliphatic)
  • polyphosphazenes that are particularly preferred as immunoadjuvants are: Both of these compounds exhibited significantly better adjuvant activity in comparison to poly[di(carboxylatophenoxy)phosphazene] (PCPP), as is shown in FIG. 1
  • Preferred polyphosphazenes of the present invention have a molecular weight of at least 5,000 g/mol while generally not exceeding 10,000,000 g/mol.
  • Polyphosphazenes of present invention can be homopolymers, having one type of side groups, or mixed substituent copolymers, having two or more types of side groups.
  • mixed substituent copolymers there is at least one type of side group that contains carboxylic acid functionality and one type of side groups that does not contain carboxylic acid functionality.
  • Side groups that do not contain carboxylic acid functionalities can be introduced in a polyphosphazene copolymer to modulate physical or physico-chemical properties of the polymer.
  • Such side groups can be used, for example, to improve water-solubility, to modulate biodegradability, to increase hydrophobicity, or to change chain flexibility of the polymer.
  • non-carboxylic acid functionality containing physical or physico-chemical property modulating side groups there may be mentioned phenoxy, alkoxy, hydroxy, halogen, and methoxyethoxyethoxy.
  • the polyphosphazenes of the present invention are polymers that are preferably biodegradable when administered to either humans or animals. Biodegradability of the polymer prevents eventual deposition and accumulation of polymer molecules at distant sites in the body, such as the spleen.
  • 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.
  • Polyphosphazenes for use as immunoadjuvants can be cross-linked.
  • Ionically-crosslinked polyphosphazenes for example, can be prepared by combining a phosphazene polymer with a multivalent metal cation such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, or other multivalent metal cation known in the art; or with a multivalent organic cation such as spermine, spermidine, poly(ethyleneimine), poly(vinylamine), or other multivalent organic cation known in the art.
  • a multivalent metal cation such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, or other multivalent metal cation known in the art
  • a multivalent organic cation such as spermine, spermidine, poly(ethyleneimine), poly(vinylamine), or other multivalent organic cation known in the art.
  • Polymers of the present invention can be used in combination with an antigen; the antigen may be any one of a wide variety of antigens against which an immune response is desired.
  • the polymer upon mixing with the antigen can form non-covalent complexes.
  • Such complexes can be water-soluble or in the form of microgels or other microparticulates, including microspheres.
  • Complexes can contain single polyphosphazene molecules and multiple antigen molecules or single antigen molecules and multiple polyphosphazene molecules. Alternatively, complexes can contain multiple molecules of both polyphosphazenes and antigens.
  • Such complexes have the ability to present the antigen to the corresponding immune competent cell or to release, antigen over an extended period of time.
  • the immunogenic response may be humoral, muscosal, and/or cell mediated.
  • the polymer in combination with an antigen is used in an amount effective to provide the desired immune response.
  • 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 parenteral, oral, or transmembrane or transmucosal administration.
  • the vaccine is administered parenterally (intravenously, intramuscularly, subcutaneously).
  • routes of delivery to mucosal surfaces are intranasal (or generally, the nasal associated lymphoid tissue), oral, respiratory, vaginal and rectal.
  • An immunogenic 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 polymeric adjuvant of the invention is a polyphosphazene that is preferably soluble in water at physiological pH, i.e., preferably has a solubility of at least 0.0001% (w/w).
  • the antigen with which the adjuvants of the invention are used can be derived from a cell, a bacteria or virus particle or a portion thereof.
  • the antigen can be a protein, peptide, polysaccharide, glycoprotein, glycolipid, or combination thereof which elicits an immunogenic response in a human; or in an animal, for example, a mammal, bird, or fish.
  • the immunogenic response can be humoral, mucosal, or cell mediated.
  • such material against which an immune response is directed is poorly antigenic, such material may be conjugated to a carrier such as albumin, or to a hapten, using standard covalent binding techniques. Such conjugation can be effected with commercially available reagent kits that are well known in the art.
  • the polymer is used to deliver nucleic acid, which encodes antigen to a mucosal surface where the nucleic acid is expressed.
  • antigens that may be contained in the polyphosphazene complexes, hydrogels, micropraticles, or microspheres there may be mentioned viral proteins, such as influenza proteins, human immunodeficiency virus (HIV) proteins, Herpes virus proteins, and hepatitus A and B proteins. Additional examples include antigens derived from rotavirus, measles, mumps, rubella, and polio; or from bacterial proteins and lipopolysaccharides such as Gram-negative bacterial cell walls. Further antigens may also be those derived from organisms such as Haemophilus influenza, Clostridium tetani, Corynebacterium diphtheria, and Nesisseria gonhorrhoae.
  • An immunogenic composition, or vaccine is prepared by combining the polymer adjuvant with an antigen. Approximately 0.0001-5 parts of antigen is added to one part of polymer, preferably by stirring a solution of polymer and antigen until a solution or suspension is obtained, preferably for 10 minutes or more at about 25° 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; or 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 methods of U.S. Pat. No. 5,149,543 issued to Cohen et al.; or U.S. Pat. No. 5,807,757 issued to Andrianov et al., the teachings of which are hereby incorporated by these references thereto, or by spray drying a solution of polymer and antigen.
  • 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, as is described in U.S. Pat. No. 5,500,161 issued to Andrianov et al.
  • 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 preferred polymers of the present invention are soluble in physiologically buffered saline (PBS, pH 7.4).
  • the immunogenic vaccine composition can contain other physiologically acceptable ingredients such as water; saline; and or surfactants.
  • the polymer can be combined with other adjuvants.
  • the polymer of present invention can be combined with an amphiphilic compound.
  • the amphiphilic compound may or may not function as an adjuvant in the absence of the water soluble polymer.
  • amphiphilic compound as known in the art means that the compound includes both a hydrophobic portion and a hydrophilic portion.
  • amphiphilic compounds suitable for producing the adjuvant of the present invention there may be mentioned dimyristoyl phosphatidylcholine, dimethyldioctadecylammonium bromide, N,N-dioctadecyl-N′,N′-bis(2-hydroxyethyl)propanediamine, N-(2-Deoxy-2-L-leucylamino- ⁇ -D-glucopyranosyl)-N-octadecydodecanoylamide hydroacetate, dimyristoyl phosphatidylglycerol, N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glycerol dipalmitate, sorbitan trioleate, deoxy
  • the adjuvant can be prepared by mixing the water soluble polymer and the amphiphilic compound at room temperature. Antigen is then added to the adjuvant combination. Alternatively, the antigen is first mixed with one of the adjuvant components, and then the second adjuvant component is added.
  • the immunogenic composition of the present invention is prepared by either mixing or conjugating the water soluble polymer with the antigen prior to administration. The water soluble polymer/antigen combination is then combined with the amphiphilic compound to form the immunogenic composition.
  • polyphosphazenes may be used to comprise vaccines, which vaccines may be directed to the treatment, or prevention, of disease.
  • 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.
  • polymer antigen mixture is administered simultaneously
  • polymer and antigen are administered separately to the same or nearby site.
  • polyphosphazene adjuvants and methods of synthesis will be further understood by reference to the following non-limiting examples.
  • the reaction mixture was transferred into a large separating funnel.
  • the polymer-containing aqueous layer was removed and collected.
  • 0.5 L of deionized water was added to the organic layer, shaken, and the aqueous phase was separated and collected.
  • the procedure was repeated two times.
  • Aqueous solution was then acidified with 4 N hydrochloric acid to pH 2.
  • the precipitate was collected and then dissolved in 0.4 L of 0.5 N potassium hydroxide. Upon dissolution, the pH was adjusted by the addition of hydrochloric acid to pH 9-10.5.
  • the polymer was then purified by size-exclusion chromatography using a Biocad workstation. Polymer fractions were collected and then precipitated by the addition of 0.5 N hydrochloric acid until pH of 2 was reached.
  • the precipitate was filtered, washed with water until the rinse became neutral, and dried in vacuum.
  • the yield was 3 g.
  • Polymers were synthesized as described in Example 1. Polymer structures, reagents, reagent quantities, and reaction conditions are shown in Table 1. TABLE 1 Polymer structures, reagents, and reaction conditions for examples 2-12. Molecular Reagent(s) NaH,* PDCP, Weight Ex. Amount, G g ⁇ 10 ⁇ 3 , No Polymer Structure Name g (mmol) (mmol) (meq) T ° C.
  • Polymers synthesized as described in examples 1 (Polymer 1) and example 2 (Polymer 2) were evaluated in vivo for their ability to enhance the immune response (adjuvant activity) to Hepatitis B surface antigen—HBsAg (Biodesign International).
  • BALB/c mice were used (5 mice per group). 1 ⁇ g of HBsAg mixed with 50 ⁇ g of the polymer in solution (final volume 100 ⁇ l) was injected in each mouse.
  • Formulations containing antigen alone, antigen formulated with 50 ⁇ g of PCPP, and antigen formulated with Alum were used as controls. Mice were immunized with a single intramuscular injection. Blood samples were collected 16 weeks post-immunization and serum stored until analysis.
  • Antigen-specific antibodies (IgG) in mouse serum were determined by ELISA in 96-well Immunolon II plates coated with HBsAg in sodium carbonate buffer, pH 9.6. The plates were washed six times with PBS containing 0.05% Tween 20 (PBST). Two-fold serial dilutions of sera in PBST containing 0.5% gelatin were added to the wells and the plate was incubated 2 hours at ambient temperature. Unbound serum was removed by washing the plates six times with PBST. Biotinylated Goat Anti-Mouse IgG (Caltag Laboratories) was added and the plates were incubated for 1 hour at ambient temperature.
  • PBST PBS containing 0.05% Tween 20
  • the plates were washed six times with PBST and alkaline phosphatase conjugated Streptavidin (BioCan Scientific) was added and plates were incubated for 1 hour at ambient temperature. Unbound conjugate was removed by washing eight times with deionized water and serum antibodies were detected by adding 1 mg/mL of p-nitrophenyl phosphate di(Tris) salt in 1% Diethanolamine-0.5 mM magnesium chloride buffer, pH 9.8. The reaction was allowed to run for 15 minutes and the absorbance was measured at 405 nm using BenchmarkTM Microplate Reader (Bio-Rad Laboratories, Hercules, Calif.).
  • the endpoint titers were the reciprocal of the highest sample dilution producing a signal identical to that of an antibody-negative sample at the same dilution plus three times standard deviation.
  • the average antibody titers for a group of mice were expressed as geometric mean titers (GMT).
  • Polymers 1 and 2 were evaluated in mice similarly as described in Example 13, except that 5 ⁇ g X-31 influenza (Charles River Laboratories) was used instead of HBsAg. The results are presented in FIG. 2 . Both Polymer 1 and Polymer 2 showed significantly higher adjuvant activity than PCPP and alum.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Immunology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Mycology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Communicable Diseases (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Polymers & Plastics (AREA)
  • Oncology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Polymers With Sulfur, Phosphorus Or Metals In The Main Chain (AREA)
  • Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
US11/355,737 2005-02-18 2006-02-16 Immunostimulating polyphosphazene compounds Abandoned US20060193820A1 (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
US11/355,737 US20060193820A1 (en) 2005-02-18 2006-02-16 Immunostimulating polyphosphazene compounds
US12/806,941 US20100322891A1 (en) 2005-02-18 2010-08-24 Immuniostimulating polyphosphazene compounds
US13/743,824 US20130136758A1 (en) 2005-02-18 2013-01-17 Immunostimulating Polyphosphazene Compounds
US14/323,083 US20140314707A1 (en) 2005-02-18 2014-07-03 Immunostimulating Polyphosphazene Compounds
US14/710,798 US20150238600A1 (en) 2005-02-18 2015-05-13 Immunostimulating Polyphosphazene Compounds
US14/977,848 US9687546B2 (en) 2005-02-18 2015-12-22 Immunostimulating polyphosphazene compounds
US15/610,747 US20170258900A1 (en) 2005-02-18 2017-06-01 Immunostimulating Polyphosphazene Compounds

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US65456705P 2005-02-18 2005-02-18
US11/355,737 US20060193820A1 (en) 2005-02-18 2006-02-16 Immunostimulating polyphosphazene compounds

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US12/806,941 Continuation US20100322891A1 (en) 2005-02-18 2010-08-24 Immuniostimulating polyphosphazene compounds

Publications (1)

Publication Number Publication Date
US20060193820A1 true US20060193820A1 (en) 2006-08-31

Family

ID=36917109

Family Applications (6)

Application Number Title Priority Date Filing Date
US11/355,737 Abandoned US20060193820A1 (en) 2005-02-18 2006-02-16 Immunostimulating polyphosphazene compounds
US12/806,941 Abandoned US20100322891A1 (en) 2005-02-18 2010-08-24 Immuniostimulating polyphosphazene compounds
US13/743,824 Abandoned US20130136758A1 (en) 2005-02-18 2013-01-17 Immunostimulating Polyphosphazene Compounds
US14/323,083 Abandoned US20140314707A1 (en) 2005-02-18 2014-07-03 Immunostimulating Polyphosphazene Compounds
US14/710,798 Abandoned US20150238600A1 (en) 2005-02-18 2015-05-13 Immunostimulating Polyphosphazene Compounds
US14/977,848 Active US9687546B2 (en) 2005-02-18 2015-12-22 Immunostimulating polyphosphazene compounds

Family Applications After (5)

Application Number Title Priority Date Filing Date
US12/806,941 Abandoned US20100322891A1 (en) 2005-02-18 2010-08-24 Immuniostimulating polyphosphazene compounds
US13/743,824 Abandoned US20130136758A1 (en) 2005-02-18 2013-01-17 Immunostimulating Polyphosphazene Compounds
US14/323,083 Abandoned US20140314707A1 (en) 2005-02-18 2014-07-03 Immunostimulating Polyphosphazene Compounds
US14/710,798 Abandoned US20150238600A1 (en) 2005-02-18 2015-05-13 Immunostimulating Polyphosphazene Compounds
US14/977,848 Active US9687546B2 (en) 2005-02-18 2015-12-22 Immunostimulating polyphosphazene compounds

Country Status (5)

Country Link
US (6) US20060193820A1 (de)
EP (1) EP1858530B1 (de)
JP (1) JP5112081B2 (de)
CA (1) CA2642734A1 (de)
WO (1) WO2006089161A2 (de)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080166390A1 (en) * 2006-04-11 2008-07-10 Andrianov Alexander K Biodegradable polyphosphazenes containing pyrrolidone side groups
WO2009009004A1 (en) * 2007-07-09 2009-01-15 Apogee Technology, Inc. Coating formulations including polyphosphazene polyelectrolytes and biologically active agents and asperities coated with such formulations
EP2173431A1 (de) * 2007-07-09 2010-04-14 Apogee Technology, Inc. Immunstimulierende polyphosphazen-verbindungen zur intradermalen immunisierung
US20100256018A1 (en) * 2009-04-03 2010-10-07 Halliburton Energy Services, Inc. Methods of Using Fluid Loss Additives Comprising Micro Gels
US11472927B2 (en) 2015-11-18 2022-10-18 University Of Maryland, College Park Polyphosphazenes, methods of making, and uses thereof
WO2023158805A1 (en) * 2022-02-18 2023-08-24 Georgia Tech Research Corporation Lithiated polyphosphazene solid polymer electrolyte compositions and methods of manufacture thereof

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060193820A1 (en) * 2005-02-18 2006-08-31 Andrianov Alexander K Immunostimulating polyphosphazene compounds
ATE533517T1 (de) * 2005-04-15 2011-12-15 Parallel Solutions Inc Biologisch abbaubare polyphosphazene mit pyrrolidonseitengruppen
KR100784485B1 (ko) 2006-01-18 2007-12-11 한국과학기술연구원 생분해성 온도 감응성 폴리포스파젠계 하이드로젤, 그의제조방법 및 그의 용도
US11939429B1 (en) * 2018-06-29 2024-03-26 Hrl Laboratories, Llc Infrared-transparent polymers and methods of making and using the same

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6015563A (en) * 1995-06-07 2000-01-18 Avant Immunotherapeutics, Inc. Polyphosphazene polyelectrolyte immunoadjuvants

Family Cites Families (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4258173A (en) 1978-09-08 1981-03-24 The Firestone Tire & Rubber Company Polyphosphazene polymers containing monoetheroxy and polyetheroxy substituents
JPS6015670B2 (ja) * 1982-11-15 1985-04-20 森六商事株式会社 植物培地調整方法
US4640849A (en) 1986-01-31 1987-02-03 Loctite (Ireland) Limited Meta-bridged styryloxy resins
JPH0816159B2 (ja) * 1986-12-26 1996-02-21 大塚化学株式会社 スルホン基を有するオリゴエチレンオキシポリホスフアゼン及びその製法
JPS63186737A (ja) * 1987-01-29 1988-08-02 Teijin Ltd 陰イオン固定型の高分子固体電解質
JP3181902B2 (ja) * 1989-01-20 2001-07-03 大塚化学株式会社 ポリホスフアゼン系電解質及びその用途
JPH03140330A (ja) * 1989-10-27 1991-06-14 Teijin Ltd 生物学的活性物質固定体及びその製造方法並びにその製造用担体及びその使用方法
US5149543A (en) 1990-10-05 1992-09-22 Massachusetts Institute Of Technology Ionically cross-linked polymeric microcapsules
US5562909A (en) * 1993-07-12 1996-10-08 Massachusetts Institute Of Technology Phosphazene polyelectrolytes as immunoadjuvants
US5500161A (en) 1993-09-21 1996-03-19 Massachusetts Institute Of Technology And Virus Research Institute Method for making hydrophobic polymeric microparticles
US5807757A (en) * 1996-07-02 1998-09-15 Virus Research Institute, Inc. Preparation of ionically cross-linked polyphosphazene microspheresy by coacervation
US20040265326A1 (en) * 1996-07-12 2004-12-30 Cates George A. Subunit respiratory syncytial virus vaccine preparation
US5707597A (en) * 1996-11-13 1998-01-13 Virus Research Institute, Inc. Polyhalophosphazene solutions stable against gelation
US5814704A (en) * 1997-03-04 1998-09-29 Virus Research Institute, Inc. Recovery of polyphosphazene polyacids or acids salts thereof
US5842471A (en) * 1997-05-14 1998-12-01 Virus Research Institute, Inc. Purification of polyphosphazene polyacids
US5869016A (en) * 1997-06-18 1999-02-09 Virus Research Institute, Inc. Production of polyorganophosphazenes having desired molecular weights
US5958414A (en) 1997-09-03 1999-09-28 Heska Corporation Composition to protect a mammal against Bartonella henselae infection
ES2272069T3 (es) * 1998-05-22 2007-04-16 Ottawa Health Research Institute Metodos y productos para inducir inmunidad en mucosas.
US6261573B1 (en) * 1998-10-30 2001-07-17 Avant Immunotherapeutics, Inc. Immunoadjuvants
US7169499B2 (en) * 2002-06-03 2007-01-30 Parallel Solutions, Inc. Sulfonated polyphosphazenes, uses thereof, and methods for preparing same
KR100517643B1 (ko) * 2003-07-25 2005-09-28 한국과학기술연구원 온도 감응성 폴리포스파젠계 고분자, 이의 제조방법 및이를 이용한 주입형 온도 감응성 폴리포스파젠 하이드로젤
US20060193820A1 (en) * 2005-02-18 2006-08-31 Andrianov Alexander K Immunostimulating polyphosphazene compounds
ATE533517T1 (de) * 2005-04-15 2011-12-15 Parallel Solutions Inc Biologisch abbaubare polyphosphazene mit pyrrolidonseitengruppen

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6015563A (en) * 1995-06-07 2000-01-18 Avant Immunotherapeutics, Inc. Polyphosphazene polyelectrolyte immunoadjuvants

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080166390A1 (en) * 2006-04-11 2008-07-10 Andrianov Alexander K Biodegradable polyphosphazenes containing pyrrolidone side groups
WO2009009004A1 (en) * 2007-07-09 2009-01-15 Apogee Technology, Inc. Coating formulations including polyphosphazene polyelectrolytes and biologically active agents and asperities coated with such formulations
EP2167131A1 (de) * 2007-07-09 2010-03-31 Apogee Technology, Inc. Beschichtungsformulierungen mit polyphosphazen-polyelektrolyten und biologischen wirkstoffen und mit solchen formulierungen beschichtete unebenheiten
EP2173431A1 (de) * 2007-07-09 2010-04-14 Apogee Technology, Inc. Immunstimulierende polyphosphazen-verbindungen zur intradermalen immunisierung
EP2167131A4 (de) * 2007-07-09 2013-06-12 Apogee Technology Inc Beschichtungsformulierungen mit polyphosphazen-polyelektrolyten und biologischen wirkstoffen und mit solchen formulierungen beschichtete unebenheiten
EP2173431A4 (de) * 2007-07-09 2013-06-12 Apogee Technology Inc Immunstimulierende polyphosphazen-verbindungen zur intradermalen immunisierung
US20100256018A1 (en) * 2009-04-03 2010-10-07 Halliburton Energy Services, Inc. Methods of Using Fluid Loss Additives Comprising Micro Gels
US20110168393A1 (en) * 2009-04-03 2011-07-14 Halliburton Energy Services, Inc. Methods of Using Fluid Loss Additives Comprising Micro Gels
US8685900B2 (en) * 2009-04-03 2014-04-01 Halliburton Energy Services, Inc. Methods of using fluid loss additives comprising micro gels
US8697609B2 (en) 2009-04-03 2014-04-15 Halliburton Energy Services, Inc. Methods of using fluid loss additives comprising micro gels
US11472927B2 (en) 2015-11-18 2022-10-18 University Of Maryland, College Park Polyphosphazenes, methods of making, and uses thereof
WO2023158805A1 (en) * 2022-02-18 2023-08-24 Georgia Tech Research Corporation Lithiated polyphosphazene solid polymer electrolyte compositions and methods of manufacture thereof

Also Published As

Publication number Publication date
EP1858530A4 (de) 2008-07-02
US20160106833A1 (en) 2016-04-21
US9687546B2 (en) 2017-06-27
WO2006089161A3 (en) 2007-05-31
US20130136758A1 (en) 2013-05-30
WO2006089161A2 (en) 2006-08-24
JP2008530347A (ja) 2008-08-07
US20140314707A1 (en) 2014-10-23
US20150238600A1 (en) 2015-08-27
EP1858530B1 (de) 2018-04-11
JP5112081B2 (ja) 2013-01-09
CA2642734A1 (en) 2006-08-24
US20100322891A1 (en) 2010-12-23
EP1858530A2 (de) 2007-11-28

Similar Documents

Publication Publication Date Title
US9687546B2 (en) Immunostimulating polyphosphazene compounds
AU691824B2 (en) Phosphazene polyelectrolytes as immunoadjuvants
US5855895A (en) Polyphosphazene polyelectrolyte immunoadjuvants
CA1323566C (en) Biodegradable polymeric drug delivery system with adjuvant activity
Dmour et al. Recent advances on chitosan as an adjuvant for vaccine delivery
JP3242118B2 (ja) 生体内分解性標的指向性マイクロパーティクル送達システム
JP6228967B2 (ja) 抗原性組成物および方法
JP2009524584A (ja) ポリマー−生物製剤送達組成物を構築するための方法
CA2676601A1 (en) Vaccine delivery compositions and methods of use
US20170258900A1 (en) Immunostimulating Polyphosphazene Compounds
JP2023545466A (ja) 免疫応答を調節することができる、金属イオンと会合したｓｔｉｎｇアゴニストの結晶多形体
WO2001015727A2 (en) Compositions of immunostimulatory polymers and saponin adjuvants and uses thereof
AU738698B2 (en) Polyphosphazene polyelectrolyte immunoadjuvants
WO2023032891A1 (ja) 免疫原性増強用組成物
Rosales-Mendoza et al. Silica-based mucosal nanovaccines
Adams Novel Cationic Pentablock Copolymers as a Nanovaccine Delivery Platform

Legal Events

Date Code Title Description
AS Assignment

Owner name: PARALLEL SOLUTIONS, INC., MASSACHUSETTS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:ANDRIANOV, ALEXANDER K.;MARIN, ALEXANDER;REEL/FRAME:017567/0688;SIGNING DATES FROM 20060329 TO 20060428

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION