U.S. patent application number 11/355737 was filed with the patent office on 2006-08-31 for immunostimulating polyphosphazene compounds.
Invention is credited to Alexander K. Andrianov, Alexander Marin.
Application Number | 20060193820 11/355737 |
Document ID | / |
Family ID | 36917109 |
Filed Date | 2006-08-31 |
United States Patent
Application |
20060193820 |
Kind Code |
A1 |
Andrianov; Alexander K. ; et
al. |
August 31, 2006 |
Immunostimulating polyphosphazene compounds
Abstract
Polyphosphazene polymers having immunomodulating activity, and
the biomedical use of such polyphosphazene polymers, in conjunction
with an antigen or an immunogen are disclosed.
Inventors: |
Andrianov; Alexander K.;
(Belmont, MA) ; Marin; Alexander; (Newton,
MA) |
Correspondence
Address: |
RAYMOND E. STAUFFER, ESQ.;c/o Carella, Byrne, Bain, Gilfillan,
Cecchi, Stewart & Olstein
5 Becker Farm Road
Roseland
NJ
07068
US
|
Family ID: |
36917109 |
Appl. No.: |
11/355737 |
Filed: |
February 16, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60654567 |
Feb 18, 2005 |
|
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Current U.S.
Class: |
424/78.17 ;
525/54.1 |
Current CPC
Class: |
A61P 37/00 20180101;
C12N 7/00 20130101; A61P 37/04 20180101; A61K 39/292 20130101; A61K
31/74 20130101; A61K 39/35 20130101; A61K 2039/55511 20130101; A61K
2039/55555 20130101; A61P 31/12 20180101; C12N 2730/10134 20130101;
A61K 39/00 20130101; A61P 31/04 20180101; A61K 31/80 20130101; Y02A
50/30 20180101; Y02A 50/464 20180101; A61K 39/39 20130101; C08G
79/025 20130101; A61K 31/80 20130101; A61K 2300/00 20130101; A61K
39/00 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/078.17 ;
525/054.1 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C08G 63/91 20060101 C08G063/91 |
Claims
1. A polyphosphazene polymer comprising the following units:
##STR20## wherein at least one R has the formula: ##STR21## wherein
at least one of C, C', or C'' includes --COOH; each of d, f, f',
f', is from 0 to 3; each of e, e', g, g', g'' is from 0 to 1 and if
f=0, such that C.dbd.--COOH then at least one of C', C'', Y, or Y'
is other than hydrogen; or X is other than --O--; --NH--; or --S--;
and the sum of d+e+e'+f'+f'+g'+g'' equals from 1 to 8.
2. The polyphosphazene polymer of claim 1 wherein:
X.dbd.--OCH.sub.2.sub.d; Y.dbd.O--CH.sub.2.sub.eH;
Y'.dbd.O--CH.sub.2.sub.e'H; C.dbd.CH.sub.2.sub.fCOOH;
C'.dbd.O--CH.sub.2.sub.g'H; C''.dbd.O--CH.sub.2.sub.g''H; and f is
2; d, e, e', g' and g'' are 0, and the compound is: ##STR22##
3. The polyphosphazene polymer of claim 1 wherein:
X.dbd.--OCH.sub.2.sub.d; Y.dbd.O--CH.sub.2.sub.eH;
Y'.dbd.O--CH.sub.2.sub.e'H; C.dbd.CH.sub.2.sub.fCOOH;
C'.dbd.O--CH.sub.2.sub.g'H; C''.dbd.O--CH.sub.2.sub.g''H; and f is
1; d, e, e', g' and g'' are 0, and the compound is: ##STR23##
4. The polyphosphazene polymer of claim 1 wherein:
X.dbd.--OCH.sub.2.sub.d; Y.dbd.O--CH.sub.2.sub.eH;
Y'.dbd.O--CH.sub.2.sub.e'H; C.dbd.CH.sub.2.sub.fCOOH;
C'.dbd.O--CH.sub.2.sub.g'H; C''.dbd.O--CH.sub.2.sub.g''H; and d is
1; e, e', f, g', g'' are 0, and the compound is: ##STR24##
5. The polyphosphazene polymer of claim 1 wherein:
X.dbd.--OCH.sub.2.sub.d; Y.dbd.O--CH.sub.2.sub.eH;
Y'.dbd.O--CH.sub.2.sub.e'H; C.dbd.CH.sub.2.sub.fCOOH;
C'.dbd.O--CH.sub.2.sub.g'H; C''.dbd.O--CH.sub.2.sub.g''H; and e and
f are 1; d, e', g', and g'' are 0, and the compound is:
##STR25##
6. The polyphosphazene polymer of claim 1 wherein:
X.dbd.--OCH.sub.2.sub.d; Y.dbd.O--CH.sub.2.sub.eH;
Y'.dbd.0--CH.sub.2.sub.e'H; C.dbd.CH.sub.2.sub.fCOOH;
C'.dbd.O--CH.sub.2.sub.g'H; C''.dbd.O--CH.sub.2.sub.g''H; and e is
1; d, e', f, g', and g'' are 0, and the compound is: ##STR26##
7. The polyphosphazene polymer of claim 1 wherein: ##STR27## and d,
e, e', g', and g'' are 0, and the compound is: ##STR28##
8. The polyphosphazene polymer of claim 1 wherein:
X.dbd.--OCH.sub.2.sub.d; Y.dbd.O--CH.sub.2.sub.eH;
Y'.dbd.(O--CH.sub.2.sub.e'H; C.dbd.O--CH.sub.2.sub.gH;
C'.dbd.CH.sub.2.sub.f'COOH; C''.dbd.CH.sub.2.sub.f''COOH; and d, e,
e', g, f', and f'' are O, and the compound is: ##STR29##
9. The polyphosphazene polymer of claim 1 wherein:
X.dbd.--OCH.sub.2.sub.d; Y.dbd.O--CH.sub.2.sub.eH;
Y'.dbd.O--CH.sub.2.sub.e'H; C.dbd.CH.sub.2.sub.fCOOH;
C'.dbd.O--CH.sub.2.sub.g'H; C''.dbd.O--CH.sub.2.sub.g''H; and f is
2; d, e, e', g' and g'' are 0, and at least a portion of the R side
groups are phenoxy groups and the compound is: ##STR30## wherein
and a+b=2.
10. The polyphosphazene polymer of claim 1 wherein:
X.dbd.--OCH.sub.2.sub.d; Y.dbd.O--CH.sub.2.sub.eH;
Y'.dbd.O--CH.sub.2.sub.e'H; C.dbd.CH.sub.2.sub.fCOOH;
C'O--CH.sub.2.sub.g'H; C'.dbd.O--CH.sub.2.sub.g''H; and f is 2; d,
e, e', g' and g'' are 0, and at least a portion of the R side
groups are decoxy groups and the compound is: ##STR31## and wherein
a+b=2.
11. A composition for inducing an immunogenic response in a human
or in an animal comprising at least one polyphosphazene polymer
according to claim 1 and an antigen.
12. A composition for inducing an immunogenic response in a human
or in an animal according to claim 11 wherein the antigen is
selected from the group consisting of protein, peptide,
polysaccharide, glycoprotein, glycolipid, nucleic acid, and
combinations thereof, said protein, peptide, polysaccharide,
glycoprotein, glycolipid, nucleic acid, and combinations thereof,
being derived from the group consisting of cell, bacteria, virus
particle, and portions thereof.
13. A composition for inducing an immunogenic response in a human
or in an animal according to claim 11 wherein the polyphosphazene
polymer and the antigen form a non-covalent water-soluble
complex.
14. A composition for inducing an immunogenic response in a human
or in an animal according to claim 13 wherein the polyphosphazene
polymer and the antigen form a non-covalent water-soluble complex
capable of multimeric antigen presentation.
15. A composition for inducing an immunogenic response in a human
or in an animal according to claim 11 wherein the antigen is
covalently conjugated to the polyphosphazene polymer.
16. A composition for inducing an immunogenic response in a human
or in an animal according to claim 11 wherein the polyphosphazene
polymer is ionically cross-linked with multivalent ions to form
microparticulate or microgel.
17. A polyphosphazene polymer comprising the following units:
##STR32## wherein at least one R has the following formula:
##STR33## d is from 0 to 15.
18. A polyphosphazene polymer according to claim 17 wherein:
X.dbd.--OCH.sub.2.sub.d; Z=-COOH; and d is 5, and the compound is:
##STR34##
19. A polyphosphazene polymer according to claim 17 wherein:
##STR35## and d is 1, and the compound is: ##STR36##
20. A polyphosphazene polymer according to claim 17 wherein:
##STR37## and d is 0, and the compound is: ##STR38##
21. A polyphosphazene polymer according to claim 17 wherein:
##STR39## and d is 0, and the compound is: ##STR40##
22. A composition for inducing an immunogenic response in a human
or in an animal comprising at least one polyphosphazene polymer
according to claim 17 and an antigen.
23. A composition for inducing an immunogenic response in a-human
or in an animal according to claim 22 wherein the antigen is
selected from the group consisting of protein, peptide,
polysaccharide, glycoprotein, glycolipid, nucleic acid, and
combinations thereof said protein, peptide, polysaccharide,
glycoprotein, glycolipid, nucleic acid, and combinations thereof,
being derived from the group consisting of cell, bacteria, virus
particle, and portions thereof.
24. A composition for inducing an immunogenic response in a human
or in an animal according to claim 22 wherein the polyphosphazene
polymer and the antigen form a non-covalent water-soluble
complex.
25. A composition for inducing an immunogenic response in a human
or in an animal according to claim 24 wherein the polyphosphazene
polymer and the antigen form a non-covalent water-soluble complex
capable of multimeric antigen presentation.
26. A composition for inducing an immunogenic response in a human
or in an animal according to claim 22 wherein the antigen is
covalently conjugated to the polyphosphazene polymer.
27. A composition for inducing an immunogenic response in a human
or in an animal according to claim 22 wherein the polyphosphazene
polymer is ionically cross-inked with multivalent ions to form
microparticulate or microgel.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority of U.S. Provisional
Application Ser. No. 60/654,567 filed on Feb. 18, 2005, the
disclosures of which are hereby incorporated by reference in their
entireties.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not Applicable
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT
DISC
[0003] Not Applicable
BACKGROUND OF THE INVENTION
[0004] Not Applicable
BRIEF SUMMARY OF THE INVENTION
[0005] Not Applicable
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0006] 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 .mu.g/mouse;
polymers: 50 .mu.g/mouse; single dose intramuscular injection; 16
week data).
[0007] 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 .mu.g/mouse;
polymers: 50 .mu.g/mouse; single dose intramuscular injection; 16
week data).
DETAILED DESCRIPTION OF THE INVENTION
[0008] 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.
[0009] A wide variety of 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.
[0010] 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. 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. 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 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.
[0011] Some synthetic polymers have been found to provide
immunostimulation when combined with an antigen. For example, the
adjuvant activities 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, have been studied (V. A.
Kabanov, From Synthetic Polyelectrolytes to Polymer--Subunit
Vaccines, Pure Appl. Chem., 2004, 76, 9, 1659-1677). However, these
polymers require covalent attachment of antigen to the polymer in
order to elicit effective immune responses, which can present
manufacturing and regulatory challenges. In addition, 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.
[0012] Poly[di(carboxylatophenoxy)phosphazene] (PCPP),
polyphosphazene polymer is described as an immunoadjuvant that does
not require covalent attachment of antigen to the polymer in order
to elicit effective immune responses (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)). However, more potent immunoadjuvants than PCPP
are needed.
[0013] 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.
[0014] The present invention provides polyphosphazenes that may be
used as adjuvants and are superior in their immunoadjuvant activity
to PCPP.
[0015] 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").
[0016] The repeat unit of a polyphosphazene has the following
formula: ##STR1##
[0017] wherein each "A" may be the same, or different, and wherein
the unit is repeated "n" times.
[0018] 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.
[0019] 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.
[0020] 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. For example, 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. After
the completion of the reaction, the 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.
[0021] Thus, in one aspect, the present invention provides a
polyphosphazene polymer that contains repeating units of the
following formula: ##STR2## 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." Wherein "W" is: ##STR3## wherein at least one of C,
C', or C'' includes --COOH; each of d, f, f', f', are from 0 to 3;
and [0022] each of e, e', g, g', g'' are from 0 to 1; [0023] and if
[0024] f=0, such that [0025] C.dbd.--COOH [0026] then at least one
of C', C'', Y, or Y' is other than hydrogen; or X is other than
--O--, --NH--, or --S--; [0027] and the sum of d+e+e'+f'+f'+g'+g''
equals from 1 to 8. The carboxylic acid groups can be either in
ionized or non-ionized form.
[0028] It is an aspect of the instant invention that 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. While it is within the ordinary skill that prevails in the
art when considered in conjunction with the instant disclosure to
determine how many of the hereinabove-described and
hereinbelow-described monomeric units will result in an
immunostimulating polyphosphazene polymer, it is an aspect of the
instant invention that the polyphosphazene polymer contains at
least 10 of the above-described carboxylic acid containing
monomeric units.
[0029] The remaining R groups (those other than the
immunostimulating carboxylic acid containing groups as described
above) may be one or more of a wide variety of substituent groups.
As representative, non-limiting examples of such 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.sub.2H, -oxyphenylSO.sub.3H, -oxyphenylhydroxyl and
-oxyphenylPO.sub.3H; -oxyaliphatic including -oxyalkyl,
-oxy(aliphatic)CO.sub.2H, -oxy(aliphatic)SO.sub.3H,
-oxy(aliphatic)PO.sub.3H, and -oxy(aliphatic)hydroxyl, including
oxy(alkyl)hydroxyl; -oxyalkaryl, -oxyaralkyl; -thioaryl;
thioaliphatic including -thioalkyl; -thioalkaryl; thioaralkyl;
--NHC(O)O-- (aryl or aliphatic);
--O--[(CH.sub.2)xO]y-CH.sub.2)--O--[(CH.sub.2)xO]y(CH.sub.2)xNH(CH.sub.2)-
xSO.sub.3H; and --O--[(CH.sub.2)xO]y-(aryl or aliphatic); wherein x
is 1-8 and y is an integer of 1 to 20. The groups can be bonded to
the phosphorous atom through, for example, an oxygen, sulfur,
nitrogen, or carbon atom.
[0030] In yet another aspect, the present invention provides a
polyphosphazene polymer that contains repeating units of the
following formula: ##STR4## 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: ##STR5## and wherein d is from 0 to 15.
[0031] It is an aspect of the instant invention that 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. While it is within the ordinary skill that prevails in the
art when considered in conjunction with the instant disclosure to
determine how many of the hereinabove-described and
hereinbelow-described monomeric units will result in an
immunostimulating polyphosphazene polymer, it is an aspect of the
instant invention that the polyphosphazene polymer contains at
least 10 of the above-described carboxylic acid containing
monomeric units.
[0032] The remaining R groups (those other than the
immunostimulating carboxylic acid containing groups as described
above) may be one or more of a wide variety of substituent groups.
As representative, non-limiting examples of such 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.sub.2H, -oxyphenylSO.sub.3H, -oxyphenylhydroxyl and
-oxyphenylPO.sub.3H; -oxyaliphatic including -oxyalkyl,
-oxy(aliphatic)CO.sub.2H, -oxy(aliphatic)SO.sub.3H,
-oxy(aliphatic)PO.sub.3H, and -oxy(aliphatic)hydroxyl, including
oxy(alkyl)hydroxyl; -oxyalkaryl, -oxyaralkyl; -thioaryl;
thioaliphatic including -thioalkyl; -thioalkaryl; thioaralkyl;
--NHC(O)O-- (aryl or aliphatic);
--O--[(CH.sub.2)xO]y--CH.sub.2)--O--[(CH.sub.2)xO]y(CH.sub.2)xNH(CH.sub.2-
)xSO.sub.3H; and --O--[(CH2)xO]y-(aryl or aliphatic); wherein x is
1-8 and y is an integer of 1 to 20. The groups can be bonded to the
phosphorous atom through, for example, an oxygen, sulfur, nitrogen,
or carbon atom.
[0033] Two examples of polyphosphazenes that are particularly
preferred as immunoadjuvants are: ##STR6## Both of these compounds
exhibited significantly better adjuvant activity in comparison to
poly[di(carboxylatophenoxy)phosphazene] (PCPP), as is shown in FIG.
1
[0034] 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.
[0035] 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. In 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. As non-limiting examples of such non-carboxylic acid
functionality containing physical or physico-chemical property
modulating side groups there may be mentioned phenoxy, alkoxy,
hydroxy, halogen, and methoxyethoxyethoxy. Examples of copolymers
that are particularly preferred as immunoadjuvants are polymer that
contain monomeric units such as the following: ##STR7## Wherein a
is between 0.1 and 1.9, b is between 1.9 and 0.1, and a+b =2.
[0036] 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. 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.
[0037] 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.
[0038] 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.
[0039] 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. Preferably, the vaccine is
administered parenterally (intravenously, intramuscularly,
subcutaneously). Non-limiting examples of routes of delivery to
mucosal surfaces are intranasal (or generally, the nasal associated
lymphoid tissue), oral, respiratory, vaginal and rectal.
[0040] 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.
[0041] 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).
[0042] 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. Where the 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.
[0043] In one embodiment, the polymer is used to deliver nucleic
acid, which encodes antigen to a mucosal surface where the nucleic
acid is expressed.
[0044] As non-limiting examples of 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.
[0045] 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.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; 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. 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, 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.
[0046] The preferred polymers of the present invention are soluble
in physiologically buffered saline (PBS, pH 7.4).
[0047] 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; and or surfactants.
The polymer can be combined with other adjuvants.
[0048] 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.
[0049] The term amphiphilic compound as known in the art means that
the compound includes both a hydrophobic portion and a hydrophilic
portion. As non-limiting examples of the 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-.beta.-D-glucopyranosyl)-N-octadecydodecanoyla-
mide hydroacetate, dimyristoyl phosphatidylglycerol,
N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glycerol
dipalmitate, sorbitan trioleate, deoxycholic acid sodium salt,
dicetyl phosphate, mono-palmitoyl-rac-glycerol,
N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-dipalmitoxy
propylamide, octadecyl tyrosine hydrochloride, D-murapalmitine,
3-O-desacyl-4'-monophosphoryl lipid A, mannide oleate,
1a,25-dihydroxyvitamin D.sub.3, phosphatidic acid,
phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine,
phosphatidylinositol, sphingosine (D-4-sphingenine, ceramides,
sphingomyelin, galactosylceramide, GM.sub.2 (ganglioside), salts of
fatty acids including oleic acid, palmitic acid, capric acid,
lauric acid, myristic acid, stearic acid, arachidic acid, behenic
acid, lignoceric acid, cerotic acid, linoleic acid, linolenic acid,
arachidonic acid, and the like.
[0050] In one aspect, 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.
Preferably 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.
[0051] In one aspect of the present invention the polyphosphazenes
may be used to comprise vaccines, which vaccines may be directed to
the treatment, or prevention, of disease.
[0052] 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.
[0053] 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.
[0054] The polyphosphazene adjuvants and methods of synthesis will
be further understood by reference to the following non-limiting
examples.
EXAMPLE 1
[0055] Synthesis of poly{di[4-(2-carboxyethyl)phenoxy]phosphazene}.
In a nitrogen atmosphere, methyl 3-(4-hydroxyphenyl)propionic acid
(25.0 g; 0.139 moles) was dissolved in diethylene glycol dimethyl
ether, diglyme (0.45 L). 1.55 g of 65% sodium hydride (0.042 moles)
was added to the mixture over a ten minute period. To the resulting
mixture polydichlorophosphazene (1.25 g; 0.011 moles) was slowly
added via syringe under nitrogen atmosphere at 110.degree. C. while
stirring. The reaction was stirred for three hours at 110.degree.
C. and then cooled to 85.degree. C. To this mixture 0.1 L of 12.7 N
potassium hydroxide was added. The reaction was stirred for one
hour at 85.degree. C., and then was cooled to ambient
temperature.
[0056] 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.
EXAMPLE 2-12
[0057] Polymers were synthesized as described in Example 1. Polymer
structures, reagents, reagent quantities, and reaction conditions
are shown in Table 1. TABLE-US-00001 TABLE 1 Polymer structures,
reagents, and reaction conditions for examples 2-12. Molecular
Reagent(s) NaH,* PDCP, Weight Ex. Amount, G g .times.10.sup.-3, No
Polymer Structure Name g (mmol) (mmol) (meq) T .degree. C. g/mol 1
##STR8## Methyl 3-(4- hydroxyphenyl) propionate 25 (139) 1.55 (42)
1.25 (21.6) 110 200 2 ##STR9## 4-hydroxybenzoic acid meethyl ester
4.21 (25.36) 0.47 (12.68) 0.150 (2.58) 120 300 3 ##STR10## Methyl
4-hydroxy phenylacetate 1.33 (8.00) 0.15 (4.00) 0.116 (2.00) 120
990 4 ##STR11## Ethyl 4'-hydroxy-4- biphenylcarboxylate 4.41
(18.19) 0.34 (9.09) 0.210 (3.62) 90 340 5 ##STR12## Ethyl
homovanillate 1.68 (7.97) 0.15 (3.98) 0.210 (3.62) 90 340 6
##STR13## Methyl vanillate 3.31 (18.19) 0.34 (9.09) 0.210 (3.62) 90
1100 7 ##STR14## Methyl 4-hydroxy Phenylacetate (90%) 1-Decanol
(10%) 2.72 (16.37) 0.29 (1.82) 0.30 (8.18) 0.03 (0.91) 0.21 (3.62)
90 510 8 ##STR15## Methyl 3-(4- Hydroxyphenyl) Propionate (90%)
Phenol (10%) 1.1 (6.08) 0.064 (0.675) 0.15 (4.05) 0.017 (0.45) 120
600 9 ##STR16## Ethyl lactate 4.30 (36.38) 0.34 (9.09) 0.210 (3.62)
90 10 10 ##STR17## Methyl 2,2- dimethyl-3- hydroxypropionate 1.60
(12.13) 0.11 (3.03) 0.140 (2.41) 120 50 11 ##STR18## Ethyl 6-
hydroxyhexanoate 5.08 (31.69) 0.59 (15.85) 0.132 (2.28) 130 230 12
##STR19## Ethyl 4- hydroxycyclohexane- carboxylate 2.18 (12.68)
0.23 (6.34) 0.075 (1.30) 120 60 *65% suspension of sodium hydride
in oil was used. The amounts are shown for a suspension (in g) and
for sodium hydride (in mmole).
EXAMPLE 13
[0058] 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 .mu.g of HBsAg mixed with 50 .mu.g of
the polymer in solution (final volume 100 .mu.l) was injected in
each mouse. Formulations containing antigen alone, antigen
formulated with 50 .mu.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.
[0059] 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. 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 Benchmark.TM. 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).
[0060] The results are presented in FIG. 1. Both Polymer 1 and
Polymer 2 showed significantly higher adjuvant activity than PCPP
and alum.
EXAMPLE 14
[0061] Polymers 1 and 2 were evaluated in mice similarly as
described in Example 13, except that 5 .mu.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.
* * * * *