U.S. patent application number 09/845156 was filed with the patent office on 2001-10-25 for therapeutic composition comprising an antigen or an in vivo generator of a compound comprising an amino acid sequence.
Invention is credited to Ganne, Vincent.
Application Number | 20010033845 09/845156 |
Document ID | / |
Family ID | 9478297 |
Filed Date | 2001-10-25 |
United States Patent
Application |
20010033845 |
Kind Code |
A1 |
Ganne, Vincent |
October 25, 2001 |
Therapeutic composition comprising an antigen or an in vivo
generator of a compound comprising an amino acid sequence
Abstract
A therapeutic composition comprising (i) at least one antigen or
at least one in vivo generator of a compound comprising an amino
acid sequence and (ii) at least one adjuvant comprising at least
one pharmaceutically acceptable and water-soluble salt of an
organic anion and a metal cation.
Inventors: |
Ganne, Vincent; (La Varenne
Saint Hillaire, FR) |
Correspondence
Address: |
Robert M. Schulman, Esq.
Hunton & Williams
Suite 1200
1900 K Street, NW
Washington
DC
20006
US
|
Family ID: |
9478297 |
Appl. No.: |
09/845156 |
Filed: |
May 1, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09845156 |
May 1, 2001 |
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08478091 |
Jun 7, 1995 |
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6274149 |
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Current U.S.
Class: |
424/278.1 ;
424/184.1; 424/283.1; 424/600; 424/617; 514/23; 536/23.1 |
Current CPC
Class: |
Y02A 50/30 20180101;
A61P 31/12 20180101; A61K 9/107 20130101; A61K 39/39 20130101; A61P
31/00 20180101; Y10S 514/937 20130101; A61K 2039/55566 20130101;
A61K 47/183 20130101; A61K 47/26 20130101; A61P 35/00 20180101;
A61P 31/04 20180101; A61P 43/00 20180101; A61K 2039/55505 20130101;
A61K 9/0019 20130101; Y10S 514/938 20130101 |
Class at
Publication: |
424/278.1 ;
514/23; 536/23.1; 424/184.1; 424/283.1; 424/600; 424/617 |
International
Class: |
A61K 047/14; A61K
047/26; C07H 021/04; A01N 043/04 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 20, 1995 |
FR |
95.04739 |
Claims
What is claimed is
1. A therapeutic composition comprising (i) at least one antigen or
at least one in vivo generator of a compound comprising an amino
acid sequence and (ii) at least one adjuvant comprising at least
one pharmaceutically acceptable and water-soluble salt of an
organic anion and a metal cation.
2. The therapeutic composition of claim 1, wherein the metal cation
is divalent.
3. The therapeutic composition of claim 2 wherein the metal cation
is zinc, manganese or calcium.
4. The therapeutic composition of claim 3, wherein the divalent
metal cation is manganese.
5. The therapeutic composition of claim 1, wherein the organic
anion is derived from a compound comprising at least one oxygenated
functional group.
6. The composition of claim 5, wherein the oxygenated functional
group is a carboxylic group or a phosphoric group.
7. The therapeutic composition of claim 6, wherein the organic
anion is an amino acid, an acid saccharide including from 5 to 7
carbon atoms, or glycerophosphoric acid.
8. The therapeutic composition of claim 7, wherein the amino acid
is aspartic acid.
9. The therapeutic composition of claim 7, wherein the acid
saccharide is gluconic acid, fructoheptonic acid or glucoheptonic
acid.
10. The therapeutic composition of claims 1, wherein the
pharmaceutically acceptable salt is manganese gluconate.
11. The therapeutic composition of claim 1 in an emulsion form
comprising at least one aqueous phase and at least one oily phase,
the oily phase including said adjuvant.
12. The therapeutic composition of claim 11, wherein the emulsion
is of the W/O/W, O/W or microemulsion type.
13. The therapeutic composition of claim 11, wherein said oily
phase including the adjuvant further comprises a surface-active
agent.
14. The therapeutic composition of claim 13 wherein said
surface-active agent comprises (i) an ester obtained by
condensation of a fatty acid with a sugar, a poly-ethylene glycol,
sorbitol, glycerol, or a derivative of such an ester whose
hydrophilicity has been modified, and (ii) an ethoxylated plant
oil.
15. The therapeutic composition of claim 11, wherein the oily
adjuvant is a self-emulsifiable oil.
16. The therapeutic composition of claim 15, wherein the oily
adjuvant is an ester of ethoxylated fatty acids corresponding to
one of the following formulae: 3in which R.sub.1, R.sub.3, R.sub.5,
R.sub.6, R.sub.8 and R.sub.10 represent a saturated or unsaturated,
linear or branched hydrocarbon chain having from 5 to 30 carbon
atoms; R.sub.2, R.sub.4, R.sub.7 and R.sub.9 represent a saturated
or unsaturated, linear or branched hydrocarbon chain having from 1
to 5 carbon atoms; the total number of ethylene oxide molecules
represented in the abovementioned formulae II, III and IV by k, 1+m
and n+p+q, respectively, being an integer such that said compounds
have an HLB value between about 4 and about 10.
17. The therapeutic composition of claim 16 wherein said HLB value
is between about 5 and about 9.
18. The therapeutic composition of claim 1, further comprising a
surface-active agent, said composition being in a micellar solution
form.
19. The therapeutic composition of claim 18, wherein the
surface-active agent is (i) an ester obtained by condensation of a
fatty acid with a sugar or glycerol, or a derivative of such an
ester whose hydrophilicity has been modified, or (ii) an
ethoxylated plant oil.
20. A method of making a composition intended for the prevention or
treatment of infectious diseases comprising combining (a) at least
one adjuvant comprising at least one pharmaceutically acceptable
and water-soluble salt of an organic anion and a metal cation and
(b) at least one antigen or at least one in vivo generator of a
compound comprising an amino acid sequence.
21. The method of claim 20, further comprising the step of
combining the salt with (a) an oily adjuvant, (b) a surfactant or
(c) an oily adjuvant combined with a surface-active agent.
22. A method for treating a functional disease comprising
administering to a patient the therapeutic composition of claim
1.
23. The method of claim 22, wherein the salt of said therapeutic
composition is combined with (a) an oily adjuvant, (b) a
surface-active agent or (c) an oily adjuvant combined with a
surface-active agent.
24. An adjuvant composition comprising a pharmaceutically
acceptable and water-soluble salt and (a) an oily adjuvant, (b) a
surfactant and/or (c) an oily adjuvant combined with a
surfactant.
25. The composition of claim 22 further comprising at least one
aqueous phase.
26. The composition of claim 25 in an emulsion form.
27. The composition of claim 26 in a micellar solution form.
28. A therapeutic composition comprising (i) at least one compound
capable of eliciting an immune response and (ii) at least one
adjuvant comprising at least one pharmaceutically acceptable and
water-soluble salt of an organic anion and a metal cation.
Description
BACKGROUND OF THE INVENTION
[0001] (i) Field of the Invention
[0002] The present invention relates to a therapeutic composition
comprising at least one antigen, in particular an antigen of viral,
bacterial or parasitic origin, or at least one in vivo generator of
a compound comprising an amino acid sequence, and at least one
adjuvant.
[0003] (ii) Description of Related Art
[0004] The use of adjuvants in therapeutic compositions of the
vaccine type has been known for a long time. The main objective of
these adjuvants is to allow an increase in the immune response.
These adjuvants are diverse in nature. They may, for example,
consist of liposomes, oily phases, for example the Freund type of
adjuvants, generally used in the form of an emulsion with an
aqueous phase, or, more commonly, may consist of water-insoluble
inorganic salts. These inorganic salts may consist, for example, of
aluminum hydroxide, zinc sulfate, colloidal iron hydroxide, calcium
phosphate or calcium chloride. Aluminum hydroxide (Al(OH).sub.3) is
the most commonly used adjuvant. These adjuvants are described in
particular in the article by Rajesh K. Gupta et al "Adjuvants,
balance between toxicity and adjuvanticity", Vaccine, Vol. 11,
issue 3, 1993, pages 993-306.
[0005] The adjuvants mentioned above have the drawback of limited
efficiency. Moreover, they may induce a certain toxicity with
regard to individuals treated. More particularly, when these
therapeutic compositions are injected, the formation of lesions and
other local reactions such as granulomae is observed at the point
of injection. These drawbacks are less pronounced when the adjuvant
in aluminum hydroxide. Accordingly, the latter compound is one of
the most commonly used adjuvants. Recently, however, aluminum
hydroxide, like all aluminum-based compounds, has come to be
suspected of being a factor promoting the appearance of certain
diseases, such as renal dysfunctions or Alzheimer's disease. In
addition, it is known that aluminum hydroxide efficiently induces
only humoral immunity and not cell immunity.
SUMMARY AND OBJECTS OF THE INVENTION
[0006] A first object of the invention is to provide therapeutic
compositions comprising an adjuvant which allows an increase in the
immune response which is at least equal to that imparted by
aluminum hydroxide, without causing lesions or local reactions of
the granuloma type and which is not liable to promote the
appearance of diseases in the individual treated.
[0007] Another object of the invention is to provide a therapeutic
composition comprising an adjuvant which efficiently induces both
cell immunity and humoral immunity.
[0008] Yet another object of the invention is to provide a method
for making a therapeutic composition using the adjuvant of the
invention.
[0009] In a first aspect, the present invention relates to a
therapeutic composition comprising (i) at least one antigen or at
least one in vivo generator of a compound comprising an amino acid
sequence and (ii) at least one adjuvant comprising at least one
pharmaceutically acceptable and water-soluble salt of an organic
anion and a metal cation.
[0010] In a second aspect, the present invention relates to a
method of making a composition intended for the prevention or
treatment of infectious diseases comprising combining (a) at least
one adjuvant comprising at least one pharmaceutically acceptable
and water-soluble salt of an organic anion and a metal cation and
(b) at least one antigen or at least one in vivo generator of a
compound comprising an amino acid sequence.
[0011] In a third aspect, the present invention relates to a method
for treating a functional disease comprising administering to a
patient the therapeutic composition as described above.
[0012] In another aspect, the invention relates to an adjuvant
composition comprising a pharmaceutically acceptable and
water-soluble salt and (a) an oily adjuvant, (b) a surfactant
and/or (c) an oily adjuvant combined with a surfactant.
[0013] With the foregoing as well as other objects advantages and
features of the invention that will become hereinafter apparent,
the nature of the invention may be better understood by reference
to the following detailed description of the preferred embodiments
and to the appended claims.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0014] In the sense of the present invention, a water-soluble salt
may be such that its solubility in water is greater than or equal
to 10 g/l, preferably between 10 and 2000 g/l.
[0015] The metal cation constituting said pharmaceutically
acceptable salt is preferably a divalent cation. This cation is
advantageously a cation of a metal chosen from the group consisting
of manganese, calcium and zinc. Manganese is a very particularly
preferred metal within the context of the present invention. The
reason for this is that it has been observed that pharmaceutically
acceptable and water-soluble salts according to the invention
comprising an Mn.sup.2+ cation allow the induction of a
particularly large immune response, while at the same time being
low in toxicity.
[0016] The organic anion constituting said pharmaceutically
acceptable salt is advantageously an anion of a compound comprising
at least one oxygenated functional group, preferably a phosphoric
group --PO.sub.4H.sub.2, or a carboxylic group --COOH.
[0017] Glycerophosphoric acid is a preferred anion containing a
phosphoric group.
[0018] The preferred anions comprising at least one carboxylic
group are derived from compounds chosen from:
[0019] acid saccharides, preferably acid saccharides having from 5
to 7 carbon atoms, more preferably those having 6 or 7 carbon
atoms,
[0020] mono- or polycarboxylic acids,
[0021] amino acids.
[0022] The preferred mono- or polycarboxylic acids are fumaric acid
and the compounds of the formula (I): 1
[0023] where: R represents COOH, CH.sub.3CO, CH.sub.3 or
CH.sub.2OH,
[0024] R' represents H or COOH and
[0025] s, t and u, which may be identical or different, are between
0 and 3.
[0026] Preferred compounds of formula (I) are acetic acid, lactic
acid, tartaric acid, malic acid, citric acid and pyruvic acid.
[0027] In the sense of the present invention, an acid saccharide is
of a saccharide comprising at least one carboxylic function, a
saccharide being a glucide consisting of reducing sugars. These
acid saccharides are advantageously aldose derivatives obtained by
oxidation of the primary alcohol function or of the aldehyde
function into a carboxylic function. Such compounds may most
particularly include of gluconic acid, glucuronic acid,
fructoheptonic acid, gluconoheptonic acid and glucoheptonic acid.
When the organic anion is derived from an amino acid, this amino
acid may be an .alpha.-amino acid such as glutamic acid, methionine
and, most particularly, aspartic acid.
[0028] A pharmaceutically acceptable, water-soluble salt according
to the invention may comprise a polyvalent cation, especially a
divalent cation, combined with an organic anion or with several
organic anions of different nature. Thus, by way of example, a
divalent cation such as the calcium cation may he combined with an
anion derived from gluconoheptonic acid and an anion derived from
gluconic acid.
[0029] A water-soluble adjuvant most particularly preferred within
the context of the present invention consists of manganese
gluconate.
[0030] A therapeutic composition according to the present invention
may comprise between 0.01 and 1000 mg/ml, preferably between 0.1
and 150 mg/ml, of an adjuvant as defined above.
[0031] A therapeutic composition according to the invention may be
prepared by simple mixing of an aqueous suspension containing the
antigen or said in vivo generator with an aqueous solution of the
salt defined above.
[0032] Besides the adjuvant comprising a pharmaceutically
acceptable and water-soluble salt as defined above, the therapeutic
composition according to the invention may also comprise an oily
adjuvant. In such a case, the therapeutic composition according to
the invention is advantageously in the form of an emulsion
combining at least one aqueous phase and at least one oily
phase.
[0033] This emulsion may be of the water-in-oil (W/O) type or,
preferably, of the oil-in-water (O/W), water-in-oil-in-water
(W/O/W) or microemulsion type. Such an emulsion may be prepared
according to the standard methods for the preparation of an
emulsion, in particular according to the processes described in
patent applications EP-A-489,181 and EP-A-481,982. Thus, the oil
constituting the oily phase may be emulsified, with stirring, with
the aqueous phase including an aqueous solution or suspension
containing the antigen.
[0034] An emulsion according to the invention may contain, by
weight, from 0.5% to 99.5% of oily phase per 99.5% to 0.5% of
aqueous phase, preferably from 5% to 95% of oily phase per 95 to 5%
of aqueous phase and, more preferably, from 25 to 27% of oily phase
per 75 to 25% of aqueous phase. The emulsion must be stable
preferably for at least 12 months when it is stored at 4.degree.
C.
[0035] The oily adjuvant may be a mineral oil, a non-mineral oil or
a mixture of a mineral oil and a non-mineral oil. Said mineral oils
may be natural or synthetic. Said non-mineral oils may be of plant,
animal or synthetic origin. The non-mineral oils are advantageously
metabolizable. All these oils are devoid of toxic effects with
regard to the host organism into which the composition of the
invention is administered. They are preferably liquid at the
storage temperature (about +4.degree. C.) or at least make it
possible to give emulsions which are liquid at this temperature. An
advantageous mineral oil according to the invention may include an
oil comprising a linear carbon chain having a number of carbon
atoms preferably greater than 16, and free of aromatic compounds.
Such oils may, for example, be those marketed under the name
"MARCOL 52" (produced by Esso France) or "DRAKEOL 6VR" (produced by
Penreco USA).
[0036] Examples of synthetic non-mineral oils which may be
mentioned are polyisobutenes, polyisopropenes, esters of alcohols
and fatty acids, such as, for example, ethyl oleate and isopropyl
myristate, mono-, di- or triglycerides, propylene glycol esters,
partial glycerides such as corn oil glycerides, for instance those
marketed by the company SEPPIC under the name LANOL TM, maisin and
oleyl oleate. Among the plant oils which may be mentioned are
unsaturated oils rich in oleic acid which are biodegradable, for
example groundnut oil, olive oil, sesame oil, soya oil or wheatgerm
oil.
[0037] The animal oils may include in particular squalene, squalane
or spermaceti oil.
[0038] Moreover, when it is in the form of an emulsion as defined
above, the therapeutic composition according to the invention may
also advantageously contain one or more surface-active agents. The
latter agent has a lipophilic or hydrophilic nature characterized
by an HLB (hydrophilic-lipophilic balance) value between 1 and
19.
[0039] Such a surfactant may include:
[0040] an alkylpolyglycoside or a mixture of alkyl-polyglycosides
of formula Ra-(O)-Zn where Ra represents a linear or branched
saturated aliphatic radical comprising from 4 to 24 carbon atoms,
preferably from 8 to 22 carbon atoms, Z is a sugar residue,
preferably glucose, and n is between 1 and 5, preferably between
1.1 and 2,
[0041] saponins,
[0042] lecithins,
[0043] polyoxyethylated alkanols such an those marketed under the
name BRIJ by the company ICI,
[0044] polymers comprising polyoxyethylene and polyoxypropylene
blocks, such as those marketed under the name PLURONIC by the
company BASF.
[0045] Particularly preferred surfactants are polyethylene glycol
esters obtained by condensation of a fatty acid, in particular a
fatty acid which is liquid at 20.degree. C., with a polyethylene
glycol of molecular weight between 80 and 2000; such a surfactant
is marketed by the company SEPPIC under the tradename SIMULSOL
2599.
[0046] Another surface-active agent preferred within the context of
the present invention is an ester obtained by condensation of a
fatty acid, advantageously a fatty acid which is liquid at
20.degree. C., with a sugar, sorbitol, glycerol or a polyglycerol,
preferably a polyglycerol comprising from 2 to 5 glycerol units.
Said sugar may be glucose, sucrose or, preferably, mannitol. By way
of particularly preferred mannitol ester, there may be mentioned
mannitol oleates obtained by dehydration of the polyhydroxylated
carbon chain of mannitol which undergoes 1-4 or 2-6
cyclization.
[0047] Derivatives of these sugar esters, of polyethylene glycol,
of sorbitol, of glycerol or of polyglycerol may also be used. These
derivatives have a hydrophilicity which is modified in particular
by grafting hydrophilic functions such as alcohol, polyol, ethylene
oxide, propylene oxide, carboxylic acid, amine or amide. Such
derivatives may, for example, include polyoxyethylated fatty esters
of sorbitan, such as the TWEENs (conf. International Cosmetic
Ingredient Dictionary, 5th ed. 1993).
[0048] Another preferred type of surfactant is ethoxylated plant
oils such as, for example, ethoxylated castor oil, this oil being
optionally hydrogenated.
[0049] A surface-active agent according to the invention is
preferably pharmaceutically acceptable for an injectable use; it
must in particular be free of heavy metals and have very low acid
numbers or peroxide numbers. It is also desirable for it to satisfy
the harmlessness test standards such as, for example, those
described by S. S. Berlin, Annals of Allergy, 1962, 20, 473 or the
tests of abnormal toxicity described in the European Pharmacopoeia.
The surface-active agent is preferably combined with the oily
adjuvant before formation of the emulsion.
[0050] The concentration of surface-active agent in the therapeutic
composition may be between 0.01 and 500 mg/ml, preferably between
0.1 and 200 mg/ml.
[0051] Oils associated with a surface-active agent (mannitol ester)
which are most particularly suitable within the context of the
present invention are those marketed by the company SEPPIC under
the tradename "MONTANIDE". The nature of these oils, the type of
emulsion which can be obtained with them and the properties
(viscosity and conductivity) of these emulsions are featured in
Table 1 below:
1TABLE I Con- duc- Aqueous tivity Type phase/ at Oil/ of emulsion
Visco- 25.degree. C. Commercial manitol emul- (% by sity (.mu.s No.
name esters sion weight) (mPs s) cm.sup.-1) 1 MONT- Mineral O/W 75%
20 5000 ANIDE ISA 25 2 MONT- Mineral + O/W 75% 20 5000 ANIDE
avridine ISA 25A 3 MONT- Mineral + O/W 75% 25 1000 ANIDE
ethyloleate ISA 28 4 MONT- Mineral W/O/W 50% 50 1000 ANIDE ISA 206
5 MONT- Mineral W/O 50% 200 1 ANIDE ISA 50 6 MONT- Plant W/O 30% 70
1 ANIDE ISA 708 * Avridine =
N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl)-propane- diamine.
[0052] The oily adjuvant may also include a self-emulsifiable oil,
that is to say an oily preparation capable of forming a stable
emulsion with an aqueous phase, with virtually no energy input, for
example by dispersion in the aqueous phase by slow mechanical
stirring. In this respect, self-emulsifiable oils such as those
known in the European Pharmacopoeia under the names Labrafil and
Simulsol may be mentioned. These oils are polyglycolyzed
glycerides.
[0053] Preferred self-emulsifiable oils are those described in
French patent application No. 9500497 filed on Jan. 18, 1995, in
the name of the Applicant, entitled "Utilisation d'esters d'acides
gras thoxyls comme composants auto-mulsionnables notamment utiles
pour la prparation de compositions phytosanitaires ou de mdicaments
usage vtrinaire ou humain" [Use of ethoxylated fatty acid esters as
self-emulsifiable components which are particularly useful for the
preparation of plant-protection compositions or medicinal products
for human or veterinary use], reference of which is included in the
present description. These oils include ethoxylated fatty acid
esters corresponding to one of the following formulae: 2
[0054] in which:
[0055] R.sub.1 R.sub.3, R.sub.5, R.sub.6, R.sub.8 and R.sub.10
represent a saturated or unsaturated, linear or branched
hydrocarbon chain having from 5 to 30 carbon atoms;
[0056] R.sub.2, R.sub.4, R.sub.7, and R.sub.9 represent a saturated
or unsaturated, linear or 5 branched hydrocarbon chain having from
1 to 5 carbon atoms;
[0057] the total number of ethylene oxide molecules represented in
the abovementioned formulae II, III and IV by k, 1+m and n+p+q,
respectively, being an integer such that the HLB value of said
compounds is between about 4 and about 10, preferably between about
5 and about 9.
[0058] R.sub.1 is preferably chosen from palmitic, stearic,
ricinoleic, oleic, linoleic and linolenic acid residues, R.sub.2
represents a methyl radical and k is an integer between 1 and 5,
preferably equal to 2, and moreover the preferred ethoxylated fatty
acid esters of formula III are those in which:
[0059] (i) R.sub.6, R.sub.8 and R.sub.10 represent hydrocarbon
chains having from 16 to 22 carbon atoms, corresponding in
particular to the fatty chains of rapeseed oil, of corn oil, of
soya oil, of groundnut oil and of apricot kernel oil,
[0060] R.sub.7 and R.sub.9 represent a methylene group,
CH.sub.2;
[0061] n, p and q represent integers such that their sum is between
3 and 30, and preferably equal to 20; or
[0062] (ii) R.sub.6, R.sub.8 and R.sub.10 represent hydrocarbon
chains corresponding to the fatty chains of castor oil;
[0063] R.sub.7 and R.sub.9 represent a methylene radical,
CH.sub.2;
[0064] n, p and q represent integers such that their sum is between
5 and 7.
[0065] The concentration of self-emulsifiable oil in the
therapeutic composition according to the invention may be
approximately between 5 and 700 g/l, preferably approximately
between 10 and 500 g/l.
[0066] Besides the oily phase and the aqueous phase, the
composition according to the invention may contain a conventional
immunostimulatory agent such as Avridine.RTM., i.e.
N,N-dioctadecyl-N',N'-bis(2-hydroxyethy- l)propanediamine, MDP
(muramyl dipeptide) derivatives, especially threonyl-MDP, mycolic
acid derivatives or Lipid A derivatives.
[0067] The therapeutic composition according to the invention may
also comprise one or more surface-active agents, in the absence of
any oily adjuvant.
[0068] The therapeutic composition is then in the form of a
micellar solution. This may be prepared by simple mixing of the
surface-active agent with a dispersion in water of the antigen or
of the in vivo antigen generator.
[0069] The surface-active agent may be chosen from the
surface-active agents described above, in combination with an oily
adjuvant.
[0070] Said micellar solution may contain from 0.5 to 500 mg/ml,
preferably from 1 to 250 mg/l, of surface-active agent.
[0071] A therapeutic composition according to the present invention
may comprise an antigen such as a virus, a microorganism, more
particularly a bacterium or parasite, or a compound comprising a
peptide chain. Such a compound may include a protein or a
glycoprotein, especially a protein or glycoprotein obtained from a
microorganism, a synthetic peptide or a protein or a peptide
obtained by genetic engineering. Said virus and microorganism may
be totally inactivated or live and attenuated. By way of virus
which may constitute an antigen according to the present invention,
there may be mentioned the rabies virus, Aujeszky viruses,
influenza viruses, the virus of foot-and-mouth disease or HIV
viruses. By way of microorganism of bacterial type which may
constitute an antigen according to the present invention, there may
be mentioned E. coli and those of the genera Pasteurella,
Furonculosis, Vibriosis, Staphylococcus and Streptococcus. By way
of parasite, there may be mentioned those of the genera
Trypanosoma, Plasmodium and Leishmania.
[0072] A therapeutic composition according to the invention
comprises an antigen concentration which depends upon the nature of
this antigen and on the nature of the individual treated. It is,
however, particularly noteworthy that an adjuvant according to the
invention, which may or may not be combined with an oily adjuvant
and/or a surface-active agent as are defined above, makes it
possible to decrease in an appreciable manner the usual dose of
antigen required. The appropriate antigen concentration may be
determined conventionally by those skilled in the art. This dose is
generally of the order of 0.1 .mu.g/ml to 1 g/ml, more generally
between 1 .mu.g/ml and 100 mg/l.
[0073] A therapeutic composition according to the invention may
also comprise an in vivo generator of a compound comprising an
amino acid sequence, that is to say a biological compound capable
of expressing such a compound in the host organism into which said
in vivo generator has been introduced. The compound comprising the
amino acid sequence may be a protein, a peptide or a
glycoprotein.
[0074] These in vivo generators are generally obtained by genetic
engineering processes.
[0075] More particularly, they may comprise living microorganisms,
generally a virus, acting as a recombinant vector, into which is
inserted a nucleotide sequence, in particular an exogenous gene.
Those compounds are known as such and are used in particular as
recombinant sub-unit vaccines.
[0076] In this regard, reference may be made to the article by M.
Eloit et al., Journal of virology (1990) 71, 2925-2431, to
International Application WO-A-91.00107 or to International
Application WO-A-94/16681.
[0077] The microorganism constituting a recombinant sub-unit
vaccine is advantageously a non-sheathed recombinant virus chosen,
for example, from adenoviruses, the virus of the vaccine, canarypox
virus, herpes viruses and baculoviruses. The exogenous gene
inserted into the microorganism may, for example, be derived from
an Aujeszky virus or HIV.
[0078] The in vivo generators according to the invention may also
comprise a recombinant plasmid comprising an exogenous nucleotide
sequence capable of expressing in a host organism a compound
comprising an amino acid sequence. Such recombinant plasmids and
their mode of administration into a host organism were described in
1990 by Lin et al., circulation 82:2217,2221; Cox et al., J. of
Virol., September 1993, 67, 9, 5664-5667 and in International
Application WO/FR 95/00345 dated Mar. 21, 1995, in the name of the
Applicant, entitled "Une composition comprenant un plasmide
recombinant et ses utilisations comme vaccin et mdicament" [A
composition comprising a recombinant plasmid and uses thereof as
vaccine and medicinal product].
[0079] Depending on the nature of the nucleotide sequence comprised
within the in vivo generator, the compound comprising the amino
acid sequence which is expressed within the host organism may:
[0080] (i) be an antigen, and permit triggering of an immune
reaction,
[0081] (ii) have a curative action with respect to a disease,
essentially a functional disease, which has been triggered in the
host organism. In this case, the in vivo generator allows a
therapeutic treatment of the host, of the gene therapy type.
[0082] By way of example, such a curative action may comprise the
in vivo cytokine generator, such as the interleukins, in particular
interleukin 2. The latter permit triggering or reinforcement of an
immune reaction aimed at the selective removal of cancer cells.
[0083] The concentration of said in vivo generator in the
therapeutic composition according to the invention depends, here
also, in particular on the nature of said generator and on the host
into which it is administered. This concentration may readily be
determined by those skilled in the art, on the basis of routine
experiment.
[0084] As a guide, it may, however, be pointed out that when the in
vivo generator a recombinant microorganism, its concentration in
the therapeutic composition according to the invention may be
between 10.sup.2 and 10.sup.15 microorganisms/ml, preferably
between 10.sup.5 and 10.sup.12 microorganisms/ml.
[0085] When the in vivo generator is a recombinant plasmid, its
concentration in the therapeutic composition according to the
invention may be between 0.01 and 100 g/l.
[0086] A therapeutic composition according to the invention may be
used as a preventive or curative medicinal product. Depending on
the nature of the antigen or of the in vivo generator, a
therapeutic composition according to the invention may be
administered to fish, to crustaceans such as shrimps, to poultry,
in particular geese, turkeys, pigeons and chickens, to canines such
as dogs, to felines such as cats, to pigs, primates, cattle, sheep
and horses. The therapeutic composition according to the invention
comprising a pharmaceutically acceptable water-soluble salt as
defined above may also be administered to man. The therapeutic
composition may be administered in a conventional manner, in
particular by subcutaneous, intramuscular or intraperitoneal
injection or via the oral or mucosal route.
[0087] Another aspect of the invention is use of an adjuvant
comprising a pharmaceutically acceptable water-soluble salt as
defined above for the preparation of a vaccine intended for the
prevention or treatment of an infectious disease, in particular an
infectious disease generated by a virus or a microorganism such as
those mentioned above.
[0088] Another aspect of the invention is the use of a
pharmaceutically acceptable water-soluble salt for the preparation
of a therapeutic composition intended to treat a functional
disease, such as cancer or mucoviscidosis.
[0089] In one other of these uses, said pharmaceutically acceptable
salt may be combined with at least one of: an oily adjuvant, a
surface-active agent and one oily adjuvant which is itself combined
with a surface-active agent; these oily adjuvants and surfactants
being as defined above.
[0090] Adjuvant compositions comprising said pharmaceutically
acceptable salt and the oily adjuvant and/or the surfactants
mentioned above constitute yet another aspect of the invention.
Where appropriate, these adjuvant compositions comprise at least
one aqueous phase.
[0091] In the latter case, the adjuvant compositions according to
the invention, comprising at least one oily phase and, where
appropriate, a surfactant, may be in the form of an emulsion. This
emulsion may be of the W/O, O/W, W/O/W or microemulsion type.
[0092] These emulsions may comprise, by weight, from 0.5% to 99.5%
of oily phase per 99.5% to 0.5% of aqueous phase, preferably from 5
to 95% of oily phase per 95 to 5% of aqueous phase and, more
preferably, from 25 to 75% of oily phase per 75 to 25% of aqueous
phase.
[0093] Where appropriate, they may comprise from 0.01 to 500 mg/ml,
preferably from 0.1 to 200 mg/ml, of at least one surfactant.
[0094] When the adjuvant composition according to the invention
comprises, besides the pharmaceutically acceptable salt and an
aqueous phase, only one or more surfactants, it is then in the form
of a micellar solution. The surfactant content of this micellar
solution may be between 0.01 and 900 mg/ml, preferably between 1
and 250 mg/ml.
[0095] An adjuvant composition according to the invention usually
comprises from 0.02 to 3000 mg/ml, preferably 0.1 to 1000 mg/ml and
more preferably from 0.1 to 150 mg/ml, of a pharmaceutically
acceptable salt according to the invention.
[0096] These compositions are useful for preparing the therapeutic
compositions according to the invention.
[0097] The latter compositions may then be prepared by simple
mixing of the adjuvant composition with a composition comprising an
antigen or an in vivo generator of a compound comprising an amino
acid sequence.
[0098] The invention will be better understood with regard to the
examples and figures below.
[0099] The examples were performed on OF1 mice whose average weight
was between 18 and 20 g.
[0100] The results expressed are an average of the results obtained
on 10 mice.
[0101] The therapeutic compositions comprised Sigma Grade V bovine
serum albumin (BSA) as antigen.
[0102] The therapeutic compositions were injected
subcutaneously.
[0103] The humoral immune response was determined by assay of the
total IgG and of the total IgG.sub.1, according to the ELISA
method.
[0104] The cell immune response was determined by assay of the
IgG.sub.2a, according to the ELISA method.
[0105] The antibody levels mentioned in the examples correspond to
the last dilution above the background noise.
[0106] The following abbreviations are used in the examples:
[0107] Glu=gluconate
[0108] Fruhp=fructoheptonate
[0109] Gly=glycerophosphate
[0110] Gluhp=glucoheptonate
[0111] Al(OH).sub.3=aluminum hydroxide
[0112] Asp=aspartic acid
[0113] Thus, by way of example, GluMn is manganese gluconate.
EXAMPLE 1:
[0114] Therapeutic compositions (or doses) of 100 .mu.l each,
comprising 50 .mu.g of BSA, are prepared.
[0115] These therapeutic compositions were injected into the mice
on D0 (i.e. the day of injection).
[0116] The level of anti-BSA antibodies was determined 14 and 28
days after the injection (primary response).
[0117] On D28, the same therapeutic composition was injected into
the same mice.
[0118] The level of anti-BSA antibodies was determined on D42 and
D56.
[0119] The results obtained are featured in Table II below.
2TABLE II Doses of Vac- adju- anti-BSA anti-BSA anti-BSA anti-BSA
cine vant antibody antibody antibody antibody com- (in primary/
primary/ primary/ primary/ posit- Ajdu- mg/ response response
response response ions vant dose) (D14) (D28) (D42) (D56) 1 -- --
<500 <500 <500 <500 2 GluMn 1 64,000 32,000 256,000
256,000 3 GluCa 3 16,000 8000 128,000 96,000 4 Fruhp- 1 4000 2000
64,000 48,000 Ca 5 GlyCa 2 4000 2000 64,000 64,000 6 GluZn 0.5
16,000 6000 96,000 96,000 7 Gluph- 1 8000 6000 64,000 64,000 Zn
Con- -- -- <500 <500 <500 <500 trol
[0120] The control composition corresponds to mice which were not
vaccinated.
[0121] The results obtained show that the addition of the adjuvants
according to the invention to a therapeutic composition induces a
significant immunostimulatory activity as compared with the
non-vaccinated control and to the therapeutic compositions not
comprising any adjuvants.
[0122] It may also be noted that GluMn, although used at a low
concentration, allows a particularly high level of antibodies to be
obtained.
EXAMPLE 2
[0123] The immunostimulatory effect of a water-soluble salt
according to the invention, GluMn was compared with that of an
insoluble salt, Al(OH).sub.3.
[0124] The therapeutic compositions (dose/mouse) used, 100 .mu.l in
volume, comprised 50 .mu.g of antigen and 1 mg of Al(OH).sub.3.
[0125] Doses not comprising any adjuvants were also injected.
[0126] One batch of mice was not vaccinated (control). The results
obtained are featured in Table III below.
3TABLE III IgG1 IgG1 IgG2.sub.a IgG2.sub.a IgG1 IgG1 secon- secon-
IgG2 IgG2.sub.a secon- secon- primary primary dary dary primary
primary dary dary response response response response response
response response response Adjuvant D14 D28 D42 D56 D14 D28 D42 D56
-- <500 <500 <500 <500 <500 <500 <500 <500
GluMn 32,000 64,000 256,000 256,000 4000 8000 24,000 64,000 Al
(OH).sub.3 16,000 64,000 128,000 256,000 1000 6000 8000 32,000
Control <500 <500 <500 <500 <500 <500 <500
<500
[0127] The IgG1s are representative of the humoral response.
[0128] The IgG2s are representative of the cell response.
[0129] The humoral and cell responses obtained with an adjuvant
according to the invention are greater than those obtained with a
conventional adjuvant such as Al(OH).sub.3.
EXAMPLE 3:
[0130] In order to demonstrate the synergy effect between an
adjuvant of the water-soluble salt type with an oily adjuvant
(Montanide ISA 25 defined in Table I), the effect of various
therapeutic compositions was compared. Each of them comprised 50
.mu.g of antigen and had a volume of 100 .mu.l (dose/mouse).
[0131] The results obtained are featured in Table IV below:
4TABLE IV Water Amount of Montanide Humoral Cell soluble salt ISA25
immunity immunity salt (mg/dose) (in mg/dose) (D14) (D14) -- -- 25
24,000 3000 GluMn 1 -- 64,000 4000 GluMn 1 25 96,000 8000 GluMn 0.5
-- 16,000 <2000 GluMn 0.5 25 96,000 6000
[0132] The combination of an oily adjuvant with a soluble salt
according to the invention makes it possible to obtain an antibody
level which is superior to that obtained by the simple addition of
the antibody levels obtained with each of these adjuvants used
individually. Synergy is indeed demonstrated. This synergy is even
more pronounced for a low amount (0.5 mg/dose) of GluMn.
EXAMPLE 4:
[0133] In order to demonstrate the importance of the organic nature
of the anion, the immunostimulatory effect of various calcium salts
was compared.
[0134] Each therapeutic composition, 100 .mu.l in volume, contained
50 .mu.g of antigen and, except for the control, 0.5 mg of a
calcium salt, such that the calcium concentration of each
composition was 2.7 mg.
[0135] The antibodies were assayed 42 days after the
vaccination.
[0136] Injection of the therapeutic composition was repeated on
D28. The antibodies were assayed on D42.
[0137] The results obtained are featured in Table V:
5 TABLE V Salts Humoral reponse Cell response CaCO.sub.3 32,000
4000 CaCl.sub.2 64,000 24,000 CaHPO.sub.4 64,000 10,000 GluCa
128,000 32,000 -- 3000 <2000
[0138] It was observed during these tests that CaCl.sub.2 induced
very large lesions.
EXAMPLE 5:
[0139] A therapeutic composition 100 .mu.l in volume, comprising 50
.mu.g of antigen, was injected into various batches of mice each
comprising 5 mice. The local reactions (lesions and granulomae)
were evaluated on D8 and D35.
[0140] The results obtained are featured in Tables VI and VII
respectively.
6TABLE VI (D8) Mouse No. Salt concentration (mg/dose) 1 2 3 4 5
GluMn 1 + + - - + GlyMn 1 L + ++ + - GluMn.sup.+ 1 ++ +++ + - +
GluK 1 - - - - - Asp K and 1 - - - - - Asp Mg GluCa 1 - - - - -
MnCl.sup.2+ 1 +++ L L L L Al(OH).sub.3 1 +++ +++ ++ +++ ++++
[0141]
7TABLE VII (D35) Mouse No. Salt concentration (mg/dose) 1 2 3 4 5
GluMn 1 - + + + - GlyMn 1 - + + + + GluMn.sup.+ 1 ++ ++ ++ +++ +
GluK 1 - - - - - Asp K and 1 - - - - - Asp Mg GluCa 1 - - - - -
MnCl.sup.2+ 1 +++ LL LL L L Al(OH).sub.3 1 ++++ +++ ++ +++ ++++
(*): GluMn combined with 25 mg of MONTANIDE ISA 25 - = no granuloma
+ = small granuloma ++ = medium granuloma +++ = large granuloma
++++ = very large granuloma - = no lesions L = small lesion LL =
medium lesion LLL = large lesion LLLL = very large lesion DCD =
mice that died during the experiment
[0142] The chosen concentration of Al(OH).sub.3 corresponds to the
concentration at which this salt allows the largest immune response
to be obtained. This concentration of Al(OH).sub.3 is that used in
the above examples.
[0143] Although only preferred embodiments of the invention are
specifically illustrated and described above, it will be
appreciated that many modifications and variations of the present
invention are possible in light of the above teachings and within
the purview of the appended claims without departing from the
spirit and intended scope of the invention.
* * * * *