Pharmaceutical composition comprising a solid nanoparticle and at least an antigen for the treatment against an intracellular pathogenic agent

Abstract

A pharmaceutical composition for its use in the prophylactic treatment of a patient against an intracellular pathogen, said composition comprising a solid nanoparticle comprising a porous cationic-polysaccharide solid core, loaded with at least an anionic phospholipid, without said cationic-polysaccharide core being surrounded by any phospholipidic layer, and at least an antigen obtained from said pathogen.

Claims

1. A pharmaceutical composition comprising, as an active ingredient, a mixture of: a solid nanoparticle comprising a porous cationic-polysaccharide core loaded with at least an anionic phospholipid and without any phospholipidic layer surrounding said porous cationic-polysaccharide core; at least an antigen obtained from an intracellular pathogenic agent; and a pharmaceutically acceptable solvent.

2. The pharmaceutical composition according to claim 1, wherein said porous cationic polysaccharide core comprises a crosslinked polymer obtained by a reaction between: a polysaccharide selected from the group consisting of a starch, a dextran, a dextrin, and a maltodextrin, and at least one cationic ligand selected from the group consisting of a primary amine, a secondary amine, a tertiary amine and a quaternary ammonium.

3. The pharmaceutical composition according to claim 1, wherein said anionic phospholipid is comprises glycerol phospholipid.

4. The pharmaceutical composition according to claim 1, wherein said intracellular pathogenic agent is selected from the group consisting of a virus, a bacteria, a mycobacteria, and a fungus.

5. The pharmaceutical composition according to claim 1, wherein said intracellular pathogenic agent is an intracellular parasite.

6. The pharmaceutical composition according to claim 1, wherein said antigen is obtained from a previously killed pathogenic agent.

7. The pharmaceutical composition according to claim 6, wherein said antigen is obtained from a tachyzoite form of said previously killed pathogenic agent.

8. A vaccine against an intracellular pathogen comprising the pharmaceutical composition according to claim 1 and further comprising a pharmaceutically acceptable additive selected from the group consisting of a suitable excipient, a suitable carrier, and a suitable vehicle.

9. A vaccine adjuvant comprising a solid nanoparticle comprising a porous cationic-polysaccharide core loaded with at least an anionic phospholipid and without any phospholipidic layer surrounding said core.

10. A method for eliciting an immune response against an intracellular pathogen in a patient in need of such treatment, said method comprising administrating to said patient a therapeutic amount of a composition according to claim 1.

11. The method according to claim 10, wherein said composition is intranasally administered.

12. The method according to claim 10, wherein said administering to said patient the therapeutic amount of the composition according to claim 1 is effective to cause a Th1 type immune response in said patient.

13. The method according to claim 10, wherein said patient is in need of a prophylactic treatment against said intracellular pathogenic agent.

14. The method according to claim 10, wherein said patient is selected from the group consisting of a human, a non-human primate, an ovine, a canine, a feline, a murine, a bovine, an equine, a porcine, and a bird.

15. The pharmaceutical composition according to claim 3, wherein said anionic phospholipid is a diacylphosphatidyl glycerol.

16. The pharmaceutical composition according to claim 15, wherein said diacylphosphatidyl glycerol is selected from the group consisting of dipalmitoylphosphatidylglycerol, diacylphosphatidyl serine and diacylphosphatidylinositol.

17. The pharmaceutical composition according to claim 4, wherein said intracellular pathogenic agent is selected from the group consisting of herpes simplex virus 1, herpes simplex virus 2, Human papilloma virus, Epstein-Barr virus, cytomegalovirus, Mycobacterium tuberculosis, dengue fever virus, human immunodeficiency virus, Human respiratory syncytial virus (RSV), hepatitis A virus, hepatitis B virus, and hepatitis C virus.

18. The pharmaceutical composition according to claim 5, wherein said intracellular pathogenic agent is selected from the group consisting of Toxoplasma gondii, Emeria spp., Neospora caninum, Sarcocystis spp., Plasmodium spp., Cryptosporidium spp., Acanthamoeba spp., Babesia spp., Balantidium coli, Blastocystis, Dientamoeba fragilis, Entamoeba histolytica, Giardia lamblia, Isospora belli, Leishmania spp., Naegleria fowleri, Rhinosporidium seeberi, Trichomonas vaginalis, Trypanosoma brucei, and Trypanosoma cruzi.

19. The pharmaceutical composition according to claim 1, comprising a dose having between 5 μg and 1 mg of the at least an antigen.

20. The pharmaceutical composition according to claim 1, wherein the solid nanoparticle is 60 nm.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the ELISA analysis of T. gondii IgG antibodies in serum of Swiss mice treated with three intranasal administrations (15-days interval between each administration) of DGNP (dipalmitoyl phosphatidyl glycerol nanoparticles), TE (total antigen extract), a mixture of TE and NPS (nanoparticles), and a mixture of TE and DGNP, for which the analyses are performed the day of the first administration, 14 days after the first administration (second administration), 28 days after the first administration (third administration), and finally 42 days after the first administration, respectively;

(2) FIG. 2 shows ELISA analysis of IFN-γ in supernatants of cultured splenocytes obtained from the groups of treated mice as explained in FIG. 1, after the third intranasal administration;

(3) FIG. 3 shows the parasite loads of mice brains obtained by microscopic counting after brain homogenization in the groups of treated mice as referred to in FIG. 1 and after infection with T. gondii cysts, for which the results are obtained 6 weeks after T. gondii infection.

(4) FIG. 4 shows the results of the humoral analysis of Toxoplasma IgG antibodies in serum of CBA/J mice treated with three intranasal administrations of TE alone, DGNP alone, a mixture of TE and DGNP, cholera toxin (CT) alone, and a mixture of TE and CT, respectively, said results being obtained 14 days after the third intranasal administration, in which the crosses refer to mice whereas the dashes refer to the average value obtained for each group of mice; the crosses referring to the mice having the stronger response in the groups treated with a mixture of TE and DGNP and with a mixture of TE and CT, respectively, are surrounded; the crosses referring to mice showing an average humoral response for each of the previously cited groups are also surrounded.

(5) FIG. 5 shows the ELISA analysis of IFN-γ (pg/ml) in supernatants of cultured splenocytes for each group as referred to in FIG. 4 and after the third intranasal administration;

(6) FIG. 6 shows the production of IFN-γ by splenocytes produced in each group of mice as defined in reference to FIG. 4, two weeks after the third intranasal administration; the black columns refer to the IFN-γ production of the mice having the stronger humoral response, in the TE-DGNP and in the TE-CT group, respectively, whereas the white columns refer to the IFN-γ production of the mice having an average humoral response, in the TE-DGNP and in the TE-CT group, respectively;

(7) FIG. 7 shows the number of cysts in mice brain for the group of mice untreated, mice treated with three intranasal administrations of DGNP alone, mice treated with three intranasal administrations of TE alone, mice treated with three intranasal administrations of a mixture of TE and DGNP, mice treated with three intranasal administrations of CT alone, and mice treated with three intranasal administrations of a mixture of TE and CT, respectively, said number of cysts being obtained two weeks after the third intranasal administration.

DESCRIPTION OF THE EMBODIMENTS

Experimental Part

(8) Preparation of Nanoparticles

(9) Polysaccharide particles are prepared from US Pharmacopoeia maltodextrin, as described previously (Paillard, A., et al, “Positively-charged, porous, polysaccharide nanoparticles loaded with anionic molecules behave as ‘stealth’ cationic nanocarriers” Pharm. Res. 27:126-33, 2010). Briefly, 100 g of maltodextrin were dissolved in 2N sodium hydroxide with magnetic stirring at room temperature. Further, 1-chloro-2,3-epoxy propane (epichlorhydrin) and glycidyl-trimethylammonium chloride (hydroxycholine, cationic ligand) is added to make cationic polysaccharide gel. The gel is then neutralized with acetic acid and sheared under high pressure in a Minilab homogenisor (Rannie; APV Baker, Evreux, France). The 60 nm polysaccharide nanoparticles obtained are ultra-filtered on an SGI Hi-flow system (hollow fiber module: 30 UFIB/1 S.6/40 kDa; Setric Génie Industriel, France) to remove low molecular weight reagents and salts. The obtained nanoparticles are hereinafter called NPS.

(10) Some of the previous NPS are loaded with anionic phospholipids. Anionic phospholipids are loaded into these porous NPS by injecting an ethanol solution of dipalmitoyl-phosphatidyl glycerol (DPPG). The porous nanoparticles containing a phospholipid are hereinafter called DGNP. The core of these nanoparticles is not surrounded by any phospholipid layer.

(11) Synthesis and Purification of Total Antigen Extract (TE) of T. gondii

(12) Tachyzoites are obtained from successive splitting of infected HFF cells (Human Foreskin Fibroblasts). About 1×10.sup.8 tachyzoites derive from one 225 cm.sup.2 culture flask corresponding to 200 μg of TE. Lysis of tachyzoites is then performed by freeze/thaw cycles, pooled, sonicated (2×10 min, 60 W in ice) and protein amount is evaluated by micro BCA method.

(13) TE refers to the product obtained from tachyzoites according to the process as above described. TE is used for mice immunization in combination with nanoparticles (NPS and DGNP), Elisa coating and cellular restimulation test. TE is a mixture of several antigens.

(14) Vaccination and Challenge Protocol-Choice Between NP and DGNP

(15) The most effective nanoparticles as antigen carriers were determined on the basis of intensity of humoral and cellular responses and protection.

(16) Adult females Swiss and CBA/J mice of 20-25 g and 6-8 weeks were purchased from Janvier (France). The animal experiments comply with the French Government's ethical and animal experiment regulations.

(17) Swiss mice received an intranasal treatment, three times at 15-day intervals, with TE (10 μg) and DGNP nanoparticles (30 μg) alone (defined as control groups) or with the combination TE+NPS, TE+DGNP (10 μg of TE and 30 μg of NPS or DGNP).

(18) Each dose of the above-mentioned total extract (TE), nanoparticles and mixtures thereof was diluted to a final volume of 10 μl in phosphate-buffered saline (10 mM phosphate, 140 mM NaCl [PBS]) and instilled into the nostrils of non-anesthetized mice with a micropipettor (5 μl/nostril). Treated mice were infected per os with 50 cysts of 76 K Toxoplasma strain, 1 month post-treatment, and followed up by clinical examination for a further period of 6 weeks.

(19) Study of Humoral Immune Responses

(20) Specific Toxoplasma IgG were quantified in the sera of treated mice by ELISA. IgG synthesis against Toxoplasma antigens was monitored sequentially in sera. The results are shown in FIG. 1.

(21) FIG. 1 shows the optical density (DO), measured in the sera of the treated mice. The optical density shows the level of serum IgG against T. gondii. DGNP refers to the mice treated with DGNP alone before infection, TE refers to the mice treated with the total antigen extract (TE) alone before infection, TE-NPS refers to the mice treated with the mixture of TE and NPS before infection and TE-DGNP refers to the mice treated with the mixture of TE and DGNP, before infection. DO refers to the optical density measured before treatment for each group of treated mice. D14 refers to the optical density measured 14 days after the first intranasal administration, D28 refers to the optical density measured 14 days after the second intranasal administration and D42 refers to the optical density measured 14 days after the third intranasal administration.

(22) As shown in FIG. 1, specific Toxoplasma IgGs could be detected after the second intranasal administration in the groups of mice immunized with TE-NPS or TE-DGNP. The boost effect due to the third intranasal administration resulted in a strong induction of IgG expression, but no significant difference was observed between the two nanoparticles. No IgGs were detected in the group of mice treated with TE alone.

(23) Study of Cellular Immune Responses:

(24) To investigate cellular immune responses, splenocyte cytokines, a strong immunogenicity biomarker of vaccine efficacy, were analyzed in supernatants of Toxoplasma-stimulated splenocytes from the previously-mentioned treated mice, 3 weeks after the third intranasal administration. Cytokines (IFN-γ, IL-12, IL-10, IL-13, TNF-α, IL-5) were quantified by ELISA.

(25) As T-cell-derived IFN-γ was also a valuable hallmark of protective immunity in toxoplasmosis, IFN-γ was determined by ELI spot analysis after Toxoplasma antigen stimulation.

(26) To investigate the cellular immune response induced after treatment with TE-NPS, TE-DGNP, TE alone, or nanoparticles alone, the supernatants of cultured cells from the spleen of 2 mice from the different groups were evaluated for the production of IFN-γ, IL-10, and IL-12 in response to TE restimulation (10 μg×ml.sup.−1).

(27) FIG. 2 shows the concentration of IFN-γ (pg/mL) in supernatants of cultured splenocytes obtained from the above-mentioned groups of treated mice.

(28) Each column referred to as DGNP corresponds to a mouse treated with DGNP alone. Each column referred to as TE corresponds to a mouse treated with TE alone. Each column referred to as TE-NPS corresponds to a mouse treated with a mixture of TE and NPS. Each column referred to as TE-DGNP corresponds to a mouse treated with a mixture of TE and DGNP.

(29) As shown in FIG. 2, only one mouse immunized with the mixture of TE and NPS responded to TE stimulation by the production of IFN-γ by spleen cells (175 pg/ml). On the other hand, as shown in FIG. 2, a specific production of IFN-γ by spleen cells of the 2 mice immunized with the mixture of TE and DGNP was observed (237 and 375 pg/ml, respectively). Consequently, DGNP seems to be more efficient at inducing a cellular immune response.

(30) Evaluation of the Amount of Cysts in the Brain of Mice Treated with a Mixture of TE and DGNP

(31) Six weeks after T. gondii infection, mice treated with a mixture of TE and DGNP and then infected with T. gondii were killed and their brains were collected.

(32) Brains were harvested 6 weeks after infection from surviving mice and homogenized in 5 mL of RPMI 1640 with a pestle and mortar. The cysts in each brain homogenate were counted under a microscope (10 counts, each on 10 μl). The results are expressed as Mean±SEM for each group. The data were statistically analysed using the Mann-Whitney U test (GraphPad prism software), (p<0.05). The results are shown in FIG. 3.

(33) FIG. 3 shows the number of cysts in mice brain for the mice treated before infection by T. gondii, with DGNP alone (referred as DGNP), TE alone (referred as TE), with the mixture of TE and NPS (referred as TE-NPS), and with the mixture of TE and DGNP (referred to as TE-DGNP).

(34) As shown in FIG. 3, mice treated with the mixture of TE and DGNP had significantly fewer cysts than mice treated with DGNP, TE, and the mixture of TE and NPS, respectively (672, 1333, 1180 and 1072, respectively; p<0.05). Mice treated with the mixture of TE and DGNP have 56% less cysts in their brains. These results suggest that treatment (vaccination) with the mixture of TE and DGNP induces a cellular immune response and then reduces the spread of parasites and the formation of cysts in the brain.

(35) According to the obtained results, DGNP nanoparticles were used for the following experiments of vaccination and to compare the vaccination protocol with DGNP, to the vaccination protocol with cholera toxin (CT).

(36) Comparison Between DGNP and Cholera Toxin (CT)

(37) Six groups of 10 CBA/J mice were treated with three intranasal administrations at 15-day intervals with TE (10 μg), nanoparticles (DGNP—30 μg), Cholera Toxin (CT—0.5 μg) alone (defined as control groups), with the combination TE+DGNP, and TE+CT (10 μg of TE and 30 μg of DGNP or 0.5 μg of CT), respectively. The experimental design includes a group of untreated mice.

(38) Each dose of the above-mentioned toxin, nanoparticles and combinations (mixtures) was diluted to a final volume of 10 μl in phosphate-buffered saline (10 mM phosphate, 140 mM NaCl [PBS]) and instilled into the nostrils of non-anesthetized mice with a micropipettor (5 μl/nostril). Five independent experiments were performed.

(39) Analysis of the Humoral Response:

(40) IgG synthesis against Toxoplasma antigens was monitored in sera 14 days after the third intranasal administration. The experimental protocol is described in reference to FIG. 1. The results are shown in FIG. 4. FIG. 4 shows the optical density of sera obtained from the above-mentioned treated mice. The specific antibody titer is given as the reciprocal of the highest dilution producing an optical density (OD) that was 2.5-fold greater than that of untreated mice sera at the same dilution. Results are expressed as the mean log titers and standard deviation (S.D).

(41) As shown in FIG. 4, specific Toxoplasma IgGs are detected in the groups of mice treated with the mixture of TE and DGNP and the mixture of TE and CT. As shown in FIG. 4, no significant difference was observed between the two groups. No IgGs were detected in the group of mice immunized with TE, CT, or DGNP alone.

(42) Analysis of the Cellular Response:

(43) To investigate the cellular immune response induced after treatment as above-described, the supernatants of cultured cells from the spleens of 2 mice from the hereinbefore identified groups were evaluated for the production of IFN-γ, IL-10, IL-13, TNF-α, IL-5, and IL-12 in response to TE restimulation with an ELISA analysis and IFN-γ with an Elispot. The results are shown in FIG. 5.

(44) FIG. 5 shows the concentration of IFN-γ (pg/mL) in supernatants of cultured splenocytes for two mice of each group as previously defined.

(45) As shown in FIG. 5, both mice immunized with the mixture of TE and DGNP; and mice treated with the mixture of TE and TC, responded to TE stimulation by the production of IFN-γ by spleen cells. Mice immunized with a mixture of TE and DGNP produce more than 400 pg/mL of IFN-γ. Mice treated with TE and CT produce around 1400 pg/mL of IFN-γ.

(46) The IFN-γ production by splenocytes was also measured two weeks after the third intra-nasal administration. The results are shown in FIG. 6.

(47) FIG. 6 shows the IFN-γ production for two mice of each group shown in FIG. 4. As regards the group treated with a mixture of DGNP and TE (DGNP-TE), and the group treated with a mixture of TE and CT (TE-CT), the results are obtained for the mice referred to by surrounded crosses. As shown in FIG. 6, the production of IFN-γ by splenocytes is increased in the mice vaccinated with a mixture of DGNP and TE. IL-12, IL-13, TNF-α, and IL-5 release was not detected in any of the samples analyzed.

(48) Test of Infection

(49) Mice treated with three intranasal administrations of DGNP alone, TE alone, the mixture of TE and DGNP, CT alone, and the mixture of CT and DGNP, respectively, were orally infected with cysts of the 76K strain of T. gondii. A group of non-treated mice was also infected as a control.

(50) Percentage of Survival

(51) The survival of each group was followed up during 30 days after the infection with 80 cysts. The total number of tested animals in each group is n=8.

(52) Except one mouse in the group control, all mice rapidly show clinical symptoms of disease, lost body weight and were dead within 11 days after infection, while 100% of the mice vaccinated with the mixture of TE and DGNP survived over the experimental period of 30 days.

(53) Number of Cysts in Mice Brain

(54) Mice of each treated group were orally infected with 50 cysts and sacrificed one month after the oral infection. Protection against T. gondii was evaluated by measuring mouse brain cyst number (three experiments with 50 cysts). The total number of tested animals in each group is n=8. The protocol is as described in reference of FIG. 3. The results are shown in FIG. 7.

(55) FIG. 7 shows the number of cysts in the mice brains of each group. As shown in FIG. 8, mice treated with the mixture of TE and DGNP before infection with T. gondii, have significantly fewer cysts than control mice (611 versus 1980 cysts, respectively; p<0.01), which represents a 70% reduction. The group of mice treated with a mixture of TE and DGPN has fewer cysts than the group treated with the mixture of TE and CT. The above-mentioned results suggest that vaccination with the mixture of DGNP and TE provides a long term protection.