DRY PHARMACEUTICAL COMPOSITION FOR INHALATION

Abstract

The present invention relates to a dry pharmaceutical composition for inhalation comprising an antigen and an amphiphilic immune stimulant, wherein said pharmaceutical composition was produced by spray-drying; and to a dry pharmaceutical composition obtained or obtainable by spray-drying a solution comprising an antigen, an amphiphilic immune stimulant, and, optionally, a bulking agent. The present invention also relates to said dry pharmaceutical composition for use in medicine, in particular for use in vaccination of a subject; and to methods and kits related thereto.

Claims

1. A dry pharmaceutical composition for inhalation comprising an antigen and an amphiphilic immune stimulant, wherein said pharmaceutical composition was produced by spray-drying.

2. The dry pharmaceutical composition of claim 1 further comprising at least one bulking agent.

3. The dry pharmaceutical composition of claim 1, wherein said antigen is comprised at a proportion of from 0.1% (w/w) to 10% (w/w) in said dry pharmaceutical composition.

4. The dry pharmaceutical composition of claim 1, wherein said antigen is comprised at a proportion of from 0.3% (w/w) to 2.5% (w/w), preferably of about 2% (w/w) in said dry pharmaceutical composition.

5. The dry pharmaceutical composition of claim 1, wherein said amphiphilic immune stimulant is comprised at a proportion of from 0.1% (w/w) to 10% (w/w) in said dry pharmaceutical composition.

6. The dry pharmaceutical composition of claim 1, wherein said amphiphilic immune stimulant is an agonist of a toll-like receptor (TLR).

7. The dry pharmaceutical composition of claim 1, wherein said amphiphilic immune stimulant is monophosphoryl lipid A.

8. The dry pharmaceutical composition of claim 1, wherein said spray-drying comprises spraying a solution comprising the compounds as specified at elevated temperature into a stream of a gaseous drying agent.

9. The dry pharmaceutical composition of claim 1, wherein said antigen comprises at least one peptide comprising an amino acid sequence corresponding to amino acids 20 to 38 of the HPV16 L2 polypeptide.

10. A dry pharmaceutical composition obtained or obtainable by spray-drying a solution comprising an antigen, an amphiphilic immune stimulant, and, optionally, a bulking agent.

11. (canceled)

12. (canceled)

13. (canceled)

14. A method for manufacturing a dry pharmaceutical composition comprising spray-drying a solution comprising an antigen, an amphiphilic immune stimulant, and, optionally, a bulking agent.

15. A kit comprising the dry pharmaceutical composition according to claim 1 in a housing.

16. The dry pharmaceutical composition of claim 1, wherein said antigen is a thermostable polypeptide, wherein said antigen comprises a thioredoxin, and/or wherein said antigen comprises at least one antigenic epitope of a papillomavirus.

17. The dry pharmaceutical composition of claim 2, wherein the at least one bulking agent is selected from mannitol, lactose, and trehalose.

18. The dry pharmaceutical composition of claim 1, wherein said amphiphilic immune stimulant is an agonist of TLR4.

19. The dry pharmaceutical composition of claim 18, wherein said amphiphilic immune stimulant is selected from the list consisting of monophosphoryl lipid A, synthetic lipid A, lipid A analogs, lipid A mimetics, cytokines, saponins, lipopolysaccharide (LPS) of gram-negative bacteria, and endotoxins.

20. A method of treating and/or preventing an infection in a subject, comprising contacting said subject with a dry pharmaceutical composition according to claim 1, thereby treating and/or preventing an infection.

21. The method of claim 20, wherein said contacting comprises inhalation of said dry pharmaceutical composition.

Description

FIGURE LEGENDS

[0042] FIG. 1. SDS-PAGE fractionation and fluorescence analysis of soluble tissue supernatants (20,000×g, 15 min) derived from lung (L) and trachea (T) explants from mice injected intro-tracheally with the dry-powder formulated vaccine containing the PfTrx-HPV16-L2x3 antigen pre-labeled with Alexa Fluor 750. The results obtained with biological replicate tissue samples derived from two mice (L1, T1; L2, T2) arc shown; the Alexa Fluor 750-labeled input antigen (C+) and a lung tissue sample derived from a mouse not injected with the fluorescently labeled dry-powder vaccine (C−) served as positive and negative controls, respectively.

[0043] FIG. 2. Evaluation of the immunogenicity of the dry-powder formulated HPV-L2 vaccine. (A) Immunization and blood collection schedule. (B) Results obtained from GST-L2 ELISA testing of individual mice (represented by the indicated symbols) injected: i) subcutaneously with the antigen-lacking powder (#1; negative control); ii) subcutaneously with the soluble, doubly-adjuvanted vaccine (20 μg of PfTrx-HPV16-L2x3 antigen) (#2; positive control); intra-tracheally with the dry-powder formulated HPV-L2 vaccine (1 mg total powder corresponding to approximately 20 μg of the PfTrx-HPV16-L2x3 antigen) (#3; test sample); subcutaneously with the mono-adjuvanted, dry-powder pre-formulated vaccine (#4; same amount as in #3) dissolved in PBS. The measured anti-L2 antibody titers, determined with the use of a horseradish peroxidase-conjugated secondary antibody and the chromogenic substrate o-phenylenediamine (read spectrophotometrically at 450 nm), are shown on the y-axis. (C) Immunoglobulin isotypes determined at a fixed immune-serum dilution using rat anti-mouse Ig subclass-specific, horseradish peroxidase-conjugated secondary antibodies.

[0044] FIG. 3. Comparative analysis of immune sera derived from mice injected intra-tracheally with dry-powder formulated vaccines containing either the reference antigen PfTrx-HPV16-L2x3 (#3) or the modified OVX313-PfTrx-HPV-L2x3 antigen (#5) as active ingredients. Immunogenicity, expressed as anti-HPV-L2 antibody titers, was determined by GST-L2 capture ELISA as described in FIG. 2 legend.

[0045] The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

EXAMPLE 1

[0046] The vaccine was produced by spray-drying, using mannitol as a bulking agent, starting from a 70:30 v/v water ethanol solution. The antigen was typically dissolved in a potassium phosphate solution at a concentration ranging from 1 to 25 mM. To prepare 100 mL of feed solution to be spray-dried, 58.7 mg of mannitol were dissolved in 69 mL of purified water, to which 1 mL of a potassium phosphate aqueous solution typically containing 10 mg of antigen was added. Monophosphoryl lipid A (MPLA; typically, 1.04 mg) was dissolved in 30 mL of ethanol (95% v/v) and the resulting solution was added to the pre-mixed solution containing mannitol and the antigen.

[0047] The final, complete solution was then sprayed with a Buchi 290 spray-drier set to the following process parameters: inlet temperature 125° C.; feed rate 3.5 ml/min; air flow 601 L/h; aspiration 37 m.sup.3/h.

[0048] The antigen concentration may range from 0.31 to 2.00% w/w with respect to the final formulation. When dissolved in water the powder yields a pH of 7.75. The particle size distribution of the resulting powder was Dv10 1.43+/−0.09 μm; Dv50 2.65+/−0.17 μm; Dv90 4.78+/−0.55 μm. The powder is stable at room temperature for at least 1 year.

[0049] The aerodynamic performance of the vaccine, measured upon in vitro/laboratory aerosolization according to Ph. Eur 0.9.0 with a RS01® (Plastiape) inhalation device, results in an emitted fraction of 81.35+/−11.2% of the loaded dose and a respirable fraction of 74.86+/−12.04% of the emitted dose.

EXAMPLE 2

[0050] To monitor effective delivery of the dry-powder vaccine to the lungs, the antigenic protein (i.e., Pyrococcus furiosus thioredoxin displaying three tandem repeats of a HPV16-L2 peptide epitope spanning amino acid positions 20-38 of minor capsid protein L2; hereafter designated as PfTrx-HPV16-L2x3) was labeled with Alexa Fluor 750 prior to incorporation into the inhalator powder formulation by spray-drying. The vaccine-containing powder (1.7 and 2.0 mg corresponding to 10 μg and 12 μg of PfTrx-HPV16-L2x3 antigen, respectively) was then administered to two Balb/c mice with the use of a Penn Century microsprayer device. This was followed by mouse sacrifice and organ (lungs, trachea) explant after 15 min, tissue homogenization, centrifugation at 20,000×g for 15 min at 4° C., and fractionation of the resulting soluble supernatant on a denaturing, SDS-containing polyacrylamide gel (11%), which was then visualized by near-infrared fluorescence (NIR) imaging (Odyssey, Li-Cor). As shown in FIG. 1, a specific NIR signal associated to a polypeptide band displaying the molecular weight (18294.3 Da) expected for PfTrx-HPV16-L2x3 was detected in the lung (L) and in the trachea (T) samples.

EXAMPLE 3

[0051] Having shown that the dry-powder formulated PfTrx-HPV16-L2x3 antigen can reach the respiratory tract upon mouse intra-tracheal delivery (a surrogate of autoinhaler-assisted self-administration in humans), the MPLA-adjuvanted dry-powder vaccine was evaluated for immunogenicity. To this end, the PfTrx-HPV16-L2x3 antigen was exchanged into PBS buffer and detoxified by Triton X-114 treatment, prior to spray-drying-mediated incorporation into the MPLA-containing powder and intra-tracheal administration with the use of a Penn Century microsprayer device to 6-8 weeks-old female BALB/c mice. These were subdivided into four groups:

[0052] 1) A negative control group, consisting of 5 mice to which the empty powder (i.e., the MPLA-containing powder lacking the PfTrx-HPV16-L2x3 antigen as the ‘active ingredient’ (1 mg) was administered subcutaneously.

[0053] 2) A positive control group consisting of 7 mice to which the soluble, Alum (50+MPLA (10 μg) adjuvanted PfTrx-HPV16-L2x3 vaccine (20 μg) was administered subcutaneously.

[0054] 3) A test group consisting of 10 mice to which the dry-powder formulated PfTrx-HPV16-L2x3 vaccine (1 mg total powder corresponding to approximately 20 μg of protein antigen) was administered intra-tracheally.

[0055] 4) An internal comparison group consisting of 7 mice to which the dry-powder pre-formulated vaccine (same amount as in #3) dissolved in PBS immediately prior to subcutaneous administration, was administered.

[0056] The immunization protocol consisted of a priming immunization, followed by two boosts at weekly intervals (FIG. 2a) and was preceded by the sampling of pre-immune scrum to be used as a background reference in subsequent immuno-assays. Two weeks after the last immunization, mice were sacrificed, blood was collected via cardiac puncture, and used for the preparation of immune-sera, which were stored at −80° C. till subsequent immunological analyses. These were conducted by an indirect GST-L2 capture enzyme-linked immunosorbent assay (ELISA) to measure total anti-L2 scrum antibody titers, and by a quantitative isotype-specific, indirect double sandwich ELISA to determine the specific immunoglobulin (Ig) isotypes and sub-types elicited by each type of immunization.

[0057] As shown in FIG. 2b, anti-HPV L2 antibody titers measured in group #3 (dry-powder vaccine delivered intra-tracheally) ranged from 25 to 800, with an average value of 1:255, which was not significantly different (P<0.2790) from the titer elicited by a standard subcutaneous immunization conducted with the doubly adjuvanted (Alum+MPLA) PfTrx-HPV16-L2x3 vaccine (group #2). Under the same experimental conditions, no above background immune response could be detected in the antigen-lacking negative control group (#1), whereas antibody titers similar to (or slightly higher than, but without statistical significance) those measured in the doubly-adjuvanted positive control group (#2) were detected upon subcutaneous immunization with the mono-adjuvanted, PBS-dissolved dry-powder vaccine.

[0058] As further shown in FIG. 2 (panel c), overall similar 1g isotypes (and sub-types) were measured in the three immunized groups (#2, #3 and #4), but with a reproducible skewing toward a prevalent IgG2b sub-type and a concomitant reduction of the early formed (and poorly matured) pentameric IgM isotype in the case of the dry-powder formulated vaccine, either administered directly (i.e., intra-tracheally; group #3) or after solubilization and subcutaneous injection (group #4), compared to the soluble, doubly-adjuvanted vaccine administered subcutaneously.

EXAMPLE 4

[0059] To evaluate the versatility of the above described dry-powder vaccine formulation procedure, an alternative HPV-L2x3 antigen (designated as OVX313-PfTrx-HPV-L2x3) sharing the same immune-epitope, but differing both in size (247560 Da vs. 18294 Da) and total net charge (+6 vs. +2) from the reference PfTrx-HPV16-L2x3 antigen and with a reportedly higher immunogenicity, was subjected to spray-drying and converted into an MPLA-adjuvanted powder form. To test the immune performance of this alternative dry-powder vaccine, 1 mg of OVX313-PfTrx-HPV-L2x3-containing powder (corresponding to approximately 20 μg of protein antigen) were administered intra-tracheally to 13 Balb/c mice following the same immunization schedule described in Example 2 (sec FIG. 2a). After mouse sacrifice and blood collection, the immune-sera were analyzed by GST-L2 capture ELISA and the resulting anti-HPV L2 titers were compared with those obtained with the dry-powder formulated and intra-tracheally administered PfTrx-HPV-L2x3 vaccine. As shown in FIG. 3, a slightly higher anti-HPV L2 antibody titer (average value of 1:500, compared to the average 1:255 titer achieved with PfTrx-HPV-L2x3), was obtained upon pulmonary vaccination with the dry-powder formulated OVX313-PfTrx-HPV-L2x3 vaccine.

NON-STANDARD LITERATURE CITED

[0060] Nardelli-Haefliger et al. (2005), Vaccine, 23:3634-3641 [0061] WO 2010/003465 [0062] WO 2010/070052 [0063] WO 2017/211886