Oil-in-water emulsions including retinoic acid

Abstract

Retinoic acid, or an analogue thereof, is delivered within an oil-in-water emulsion to provide an improved immunological adjuvant.

Claims

1. An oil-in-water emulsion including squalene; polysorbate 80; sorbitan trioleate and retinoic acid, wherein at least 80% (by number) of the emulsion's oil droplets are less than 220 nm in diameter and wherein the oil-in-water emulsion has 4-6% by volume squalene; 0.4-0.6% by volume polysorbate 80; 0.5% ±0.05 by volume sorbitan trioleate; and the retinoic acid is present at a concentration between 2 μg/mL to 2 mg/mL.

2. The oil-in-water emulsion of claim 1, wherein the emulsion is injectable.

3. An immunogenic composition comprising (i) the oil-in-water emulsion of claim 1, and (ii) at least one immunogen.

4. The oil-in-water emulsion of claim 1, wherein the retinoic acid is all-trans retinoic acid.

5. The oil-in-water emulsion of claim 1, wherein the emulsion does not contain one or more of oleic acid butylated hydroxytoluene, lecithin, medium-chain triglycerides, soybean oil, and/or cetyl palmitate.

6. The immunogenic composition of claim 3, wherein the retinoic acid is all-trans retinoic acid.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the proportion of (a) CD4.sup.+ and (b) CD8.sup.+ antigen-specific T cells after administration of (i) medium alone (ii) retinoic acid (iii) MF59 or (iv) retinoic acid and MF59. The y-axis shows the % of CCR9+ cells.

EXAMPLES

(2) This invention is further illustrated by the following examples, which should not be construed as limiting.

(3) Oil-in-Water Emulsions for Up-Regulating CCR9 on Antigen-Specific T Cells

(4) CCR9 is a key homing receptor involved in regulating effector T-cell migration to intestinal mucosa. MF59 and/or retinoic acid were tested for their effect on expression of CCR9 on CD4.sup.+ and CD8.sup.+ T-cells. A negative control was also tested.

(5) Antigen-presenting cells isolated from bone marrow were cultured with CD4.sup.+ or CD8.sup.+ antigen-specific T cells from transgenic mice with ovalbumin-specific T-cells (DO-11.10 or OT-I mice) in the presence of ovalbumin and adjuvant. FIG. 1 shows the proportion of (a) CD4.sup.+ and (b) CD8.sup.+ antigen-specific T cells which are CCR9-positive, with the highest proportion being seen when MF59 and retinoic acid were both administered (i.e. group (iv) in the FIGURES).

(6) Adoptive T-Cell Transfer

(7) Reference [57] describes experimental protocols for studying the homing of T-cells in mice who receive retinoic acid. In brief, T-cells specific for test antigens (in the present case, ovalbumin) are adoptively transferred into mice and their behaviour in response to specific treatments is studied. This general experimental procedure was used to assess the effect of MF59 and retinoic acid on CCR9 expression in T-cells, and on their in vivo migration.

(8) Mice were injected intravenously with 2×10.sup.6 CD4.sup.+ T-cells which react to ovalbumin peptides (DO-11.10 cells). These cells were labelled with CFSE before being administered. At around the same time they were given either retinoic acid or a plain vehicle. The next day they received MF59-adjuvanted ovalbumin, and the next day they again received RA or plain vehicle, plus fingolimod (which blocks the egress of lymphocytes from lymph nodes). A few days later their draining lymph nodes were drained and CD4.sup.+ T-cells expressing the DO-11.10 T-cell receptor were tested for CCR9 expression by FACS. The CFSE label was used to trace cell proliferation.

(9) CFSE labelling confirmed that T cells proliferated in mice receiving MF59, with no real difference between mice receiving RA or vehicle. In relation to CCR9 expression, however, a big difference was seen: in the mice who had received MF59 and the plain vehicle CCR9 expression was essentially the same as seen in naïve mice, but in the mice who had received retinoic acid the number of CCR9-positive cells was about 5× higher.

(10) In separate experiments a similar protocol was used, but without fingolimod administration, and antigen-specific CD4.sup.+ T-cells were assessed in both the draining lymph nodes and lungs 7 days after T-cells cells were injected. In both of the MF59-treated groups the proportion of ovalbumin-specific T-cells in the draining lymph nodes was essentially the same, but in lungs the levels were about twice as high for animals who received retinoic acid. Thus retinoic acid did not cause a general change in T-cell responses, but it did increase their migration towards mucosal tissues.

(11) Mucosal Antibody Responses

(12) To assess antibody responses mice were injected with 3 doses of either ovalbumin or H56 (a malaria antigen) on days 0, 21, and 42, adjuvanted with MF59. The day before and after each immunization one group of mice were given retinoic acid whereas control mice received a plain vehicle instead. Serum IgG and vaginal wash IgA antibody responses were assessed at day 56. IgA-secreting cells were also measured in the bone marrow.

(13) Serum IgG titres against ovalbumin or H56 were essentially the same in mice who received retinoic acid or the plain vehicle. In contrast, antigen-specific IgA titres in vaginal washes were essentially zero in mice who received plain vehicle, but were about 1250 in mice who had received retinoic acid. Moreover, the proportion of IgA-secreting cells was much higher in mice who had received retinoic acid: about 7× higher for ovalbumin, and around 12× higher for H56.

(14) Formation of Emulsions Including Retinoic Acid

(15) Three oil-in-water emulsions (A, B, C) were made with or without all-trans retinoic acid in the oil phase. Emulsion A is similar to ‘MF59’ [13]; emulsion B is similar to ‘AS03’ [14]; and emulsion C is similar to ‘SE’ [aka. Stable Emulsion of Immune Design—see above].

(16) The components for test batches of each emulsion were as follows, with 60 mg retinoic acid (RA) also being included in the oil phase where appropriate: (A) oil phase: 100 mg Span 85, 860 mg squalene; aqueous phase: 100 mg Tween 80, 19.5 ml 10 mM citrate buffer. (B) oil phase: 855.2 mg squalene, 948.8 mg±-α-tocopherol; aqueous phase: 388.8 mg Tween 80, 19.5 ml 1× phosphate-buffered saline. (C) oil phase: 380 mg lecithin, 2.02 mL squalene, 12 mg±-α-tocopherol; aqueous phase: 454 mg glycerol, 18 mg Pluronic F68, 2 mL 250 mM ammonium phosphate pH 5.1, 15.64 ml ultrapure water.

(17) Where retinoic acid did not dissolve in the oil phase it was first dissolved in dichloromethane (DCM) at 5 mg/mL and then added to the oil. The DCM was then allowed to evaporate at room temperature.

(18) To form the emulsions, the oil and aqueous phases were homogenised for about 2 minutes, and then up to 32 chums through a microfluidiser were used. 12 passes at 30,000 psi was generally fine.

(19) The emulsions without retinoic acid formed easily, and had the expected milky visual appearance. They were easily filtered. The emulsions with retinoic acid were yellow in colour and were more difficult to filter. The colour of emulsion B.sup.+RA changed from yellow to off-white after filtration. Precipitates were observed in emulsion A.sup.+RA. Emulsion C.sup.RA was very difficult to filter.

CONCLUSIONS

(20) MF59 in combination with retinoic acid (RA) induces expression of mucosal homing receptors on antigen-specific T lymphocytes in vitro, and also in vivo in draining lymph nodes.

(21) Antigen-specific T lymphocytes are able to reach mucosal compartments after immunisation in the presence of retinoic acid.

(22) Systemic and mucosal antigen-specific antibody responses are induced following systemic immunization with an oil-in-water emulsion adjuvant in the presence of retinoic acid.

(23) Thus mucosal homing capacity and mucosal antigen-specific responses induced by oil-in-water emulsion adjuvants can be improved by including retinoic acid. Such emulsions can be made using the methods disclosed herein.

(24) It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

REFERENCES

(25) [1] Lisulo et al. (2013) Clin Exp Immunol 175:468-75. [2] Ma et al. (2005) J Immunol 174:7961-69. [3] Ma & Ross (2005) PNAS USA 102:13556-61. [4] Tan et al. (2011) J Virol 85:8316-27. [5] Kusmartsev et al. (2003) Cancer Res 63:4441-49. [6] U.S. Pat. No. 6,461,622. [7] Simoni et al. (2001) Pure Appl. Chem. 73:1437-44. [8] Alvarez et al. (2004) Bioorg Medic Chem Lett 14:6117-22. [9] Muccio et al. (1998) J Med Chem 41:1679-87. [10] Kagechika (2002) Curr Med Chem. 9:591-608. [11] Le Doze et al. (2000) Drug Metab Dispos 28:205-8. [12] Sandberg et al. (1994) Drug Metab Dispos 22:154-60. [13] WO90/14837. [14] Garçon et al. (2012) Expert Rev Vaccines 11:349-66. [15] WO2010/023551 [16] Brito et al. (2011) Vaccine 29:6262-6268. [17] WO94/26683. [18] He et al. (2002) J Agric Food Chem 50:368-72. [19] WO2011/141819 [20] U.S. Pat. No. 8,092,813. [21] Light Scattering from Polymer Solutions and Nanoparticle Dispersions (W. Schartl), 2007. ISBN: 978-3-540-71950-2. [22] WO2011/067669 [23] WO2011/067673 [24] WO2011/067672 [25] WO90/14837. [26] Podda & Del Giudice (2003) Expert Rev Vaccines 2:197-203. [27] Podda (2001) Vaccine 19: 2673-2680. [28] Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X). [29] Vaccine Adjuvants: Preparation Methods and Research Protocols (Volume 42 of Methods in Molecular Medicine series). ISBN: 1-59259-083-7. Ed. O'Hagan. [30] WO2008/043774. [31] US-2007/014805. [32] US-2007/0191314. [33] Fox et al. (2013) Vaccine 31:1633-1640. [34] Allison & Byars (1992) Res Immunol 143:519-25. [35] Hariharan et al. (1995) Cancer Res 55:3486-9. [36] Suli et al. (2004) Vaccine 22(25-26):3464-9. [37] WO95/11700. [38] U.S. Pat. No. 6,080,725. [39] WO2005/097181. [40] WO2006/113373. [41] WO2007/052155. [42] WO01/22992. [43] Hehme et al. (2004) Virus Res. 103(1-2):163-71. [44] Treanor et al. (1996) J Infect Dis 173:1467-70. [45] Keitel et al. (1996) Clin Diagn Lab Immunol 3:507-10. [46] Banzhoff (2000) Immunology Letters 71:91-96. [47] WO02/097072. [48] Chinsriwongkul et al. (2010) PDA J Pharm Sci Tech 64:113-23. [49] Chinsriwongkul et al. (2011) AAPS Phar Sci Tech DOI: 10.1208/s12249-011-9733-8. [50] Greenbaum et al. (2004) Vaccine 22:2566-77. [51] Zurbriggen et al. (2003) Expert Rev Vaccines 2:295-304. [52] Piascik (2003) J Am Pharm Assoc (Wash DC). 43:728-30. [53] Mann et al. (2004) Vaccine 22:2425-9. [54] Halperin et al. (1979) Am J Public Health 69:1247-50. [55] Herbert et al. (1979) J Infect Dis 140:234-8. [56] Chen et al. (2003) Vaccine 21:2830-6. [57] Hammerschmidt et al. (2011) J Clin Invest 121:3051-61.