Intradermal delivery of immunological compositions comprising toll-like receptor 7 agonists

Abstract

An intradermal delivery system comprises an immunogenic composition comprising a TLR agonist and immunogen and a microneedle. The immunogenic composition may comprise a solid biodegradable microneedle or a solid coated microneedle. The intradermal delivery system may be formulated into a skin patch.

Claims

1. An intradermal delivery system comprising a microneedle, wherein the microneedle comprises a Toll-like Receptor 7 (TLR7) agonist and a bacterial antigen or a viral antigen, wherein the TLR7 agonist is a TLR7 agonist having formula T1: ##STR00014## wherein R.sup.1 is C.sub.1-C.sub.6alkyl, -L.sup.2R.sup.5, -L.sup.2R.sup.6, —OL.sup.2R.sup.5, or —OL.sup.2R.sup.6; R.sup.2 is C.sub.1alkyl; R.sup.3 is selected from C.sub.1-C.sub.4alkyl, -L.sup.3R.sup.5, -L.sup.3R.sup.7, —OL.sup.3R.sup.5, —OL.sup.3R.sup.7, —OR.sup.8, and —C(R.sup.5).sub.2OH; L.sup.2 is C.sub.1-C.sub.6alkylene, or —((CR.sup.4R.sup.4).sub.pO).sub.q(CH.sub.2).sub.p—, wherein the C.sub.1-C.sub.6alkylene of L.sup.2 is optionally substituted with 1 to 4 fluoro groups; each L.sup.3 is independently selected from C.sub.1-C.sub.6alkylene and —((CR.sup.4R.sup.4).sub.pO).sub.q(CH.sub.2).sub.p— wherein the C.sub.1-C.sub.6alkylene of L.sup.3 is optionally substituted with 1 to 4 fluoro groups; R.sup.4 is H; R.sup.5 is —P(O)(OR.sup.9).sub.2, R.sup.6 is —CF.sub.2P(O)(OR.sup.9).sub.2 or —C(O)OR.sup.10; R.sup.7 is —CF.sub.2P(O)(OR.sup.9).sub.2 or —C(O)OR.sup.10; R.sup.8 is H or C.sub.1-C.sub.4alkyl; each R.sup.9 is independently selected from H and C.sub.1-C.sub.6alkyl; R.sup.10 is H or C.sub.1-C.sub.4alkyl; each p is independently selected from 1, 2, 3, 4, 5 and 6, and q is 1, 2, 3 or 4.

2. The intradermal delivery system of claim 1, wherein the microneedle is a hollow microneedle.

3. The intradermal delivery system of claim 1 comprising a solid biodegradable microneedle.

4. The intradermal delivery system of claim 1, wherein the microneedle is a solid microneedle.

5. A process for preparing the intradermal delivery system of claim 4, wherein the method comprises the steps of a) mixing the bacterial antigen or the viral antigen and the TLR7 agonist to form an immunogenic composition in which the immunogen has a concentration of 10 mg/ml-50 mg/ml and the TLR7 agonist has a concentration of 0.1 mg/ml-10 mg/ml and b) drying the immunogenic composition to form a solid microneedle.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: anti-H1N1 hemagglutination inhibition (HI) titers provided by intradermal and intramuscular delivery of influenza antigen and TLR agonists following two immunizations.

(2) FIG. 2: anti-H3N2 HI titers provided by intradermal and intramuscular delivery of influenza antigen and TLR agonists following two immunizations.

(3) FIG. 3: anti-B HI titers provided by intradermal and intramuscular delivery of influenza antigen and TLR agonists following two immunizations.

(4) FIG. 4: bactericidal titers provided by intradermal and intramuscular delivery of meningitidis antigens and TLR agonists.

MODES FOR CARRYING OUT THE INVENTION

(5) Immunogen Concentration

(6) Centrifugal Filtration

(7) Centrifugal filtration used a Millipore™ device with a 10 kDa cut-off, operated at 5000 rpm.

(8) Three centrifugation durations were tested: 15, 30 and 45 minutes. The retentate (concentrate) and filtrate were checked to see the location of an influenza virus hemagglutinin. FIG. 1 shows that the antigen is still in the retentate after 45 minutes. Antigen concentration was 3-fold after 15 minutes, 6-fold after 30 minutes, and 13-fold after 45 minutes. Antigen recovery was 40% after 15 minutes, 41% after 30 minutes, and 55% after 45 minutes. Thus 45 minutes was chosen for further work.

(9) In further work, antigen was lyophilised after centrifugation, to provide further concentration. Sucrose was used as the lyoprotectant, alone (at two different concentrations) or with mannitol. Lyophilised material was reconstituted. The reconstituted samples contained visible aggregates. Relative to the starting material, HA content (measured by ELISA) was concentrated as follows:

(10) TABLE-US-00001 Treatment Concentration (x) Starting material 1.0 x Addition of sucrose 2.3 x Addition of sucrose (higher concentration) 1.3 x Addition of sucrose + mannitol 0.8 x Sucrose, lyophilise, reconstitute 13.3 x  Sucrose + mannitol, lyophilise, reconstitute 8.5 x Centrifuge, sucrose, lyophilise, reconstitute 25.2 x  Centrifuge, sucrose (higher), lyophilise, reconstitute 28.4 x 

(11) Thus the combination of centrifugation and lyophilisation can provide a >25-fold concentration in influenza virus HA content. The two centrifuged samples were also assessed by SRID and they showed a 21.1× and 35.1× increase in HA content, with the higher sucrose level again giving better results.

(12) Ultrafiltration

(13) Ultrafiltration used an Amicon™ stir cell concentrator with a 10 kDa cut-off membrane made from regenerated cellulose, operated under pressurised nitrogen for 1 hour.

(14) If a lyophilisation was added, followed by reconstitution back into the pre-lyophilisation volume, the reconstituted material had a HA concentration (as measured by SRID) comparable to the starting material, indicating no loss of functional antigen. The reconstituted material was stable for >2 weeks.

(15) Intradermal Delivery of Immunogenic Compositions

(16) Influenza

(17) Immunization of Balb/c mice with 100 μl or 20 μl immunogenic composition was carried out using both intramuscular delivery and intradermal delivery. The composition included a trivalent influenza vaccine with a 1 μg HA dose of each of X181 H1N1 Cal, X187 H3N2 Perth and B/Brisbane and one of a group of adjuvants including TLR agonists. Mice were anaesthetized before immunization. For the mice receiving intradermal immunization, an area on the back of the mouse was shaved or plucked to remove the hair at the injection site. The site was swabbed with 70% ethanol. The needle was inserted, bevel up, with the needle held nearly parallel to the plane of the skin. Both the mice receiving intramuscular and intradermal immunization, a number of different TLR agonists were used and the doses administrated, the total volume of the composition used and the route of administration are shown in Table 1 below. Using volumes of 50 μl or less per site for intradermal delivery avoids tissue trauma.

(18) TABLE-US-00002 TABLE 1 Group Adjuvant Dose Total volume Route 1 — — 100 ul  IM 2 MF59 (1:1) 100 μl  IM 3 — — 100 μl  IM 4 — — 20 μl ID 5 T1a 50 μg 20 μl ID 6 T1b 50 μg 20 μl ID 7 T1b 100 μg  20 μl ID 8 T2 50 μg 20 μl ID 9 LTK63  5 μg 20 μl ID 10 α-GalCer  5 μg 20 μl ID 11 MPLA 25 μg 20 μl ID 12 MF59 (1:1) 20 μl ID

(19) The T1a adjuvant was formulated by dispersion in 0.05% carboxymethyl cellulose or 0.05% Tween80 and sonicated in a water bath. The T1b adjuvant was formulated by dispersion in 1× and sonicated in a water bath. The T2 adjuvant was formulated by dispersion in 10 mM ammonia solution and sonicated in a water bath. The LTK63 adjuvant [114] was formulated in 0.05M sodium phosphate and 0.2M L-arginine. The α-GalCer adjuvant was formulated in water and 0.05% Tween20 and sonicated in a water bath for 30 minutes at 37° C. The MPL adjuvant was formulated by aqueous dispersion using 0.5% TEoA/WFI.

(20) Two immunizations were carried out 28 days apart, and individual samples were analyzed for anti-H1N1, anti H3N2 and anti-B hemagglutination inhibition (HI) titers 14 and 28 days after the first immunization and 14 days after the second immunization. FIGS. 1-3 show the HI titers for anti-H1N1, anti-H3N2 and anti-B respectively.

(21) When administered intradermally, the influenza antigens induce comparable HI titers to those induced following intramuscular administration. An improved immune response was provided following a second intradermal immunization in the presence each of the TLR agonists compared to intradermal immune in the absence of a TLR agonist. The immune response provided by intradermal immunization with antigen alone or antigen plus MF59 is comparable to the immune response provided by intramuscular immunization with antigen alone or antigen plus MF59.

(22) Intradermal immunization with T1b (100 μg) or LTK63 (5 μg) provided a significantly improved immune response compared to both intradermal or intramuscular immunization using antigen alone.

(23) Neisseria meningitidis

(24) Immunization of CD1 mice with 100 μl immunogenic composition comprising a 10 μg dose of three N. meningitidis B antigens [74] and one of a group of adjuvants including TLR agonists was carried out using both intramuscular delivery and intradermal delivery. Mice were anaesthatized before immunization. An area on the back of the mouse was shaved or plucked to remove the hair at the injection site. The site was swabbed with 70% ethanol. The needle was inserted, bevel up, with the needle held nearly parallel to the plane of the skin. A number of different TLR agonists were used and the doses administrated, the total volume of the composition used and the route of administration are shown in Table 2 below.

(25) TABLE-US-00003 Group Adjuvant Dose Total volume Route 1 — — 100 ul IM 2 — — 100 μl ID 3 T1a 100 μg 100 μl IM 4 T1a 100 μg 100 μl ID 5 T1b 100 μg 100 μl IM 6 T1b 100 μg 100 μl ID 7 T2 100 μg 100 μl IM 8 T2 100 μg 100 μl ID 9 Alum/T1c 100 μg 100 μl IM 10 Alum/T1c 100 μg 100 μl ID

(26) The T1a adjuvant was formulated by dispersion in 0.05% carboxymethyl cellulose or 0.05% Tween80 and sonicated in a water bath. The T1b adjuvant was formulated by dispersion in 1× and sonicated in a water bath. The T2 adjuvant was formulated by dispersion in 10 mM NH.sub.3 solution and sonicated in a water bath.

(27) Immunizations were carried out, and individual samples were analysed for bactericidal activity. FIG. 4 shows the bactiericidal titers for each of the compositions tested.

(28) When administered intradermally, the MenB antigens induce higher bactericidal titers to those induced following intramuscular administration even without the presence of TLR agonists. Intradermal immunization with T2 provides an improved immune response compared to combination of the antigens with other adjuvants. The immune response provided following intradermal administration of meningitidis antigens and TLR agonists is comparable to the immune response provided following intramuscular administration of meningitidis antigens and TLR agonists.

(29) 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

(30) [1] Prausnitz et al. (2009) Curr Top Microbiol Immunol. 333:369-93. [2] Kang et al. (2012) Expert Rev Vaccines. 11(5):547-60. [3] Davidson et al. (2008) Chemical Engineering Research and Design 86:1196-1206. [4] Carey et al. (2011) PLoS ONE 6(7): e22442. [5] Bal et al. (2010) J Control Release. 147:218-24. [6] Donnelly et al. (2011) Pharm Res 28:41-57. [7] Koutsonanos et al. (2009) PLoS ONE 4(e): e4773. [8] Quan et al. (2009) PLoS ONE 4(9):e7152. [9] Matsuo et al. (2012) J Control Release 161:10-17. [10] US-2011/112509. [11] WO2009/040548. [12] Lee et al. (2008) Biomaterials 29(13):2113-24. [13] WO2007/030477. [14] U.S. Pat. No. 6,945,952. [15] U.S. Pat. No. 7,211,062. [16] Sullivan et al. (2010) Nature Med 16:915-920. [17] US-2009/0182306 [18] U.S. Pat. No. 7,182,747. [19] Oh et al. (2006) American Association of Pharmaceutical Scientists, 2006 Annual Meeting and Exposition. The AAPS Journal. 8(S2). [20] WO2007/127976. [21] Matsuo et al. (2012) J Control Release. 160(3):495-501. [22] Koutsonanos et al. (2012) Sci Rep. 2:357. [23] EP-A-2289843. [24] Gill & Prausnitz (2007) J Control Release 117:227-37. [25] WO2007/124393. [26] WO2007/061964. [27] WO2007/059289. [28] Jin et al. (2009) Biomed Microdevices. 11(6):1195-203. [29] Vrdoljak et al. (2012) J Control Release 159:34-42. [30] Rosenberg et al. (2010) J Immunol 184:136.20. [31] U.S. Pat. No. 4,666,886. [32] WO2009/118296. [33] WO2008/005555. [34] WO2009/111337. [35] WO2009/067081. [36] WO2007/040840. [37] WO2010/014913. [38] WO2011/049677. [39] WO2012/031140 [40] WO2011/119759. [41] US2010/0143301. [42] GB-A-2220211. [43] Myers et al. (1990) pages 145-156 of Cellular and molecular aspects of endotoxin reactions. [44] Ulrich (2000) Chapter 16 (pages 273-282) [45] Johnson et al. (1999) J Med Chem 42:4640-9. [46] Baldrick et al. (2002) Regulatory Toxicol Pharmacol 35:398-413. [47] Coler et al. (2011) PLoS ONE 6(1):e16333. [48] Johnson et al. (1999) Bioorg Med Chem Lett 9:2273-2278. [49] Evans et al. (2003) Expert Rev Vaccines 2:219-229. [50] Bazin et al. (2006) Tetrahedron Lett 47:2087-92. [51] Wong et al. (2003) J Clin Pharmacol 43(7):735-42. [52] US2005/0215517. [53] WO03/011223. [54] WO2007/053455. [55] De Libero et al, Nature Reviews Immunology, 2005, 5: 485-496. [56] U.S. Pat. No. 5,936,076. [57] Oki et al, J. Clin. Investig., 113: 1631-1640. [58] US2005/0192248. [59] Yang et al, Angew. Chem. Int. Ed., 2004, 43: 3818-3822. [60] WO2008/047174. [61] WO2008/047249. [62] WO2005/102049. [63] Goff et al, J. Am. Chem., Soc., 2004, 126: 13602-13603. [64] WO03/105769. [65] Peppoloni et al. (2003) Expert Rev Vaccines 2:285-93. [66] WO95/17211. [67] da Hora et al. (2011) Vaccine 29:1538-44. [68] WO93/13202. [69] Pizza et al. (2000) Int J Med Microbiol 290:455-61. [70] WO98/18928. [71] Feng et al. (2005) Acta Biochim Biophys Sin (Shanghai). 37(2):126-32. [72] Treanor et al. (1996) J Infect Dis 173:1467-70. [73] Keitel et al. (1996) Clin Diagn Lab Immunol 3:507-10. [74] Giuliani et al. (2006) Proc Natl Acad Sci USA. 103:10834-9. [75] WO03/097091. [76] Cassone & Torosantucci (2006) Expert Rev Vaccines 5:859-67. [77] Research Disclosure, 453077 (January 2002). [78] EP-A-0372501. [79] EP-A-0378881. [80] EP-A-0427347. [81] WO93/17712. [82] WO94/03208. [83] WO98/58668. [84] EP-A-0471177. [85] WO91/01146. [86] Falugi et al. (2001) Eur J Immunol 31:3816-3824. [87] Baraldo et al. (2004) Infect Immun 72(8):4884-7. [88] EP-A-0594610. [89] Ruan et al. (1990) J Immunol 145:3379-3384. [90] WO00/56360. [91] Kuo et al. (1995) Infect Immun 63:2706-13. [92] Michon et al. (1998) Vaccine. 16:1732-41. [93] WO02/091998. [94] WO01/72337. [95] WO00/61761. [96] WO00/33882 [97] U.S. Pat. No. 4,761,283. [98] U.S. Pat. No. 4,356,170. [99] U.S. Pat. No. 4,882,317. U.S. Pat. No. 4,695,624. Mol. Immunol., 1985, 22, 907-919 EP-A-0208375. Bethell G. S. et al., J. Biol. Chem., 1979, 254, 2572-4 Hearn M. T. W., J. Chromatogr., 1981, 218, 509-18 WO00/10599. Gever et al., Med. Microbiol. Immunol, 165: 171-288 (1979). U.S. Pat. No. 4,057,685. U.S. Pat. Nos. 4,673,574; 4,761,283; 4,808,700. U.S. Pat. No. 4,459,286. U.S. Pat. No. 4,965,338. U.S. Pat. No. 4,663,160. WO2007/000343. Remington: The Science and Practice of Pharmacy (Gennaro, 2000; 20th edition, ISBN: 0683306472) Tritto et al. (2007) J. Immunol. 179:5346-5357.