Formulations of Viable Cells for Oral Delivery
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(19) TZZ ¥_Z_T (11) EP 2 384 190 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: A61K 9/20 (2006.01) A61K 9/50 (2006.01) 26.07.2017 Bulletin 2017/30 A61K 35/74 (2015.01) (21) Application number: 10700756.9 (86) International application number: PCT/GB2010/000035 (22) Date of filing: 11.01.2010 (87) International publication number: WO 2010/079343 (15.07.2010 Gazette 2010/28) (54) FORMULATIONS OF VIABLE CELLS FOR ORAL DELIVERY FORMULIERUNGEN AUS LEBENSFÄHIGEN ZELLEN FÜR ORALE FREISETZUNG FORMULATIONS DE CELLULES VIABLES POUR UNE ADMINISTRATION ORALE (84) Designated Contracting States: WO-A1-2007/140613 WO-A2-2004/076657 AT BE BG CH CY CZ DE DK EE ES FI FR GB GR WO-A2-2007/079147 GB-A- 825 480 HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL US-A- 5 942 242 US-A1- 2006 093 591 PT RO SE SI SK SM TR • DATABASE WPI Section Ch, Week 200377 (30) Priority: 09.01.2009 GB 0900350 Thomson Scientific, London, GB; Class D13, AN 2003-820419 XP002587845 KAJIYA T; MORI H; (43) Date of publication of application: OKADA H; ONISHI H; SAWADA S; TAKAGI S; 09.11.2011 Bulletin 2011/45 TASHIROM: "Foodstuff containing Lactobacillus for use as health food for reducing (73) Proprietors: hypercholesterol level, comprises mixture of • Cambridge Enterprise Limited microbial cells belonging to Lactobacillus genus Cambridge having bile acid binding capacity and Cambridgeshire CB2 1TN (GB) cholestyramine" -& JP 2003 235501 A • Prokarium Limited (ANDERSEN INST BREAD & LIFE CO LTD; Keele Science Park TAKAKI BAKERY KK) 26 August 2003 Keele (2003-08-26) Staffordshire ST5 5SP (GB) • DATABASE WPI Section Ch, Week 200461 Thomson Scientific, London, GB; Class B04, AN (72) Inventors: 2004-630668 XP002587846 MIYAO M: • EDWARDS, Alexander "Formulation such as tablet and capsule Reading containing drug and foodstuff, comprises enteric RG6 6AD (GB) solublecoating on composition which comprises • SLATER, Nigel beneficial live microbial powder and bile Cambridge CB2 3RA (GB) adsorption components" -& JP 2004 250338 A (SUNSTAR CHEM IND CO LTD) 9 September 2004 (74) Representative: Sutcliffe, Nicholas Robert et al (2004-09-09) Mewburn Ellis LLP City Tower 40 Basinghall Street London EC2V 5DE (GB) (56) References cited: EP-A1- 1 281 752 EP-A1- 1 421 945 WO-A1-97/41741 WO-A1-2007/112747 Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 384 190 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 384 190 B1 • FORD R P ET AL: "The effect of a rat plasma • EDWARDS ALEXANDER D; SLATER NIGEL K H: high-density lipoprotein subfraction on the "Formulation of a live bacterial vaccine for stable synthesis of bile salts by rat hepatocyte room temperature storage results in loss of acid, monolayers" FEBS LETTERS, ELSEVIER, bile and bile salt resistance" VACCINE, vol. 26, AMSTERDAM, NL LNKD- no. 45, 23 October 2008 (2008-10-23), pages DOI:10.1016/0014-5793(85)80215-5, vol. 179, no. 5675-5678, XP002587847 cited in the application 1, 1 January 1985 (1985-01-01), pages 177-180, XP025597771 ISSN: 0014-5793 [retrieved on 1985-01-01] • EDWARDS A D ET AL: "Protection of live bacteria from bile acid toxicity using bile acid adsorbing resins"VACCINE, ELSEVIER LTD, GB, vol.27, no. 29, 12 June 2009 (2009-06-12) , pages 3897-3903, XP026802345 ISSN: 0264-410X [retrieved on 2009-04-23] cited in the application 2 1 EP 2 384 190 B1 2 Description ing buffer is currently the standard mode of delivery in ongoing human clinical trials of vaccine strains for dis- [0001] This invention relates to formulations for the oral eases such as Cholera [9] and Enterotoxic E. coli [10]. delivery of viable bacterial vaccine cells. [0008] The stomach has the ability to secrete hydro- [0002] Oral live bacteria delivery is currently used in 5 chloric acid and reduce the contents below pH 1. Few two main types of therapeutic application. Firstly, un- bacteria can survive to this pH [11] and dried live bacteria modified enteric bacteria strains identified from gut iso- have been shown to be even more sensitive than bacteria lates are of therapeutic interest in re-colonisation of the from fresh cultures [12]. An ’enteric’ coating may be used intestine after antibiotic treatment [1], in symptomatic re- to avoid the death of the live bacterial formulations in lief for sufferers of inflammatory intestinal disease [2], 10 gastric acid. An enteric coating is a polymer layer that and also in genetically engineered forms for delivery of will not dissolve in acidic conditions, but will dissolve in biological therapies (e.g. IL-10 [3]). Secondly, live bac- the intestine after stomach emptying, releasing the dry teria are attractive candidate vaccines. Currently, a bacteria into the upper small intestine. As noted above, number of human clinical trials are in progress testing existing enteric capsules of a live bacterial vaccine are various strains of attenuated live bacteria for protection 15 relatively ineffective, in spite of their ability to protect bac- against diseases such as Cholera, enterotoxic E. coli and teria from stomach acid [8]. Typhoid fever ([4]). Live bacterial vaccines have the ad- [0009] The intestine contains a complex mixture of en- vantage over conventional vaccines that they are admin- zymes and other microbicidal agents. Bile is secreted istered orally, avoiding injections and needles. Geneti- into the duodenum from the gall bladder. Bile generally cally engineered live bacterial vaccines may also carry 20 comprises at least 50% bile acids, which act as deter- heterologous DNA or antigens from pathogens or tu- gents to dissolve and digest fatty food components. Bile mours to stimulate or prime immune responses against acids are also potent at killing bacteria. Bacterial strains pathogen or tumour cells. that typically reside in intestinal sites, such as Salmonella [0003] Bacteria for live delivery are generally dried for spp, have evolved resistance mechanisms to detergents stability reasons. The alternative to dry formulation for 25 such as bile salts, allowing them to survive and grow in live bacterial delivery is a ’wet’ format, e.g. a food product the intestine. such as yoghurt. Live bacteria have significant stability [0010] Bacteria generally lose their resistance to bile problems in liquid forms, even if refrigerated. acids after drying or freezing. Dried bacteria which are [0004] A number of microencapsulation systems have released into the intestine from an enteric formulation are been proposed for oral delivery of bacteria [5], but all30 sensitive to bile acids and do not survive in the intestinal suffer from the same issue of degradation of the bacteria environment [12]. during storage, and a requirement for costly refrigeration [0011] The use of formulations comprising a first im- to reduce degradation. munogen and one or more second immunogens to de- [0005] Various techniques for producing dried bacteria liver the first immunogen to particular locations within the are known in the art. Most commonly, conventional35 gastrointestinal tract has been reported [18]. freeze-drying is used. In addition, there has been recent [0012] The present invention relates to the finding that focus on the use of di-saccharides such as trehalose and the incorporation of small amounts of a bile binding agent sucrose, which stabilise biomolecules and make dried intoformulations of dried viable cellssignificantly increas- bacteria more stable at room temperature [6], [7]. Disac- es the survival of the cells in the intestinal tract. According charide-stabilised dry bacteria, for example, may be con- 40 to the invention, the bile binding agent is cholestyramine veniently stored at room temperature. and the dried viable cells are dried viable bacterial vac- [0006] Currently, dried live bacteria are administered cine cells. This applies to the rest of the specification, orally in two common formats: enteric capsules of freeze- whenever reference is made to the invention. These for- dried bacteria, and sachets of freeze-dried bacteria to be mulations may therefore be useful for the oral delivery of resuspended in a bicarbonate buffer. Often, very high 45 live bacterial vaccine cells to the intestine. A first aspect doses of bacteria are given, and multiple doses are need- of the invention provides a solid formulation comprising ed to give only modest effects. Interestingly, clinical data dried viable bacterial vaccine cells and a bile binding indicates that the enteric capsule formulation is signifi- agent which is cholestyramine for use in a method of cantly less effective than the bicarbonate buffer system immunotherapy, as set out in claim 1. A second aspect [8][15], although little is known about why the enteric cap- 50 of the invention provides a method of producing the solid sules are relatively ineffective. formulation for use according to claim 1, said method [0007] Little is known about the mechanism of action comprising the steps according to claim 12. of the bicarbonate buffer system. The quantity of bicar- [0013] The bile binding agent allows aqueous liquids bonate buffer swallowed is not likely to absorb the acid to permeate the formulation but absorbs bile acids and secreted into the stomach, and so it seems likely that 55 retards their permeation through the formulation. This bacteria administered in this way will encounter some protects the dried viable cells from bile acid toxicity until acidity.