(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

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• 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. In spite of these potential limitations, co-admin- they are rehydrated by the permeating aqueous liquid istration with bicarbonate buffer or similar acid neutralis- and recover their bile tolerance. This may, for example,

3 3 EP 2 384 190 B1 4 increase the efficacy of delivery of the cells to the intes- cially, serovar Typhi). tine. Dried viable cells may be microbial cells. Enteric [0018] Live bacterial vaccines for use in the therapeutic bacterial are well-known in the art and include pathogen or prophylactic treatment of typhoid are well-known in and non-pathogenic species, for example, Enterobacte- the art and include Salmonella typhi strains Ty21a (Viv- riaceae such as Escherichia, Salmonella, Klebsiella, En- 5 otif™), Ty800, CVD908, CVD908-htr, CVD915, and ZH9. terobacter, Serratia, Proteus spp, and also Bifidobacte- [0019] Live bacterial vaccines for use in the therapeutic rium, Bacteroides, Clostridium, Pseudomonas, Rumino- or prophylactic treatment of cholera are also well known coccus, Lactobacillus, Lactococcus, Campylobacter, in the art and include JBK-70, CVD 101, CVD 102, CVD and Streptococcus spp. 104, CVD 105,395N1, JJM43, TCP2, CVD 103, CVD [0014] The delivery of enteric bacterial cells to the in- 10 103-RM, CVD 103-HgR (Mutachol®), CVD110, Bahrain- testine may be useful, for example, in restoring the gut 3, Peru-3, Peru-5 and Peru-15. flora of a patient, e.g. after antibiotic treatment, and in [0020] Live bacterial vaccines for use in the therapeutic the symptomatic relief of patients with inflammatory in- or prophylactic treatment of shigellosis and bacterial dys- testinal disease. Dried viable microbial cells may be eu- entryare also well knownin theart and include attenuated karyotic cells, for example yeast or fungal cells such as 15 vaccine strains of attenuated Shigella, such as CVD 1204 Saccharomyces, Aspergillus spp or Candida spp. The and WRSS1. delivery of microbial cells with specific enzymatic or other [0021] Live bacterial vaccines for use in the therapeutic properties to the intestine may be useful, for example, in or prophylactic treatment of diarrhoea are also well the breakdown or metabolism of disease associated known in the art and include attenuated enterotoxicE. compounds or toxins, such as oxalate, which is a risk 20 coli cells, such as PTL002 and PTL003. factor for renal stone formation, or in reducing cholesterol [0022] Live bacterial vaccines for use in the therapeutic levels, by influencingintestinal absorption. Microbial cells orprophylactic treatment of listeriosisare also well known may express a therapeutic factor, such as a recombinant in the art and include attenuated strains of Listeria mono- protein. Suitable recombinant polypeptides may exoge- cytogenes, such as Lmdd. nous to the bacterial cell (i.e. a polypeptide not naturally 25 [0023] A bacterial vaccine may comprise heterologous expressed by the bacterial cell, such as a human nucleic acid which encodes an exogenous antigen i.e. polypeptide), and may have a therapeutic effect on the an antigen not naturally expressed by the vaccine cell patient. Suitable recombinant polypeptides include cy- (for example a human or viral antigen or an antigen from tokines, including interleukins such as IL-10; chemok- different bacterial species). The bacterial vaccine may ines; and antibodies, antibody fragments or related bind- 30 stimulate or prime a protective immune response against ing molecules that bind to mediators of the immune re- the exogenous antigen, for example when the antigen is sponse, such as cytokines (e.g. to neutralise TNF-alpha displayed on the surface of a pathogen or a diseased in inflammatory bowel diseases). cell, such as cancer cell or a virally infected cell. [0015] Suitable microbialcells for expressionof recom- [0024] The term "heterologous" indicates that a nucleic binant polypeptides include cells from lactic acid bacterial 35 acid sequence is a recombinant sequence which has species, e.g. Lactococci such as L.lactis (European Jour- been introduced into a construct, vector or cell, artificially nal of Pharmaceutics and Biopharmaceutics (2005) using genetic engineering or recombinant means, i.e. by 60:349), Enterobacteriaceae and other enteric bacteria human intervention. Nucleotide sequences which are as described above, and other microbial cells such as heterologous do not occur together in nature. Nucleotide yeasts and fungi, for example Saccharomyces or Cand- 40 or amino acid sequences which are heterologous to a ida spp. cell are non-naturally occurring in cells of that type, va- [0016] In the invention, the dried viable cells are cells riety or species (i.e. exogenous or foreign). A nucleic acid of a live bacterial vaccine. Live bacterial vaccines are sequence which is heterologous to a cell may encode a generally bacterial cells whose virulence has been atten- heterologous amino acid sequence i.e. an amino acid uated, for example by the inactivation of one or more45 sequence which is exogenous or foreign to the cell. virulence genes. Many examples of attenuated bacterial [0025] In some embodiments, the heterologous nucle- cells suitable for use as vaccines are known in the art. ic acid may be expressed by the microbial cell to produce [0017] A bacterial vaccine may be an attenuated cell the exogenous antigen. The exogenous antigen may be of a bacterial pathogen. The expression of native endog- secreted from the microbial cell, displayed on the surface enous antigens of the pathogen by the attenuated vac- 50 of the microbial cell or remain intracellular within the mi- cine may stimulate or prime a protective immune re- crobial cell. sponse against virulent non-vaccine strains of the path- [0026] Inother embodiments, the heterologousnucleic ogen. Suitable bacterial vaccines include attenuated acid may not be expressed by the microbial cell. For ex- cells of cells of Vibrio cholerae, Enterotoxic strains of E. ample, the heterologous nucleic acid may be transferred coli, such as 0157:H7, 0121 and 0104:H21, Shigella spe- 55 to a host cell of the individual following delivery to the cies, such as S. dysenteriae, S. sonnei, S. boydii and S. intestine and expressed in the host cell. The heterolo- flexneri, Campylobacter species such as C. jejuni, and gous nucleic acid may be expressed to produce the ex- Salmonella species, such as Salmonella enterica (espe- ogenous antigen, for example in a method of DNA vac-

4 5 EP 2 384 190 B1 6 cination or DNA transfer, or may be expressed to produce gibbon), a bird (e.g. chicken), or a fish (e.g. salmon, trout). a sense or anti-sense RNA molecule, for example in a [0033] For example, formulations as described herein method of RNA interference. comprising dried viable salmonella cells may be useful [0027] Suitable exogenous antigens which may be en- as vaccines in the veterinary treatment of non-human coded by a heterologous nucleic acid include disease- 5 animals, such as pigs and horses. related antigens such as tumour antigens and antigens [0034] Suitable individuals may be identified or select- of viral, bacterial and other pathogens, for example an- ed as being in need of treatment. tigens derived from Streptococcus mutans, Shigella son- [0035] The formulation preferably comprises a thera- nei, Escherichia coli, Campylobacter jejuni, Mycobacte- peutically-effective or prophylactically-effective amount rium leprae, Mycobacterium tuberculosis, Vibrio choler- 10 of dried viable cells. ae, Yersinia pestis, Bacillus anthracis, HIV, Influenza vi- [0036] The terms "therapeutically-effective amount" or rus, hepatitis virus, papilloma virusPlasmodium falci- "prophylactically-effective amount" as used herein, per- parum, Francisella tularensis, and Schistosoma manso- tains to that amount of a dried viable cells which is effec- ni, or antigens derived from tumours such as Her2/neu tive for producing some desired therapeutic or prophy- or prostate specific antigen (PSA). 15 lactic effect, respectively, commensurate with a reason- [0028] Other suitable exogenous antigens include mol- able benefit/risk ratio. ecules from the individual such as antibody idiotypes, for [0037] A formulation described here may for example example for the generation of an anti-idiotypic immune comprise 103 or more, 104 or more, 105 or more, 106 or response. more, 107 or more, 108 or more, 109 or more, or 1010 or [0029] Optionally, the bacterial vaccine may also co- 20 more dried viable cells. express a recombinant heterologous immunomodulatory [0038] Dried viable cells in the formulations described polypeptide, for example a cytokine, such as TNF-alpha, herein retain at least 0.1%, at least 1%, at least 5%, at GM-CSF, IL-1, IL-6, IL-12, IL-18, IFN-alpha, IFN-gamma, least 10%, at least 20%, at least 40% or at least 60% a chemokine such as CCL-25, or an antibody or antibody viability following rehydration. A significant number of fragment or other binding polypeptide that inhibits cy- 25 cells within the formulation are therefore capable of met- tokines (e.g. IL-10, TGF-beta) or other immune response abolic activity and cell division, following rehydration. mediators or immune cells (such as regulatory or sup- Methods of assessing the viability of cells are well-known pressor T cells). This may be useful in increasing the in the art and include, for example plate assays for colony immune response to the exogenous antigen. forming units (CFU). [0030] Suitable bacterial vaccines for the expression 30 [0039] Preferably, the dried viable cells in the formu- of exogenous antigens include live bacterial vaccine lations described herein retain viability as set out above strains, such as attenuated pathogenic bacteria, such as when the formulation is stored at room temperature for Salmonella enteric, Vibrio cholerae, Enterotoxic E. coli, prolonged periods (e.g. 15°C to 30°C for 24 months or Shigella, and Campylobacter jejuni and non-pathogenic longer). bacterial cells, including enteric bacteria such as Entero- 35 [0040] Dried cells have reduced moisture content rel- bacteriaceae, and other bacteria such as Bacillus subtlis ative to undried cells. For example, dried cells may con- and lactic acid bacterial species, e.g. Lactococci spp tain 10% or less, 5% or less, 4% or less, 3% or less, or such as L. lactis (Nature Reviews Microbiology (2008) 1% or less residual moisture, relative to the same cells 6:349). before drying. [0031] The dried viable cells in the solid formulation 40 [0041] Cells suitable for use as described herein may may be a homogeneous population of the same type of be dried by any convenient method which does not sig- cells (for example, cells of the same strain of micro-or- nificantly affect cell viability. A range of suitable methods ganism). Alternatively, the dried viable cells in the solid are known in the art, including freeze-drying, ambient formulation may be a heterologous population containing temperature drying, spray drying, fluidised bed drying, a mixture of different types of cells. For example, the45 foam drying, directly drying during granulation for tablet- formulation may comprise cells of two or more, three or ting, and freezing with cryoprotectants (Potts (2004) Mi- more, four or more, or five or more different types. Dif- cro Rev 58:755-805; Crowe et al (1990) Cryobiol. ferent types of cells may include cells of different strains 27:219-231; Lievense et al (1994) Adv Biochem Eng Bi- or species of micro-organism and/or cells with different otechnol.51 45-89). Optionally, drying may be carried out genetic modifications. 50 in the presence of a stabilising agent, for example a dis- [0032] The formulations described herein may be ad- accharide, and/or a pharmaceutical excipient, for exam- ministered to any suitable individual. For example, the ple, a polymer such as hydroxy propyl methyl cellulose. individual may be a human or a non-human animal, such [0042] In some embodiments, the cells may be as a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), lyophilised by freeze-drying. Freeze-drying involves the murine (e.g. a mouse), canine (e.g. a dog), feline (e.g. a 55 removal of water from a frozen material by sublimation cat), equine (e.g. a horse), porcine (e.g. a pig), a primate, and desorption at reduced pressure. Suitable freeze-dry- simian (e.g. a monkey or ape), a monkey (e.g. marmoset, ing techniques are well known in the art. (F. Franks Eu- baboon), an ape (e.g. gorilla, chimpanzee, orang-utan, ropean Journal of Pharmaceutics and Biopharmaceutics

5 7 EP 2 384 190 B1 8

1997 (45)221-229; Fundamentals of freeze-drying JD amount of bile binding agent is sufficient to protect dried Mellor Academic Press 1978). Optionally, cells may be viable cells from bile acid toxicity. The amount of bile freeze-dried in the presence of a stabilising agent, for binding agent in a formulation described herein will gen- example a disaccharide such as trehalose (Israeli et al erally be significantly less than the amount required to (1993) Cryobiol 30 519-523; Leslie et al (1995) Appl. Env. 5 bind all the bile acids to which the formulation is exposed Microbiol. 61 3592-3597) and/or a pharmaceutical excip- in the intestine of an individual. ient or other components of the formulation. [0051] A solid formulation may be in a unit dosage form [0043] In some embodiments, the cells may be sub- and may comprise 5g or less, 2g or less, 1g or less or jected to disaccharide-stabilised drying. This may com- 0.5g or less of solid material. prise increasing the intracellular level of a disaccharide, 10 [0052] A solid formulation as described herein may such as trehalose, in the cells and then drying the cells comprise less than 95% (w/w), less than 90% (w/w), less in the presence of a carbohydrate stabilising agent, for than 80% (w/w), less than 70% (w/w), less than 60% example, by evaporation and at reduced pressure and (w/w), less than 50% (w/w), less than 40% (w/w), less non-freezing temperatures. Suitable carbohydrate sta- than 30% (w/w), less than 20% (w/w), less than 10% bilising agents include disaccharides such as trehalose, 15 (w/w), less than 1% (w/w) or less than 0.1% (w/w) of said maltitol, lactitol, palatinit, GPS (alpha-D-glucopyranosyl- bile binding agent. 1->6-sorbitol), GPM (alpha-D-glucopyranosyl-1->6- [0053] A solid formulation as described herein may mannitol), maltooligosaccarides, and hydrogenated mal- comprise greater than 5% (w/w), greater than 10% (w/w), tooligosaccarides. Suitable disaccharide-stabilised dry- or greater than 20% (w/w), greater than 30% (w/w), great- ing methods are well known in the art and are described, 20 er than 40% (w/w), greater than 50% (w/w), greater than for example in Crowe JH et al Annu Rev Physiol 1998; 60% (w/w), greater than 70% (w/w), greater than 80% 60:73-103. Bullifent HL, et al. Vaccine 2000 Dec 8; (w/w), greater than 90% (w/w), or greater than 95% (w/w) 19(9-10):1239-45; Garcia De Castro A, et al Appl Environ of said bile binding agent. Microbiol 2000 Sep; 66(9):4142-4; US6468782 and [0054] The ratio by weight of cells to bile binding agent US6610531. 25 in the formulation may be 5 or less, 1 or less, 0.5 or less, [0044] Bile acids are steroid acids which are produced 0.1 or less, 0.01 or less, 0.001 or less, or 0.0001 or less. in the liver by the oxidation of cholesterol and secreted [0055] The ratio by weight of bile binding agent to cells into the intestine as components of bile. Bile acids include in the formulation may be 0.2 or more, 1 or more, 10 or cholic acid, deoxycholic acid and chenodeoxycholic acid. more, 100 or more, 1000 or more, or 10000 or more. [0045] A bile acid binding agent or sequestrant is a30 [0056] In some preferred embodiments, the dried via- compound which binds, absorbs or sequesters bile acids. ble cells, bile binding agent and other components of the Suitable bile binding agents include positively charged formulation may be encapsulated in a polymeric shell. polymers which bind to the negatively charged carboxylic [0057] The polymeric shell may be useful in further in- acid head group of bile acids. Positively charged poly- creasing the bile resistance of the formulation. mers which bind to bile acids include anion-exchanging 35 [0058] Preferably, the polymeric shell comprises an resins, such as cholestyramine (e.g. Questran™ or amphiphilic hydrophobic-hydrophilic polymer, such as Dowex 1X2™), colestilan, colestimide, colextran hydro- hydroxypropyl methylcellulose (HPMC). Suitable poly- chloride, colesevelam HCl, colestipol, DEAE-dextran, meric shells are commercially available. aminated cellulose, DEAE-cellulose, TEAE-cellulose, Q- [0059] In some preferred embodiments, a solid formu- sepharose, QAE Sephadex, quaternised hydroxyethyl 40 lation as described herein may be covered with an enteric cellulose ethoxylate and aminated chitosan. coating. [0046] Suitable positively charged polymers are known [0060] An enteric coating is impermeable outer layer in the art for use as cholesterol lowering agents, and are that protects the dried viable cells from acid damage as described, for example in US4557930, US3308020, the formulation passes through the stomach and then US3383281, US3499960, US3780171, WO2005023305 45 allows the release of the cells when the formulation and GB929,391. reaches the intestine. The use of enteric coatings is well- [0047] Other suitable bile binding agents include hy- known in the art. drophobic polymers which bind to the hydrophobic cho- [0061] Enteric coatings may be pH sensitive. For ex- lesterol tail of bile acids. Suitable hydrophobic polymers ample, an enteric coating may be non-labile under acidic include polystyrene-related polymers. 50 conditions within the stomach (e.g. pH 1 to 3) but may [0048] Other suitable agents may also include natural- be labile at higher pH within the intestine (e.g. pH above ly occurring agents, such as dietary fibre, activated car- 5.5). bon, and bone char. In the invention, the bile binding [0062] Suitable polymeric materials for use in enteric agent is cholestyramine. coatings are well known in the art and may, for example, [0049] The bile binding agent may be in any shape or 55 include fatty acids, waxes, shellac™, cellulose acetate form within the formulation, for example granules, pow- phthalate (CAP), methacrylic acid copolymers, methyl ders, beads, membranes or porous monoliths. acrylate-methacrylic acid co-polymers, cellulose acetate [0050] The data set out herein shows that a small succinate, hydroxypropyl methylcellulose acetate succi-

6 9 EP 2 384 190 B1 10 nate, hydroxylpropylmethyl cellulose phthalate, polyvinyl other ingredients of the formulation. Suitable carriers, ex- acetate phthalate (PVAP), cellulose acetate trimellitate, cipients, etc. can be found in standard pharmaceutical carboxymethyl ethylcellulose, and methyl methacrylate- texts, for example, Remington: The Science and Practice methacrylic acid copolymers. of Pharmacy, 21st edition, Mack Publishing Company, [0063] A solid formulation as described herein may fur- 5 Easton, Pa., 2005; and Handbook of Pharmaceutical Ex- ther comprise one or more additional components, for cipients, 5th edition, 2006, Pharmaceutical Press, Lon- example to modify or adjust the properties of the formu- don. The formulation of the invention is produced by dry- lation for specific applications. ing viable bacterial cells and then admixing the dried vi- [0064] For example, a formulation may further com- able cells with a bile binding agent and optionally a phar- prise a delayed release agent which controls the release 10 maceutically acceptable carrier to produce a solid formu- of the cells from the formulation into the intestine, after lation. Alternatively, the formulation may be produced by dissolution of the enteric coating, for example by forming admixing viable bacterial cells with the bile binding agent a stable gelatinous layer that slowly erodes to release and optionally a pharmaceutically acceptable carrier to the cells. produce an admixture and the admixture may then be [0065] Suitable delayed release agents include hy-15 driedto producea solid formulation;wherein the bile bind- droxypropyl methylcellulose (HPMC), hydroxyethyl cel- ing agent is cholestyramine and the viable bacterial cells lulose, other modified celluloses, natural gums such as are bacterial vaccine cells. Suitable methods of drying guar gum, polyethylene oxide, polyvinyl alcohol, and poly are described in more detail above. (lactic-co-glycolic acid). [0071] The dried viable cells and bile binding agent [0066] Dried viable cells may be sensitive to toxins in 20 may be admixed with further pharmaceutically accepta- the intestine other than bile acids, for example lysozyme; ble carriers, adjuvants, excipients and/or other materials anti-microbial peptides such as defensins; and digestive described above; compressed, moulded or otherwise enzymes, such as pepsin, trypsin and lipase. A formula- made into a suitable formulation for oral delivery; and/or tion may further comprise agents to protect the cells from covered with an enteric coating to produce the formula- such toxins. For example, a formulation may comprise a 25 tion. cation-exchanging resin to bind and protect dried viable [0072] The resistance of the cells in the formulation to cells from positively charged enzymes, such as lys- exposure to bile may be determined. The amount of cells ozyme. and/or bile binding agent in the formulation may be al- [0067] Suitable cation-exchanging resins include ion tered to optimise the resistance of the cells to bile. For exchange resins such as Dowex 50wX1 resin, sodium 30 example, if reduced numbers of viable cells can be re- polystyrene sulfonate, or polymers such as car-covered from the formulation after bile exposure, relative boxymethylcellulose or alginic acid. to controls, then the amount of bile binding agent in the [0068] A formulation may further comprise stabilisers formulation may be increased. to maintain the viability of the dried cells. Examples of [0073] Suitable solid formulations for oral administra- suitable stabilisers include carbohydrates as described 35 tion may consist of one or more discrete units, such as above. tablets, pellets, pills or capsules. In some embodiments, [0069] A formulation may further comprise one or more a discrete unit such as a pill or capsule may comprise pharmaceutically acceptable carriers, adjuvants, excipi- multiple small sub-units, such as granules or powders. ents, binders, disintegrants, bulk agents, glidants, dilu- [0074] Solid formulations for oral administration may ents, fillers, buffers, stabilisers, preservatives, lubricants, 40 conveniently be presented in unit dosage form and may or other materials well known to those skilled in the art be prepared by any methods well known in the art. Such and optionally other therapeutic or prophylactic agents. methods may include the step of bringing into association In some embodiments, a formulation may further com- the dried viable cells and the bile binding agent with the prise one or more mucoadhesives which hold the formu- carrier which constitutes one or more accessory ingredi- lation in the intestine during release of the bacteria. Suit- 45 ents. In general, the formulations are prepared by uni- able mucoadhesives include polyvinyl pyrrolidone, poly- formly and intimately bringing into association the admix- acrylates, chitosan, methyl cellulose, carboxymethylcel- ture of dried viable cells and the bile binding agent with lulose, hydroxypropylcellulose and other cellulose deriv- finely divided solid carriers, and then if necessary shap- atives. ing the product. [0070] The term "pharmaceutically acceptable" as50 [0075] Formulations suitable for oral administration used herein pertains to compounds, materials, compo- (e.g. by ingestion) may be presented as discrete units sitions, and/or dosage forms which are, within the scope such as capsules, cachets or tablets, each containing a of sound medical judgement, suitable for use in contact predetermined amount of the dried viable cells and the with the tissues of a subject (e.g. human) without exces- bile binding agent; for example as a powder or granules. sive toxicity, irritation, allergic response, or other problem 55 [0076] A tablet may be made by conventional means, or complication, commensurate with a reasonable ben- e.g. compression, moulding, wet granulation or dry gran- efit/risk ratio. Each carrier, excipient, etc. must also be ulation, optionally with one or more accessory compo- "acceptable" in the sense of being compatible with the nents, such as binders (e.g. povidone, gelatin, acacia,

7 11 EP 2 384 190 B1 12 sorbitol, tragacanth, hydroxypropylmethyl cellulose); fill- [0083] Optionally, the layer of bile binding agent may ers or diluents (e.g. lactose, microcrystalline cellulose, further comprise a delayed release material. Optionally, calcium hydrogen phosphate); lubricants (e.g. magnesi- the core and the layer of bile binding agent may be en- um stearate, talc, silica); disintegrants (e.g. sodium capsulated in a polymeric shell. Optionally, the formula- starch glycolate, cross-linked povidone, cross-linked so- 5 tion may further comprise an enteric coating which sur- dium carboxymethyl cellulose); surface-active or dis- rounds the layer of bile binding agent. persing or wetting agents (e.g. sodium lauryl sulfate); and [0084] Formulations as described herein are stable at preservatives (e.g. methyl p-hydroxybenzoate, propyl p- room-temperature i.e. cells remain viable after prolonged hydroxybenzoate, sorbic acid). For example, com- storage at room-temperature (e.g. 15°C to 30°C). pressed tablets may be prepared by compressing in a 10 [0085] Combinations of the bile binding agent and the suitable machine the active compound in a free-flowing dried viable cells, e.g. in a formulation as described form such as a powder or granules, optionally mixed with above, are useful in delivering live cells to the intestine one or more accessory components. Moulded tablets of an individual. maybe made by moulding in a suitable machinea mixture [0086] Formulations as described herein may be use- of the powdered compound moistened with an inert liquid 15 ful for medicinal, therapeutic or prophylactic applications. diluent. The tablets may optionally be coated or scored For example, a combination of the bile binding agent and and may be formulated so as to provide slow or controlled the dried viable cells, e.g. in formulation as described release of the active compound therein using, for exam- above, may be useful in the therapeutic or prophylactic ple, hydroxypropylmethyl cellulose or other delayed re- treatment of the human or animal body. lease agents in varying proportions to provide the desired 20 [0087] Theterm "therapeutic treatment" asused herein release profile. As described above, tablets, pellets, pills, in the context of treating an individual for a condition, capsules, and other solid formulations may optionally be pertains generally to treatment and therapy, whether of provided with an enteric coating, to provide release in a human or a non-human animal (e.g. in veterinary ap- parts of the gut other than the stomach. plications), in which some desired therapeutic effect is [0077] Capsules comprising the dried cells and the bile 25 achieved, for example, the inhibition of the progress of binding agent in a powder or granule form encapsulated the condition, and includes a reduction in the rate of by a polymeric shell may be made by conventional progress, a halt in the rate of progress, amelioration of means. the condition or one or more symptoms thereof, and cure [0078] The components may be disposed in various of the condition. different arrangements within the formulations described 30 [0088] The term "prophylactic treatment" as used here- herein and the precise arrangement of the components in in the context of a treating an individual for a condition, will depend on the specific properties which are required pertains generally to treatment which prevents or reduc- for a given application. For example, a formulation may es the risk of the occurrence or recurrence of the condi- be homogenous i.e. the dried viable cells and bile binding tion or one or more symptoms thereof. Prophylactic treat- agents may be distributed homogenously within the for- 35 ment may, for example, be carried out on an individual mulation, or it may be heterogeneous i.e. the dried viable (e.g. a human or a non-human animal) who is not at the cells and bile binding agents may be distributed hetero- time of treatment suffering from the condition or who does geneously within the formulation. not display symptoms of the condition. [0079] Examples of different arrangements of compo- [0089] In particular, a combination of a bile binding nents in the formulations described herein are shown in 40 agent and dried viable cells may be useful in the thera- figure 6. peutic or prophylactic treatment of pathogen infection in [0080] In some embodiments, a formulation may com- an individual. A method of treating a pathogen infection prise; a core which comprises the live dried bacteria and in an individual may comprise orally administering a com- the bile binding agent, and; a coating, bination of a bile binding agent and dried viable cells to preferably an enteric coating, which surrounds the core. 45 an individual in need thereof. Optionally, the coating may be a polymeric shell which [0090] Pathogen infections include bacterial infec- encapsulates the core. tions, such as cholera (V. cholerae), typhoid (S. typhi), [0081] Alternatively, a formulation may comprise a dysentery (Shigelia spp), anthrax (Bacillus anthracis), core of pharmaceutically acceptable excipient which plague (Yersinia pestis), tuberculosis (Mycobacterium comprises granules of the dried viable bacteria and gran- 50 tuberculosis), leprosy (Mycobacterium leprae), campy- ules of the dry bile binding agent. Optionally, the core lobacteriosis (Campylobacter spp) and gastroenteritis may further comprise granules of delayed release mate- (enterotoxic E. coli and Salmonella spp); viral infections rial. Optionally, the core may be encapsulated in a poly- such as influenza, HIV-AIDS, smallpox and SARS, and meric shell. Optionally, the formulation may further com- parasitic infections such as malaria (Plasmodium spp), prise an enteric coating which surrounds the core. 55 schistosomiasis (Schistosoma spp). [0082] Alternatively, a formulation may comprise a [0091] A combination of a bile binding agent and dried core of the viable dried bacteria which is surrounded by viable cells may also be useful in methods of immunisa- a layer of the bile binding agent. tion and/or immunotherapy, for example by stimulating

8 13 EP 2 384 190 B1 14 or priming an immune response to a disease-related an- need thereof. tigen which is encoded and preferably expressed by the [0098] A combination of a bile binding agent and dried cells. A method of treating an individual by immunisation viable cells may also be useful in reducing cholesterol or immunotherapy may comprise orally administering a levels in an individual. A method of reducing cholesterol combination of a bile binding agent and dried viable cells 5 levels may comprise orally administering a combination to an individual in need thereof. of a bile binding agent and dried viable cells to an indi- [0092] As described above, the disease-related anti- vidual in need thereof. gen may be naturally expressed by the cells (e.g. in an [0099] A bile binding agent and viable dried cells may attenuated bacterial vaccine) or the disease-related an- be used in the manufacture of a medicament for use in tigen may be an exogenous antigen which is expressed 10 any of the therapeutic and prophylactic applications and in the cell from heterologous nucleic acid. treatments described above. [0093] The cells may further comprise heterologous [0100] Suitable formulations, bile binding agents and nucleic acid encoding the an immunomodulatory viable dried bacterial cells are described in more detail polypeptide, for example a cytokine such as TNF-alpha, above. GM-CSF, IL-1, IL-6, IL-12, IFN-alpha or IFN-gamma; a 15 [0101] The methods described herein may be useful, chemokine such as CCL-25, or an antibody or other bind- for example, in improving the efficacy of a therapeutic ing molecule which inhibit cytokines (e.g. IL-10, TGF- agent which comprises dried viable cells, such as a bac- beta) or other immune response mediators or cells (such terial vaccine. A method of improving the efficacy of such as regulatory or suppressor T cells), leading to enhanced an agent may comprise formulating the agent with a bile immune responses. 20 binding agent, as described herein. [0094] Disease-related antigens include tumour anti- [0102] Themethods described hereinmay also be use- gens and antigens of viral, bacterial and other pathogens ful, for example, in increasing the survival of dried viable as described above. For example, the cells may express cells in the intestine of an individual. A method of increas- an antigen from a pathogen such as Streptococcus mu- ing the survival of dried viable bacteria cells in the intes- tans, Shigeila sonnei, Campylobacter jejuni, Escherichia 25 tine may comprise formulating the cells with a bile binding coli, Mycobacterium leprae, Mycobacterium tuberculo- agent, as described herein. sis, Vibrio cholerae, Yersinia pestis, Bacillus anthracis, [0103] The disclosure also provides the use of a bile HIV, Influenza virus, hepatitis virus,Plasmodium falci- binding agent as described herein in a method described parum, Francisella tularensis, or Schistosoma mansoni, herein, for example, a method of producing a solid for- or an antigenfrom a tumour, suchas Her2/neu or prostate 30 mulation comprising dried viable cells for oral adminis- specific antigen (PSA) in order to elicit or prime an im- tration; increasing the survival of dried viable bacteria mune response to the pathogen or tumour. Formulations cells in the intestine of an individual or improving the ef- as described herein may thus be useful in the therapeutic ficacy of a therapeutic agent which comprises dried via- and prophylactic treatment of pathogen infection and ble cells. cancer. 35 "and/or" where used herein is to be taken as specific [0095] A combination of a bile binding agent and dried disclosure of each of the two specified features or com- viable cells may also be useful in delivering viable cells ponents with or without the other. For example "A and/or to the intestine, for example to restore the microbial flora B" is to be taken as specific disclosure of each of (i) A, of the intestine in an individual. This may be useful, for (ii) B and (iii) A and B, just as if each is set out individually example, following treatment with antibiotics. A method 40 herein. of restoring the microbial flora of the intestine may com- [0104] Certain aspects and embodiments of the inven- prise orally administering a combination of a bile binding tion will now be illustrated by way of example and with agent and dried viable cells to an individual in need there- reference to the figures described below. of e.g. following treatment with antibiotics. [0096] A combination of a bile binding agent and dried 45 Figure 1 shows the rapid recovery of bile tolerance viable cells may also be useful in the therapeutic and after rehydration. prophylactic treatment of inflammatory intestinal disease or one or more symptoms thereof in an individual. A meth- Figure 2 shows that removing bile acids from bile od of treating inflammatory intestinal disease or one or solutions using BAR prevents the toxicity of bile so- more symptoms thereof may comprise orally administer- 50 lutions to dry bacteria. ing a combination of a bile binding agent and dried viable cells to an individual in need thereof. Figure 3 shows that cholestyramine prevents bile [0097] A combination of a bile binding agent and dried toxicity. viable cells may also be useful in the therapeutic and prophylactic treatment of renal stone formation in an in- 55 Figure 4 shows that adding BAR to drying excipient dividual. A method of treating renal stone formation may prior to drying bacteria provides significant protec- comprise orally administering a combination of a bile tion against toxicity when dried cells are resuspend- binding agent and dried viable cells to an individual in ed in bile solutions.

9 15 EP 2 384 190 B1 16

Figure 5 shows that adding BAR to compressed ma- an enzymatic assay (Randox Total Bile Acid Assay, Ran- trix tablets provides significant protection against dox labs, Co. Antrim) demonstrated that the concentra- toxicity of a 1% bile solution tion of bile salts was reduced from 130mM to 0.8mM by cholestyramine treatment. The resulting solutions were Figure 6 shows three examples of possible formula- 5 added to replicate samples of dried bacteria and cultured tions for oral dosage forms. for 1h, followed by dilution, plating, counting overnight colonies and calculation of relative cfu/ml. Figure 7 shows the validation of optimised formula- [0109] Removal of bile acids by BAR was found to ef- tion for protecting cells of a dried live bacterial vac- fectively prevent toxicity of bile solutions to dried bacteria cine from bile 10 (fig 2). Figure 2 also demonstrates high bile toxicity to dried samples of a different strain of bacteria, E. coli K12, Figure 8 shows the protection of a dried live Salmo- and shows that pre-treatment with cholestyramine pro- nellavaccine from acid andbile using an entericcoat- tects these dried cells too. 10m l dried samples of SL- ed capsule and the BAR cholestyramine. DAPD pUC18I (containing the ampicillin resistance 15 gene) were dried in 6 ml test tubes. To replicate samples, Figure 9 shows the protection of a dried lactic acid either nothing or 150 mg of cholestyramine were added bacteria from bile solutions in capsules using the on top of the dried cells. Then, 2 ml of a 2% solution of BAR cholestyramine. These results are not accord- bile in PBS or control PBS were added onto the choles- ing to the invention. tyramine, followed by incubation for 1h at 37 degrees. 20 Afterincubation, samples were taken, diluted, plated,and Experiments overnight colonies were counted on agar plates with or without ampicillin and relative cfu/ml calculated. Choles- [0105] We used the live bacterial vaccine strain SL- tyramine was sterilised prior to adding to bacterial sam- DAPD pUC18I as a model for oral bacterial delivery [13]. ples by pretreatment with 70% Ethanol and extensive Bacteria were grown overnight in M9 medium (with es- 25 drying. Furthermore, the same number of colonies were sential amino acids and nutrients) plus 250mM NaCl, found with or without ampicillin, indicating that only the then 10 microlitre samples were dried in phosphate buff- test strain of bacteria were present and no contamination ered saline (PBS) plus 40% trehalose and 1.5% PVP in had occurred. adesiccator, asdescribed ([14]). Replicate sampleswere [0110] The addition of BAR on top of dried bacteria kept dry or rehydrated with 200 microlitres PBS or water 30 followed by bile solution addition was found to block the and left for 1, 5 or 20 minutes as indicated. Dry or recov- toxicity of the bile solution. This demonstrates that se- eredsamples or controlcells from richbroth cultures were quential removal of bile followed by administration of bile then tested by incubation for 1h diluted extensively in acid-cleared liquid to dry bacteria is not required to pro- acid or bile solution or control buffer, as indicated, fol- tect bacteria, and demonstrates that bile acids com- lowed by dilution, plating on agar plates, and counting 35 plexed to BAR have no toxicity to dried bacteria (figure 3). overnight colonies. Bacterial counts were corrected for [0111] Replicate 10 ml samples of SLDAPD pUC18I the dilution during recovery and testing, and expressed were suspended in either control excipient (40% treha- as cfu/ml relative to the original 10 ul sample volume. lose, 1.5%PVP in PBS) or BAR-containing excipient [0106] Dried bacteria were found to recover bile toler- (40% trehalose, 1.5% PVP plus 5 or 10% cholestyramine ance very rapidly when rehydrated in water or buffer prior 40 in PBS) and dried in 6ml test tubes. To replicate samples, to exposure to bile. A dried preparation of bacteria that 2ml of 2% or 0.4% bile solutions or control buffer were lost 50x viability when rehydrated in bile, gained 5x better added and the tubes were incubated for 1h at 37 degrees, survival within 5 minutes of rehydration, and 20x better followed by sampling, dilution and plating on agar plates. survival after 20 minutes (fig 1). Inclusion of BAR in the drying excipient was found to [0107] Similar results were obtained by rehydration in 45 provide effective protection of dried bacteria from bile, buffer and water. By 1h after rehydration, bacteria be- even at quantities unable to bind the total amount of bile haved indistinguishably from control cells from liquid cul- acid present in a test solution (fig 4). tures. [0112] Crucially, simply drying bacteria with small [0108] The live bacterial vaccine strain SLDAPD quantities of cholestyramine added to the drying excipi- pUC18I[13] and E.coli strain K12 DH5a (Invitrogen)were 50 ent was found to provide significant protection in condi- grown overnight in M9 medium plus 250mM NaCl then tions where no protection would be expected if the BAR 10 ml samples were dried in PBS/40% trehalose/1.5% was simply binding and sequestering bile acids. In figure PVP in a desiccator, as described [12]. A 5% w/v solution 4, the amount of cholestyramine present is far smaller of bile (Sigma, Ox-bile) in PBS was mixed with or without than the total amount of bile acids present in the test 10% w/v of cholestyramine (Dowex 1x2, Sigma, Poole 55 medium. Cholestyramine has a capacity of 1.2 mg bile UK) for 1 hour at room temperature. The cholestyramine salts per 1 mg (typical value, exact value depends on was removed by centrifugation followed by filtration which bile acid is bound, [14]). Therefore, in the 10m l (0.2micron, millipore). Measurement of bile salts using dried bacteria sample there is 1 mg cholestyramine with

10 17 EP 2 384 190 B1 18 a capacity for 1.2mg bile acids, yet in the 2 ml 2% bile cholestyramine having the capacity to bind a maximum solution a total of 20mg bile acids are present (typically of 29mg bile acids but 17ml of 1% bile solution contains the ox-bile used contains in excess of 50% bile acids by approximately 85mg bile acids, therefore the cholesty- weight), greater than 15-fold excess of bile acids. ramine is not simply sequestering the total bile acids [0113] SLDAPD pUC18I cells were dried in standard 5 which are in excess in the test solution. excipient(40% trehalose, 1.5%PVP in PBS),then ground [0116] Without being bound in any way by theory, the in a mortar to make a powder. The powdered dried bac- formulation may work as follows: the cholestyramine in teria were mixed in carefully weighed proportions as fol- the excipient binds the bile acids present in the limited lows: volume of buffer which is absorbed into the dry sample, 10 and thus the dried bacteria are rehydrated in ’cleaned’ Control tablets- sucrose filler: 9% powdered dried water, allowing rapid recovery, as shown in figure 1. Sub- bacteria/91% sucrose powder sequently, when the rehydrated sample disintegrates Control tablets- MCC filler: 8% powdered dried bac- and disperses into the bile solution, the bacteria have teria/92% microcrystalline cellulose (MCC, Avicel already recovered significant bile resistance. PH101) 15 [0117] Even with the simple experimental formulation BAR tablets- Cholestyramine: 8% powdered dried consisting of BAR added to the drying excipient, 50-fold bacteria/33% cholestyramine/58% MCC filler more live bacteria were recovered with the BAR formu- BAR tablets- Dowex 1X2: 6% powdered dried bac- lation (10% cholestyramine) than the control formulation teria/63% Dowex 1X2 400 mesh resin/32% MCC fill- when resuspended in a 2% bile solution, representing a er 20 significant improvement. This represents a 5-fold reduc- tion in live cell recovery with the formulation when resus- [0114] 70-90mg samples of the mixed powders were pended in 2% bile, as compared to 270-fold reduction in then pressed in a 7mm die in a press (Port-A-Press from live cell recovery with the conventional control formula- International Crystal Laboratories, supplied by Crystan tion with conventional drying excipient. Similarly, with the Ltd, using 10 newton metres torque) to make com-25 compressed matrix tablet formulation consisting of BAR pressed matrix tabletsapproximately 1mm thick. The tab- mixed with dried bacteria prior to tablet compression, 208 lets were then tested in simulated intestinal conditions to 254-fold more live bacteria were recovered with the as follows: The tablets were weighed and individually BAR containing tablet formulation than with the control added to 17ml portions of International Pharmacopoeia formulation when resuspended in a 2% bile solution, rep- simulated intestinal fluid, i.e. sodium/potassium phos- 30 resenting a significant improvement. This represents a phate buffer pH7.0 either alone or plus 1% ox bile solu- 2.5 to 3.1-fold reduction in live cell recovery when disso- tion, and incubated for 45 minutes at 37°C. Samples were lution in 1% bile, as compared to 1228 to 638-fold reduc- taken, diluted, plated overnight, and live cfu counted. The tion in live cell recovery with the conventional control tab- bacterial count recovered was expressed in terms of the let formulation. original dried bacteria weight, i.e. in cfu/mg of dried bac- 35 [0118] The ability of an optimal formulation to protect teria, and each bar represents a single tablet, with the a dried live bacterial vaccine from bile was tested. error bar representing multiple bacterial counts for this [0119] In these experiments, the mouse plague vac- tablet. cine strain SL3261/pAHL was used [17]; similar results [0115] Prototype human tablets containing BAR plus were obtained using the antibiotic-free plasmid mainte- dried bacteria were found to offer significant bile protec- 40 nance strain SLDAPD/pUC18I [12, 16, 13]. Bacteria were tion in a simulated intestinal release model (figure 5). We counted using LB agar plates containing either ampicillin tested tablets containing live dried bacteria with or with- for SLDAPD/pUC18I or kanamycin for SL3261/pAHL to out addition of the BAR cholestyramine, or the equivalent ensure only vaccine bacteria were counted. Bacteria ion exchange resin Dowex 1x2, by dissolution in buffer were grown and dried as described in [12, 16], and then vs bile solutions. With control tablets containing dried 45 several replicate dried preparations were pooled and bacteria diluted in sucrose as a filler, 1228-fold fewer ground to a homogeneous powder in a mortar and mixed cells were recovered after dissolution in 1% bile solution with dry MCC to make a free-flowing powder for formu- compared to dissolution in buffer alone, indicating signif- lation and filling into capsules. This powder typically con- icant bile toxicity. Similarly, tablets made using MCC as tained 2-5x106 cfu/mg and was stored in a vacuum des- a filler, 638-fold fewer cells were recovered after disso- 50 iccator at room temperature until testing; the preparation lution in a 1% bile solution compared to buffer alone. In showed less than 10% loss of viability over a 2 month contrast, with tablets containing cholestyramine or period. Dowex 1X2 resin in addition to the dried bacteria and [0120] This powder was mixed with MCC filler plus MCC filler, dissolution in bile only gave a 2.5 to 3.1-fold BAR at 25%, then either weighed directly into test tubes, reduction in live cell recovery. This represents an im- 55 or filled into size 00 capsules of the indicated shell ma- provement of 208 to 254-fold more live cells with the BAR terialand weighed into testtubes. Between20-30mg bac- tablet compared to control tablets. Importantly, the teria were added per 1g total powder, to ensure that cholestyramine tablets contain a maximum of 24mg 6-10mg powdered dried bacteria were included per cap-

11 19 EP 2 384 190 B1 20 sule. The mixed powder was manually filled into size 00 ments, the total amount of cholestyramine was 75mg capsules of the indicated shell material using a Cap-M- (25% of 300mg powder), which can bind a maximum of Quick (Value Healthcare Company, Sheffield UK); a 218-292 mmole bile acids [4]. This was added to 25ml of 6.4mm diameter steel ball bearing was added to the cap- a 4% bile solution that contained a total of 2500umole sule to sink it during dissolution. 5 bile acids, representing an 8-11 fold excess of bile acids [0121] 25ml of buffer or 1% or 4% ox bile were added over BAR capacity, indicating that as with tablets, cap- and incubated at 37°C for 50 minutes, then the tubes sules containing BAR provide temporary protection were mixed thoroughly and samples taken, diluted, plat- whilst dry bacteria recover bile tolerance, rather than sim- ed on agar plates with kanamycin, and incubated over- ply sequestering all bile acids within the test sample. night. Colonies were counted and used to determine live 10 [0126] The ability of an enteric coated capsule and the cell number, expressed as CFU per mg of the initial dried BAR cholestyramine to protect dried live Salmonella vac- vaccine powder. Dried stabilised cells of the mouse cine from acid and bile was tested. plague vaccine strain SL3261/pAHL were employed; [0127] SL3261/pAHL bacteria were grown and dried similar protection was seen in separate experiments us- in the same manner as previous experiments. Powdered ing dried cells of the vaccine strain SLDAPD/pUC18I. 15 dried bacteria were mixed with microcrystalline cellulose [0122] The effect of differing BAR types on bile protec- and cholestyramine in the ratio of 1:75:25 tion was studies within the Quick-release HPMC capsule cells:MCC:cholestyramine. This powder was filled into shell. Quick-releaseHPMC capsules aremodified HPMC size 00 standard HPMC capsules, approximately 300mg capsules designed to give far faster release compared per capsule, with a ball bearing to sink during dissolution to conventional HPMC capsules. Imaging studies pre- 20 testing. The capsules were either uncoated or dipped dicted that protection given by 400 mesh Dowex 1x2 twice from each side into a 10% w/v solution of enteric could be significantly improved simply by removing mois- polymer methacrylic acid:ethyl acrylate copolymer (Kol- ture to generate a free-flowing non-aggregated powder, licoat MAE 100 P, BASF, Mannheim Germany) dissolved and indeed when dried 400mesh Dowex 1x2 was added in a 3:2 v/v mix of methanol and dichloromethane, then at 25% to dried LBV and MCC filler an impressive differ- 25 dried overnight. ence was seen, with dried 400 mesh Dowex 1x2 provid- [0128] 5 enteric coated capsules were individually im- ing 92-fold protection compared to powder without cap- mersed for 40 minutes in 20ml 0.1M HCl at 37°C to sim- sules. In contrast the wet preparation of 400 mesh Dowex ulate gastric acid in the stomach; as expected the enteric 1x2 gave no significant protection (fig. 7a). Furthermore, coating prevented dissolution or penetration of the cap- cholestyramine gave even better protection than the30 sule shell by the acid. These 5 capsules were then trans- dried 400 mesh Dowex 1x2, with 120-fold protection com- ferred into 2% ox bile dissolved in phosphate buffer pH pared to powder without capsules, and showing no sig- 7.0. At the same time 2 coated and 3 uncoated capsules nificant loss of bacteria in 1% bile compared to buffer (fig. were individually added to 20ml phosphate buffer pH7.0 7a). (buffer); at the same time 2 uncoated capsules were add- [0123] Next, we made and tested an optimised formu- 35 ed individually to 20ml 0.1M HCl. All capsules were then lation in an increased bile concentration. This formulation incubated at 37oC for 1h, then mixed thoroughly. The incorporated theprotective features predictedby imaging uncoated capsules completely dissolved in the acid with- and theoretical work - 25% cholestyramine, an increased in 1h. After a further 1h (2h total incubation), samples dose size and capsule geometry - and was also tested were taken, diluted, and plated on agar plates containing using conventional HPMC capsules 40 kanamycin then incubated overnight. Colonies were [0124] Adding 25% cholestyramine BAR resulted in counted and the live cell recovery calculated and ex- 100% recovery of viable cells in HPMC capsules under pressed as colony forming units per ml; each bar repre- 4% bile conditions, with no difference in cell recovery sents 1 capsule and the error bars indicate 1 standard between bile and buffer (fig. 7b). Furthermore no signif- deviation. icant loss of bacteria was detected in 4% bile compared 45 [0129] Uncoated capsules were found to release high to buffer when Quick -release HPMC capsules were also numbers of viable cells into the buffer, but no live cells filled with 25% cholestyramine and dried LBV (fig. 7b). were recovered in acid. In contrast, similar numbers of Therefore simply adding 25% cholestyramine into dried viable cells were recovered into buffer and into bile after LBV preparations can provide 4200-fold protection from acid immersionwith theenteric coated capsules (seeFig- 4% bile. The composition of pig bile is closer to that of 50 ure 8). This demonstrates that an enteric-coated solid humans [8], and a similar high degree of protection from dose containing the BAR cholestyramine protects cells pig bile at 1%, 4% and 8% was also observed, confirming of a live dried heterologous antigen bacterial vaccine that this optimised formulation can protect dried vaccine from gastric acid followed by intestinal bile. bacteria from excess concentrations of bile acids. [0130] The ability of capsules containing the BAR [0125] The inclusion of BAR in tablets provides protec- 55 cholestyramine to protect dried lactic acid bacteria from tion from a solution of bile that contains a quantity of bile bile solutions was tested. These tests are not according acids that far exceeds the maximal capacity of the BAR to the invention. Live lactic acid bacteria were commer- within the tablet [16]. Similarly, in the capsule experi- cially manufactured in powder form through a fermenta-

12 21 EP 2 384 190 B1 22 tion process and freeze drying. The culture was tested [0136] The data set out above also shows that if dry for salmonella and other pathogens by the manufacturer bacteria and bile acid binding agents are present togeth- and supplied as a food supplement that is suitable for er, that the toxicity of bile acids is blocked -i.e. the bile human consumption. acids cannot kill the dried bacteria in the presence of bile [0131] The dried lactic acid bacterial powder contain- 5 acid binding agents. This shows that dried bacteria may ing Lactobacillus caseii was mixed with microcrystalline be protected in vivo from the toxicity of bile acids. cellulose (MCC) filler either with or without 50% w/w of [0137] The data set out above also show that if bacteria the BAR cholestyramine, and then filled into HPMC cap- are dried in the presence of bile binding agents, then sules containing a 1g ball bearing to sink the capsules. sufficient protection from bile toxicity is provided even in 13-15% w/w of capsule content was bacteria which cor- 10 a situation where the total quantity of bile acid in the test relates to 39-45mg of a 300mg capsule i.e. in the range tube is in great excess of the total capacity of the bile 9.8x10^9 - 11x10^9 CFU/capsule. Individual capsules binding agents present in the dried bacterial formulation. were added to 50ml tubes containing 25ml of either con- [0138] Bile acid sequestrants may therefore be incor- trol phosphate buffer pH7.0, or the indicated concentra- porated into dose formulations to increase the efficacy tions of ox bile and porcine bile in phosphate buffer for 15 of delivery of live bacteria to the intestine. When released 1h at 37oC. Samples were taken, diluted and portions into the intestine by a conventional enteric delivery sys- plated on agar culture plates, incubated for 72h at 37oC tem, the bile acid sequestrant will protect the dried bac- after which colonies were counted and colony forming teria from bile acids giving the bacteria time to rehydrate units (CFU) were calculated to indicate the number of without bile acid toxicity, thereby allowing recovery of bile live cells relative to the original weight of dried lactic acid 20 tolerance and improving live bacterial delivery to the in- powder added to the capsules. Each bar represents the testine. For example, a bacterial vaccine (strain mean of 4 individually tested capsules. SL3261/pAHL) containing a recombinant antigen [0132] When dispersed in control buffer, the control (plague F1 antigen in a plasmid) (according to the inven- capsules containing cells mixed with MCC filler alone tion) and a commercial dried preparation of the lactic acid were found to release the expected high numbers of live 25 bacterium Lactococcus caseii (not according to the in- L. caseii cells; in contrast, increasing doses of bile gave vention) are protected from bile by the formulations de- reductions in live cell recovery, with 4% ox-bile giving scribed herein. Furthermore, the presence of an enteric over 30-fold killing. When cholestyramine was added into coating as well as a bile acid sequestrant protects dose the capsule mixed with the bacteria and filler powders, formulations from sequential exposure to acid followed less than 3-fold loss was seen with 4% ox-bile, giving 30 by bile solutions (simulated ingestion). over 10-fold protection against this high dose of bile. Sim- ilar results were seen with pig bile (Figure 9). This dem- References onstrates that a commercial, freeze-dried, food grade preparation of lactic acid bacteria may be protected from [0139] bile using the BAR cholestyramine. 35 [0133] The experimental data set out above shows that [1] Bergogne-Berezin E. Int J Antimicrob Agents bile sensitive dried bacteria rapidly recover tolerance to 2000 Dec;16(4):521-6. bile,if rehydrated priorto bileexposure and pre-treatment [2] Bohm SK, Kruis W. Ann N Y Acad Sci 2006 of bile solutions with a bile acid sequestrant prevents bile Aug;1072:339-50. toxicity to dried bacteria. Furthermore, adding the bile 40 [3] Huyghebaert N et al Eur J Pharm Biopharm 2005 acid sequestrant on top of dried bacteria is shown to pre- Aug;60(3):349-59. vent bile toxicity and adding the bile acid sequestrant to [4] Levine MM. Vaccine 2006 May 1;24(18):3865-73. the excipient prior to drying also provides significant pro- [5] Prakash S et al Appl Biochem Biotechnol 2006 tection against bile toxicity compared to bacteria dried in Jan;128(1):1-22. the conventional excipient. Matrix tablets and capsules 45 [6] Crowe JH, et al Annu Rev Physiol containing the bile acid sequestrant also show increased 1998;60:73-103. viable bacteria release when added to bile solutions, [7] Bullifent HL et al. Vaccine 2000 Dec compared to control tablets or capsules without the bile 8;19(9-10):1239-45. acid sequestrant. [8] Fraser A et al Vaccine 2007 Nov [0134] After rehydration without bile acids, dried bac- 50 7;25(45):7848-57. teria were found to recover bile resistance very rapidly. [9] Sack DA et al Infect Immun 1997 This demonstrates that dried bacteria may be allowed to Jun;65(6):2107-11. recover in an oral delivery form in the intestine through [10] McKenzie R et al. Vaccine 2008 Aug rehydration with bile protection. 26;26(36):4731-9. [0135] BAR are used conventionally to sequester bile 55 [11] Foster JW. Nat Rev Microbiol 2004 acids and block reabsorbtion, but have not been previ- Nov;2(11):898-907. ously shown to modulate the effect of bile acids in the [12] Edwards AD, Slater NK. Vaccine 2008 Aug 30; intestine. 26 (45) : 5675-8.

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[13] Garmory HS et al. Infect Immun 20059. A solid formulation for use according to claim 8 Apr;73(4):2005-11. wherein the polymeric shell comprises hydroxypro- [14] Honda Y, Nakano M. Chem Pharm Bull (Tokyo) pyl methylcellulose. 2000 Jul;48(7):978-81. [15] Levine MM, et al Lancet 1990 Oct 5 13;10. A solid formulation for use according to any one of 336(8720):891-4. the preceding claims which is covered with an enteric [16] A.D. Edwards, N.K. Slater Vaccine 27(29) coating. (2009) 3897-3903. [17] H.L. Bullifent et al Vaccine 19(9-10) (2000) 11. A solid formulation for use according to claim 10 1239-1245. 10 wherein the formulation comprises; [18] WO2007/112747 a core which comprises dried viable bacterial cells and bile binding agent, optionally wherein the core is encapsulated in a polymeric shell, and; Claims an enteric coating which surrounds the core and op- 15 tional polymeric shell. 1. A solid formulation comprising dried viable bacterial cells and a bile binding agent for use in a method of 12. A method of producing a solid formulation for use immunotherapy which comprises oral administration according to claim 1 comprising; of said formulation to an individual in need thereof, drying viable bacterial cells, and wherein the bile binding agent is cholestyramine and 20 admixing the dried viable cells with a bile binding the viable bacterial cells are bacterial vaccine cells. agent and optionally a pharmaceutically acceptable excipient to produce a solid formulation; or, 2. A solid formulation for use according to claim 1 admixing viable bacterial cells with a bile binding wherein the viable cells are dried by increasing the agent and optionally a pharmaceutically acceptable intracellular level of disaccharide in the cells and dry- 25 carrier to produce an admixture and drying the ad- ing the cells in the presence of a carbohydrate sta- mixture to produce a solid formulation; bilising agent. wherein the bile binding agent is cholestyramine and the viable bacterial cells are bacterial vaccine cells. 3. A solid formulation for use according to any one of the preceding claims comprising either; 30 Patentansprüche (i) a core which comprises granules of dried vi- able bacterial cells and granules of dry bile bind- 1. Feste Formulierung, umfassend getrocknete le- ing agent; or, bensfähige Bakterienzellen und ein Galle-binden- (ii) a core which comprises viable dried bacterial 35 des Mittel, zur Verwendung in einem Verfahren der cells; said core being surrounded by a layer Immuntherapie, das die orale Verabreichung der comprising the bile binding agent. Formulierung an ein Individuum mit Bedarf daran umfasst, 4. A solid formulation for use according to any one of wobei das Galle-bindende Mittel Cholestyramin ist the preceding claims wherein the dried viable cells 40 und die lebensfähigen Bakterienzellen bakterielle are lysozyme sensitive. Vakzinenzellen sind.

5. A solid formulation for use according to claim 4 which 2. Feste Formulierung zur Verwendung nach Anspruch further comprises a cation exchange resin. 1, wobei die lebensfähigen Zellen durch Erhöhen der 45 intrazellulären Menge von Disaccharid in den Zellen 6. A solid formulation for use according to any one of und Trocknen der Zellen in der Gegenwart eines the preceding claims wherein the dried viable cells Kohlenhydrat-Stabilisierungsmittels getrocknet wer- comprise heterologous nucleic acid. den.

7. A solid formulation for use according to claim 50 6 3. Feste Formulierung zur Verwendung nach einem wherein the heterologous nucleic acid encodes an der vorangegangenen Ansprüche, die eines der bei- exogenous antigen. den Folgenden umfasst:

8. A solid formulation for use according to any one of (i) einen Kern, der Granulat aus getrockneten the preceding claims wherein the dried viable cells 55 lebensfähigen Bakterienzellen und Granulat of the bacterial vaccine and the bile binding agent austrockenem Galle-bindendemMittel umfasst; are encapsulated in a polymeric shell. oder (ii) einen Kern, der lebensfähige getrocknete

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Bakterienzellen umfasst; wobei der Kern von ei- mit einem Galle-bindenden Mittel und gegebe- ner Schicht umgeben ist, die das Galle-binden- nenfalls einem pharmazeutisch annehmbaren de Mittel umfasst. Träger, um ein Gemisch zu erzeugen, und Trocknen des Gemischs, um eine feste Formu- 4. Feste Formulierung zur Verwendung nach einem 5 lierung zu erzeugen; der vorangegangenen Ansprüche, wobei die ge- wobei das Galle-bindende Mittel Cholestyramin trockneten lebensfähigen Zellen empfindlich gegen- ist und die lebensfähigen Bakterienzellen bak- über Lysozym sind. terielle Vakzinenzellen sind.

5. FesteFormulierung zurVerwendung nach Anspruch 10 4, die weiters ein Kationenaustauschharz umfasst. Revendications

6. Feste Formulierung zur Verwendung nach einem 1. Formulation solide comprenant des cellules bacté- der vorangegangenen Ansprüche, wobei die ge- riennes viables séchées et un agent de liaison à la trocknetenlebensfähigen Zellen eineheterologe Nu- 15 bile pour une utilisation dans un procédé d’immuno- cleinsäure umfassen. thérapie qui comprend une administration orale de ladite formulation à un individu en ayant besoin, 7. FesteFormulierung zurVerwendung nach Anspruch dans laquelle l’agent de liaison à la bile est la cho- 6, wobei die heterologe Nucleinsäure für ein exoge- lestyramine et les cellules bactériennes viables sont nes Antigen kodiert. 20 des cellules de vaccin bactérien.

8. Feste Formulierung zur Verwendung nach einem 2. Formulation solide à utiliser selon la revendication der vorangegangenen Ansprüche, wobei die ge- 1, dans laquelle les cellules viables sont séchées en trockneten lebensfähigen Zellen der bakteriellen augmentant le niveau intracellulaire de disaccharide Vakzinenzellen und das Galle-bindende Mittel in ei- 25 dans les cellules et en séchant les cellules en pré- ner Polymerhülle eingekapselt sind. sence d’un agent stabilisateur de carbohydrates.

9. FesteFormulierung zurVerwendung nach Anspruch 3. Formulation solide à utiliser selon l’une quelconque 8, wobei die Polymerhülle Hydroxypropylmethylcel- des revendications précédentes, comprenant ; lulose umfasst. 30 (i) un noyau qui comprend des granules de cel- 10. Feste Formulierung zur Verwendung nach einem lules bactériennes viables séchées et des gra- der vorangegangenen Ansprüche, die mit einer ma- nules d’agent de liaison à la bile sec ; ou, gensaftresistenten Beschichtung bedeckt ist. (ii) un noyau qui comprend des cellules bacté- 35 riennes séchées viables ; ledit noyau étant en- 11. FesteFormulierung zurVerwendung nach Anspruch touré par une couche comprenant l’agent de 10, wobei die Formulierung Folgendes umfasst: liaison à la bile.

einen Kern, der getrocknete lebensfähige Bak- 4. Formulation solide à utiliser selon l’une quelconque terienzellen und ein Galle-bindendes Mittel um- 40 des revendications précédentes, dans laquelle les fasst, gegebenenfalls wobei der Kern in einer cellules viables séchées sont sensibles au lysozy- Polymerhülle eingekapselt ist, und me. eine magensaftresistente Beschichtung, die den Kern und gegebenenfalls die Polymerhülle 5. Formulation solide à utiliser selon la revendication umgibt. 45 4, qui comprend en outre une résine échangeuse de cations. 12. Verfahren zur Herstellung einer festen Formulierung zur Verwendung nach Anspruch 1, das Folgendes 6. Formulation solide à utiliser selon l’une quelconque umfasst: des revendications précédentes, dans laquelle les 50 cellules viables séchées comprennent un acide nu- Trocknen von lebensfähigen Bakterienzellen cléique hétérologue. und Vermengen der getrockneten lebensfähigen 7. Formulation solide à utiliser selon la revendication Bakterienzellen mit einem Galle-bindenden Mit- 6, dans laquelle l’acide nucléique hétérologue code tel und gegebenenfalls einem pharmazeutisch 55 pour un antigène exogène. annehmbaren Exzipienten, um eine feste For- mulierung zu erzeugen; oder 8. Formulation solide à utiliser selon l’une quelconque Vermengen von lebensfähigen Bakterienzellen des revendications précédentes, dans laquelle les

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cellules viables séchées du vaccin bactérien et de l’agent de liaison à la bile sont encapsulées dans une enveloppe polymère.

9. Formulation solide à utiliser selon la revendication 5 8, dans laquelle l’enveloppe polymère comprend de l’hydroxypropylméthylcellulose.

10. Formulation solide à utiliser selon l’une quelconque des revendications précédentes, qui est recouverte 10 d’un revêtement entérique.

11. Formulation solide à utiliser selon la revendication 10, dans laquelle la formulation comprend : 15 un noyau qui comprend des cellules bactérien- nes viables séchées et un agent de liaison à la bile, éventuellement dans laquelle le noyau est encapsulé dans une enveloppe polymère, et ; un revêtement entérique qui entoure le noyau 20 et l’enveloppe polymère facultative.

12. Procédé de production d’une formulation solide à utiliser selon la revendication 1, comprenant les éta- pes consistant à : 25

sécher des cellules bactériennes viables, et mélanger les cellules viables séchées avec un agent de liaison à la bile et éventuellement un excipient pharmaceutiquement acceptable pour 30 produire une formulation solide ; ou, mélanger des cellules bactériennes viables avec un agent de liaison à la bile et éventuelle- ment un support pharmaceutiquement accepta- ble pour produire un mélange, et 35 sécher le mélange pour produire une formula- tion solide ; dans lequel l’agent de liaison à la bile est la cho- lestyramine, et les cellules bactériennes viables sont des cellules de vaccin bactérien. 40

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

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