(19) TZZ _Z¥_T (11) EP 2 170 386 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: A61K 39/39 (2006.01) A61P 37/04 (2006.01) 10.08.2016 Bulletin 2016/32 (86) International application number: (21) Application number: 08776561.6 PCT/IB2008/052629 (22) Date of filing: 30.06.2008 (87) International publication number: WO 2009/004569 (08.01.2009 Gazette 2009/02) (54) USE OF INTRANASALLY ADMINISTERED PIDOTIMOD TO ENHANCE ANTIGEN-SPECIFIC HUMORAL AND CELL-MEDIATED IMMUNE RESPONSE VERWENDUNG VON INTRANASAL VERABREICHTEM PIDOTIMOD ZUR VERSTÄRKUNG DER ANTIGEN-SPEZIFISCHEN HUMORALEN UND ZELLVERMITTELTEN IMMUNANTWORT UTILISATION DE PIDOTIMODE ADMINISTRÉ PAR VOIE INTRA-NASALE POUR AMÉLIORER LA RÉPONSE HUMORALE SPÉCIFIQUE À UN ANTIGÈNE ET LA RÉPONSE IMMUNITAIRE À MÉDIATION CELLULAIRE (84) Designated Contracting States: (56) References cited: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR EP-A- 0 510 561 HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT RO SE SI SK TR • CAREDDU P: "Role of immunoactivation with pidotimod in recurrent respiratory infections in (30) Priority: 02.07.2007 IT TO20070480 childhood" ARZNEIMITTEL-FORSCHUNG, vol. 44, no. 12A, 1994, pages 1506-1511, XP008097246 (43) Date of publication of application: ISSN: 0004-4172 07.04.2010 Bulletin 2010/14 • CIACCIA A ET AL: "Pidotimod activity against chronic bronchitis exacerbations" (60) Divisional application: ARZNEIMITTEL-FORSCHUNG, vol. 44, no. 12A, 14152382.9 / 2 724 726 1994, pages 1516-1520, XP008097247 ISSN: 0004-4172 (73) Proprietor: Polichem SA • DATABASE WPI Week 200721 Thomson 1526 Luxembourg (LU) Scientific, London, GB; AN 2007-201303 XP002498876 & CN 1 840 172 A (SUZHOU (72) Inventor: CARUSO, Arnaldo CHANGZHENG XINKAI PHARM CO LTD) 4 I-20124 Brescia (IT) October 2006 (2006-10-04) • COPPI G ET AL: "Experimental immunological (74) Representative: Pistolesi, Roberto et al screening tests on pidotimod" Dragotti & Associati Srl ARZNEIMITTEL-FORSCHUNG, vol. 44, no. 12A, Via Nino Bixio, 7 1994, pages 1411-1416, XP008097245 ISSN: 20129 Milano (IT) 0004-4172 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 2 170 386 B1 Printed by Jouve, 75001 PARIS (FR) 1 EP 2 170 386 B1 2 Description discloses the use of Pidotimod in the preparation of med- icament for reducing the exacerbations of chronic bron- [0001] The present invention relates to a novel use in chitis. the therapeutic field of the active ingredient Pidotimod, [0011] EP 510561 discloses a liquid pharmaceutical and to pharmaceutical formulations comprising it. 5 composition in the form of foam, said composition con- [0002] Pidotimod, ((R)-3-[(s)-(5-oxo-2-pyrrolydi- sisting of: a) one or more natural or synthetic ionic or non nyl)]carbonyl]-thiazolidine-4-carboxylic acid), is a syn- ionic, acid, basic or neutral surfactants; b) a solvent or a thetic dipeptide provided with an in vitro and in vivo im- solvent mixture; c) an active ingredient or a combination munomodulating action. of active ingredients; d) optional adjuvants or excipients, [0003] Immune system intervenes in maintaining a10 wherein the active ingredient can be Pidotimod base or homeostatic balance between our body and all foreign a salt or a derivative thereof. substances; therefore, an abnormality in this function [0012] CN 1 840 172 A (SUZHOU CHANGZHENG may cause a defective response towards non-self struc- XINKAI PHARM CO LTD, 2006-10-04) discloses the use tures, or loss of tolerance toward self antigens: in both of Pidotimod in the preparation of medicament in the form cases, the immune system error exhibits clinically as15 of solution for intravenous injection. signs of disease. [0013] The present invention originates from an exper- [0004] Pidotimod proved to be able to exert a protective imental research which allowed highlighting a direct ac- action towards infectious processes (although it is free tion of Pidotimod on human dendritic cells, and the ability from a direct antimicrobial and viral action), as it has been thereof to enhance the communication between such in- confirmed in experimental bacterial and viral infection 20 nate immune system cells and the acquired immune sys- models, and confirmed by a number of controlled clinical tem cells; the results of such research further indicate trials. that Pidotimod administered through the intranasal mu- [0005] Particularly, Pidotimod increases the lym- cosal route acts in vivo as an adjuvant drug, that is, able phocyte proliferation and the production of pro-inflamma- to enhance the specific both humoral, and cell-mediated tory cytokines by pre-activated lymphocytes withdrawn 25 immune response towards a specific antigen. from older subjects. Furthermore, it is able to increment [0014] In view of the results of such study, a first object the release of antiviral molecules (IFN- α) induced by spe- of the invention is the use of Pidotimod, for the prepara- cific stimuli (Poly I:C). E x vivo studies highlighted the Pi- tion of a medicament in a form which can be administered dotimod ability to increment phagocytes stimulation and through the intranasal mucosal route, useful as an adju- relative phagocyte action, as well as the cytotoxic action 30 vant in enhancing both a humoral, and cell-mediated spe- of NK cells. cific immune response toward a specific antigen. [0006] Pidotimod, administered in tablets via oral [0015] In the study which has been carried out within route, proved to be able to induce, in animal models, a the scope of the invention, it has been high-lighted that higher resistance to viral infections and a higher efficien- Pidotimod, when administered instead via oral route, is cy in the treatment of recurrent respiratory infections in 35 not able to enhance either the local, or the systemic im- the paediatric patient. mune response towards a specific antigen; this observa- [0007] Recent studies demonstrate how the vaccina- tion suggests that Pidotimod acts on mucosal cells of the tions through the mucosal membranes are able to en- upper respiratory tract, triggering a specific immune re- hance the body immune defences, locally -therefore at sponse. the site of entry of the external pathogen- stimulating an 40 [0016] Particularly, Pidotimod in the form of a nasal efficient inflammatory response. spray can be used as a drug capable to enhance the [0008] However, the administration of the antigen mol- immune responses mucosally and systemically, in order ecule alone is sometimes not able to trigger an efficient to both prevent the respiratory tract infections, and to inflammatory response mucosally, and this causes a par- enhance immune responses during respiratory tract tial enzymatic degradation of the molecules, which oc- 45 acute infections. Furthermore, Pidotimod can be used as curs locally. Therefore, co-administration of so-called an adjuvant in mucosal vaccines intranasally. auxiliary substances represents an extremely attractive, [0017] The preferred administration form is a solution widely-used strategy, able to enhance the local and sys- which is suitable for an administration as a spray or an temic immune response. aerosol, consisting in Pidotimod and a pharmaceutically [0009] CAREDDU P: "Role of immunoactivation with 50 acceptable liquid carrier. Preferably, the administration Pidotimodin recurrent respiratory infectionsin childhood" form consists in Pidotimod in aqueous brine solution, op- ARZNEIMITTEL-FORSCHUNG, vol. 44, no. 12A, 1994, tionally including preservatives, antioxidants, antiseptic pages 1506-1511, discloses the use of Pidotimod in the agents, and/or agents promoting adhesion to the mucos- preparation of medicament for decreasing the incidence al membrane, or penetration into the mucosal mem- rate of recurrent respiratory infections (RRI) of children. 55 brane. Typically, Pidotimod is used in a concentration [0010] CIACCIA A ET AL: "Pidotimod activity against from 1 mg/liter to 10 g/liter, preferably from 500 mg/ liter chronic bronchitis exacerbations" ARZNEIMITTEL- to 5 g/liter. FORSCHUNG,vol. 44, no.12A, 1994, pages 1516-1520, [0018] The administration dose in the human being is 2 3 EP 2 170 386 B1 4 typically of about 0.5 mg/spray (corresponding to about CD4+ T-cells were purified by negative selection using a 0.130 mL). "CD4+ T-cell isolation kit" in conjunction with CD45RO- [0019] Also within the scope of the invention is a phar- specific beads (Miltenyi). The so-obtained lymphocytes maceutical product in the form of a combined prepara- were marked with Cell-Trace CFSE Cell proliferation kit tion, containing a mucosal vaccine and Pidotimod, in a 5 (Molecular Probes Eugene, OR), then cultured at differ- form which can be administered through the intranasal ent cell ratios with Pidotimod-treated DCs in flat-bottom mucosal route, for the concomitant, separated, or se- 96-well plates. The proliferative ability of CD4 lym- quential use for the prevention of respiratory tract infec- phocytes was established after 4 days by cytofluorimetric tions. analysis. [0020] Pidotimod action, which justifies the use thereof 10 in accordance with the invention, has been shown by the e) Production of cytokines intracellularly following experimental tests. [0025] In order to analyze the cytochemical polariza- 1- Pidotimod action on human dendritic cells tion of the CD4 + T lymphocytes, DCs and T lymphocytes 15 were co-cultured for 6 days in the presence and absence a) Culture of human dendritic cells of Pidotimod, then subjected to re-stimulation with PMA and ionomicyn. The presence of IL-2, IFN-γ, and IL-4 [0021] Immature human dendritic cells (DCs) have intracellularly was analyzed by flow cytofluorimetry using been generated in vitro from circulating monocytes, as fluorochrome-marked monoclonal antibodies as specific described in the literature (1-2).