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WO2023281430A1 - Complexe à base de lactoferrine et/ou d'un hydrolysat de celle-ci, son procédé de production et ses utilisations - Google Patents

Complexe à base de lactoferrine et/ou d'un hydrolysat de celle-ci, son procédé de production et ses utilisations Download PDF

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Publication number
WO2023281430A1
WO2023281430A1 PCT/IB2022/056274 IB2022056274W WO2023281430A1 WO 2023281430 A1 WO2023281430 A1 WO 2023281430A1 IB 2022056274 W IB2022056274 W IB 2022056274W WO 2023281430 A1 WO2023281430 A1 WO 2023281430A1
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Prior art keywords
lactoferrin
hydrolyzate
complex according
complex
pullulan
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English (en)
Inventor
Claudio ANGELINETTA
Lucio Lenzi
Mirco GROSSI
Paolo RUBINACCI
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Priority to EP22743589.8A priority Critical patent/EP4366760A1/fr
Publication of WO2023281430A1 publication Critical patent/WO2023281430A1/fr
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Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/40—Transferrins, e.g. lactoferrins, ovotransferrins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/719—Pullulans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/01—Hydrolysed proteins; Derivatives thereof
    • A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
    • A61K38/018—Hydrolysed proteins; Derivatives thereof from animals from milk
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0034—Urogenital system, e.g. vagina, uterus, cervix, penis, scrotum, urethra, bladder; Personal lubricants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/0063—Periodont
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/02—Drugs for genital or sexual disorders; Contraceptives for disorders of the vagina
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/005—Antimicrobial preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin

Definitions

  • the present invention relates to a complex based on lactoferrin and/or a hydrolyzate thereof, a process for its production, and uses thereof.
  • Lactoferrin is an antimicrobial and iron-carrying glycoprotein, naturally present in various biological fluids of mammals, such as milk, saliva, tears. It is also present in neutrophil granulocytes, which are immune cells with defence functions against bacterial and fungal infections. Lactoferrin is considered a particularly useful product for its antioxidant, immunomodulatory and anti-infective properties.
  • lactoferrin Several scientific papers have recently been published demonstrating the antiviral properties of lactoferrin. See for example the article by E. Campione et al, published in Int. J. Mol. Sci. 2020, 21(14), 4903; https;//doi.org/10.3390/ i ms21144903, where the protective effect exerted by lactoferrin in cases of coronavirus infections and inflammations is demonstrated, acting both as a natural barrier for respiratory and intestinal mucous membranes and to counteract disorders in iron metabolism due to viral colonisation.
  • lactoferrin Through pepsin digestion of lactoferrin, it is possible to obtain peptides, which are commonly indicated as lactoferrin hydrolyzates or even, more simply, as lactoferricin which, although having a rather lower molecular weight with respect to the starting protein, contain the N-terminal sequence of lactoferrin and maintain the biological activity thereof; in some cases such activity is even reinforced.
  • lactoferricin which, although having a rather lower molecular weight with respect to the starting protein, contain the N-terminal sequence of lactoferrin and maintain the biological activity thereof; in some cases such activity is even reinforced.
  • lactoferricin and its antimicrobial, antiviral, antitumor and immunological properties are described.
  • Patent application WO 2016/046108 describes a composition containing a lactoferrin hydrolyzate and glycerophosphoinositol or salts thereof and/or verbascoside or extracts that contain it, and the use thereof for the prevention and/or treatment of dermatological, otologic and stomatological inflammations and infections.
  • Such composition can be applied for the treatment of different infections, especially in the veterinary field, which can cause diseases of the ears, skin or of the stomatological type.
  • Lactoferrin and hydrolyzates thereof have not yet been widely used, mainly because of their poor stability and compatibility with excipients normally used in the pharmaceutical or para-pharmaceutical field (e.g. toothpaste, mouthwash, dietary supplements and the like). Lactoferrin and hydrolyzates thereof also have relatively low bioavailability when applied topically and are subject to oxidative degradation phenomena.
  • the Applicant has therefore faced the problem of obtaining a lactoferrin and/or a hydrolyzate thereof in a physiologically acceptable form with high stability and bioavailability, so as to exert a bacteriostatic, virustatic and/or antioxidant action that is effective and long-lasting.
  • the present invention thus relates to a complex between at least one lactoferrin and/or a hydrolyzate thereof with at least one pullulan.
  • the present invention relates to a pharmaceutical or nutraceutical composition, which comprises said complex and at least one physiologically acceptable excipient.
  • the pharmaceutical composition is for oral use or for topical use.
  • the present invention relates to a composition for oral hygiene, which comprises said complex and at least one physiologically acceptable excipient.
  • the present invention relates to a complex as defined above for use, by topical application, in the prevention or treatment of pathologies of dental apparatus, in particular for use in the prevention or treatment of periodontitis or perimplantitis .
  • the present invention relates to a complex as defined above for use, by topical application, in the prevention or treatment of viral and/or bacterial infections of nasal, oral and/or ocular mucous membranes.
  • This complex is indeed able to effectively exert a barrier effect on these mucous membranes, so as to inhibit viral and/or bacterial proliferation.
  • the present invention relates to a complex as defined above for use, by topical application or oral administration, in the prevention or treatment of inflammations of urogenital apparatus, in particular of vagina, such as vaginosis or vaginitis of bacterial or viral origin, or yeast infections (candidiasis).
  • the present invention relates to a complex as defined above for use, by oral administration, in the prevention or treatment of intestinal dismicrobism, i.e. for restoring the balance of the intestinal microbiome.
  • the present invention relates to a complex as defined above for use, by topical application, in the treatment of skin lesions, in particular skin sores, ulcers or erosions.
  • complex refers to an association between two different molecules in which intermolecular interactions (of electrostatic or van der Waals type) are established that modify release and biological activity characteristics of the molecules.
  • pullulan interacts with lactoferrin and/or a hydrolyzate thereof so as to modulate their release over time, as thus to prolong their effectiveness.
  • pullulan is a water-soluble polysaccharide formed by the repetition of maltotriose units, connected to each other by an -1,6 glycosidic bond, having the following structural formula:
  • the pullulan has a number average molecular weight (M n) comprised between 30,000 and 800,000 g/mol, more preferably between 50,000 and 200,000 g/mol.
  • M n number average molecular weight
  • lactoferrin this can be of natural or synthetic origin, and can include different forms, both monomeric and oligomeric, possibly already bound to iron (ololactoferrin) or not bound to iron (apolactoferrin) .
  • lactoferrin hydrolyzates they are products well known in the art, which can be prepared according to known techniques, in particular through treatment of a lactoferrin with a proteolitic enzyme, e.g. pepsin.
  • the starting lactoferrin can be of natural or synthetic origin, and can include different forms, both monomeric and oligomeric, possibly already bound to iron (ololactoferrin) or not bound to iron (apolactoferrin) . Further details on lactoferrin hydrolyzates and on the preparation thereof are reported, for example, in the following articles: Wakabayashi H., et al, Current Pharm.
  • the peptides resulting from lactoferrin proteolysis generally have a number of amino acid units from 5 to 30, preferably from 5 to 15.
  • the weight ratio between lactoferrin and/or its hydrolyzate and pullulan is from 0.01:1 to 1:1, more preferably from 0.04:1 to 0.4:1.
  • the complex according to the present invention comprises a mixture of at least one lactoferrin and at least one lactoferrin hydrolyzate, the weight ratio between said at least one lactoferrin and said at least one lactoferrin hydrolyzate being between 5:95 and 95:5, preferably between 50:50 and 90:10.
  • the Applicant has found that the combination of a lactoferrin and/or a hydrolyzate thereof with a pullulan as defined above, which is belived to be in the form of a stable complex in which the lactoferrin and/or its hydrolyzate are incorporated into a pullulan matrix, allows to obtain an improved antibacterial and antioxidant effect compared to the lactoferrin and/or its derivative used alone at the same concentrations, which is exerted for longer periods of time, so that the effect is prolonged without having to repeat the application of the composition too often.
  • pullulan is able to perform a protective function for lactoferrin and/or its derivative, so as to hinder or prevent any degradation of the protein structure due to, for instance, pH changes.
  • a protective function for lactoferrin and/or its derivative so as to hinder or prevent any degradation of the protein structure due to, for instance, pH changes.
  • Such variations may be caused, for example, by the addition of particular excipients suitable for particular formulations.
  • the complex can be formulated to have a pH value from 4 to 7.
  • the complex according to the present invention is preferably prepared through a process which comprises: preparing an aqueous solution of at least one pullulan; mixing the aqueous solution of at least one pullulan with at least one lactoferrin and/or a hydrolyzate thereof; subjecting the mixture thus obtained to a drying step.
  • the concentration of pullulan in the aqueous solution is preferably comprised between 0.1% and 10% by weight, more preferably between 0.3% and 5% by weight.
  • the aqueous solution may comprise water alone as a solvent or a mixture of water with a water-soluble organic solvent, in particular with glycerol.
  • Mixing between the aqueous solution of pullulan and the lactoferrin and/or its hydrolyzate is preferably accomplished by gradual addition of the lactoferrin and/or its hydrolyzate to the solution until a homogeneous dispersion of the lactoferrin and/or its hydrolyzate in the aqueous phase is achieved.
  • the mixing temperature is generally maintained between 15°C and 50°C.
  • the drying phase is carried out by atomisation (spray drying), according to known techniques.
  • atomisation spray drying
  • the mixture between pullulan and lactoferrin and/or its hydrolyzate in aqueous solvent is introduced into a high-pressure atomiser, which comprises a distribution ring provided with a plurality of nozzles through which the mixture exits in the form of tiny droplets (spray) in order to promote heat exchange due to increase in specific surface area.
  • the droplets come into contact with a hot air flow, which causes the aqueous solvent to evaporate very quickly, which is then removed in the form of vapour, while the solid components form dried particles, which generally have an average size (dm) varying from 20 pm to 200 pm, which depends mainly on sizes of the nozzle holes and characteristics of the atomiser.
  • the dried particles generally contain an amount of residual moisture from 1% to 8% by weight, preferably from 3% to 5% by weight.
  • the oral hygiene composition according to the present invention comprises, in addition to the complex, at least one physiologically acceptable excipient. This is in order to obtain a product for oral hygiene in different forms, for example toothpaste, mouthwash, gel, chewing gum, orodispersible tablet, gingival dyeing, and the like.
  • composition according to the present invention can comprise ingredients commonly used in the field of dental hygiene products, such as for example: abrasive agents in particulate form, humectants, binders, thickeners, viscosity regulators, surfactants, sweeteners, flavourings, preservatives, anti-plaque agents, dyes, etc.
  • ingredients commonly used in the field of dental hygiene products such as for example: abrasive agents in particulate form, humectants, binders, thickeners, viscosity regulators, surfactants, sweeteners, flavourings, preservatives, anti-plaque agents, dyes, etc.
  • compositions which include the complex according to the present invention may be presented in different forms, such as tablets, capsules, powders, aqueous suspensions, syrups, suppositories, and contain the common excipients used for such products.
  • nutraceutical compositions including the complex according to the present invention may be in the form of capsules, tablets, liquid formulations, bars, gels, powders, and the like.
  • they can include water and other products with nutritional value such as carbohydrates, lipids, proteins, vitamins, minerals and the like.
  • the complex in accordance with the present invention may be combined with other biologically active products in order to increase, for example, bacteriostatic and/or virustatic effect.
  • nutraceutical compositions particularly suitable in the prevention or treatment of intestinal dismicrobism, they may contain, in addition to the complex, one or more microorganism strains useful for intestinal microbiome, such as Lactobacilli and the like (e.g. Lactobacillus easel).
  • the complex may be formulated in the form of a suspension, gel, tincture, cream and the like, with excipients commonly used for such products.
  • the formulation may possibly contain a pH adjuster, in order to guarantee a physiological pH, e.g. lactic acid which provides a pH around 3.4/4, typical of vaginal environment.
  • a solution of a pullulan in water at a concentration of 1% by weight was prepared.
  • the weight ratio of (lactoferrin + hydrolyzate) to pullulan was equal to 0.08:1.
  • the weight ratio of lactoferrin to hydrolyzate was equal to 80:20.
  • the resulting mixture was subjected to atomisation (spray drying) in order to obtain the complex between pullulan and (lactoferrin + hydrolyzate) (PLL - Example 1), in the form of particles with an average diameter (d 9 o) equal to 100 pm.
  • the particles were dispersed in water at a concentration of either 1% by weight or 10% by weight.
  • compositions were tested to verify antimicrobial activity with respect to E. coli.
  • An amount equal to 9 g of the composition was added to 1 ml of inoculum of E. coli (ATCC 8739) so as to obtain a final charge of about 10 5 CFU.
  • 0.5 ml of the suspension was taken to carry out plate count (surface spatulation method). Percentage variation of the bacterial load was therefore determined with respect to the initial one.
  • the PLL complex both in 1% solution and in 10% solution, has an antibacterial activity that grows more slowly but is prolonged over time and remains at higher values even 24 hours after application, compared to the LL mixture.

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  • Bioinformatics & Cheminformatics (AREA)
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  • Nutrition Science (AREA)
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  • Urology & Nephrology (AREA)
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  • Oral & Maxillofacial Surgery (AREA)
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  • Cosmetics (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Compounds Of Unknown Constitution (AREA)

Abstract

La présente invention concerne un complexe entre au moins une lactoferrine et/ou un hydrolysat de celle-ci avec au moins un pullulane, et une composition comprenant ledit complexe et au moins un excipient physiologiquement acceptable, pour une utilisation orale ou pour une utilisation topique. Le complexe présente une stabilité et une biodisponibilité élevées, de sorte qu'il exerce une action bactériostatique, virostatique et/ou antioxydante qui est efficace et durable. Il peut avoir diverses utilisations, en particulier dans la prévention ou le traitement : des pathologies de l'appareil dentaire; des infections virales et/ou bactériennes des muqueuses nasales, orales et/ou oculaires ; des inflammations du système urogénital ; du dysmicrobisme intestinal ; des lésions cutanées, en particulier des plaies, des ulcères ou des érosions cutanées.
PCT/IB2022/056274 2021-07-08 2022-07-07 Complexe à base de lactoferrine et/ou d'un hydrolysat de celle-ci, son procédé de production et ses utilisations Ceased WO2023281430A1 (fr)

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EP22743589.8A EP4366760A1 (fr) 2021-07-08 2022-07-07 Complexe à base de lactoferrine et/ou d'un hydrolysat de celle-ci, son procédé de production et ses utilisations

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IT102021000018008 2021-07-08
IT102021000018008A IT202100018008A1 (it) 2021-07-08 2021-07-08 Complesso a base di lattoferrina e/o un suo idrolizzato, processo di produzione dello stesso, e relativi usi.

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Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2019026997A1 (fr) * 2017-08-03 2019-02-07 森永乳業株式会社 Film comestible
WO2020250195A1 (fr) * 2019-06-13 2020-12-17 L.I.Co.Rice S.R.L. Complexe entre un hydrolysat de lactoferrine et une silice, procédé de production de celui-ci, et utilisations associées

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019026997A1 (fr) * 2017-08-03 2019-02-07 森永乳業株式会社 Film comestible
WO2020250195A1 (fr) * 2019-06-13 2020-12-17 L.I.Co.Rice S.R.L. Complexe entre un hydrolysat de lactoferrine et une silice, procédé de production de celui-ci, et utilisations associées

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Title
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KUMAR DEEPAK ET AL: "An Insight To Pullulan: A Biopolymer in Pharmaceutical Approaches", INTERNATIONAL JOURNAL OF BASIC AND APPLIED SCIENCES, vol. 1, no. 3, 1 January 2012 (2012-01-01), pages 202 - 219, XP055905176, DOI: 10.14419/ijbas.v1i3.101 *
MOCANU G ET AL: "Associative pullulan gels and their interaction with biological active substances", JOURNAL OF CONTROLLED RELEASE, ELSEVIER, AMSTERDAM, NL, vol. 83, no. 1, 18 September 2002 (2002-09-18), pages 41 - 51, XP004380058, ISSN: 0168-3659, DOI: 10.1016/S0168-3659(02)00169-4 *
PARK TAE HOON ET AL: "Development and characterization of a superabsorbing hydrogel film containing Ulmus davidiana var. Japonica root bark and pullulan for skin wound healing", SAUDI PHARMACEUTICAL JOURNAL, ELSEVIER, AMSTERDAM, NL, vol. 28, no. 7, 3 June 2020 (2020-06-03), pages 791 - 802, XP086206885, ISSN: 1319-0164, [retrieved on 20200603], DOI: 10.1016/J.JSPS.2020.05.007 *
TIAN YU ET AL: "Orodispersible films based on blends of trehalose and pullulan for protein delivery", EUROPEAN JOURNAL OF PHARMACEUTICS AND BIOPHARMACEUTICS, ELSEVIER SCIENCE PUBLISHERS B.V., AMSTERDAM, NL, vol. 133, 29 September 2018 (2018-09-29), pages 104 - 111, XP085537699, ISSN: 0939-6411, DOI: 10.1016/J.EJPB.2018.09.016 *

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