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WO2011138050A1 - Procédé de vaccination - Google Patents

Procédé de vaccination Download PDF

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Publication number
WO2011138050A1
WO2011138050A1 PCT/EP2011/002294 EP2011002294W WO2011138050A1 WO 2011138050 A1 WO2011138050 A1 WO 2011138050A1 EP 2011002294 W EP2011002294 W EP 2011002294W WO 2011138050 A1 WO2011138050 A1 WO 2011138050A1
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WO
WIPO (PCT)
Prior art keywords
compound
epitope
carrier
vaccination
adjuvant
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2011/002294
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English (en)
Inventor
Claus-Michael Lehr
Steffi Hansen
Ulrich Friedrich SCHÄFER
Carlos Guzman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
Universitaet des Saarlandes
Original Assignee
Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
Universitaet des Saarlandes
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Publication date
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Publication of WO2011138050A1 publication Critical patent/WO2011138050A1/fr
Anticipated expiration legal-status Critical
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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6093—Synthetic polymers, e.g. polyethyleneglycol [PEG], Polymers or copolymers of (D) glutamate and (D) lysine

Definitions

  • the present invention relates in a first aspect to means, in particular, to transdermal or transcutaneous systems, for delivery of a compound, like an antigen for eliciting or modulating an immune response or a therapeutical effect in an individual.
  • Said means or systems comprise a carrier or particle containing a compound, like said antigen optionally in combination with an adjuvant.
  • the present invention relates to methods for transcutaneous or transdermal application of compounds, like vaccination, in particular, transappendageal vaccination.
  • the present invention relates to a pharmaceutical composition comprising carrier or particles containing the compound, like antigen and, optionally, adjuvant, whereby the compound, like antigen is released by the carrier or particles in situ.
  • the present invention relates to the use of said pharmaceutical composition in transdermal or transcutaneous delivery of compounds, like for vaccination of an individual, in particular, for use in transappendageal vaccination of an individual.
  • microbial pathogens enter the body through mucosal surfaces or the (damaged) skin, causing local infections or transiting across them before systemic dissemination.
  • Mucosal vaccination is also characterised by an easy administration logistics and being highly accepted by the public.
  • antigens administered by the mucosal route are usually poor or non immunogenic. This may be due to accelerated antigen elimination by the non-specific host clearance mechanisms, antigen degradation by local enzymes, antigen alteration and antigen modification as a result of the environment, poor antigen penetration through the mucosa, limited access of vaccine antigen to antigen presenting cells and local peripheral tolerance, respectively.
  • adjuvants are those which are added into or co-formulated with the actual antigen, i.e. the substance which provokes the desired immune response, in order to enhance the humoral and/or cell mediated immune response.
  • adjuvants are compounds having immunopotentiating properties, in particular, when coadministered with antigens. While the adjuvant itself does not initiate an immune response, the adjuvant promote the immune response against the antigen administered with the vaccine.
  • the present invention aims to provide new means, new methods and uses allowing transcutaneous or transdermal delivery of compounds, e.g. for vaccination, in particular, transappendageal vaccination.
  • the present invention relates to means for delivery of a compound, like an epitope-containing compound, in particular, of an antigen, comprising a carrier or particle containing said compound, like epitope-containing compound, optionally, in combination with an adjuvant, for eliciting or modulating an immune response or a therapeutical effect in an individual.
  • the carrier or particle are characterised in being carrier or particles releasing in situ the compound, like an epitope-containing compound, upon triggering.
  • Triggering events include humidity, pH and/or salt concentration.
  • Other triggering events include changes in temperature or energy irradiation, like energy, like electromagnetic irradiation, e.g. UV, IR etc but also applying a magnetic field or ultra-sound. In particular, triggering occurs at a desired place and at a desired time-point, thus, releasing the compounds contained in the carrier or particles.
  • the means are in form of a transappendageal system for delivering of a compound, like an epitope-containing compounds via the microenvironment of hair follicles or the sweat gland.
  • the triggering event may be changes in salt, humidity or pH e.g. by sweat or sebum.
  • the present invention relates to a method for transcutaneous or transdermal delivery of a compound, in particular for vaccination, in particular, for transappendegeal vaccination.
  • Said method comprises the step of administering transcutaneously or transdermal ⁇ , in particular, transappendageally to an individual means for delivery a compound, like an epitope-containing compound, in particular, an antigen, and, optionally, an adjuvant, present in the carrier or particle to an individual whereby the carrier or particle release the compound, like the epitope-containing compound in situ.
  • a triggering event like exposure to humidity, pH and/or salt concentration as well as temperature or energy rich irradiation.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising said means or systems or comprising carrier or particles containing the compound, like the epitope-containing compounds, like antigens, and, optionally, an adjuvant, whereby the carrier or particles release the compounds, like the epitope-containing compound in situ upon trigger- ing.
  • another embodiment of the present invention relates to a pharmaceutical composition for use in transdermal or transcutaneous delivery of compounds, in particular, for vaccination of an individual, in particular, for use in transappendageal vaccination of an individual whereby said pharmaceutical composition contains the means and/or systems according to the present invention, in particular, the antigen containing carrier and particles according to the present invention.
  • the present inventors recognised that transcutaneous or transdermal delivery of compounds, like vaccination, in particular, transappendageal vaccination is possible when using carrier or particles containing said compounds, like an epitope-containing compound, in particular, antigens. Said carrier or particles are characterised in releasing the compounds in situ only.
  • the present invention relates in a first aspect to means for delivery of a compound, like an epitope-containing compound, in particular, of an antigen, comprising a carrier or particle containing said compound, like the epitope-containing compound, optionally, in combination with an adjuvant, for eliciting or modulating an immune response or a therapeutical effect in an individual whereby the carrier or particles are characterised in releasing said compound, like the epitope-containing compound in situ upon triggering.
  • the triggering event may be based on humidity, pH, and/or salt concentration. Said triggering events include temperature or irradiation with energy, like electromagnetic irradiation but also ultra-sound or the use of magnetic-field.
  • the means according to the present invention are in form of a transdermal or transcutaneous system, in particular, in form of a transappendageal system, for delivering of the compounds like the epitope-containing compound into an individual.
  • transdermal As used herein, the terms “transdermal” or “transcutaneous” are used synonomously.
  • transappendageal refers to transfoHicular as well as transglan- dular application.
  • the application route include the route via the hair follicle or the sweat glands.
  • the term "compound” as used herein refers to any type of compounds including therapeutically or cosmetically active compounds.
  • the compound is a immune response eliciting or modulating compound or a therapeutically effective compound, e.g. a drug or prodrug, unless otherwise indicated.
  • carrier or particle containing a compound refers to carrier or particles which encapsulate compounds, like epitope-containing compounds and/or which carry the compounds, like epitope-containing compounds, on their surface or adsorb the compounds in a way that the compounds, like the antigen/epitope, are not released before triggering or not inactivated or do not elicit or modulate an immune response or is therapeutically effective before triggering.
  • the carrier or particles containing the compounds, like the epitope-containing compound do release the compounds, like the epitope-containing compound, after exposure to the triggering event.
  • epitope-containing compound refers to any compound or molecule either naturally or artificially, preferably provided in isolated form, containing an epitope allowing eliciting a desired immune response e.g. vaccination, in an individual.
  • the epitope may be combined with foreign moieties depending on the desired characteristics.
  • suitable epitope-containing compounds useful for eliciting an immune response in an individual.
  • the epitope- containing compound is an antigen which may be modified. Modification includes solvatisation, the antigen in form of suitable salts, etc. as well as modifications improving stability, bioavailability, etc.
  • the compound may encode an antigen containing the desired epitope which will be expressed in the cells. That is, the epitope-containing compounds include DNA/RNA based vaccines as well.
  • the compounds present in the carrier or particles may be immune modulators.
  • immune modulators modulate, e.g. direct the immune response in an individual, e.g. from a Th1 to a Th2 response or vice versa.
  • the modulators may modulate the response from a humoral to a cellular or vice versa.
  • the carrier or particles are nanometer-sized carrier or particles. It is particularly preferred that the particles or carriers are composed of biodegradable and biocompatible material, like polymers. Of course, other materials are possible including organic or inorganic polymers, lipids or natural occurring, isolated material.
  • the most studied polymers for vaccine delivery are undoubtedly poly(lactic-co-glycolic acid) (PLGA) and chitosan (derivatives). Both polymers share the properties to be safe, biodegradable and suitable to prepare nanoparticles.
  • PLGA poly(lactic-co-glycolic acid)
  • chitosan derivatives
  • the means and systems according to the present invention are particularly adapted to deliver the compound, like the epitope-containing compounds transappendageally, that is, into the microenvironment of hair follicles, in particular, hair bulbs, or into the microenvironment of sweat glands.
  • the present invention relates in a further aspect to a method for transcutaneous or transdermal delivery of compounds, like vaccination comprising the step of providing a compound, e.g. an antigen and, optionally, an adjuvant present in a carrier or particle whereby the carrier or particle releases the compound, e.g. the antigen and, optionally, adjuvant in the microenvi- ronment of a hair follicle, like hair bulbs, or sweat glands.
  • the transappendageal like the transfollicular or the transglandular, route appears as a very attractive and safe strategy for the delivery of compounds, like vaccine antigens to enhance dendritic cells accumulating around hair bulbs of glands. Hair bulbs are also an excellent reservoir being only slowly cleared by hair growth and sebum production.
  • the follicles are covered by an epidermal barrier and in deeper regions tight junctions prevent even small nanoparticles having a size of below 30 nm from transfer into the living skin tissue. It is known that ⁇ -large pollen accumulates in the follicle opening, sebaceous gland or dermatoglyphs very rapidly so that pollen antigens can enter the sebum- filled follicle.
  • the process is enabled by pollen-bursting in a moist atmosphere with sufficient humidity, e.g. provided by sweat, and the subsequent delivery of antigens into the follicles.
  • the antigens are then ingested by Langerhans cells that are found in the surrounding of the hair follicles. This in turn triggers immune responses in susceptible persons, being the central mechanisms of allergic contact dermatitis.
  • the underlying strategy of the present invention is to mimetic pollen delivery to vaccinate across the intact skin barrier. That is, a pollen- mimetic carrier of a size of typically 50 to 5,000 nm allows to transport the antigen optionally together with adjuvants into the lower parts of the skin to allow delivery of the vaccine antigens to Langerhans and dendritic cells.
  • the carrier is typically composed of a biocompatible and biodegradable polymer whereby the optimal size for penetration into hair follicles is about 300 to 1 ,000 nm, like 400 to 700 nm. This size matches the size of hair cuticula surface structures.
  • the carrier maintains its integrity during manufacturing, storage and application to the skin surface, but will release the antigen and, optionally, the mucosal adjuvants, in situ.
  • the release can be triggered e.g. upon contact with sweat (80 to 185 mosmol/kg pH 6.5) which creates swelling and/or bursting of the carrier or upon contract with sebum.
  • the triggering event is in particular the exposure to changes in humidity, pH, temperature and/or salt concentration.
  • Another possibility to trigger the release of the epitope-containing compound is energy-rich irradiation, e.g. electromagnetic irradiation. Typical examples of irradiation include visible and non-visible light, like UV, IR, e.g. laser induced release.
  • triggering may a applying a magnetic field or ultra-sound.
  • the triggering event may be one or more of the events described above.
  • Exposure to pH include change of the pH either into more basic or more acid pH range. Changes in salt concentration may include decrease or increase of the salt concentration.
  • the triggering event may be exposure to sweat or sebum as present on the skin and in hair follicles or sweat glands, e.g. as described for pollen.
  • a pollen mimetic transcutaneous vaccination allows vaccination of individuals. This is in particular useful for antigens showing low mucosal vaccination properties.
  • the antigen presenting cells present in the skin represents useful cells for eliciting or modulating an immune response allowing vaccination against a desired antigen.
  • the particles or carriers are composed of biodegradable polymers, like polylactide gly- colic acid polymers (PLGA), starch derivatives or other types of polysaccharides, like chitosan.
  • PLGA polylactide gly- colic acid polymers
  • polyacrylates may be used or other polymers known to be biocompatible and biodegradable.
  • the particles or carriers are characterised in showing a swelling, burst-out or triggered re- lease upon contact with the specific microenvironment in hair follicles or sweat glands, e.g. when be in contact with sweat.
  • the microenvironment in the hair follicle is characterised in the specific conditions of humidity, pH, salt concentration etc.
  • sebum is present.
  • the microenvironment present in the hair follicle or the sweat glands triggers the particles or carriers containing the antigen and, optionally, the mucosal adjuvants, thus, enabling transdermal, in particular, transapendageal vaccination.
  • the particles containing the antigen and, optionally, the mucosal adjuvants are triggered upon entry into the hair follicle, thus, enabling to cross the stratum corneum and eliciting or modulating an immune response or a therapeutic effect similar to the immune response which can be observed in case of collagenic contact dermatitis.
  • the present invention provides a method which does not harm the skin resulting in injuries of the stratum corneum, as it is the case when using needles or other types of stratum corneum destructing measures.
  • the antigen optionally together with a mucosal adjuvant to Langerhans and dendritic cells present in the skin around the hair bulb or hair follicle or the sweat glands allows to trigger an immune response.
  • said immune response includes a local as well as a systemic immune response, thus, providing protection against the antigen.
  • the method of the present invention relates in another aspect to means for transdermal vaccination comprising a composition of the particle or carrier of biodegradable and biocompatible polymers having a size of typically 50 to 5,000 nm containing antigen and, optionally, adjuvants.
  • Said particles or carriers present in said composition allows to cross the skin barrier through the hair bulbs after triggering the hair bulbs resulting in a swelling and/or bursting of said particles and carriers.
  • the triggering event is the contact with sweat and/or sebum.
  • the contacting medium may be a suitable medium providing the necessary humidity, pH and/or salt concentration for bursting said particles and carriers.
  • the present invention relates to a transdermal system comprising the particles describes particles or carriers containing the antigen.
  • Said transdermal system may be in form of a transdermal system containing adhesive components, e.g. in form of a plaster or patches or pavement e.g. silicone-based transdermal system.
  • the particles or carriers containing the antigen and, optionally, the adjuvants may be semi-liquid form e.g. in form of ointments, salves, cream or gel, etc. or in a liquid form or as a dispersion, like suspension, emulsion, solution, shampoo, etc allowing to migrate into the hair follicle of the individual to be treated.
  • the present invention relates to the use of the particles or carriers as described herein for transcutaneous vaccination whereby said particles or carriers contain an antigen and, optionally, an adjuvant, like a mucosal adjuvant.
  • the skilled person is well aware of methods for providing said particles or carriers containing the compounds, like the antigens and adjuvants, respectively.
  • the skilled person is well aware of suitable methods for administering and applying the same on the skin of the individuals to be treated.
  • the present invention is characterised in that the skin of the person to be treated is not impaired or damaged by applying the composition containing the particles or carriers.
  • the particles or carriers are composed of biodegradable and biocompatible material, like polymers, which do not irritate or harm the skin or which would elicit or modulate an immune response or a therapeutical effect.
  • said particles or carriers are suitable transdermal systems for delivery of compounds, like vaccine compositions containing the compounds, e.g. the antigen and an adjuvant, preferably a mucosal adjuvant, respectively.
  • a pharmaceutical composition e.g. in form of a transcutaneous or transdermal system, in particular, in form of a transap- pendageal system.
  • Said pharmaceutical composition comprises carriers or particles containing epitope-containing compounds and, optionally, an adjuvant, characterised in that the carrier or particles release the compound, like an epitope-containing compound upon triggering in situ.
  • the triggering event may be any event as described above.
  • the pharmaceutical composition may be provided solid, semi-solid or liquid form, e.g. in form of a plaster, film, patch or pavement or in form of an ointment, salve, lotion, cream or gel, as a dispersion, suspension, emulsion, solution or shampoo.
  • the present invention is particularly useful for vaccination including vaccination against infection, cancer or autoimmunity e.g.for hyposensibilisation against allergy inducing compounds. It may be envisaged to use the means and systems as well as the pharmaceutical compositions according to the present invention to treat or prevent atophic dermatitis or other antigen, in particular, allergy induced diseases or conditions.
  • Ovalbumin was used as a model antigen due to the availability of reagents for performing an in depth characterisation of immune responses including known dominant, subdominant and cryptic epitopes; TCR transgenic animals; specific antibodies; LPS free antigen; etc.
  • As adjuvant a Toll like receptor agonist promoting mixed T helper (Th1) and Th2 responses was included, since it promotes broader responses and target receptors and mechanisms of action are well-known.
  • the particles or carriers according to the present invention were evaluated for the in vitro capacity to affect the activation, maturation, and antigen processing/ presentation functions of bone marrow-derived murine DC.
  • the process of differentiation was followed by analysing the expression of surface molecules by FACS.
  • the secreted cytokines and chemokines were evaluated by using flow cytometric bead arrays or ELISA.
  • the functional capacity was assessed by measuring DC ability to take up, process and present antigens to CD4+ and CD8+ T cells from TCR transgenic animals for OVA (ovalbumin).
  • mice were used to immunise transcutaneously mice via the skin where the hair has been clipped before treatment.
  • the control animals received pollen-mimetic empty carriers or carriers containing only antigen.
  • standing groups of mice received transcutaneous vaccines based on A-B moieties toxins as adjuvants, and antigens co-delivered with established mucosal adjuvants by either oral (CTB) or intranasal (CpG, TLR agonists) route.
  • CTB oral
  • CpG, TLR agonists intranasal
  • Immunisation protocols based on one or multiple doses were tested.
  • Humoral and cellular immune responses were characterised at systemic and mucosal level, e.g. lungs, nasal cavities, vaginal tract, intestinal tract.
  • Said vaccine- relevant antigens may be derived from hemagglutinin from the influenza virus and PspA/ PscB from Streptococcus pneumoniae for respiratory pathogens; VP6/ VP4 from Rotavirus and Psn/ LcrV for Yersinia spp; for enteric pathogens; Tat/ Gag of HIV and HBsAg from HBV for sexual transmitted pathogens.
  • the immune response can be evaluated and tailored further due to incorporation of different adjuvants allowing promotion of polarised Th1 , Th2 or Th17 responses. Suitable mucosal adjuvants are described e.g. in EP 1 973 571. Further, the responses are improved by incorporating DC/ Langerhans cells targeting moieties. If appropriate, the transcutaneous vaccination may be accompanied by a mucosal challenge or boosts.
  • Loading of the carrier and particles with the epitope-containing compounds may be effected by known methods for encapsulation or adsorption of compounds.
  • Suitable adjuvants include known systemic and/or mucosal adjuvants, e.g. MALP-2 or derivatives thereof or cyclic-dinucleotides, like c-diAMP.
  • PLGA nanoparticles were prepared by an adapted double emulsion method. Briefly, 100 mg of PLGA were dissolved in 2.5 ml of ethyl acetate at room temperature. Then 5 mg (in 200 ⁇ , 400 ⁇ , 600 ⁇ ) of Ova solution in water was added to it and homogenized (Ultra-Turrax TP18/10, IKA, Germany) at 18.000 rpm for 2 min. Then 5 ml of an aqueous phase containing the stabilizer (1 %, 2%, 3% w/v) was added to it and again homogenized at 18.000 rpm for 2 min.
  • the resulting w/o/w emulsion was poured into 25 ml of water under constant stirring to diffuse and finally evaporate the organic solvent. This resulted in nanoprecipitation and formation of nanoparticles.
  • the nanoparticles were freeze-dried after mixing with trehalose (0.5% w/v). Optimization of formulation and process parameters
  • Nanoparticles of PLGA were prepared by using different types of stabilizers like PF-68, sodium cholate, and PVA. The right stabilizer was identified based on the optimum particle size and entrapment efficiency.
  • the best suitable stabilizer in the above steps was then screened for the concentration of the stabilizer used in the preparation of the nanoparticles.
  • the optimum concentration was determined on the basis of particle size, size distribution, zeta potential.
  • the nanoparticles were characterized for their particle size and size distribution (mean diameter and polydispersity index) as well as their electro- phoretic mobility using a Zetasizer nano ZS (Malvern Instruments Ltd., Worcestershire, UK).
  • the entrapment efficiency of the nanoparticles with OVA was determined after their hydrolysis using the QuantiPro bicinchoninic acid (BCA) assay (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. Briefly, lyophilized samples of purified nanoparticles were hydrolyzed in 0.1 N NaOH (37°C) for 2 h and neutralized with 0.1 N HCI containing 3.3 m M Na 2 HP0 4 and KH 2 P0 and determined with the QuantiPro BCA method.
  • BCA QuantiPro bicinchoninic acid
  • the surface morphology of nanoparticles was analyzed by atomic force microscope (AFM) and scanning electron microscopy (SEM).
  • the particles obtained after freeze drying were re-dispersed in double distilled water (dd. water) to see the effect on size and PDI after freeze dry- ing.
  • PVA was identified as a good stabilizer for the freeze drying with respect to stability in size and PDI.
  • Entrapment efficiency of the compound in the nanoparticles showed an increase with increase in compound loading up to 5% loading after which possibly the polymer was saturated with the compound.
  • SEM & AFM image of nanoparticles shows distinct spherical and particles with a narrow size distribution and a smooth surface.
  • the optimized nanoparticle batch with initial drug loading (5 % w/w of polymer) resulted in particles with a mean diameter of 244.1 ⁇ 4.55 nm, narrow size distribution (PDI) of 0.084 ⁇ 0.016, zeta potential of -20.58 ⁇ 0.17 with % EE of 47.31 ⁇ 3.64.
  • PDI narrow size distribution
  • Chitosan-PLGA nanoparticles were prepared by an adapted double emul- sion method. Briefly, 100 mg of PLGA were dissolved in 2.5 ml of ethyl acetate at room temperature. Then 5 mg (in 200 ⁇ , 400 ⁇ , 600 ⁇ ) of Ova solution was added to it and homogenized (Ultra-Turrax TP18/10, IKA, Germany) at 18.000 rpm for 2 min. Then 5 ml of an aqueous phase containing the PVA stabilizer (2% w/v) and 0.3% w/v Chitosan (ProtasanTM UP, NovaMatrix, USA) was added to it and again homogenized at 18.000 rpm for 2 or 4 min. The resulting w/o/w emulsion was poured into 25 ml of water under constant stirring to diffuse and finally evaporate the organic solvent. This resulted in nanoprecipitation and formation of nanoparticles.
  • the in vitro release of OVA from the nanoparticles has been carried out by a centrifugation method. Briefly, for each pre-determined time point in the release profile an Eppendorf cup containing 1 ml of nanoparticles suspension in PBS (pH 5.5) was incubated at 37 °C for 3 days under continuous stirring at 100 rpm. At those pre-determined time points, the respective eppendorff cup was centrifuged for 20 min at 24,000 g and the supernatant wasd analyzed by using the QuantiPro BCA assay.
  • OVA encapsulated PLGA and Chitosan-PLGA nanoparticles with or without adjuvants was evaluated on the efficacy of the particulate system in provoking the immune response in SHK-1 mice.
  • a dose of 10 pg OVA (encapsulated in nanoparticles) was applied as aqueous gel formulation (1.5% w/v Natrasol Typ 250 M) to the skin of SHK-1 mice.
  • the present invention provides suitable methods for trans- dermal vaccination, suitable systems and means for transdermal vaccination as well as its use for the same.
  • the method comprises administering or applying particles or carriers containing the antigen and, optionally, adjuvants, like mucosal adjuvants, on the skin of an individual.
  • Said particles or carriers are suitable to enter into hair follicles.
  • said particles or carriers are characterised in being composed of biodegradable and biocompatible polymers. Said polymers are characterised in displaying release of the epitope-containing compounds, like antigen, e.g. after swelling and bursting after coming in contact with sweat or sebum, etc., i.e.
  • the mi- croenvironment present in the hair bulbs or hair follicles of said individuals.
  • the antigens and adjuvants cross the skin barrier and can be delivered to Langerhans and dendritic cells which are present around the hair bulbs, thus, eliciting a local as well as a systemic immune response.
  • the vaccination according to the present invention by tropical application represent an approach having the following advantages: needle-free method which ensures patient compliance
  • the vaccines according to the present invention stimulates both systemic and mucosal protective immune responses in contrast to most state of the art vaccine formulations.
  • Embodiments of the present invention include:
  • a method for transcutaneous or transdermal vaccination of an individual comprising the step of providing an antigen and, optionally, an adjuvant present in a carrier or particle whereby the carrier or particle releases the antigen and the adjuvant in the microenvironment of a hair follicle, like hair bulbs.
  • Means, in particular, a Transdermal System, for delivery of a vaccine comprising a carrier or particle containing the antigen and, optionally adjuvant for eliciting an immune response, the carrier or particle are characterized by showing swelling and/or bursting upon triggering with humidity, pH and/or salt concentration, in particular, after triggering with sweat, e.g. in the microenvironment of hair follicles.
  • transdermal system of embodiment in form of a plaster, patch or pavement or in form of an oitment, salve etc.

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  • Dermatology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicinal Preparation (AREA)

Abstract

Selon un premier aspect, la présente invention concerne des moyens, en particulier, des systèmes transdermiques ou transcutanés pour l'administration d'un composé, tel qu'un antigène, en vue de provoquer ou de moduler une réponse immunitaire ou un effet thérapeutique chez un individu. Lesdits moyens ou systèmes comprennent un support ou une particule contenant un composé, tel que ledit antigène facultativement en combinaison avec un adjuvant. Selon un autre aspect, la présente invention concerne des procédés pour l'application transcutanée ou transdermique de composés, tels qu'une vaccination, en particulier, une vaccination trans-appendice. En outre, la présente invention concerne une composition pharmaceutique comprenant un support ou des particules contenant le composé, tel qu'un antigène, et, facultativement, un adjuvant, le composé, tel qu'un antigène, étant libéré par le support ou les particules in situ. Enfin, la présente invention concerne l'utilisation de ladite composition pharmaceutique dans l'administration transdermique ou transcutanée de composés, telle que pour une vaccination d'un individu, en particulier, pour une utilisation lors d'une vaccination trans-appendice d'un individu.
PCT/EP2011/002294 2010-05-07 2011-05-09 Procédé de vaccination Ceased WO2011138050A1 (fr)

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