Classifications

• • 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
  • A61K9/0036—Devices retained in the vagina or cervix for a prolonged period, e.g. intravaginal rings, medicated tampons, medicated diaphragms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
  • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers

Definitions

• the present disclosure relates to the prevention of sexual transmission of human immunodeficiency virus (HIV) subtypes 1 and/or 2, namely by the vaginal route upon penile sexual penetration. It is intended to deliver antiretroviral compounds upon insertion into the vaginal canal. Antiretroviral compounds can inactivate the virus directly or interfere with their cellular cycle at the mucosal level.
• HIV human immunodeficiency virus
• the present subject-matter discloses a new microbicide pharmaceutical formulation for topical pre-exposure prophylaxis.
• HIV/AIDS epidemic is an increasing global concern, responsible for millions of deaths every year. Since no effective curative therapy is available, these numbers can only be hindered by effective preventive strategies of HIV transmission, such as anti-HIV vaccines or condoms; however, for different reasons, both strategies have not been able to stop HIV infection spreading.
• topical pre-exposure prophylaxis (PrEP) emerged in the battle against sexual HIV transmission.
• This strategy comprises the use of vaginal products containing anti-infective agents, termed microbicides, in order to protect against HIV and possibly other pathogens [1].
• topical PrEP may be particularly important in those settings where women do not have the possibility to negotiate condom use, since usage of these products do not require the cooperation, consent or even knowledge of male partners.
• Proof of concept for vaginal microbicides has been recently achieved for vaginal microbicides with the CAPRISA 004 study [2]. After several initial failures using products based on non-specific antiviral compounds, this Phase 2b clinical trial found partial but significant protection by a tenofovir containing gel upon vaginal application around the time of sexual intercourse.
• This nanosize dendrimer is a highly branched synthetic polymeric macromolecule obtained by controlled polymerization of polylysine branches from a reactive central core (benzhydrylamine amide) and terminally derivatised with naphthalene disulfonate groups, conferring an outer polyanionic surface allegedly responsible for its activity.
• the dendrimer surface groups bind to gpl20 glycoprotein on HIV's envelope, thus blocking virus attachment to CD4 cellular receptors.
• poly(lactide-co-glycolide) (PLGA) nanoparticles are feasible for the delivery of PSC-RANTES, an antiretroviral chemokine, to the vaginal epithelium [4].
• PLGA nanoparticles can retain the anti-HIV activity of PSC-RANTES, while allowing increased tissue uptake, permeation, drug targeting to the site of action, and anti-HIV-1 activity over extended periods of time.
• microbicides namely to HIV-target cells
• capability to protect anti-HIV agents from enzymatic degradation, and wide distribution through the genital tract upon vaginal administration are interesting features that may be advantageous for microbicide development [3].
• Different active compounds have been incorporated in different type of nanocarriers for the development of microbicides, namely tenofovir, dapivirine, zidovudine, efavirenz, raltegravir, saquinavir, RANTES derivatives and MC1220.
• Useful nanocarriers include systems based in polymeric nanoparticles (nanospheres or nanocapsules), polymeric micelles, dendrimers, nanogels, polymersomes, polymer-modified nanocarriers, solid lipid nanoparticles, and liposomes [3]. [0006] Nonetheless, substantial work is still required to transform the concept of nanotechnology-based microbicides in products that can be used in real life situations. In particular, developed nanosystems require adequate formulation to be used by women. Vaginal films are solid and comprise thin sheets, usually of polymeric nature. These present different forms although rectangular or square versions with sides measuring 5-10 cm are more frequent. Films are usually obtained by casting methods although others may apply [6].
• Vaginal films are among the most acceptable vaginal dosage forms and present advantageous technological features such as good physical-chemical stability, ease of manufacture, and reduced price. Also, these dosage forms allow for prolonged in situ retention and release of incorporated drugs. Films are intended to be self-administered in the vagina, usually with the aid of one or more fingers, and disperse/dissolve therein upon contact with mucosal fluids. Films provide interesting platforms for the incorporation and human administration of drug-loaded nanocarriers. The present invention relates to the use of antiretroviral-loaded nanoparticles-in- vaginal films for prevention of sexual HIV transmission.
• the hereby described invention provides an innovative way for the single or combined administration of antiretroviral compounds by means of their incorporation in nanoparticles and subsequent inclusion in vaginal polymeric films.
• the incorporation of nanoparticles into films presents synergistic advantages over the administration of active compound(s) in films alone or active compound(s) in nanoparticles alone.
• mucoadhesive films allow nanoparticles to retain for prolonged time periods in the vagina. This is in contrast with currently available rapid dissolving films. This long lasting ability circumvents an important limitation of nanoparticles, namely the rapid vaginal clearance after administration [3].
• the large surface of films contributes to a better distribution of nanoparticles throughout the vaginal milieu.
• Specific formulation of films allows releasing nanoparticles in a controlled fashion, and controlled rate that nanocarriers can reach the mucosal tissues.
• the use of nanoparticles as carriers allows improving the biodistribution and pharmacokinetics profile of active compound(s), which are related with enhanced mucosal accumulation and cell-targeting within the tissue [16].
• the use of nanoparticles allow for the combination of physicochemically incompatible active compounds which could not be previously formulated within the same film.
• the hereby invention is particularly suited for drugs presenting distinct solubility profiles and to which no common solvent system is available. Nanoparticles can also abbreviate stability problems of active compound(s) with poor solubility within film matrices.
• the present disclosure provides a new microbicide pharmaceutical composition for topical pre-exposure prophylaxis or topical post-exposure prophylaxis.
• the present disclosure relates to the incorporation of nanoparticles loaded with different antiretroviral compounds in soft and flexible vaginal films.
• Antiretroviral compounds may be present in films either as single agent or in combination with nanoparticles.
• Antiretroviral-loaded nanoparticles-in-vaginal films are to be administered in the vagina around the time of sexual intercourse in order to prevent the sexual transmission of HIV.
• the technical problem underlying the invention was to develop a composition that provides a way for combined antiretroviral compounds, even when incompatibilities, such as the inability to find a common solvent for the production of other dosage forms (e.g. gels).
• composition of the present disclosure also provides a suitable way to administer antiretroviral compounds that can be useful in preventing the vaginal transmission of HIV.
• the composition of the present disclosure comprises retroviral loaded nanoparticles, this form allowed the treatment of the patient with an increase dosage of antiretroviral.
• antiretroviral compounds into nanoparticles allows obtaining significant advantages over non-formulated compounds, such as modified antiretroviral compound(s) release, increased antiretroviral compound(s) solubility, protection of incorporated antiretroviral compound(s), intracellular antiretroviral compound(s) delivery and targeting of HIV-susceptible cells, enhanced antiretroviral compound(s) safety/toxicity and activity profiles, modulation of mucoadhesion, allow effective genital distribution and mucosal coating and influence antiretroviral compound(s) pharmacokinetics.
• nanoparticles in the film of the present disclosure allows reducing leakage after administration as it forms a gel like fluid that, contrary to fast dissolving gels, enhances retention in the vagina.
• composition of the present disclosure allows incorporation of antiretroviral compounds with different properties, including those thermo-sensitive and/or moisture-sensitive.
• composition of the present disclosure are readily available and generally regarded as safe.
• composition of the present disclosure relates for the use in a method for prevention or treatment of human immunodeficiency virus infections comprising antiretroviral compound-loaded nanoparticles, wherein said composition is administrated in a vaginal film; wherein said film comprises:
• an antiretroviral compound selected from a list consisting of as tenofovir, dapivirine, zidovudine, efavirenz, raltegravir, saquinavir, emtricitabine, ANTES derivatives MC1220, or mixtures thereof;
• polymeric nanoparticles comprising a compound selected from a list consisting of: poly(lactide-co-glycolide), poly(lactide), poly(beta-hydroxybutyric acid), poly(beta-hydroxyvaleric acid), polycaprolactone, polyacrylates, poly(alkyl cyanoacrylates), popvalyanhydrides, polyphosphoesters, poly-L-lysine, poly(ortho esters), polyphosphazenes, poly(amidoamine), polysaccharides such as chitosan, alginates, cellulose derivatives; proteins such us albumin and/or gelatine; copolymers of poly(ethylene glycol) , or poly(ethylene oxide), or mixtures thereof;
• a polymeric film comprising a compound selected from a list consisting of: PVA, cellulose derivatives, carrageenan, xanthan gum, arabic gum, agar gum, guar gum, alginate, tragacanth gum, karaya gum, locust bean gum, gellan gum, glucomannan, gallactomannan, pectin, collagen, gelatin, hyaluronic acid, chitosans, starch, pullulan, polyvinylpyrrolidone, polyethylene, polypropylene, PEO, copovidone, poloxamers, polyacrylates, polyvinyl amine), boronate-containing polymers, or mixtures thereof.
• the antiretroviral compound is dissolved, entrapped, encapsulated or bound to the surface or throughout the nanoparticles (nanoparticle matrix).
• the composition may comprise 2-4 % (w/v) of an antiretroviral compound, 4-10 % (w/v) of a polymeric nanoparticles, 90-94 % (w/v) of a polymeric film.
• the loaded nanoparticles may comprise at least two antiretroviral compounds.
• the polymeric film may further comprise free antiretroviral.
• the composition may comprise a first antiretroviral loaded in the nanoparticle and a second antiretroviral free in the film, and the second antiretroviral is different from the first antiretroviral, preferably the first and second retroviral are least two antiretroviral.
• the composition may comprise the combination of the following antiretroviral tenofovir and dapivirine, tenofovir and zidovudine, tenofovir and efavirenz, tenofovir and raltegravir , tenofovir and saquinavir, tenofovir and emtricitabine, tenofovir and MC1220, tenofovir and RANTES derivatives, dapivirine and zidovudine, dapivirine and efavirenz, dapivirine and raltegravir, dapivirine and saquinavir, dapivirine and emtricitabine, dapivirine and RANTES derivatives, dapivirine and MC1220, zidovudine and efavirenz, zidovudine and raltegravir, zidovudine and saquinavir, zidovudine and saquina
• the nanoparticles may further comprise a specific drug-targeting, preferably bound to the nanoparticle surface.
• the specific drug-targeting may be: peptides, proteins, antibodies, enzymes, polymers, fatty acids, sugars, polysaccharides, RNA and/or DNA, or mixtures thereof.
• the nanoparticles may be nanospheres, nanocapsules, polymeric micelles, dendrimers, nanogels, polymersomes, polymer-modified nanocarriers, solid lipid nanoparticles, nanostructured lipid carriers, liposomes, or mixtures thereof.
• the nanoparticle size is between 1-1,000 nanometers, preferably between 5-100 nanometers.
• the size of the nanoparticle may be determined by Micro-CT 3D analysis or light-scattering techniques.
• the polymeric nanoparticles are PEG/PEO-b- poly(3-[(3-aminopropyl)amino]propylaspartamide), PEG/PEO-b-poly(amino acids), PEG/PEO-b- poly[(2-dimethylamino)ethyl methacrylate], PEG/PEO-b-poly(alpha,beta-aspartic acid), PEG/PEO-b-P(Asp) processing the hydrazide groups in the side chains, PEG/PEO-b-poly(beta- benzyl L-aspartate), PEG/PEO-b-PCL, PEG/PEO-b-PLA, PEG/PEO-b-phosphatidylethanolamine, PEG/PEO-b-polyethylethylene, PEG/PEO-b-poly(glutamic acid), PEG/PEO-b-poly
• the polymeric films may be obtainable by solvent- casting or hot-melt extrusion methods.
• the nanoparticles may be obtainable from monomers using standard polymerization methods such as emulsion polymerization, suspension polymerization, interfacial polymerization, or their combinations.
• the nanoparticles may be obtainable from preformed polymers by using methods such as emulsification/solvent evaporation, nanoprecipitation or solvent displacement, phase-inversion nanoencapsulation, coacervation/precipitation, extrusion, ionic gelation, spray-drying/nebulization, interfacial deposition, supercritical fluid technology, sieving method, thermal denaturation of proteins, desolvation of proteins, salting out, hot melting, fluid bed coating, spray-freezing, spray-cooling, high pressure homogenization, microemulsion technique, microwave-assisted microemulsion technique, solvent evaporation, double emulsion, solvent diffusion, solvent injection, high shear homogenization and/or ultrasound, membrane contactor method, supercritical fluid extraction of emulsions, phase inversion temperature technique, micellar self-assembling, lithographic techniques, nanoimprinting, electrospinning, freeze-drying of monophase solutions.
• methods such as emulsification/solvent evaporation,
• the nanoparticles may be incorporated in the film during film production, preferably by dipping or spraying pre-formed films.
• compositions for use according to any of the previous claims my further comprising preservatives, antioxidants, stabilizers, colouring agents, flavouring agents, fillers, pH buffering agents, fragrances, disintegration or dissolution modifiers, or combinations thereof.
• preservatives antioxidants, stabilizers, colouring agents, flavouring agents, fillers, pH buffering agents, fragrances, disintegration or dissolution modifiers, or combinations thereof.
• the preservatives may be benzoic acid and salts, benzyl alcohol, boric acid and salts, chlorobutanol, chlorocresol, chorhexidine salts, imidazolidinyl urea, parabens, sodium benzoate, sorbic acids and salts or mixtures thereof.
• the antioxidants may be alpha-tocopherol acetate, acetylcysteine, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, cysteine, cysteine hydrochloride, d-alpha-tocopherol, monothioglycerol, propyl gallate, tocopherols or mixtures thereof.
• the stabilizer agents may be lactic acid, EDTA, or mixtures thereof.
• the colouring agents may be titanium dioxide, indigo carmine, caramel, or mixtures thereof.
• flavouring agents may be raspberry, honey, spearmint oil, vanilla, cocoa, chocolate, or mixtures thereof.
• the fillers may be lactose, starch, cellulose derivatives, dibasic calcium phosphate dehydrate, mannitol, sorbitol, sucrose, calcium sulphate dehydrate, dextrose or mixtures thereof.
• the pH buffering agents may be lactic acid, tartaric acid, citric acid, mixtures thereof.
• the fragrances may be aromatic essential oils, aqueous extracts or mixtures thereof.
• the disintegration/dissolution modifiers may be starch derivatives, clay, alginates, polyvinyl pyrrolidone or mixtures thereof.
• the dose or dosage form may be administered to the subject, for example, once a day, twice a day, or three times a day. In other embodiments, the dose is administered to the subject once a week, once a month, once every two months, four times a year, three times a year, twice a year, or once a year.
• Figure 1 Schematic representation of different polymer-based nanoparticles. Polymeric parts are generally presented in black. Hydrophilic and hydrophobic components of amphiphilic polymers in micelles and polymersomes are presented in black blue and grey, respectively. Liquid (aqueous) content of nanocapsules, polymersomes and nanogels is depicted in grey. Reprinted with permission from [16].
• Figure 2 Schematic representation of typical dimensions and thickness are indicated.
• An aspect of the present disclosure is to provide a soft, flexible film containing antiretroviral compound-loaded nanoparticles to be administered in the human vagina in order to prevent sexual transmission of HIV.
• the manufacturing process of the film composition of the present subject-matter involves two steps: (1) production of nanoparticles loaded with antiretroviral compounds; and (2) production of vaginal films with the incorporation of nanoparticles.
• Nanoparticles comprise specifically engineered systems (or materials) of dimensions in the nanometer scale and produced by various methods in order to incorporate one or more antiretroviral compounds.
• the nanometer scale is typically used in the vast framework of nanotechnology by EU and other worldwide authorities for systems or materials having one or more dimensions of the order of 100 nm or less (usually 1-100 nm).
• nanomedicine i.e., application of nanotechnology to medicine
• nanotechnology typically encompassing several hundred nanometers and up to a limit of 1,000 nm [17].
• This last broader definition is used for the purpose of the hereby described the composition of the present disclosure - the suffix "nano" is used to describe objects having one or more dimensions in the range of 1-1,000 nm even if some of these may fail to comply with more strict legal definitions of nanotechnology.
• Nanoparticles are typically of polymeric nature and can be nanospheres, nanocapsules, polymeric micelles, dendrimers, nanogels, polymersomes or polymer-modified nanocarriers (Figure 1). Nanospheres are characterized by a continuous polymeric matrix and nanocapsules comprise an outer polymeric shell of variable thickness surrounding an oily or aqueous core. Lipid nanocarriers, namely solid lipid nanoparticles, nanostructured lipid carriers and liposomes can also be incorporated in films. Nanosystems of mixed nature, for example combining polymers and lipids, are also useful.
• One or several polymers of synthetic or natural origin are used to produce nanoparticles, including polyesters (for example, various grades of poly(lactide- co-glycolide) (PLGA), poly(lactide) (PLA), poly(beta-hydroxybutyric acid), poly(beta- hydroxyvaleric acid) and polycaprolactone (PCL)), polyacrylates, poly(alkyl cyanoacrylates), polyanhydrides, polyphosphoesters, poly-L-lysine, poly(ortho esters), polyphosphazenes, poly(amidoamine), polysaccharides (for example, various grades of chitosan, alginates, cellulose derivatives), and proteins (for example, albumin and gelatin), among others.
• polyesters for example, various grades of poly(lactide- co-glycolide) (PLGA), poly(lactide) (PLA), poly(beta-hydroxybutyric acid), poly(beta- hydroxyvaleric acid) and poly
• Copolymers of poly(ethylene glycol) (PEG) or poly(ethylene oxide) (PEO) may also be used, namely PEG/PEO-b- poly(3-[(3-aminopropyl)amino]propylaspartamide), PEG/PEO-b-poly(amino acids), PEG/PEO-b- poly[(2-dimethylamino)ethyl methacrylate], PEG/PEO-b-poly(alpha,beta-aspartic acid), PEG/PEO-b-P(Asp) processing the hydrazide groups in the side chains, PEG/PEO-b-poly(beta- benzyl L-aspartate), PEG/PEO-b-PCL, PEG/PEO-b-PLA, PEG/PEO-b-phosphatidylethanolamine, PEG/PEO-b-polyethylethylene, PEG/PEO-b-poly(gluta
• Materials used for producing nanoparticles of lipid-origin include phosphatidylcholine, cholesterol, stearylamine, dilauroylphosphatidylcholine, dipalmitoyl- phosphatidylcholine, l,2-dioleoyl-sn-glycero-3-phosphocholine, dimyristoylphosphatidic sodium, l,2-dioleoyl-3-trimethylammoniumpropane, N-[2,3-(dioleyloxy)propyl]-N,N,N- trimethylammonium chloride, 3- -[N-(N',N'-dimethylaminoethyl)carbamoyl]-cholesterol, 2,3- dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,Ndimethyl-l-propanaminium trifluoroacetate, dioleoyl phosphatidylethanolamine,
• Stabilizers are frequently used in order to allow nanoparticle production. These last include lecithin of different origins, poloxamers of different grades, sodium cholate, polyvinyl alcohol) (PVA) of different grades, sodium lauryl sulfate, cetrimide, cetyl trimethylammonium bromide, polysorbates of different grades, among others.
• Nanoparticles may be produced from monomers by using standard polymerization methods such as emulsion polymerization, suspension polymerization and interfacial polymerization. Nanoparticles may be produced from pre-formed polymers by using standard manufacturing methods such as emulsification/solvent evaporation, nanoprecipitation or solvent displacement, phase-inversion nanoencapsulation, coacervation/precipitation, extrusion, ionic gelation, spray-drying/nebulization, interfacial deposition, supercritical fluid technology, sieving method, thermal denaturation of proteins, desolvation of proteins, salting out, hot melting, fluid bed coating, spray-freezing, spray-cooling, high pressure homogenization, microemulsion technique, microwave-assisted microemulsion technique, solvent evaporation, double emulsion, solvent diffusion, solvent injection, high shear homogenization and/or ultrasound, membrane contactor method, supercritical fluid extraction of emulsions, phase inversion temperature technique, micellar self-
• nanoparticles are well characterized and have been fully described previously [18]. The choice of method is dependent on the material(s) used to form nanoparticles and the antiretroviral compound(s) to be incorporated in the previous. Antiretroviral compound(s) can be incorporated during or after nanoparticles formation, either alone or in combination with one or more compounds in the same nanoparticle system.
• Nanoparticles may present surface modification for specific drug-targeting.
• Surface modification can be performed by means of covalent or non-covalent binding of targeting moieties such as peptides, proteins, antibodies, enzymes, polymers, fatty acids, sugars and polysaccharides, and genetic material ( NA and DNA).
• Antiretroviral compounds to be loaded in nanoparticles include molecules belonging to different classes such as entry inhibitors, fusion inhibitors, nucleoside reverse transcriptase inhibitors (N TI), nucleotide reverse transcriptase inhibitors (NtRTI), non-nucleoside reverse transcriptase inhibitors (NNRTI), integrase inhibitors, protease inhibitors, and maturation inhibitors.
• Biopharmaceuticals such as neutralizing antibodies, RNA-based inhibitors (antisense RNA, ribozymes, aptamers, small interfering RNA) targeting viral or host ligands, and protein-based inhibitors (dominant-negative proteins, intrabodies, intrakines, fusion inhibitors, zinc-finger nucleases) targeting viral or host ligands can also be incorporated in nanoparticles.
• Antiretroviral compounds can be dissolved, entrapped, encapsulated or attached (covalent bonding, electrostatic interaction, hydrophobic interaction, hydrogen bonding, and/or van der Waals forces) to the surface or throughout the matrix of nanoparticles depending on the method of preparation and type of nanosystem.
• different PCL nanoparticles loaded with the NNRTI dapivirine used as a model microbicide drug are produced by nanoprecipitation.
• the laboratory-scale production of nanoparticles comprises the initial dissolution of 40 mg of PCL, 10 mg of poloxamer 338 NF (Pluronic ® F108 NF) as surface modifier, and variable amounts of dapivirine (0-10 mg) in 2 mL of acetone or a variable mixture of acetone:ethanol with the aid of mild heating (37°C) and vortexing. This last mixture is added dropwise (approximately one drop per second) to 20 mL of stirring water (200-600 rpm).
• Nanoparticles are immediately formed due to the rapid diffusion of acetone into water, resulting in the precipitation of PCL and spontaneous formation of nanoparticles. Stirring is continued overnight to allow partial organic solvent evaporation. Obtained nanoparticles are then recovered by ultracentrifugation at 50,000- 90,000xg for 30 min, and the obtained pellet washed twice with 20 mL of deionized water and re-recovered by ultracentrifugation. Ultracentrifugation can be replaced by low speed centrifugation (2,000xg, 30 min) using Amicon ® filter tubes. Samples can be freeze-dried or used in aqueous dispersion.
• Obtained nanoparticles are spheroid in shape, monodisperse and present typical mean hydrodynamic diameter values of 230-260 nm when acetone is used (zeta potential of -39 to -45 mV for nanoparticles produced using poloxamer 338 NF).
• hydrodynamic diameter values are reduced to 180-210 nm and zeta potential values are -29 ⁇ 6 mV.
• dapivirine-loaded PLGA nanoparticles are prepared at the laboratory scale by single oil-in-water emulsion/solvent evaporation method. Fifty milligrams of PLGA (Purasorb ® PDLG 5004A, 50:50 D,L-lactide:glycolide ratio, 0.4 dL/g inherent viscosity) and dapivirine (0-10 mg) are dissolved in ethyl acetate (2-6 mL) and mixed with 10 mL of a 1-5% PVA solution using a Vibra-CellTM VCX 130 ultrasonic processor equipped with a standard 6x113 mm probe (Sonics & Materials, Inc., Newtown, CT, USA) at 60-90% intensity for one minute.
• PLGA Purasorb ® PDLG 5004A, 50:50 D,L-lactide:glycolide ratio, 0.4 dL/g inherent viscosity
• dapivirine (0-10 mg) are dissolved in e
• Nanoparticles are monodisperse and spheroid in shape. Typical values of 115-172 nm and of -20 to -22 mV are obtained for hydrodynamic diameter and zeta potential, respectively. Drug association efficiency percentages values are 85-92% for a theoretical drug loading of 2%.
• the NN TI drug efavirenz is loaded in PLGA nanoparticles by using a single oil-in-water emulsion/solvent evaporation method.
• Efavirenz (5 mg) is dissolved in 1 mL of ethyl acetate and added to the polymer (Purasorb ® PDLG 5002, 50:50 D,L-lactide:glycolide ratio, 0.2 dL/g inherent viscosity) previously dissolved in 1 mL ethyl acetate.
• the aqueous phase (5 mL of 0.1% poloxamer 407 NF) is added, and sonicated for 60 seconds at 70% intensity (Vibra Cell, Sonics & Materials Inc., Danbury, CT, USA).
• the resulting emulsion is promptly poured into an additional 15 mL aqueous solution of 0.1% poloxamer 407 NF, and left under magnetic stirring for 4 hours at 200 rpm to allow organic solvent evaporation. Nanoparticles are then washed by centrifugation at 30,000xg for 50 minutes, and washed twice with water. High speed centrifugation can be replaced by low speed centrifugation (2,000xg, 30 min) using Amicon ® filter tubes.
• Nanoparticles are spheroid in shape.
• PLGA nanoparticles loaded with efavirenz present hydrodynamic diameter and zeta potential of 145 nm and -17 mV, respectively.
• Drug association efficiency is 96% at a theoretical drug loading of 11%.
• PLGA nanoparticles loaded with the NtRTI tenofovir or the NNRTI emtricitabine are also prepared at the laboratory scale by using a double water-in-oil-in-water emulsion/solvent evaporation method.
• Tenofovir (5 mg) is dissolved in 1 mL of water and added to 40 mg of PLGA (Purasorb ® PDLG 5002, 50:50 D,L-lactide:glycolide ratio, 0.2 dL/g inherent viscosity) previously dissolved in 4 mL ethyl acetate, under 30 seconds of vortexing in order to obtained the primary emulsion.
• PLGA may be partially replaced by stearylamine (1-10 mg) in order to increase the association efficiency of tenofovir.
• the primary emulsion is immediately transferred to an aqueous phase, containing 10 mL of 0.5% poloxamer 407 NF, and sonicated for 60 seconds at 70% intensity (Vibra Cell, Sonics & Materials Inc., Danbury, CT, USA).
• the resulting secondary emulsion is promptly poured into an additional 10 mL aqueous solution of 0.5% poloxamer 407 NF, and left overnight under magnetic stirring at 300 rpm to allow organic solvent evaporation. Nanoparticles are then washed by centrifugation at 30,000xg for 50 minutes, and washed twice with water.
• High speed centrifugation can be replaced by low speed centrifugation (2,000xg, 30 min) using Amicon ® filter tubes.
• Samples can be freeze-dried or used in aqueous dispersion.
• Nanoparticles are spheroid in shape. Typical values of 119-196 nm and of +31 to +50 mV are obtained for hydrodynamic diameter and zeta potential, respectively, of tenofovir-loaded PLGA nanoparticles containing stearylamine.
• the increasing amount of stearylamine increases the amount of associated tenofovir (from 19% at no stearylamine to 59% at 10 mg of stearylamine) but also polydispersion.
• PLGA nanoparticles loaded with emtricitabine and no stearylamine present hydrodynamic diameter and zeta potential of 104 nm and -22 mV, respectively.
• Drug association efficiency is 16% at a theoretical drug loading of 11%.
• films are defined as homogeneous, solid, soft, flexible, thin sheets composed mainly of certain film-forming polymers, plasticizers and active pharmaceutical ingredients (APIs) [6].
• APIs are defined, according to the proposal of the World Health Organization (WHO), as "a substance used in a finished pharmaceutical product (FPP), intended to furnish pharmacological activity or to otherwise have direct effect in the diagnosis, cure, mitigation, treatment or prevention of disease, or to have direct effect in restoring, correcting or modifying physiological functions in human beings" [19].
• WHO World Health Organization
• FPP finished pharmaceutical product
• the API(s) is(are) the antiretroviral compound(s).
• the antiretroviral compound(s) is/are incorporated into nanoparticles and/or dissolved or dispersed in the film.
• Nanoparticles can be dispersed throughout the film matrix or be present only at the surface depending on the manufacturing process.
• the general aspect of the invention is presented in schematic in Figure 2. Films may bear different shapes, from simple geometric figures to complex forms, but are typically square or otherwise rectangular, with side length in the range of 5-10 cm. Thickness of the film is usually in the range of 50-500 ⁇ . Films may be colorless or present different colors, and be transparent or present different degrees of opacity.
• Films are to be self-administered, either folded or in their original format, in the vaginal cavity around the time of sexual intercourse, typically before (within a few days to hours) or immediately after.
• dispersion or dissolution of the film occurs at different rates depending on its composition, and the nanoparticles and antiretroviral compound(s) (incorporated in the film matrix) are released from the film.
• Dispersion or dissolution of the films typically originates a gel like mixture that enhances retention in the vaginal canal and decreases leakage.
• Antiretroviral compound(s) incorporated in nanoparticles are also released at different rates depending on the composition of nanoparticles.
• film-forming polymers used to produce films include the following: PVA, cellulose derivatives (methylcellulose (MC), hydroxypropyl methylcellulose (HPMC), hydroxypropylcellulose (HPC), hydroxyethylcellulose (HEC), ethyl cellulose (EC), sodium carboxymethyl cellulose (NaCMC), microcrystalline cellulose, cellulose acetate, cellulose acetate phtalate), carrageenan, xanthan gum, Arabic gum, Agar gum, Guar gum, alginate, Tragacanth gum, Karaya gum, Locust bean gum, Gellan gum, glucomannan, gallactomannan, pectin, collagen, gelatin, hyaluronic acid, chitosans with variable molecular mass and degree of deacetylation and derivatives (thiolated chitosans, N-trimethylchitosans, N-carboxymethyl chitosans, O-carboxymethyl chitosans, star
• Plasticizers are used to provide flexibility and softness to produced films and include the following materials: PEG of different grades, PEG/PEO stearates, PEG/PEO distearates, PEO alkyl ethers, glycerine, propylene glycol, sorbitol, triacetin, dibutyl phthalate, and sorbitan esters. These last can be used alone or in mixture of two or more.
• Films also contain water, typically at concentrations below 20% of the total weight of the film.
• the films of the present disclosure can be produced by two general methods: solvent-casting or hot-melt extrusion [6].
• nanoparticles loaded with antiretroviral compound(s) can be incorporated in films at different stages of the production of the films.
• the general production process by solvent-casting involves the following steps: (1) preparation of the solution/dispersion to be cast (film dope), (2) manual or automatic casting of the film dope, (3) drying, (4) cutting of individual films, and (5) packaging.
• preparation of the film dope involves the dissolution or dispersion of the components of the formulation in an adequate solvent, typically water. Heating may be required in order to aid dissolution/dispersion. Antiretroviral compound(s) and/or antiretroviral compound-loaded nanoparticles can be incorporated at this stage.
• the casting solution/dispersion may be degassed at different stages by vacuum system, centrifugation and/or rest at 2-8°C. In automatic production machines, in-line degassing can be used. Casting is performed manually by transferring the film dope into molds of appropriate dimensions. It can be performed automatically using a commercial continuous belt casting machine.
• the film dope is transferred into an endless belt that can be covered by an insoluble release inert substrate and that is moved by a continuous roll.
• the thickness of the film is controlled by a doctor blade of the dispensing mechanism during the transfer of the film dope.
• drying is performed by placing film dopes casted manually in molds in static or convection ovens with controlled temperature and humidity that may be aided by reduced pressure or vacuum. Obtained dried bulk films are collected and further processed.
• antiretroviral compound(s) and/or antiretroviral compound-loaded nanoparticles can be incorporated at this stage by briefly dipping or spraying obtained films with solutions/dispersions of antiretroviral compound(s) and/or antiretroviral compound-loaded nanoparticles and re-drying.
• drying is performed in the continuous belt by means of warmed air or infrared light in an incorporated oven or a convection chamber. Dried bulk films are collected into roll drums coupled with the casting machine.
• Antiretroviral compound(s) and/or antiretroviral compound-loaded nanoparticles can be incorporated at this stage by briefly dipping or spraying obtained films with solutions/dispersions of antiretroviral compound(s) and/or antiretroviral compound-loaded nanoparticles and re-drying. Dried bulk films are then cut into individual films of appropriate shape and dimensions either manually or automatically using a die-cutter. Obtained films can be packaged in individual or multi-dose packaging material of pharmaceutical grade.
• films obtained by hot-melt extrusion are produced by the following steps: (1) heating and extrusion of the components of the film (except nanoparticles), (2) cooling of the bulk film, (3) incorporation of nanoparticles, (4) cutting of individual films, and (5) packaging.
• components of the film including thermo-resistant antiretroviral compound(s) are transferred, melted, mixed and extruded using an extrusor machine. Melting is obtained by the heating mechanism of the extrusor, and mixing achieved by means of an endless screw. The molten mass is forced through a flat extrusion die in order to form the film. The film thickness can be controlled by the geometry of the extrusion die and the speed of elongation and collecting rolls. The hot film is cooled by means of cool air or by immersion in water and air drying.
• Thermo-sensitive antiretroviral compound(s) and/or antiretroviral compound-loaded nanoparticles can be incorporated at this stage by briefly dipping or spraying obtained films with solutions/dispersions of antiretroviral compound(s) and/or antiretroviral compound-loaded nanoparticles and re-drying. Dried bulk films are then cut into individual films of appropriate shape and dimensions either manually or automatically using a die-cutter. Obtained films can be packaged in individual or multi-dose packaging material of pharmaceutical grade.
• antiretroviral compound-loaded nanoparticles-in-films PLGA nanoparticles containing tenofovir or emtricitabine or efavirenz, as described above, are produced and incorporated in films by solvent-casting at the laboratory scale.
• Nine parts of a mixture of PVA (87-90% hydrolyzed, MW 30,000-70,000, Sigma-Aldrich) and HPMC (Methocel E4M Premium, Colorcon) are dissolved in water and one part of glycerin, used as plasticizer, is then added.
• composition of the resulting aqueous mixture is as follows (% w/w): 0.54% PVA, 2.16% HPMC, 0.3% glycerin and 97% water.
• the mixture is stored at 4°C in order to allow air bubble removal.
• unformulated tenofovir is included at 1-10% of total content.
• the film dope is obtained by dispersing 1-10% nanoparticles in water in the previous mixture under gentle magnetic stirring. The film dope is then transferred into 10x10 cm polystyrene molds and left to dry at 35°C for 72h in a static oven.
• the final composition of a 10x10 cm tenofovir-loaded PLGA nanoparticles-in-vaginal film is, for example, as follows (% w/w): 45.19% HPMC, 26.79% PLGA, 11.29% PVA, 8.79% water, 6.27% glycerin and 1.67% tenofovir.
• Tenofovir concentration is 0.4 mg/cm2.
• films obtained by standard methodologies range from translucent and colorless, to translucent and pale yellow. Films presented homogeneous surface when made of single polymers or of mixtures containing HPMC at 60% or higher. The average thickness of the films and moisture content increases with higher HPMC content and ranges from 78 to 105 ⁇ and from 12%-16%, respectively. Obtained films are easy to peel off the molds, flexible and easy to manipulate. Films possess mechanical properties considered suitable for vaginal use.
• Puncture strength tests determine the puncture or rupture characteristics of a material. This is achieved by applying a compressive force to the material until it ruptures or until an elongation limit is achieved. It gives a measure of the resistance and pliability of the films.
• the distance at burst values for the plain films with the ratios PVA:HPMC 0:100 (3.6 ⁇ 0.1 mm) and 20:80 (3.6 ⁇ 0.9 mm) are approximately half the values found for the VCF ® Vaginal Contraceptive Film, Apothecus (6.6 ⁇ 0.3 mm).
• distance at burst is similar to that of the VCF ® Vaginal Contraceptive Film, Apothecus.
• the distance at burst gives an indication of the elasticity of the films.
• the previous films are able to disintegrate at 37°C in a previously described simulated vaginal fluid (pH 4.2) [20], either containing 1.5% mucin or not.
• the disintegration time increases for films with higher content of PVA, being higher than 24 hours for contents of PVA of 60% of the film-forming polymers or higher.
• the previous results are 6 ⁇ 4 minutes, 17 ⁇ 5 minutes and 19 ⁇ 5 minutes, respectively.
• the osmolality and pH of the simulated vaginal fluid after the disintegration test for plain films with PVA:HPMC ratio values of 0:100, 20:80 and 40:60 are within the range of 245-277 mOsm and 4.2-4.3, respectively.
• the invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process.
• the invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
• any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects are excluded are not set forth explicitly herein.

Applications Claiming Priority (2)

Publications (1)

Family

ID=57629597

Family Applications (1)

Country Status (2)

Families Citing this family (1)

Family Cites Families (11)

• 2016
  • 2016-11-07 WO PCT/IB2016/056695 patent/WO2017077520A1/en active Application Filing
  • 2016-11-07 EP EP16819358.9A patent/EP3370701A1/de not_active Withdrawn

Also Published As

Similar Documents

Legal Events