WO2008049620A1

WO2008049620A1 - Semi-solid controlled release composition

Info

Publication number: WO2008049620A1
Application number: PCT/EP2007/009283
Authority: WO
Inventors: Christian Hiller; Michael Dahm
Original assignee: Hexal Gentech Forschungs Gmbh
Priority date: 2006-10-25
Filing date: 2007-10-25
Publication date: 2008-05-02
Also published as: EP2083795A1

Abstract

The invention provides semi-solid compositions for the controlled release of macromolecules having a molecular weight of at least 1.5 kDa. The compositions comprise at least one gelatinizing agent and are particularly useful as medicaments for topical administration.

Description

Semi-solid controlled release composition

The present invention relates to semi-solid compositions for the controlled release of macromolecules. The present invention also relates to the use of said compositions as medicaments for topical administration.

High molecular weight macromolecules of natural and synthetic origin, for example biological macromolecules such as polynucleotides, polypeptides and polysaccharides are increasingly important pharmaceutical and cosmetic agents. Polynucleotides such as antisense oligonucleotides and polypeptides such as DNA-binding proteins may be used, for instance, to inhibit expression of endogenous and exogenous genes, for example cellular, microbial or viral genes, and to prevent formation of undesired expression products. Such macromolecules, therefore, may be useful for the treatment of conditions or diseases caused by the endogenous production of undesired proteins or by the production of excessive amounts of proteins, as well as for prevention and treatment of diseases caused by pathogenic organisms such as bacterial and viral pathogens.

One problem when using macromolecules as active ingredients for the treatment of diseases lies in the fact that these macromolecules are degraded by enzymes present in the organism to be treated. For example, biopolymers such as polynucleotides and polypeptides are quickly degraded by nucleases and peptidases, respectively, and so it is difficult to continuously maintain a therapeutically effective concentration of the active ingredient at the target site. On the other hand, if the active ingredients are administered in higher doses in order to compensate for this biological degradation, this may result in toxic side-effects in the organism to be treated.

Thus, it is one object of the present invention to provide a composition which protects macromolecules, in particular pharmaceutically and cosmetically active macromolecules or macromolecules for diagnostic purposes, from degradation and allows a targeted delivery of these substances to the target site. It is another object of the present invention to provide a composition which allows continuous delivery of a macromolecule to the target site so as to maintain a constant and, in particular, therapeutically effective concentration of this agent without causing unacceptable side-effects for the organism to be treated.

It has now surprisingly been found that macromolecules can continuously be delivered and targeted to a target site in the desired therapeutically effective amounts if they are administered in the form of a semi-solid composition comprising a gelatinizing agent, for example a cellulose derivative or a polyacrylate, as a base of this composition.

Thus, the present invention relates to a semi-solid composition for the controlled release of macromolecules, said composition comprising at least one gelatinizing agent and at least one macromolecule to be released, wherein the macromolecule has a molecular weight of at least 1.5 kDa.

The controlled release compositions according to the present invention are capable of releasing the desired macromolecules for a long time and in the desired amounts so that a (therapeutically) effective concentration may be maintained at the release site, i.e., the target site or the site of action of the pharmaceutical or cosmetic preparation. Thus, the compositions according to the present invention are particularly useful for pharmaceutical, cosmetic and diagnostic purposes.

According to the present invention, the semi-solid composition comprises at least one gelatinizing agent. Suitable gelatinizing agents are, for example, those polymeric substances, in particular water-swellable polymeric substances, which are commonly known to be useful for producing semi-solid compositions such as hydrogels. The gelatinizing agents used according to the present invention preferably are cellulose derivatives and polyacrylates, cellulose derivatives being preferred to polyacrylates due to their lower electrolyte sensitivity.

Cellulose derivatives suitable for the present invention may be both short-chain and long- chain cellulose derivatives. Typically, these cellulose derivatives have, but are not limited to, a molecular weight of at least about 200 kDa, preferably of from about 200 kDa to 2,000 kDa. Suitable cellulose derivatives are, for example, alkylcelluloses, such as methylcellulose, ethylcellulose, propylcellulose and butylcellulose, hydroxyalkylcelluloses, such as hydroxyethylcellulose and hydroxypropylcellulose, hydroxyalkylalkylcelluloses, such as hydroxypropylmethylcellulose, carboxyalkylcelluloses, such as carboxymethylcellulose sodium, and mixtures thereof. Most preferred are hydroxyethylcellulose and hydroxypropylcellulose. Suitable cellulose derivates such as hydroxyethylcelluloses are commercially available, for example under the trade name Natrosol^® 250 G and Natrosol^® 250 HX (Caesar and Loretz GmbH, Deutschland).

Suitable polyacrylates are, in particular, high molecular weight polyacrylic acids which, in a crosslinked form, are known as carbomers, for example those of types A, B and C in accordance with the definition of the US Pharmacopeia/National Formulary (USP 29- NF24, 2006, Deutscher Apotheker Verlag). Preferred carbomers are those of types B and C, which are capable of forming highly crosslinked gels, in particular carbomers of type B. Suitable carbomers are commercially available, for example under the trade name Carbopol^®. Suitable Carbopols^® are for example Carbopol^® 971 P NF, 974 P NF, 980 NF, 981 NF and 5984 EP. Preferably, Carbopol^® 974 P NF and 5984 EP (type B-carbomers) as well as Carbopol^® 980 NF (type C-carbomer) are used. Applying standard conditions of a 0.5% solution and following neutralisation as indicated in the USP/NF, the carbomers useful according to the present invention typically have an apparent viscosity of from about 4,000 to 70,000 mPas, preferably of from about 10,000 to 60,000 mPas, and most preferably of from about 25,000 to 45,000 mPas.

Preferably, the compositions of the invention exhibit bioadhesion to the skin, in particular to the mucosa (mucoadhesion). Bioadhesion and/or mucoadhesion are conferred to the composition, for example, by appropriate gelatinizing agents such as, for example, cellulose derivatives or polyacrylates, which are capable of adhering on skin and/or mucosal surfaces, for example by non-covalent bonds such as hydrogen bonding and ionic interactions or by covalent bonds. Bioadhesive or mucoadhesive properties, respectively, of the compositions of the invention help to prolong residence time on the target site, to reduce degradation of the macromolecule to be released from the composition and to enhance permeation. The controlled release composition according to the present invention preferably is an aqueous semi-solid composition, preferably a hydrophilic gel (hydrogel). The term hydrogel means any gel produced by gelling water or aqueous solutions using water- swellable substances such as the gelatinizing agents mentioned above. The hydrogels may be single-phase gels, i.e., gels wherein the water-swellable substance is uniformly distributed throughout the liquid so that no apparent boundaries exist between the dispersed water-swellable substance and the liquid, but may also be two-phase gels (emulsion hydrogels), i.e., aqueous gels further comprising fatty or fat-like base materials and emulsifying agents. Single phase hydrogels are preferred.

Typically, the amount of gelatinizing agent used in the composition of the present invention is from about 0.5 to 10 percent by weight, preferably of from about 0.75 to 8 percent by weight, and most preferably of from about 1.0 to 7 percent by weight, based on the total weight of the semi-solid composition. Typically, cellulose derivatives will be used in an amount of from about 0.5 to 10 percent by weight, preferably of from about 1.0 to 8 percent by weight, and most preferably of from about 1.5 to 7 percent by weight, based on the total weight of the composition. Polyacrylates usually will be used in an amount of from about 0.5 to 5 percent by weight, preferably of from about 0.75 to 4 percent by weight, and most preferably of from about 1.0 to 3 percent by weight, based on the total weight of the composition.

Preferably, the amount of the gelatinizing agent is selected so as to result in a viscosity of the semi-solid compositions, for example the hydrogels, of from about 10 to 3,000 Pas, determined at a shear stress of 100 Pa and at 20 °C, for example in a rotary viscometer (for example a Physica Rheolab MC-I rotary viscometer) equipped with a DIN Z3 rotational bob. When using medium molecular weight cellulose derivatives, the viscosity typically is from about 10 to about 50 Pas, and in case of high molecular weight cellulose derivatives the viscosity typically is from about 50 to 500 Pas. Under the conditions indicated above, the viscosity of polyacrylates typically is from about 1,000 to 3,000 Pas.

The macromolecule is released from the compositions of the present invention in a controlled and substantially continuous manner. Typically, release rates of the macromolecule from the composition are from about 5 - 35 %, preferably from about 7.5 - 35 %, after 0.5 hours, from about 10 - 45 %, preferably from about 15 - 45 %, after one hour, from about 20 - 65 %, preferably from about 25 — 65 %, after 2 hours, from about 35 - 85 %, preferably from about 40 - 85 %, after 4 hours and from about 40 - 90 %, preferably from about 45 - 90 %, after 6 hours, and usually more than about 55 %, preferably more than about 60 %, after 24 hours, as determined, for example, by in vitro- release tests in a vertical diffusion cell (Franz cell) at 37 °C according to the SUPAC-SS- guidelines (see, e.g., www.fda.gov/cder/guidance/1447fnl.pdf).

The pH value of the compositions according to the present invention usually depends on the intended application. In pharmaceutical, diagnostic and cosmetic compositions, for example, it depends on the target site or the site of action to which the composition is to be applied. If these pharmaceutical, cosmetic or diagnostic compositions are to be applied onto the skin or the mucosa, the pH value of the compositions is advantageously adjusted to match the pH value of the application site. Usually, the pH value of the composition ranges of from about 2.0 to 8.5, preferably of from about 3.0 to 7.0. For example, if application is destined onto the cervical mucosa, the pH value of the composition of the present invention preferably ranges of from about 3.5 to 5.5. If necessary, the pH value can be adjusted with standard buffer systems, for example physiologically acceptable buffer systems. Buffer systems suitable for use in the compositions of the present invention are, for example, acetate buffer, citrate buffer, tartrate buffer, succinate buffer, lactic acid buffer, Tris/HCl-buffer and phosphate buffer, without being limited thereto.

The macromolecules to be released from the compositions of the present invention have a molecular weight of at least 1.5 kDa. Typically, the molecular weight of the macromolecules is from 1.5 to 40 kDa, preferably from about 2.5 to 30 kDa, more preferably from about 3 to 25 kDa and most preferably from about 3.5 to 22.5 kDa.

The term ,,macromolecule" as used herein includes any molecule containing one or more structural repeat-units of a same or a different type linked to each other, and includes "true" macromolecular compounds as well as so called oligomers, as long as the molecular weight of the molecule is at least 1.5 kDa. Thus, the term ,,macromolecule" includes unmodified macromolecules as well as modified derivatives thereof, such as chemically or physically modified macromolecules. Modifications can be naturally occurring modifications or artificial modifications, for example those which occur by covalent or non-covalent bonding to, e.g., low molecular compounds.

The macromolecule to be released from and contained in the compositions of the present invention may be a hydrophilic or a lipophilic macromolecule. Preferably, but not necessarily, the macromolecule is a hydrophilic, water soluble substance. In case of a lipophilic substance, solubility of said substance in an aqueous composition may be achieved by a solubilizer.

The macromolecule to be released from the composition according to the present invention may be an anionic, a cationic or a neutral macromolecule. In order to achieve a better controlled release, if a polyacrylate is used as the gelatinizing agent, the macromolecule advantageously, but not necessarily, is an anion- or polyanion-forming macromolecule which, at the pH of the composition, at least in part is present in an anionic form.

The macromolecule to be released from the compositions of the present invention preferably is a pharmaceutically or cosmetically active agent or a diagnostic agent.

In a preferred embodiment of the present invention the macromolecule is an unmodified or modified biopolymer (biological macromolecule) of natural or synthetic origin, for example a polynucleotide, a polypeptide or a polysaccharide.

In a preferred embodiment of the invention, the macromolecule, for example the pharmaceutically or cosmetically active macromolecule or the diagnostic agent, is a polynucleotide such as a DNA or RNA. There is no specific sequence requirement for the polynucleotide and there is no specific limit for the length of the polynucleotide. Preferably, however, the polynucleotide has a length of from 5 to 50 nucleotides, for example of from 5 to 40 nucleotides, preferably of from 7 to 35 nucleotides, more preferably of from 10 to 30 nucleotides and most preferably of from 12 to 30 nucleotides. The polynucleotide may comprise deoxyribonucleotides and ribonucleotides and/or modified deoxyribonucleotides and ribonucleotides. The polynucleotide may be a single- stranded or a partially or fully double-stranded polynucleotide or a double strand-forming polynucleotide. A partially double-stranded or double strand-forming polynucleotide may be, for example, a DNA fragment with protruding ends, such as a restriction fragment, or it may be a partially self-complementary single-stranded DNA or RNA fragment capable of forming a hairpin structure. The polynucleotide molecule does not necessarily exist from the beginning as a double strand in the composition according to the invention, but the polynucleotide molecule may form the double-stranded structure also upon its release, for example at its site of action.

In a preferred embodiment of the present invention, the unmodified or modified polynucleotide is an antisense DNA or an antisense RNA. In another preferred embodiment of the present invention, the polynucleotide is a so called small RNA, in particular an siRNA (small interfering RNA), an miRNA (microRNA) or a tnRNA (tiny noncoding RNA). In a further preferred embodiment of the present invention, the polynucleotide may be an aptamer, i.e. a DNA or RNA molecule being capable of binding other nucleic acids or protein molecules as well as small organic molecules and microorganisms such as viruses. According to a further preferred embodiment of the invention, the polynucleotide is an smRNA (small modulatory RNA), i.e. a short double- stranded RNA allowing the expression of neuron-specific genes in differentiated neurons.

Preferred polynucleotides for therapeutic use are antisense DNAs and RNAs as well as siRNAs, preferably those directed to viral targets or virus-associated cellular targets and preventing viral or cellular gene expression and, thus, virus proliferation. Preferred polynucleotides are antisense DNAs, antisense RNAs and siRNAs directed to viral targets on viruses infecting the skin and/or the mucosa, for example polynucleotides directed to the early El, E6 and E7 genes of human papillomavirus or to genes of herpes simplex virus, for example genes having transactivating function or genes involved in the virus replication. Other preferred polynucleotides are antisense DNAs, antisense RNAs and siRNAs directed to virus-associated cellular targets necessary for virus proliferation, such as for example the gene for the E6-associated protein (E6-AP gene) used by human papillomavirus (HPV) types 16 and 18 (Scheffner, M. et al. (1993) Cell 75:495-505), which prevent expression of these cellular genes and, thus, proliferation of the virus.

The polynucleotides useful in the present invention may be naturally occurring or artificial modified or unmodified polynucleotides. Modifications in polynucleotides, for example in therapeutic antisense DNAs and RNAs or siRNAs, mainly serve the purpose to augment (i) binding affinity and (ii) delivery to the target or (iii) to increase the half-life of the therapeutic molecule by protecting the molecule from the action of endogenous exonucleases or endonucleases. Such modifications may be introduced at the level of the internucleotide phosphodiester bridge, for example phosphorothioate-, phosphorodithioate- or methylphosphonate-modifications, and/or at the sugar level, such as modifications at the 2'-hydroxy- position of the ribose or deoxyribose, for example 2'O-methyl- or 2'fluoro- modifications, or at the base level (for review, see, e.g., Kurreck, J. (2003), Eur. J. Biochem. 270:1628-44). Other examples of modifications are chimeras like peptide nucleic acids.

In a further preferred embodiment of the present invention, the macromolecule is a polypeptide, for example a therapeutically or cosmetically active polypeptide. The polypeptides are not specifically limited but are preferably hydrophilic, water soluble polypeptides. Polypeptides that may be used as macromolecules to be released from the composition of the present invention are, for example, polypeptides capable of binding to nucleic acids, such as DNA- or RNA-binding polypeptides, or receptor-binding proteins, for example receptor agonists and antagonists. Other examples for polypeptides that may be incorporated into the compositions of the present invention are polypeptides capable of forming complexes with other proteins. Examples for such complex-forming polypeptides are protease inhibitors, anticoagulant polypeptides such as hirudine and hirudine derivatives, as well as transcription factor inhibitors.

In another preferred embodiment of the present invention the macromolecule is a polysaccharide. Such polysaccharides may be therapeutically active polysaccharides, for example polysaccharides that may serve as enzyme substrates or that inhibit enzyme activity. The polysaccharides, however, may also be antigenic polysaccharides giving rise to a specific immune response in the target tissue. Examples for polysaccharides that may be contained in the compositions of the present invention, for example as therapeutically active ingredients, are heparine and heparine derivatives. A further polysaccharide useful in the present invention is hyaluronic acid, which is for example used in the cosmetic industry for the treatment of wrinkles, and derivatives thereof. The polynucleotides, polypeptides and polysaccharides of the compositions according to the present invention may be naturally occurring or synthetically produced or modified compounds. As mentioned above, the polynucleotides, polypeptides and polysaccharides may be attached to other inorganic or organic molecules, for example other therapeutically active compounds that are to be delivered to a predetermined site of action in the target tissue using the macromolecule as a vehicle. Similarly, the macromolecule may be coupled to a linker, ligand, signal and/or transporter molecule in order to direct the macromolecule to a desired target site. According to a further preferred embodiment of the present invention, polynucleotides, polypeptides and polysaccharides may be coupled to a marker compound for diagnostic purposes, for example they may contain a radioactive marker or a fluorescent marker. Polypeptides may be also modified with sugar residues, for instance to improve water solubility or antigenicity.

The amount of macromolecule to be released that can be incorporated into the composition of the present invention is not specifically limited. Typically, the amount of the macromolecule is in the range of from about 0.0001 to 60 percent by weight, preferably of from about 0.001 to 50 percent by weight, based on the total weight of the composition, in particular if the gelatinizing agent is a cellulose derivative. If the gelatinizing agent is a polyacrylate, the amount of the macromolecule in the composition advantageously is in the range of from about 0.0001 to 15 percent by weight, preferably of from about 0.001 to 10 percent by weight, based on the total weight of the composition. Usually, the amount of the macromolecule in the composition of the invention depends on the desired application. For example, in case of a pharmaceutical, cosmetic or diagnostic composition, the starting amount of macromolecule to be released from the composition is a therapeutically, cosmetically or diagnostically effective amount and, typically, is in the range of from about 0.001 to 5 percent by weight, based on the total weight of the composition.

The compositions according to the present invention may comprise further excipients and additives, for example physiologically acceptable excipients and additives. Excipients and additives suitable for use in the present invention may be electrolytes, emulsifying agents, emollients, buffers, antioxidants, solubilizers, preservatives, wetting agents, permeation enhancers, transfection mediators, drug carriers and mucolytics. Depending on the intended use of the compositions, these excipients may be added to the compositions of the present invention in a modular manner without affecting the advantageous properties of the compositions.

Emulsifying agents may be employed to disperse lipophilic macromolecules in the hydrophilic base of the composition, for example in aqueous solutions of the cellulose derivatives or polyacrylates used. Emulsifying agents are particularly useful for the preparation of gel-emulsion systems. Both emulsifying agents for the preparation of oil-in- water emulsions (O/W) and emulsifying agents for the preparation of water-in-oil (W/O) emulsions may be employed. Suitable O/W emulsifying agents are, for example, polyoxyethylene sorbitan fatty acid esters, such as polyethyleneglycol sorbitan monolaurate, polyethyleneglycol sorbitan monostearate and polyethyleneglycol sorbitan oleate, as commercially available under the trade name Tween^®, for example as Tween^® 20, Tween^® 60 and Tween^® 80. Suitable W/O emulsifying agents are, for example, sorbitan fatty acid esters, such as sorbitan monopalmitate or sorbitan monostearate, as commercially available under the trade name Span^®. Mixtures of O/W and W/O emulsifying agents may be used as well.

Suitable emollients useful in the semi-solid compositions are known in the art and include, for example, polyvalent alcohols such as glycerol, sorbitol and mannitol.

Suitable antioxidants are, for example, L-cysteine, gluthathione, lipoic acid, ascorbic acid, citric acid, ethylenediaminetetraacetic acid (EDTA) and tartaric acid. Citric acid, EDTA and tartaric acid also may serve as complexing agents, in particular as scavengers for heavy metal ions.

Suitable solubilizers may be emulsifying agents as described above, cyclodextrines, complexing agents such as coffeine, co-solvents such as lower alcohols, for example Ci-C-ralkanols such as ethanol, propanol and isopropanol, and salt-forming substances such as ethylenediamine.

Suitable preservatives are, for example, organic acids such as sorbic acid and benzoic acid, parabenes such as p-hydroxybenzoic acid esters (PHB), for example the methyl or propyl esters, alcoholic preservatives such as benzyl alcohol, as well as benzalkonium chloride and cetylpyridinium chloride. Of course, cationic preservatives such as the latter ones should not to be used in combination with anionic macromolecules.

Suitable wetting agents, in particular for use in gels, are for example those polyvalent alcohols which are also useful as emollients, for example ethylene glycol and glycerol.

Permeation enhancers useful in the present invention may be solvatizing and hydratizing substances, for example alcohols such as ethanol, isopropanol, propylene glycol, glycerol and sorbitol, alkylmethyl derivatives such as dimethylsulfoxide (DMSO) and keratolytics such as urea; and amphiphilic substances that temporarily affect the permeability

(microfluidity) of biological membranes, for example fatty acids such as C₁₂-C₂₀-fatty acids, in particular C₁₈-fatty acids, e.g., stearic acid or oleic acid, surfactants, such as anionic surfactants, e.g., fatty acids and alkylsulfates, such as sodium dodecyl sulfate (SDS), cationic surfactants, e.g., alkylammoniumbromide, such as cetylmethylammoniurnbromide, non-ionic surfactants, e.g., polyoxyethylene sorbitan esters (T ween), alkylethers and alkylesters, and amphotheric surfactants, for example lecithin.

Transfection mediators useful in the compositions of the present invention are in particular those acting by neutralizing the charge of macromolecules, for example cationic lipids and polypeptides having predominantly cationic character. Suitable cationic lipids are, for example, amphiphilic molecules such as quarternary compounds having at least one long- chain hydrocarbon residue, preferably monounsaturated or polyunsaturated aliphatic hydrocarbon residues, for example a C^-Qo-alkyl-, -alkenyl-, or -alkyloxy- or alkenyloxy residue, such as DOTMA (N-[l-(2,3 dioleyloxy)propyl]N,N,N-trimethylammonium chloride), DOTAP (N- 1 -(2,3-dioleyloxy)propyl]-N,N,N-trimethylammoniurn methylsulfate), DOSPA (2,3-dioleyloxy-N-[2(spermincarboxamido)ethyl]-N,N-dimethyl- 1-propanaminium-trifluoroacetate), and DMRIE (l,2-dimyristyloxypropyl-3- dimethylhydroxyethylammonium bromide), if desired in combination with phospholipids such as dioleoylphosphatidyl ethanolamine (DOPE), which destabilize the endosomal cell membrane and thereby facilitate release of the macromolecule into the cytosol (these combinations are commercially availabe, for example under the trade name Lipofectin^®); cationic cholesterol derivatives, for example DC-Choi (3β-N-(N',N'-dimethyl amino ethane)carbamoylcholesterol) and AH-chol (cholest-S-en-Sβ-yl-ό-aminohexylether); and cationic lipids having multivalent headgroups, for example lipopolyamines such as DOGS (dioctadecylamidoglycylspermine) and DPPES

(dipalmitoylphosphatidylethanolamidospermine). Suitable polypeptides with predominantly cationic character are, for example, penetratine und protamine. The transfection mediators, such as penetratine, may also be covalently attached to the macromolecule to be released.

Drug carriers useful in the compositions of the present invention serve to improve bioavailability of the macromolecule at the target site and protection against enzymatic degradation. The use of carriers which can smoothly be incorporated into hydrogels is preferred. More preferably, the carriers used are cationic, neutral and anionic liposomes, nanoparticles and micro- and nanoemulsions. Nanoparticles are solid particles having a colloidal structure and a size of up to 1 μm, preferably in the range of from about 10 to 300 run, and consisting of macromolecular substances. These may be natural polymers, for example albumines, polysaccharides, gelantine or synthetic histocompatible plastics, for example polyalkylacrylates, polycyanoacrylates, polyvinylpyrrolidone, acrylpolymers, polylactic acid (polylactide) and condensation products thereof with other polyhydroxycarboxylic acids. Positively charged nanoparticles that may be formed, for example, by use of cationic polymers such as polyethylenimine or by incorporation of e.g. DEAE (diethylaminoethyl)dextrane are preferred to deliver anionic macromolecules binding to the positive exterior of the particles. Liposomes are molecular associations of amphiphilic lipids in water comprising concentrically arranged phospholipid double layers (made of egg-lecithin, for example) alternating with aqueous intermediate layers. They effect membrane fusion by endocytosis. Cationic liposomes mix with the negatively charged macromolecule forming a complex due to the opposite charge. Anionic and neutral liposomes take up the macromolecule. Micro- and nanoemulsions are colloid systems comprising water, oil, surfactants and, optionally, a co-surfactant (for example polymeric co-surfactants such as long-chain alcohols or block copolymers such as poloxamers to increase stability and to reduce toxicity of the emulsions). Emulsions having a particle size of > 500 nm are referred to as microemulsions, emulsions having a particle size of < 500 nm are referred to as nanoemulsions. The emulsions may be both W/O-type and O/W-type emulsions. The amphiphilic and inverse hexagonal structure allows solubilization of insoluble substances by uptake into the interior of the structure. A W/O- micro- or -nanoemulsion may absorb water-soluble macromolecules and transport them across biological membranes due to their lipophilic exterior. Anionic macromolecules, for example, are taken up into the hydrophilic interior of the vesicular structure. On the other hand, the O/W-type represents a vesicular structure having an inner oil phase and an external cationic aqueous phase, formed upon proper choice of cationic surfactants. In this case, an anionic macromolecule binds to the positively charged exterior of the emulsion.

While the presence of permeation enhancers, transfection mediators and/or drug carriers is not required for a controlled release of the macromolecules of the composition of the present invention, use thereof may nevertheless be advantageous to enhance uptake of the macromolecules into target cells. For example, uptake of rigid macromolecules such as siRNAs into target cells may be considerably improved when using permeation enhancers transfection mediators and/or drug carriers.

Mucolytics useful in the compositions of the present invention are in particular reducing agents capable of cleaving disulfide bonds in mucus, for example n-acetylcysteine, or agents stimulating mucus production, for example (trans-4-(2-amino-3,5- dibrombenzylamino)-cyclohexanol), which results in a less cross-linked mucus that can easily be expectorated as its consistency is more fluid.

The compositions of the present invention may be prepared by conventional methods known in the art. Single-phase compositions according to the present invention, such as hydrogels, may be prepared by dissolving gelatinizing agents such as cellulose derivatives or polyacrylates in the aqueous phase containing the macromolecule and optionally a solubilizer and other desired excipients and additives, and allowing the mixture to swell. The person skilled in the art will realize that the conditions of preparation have to be properly chosen to ensure stability of the individual components of the composition, in particular of the macromolecule. Usually, dissolving or mixing the individual components of the compositions of the invention is carried out at a temperature of from ambient temperature to 60 °C, while swelling of the gel is typically performed at lower temperatures of from 2 to 10 °C, preferably at about 4 °C. Other temperatures, however, may be equally appropriate depending on the components used. Emulsion gels are formed by dissolving gelatinizing agent as well as macromolecule(s) and, optionally, solubilizers and other desired excipients and additives in the aqueous phase, followed by combining and emulsifying the mixture with the desired lipophilic phase and swelling.

The compositions of the present invention are particularly useful for the preparation of human and veterinary medicaments as well as for the preparation of diagnostics and cosmetics, in particular for topical administration. These preparations are especially useful for local application, for example on the skin and preferably on the mucosa, in particular the oral, anal and genital mucosa, such as the mucosa of the human reproductive tract, for example the cervical mucosa, the mucosa of the inner foreskin layer and the glans penis. In a preferred embodiment of the invention the compositions are pharmaceutical preparations or medicaments for the treatment of diseases of the skin and/or the mucosa, in particular of the oral, anal and genital mucosa, caused by infections with DNA or RNA viruses, for example viruses of the family papovaviridae and herpesviridae. Viruses of the family papovaviridae causing diseases of the skin and/or the mucosa are, e.g., papillomaviruses such as low risk human papillomaviruses (HPV), for example HPV 6 and 11, causing, e.g., genital warts, or high risk HPV, for example HPV 16 and 18, causing, e.g., cervival dysplasia followed by cervical carcinoma. Viruses of the family herpesviridae causing diseases of the skin and/or the mucosa are, e.g., α-herpesviruses, for example herpes simplex viruses (HSV) causing, e.g., herpes labialis or herpes genitalis, or varicella-zoster viruses causing, e.g., varicella and shingles. In all of these cases, the medicament may be used both for prevention and treatment of the diseases. If desired, the retention time of the applied active ingredient may be prolonged by occlusion of the target tissue, for example using a cervical cap or a diaphragm.

The compositions according to the present invention are suitably provided in a device suitable for semi-solid compositions, for example a tube. Said devices have a volumetric capacity for the medicament suitable either for multiple applications or a single application. One advantage of a tube adapted for single use is that it is not necessary to add preservatives to the medicament which might lead to irritations in the target tissue. If the medicament is to be applied to a poorly accessible area of the body, it might become necessary to use an application aid. In case of a vaginal-cervical application, it is advisable to use a vaginal applicator similar to a syringe. The semi-solid medicament is transferred from the primary packing into the syringe-like applicator. Subsequently, the drug is applied onto the target tissue by means of the applicator.

In the case of a vaginal-cervical application, a directed long-term contact of the medicament with the target tissue may be facilitated by the use of a diaphragm or a portio cap. In this case, the semi-solid formulation containing the active ingredient is applied to the medical product and the medical product then is placed in the vagina or on the os uteri similar as in conception control.

The compositions of the present invention not only provide for a long-term controlled release of the macromolecules in the desired amounts, they also may be applied topically in defined intervals directly onto the target tissue. This allows maintaining a therapeutically effective concentration at the desired site of action for a long period of time. As a further advantage, the topical application of the composition of the present invention allows to build up high therapeutic concentrations of the active macromolecule at the target site without undesired systemic side-effects. The compositions of the invention remain stable over a long period of time even at a low pH value so that the macromolecules to be released are protected from degradation and a high concentration of the active ingredient can be maintained on the target tissue.

The compositions of the invention exhibit excellent spreadability and at the same time are highly bioadhesive to the skin and in particular to the mucosa. The desired active macromolecule thus will be released completely and over a prolonged period of time to the target tissue. Moreover, the compositions of the present invention may be easily washed off from the target tissue so as to allow for administration of a new therapeutic dose.

The compositions of the present invention further exhibit highly favourable rheologic properties. For example, the compositions of the present invention exhibit an advantageous plastic or pseudoplastic flow behaviour with little or no thixotropy (i.e. they show no or only very small hysteresis areas in a rheogram). Following deformation through an applied force, for example by pressing through a tube orifice, which will result in a rather fluid low viscous state of the composition, the composition thus will quickly return to its original, more viscous state. This is advantageous when administering compositions such as hydrogels onto a target site, as these will adhere much better to the application site when they are in a higher viscous state.

The present invention will now be explained in more detail with reference to the following examples and drawings, which are merely illustrative and do not in any way limit the scope of the present invention. In the following,

Fig. 1 shows the release of a 17-mer oligodeoxynucleotide from 5.0 % (Gl), 5.5 % (G2) and 6.0 % (G3) HEC gels;

Fig. 2 shows the release of a 17-mer oligodeoxynucleotide from 2.0 % (HXl), 2.5 %

(HXZ) and 3.0 % (HX3) HEC gels.

Fig. 3 shows the release of a 21 -mer siRNA from a 6.0 % (G4) HEC gel.

Fig. 4 shows the release of a 21 -mer siRNA from a 3.0 % (HX4) HEC gel.

Fig. 5 shows a representative chromatogram of a HPLC run under native conditions at

50°C for the siRNA released from a HEC gel

Fig. 6 shows a representative chromatogram of a HPLC run under denaturising conditions at 75 °C for the siRNA released from a HEC gel

Examples

Example 1

Hydrogel formulations based on hydroxyethyl cellulose containing a single-stranded oligodeoxynucleotide (ASO)

2 - 6 % (w/w) hydrogels were prepared on the basis of pharmaceutical grade hydroxyethyl cellulose (HEC). A long-chain HEC (MW approx. 1 ,000 kDa; commercially available under the trade name Natrosol^® HX) was used for the preparation of 2.0 %, 2.5 % and 3.0 % hydrogels (HXl, HX2 and HX3), and a short-chain HEC (MW approx. 300 kDa; commercially available under the trade name Natrosol^® G) was used for 5.0 %, 5.5 % and 6.0 % hydrogels (Gl, G2 and G3). The amount of HEC necessary for the preparation of 75 g gel was weighed in a mortar and then levigated with a mixture of 2.0 ml of a potassium sorbate solution (75 mg/ml) as a preservative and 7.5 g of 85% glycerol as a wetting agent. To this mixture a solution of 0.2 M citrate buffer (pH 5.0) and 5 ml of an L-cysteine solution (10 g/1, antioxidant) were added in portions while stirring with a pestle. Before addition of the last portion of buffer, 748 μl of an aqueous solution of a 17-mer deoxynucleotide having the sequence 5'-TACATCGACCGGTCCAC-S ' (SEQ ID NO:1) (50.15 mg/ml; MW approx. 5.4 kDa; in the following also referred to as ASO) was added to this formulation as the macromolecule to arrive at a final concentration of 0.5 mg ASO/g gel. These steps were carried out at 20 °C under aseptic conditions and using autoclaved equipment and solutions. The mixtures obtained were allowed to swell over night at 4 °C in a refrigerator.

Example 2

Testing ASO-release in a vertical diffusion chamber (Franz cell)

In vitro release of ASO was tested in a vertical diffusion chamber (Franz cell), which is a standard method for characterising the release properties of topical administration forms such as hydrogels. The Franz cell is a closed system with a diffusion chamber consisting of an upper donor compartment and a lower acceptor compartment. A synthetic membrane is placed on top of the acceptor compartment allowing the molecule release of which is to be tested to pass unhindered from the donor to the acceptor compartment. The composition comprising the molecule to be released, for example a hydrogel, is arranged approximately in the middle of the membrane by means of a syringe. The acceptor compartment is filled with an acceptor medium (usually a buffer) to absorb the molecule released from the composition. The acceptor medium is in contact with the membrane avoiding the formation of air bubbles under the membrane that would reduce the permeation area for the released molecule. The medium is heated to a temperature of 37 °C ± 0.5 °C by a water jacket. A Helix™ stirrer ensures that the amount of released molecule is mixed into the acceptor medium. The acceptor medium is selected depending on the type of molecule to be released so as to have Sink-conditions, i.e. the dissolution rate of the released molecule in the acceptor medium is constant over time. This is the case, for example, if the soluble amount of the molecule to be released in the respective buffer is at least 10 % higher than the maximum amount of the molecule releasable from the composition. Samples are taken from the acceptor medium at predetermined time intervals and the cell is subsequently refilled with fresh acceptor medium. The amount released of the molecule is quantified in the fractions of each time interval, for example by HPLC in combination with appropriate detection methods.

In vitro release of the 17-mer oligonucleotide (ASO) from the hydrogel formulations prepared as described in example 1 was tested in a Franz cell. The test was carried out according to the SUPAC-SS guidelines rwww.fda.gov/cder/guidance/1447fnl.pdf) at 37 ⁰C using a HANSON Microette^® system (Hanson Research Corporation, Chatsworth, California, USA). Briefly, 300 mg of each of the compositions obtained in example 1 (corresponding to 150 μg oligonucleotide) were placed in parallel with a syringe into three Franz cells and spread over the centre of a Pall - GH Polypro Hydrophilic Polypropylene Membrane Filter (0.2 μm). This membrane retains the gel in the donor compartment, but allows the ASO molecule to pass unhindered. The acceptor medium used was Hanks Balanced Salt Solution (HBSS), pH 7.4 (Biochrom AG, Germany, Art-Nr. L2035), modified with 0.01 M HEPES buffer (Carl Roth GmbH & Co.KG, Germany, Art.-Nr. 9105.4) and 0,067 M EDTA. Release of the oligonucleotide from a solution consisting of ASO dissolved in the above-described acceptor medium (ASO-solution) from which ASO is released without delay (,,burst release") served as a control.

200 μl-Samples were taken manually with glass capillaries after 30 min, 1 h, 2 h, 4 h, 6 h and 24 h and transferred into HPLC tubes with a glass insert. The amount of ASO contained in the acceptor medium at each time interval was determined by HPLC and UV- detection at 260 run. HPLC was carried out at ambient temperature using metal free columns (length: 250 mm, internal diameter: 4 mm) with DNAPac^®-PA100 (Dionex) as stationary phase and a Jasco 1550-AS-sampler, a PU-2080 pump, a DAD MD-2010- detector, a column oven Jetstream and a 2080-53 degasser. The injection volume was 20 μl each and the loop volume was 100 μl. Elution was carried out using a sodium perchlorate- gradient containing 250 mM sodium hydroxide with a flow rate of 1.0 ml/min and a total run time of 30 min. The retention time obtained for ASO was 11 min. The results are shown in the following Table 1 and Figs. 1 and 2.

Table 1

Release of a 17-mer oligonucleotide (ASO) from hydrogel formulations

* Mean value of three replicas

SD: Standard deviation (calculated with the Excel STABW function)

Fig. 1 graphically shows ASO-release from 5.0 % (Gl), 5.5 % (G2) and 6.0 % (G3) HEC gels compared to release from the control ASO-solution, expressed as percent of the starting amount of ASO present in the tested compositions, for the first 6 hours after start of the test. Fig. 2 is a corresponding graph showing ASO-release from 2.0 % (HXl), 2.5 % (HX2) and 3.0 % (HX3) HEC gels.

These results show that the compositions of the present invention release the ASO macromolecule continuously and in a controlled manner, irrespective of whether short- chain or long-chain hydroxyethyl cellulose is used. Moreover, it is apparent that the compositions of the present invention release comparable amounts of ASO per time unit irrespective of the concentration of the gelatinizing agent and, thus, the viscosity of the gels. Example 3

Release of double strand siRNA from hydrogels

3 % and 6 % (w/w) hydrogels were prepared as described in Example 1 with the exception that 750 μl of an aqueous solution of a double-stranded 21-mer small interfering RNA (siRNA; 50.0 mg/ml; MW approx. 12.7 kDa; S.M. Elbashir et al., Nature 411 :494-498, 2001) were added instead of the single-stranded ASO oligonucleotide. The siRNA duplex is formed of the complementary nucleotide sequences:

5 ' - CUUACGCUGAGUACUUCGATT- 3 ' ( SEQ ID NO : 2 ) and 3 ' -TTGAAUGCGACUCAUGAAGCU - 5 ' ( SEQ ID NO : 3 )

giving raise to protruding 3 '-ends.

As in Example 1, long-chain HEC was used for the preparation of the 3.0 % hydrogel (HX4), and short-chain HEC was used for the 6.0 % hydrogel (G4). In vitro release of siRNA was tested in a Franz cell as described in Example 2, except that unmodified HBSS, pH 7.4, was used as the acceptor medium. Burst release of siRNA from acceptor medium (siRNA-solution) served as a control.

200 μl-Samples were taken manually with glass capillaries after 30 min, 1 h, 2 h, 4 h, 6 h and 24 h and transferred into HPLC tubes with a glass insert. The amount of siRNA contained in the acceptor medium at each time interval was determined by HPLC and UV-detection at 260 nm. HPLC was carried out using a Merck Hitachi HPLC system

(Merck Hitachi, Darmstadt, Germany) with metal free columns (length: 250 mm, internal diameter: 4 mm) with DNAPac^®-PA200 (Dionex) as stationary phase and a L-7120 pump, a Diode Array Detector (DAD) L-7455, and an autosampler L-7200. Data acquisition was performed by Chromatography Data Station Software 4.0 (Merck Hitachi, Darmstadt, Germany). Release of double-stranded siRNA was quantified under native conditions at a temperature of 50°C. HPLC under denaturising conditions at 75°C for separating the single strands of the double-stranded siRNA released from the gels was performed as a control to demonstrate that it is in fact double-stranded siRNA which is released from the hydrogels. The injection volume was 20 μl each and the loop volume was 100 μl. Elution was carried out using a sodium bromide gradient containing 100 raM phosphate buffer, pH 7.0, with a flow rate of 1.0 ml/min and a total run time of 35 min. The retention time obtained for double strand siRNA was about 11.4 min and that for the individual single strands was about 16.5 min and 17.0 min, respectively. The results of the HPLC are shown in the following Table 2 and in Figs. 3 to 6.

Table 2

* Mean value of three replicas ss: Single strand ds: Double strand

SD: Standard deviation (calculated with the Excel STABW function)

Figs. 3 and 4 graphically show the release of double strand siRNA from a 6.0 % (G4) and a 3.0 % (HX4) HEC gel, respectively, compared to release from the control siRNA-solution, each expressed as percent of the starting amount of siRNA present in the tested compositions up to 24 hours after start of the test. These results show that the compositions of the present invention release double-stranded siRNA continuously and in a controlled manner, irrespective of whether short-chain or long-chain hydroxyethyl cellulose is used. The compositions of the present invention release comparable amounts of siRNA per time unit. From the chromatogram shown in Fig 5 it is seen that siRNA released from HEC gels elutes as a single peak when tested by HPLC under native conditions at 50⁰C, demonstrating that siRNA is released as a double- stranded molecule from the gels. Single strand analysis of siRNA under denaturising conditions of HPLC correlates well with the data obtained for the double strand molecule. From the chromatogram shown in Fig 6 it is seen that siRNA released from HEC gels elutes as two individual peaks (peak 1 and peak 2) when tested by HPLC under denaturising conditions at 75°C. This confirms the above result that siRNA is released as a double strand from the hydrogels.

Claims

1. A semi-solid composition for the controlled release of macromolecules, comprising at least one gelatinizing agent and at least one macromolecule to be released, wherein the macromolecule has a molecular weight of at least 1.5 kDa.

2. Composition according to claim 1, wherein the at least one gelatinizing agent is a cellulose derivative or a polyacrylate.

3. Composition according to claim 1 or claim 2, wherein the amount of the at least one gelatinizing agent in the composition is of from 0.5 to 10 percent by weight, preferably of from 0.75 to 8 percent by weight, and most preferably of from 1.0 to 7 percent by weight, based on the total weight of the composition.

4. Composition according to any one of claims 1 to 3, wherein the cellulose derivate is selected from the group consisting of alkylcelluloses, hydroxyalkylcelluloses, hydroxyalkylalkylcelluloses, carboxyalkylcelluloses and mixtures thereof.

5. Composition according to claim 4, wherein the hydroxyalkylcellulose is selected from the group consisting of hydroxy ethylcellulose, hydroxypropylcellulose and mixtures thereof.

6. Composition according to any one of claims 1 to 5, wherein the composition is a hydrogel.

7. Composition according to any one of claims 1 to 6, wherein the macromolecule to be released has a molecular weight of from 1.5 to 40 kDa, preferably of from 2.5 to 30 kDa, more preferably of from 3 to 25 kDa and most preferably of from 3.5 to 22.5 kDa.

8. Composition according to any one of claims 1 to 7, wherein the macromolecule to be released is a biopolymer.

9. Composition according to claim 8, wherein the biopolymer is a polynucleotide, a polypeptide or a polysaccharide.

10. Composition according to claim 9, wherein the length of the polynucleotide is from 5 to 50 nucleotides, preferably from 7 to 35 nucleotides, more preferably from 10 to 30 nucleotides and most preferably from 12 to 30 nucleotides.

11. Composition according to claim 9 or claim 10, wherein the polynucleotide is an antisense DNA or an antisense RNA.

12. Composition according to claim 9 or claim 10, wherein the polynucleotide is a small RNA, in particular an siRNA, an miRNA, a tnRNA or an smRNA.

13. Composition according to claim 9 or claim 10, wherein the polynucleotide is an aptamer.

14. Composition according to any one of claims 1 to 13, further comprising excipients and additives, preferably selected from the group consisting of electrolytes, emulsifying agents, emollients, buffers, antioxidants, solubilizers, preservatives, wetting agents, permeation enhancers, transfection mediators, drug carriers and mucolytics.

15. Composition according to any one of claims 1 to 14, wherein the composition is a pharmaceutical, cosmetic or diagnostic composition.

16. Composition according to any one of claims 1 to 15 as a medicament for topical administration.

17. Use of a composition according to any one of claims 1 to 15 for preparing a medicament for topical administration.

18. Use according to claim 17, wherein the medicament is for local administration.

19. Use according to claim 17 or claim 18, wherein the medicament is for administration to the skin and/or the mucosa, in particular the oral, anal and genital mucosa.

20. Use according to claim 19, wherein the medicament is for administration to the cervical mucosa.

21. Use of a composition according to any one of claims 1 to 15 for the preparation of a medicament for the prevention and/or treatment of diseases of the skin and/or the mucosa, in particular of the oral, anal and genital mucosa.

22. Use according to claim 21, wherein the disease of the skin and/or the mucosa is caused by a virus.

23. Use according to claim 22, wherein the virus is a virus of the family papovaviridae or herpesviridae.

24. Use according to claim 23, wherein the virus of the family papovaviridae is a papillomavirus and the virus of the family herpesviridae is an α-herpesvirus, in particular a herpes simplex virus or a varicella-zoster virus.

25. Application device for semi-solid compositions, containing a composition according to any one of claims 1 to 16.