Nucleic Acid Vector Tablets

Abstract

The present invention relates to a method for producing tablets containing nucleic acid vectors.

Description

[Technical Field]

[0001] The present invention relates to a method for producing tablets containing nucleic acid vectors. [Background technology]

[0002] Gene therapy uses nucleic acids (especially DNA or RNA) to treat or prevent disease. Depending on the pathology, nucleic acids are administered to regulate, repair, replace, add, or delete gene sequences. The therapeutic, preventive, or diagnostic effect depends directly on the nucleic acid sequence or the product of the gene expression of that sequence. Therapeutic nucleic acids include, for example, antisense oligonucleotides, siRNA, and shRNA. In particular, molecular interfering RNAs or siRNAs combined with small vectors constitute agents with high therapeutic potential due to their ability to specifically suppress the expression of pathological proteins. Therapeutic nucleic acids can be directly injected into cells as naked nucleic acids, but they are most often delivered to the patient's cells using nucleic acid vectors. Nucleic acid vectors can be viral vectors based on polymers or lipid systems, or synthetic vectors.

[0003] Currently, nucleic acid vectors are mainly administered parenterally in the form of colloidal suspensions. This type of formulation and administration route imposes significant limitations, such as the size of the administered vector and the need to obtain a sterile preparation. These limitations are obstacles to the development of nucleic acid vectors as potential new drugs. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication WO15023775 Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, there is a need for new formulations of nucleic acid vectors to avoid the above-mentioned limitations while maintaining the biological activity of therapeutic nucleic acids. [Means for solving the problem]

[0006] According to a first aspect, the present invention provides a method for producing a cellular membrane comprising: (i) nucleic acid; and (ii) a lipid particle comprising a cationic lipid, preferably a liposome or micelle, preferably a liposome; and a method for producing a tablet comprising the complex of The method comprises: a) - (i) a complex of a nucleic acid and (ii) a lipid particle comprising a cationic lipid, preferably a liposome or micelle, preferably a liposome; - one or more additives suitable for drying, in particular freeze-drying; drying the aqueous mixture comprising the compound, in particular by freeze-drying; b) mixing one or more compression additives with the dry mixture obtained in step a); and c) compressing the mixture obtained in step b) Includes:

[0007] In particular embodiments, the cationic lipid is selected from lipopolyamines, quaternary ammonium, and lipids with a cationic head group of the guanidine or imidazole type, preferably the cationic lipid is selected from dimyristylaminopropylaminopropyl (DMAPAP), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), [1,2-bis(oleoyloxy)-3-(trimethylammonio)propane] (DOTAP), 3β[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DCChol) and dioctadecylamidoglyceryl spermine (DOGS), preferably dimyristylaminopropylaminopropyl (DMAPAP). The cationic lipid can be optionally mixed with a neutral lipid, particularly selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, dioleoyl-sn-glycero-3-phosphocholine (DOPC), dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and N'-(rac-1-[11-(F-octyl)undec-10-enyl]-2-(hexadecyl)glycero-3-phosphoethanoyl)-sperminecarboxamide), preferably 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In particular, the mixture of cationic lipid and neutral lipid may contain 50 to 99 mol%, preferably about 50 mol%, of the cationic lipid in the mixture, or 1 to 50 mol%, preferably about 50 mol%, of the neutral lipid in the mixture.

[0008] According to certain embodiments, the complex comprises a therapeutic nucleic acid, in particular siRNA, miRNA, shRNA, plasmid, or mRNA. According to certain embodiments, the charge ratio (+ / -) of the positive charge of the cationic lipid to the negative charge of the nucleic acid is 0.5 to 10, preferably 4 to 10, and preferably about 8.

[0009] According to the method of the invention, the additive suitable for drying, in particular for freeze-drying, may be a protective agent (cryoprotectant or lyoprotectant) and / or bulking agent, in particular selected from trehalose, mannitol, sucrose, sorbitol, lactose, glucose, glycerin, glycine, alanine, lysine, polyethylene glycol, polyvinylpyrrolidone and dextran, optionally in a mixture. In particular, the additive suitable for freeze-drying is trehalose in a proportion of 10 to 100% by weight of the additive suitable for freeze-drying, optionally mixed with another additive suitable for freeze-drying, preferably mannitol, in a proportion of 0 to 90% by weight of the additive suitable for freeze-drying.

[0010] According to the method of the present invention, the compression additive protects the complex of lipid particles comprising nucleic acid and cationic lipid during compression step c), and the compression additive is in particular a lubricant (e.g., magnesium stearate, stearic acid and talc, preferably magnesium stearate), optionally mixed with another compression additive, preferably an excipient (e.g., lactose, starch, calcium phosphate and its derivatives, and cellulose and its derivatives, preferably lactose). In particular, the lubricant may represent 0.25 to 5% by weight, preferably 0.5%, of the finished tablet.

[0011] In a particular embodiment, the method according to the invention comprises a compression step (step c) carried out at a pressure between 10 and 400 MPa, preferably between 50 and 250 MPa.

[0012] In another aspect, the present invention relates to a tablet obtainable according to the above process. The tablet of the present invention comprises: - liposomes or micelles complexes containing nucleic acids and cationic lipids; - one or more suitable lyophilization additives, preferably trehalose and mannitol, to protect the complex during the lyophilization step a) and / or to obtain a lyophilisate with a texture suitable for the compression step; - one or more suitable compression additives to protect the complex during compression step c) and to maintain the biological activity of the complex; may include:

[0013] According to a particular embodiment, the tablets of the invention are intended for oral, vaginal, rectal, sublingual or transmucosal administration, preferably by the oral route.

[0014] The present invention also relates to the above tablet for use in treating gastrointestinal or vaginal disorders. [Brief explanation of the drawings]

[0015] [Figure 1] Freeze-drying cycle: Representation of product temperatures during the three stages of the cycle; (a) initiation of freezing, primary drying and secondary drying; (b) focus on freezing in liquid nitrogen. [Figure 2] Efficacy of tablets containing lipoplexes as a function of the lyophilisate (=lyoph) and the compression stress applied during the compression method (CX at X = 50, 100, 150, 200 or 250 MPa): 48 h transfection of B16-Luc cells, triplicate, 0.5 μg luciferase siRNA per well. Measurement of luciferase activity and presentation of the percentage of inhibition compared to non-transfected cells. Positive control (T+): cells transfected with lipoplexes prepared immediately before use (not lyophilised or compressed); Negative control (siCtle): cells transfected with lipoplexes containing control siRNA resuspended from the tablet. [Figure 3] SAXS profile of siRNA lipoplexes (DMAPAP / DOPE + alginate), prepared in 150 mM NaCl buffer at a + / - charge ratio of 8. Peak analysis identifies two phases, a major cubic peak (C1, q = 0.084 nm-1) and a minor lamellar peak (L1, q = 0.101 nm-1). [Figure 4]SAXS profile of siRNA lipoplexes in dry powder form after freeze-drying and subsequent compression. siRNA lipoplexes (DMAPAP / DOPE + alginate) prepared at a + / - charge ratio of 8 in 150 mM NaCl buffer are supplemented with trehalose and maltose and then freeze-dried for 30 hours. The freeze-dried material is weighed, supplemented with lactose, and compressed. SAXS analysis is performed on the freeze-dried material or tablet powder placed in a capillary tube. A single large peak (q = 0.096 nm-1) is detected. [Figure 5] SAXS profiles of siRNA lipoplexes suspended in NaCl / sugar. Lipoplexes prepared as shown in the legend to Figure 3 were lyophilized or compressed under the same conditions as the samples, supplemented first with a trehalose / mannitol mixture or second with a trehalose / mannitol / lactose mixture, respectively. Peak analysis identifies a single cubic phase, with the position of the first peak slightly shifting between the shape of the first mixture (C1, q = 0.095 nm-1) and the shape of the second mixture (C1, q = 0.097 nm-1). [Figure 6] SAXS profiles of suspended siRNA lipoplexes. The first curve corresponds to a sample that was (A) lyophilized, then (B) compressed (see legend to Figure 4), and then resuspended by adding water. The second curve corresponds to a suspended sample that was not lyophilized or compressed, but was supplemented with the same amount of trehalose / mannitol (A) as the lyophilized resuspended sample, or the same amount of trehalose / mannitol / lactose (B) as the resuspended compressed sample. Peak analysis identifies a single phase (C1, (A) q = 0.094 nm-1; (B) q = 0.096 nm-1) in the profile corresponding to the second curve, a cubic phase (C1, q = 0.093 nm-1) in the profile of the first curve in (A), and two phases, cubic (C1, q = 0.086 nm-1) and lamellar (L1, q = 0.101 nm-1) in the profile of the first curve in (B). DETAILED DESCRIPTION OF THE INVENTION

[0016] The present inventors propose to formulate a nucleic acid vector in tablet form for oral, vaginal, rectal, sublingual, or transmucosal administration. The "tablet" form facilitates administration while avoiding the formulation and administration constraints mentioned above. In addition, the tablet allows for multiple formulation options to control or regulate the release of the nucleic acid vector based on the therapeutic target. The "tablet" form also represents a breakthrough in the stability of nucleic acid vectors. In particular, it makes it possible to avoid their preparation immediately before use. In addition, the "tablet" form improves both patient comfort and treatment compliance.

[0017] Prior to the implementation of the present invention, nucleic acid vector tablets were unavailable or even unthinkable to those skilled in the art due to fears of loss of nucleic acid activity during the steps required to form the tablets. However, contrary to this prejudice, the present inventors propose an innovative means of generating nucleic acid vector tablets.

[0018] The present invention relates to a method for producing tablets containing at least one nucleic acid vector. More specifically, the proposed invention makes it possible to formulate nucleic acid vectors in a "tablet" form while maintaining their therapeutic properties despite the mechanical stresses involved in this forming operation.

[0019] definition "Tablet" means any uniform solid preparation without particular shape or size obtained from compressed powder, which may or may not be agglomerated.

[0020] "Nucleic acid vector" (otherwise referred to below as "complex") refers to any structure known to be suitable for vectorizing nucleic acids; in particular, these terms refer to a complex of (i) a nucleic acid and (ii) a lipid particle comprising a cationic lipid, preferably a liposome or micelle, preferably a liposome.

[0021] "Lipoplex" means a complex of (i) a nucleic acid and (ii) a liposome containing a cationic lipid.

[0022] "Cationic lipid" means a lipid that has an overall positive charge. A cationic lipid comprises a cationic polar head and one or more hydrophobic chains.

[0023] "Neutral lipid" means a lipid having a neutral overall charge, more specifically, a zwitterionic lipid.

[0024] "Micelle" means a spherical aggregate of amphiphilic molecules having hydrophilic polar heads and hydrophobic chains.

[0025] "Liposome" means an artificial vesicle formed by concentric lipid bilayers enclosing an aqueous compartment between them.

[0026] For example, the nucleic acid vector can be selected from liposomes, lipids, such as cationic lipids, anionic lipids, amphoteric lipids or uncharged lipids, cationic polymers, polymers, hydrogels, micro- or nanocapsules (biodegradable), microspheres (optionally bioadhesive), cyclodextrins, protein vectors, or any combination thereof. Preferably, the vector can be selected from lipid-based delivery systems, polyimine-based delivery systems, dendrimers, poly(lactide-co-glycolide) (PLGA) particles, systems described in International Publication WO15023775, and similar systems.

[0027] "About" refers to a value + or - 10%, preferably + or - 5% thereof. For example, about 50 is 45 to 55, preferably 47.5 to 52.5.

[0028] Preparation of nucleic acid vectors Nucleic acids may be, in particular, deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) of any size, single- or double-stranded, linear or circular, and chemically unmodified or modified (e.g., phosphorothioate, phosphoramidate, and / or 2'-O-methyl). Nucleic acids may vary widely in size, e.g., from 10 base pairs to tens of thousands of base pairs. For example, nucleic acids may reach approximately 10,000 base pairs or more for plasmids, approximately 19-25 nucleotides for double-stranded siRNAs, or approximately 20-30 nucleotides for single-stranded microRNAs. They may be natural, hybrid, or synthetic sequences of any origin (e.g., prokaryotes, eukaryotes, viruses, parasites, plants, etc.). Preferably, the nucleic acids are therapeutic nucleic acids, i.e., nucleic acids that exert a therapeutic effect by modulating, repairing, replacing, adding, or deleting gene sequences. Preferably, the nucleic acid is capable of regulating protein expression and can be selected from antisense oligonucleotides, oligonucleotides for exon skipping, oligonucleotides for alternative splicing modification, interfering RNA, messenger RNA, or plasmid DNA. Preferably, the nucleic acid is an interfering RNA. In the context of the present invention, "interfering RNA" refers to RNA that inhibits gene expression. In particular, interfering RNA is a small (especially 20-25 base pairs, particularly 21-23 base pairs) ribonucleic acid that interferes with a specific messenger RNA, resulting in its degradation and inhibition of its translation into protein. For example, small interfering RNA (siRNA) and microRNA (miRNA) are interfering RNAs.

[0029] In particular, the nucleic acid is selected from a plasmid, an siRNA, an miRNA, an mRNA and an shRNA, preferably the nucleic acid is an siRNA or an miRNA. Preferably, the nucleic acid is an siRNA.

[0030] The tablets produced according to the present invention comprise one or more nucleic acid vectors (or complexes) as defined above. According to a particular embodiment, the tablets comprise a combination of several different nucleic acid vectors, in particular a combination of at least two, at least three, at least four or even at least five different nucleic acid vectors. According to another particular embodiment, the tablets comprise a combination of one or more nucleic acid vectors (or complexes) and at least one probiotic.

[0031] "Probiotic" means a live microorganism that, when administered in sufficient amounts, provides a positive health benefit beyond conventional nutritional benefits, such as improved digestion or improved immune defenses.

[0032] According to the present invention, the complex is formed by electrostatic interaction between the negatively charged nucleic acid and the lipid particle containing the cationic lipid.

[0033] Preferably, the lipid particle comprising the cationic lipid is a micelle or a liposome.

[0034] Even more preferably, the lipid particle is a liposome. According to this embodiment, the complex formed between the nucleic acid and the liposome is called a "lipoplex."

[0035] According to a particular embodiment, the present invention provides: (i) nucleic acid; and (ii) lipid particles comprising cationic lipids, which are preferably liposomes or micelles, and even more preferably liposomes; The present invention relates to a method for producing a tablet comprising a complex of

[0036] According to certain embodiments, the methods of the present invention comprise a step of complexing the nucleic acid with lipid particles, preferably liposomes or micelles, comprising cationic lipids, prior to the drying step described in detail below.

[0037] According to certain embodiments, the nucleic acid can be premixed with an anionic polymer before the step of forming a complex between the nucleic acid and lipid particles. The anionic polymer is an adjuvant for improving the structure of the complex between the nucleic acid and lipid particles, thereby increasing the effectiveness of the complex. The anionic polymer can be selected from anionic polysaccharides and anionic polypeptides, and preferably, the anionic polymer is selected from sodium alginate and sodium polyglutamate. According to certain embodiments, the anionic polymer is an alginate, particularly sodium alginate. Therefore, the production method according to the present invention may also include a step of mixing the nucleic acid with an anionic polymer before the step of forming a complex between the nucleic acid and lipid particles.

[0038] In addition, the method according to the present invention may comprise a prior step of forming lipid particles, preferably liposomes or micelles, comprising cationic lipids, preferably liposomes.

[0039] Preferably, the cationic lipid is selected from cationic lipids that can form particles by combining the hydrophobic chains of several lipids.The cationic lipid according to the present invention can be selected from lipopolyamine, quaternary ammonium, and lipids with guanidine or imidazole type cationic head groups.Preferably, the cationic lipid is selected from dimyristylaminopropylaminopropyl (or DMAPAP), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (or DOTMA), 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane] (or DOTAP), 3β[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DCChol), and dioctadecylamidoglycerylspermine (or DOGS).Preferably, the cationic lipid is dimyristylaminopropylaminopropyl (or DMAPAP).

[0040] According to a particular embodiment, the cationic lipid is mixed with a neutral lipid. Preferably, the neutral lipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, dioleoyl-sn-glycero-3-phosphocholine (DOPC), dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and N'-(rac-1-[11-(F-octyl)undec-10-enyl]-2-(hexadecyl)glycero-3-phosphoethanoyl)-sperminecarboxamide). Preferably, the neutral lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0041] Thus, a tablet according to the present invention may comprise a complex of (i) a nucleic acid and (ii) lipid particles comprising a cationic lipid optionally mixed with a neutral lipid.

[0042] According to a preferred embodiment, the tablet according to the invention comprises a complex of (i) a nucleic acid and (ii) a lipid particle comprising a mixture of a cationic lipid and a neutral lipid. Preferably, the lipid particle is a micelle or a liposome, preferably a liposome.

[0043] The mixture of cationic lipids and neutral lipids contains cationic lipids and neutral lipids in a specifically defined ratio. Preferably, the ratio of cationic lipids is 50 to 99 mol% of the lipids in the mixture. In particular, the ratio of cationic lipids may be about 50 mol%, about 60 mol%, about 70 mol%, about 80 mol%, or about 90 mol% of the lipids in the mixture. Preferably, the ratio of cationic lipids is about 50 mol% of the lipids in the mixture.

[0044] Preferably, the ratio of triglycerides is 1 to 50 mol% of lipids in the mixture. In particular, the ratio of triglycerides may be about 10 mol%, about 20 mol%, about 30 mol%, about 40 mol%, or about 50 mol% of lipids in the mixture. Preferably, the ratio of triglycerides is about 50 mol% of lipids in the mixture.

[0045] According to a preferred embodiment, the cationic lipid is mixed with the neutral lipid in a 1:1 equimolar ratio.

[0046] The method according to the present invention may include a step of mixing the cationic lipids and neutral lipids prior to the step of forming lipid particles, which are then used during the step of forming complexes with nucleic acids.

[0047] According to a particular embodiment, the mixture of cationic lipid and neutral lipid is a mixture of DMAPAP and DOPE.

[0048] According to certain embodiments, the nucleic acids of the present invention are mixed with the lipid particles of the present invention according to a defined charge ratio. The "charge ratio" corresponds to the + / - ratio of the number of positive charges on the cationic lipid to the number of negative charges on the nucleic acid. This charge ratio can affect the effectiveness of the formed complex by influencing its physicochemical properties (e.g., size or surface charge). The charge ratio may vary depending on the selected lipids and nucleic acids and the desired application (target organ or cell type) and can be adapted by those skilled in the art. Preferably, the + / - charge ratio is 0.5 to 10, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Preferably, the + / - charge ratio is 4 to 10, for example, about 4, 5, 6, 7, 8, 9, or 10. Even more preferably, the charge ratio is about 8.

[0049] According to certain embodiments, the mixture of nucleic acid and lipid particles is an aqueous mixture. Thus, the method according to the present invention may comprise the step of preparing an aqueous mixture of nucleic acid and lipid particles comprising cationic lipids.

[0050] Preferably, the aqueous mixture of nucleic acid and lipid particles is carried out in a buffer solution. In particular, the pH of the buffer solution is about 4 to 8. Preferably, the selected buffer solution is a solution containing NaCl. The buffer solution may contain an NaCl concentration of about 150 mM. For example, those skilled in the art can use HEPES or Opti-MEM type buffers, or solutions containing glucose.

[0051] Thus, according to a particular embodiment, the method for producing tablets according to the invention comprises: - forming lipid particles, preferably liposomes, in a buffer solution; and / or - preparing the nucleic acid in a buffer; and / or - mixing the nucleic acid and lipid particles in a buffer solution; may include:

[0052] According to a particular embodiment, the mixing time of the nucleic acid and the lipid particles, preferably liposomes, is between 10 seconds and several days, for example between 10 seconds and 10 hours, in particular between 5 minutes and 5 hours, and in particular between 10 minutes and 1 hour. Preferably, the mixing time is greater than 30 minutes, in particular equal to about 30 minutes. Mixing can be carried out over a wide temperature range. However, according to a preferred embodiment, mixing is carried out at a temperature between 10°C and 30°C, in particular between 15°C and 25°C.

[0053] After the complex has been formed, it may be directly subjected to the following steps of the method according to the invention or may be stored, in particular for at least one day, before said following steps.

[0054] "Tablet" formulation The method according to the present invention includes the step of formulating a tablet from an aqueous mixture containing nucleic acid and lipid particles as described above. The inventors of the present application have succeeded in formulating a nucleic acid vector (or complex) into a "tablet" form for potential oral, vaginal, rectal, sublingual or transmucosal administration, while maintaining the biological properties of the nucleic acid vector.

[0055] More specifically, the method according to the present invention comprises: - drying the aqueous mixture containing the complex defined above; and - compressing the dry mixture thus obtained Includes:

[0056] Preferably, the method according to the invention comprises the steps of: a)- a complex as defined above; and - one or more additives suitable for drying drying the aqueous mixture comprising: b) mixing one or more compression additives with the dry mixture obtained in step a); and c) compressing the mixture obtained in step b) Includes:

[0057] ·Drying process a): Drying refers to any dehydration process capable of removing water contained in a substance or body. For example, drying can be achieved by spray drying, convection drying, or freeze drying. According to a preferred embodiment, drying is performed by freeze drying. According to this embodiment, the additive suitable for drying is an additive suitable for freeze drying. Freeze drying is a low-temperature, reduced-pressure drying process that removes water from a previously frozen product by sublimation.

[0058] According to a preferred embodiment, the drying step a) is freeze-drying. According to a particular embodiment, the freeze-drying process carried out comprises the following steps: - freezing an aqueous mixture comprising the complex as defined above and one or more additives suitable for freeze-drying; - Primary drying (or sublimation); and - Secondary drying Includes:

[0059] Preferably, the method according to the invention comprises, before the drying step, a step of mixing the complex as defined above with an additive suitable for drying.

[0060] Additives suitable for drying are additives that can preserve the structure of the nucleic acid vector during the drying process, thereby preserving the biological activity of the nucleic acid. Additives suitable for drying also provide a dry mixture suitable for the compression process. In particular, additives make it possible to obtain a dry product whose texture (affected by residual moisture) is suitable for the compression process. Additives also make it possible to obtain a dry product whose mass and / or mechanical strength are suitable for the compression process, for example, by avoiding the collapse of the structure of the dry product. Preferably, additives suitable for drying, preferably freeze-drying, facilitate the grinding and uniform mixing of the compressed additive with the resulting dry product. The optimization of parameters such as the texture, mass and / or mechanical strength of the dry product by selecting the nature and amount of additives suitable for drying is conventionally performed by those skilled in the art, particularly based on the physicochemical characteristics of each additive (presence of eutectic, glass transition temperature, etc.).

[0061] According to a preferred embodiment of the present invention, when freeze-drying is used in step a), the additive suitable for drying is suitable for freeze-drying, thus protecting the complex during the freeze-drying step and / or making it possible to obtain a freeze-dried product with a texture suitable for the compression step.

[0062] According to certain embodiments, additives suitable for lyophilization are protective agents (cryoprotectants or lyoprotectants) and / or bulking agents (or ballasts). Cryoprotective additives are additives that protect the structure of nucleic acid vectors during the freezing process. Lyoprotective additives are additives that protect the structure of nucleic acid vectors during the dehydration process. During lyophilization, these agents replace water molecules and interact with the polar heads of phospholipids (water displacement hypothesis), forming a glassy matrix that helps maintain the structure of the lipoplexes (vitrification hypothesis). Bulking agents (or ballasts) are additives that maintain the structure and rigidity of the lyophilizate (as opposed to a collapsed state).

[0063] According to a particular embodiment, the additives suitable for lyophilization are selected from trehalose, mannitol, sucrose, sorbitol, lactose, glucose, glycerin, glycine, alanine, lysine, polyethylene glycol, polyvinylpyrrolidone (PVP) and dextran, optionally in a mixture.

[0064] According to a particular embodiment, the additive suitable for lyophilization is trehalose in a proportion of 1 to 100% by weight, in particular 10 to 100% by weight, based on the weight of the additive suitable for lyophilization, for example, the proportion of trehalose is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% by weight of the additive suitable for lyophilization.

[0065] According to certain embodiments, the additive suitable for lyophilization is mannitol in a proportion of 1 to 90% by weight of the additive suitable for lyophilization, for example, the proportion of mannitol is about 0%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% by weight of the additive suitable for lyophilization.

[0066] Preferably, the excipient suitable for freeze-drying is trehalose optionally mixed with another excipient suitable for freeze-drying selected from mannitol, sucrose, sorbitol, lactose, glucose, glycerin, glycine, alanine, lysine, polyethylene glycol, polyvinylpyrrolidone (PVP) and dextran, preferably mannitol.

[0067] According to a specific embodiment, trehalose is mixed with mannitol in a defined ratio. In particular, the trehalose content may be 10 to 99% by weight of the trehalose / mannitol mixture. For example, the trehalose content may be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weight of the trehalose / mannitol mixture, preferably about 71% by weight of the trehalose / mannitol mixture.

[0068] According to certain embodiments, the proportion of mannitol may be 1 to 90% by weight of the trehalose / mannitol mixture, for example, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weight of the trehalose / mannitol mixture, and preferably about 29% by weight of the trehalose / mannitol mixture.

[0069] According to a particular embodiment, the method according to the invention comprises the steps of: - preparing an additive suitable for drying, preferably prepared in an aqueous medium; - mixing an additive suitable for drying with the complex defined above; and - drying the mixture thus obtained, in particular by freeze-drying. may include:

[0070] According to a particular embodiment, the freezing step carried out during lyophilization corresponds to freezing of a mixture comprising an additive suitable for drying and a complex as defined above. The freezing is carried out in such a way as to preserve the biological activity of the nucleic acid vector, preferably the supramolecular assembly of the nucleic acid vector. The freezing parameters are controlled to promote amorphization of the components of the mixture and / or to limit crystal growth.

[0071] Preferably, the freezing kinetics is fast, particularly depending on the freezing temperature. Preferably, the freezing rate is between -40°C / min and -0.5°C / min.

[0072] Preferably, to promote amorphization, freezing is carried out in liquid nitrogen, particularly at a temperature of about −190° C. Preferably, the freezing time in liquid nitrogen is 1 minute to 24 hours, particularly 1 minute to 1 hour, and the freezing time is particularly about 10 minutes.

[0073] According to certain embodiments, the parameters of the freeze-drying process make it possible to preserve the biological activity of the complexes. In particular, the operating parameters controlling the freeze-drying cycle (room or storage temperature, pressure in the drying chamber and cycle duration) are appropriately selected to maintain the biological activity of the complexes and can be adapted depending on the volume of the aqueous mixture containing the complexes (more specifically depending on the volume of water to be sublimated).

[0074] According to a particular embodiment, the duration of the lyophilization, including primary and secondary drying, is between 10 and 80 hours, preferentially between 15 and 72 hours, more preferentially between 20 and 48 hours, preferably about 30 hours. The lyophilization time can be adapted by a person skilled in the art based on the volume and formulation of the aqueous mixture to be lyophilized.

[0075] According to a particular embodiment, drying, in particular by freeze-drying, is carried out at atmospheric pressure.

[0076] ·Mixing process b): The process according to the invention may also include a step of destructuring the product obtained after drying. This may be a step of light grinding (low energy) or redispersion of the product. It can be carried out, for example, using vibration or a calibrated grid, or in a paddle mixer, for example when mixing with excipients.

[0077] Prior to compression, the optionally milled dry mixture is mixed with one or more compression additives. - to protect the complex of nucleic acid and lipid particles during the compaction step c); and / or - to reduce friction phenomena during the compression step c); and / or - delimiting the compartments in which the powder is compressed, allowing for a regular supply of the matrix used for compression; and / or - to obtain tablets of sufficient mass and sufficient mechanical strength, and / or - to control or delay the release of the nucleic acid vector, and / or - Allows tablets to disintegrate represents an optional additive.

[0078] According to a particular embodiment, the compression additives are selected from among the additives used in the granular mixture for compression.

[0079] In a preferred embodiment, the compression additive is a lubricant, optionally mixed with at least one other compression additive well known to those skilled in the art, preferably an excipient.

[0080] "Lubricant" refers to any additive that reduces adhesion of powder to the punches and dies of a compression machine and / or reduces friction between the particles and the dies during tablet compression and ejection.

[0081] According to a particular embodiment, the lubricant represents 0.25-5% by weight of the finished tablet. Preferably, the lubricant represents about 0.5% by weight of the finished tablet. According to a preferred embodiment, the lubricant is selected from magnesium stearate, talc, sodium stearyl fumarate, and stearic acid, optionally in admixture. Preferably, the lubricant is magnesium stearate.

[0082] "Excipient" refers to any additive that has a filler role when the amount of active ingredient and other additives is insufficient to produce a tablet of appropriate size, thus allowing adjustment of the tablet mass and dimensions.

[0083] According to a preferred embodiment, the excipient is selected from lactose, starch and its derivatives, calcium phosphate and its derivatives, in particular dicalcium phosphate, and cellulose and its derivatives, in particular microcrystalline cellulose, optionally in admixture. Preferably, the excipient is lactose.

[0084] The tablet may further contain pharmaceutical additives well known to those skilled in the art, such as flow agents, disintegrants, binders, additives for controlling or delaying the release of the nucleic acid vector, additives for imparting mucoadhesive properties to the tablet, and combinations thereof. For example, the tablet may contain one or more of the following additives: sucrose, glucose, mannitol, trehalose, and silica.

[0085] Preferably, the compressed additive is a dry additive, preferably in powder form.

[0086] The method of the present invention may comprise, prior to the mixing step, a step of mixing different compression additives with the dry mixture obtained after drying step a).

[0087] Compression process (c): The method according to the invention further comprises a step c) of compressing the mixture obtained at the end of step b). The compressing step is carried out under conditions that preserve the biological function of the nucleic acid vector. In particular, the compressing step is carried out in such a way as to preserve the therapeutic activity of the nucleic acid vector, e.g., in the case of siRNA, the expression of the target gene is inhibited.

[0088] The mechanical stress applied during the compression step can be controlled and monitored. More specifically, the pressure applied to the mixture containing the nucleic acid vector according to the present invention is controlled and monitored during the compression step. Preferably, the compression in step c) is carried out at a pressure of 10 to 400 MPa, preferably 50 to 250 MPa.

[0089] According to a preferred embodiment, the compression of step c) is carried out using a tablet press, such as a rotary press or an alternative press.

[0090] In certain embodiments, a wet granulation or dry granulation step is carried out before the compression step c). The "wet granulation" or "dry granulation" step consists of increasing the size of particles to obtain agglomerates to facilitate compression. More specifically, the wet granulation step involves the addition of a binder to obtain granules in the form of agglomerates, which are then dried and optionally sieved before the compression step. Dry granulation consists of strongly compressing particles to obtain agglomerates, which are then broken down to form particles, which are optionally calibrated before the final shaping step by compression.

[0091] According to a preferred embodiment, the compression step c) is a direct compression, which does not require a prior granulation step. According to this embodiment, the dry mixture obtained at the end of the drying step (step a)), optionally mixed with compression additives (step b)), has properties that make it suitable for direct compression.

[0092] In a particular embodiment, the method according to the invention comprises a step of film-coating (or coating) the tablets obtained after the compression step. The advantage of this step is the production of better protected tablets, whose stability is improved and / or whose release kinetics are adapted. The nature of the film-coating and / or coating agents and the processes used to carry out this step are well known to those skilled in the art of formulation.

[0093] In a second aspect, the present invention relates to a tablet obtainable by the process described above.

[0094] In particular, the present invention provides: - one or more nucleic acid vectors (or complexes) as above; - one or more additives suitable for drying; - one or more compression additives The present invention relates to a tablet comprising:

[0095] In particular, the tablets of the present invention: - complexes of nucleic acids with liposomes or micelles containing cationic lipids; - one or more suitable lyophilization additives to protect the complex during the lyophilization step a) and / or to obtain a lyophilisate with a texture suitable for the compression step, preferably trehalose and mannitol; - one or more suitable compression additives to protect the complex during compression step c) and to preserve the biological activity of the complex; may also include:

[0096] In particular, the finished tablet contains 1 μg to 1 mg, preferably about 10 μg, of nucleic acid.

[0097] The mass of cationic lipid in the finished tablet may be between 18.7 μg and 18.7 mg, preferably about 170 μg.

[0098] The mass of neutral fat in the finished tablet may be 16.5 μg to 16.5 mg, preferably about 150 μg.

[0099] The mass of polymer in the finished tablet may be between 1 μg and 1 mg, preferably about 10 μg.

[0100] The mass of the additives suitable for drying may be 36 mg to 720 mg, preferably about 126 mg, in the finished tablet. In particular, the mass of trehalose in the finished tablet may be 3.6 mg to 720 mg, preferably about 90 mg. In particular, the mass of mannitol in the finished tablet may be 0 mg to 648 mg, preferably about 36 mg.

[0101] The proportion of excipients suitable for compression may be 0.25 to 95% by weight of the finished tablet. In particular, the proportion of a lubricant, preferably magnesium stearate, may be 0.25 to 5% by weight, preferably about 0.5% by weight, of the finished tablet. In particular, the proportion of other compression excipients may be 0 to 94.75% by weight of the finished tablet.

[0102] According to a particular embodiment, the tablet according to the invention comprises: - complexes of nucleic acids with liposomes or micelles comprising DMAPAP and DOPE, which further comprise an alginate, in particular sodium alginate; trehalose and mannitol; and - Lactose Includes:

[0103] According to a variant of this embodiment, the tablet thus constructed has, after resuspension in water, a synchrotron radiation small-angle X-ray scattering (SAXS) profile comprising a cubic phase and a lamellar phase, optionally a liquid crystalline phase. The SAXS profile can be determined based on the protocol given in the examples. The SAXS profile of a tablet according to the invention after resuspension in water has a q=0.086 nm -1 and q = 0.101 nm -1 According to a particular embodiment, the lamellar phase predominates compared to the cubic phase.

[0104] According to a particular embodiment, the tablet according to the invention is intended for oral, vaginal, rectal, sublingual or transmucosal administration. Preferably, the tablet according to the invention is intended for oral administration.

[0105] In another aspect, the present invention relates to a tablet of the present invention for use in treating a local, locoregional, or systemic pathology. Preferably, the tablet according to the present invention is used to treat a disease of the gastrointestinal tract. For example, the disease of the gastrointestinal tract can be Crohn's disease (wherein the nucleic acid is, for example, an siRNA that inhibits an inflammatory protein such as TNF-α), ulcerative colitis (wherein the nucleic acid is, for example, an anti-ICAM-1 oligonucleotide, an IL-10 or anti-TNF-α siRNA encoding plasmid), or gastric cancer (wherein the nucleic acid is, for example, an siRNA or anti-Her2 oligonucleotide). The tablet according to the present invention can also be used to treat vaginal pathologies, in particular vulvovaginal candidiasis, or HSV, HPV, or HIV infection (for example, via administration of an antiviral siRNA).

[0106] Additionally, the tablets can be used to treat conditions such as spinal muscular atrophy (wherein the nucleic acid is, for example, the antisense oligonucleotide Nusinersen), familial hypercholesterolemia (wherein the nucleic acid is, for example, the antisense oligonucleotide Mipomersen), Duchenne myopathy, or age-related macular degeneration (wherein the nucleic acid is, for example, the aptamer Macugen).

[0107] The tablets according to the invention are stable, ie, substantially retain their biological activity, preferably at room temperature for up to 2 weeks, 1 month, 3 months, or 1 year (between formulation and administration of the tablet). [Example]

[0108] Example 1 Tablet preparation 1. Lipoplex formation The first step involves the formation of lipoplexes, i.e., complexes of nucleic acids and liposomes. The nucleic acid used in this example is luciferase-specific siRNA. The liposomes used here contain a cationic lipid (DMAPAP) and a neutral lipid (DOPE). The formation of lipoplexes was carried out using the following components:

[0109] [Table 1]

[0110] The siRNA, liposome and polymer preparations were vortexed. - Liposomes were mixed with NaCl solution to obtain a final volume of 200 μL (10.7 μL liposomes and 189 μL NaCl solution). Furthermore, the siRNA was mixed with the polymer in NaCl solution to obtain a final volume of 200 μL (7.5 μL siRNA + 6.5 μL polymer + 186 μL NaCl solution), so that the resulting mixture contains a 1:1 siRNA to polymer ratio by mass. - 200 μL of each of the two preparations obtained was mixed, vortexed, and then left at room temperature for 30 minutes to allow the formation of a complex between the siRNA and the liposomes.

[0111] The above components were mixed to form lipoplexes with a + / - charge ratio of 8.

[0112] 2. Freeze drying In this example, mannitol and trehalose were used as additives suitable for lyophilization.

[0113] The lipoplex suspension obtained previously was transferred to a 50 mL centrifuge tube. A solution containing 2.5% m / v trehalose and 1% m / v mannitol was prepared. 3.6 mL of this trehalose / mannitol solution was then added to 400 μL of the lipoplex suspension. The centrifuge tube was then immersed in liquid nitrogen for 10 minutes.

[0114] Freeze-drying was performed in a freeze-dryer with a manifold equipped with 12 valves to which the frozen sample tubes were connected. Freeze-drying was carried out for 30 hours at a pressure of less than 0.2 mbar (more specifically, 0.1-0.18 mbar) in the freeze-drying chamber. The energy required to compensate for the energy absorbed during sublimation was provided by the ambient air (room temperature, more specifically, 20-25 °C).

[0115] A temperature sensor was placed in the sample to monitor the temperature of the product during the freezing (see Figure 1b) and freeze-drying (see Figure 1a) processes.

[0116] 3. Compression In this example, magnesium stearate (lubricant) and lactose (filler) were used as additives suitable for compression.

[0117] The previously obtained lyophilized material was first powdered by lightly grinding it in a mortar, and then 36.2 mg of lactose and 0.8 mg of magnesium stearate were added to the mortar and mixed with the lyophilized material.

[0118] Compression was performed using a STYL'One Evolution compression simulator according to the following parameters: - 6mm diameter chamfer flat punch - Compression Cycle = Cycle 1 compression (default cycle), speed 2%. - Force Control - Die filling height = 18mm - Manual filling - Compression force = 1.4~7.1kN (50~250MPa)

[0119] Example 2 Example of the composition of a tablet containing lipoplexes Table 1 below shows, by way of example, the composition of a tablet containing lipoplexes, the different components being expressed in mg.

[0120] [Table 2]

[0121] Example 3 Testing the efficacy of tablets The effectiveness of the tablets is then evaluated in cultured cells expressing the luciferase gene. When these cells are exposed to a vector containing luciferase-specific siRNA, luciferase activity is inhibited. The test consists of transfecting mouse melanoma cells expressing luciferase (B16-Luc) and determining the residual luciferase activity in the cells 48 hours later. This measurement allows us to obtain the percentage of inhibition induced by the siRNA vector studied, relative to the activity of cells not treated or treated with a vector containing control siRNA (no inhibitory effect).

[0122] To assess the maintenance of this activity after compression, the tablets are solubilized before contacting the cells to be transfected. The lyophilisates (obtained before the compression step) were also rehydrated to analyse the effectiveness of the dry mixture before compression.

[0123] So the test is: - negative control: i.e. lipoplexes containing control siRNA (whose sequence is selected so as not to inhibit the protein target) resuspended from a compacted form; - positive transfection control: i.e., resuspended lipoplexes (not lyophilized, not compacted); - rehydrated lyophilisate; or - Rehydrated tablets This consisted of treating B16-Luc cells with

[0124] More precisely, resuspended lipoplexes (corresponding to the positive transfection control) were prepared as described above and then mixed with the culture medium: complete medium (DMEM + Glutamax + fetal bovine serum + penicillin / streptomycin) supplemented with geneticin (G418).

[0125] Lyophilisates and tablets were prepared as described above and then dissolved in distilled or Milli-Q water and mixed with the culture medium.

[0126] The procedure is carried out over four days (days 0, 1, 2 and 3): On day 0, cells are prepared on 24-well plates (40,000 cells / well). On day 1, replace the cell culture medium with 1 mL of transfection medium (to add 0.5 μg of siRNA per well), which is the medium containing the negative control, the positive transfection control, the lyophilisate or the tablets. On day 2, the transfection medium is replaced with culture medium (complete medium + G418). On day 3, the cells are lysed and luciferase activity is measured. Tests are performed in triplicate (three repeats per condition).

[0127] result: Testing the efficacy of tablets Cell transfection studies demonstrated superior efficacy of the lipoplex-containing tablets (efficacy expressed as the percentage of inhibition of the tablets compared to non-transfected cells). In addition, no compressive stress effect was observed on the percentage of inhibition of the luciferase gene over the entire range of applied compressive stress (Figure 2).

[0128] Example 4 supramolecular structure Small-angle X-ray scattering (SAXS) techniques were used to identify the supramolecular structure of siRNA vectors, both in suspended particles and in dry forms, such as lyophilisates or tablets. The siRNA used was a control siRNA, and each sample contained 100 micrograms of siRNA. This identification is made necessary by the fact that the structure of this type of particle is directly related to their effectiveness in administering nucleic acids. Synchrotron radiation SAXS analysis is the only technique that provides access to this structural identification in various galenic forms suspended in aqueous media or powder.

[0129] We first determined the structure of siRNA lipoplexes (DMAPAP / DOPE + alginate) prepared with a + / - charge ratio of 8. Studies performed on lipoplexes suspended in salt buffer (150 mM NaCl) showed that these lipoplexes form two coexisting structures: lamellar and cubic phases (Figure 3).

[0130] Next, we investigated the fate of this structure when the suspended vector was subjected to a freeze-drying process, a necessary prerequisite before the compression step, which can then be carried out. Figure 4 shows that a signal was detected in both types of samples (lyophilisates and tablets), with q = 0.096 nm. -1 The results show a single, somewhat broad peak at . Its position is intermediate between the values ​​of peaks L1 and C1 of the suspended lipoplexes (Figure 3), and its specificity makes it impossible to deduce the phase corresponding to this peak. The peak observed in the freeze-dried sample is more intense than that of the compressed sample, although the latter remains fully detectable.

[0131] The freeze-drying and compression process requires the addition of additives. Therefore, we evaluated the signals obtained for lipoplexes diluted with NaCl / additive mixtures under the same concentration conditions as the freeze-dried and compressed samples (Figure 5). The obtained profile shows the presence of a single cubic phase, the first peak of which is slightly offset between the "lyophilized" and "tablet" conditions. The position of this peak is close to the peak detected during powder analysis, which may indicate that the phase present in the dry form is a cubic phase. The peak corresponding to the lamellar phase is no longer detectable in the presence of additives, either in the dry form or in suspension.

[0132] Next, we analyzed the SAXS profiles of the resuspended, freeze-dried, and compressed samples. Previous analysis demonstrated the effect of additives on particle structure (Figure 5). We compared each resuspended sample with suspended lipoplexes under the same additive concentration conditions. The results are shown in Figure 6. The SAXS profile of the resuspended freeze-dried sample was found to be close to that of suspended lipoplexes under the same conditions (Figure 6A), suggesting that freeze-drying does not alter the lipoplex structure. In the presence of additives, only the cubic phase was detected. On the other hand, for the resuspended compressed sample, the profile clearly showed two phases, lamellar and cubic, with the major peaks at positions similar to those observed for the initial lipoplex suspension (Figure 3). The profile of this sample is clearly different from that of the lipoplex suspension under the same additive concentration conditions. It also differs from the initial lipoplex suspension because the relative proportions of the cubic and lamellar phases are reversed. In the initial suspension, the cubic phase predominates, while in the resuspended, compressed sample, the lamellar phase predominates. This result indicates that compression affects the structure by changing the relative proportions of one phase to another, but does not cause new phases to appear or disappear.

[0133] Example 5 In vivo efficacy assessment Model: Induction of ulcerative colitis in mice by administration of dextran sulfate (DS) in drinking water for 7 days

[0134] Assessment of vector efficacy: Oral administration of tablets containing siRNA lipoplexes. Mice under DS treatment receive tablets on days 1, 3, and 5 (starting on day 0). Each mouse receives a tablet containing 50 μg of siRNA formulated as a lipoplex.

[0135] Three groups of 10 mice were used: Group 1: Drink water and do not receive Lipoplex. Group 2: DS on days 1, 3, and 5, and tablets containing siRNA lipoplexes against TNF-α Group 3: DS on days 1, 3, and 5, plus tablets containing control siRNA lipoplex (no inhibitory effect) Group 4: DS and no Lipoplex

[0136] Fecal mass and appearance (presence of blood) are monitored throughout treatment. On day 7, animals are euthanized, colons are harvested, colon size is measured, colon lysates are assayed for inflammatory cytokines, and colon sections are histologically analyzed for inflammation and tissue damage.

[0137] Expected results: A reduction or even elimination of clinical and biochemical signs of disease after treatment, which is specific to the siRNA sequence, i.e., observed in mice treated with lipoplexes containing anti-TNF-α siRNA but not in mice treated with lipoplexes containing control siRNA.

Claims

1. (i) a nucleic acid that is mRNA; and (ii) lipid particles containing a cationic lipid consisting of dimyristylaminopropylaminopropyl (DMAPAP) mixed with a neutral lipid consisting of 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine (DOPE); 1. A method for producing a tablet comprising a complex of: a) - (i) a complex of said nucleic acid, which is mRNA, and (ii) said lipid particle; - one or more additives suitable for drying, including trehalose; drying the aqueous mixture comprising: b) mixing one or more compression excipients, including magnesium stearate, with the dry mixture obtained in step a); and c) compressing the mixture obtained in step b) A method comprising:

2. The method of claim 1 , wherein the lipid particle is a liposome or a micelle.

3. 3. The method of claim 1, wherein the drying is freeze-drying.

4. A mixture of cationic lipids and neutral lipids: - 50-99 mol% cationic lipids in the mixture - 1-50 mol% neutral fat in the mixture 4. The method of claim 1, comprising:

5. 4. The method according to claim 1, wherein the charge ratio (+ / -) of the positive charge of the cationic lipid to the negative charge of the nucleic acid is 0.5 to 10.

6. The method according to claim 1, wherein the additive suitable for drying further comprises, optionally in a mixture, mannitol, sucrose, sorbitol, lactose, glucose, glycerin, glycine, alanine, lysine, polyethylene glycol, polyvinylpyrrolidone and / or dextran.

7. The method according to claim 1, wherein the additive suitable for drying comprises trehalose in a proportion of 10 to 100% by weight of the additive suitable for drying.

8. 8. The method according to claim 7, characterized in that the additive suitable for drying further comprises mannitol in a proportion of 0 to 90% by weight of the additive suitable for drying.

9. 9. The method according to claim 1, wherein the compression additive protects the complex of the lipid particles comprising nucleic acid and cationic lipid during compression step c), and the compression additive is magnesium stearate mixed with another compression additive.

10. 10. The method of claim 9, wherein the further compression additive is an excipient selected from lactose, starch, calcium phosphate and cellulose.

11. The method according to claim 1, wherein the magnesium stearate represents 0.25 to 5% by weight of the finished tablet.

12. The method according to claim 1, wherein the magnesium stearate represents 0.5% by weight of the finished tablet.

13. 13. The method according to any one of claims 1 to 12, characterized in that the compression in step c) is carried out at a pressure of 10 to 400 MPa.

14. 14. The method according to any one of claims 1 to 13, characterized in that the compression in step c) is carried out at a pressure of 50 to 250 MPa.

15. - complexes of mRNA with lipid particles containing cationic lipids consisting of dimyristylaminopropylaminopropyl (DMAPAP) mixed with neutral lipids consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); - one or more suitable lyophilization additives for protecting the complex during the lyophilization step a) and / or for obtaining a lyophilizate with a texture suitable for the compression step, the lyophilization additive comprising trehalose; - one or more suitable compression additives for protecting the complex during compression step c) and for maintaining the biological activity of the complex, the compression additives comprising magnesium stearate; tablets, including

16. 16. The tablet of claim 15 for oral, vaginal, rectal, sublingual or transmucosal administration.

17. 17. A tablet according to claim 15 or 16 for use in the treatment of gastrointestinal or vaginal disorders.

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