HK1110205B

HK1110205B - Multiparticulate form of administration, comprising nucleic acid-containing mucoadhesive active ingredients, and method for producing said form of administration

Info

Publication number: HK1110205B
Application number: HK08100739.8A
Authority: HK
Inventors: R．利齐奥; H-U．彼得莱特; D．特鲁普提; M．古特斯查克
Original assignee: 赢创运营有限公司
Priority date: 2004-12-10
Filing date: 2005-11-05
Publication date: 2013-05-31

Description

Multiparticulate pharmaceutical dosage form comprising a mucoadhesive formulated nucleic acid active ingredient and process for preparing said pharmaceutical dosage form

Technical Field

The present invention relates to multiparticulate pharmaceutical dosage forms comprising a mucoadhesive formulated nucleic acid active ingredient and to a process for the preparation of the pharmaceutical dosage forms.

Background

WO 02/64148 describes formulations comprising mucopolysaccharides and methods for their preparation. Here, a mucopolysaccharide, such as heparin, is formulated with an adsorption enhancer, such as chitosan, which is then provided with a coating soluble in intestinal fluid so that the active ingredient can be released in the middle or lower section of the small intestine. Examples of suitable enteric fluid soluble coatings areL, S, L100-55 type anionic acrylic copolymers. The formulations may include capsules, tablets and granules.

Telomerase is an enzyme that promotes DNA doubling during cell division, particularly in the terminal region of the chromosome. The enzyme is therefore important for maintaining a perfect chromosomal structure. Telomerase activity is inhibited in most adult human cells, with increased telomerase activity being observed only in germ cells, but also in many tumor cell types. Telomerase is presumed to play an important role in the molecular control of cells up to their normal life cycle of genetically programmed cell death. High telomerase activity in tumor cells, unlike normal cells, is considered to be a sign that normal cell division control has been lost. Telomerase or a gene construct related thereto is considered a starting point for gene therapy of tumor cells.

WO 99/38964 describes nucleic acids for use in gene therapy, which comprise, inter alia, a telomerase gene promoter. Such DNA may be combined with a foreign gene, such as a cytotoxic encoding gene. The nucleic acid construct can be used as an active ingredient for the transfection of tumor cells with increased telomerase activity. This is expected to inhibit tumor cell division until these cells are specifically killed. The oral possibilities of pharmaceutical dosage forms of the type of active ingredient described in WO 99/38964 or derived therefrom are mentioned.

Roy et al (1999) in Nature Medicine, Vol.5, No.4, p.387 391, "Oral gene delivery with chitosan-DNA nanoparticles immunological protection in a protein model of paraqualloy (Oral gene delivery with chitosan-DNA nanoparticles producing immune protection against peanut allergy in murine models)" describe the Oral administration of DNA active ingredients in mice. The dominant peanut allergen gene (pcmva arah2) present on plasmid DNA was formulated by complex colonization into nanoparticles of 100 to 200 nanometers in size with chitosan having an Mw of about 390000. These nanoparticles were orally administered to AKR/J mice, where transduced gene expression in intestinal epithelial cells could be detected. The mice thus treated produced allergen-specific secretory IgA antibodies and serum IgG2a antibodies and exhibited reduced allergen-induced anaphylactic reactions compared to the control group.

Leong et al (1998) in Journal of Controlled Release 53, pp 183-193, "DNA-polynucleotides as non-viral gene delivery vectors" describe gene transfer vectors that produce foreign gene expression in BALB/c mice. Nanospheres are prepared as complexes of DNA with gelatin or chitosan of 200 to 700 nanometers in size.

WO 02/094983 describes formulations consisting of nucleic acids, antibodies specific for DNA and cationic macromolecular complexes associated therewith. The formulation was done in nanoparticle form, where increased transfection rates were detected both in vitro and in vivo. Formulations for oral use with a slow release of the active ingredient are mentioned.

WO 03/007913 describes an oral multiparticulate pharmaceutical dosage form comprising the active ingredient in the form of a plurality of so-called Patches (Patches). The membrane is a disc-shaped object made of biocompatible material with a diameter of 500 to 5 mm and a height of 100 to 1000 microns. The membrane consists of two layers or faces, one face having only low permeability to water or body fluids, for example consisting of ethylcellulose, and the second face comprising an active ingredient, for example a protein, a polysaccharide or a small molecule, which may be present in admixture with a mucoadhesive polymer, for example chitosan, CMC, polyacrylic acid or pectin. These films may be compressed to form tablets or filled into capsules which additionally have a coating soluble in intestinal fluids. The active ingredient preparation can also be combined with so-called enhancers (e.g. fatty acids, fatty alcohols, esters, surface-active substances and protease inhibitors). At the site of action, e.g. at a specific segment of the intestine, the capsule dissolves and releases the film patch. The released patches are capable of adhering to the intestinal mucosa with their mucoadhesive side and there release the active ingredient slowly and targeted to the intestinal mucosa. The only low permeable second side of the membrane sheet is intended to provide some protection for the active ingredient from chemical or enzymatic inactivation of the luminal side and to prevent the active ingredient from escaping onto this side.

WO 03/092732 describes pH sensitive polymers based on anionic (meth) acrylate copolymers having a comparatively low molecular weight Mw of 1000 to 50000. The pH sensitive polymers are also particularly suitable for complexing nucleic acids. The pH sensitive polymer has cytotoxic properties only at high concentrations or no cytotoxic properties at all in the range of pH7.0 or slightly above, but already at low concentrations below pH6.5 has cytotoxic or hemolytic or membrane lytic (membrane lysis) effects in vivo.

Disclosure of Invention

Problem and solution

WO 99/38964 describes nucleic acids and vectors relating to the human telomerase gene or the promoter of this gene. Wherein said nucleic acid can be considered as a potential active ingredient for gene therapy of tumor cells. The oral administration of the active ingredient types mentioned is only very generally proposed in WO 99/38964. There is a need to propose formulations which enable the person skilled in the art to deliver active ingredients of this type to the site of action in such a way that premature inactivation (especially by nucleases) does not occur and a sufficient proportion of the active ingredient successfully transfects the target cells. The WO 02/094983, which was mentioned at the outset and describes antibody-DNA conjugated complexes in nanoparticles, also gives only a fairly general indication of the formulation of oral pharmaceutical dosage forms.

WO 03/007913 describes a possible solution to provide an oral pharmaceutical dosage form which is released in the intestinal lumen and is intended to function therein. One disadvantage of this solution can be seen in particular in the complicated construction and production of the two-layer membrane structure. It seems particularly disadvantageous to provide the pharmaceutical dosage form in the form of capsules with a coating which is resistant to gastric juice and soluble in intestinal fluid. When the size is significantly larger than 2.5 mm, insufficient reproducibility of treatment is feared. The time for the capsule to pass through the stomach may vary greatly. In any case, it is expected that the action will take place with a delay. Furthermore, the capsule itself may dissolve rapidly or slowly already after partial dissolution of the coating has occurred. The two principles of coating and encapsulation overlap in this case in an unfavorable manner, so that the release of the film tablets is expected to be overall uncontrolled. The capsule may remain intact or substantially mechanically destroyed depending on the intestinal contents or the intestinal peristalsis at that time, at least partially accessible to intestinal fluids. Depending on the disintegration or mechanical stress of the initially coated capsule formation, a large number of film pieces may be suddenly released on the one hand or an undesired delayed release may also occur on the other hand. Thus, it is highly desirable to better control the release of the active ingredient as a whole.

The invention relates to orally administrable pharmaceutical dosage forms for nucleic acid active ingredients, in particular for gene therapy purposes. A general problem in this respect is to formulate the active ingredient in a dosage form which facilitates transfection of living cells at the site of action and at the same time ensures that the active ingredient, or at least a sufficient amount of the active ingredient, reaches the site of action in a form which enables transfection. One of the problems of the present invention is believed to be to provide a pharmaceutical dosage form suitable for targeted and efficient release of a nucleic acid active ingredient. Such pharmaceutical dosage forms are intended to provide high dose reliability and good distribution in the intestinal lumen after rapid passage through the stomach. The nucleic acid active ingredient contained therein should be protected to a large extent against physical, chemical or nucleolytic inactivation and be released at the designated site of action so that a high proportion of the active ingredient can be absorbed by the body. The release site should be variable and reliably adjustable depending on the therapeutic purpose. The pharmaceutical dosage form should contain, in addition to the DNA active ingredient, only pharmacologically acceptable, non-toxic ingredients, so that no undesirable side effects are expected initially, even upon frequent or regular ingestion of the pharmaceutical dosage form.

This problem is solved by an oral multiparticulate pharmaceutical dosage form comprising pellets having an average diameter of 50 to 2500 microns, these pellets being composed of:

a) an inner matrix layer comprising nanoparticles which contain a nucleic acid active ingredient and are embedded in a matrix of a polymer having mucoadhesive action, wherein the matrix may optionally comprise further pharmaceutically customary auxiliary substances,

b) an external film-forming coating consisting essentially of an anionic polymer or copolymer which may optionally be formulated with pharmaceutically customary auxiliaries, especially plasticizers,

it is characterized in that

The multiparticulate pharmaceutical dosage form is formulated such that the contained pellets are released in the pH range of the stomach, the outer coating is adjusted by selection of the anionic polymer or copolymer or its formulation with excipients and the layer thickness thereof such that the coating dissolves in the intestine within 15 to 60 minutes in the pH range of 4.0 to 8.0, thereby exposing the mucoadhesive matrix layer containing the active ingredient, which can adhere to the intestinal mucosa and release the active ingredient there, wherein the polymer with mucoadhesive action is selected such that it exhibits at least eta in the range of 0.5pH units based on the pH value at which the outer coating starts to dissolve_bA mucoadhesive effect of 150 to 1000mPa · s and a water absorption of 10 to 750% within 15 minutes, and the active ingredient content of the nanoparticles in the matrix layer is at most 40 wt% of the content of the polymer having mucoadhesive effect.

Embodiments of the invention

The invention relates to an oral multiparticulate pharmaceutical dosage form, in particular in the form of tablets, minitablets, pellets filled into capsules, sachets or powders for reconstitution () Which comprises average size or average diameterPellets having a diameter of 50 to 2500, preferably 100 to 1000 microns, the pellets being composed of:

b) an external film-forming coating consisting essentially of an anionic polymer or copolymer which may optionally be formulated with pharmaceutically customary auxiliaries, especially plasticizers.

The multiparticulate pharmaceutical dosage form is formulated such that the contained pellets are released within the pH range of the stomach.

The term pellet in the sense of the present invention includes round to spherical agglomerates, which may also be referred to as microparticles, beads or mini-tablets, as long as they have the structure and dimensions described in the present invention.

The outer coating is adjusted by the choice of the formulation of the anionic polymer or copolymer or of the formulation thereof with the auxiliary substances and the layer thickness thereof, such that the coating dissolves in the intestine in a pH range of from 4.0 to 8.0, preferably from 5.5 to 7.8, particularly preferably from 5.8 to 7.5 in from 15 to 60 minutes, preferably from 20 to 40 minutes, thereby exposing the mucoadhesive matrix layer containing the active ingredient, which can adhere to the intestinal mucosa and release the active ingredient there.

The polymer or copolymer having mucoadhesive action is selected so as to exhibit at least eta in a range of + -0.5, preferably + -0.3 pH units based on the pH value at which the outer coating starts to dissolve_b150 to 1000, preferably 150 to 600 mPas, and a water absorption of 10 to 750%, preferably 10 to 250%, particularly preferably 10 to 160% in 15 minutes, and the active ingredient content in the matrix layer is at most 40% by weight, in particular 0.001 to 15% by weight, or 0.05 to 5% by weight, of the content of the polymer having mucoadhesive action.

Internal matrix layer

The inner matrix layer serves as a carrier for the active ingredient. The inner matrix layer also has the function of binding the active ingredient to the intestinal mucosa by means of the mucoadhesive polymer contained, so that the active ingredient can pass there into the body. The inner matrix layer also has the function of protecting the active ingredient from physical, chemical or enzymatic inactivation.

The internal matrix may also comprise pharmaceutical adjuvants, in particular G-protein coupled receptors and ligands (see, e.g., WO 02/102407, pages 74 to 76), in particular 8-OH-DPAT, Aminoketanserin, atropine, butalamol, chlorpromazine, Chloroprozhihexin, sinaxelin, Cyanopindolol, cyproheptadine, pimiperidone, Epi-depride, epinephrine, fenoldopam, flupentixol, fluphenazine, haloperidol, Hexoclium, Himbacin, Iodomelacton (Iodomelatonin), ketanserin, ergonic acid derivatives, mesoridazine, Mesullergin (Mesumeresurus), Methocarramin (Memetriramine), Methylorgramine, Methylorgyrgid, Methylorgrid, metoclopramide, Midinerin, Molindonem, Methoderin, Piperazone, Methyloridone, Piroprione, Chloranthrin, Methylol, Perineralol, Periplandrol, Methylol, Periplanel, Piropamide, Pirop, Phentolamine, perancine, PPHT-coumarin, PPHT-rhodamine, PPHT-Texas Red, prazosin, promazine, Raclopride, 5-hydroxytryptamine, Speperone, Spiroxatrine, sulpiride, sumatriptan, tenirapine, and Trifluprimazin (trifluoropropylamine).

The inner matrix may also contain penetration enhancers such as plasticizers, for example triethyl citrate, acetyl triethyl citrate, diethyl sebacate, dibutyl sebacate; polymers, such as Carbomer (Carbomer), chitosan-cysteine, sodium carboxymethylcellulose, N-trimethylated chitosan, polycarbophil (carbophil) -cysteine, long chain fatty acids, their esters (e.g., mono-and diglycerides), and their salts, such as lauric acid, laurylsulfonic acid, palmitic acid, caprylic acid, capric acid, oleic acid, acylcarnitines; chelating agents, e.g. EDTA, salicylates, cyclodextrins, polyacrylic acid, bile acids such as cholic acid, cholic sarcosine, chenodeoxycholic acid and salts thereofSuch as sodium cholate, sodium glycocholate, sodium taurocholate, sodium glycodihydrofusrate; surfactants and emulsifiers, such as, in particular, polyethylene-660-12-hydroxystearate: (HS15, (Solutol HS15), Polysorbat 80 (Tween 80), polyoxyethylated castor oil (Cremophor EL), polyoxyethylene-polyoxypropylene glycol (C. sub.H.) (C. sub.H)F68) Toxin tight junction toxin (ZOT), and vitamins, such as vitamin E (tocopherol) or vitamin B12.

The pharmaceutical adjuvants, penetration enhancers and/or G-protein coupled receptors and ligands are preferably absent or present in the inner matrix layer in only small amounts, for example from 0.01 to 10 wt.%, preferably from 0.05 to 2 wt.%, particularly preferably from 0.1 to 1 wt.%.

Nucleic acid active ingredient

The matrix layer comprises nanoparticles comprising a nucleic acid active ingredient. The nucleic acid active ingredient has the task of initiating an interaction at a target site in the body with the DNA of mammalian cells, in particular human cells, which leads to an altered DNA structure or very generally to an altered cellular nature within the cell. In this connection, mention should first be made of so-called gene therapy, the aim of which is to repair gene structures which are defective in gene-dependent disorders. This may mean, for example, inactivation or interruption of undesirable gene activity (e.g., telomerase activity in tumor cells). It may also refer to the restoration of gene activity normally present in healthy cells (e.g., p53 gene activity, a long known and well studied tumor suppressor gene). The invention therefore relates to orally administrable pharmaceutical dosage forms for nucleic acid active ingredients, in particular for gene therapy.

The nucleic acid active ingredient may be single-stranded or double-stranded DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) or DNA-RNA chimeras, in which naturally occurring and/or non-naturally occurring synthetically modified nucleotides may be present. The nucleic acid active ingredient may be present in a linear or circular form. It may be an oligonucleotide unit, for example 10 to 200 bases or base pairs in length. It may also be a longer unit, for example more than 200 to 100000, 500 to 10000 or 1000 to 5000 bases or base pairs. In addition to the sequences which act as actual active ingredients (for example nucleic acid sequences which are present in the target cells or which should be supplemented), the nucleic acid active ingredients may, where appropriate, also comprise vector sequences which are not normally present in the target cells and which should not interact with the latter.

Vector systems based, for example, on double-stranded DNA are known, which involve plasmids or vectors based on viral systems. For example, recombinant adeno-associated virus (rAAV) vectors are known. Other double-stranded vectors may contain promoter or regulatory sequences derived from Cytomegalovirus (CMV) or SV40 viruses. Other vectors may involve single stranded DNA, which is protected from degradation by means of additional RNA units. Also known are so-called RDOI and RDOII constructs, in which short DNA fragments, for example 30 to 60 bases, are provided at the end with short RNA fragments of 1 to 4 bases. Additional improvements in half-life or nuclease resistance can be obtained by introducing non-naturally occurring nucleotides into the RNA or DNA. In this regard, for example, a single oxygen atom may be replaced by a sulfur atom, thereby obtaining a phosphorus-sulfur bridge (MSO). A variety of nucleic acid forms which can be used as active ingredients in the sense of the present invention, suitable as gene repair or gene replacement vectors, are described, for example, in Nature Reviews, Vol.4, 2003, pp.679-689, Li Liu et al. Preferably nucleic acid fragments which contain essentially only the nucleic acid sequence acting as active ingredient and only a small proportion or no vector DNA.

The nucleic acid active ingredient may be present, for example, in a complex or conjugate with a cationic polymer or protein (e.g., an antibody). The complexing or conjugate binding may be covalent, reversibly or irreversibly, by chemical bridging or non-covalent via van der waals forces, ionic bonds, hydrophobic bonds. The molecules present in the complex or conjugate, except for the nucleic acid active ingredient, do not exert a therapeutic effect themselves and are therefore considered formulation aids and not as an active ingredient or part of an active ingredient.

The nucleic acid active ingredient may be formulated with the aid of proteins or peptides, as appropriate. However, they do not exert any therapeutic effect per se and are therefore considered formulation aids and not as active ingredients or part of active ingredients.

The nucleic acid may, for example, be in the form of a complex with an antibody which specifically binds to the nucleic acid or with a cationic species according to WO 02/094983. It has been possible to show that this approach may contribute to increased transfection rates in vitro and in vivo. This may preferably mean monoclonal IgG or IgM antibodies, either intact or in the form of fragments, Fc antibody fragments, Fab 'antibody fragments, F (a, b)' 2 antibody fragments or half-antibody fragments, but which in each case must comprise at least one anti-DNA binding site. The molecular ratio of nucleic acid to anti-DNA antibody may be, for example, 1: 20 to 1: 5.

The nucleic acid active ingredient may, for example, be for the purpose of treatment of haemophilia and comprise a coagulation factor gene, for example the cDNA gene of human coagulation factor IX (see, for example, WO 03/028657 or Palmer et al, Blood, 1989, 73(2), p 438-445 or Yao et al, Proc Natl Acad Sci, USA, 1992, 89 (8): p 3357-3361). The nucleic acid active ingredient may comprise, in addition to the therapeutically effective gene portion, an immune tolerance-inducing gene, for example a Fas ligand. The co-expressed Fas ligand or Fas gene fragment can cause apoptosis in T cells, which can be specifically activated after gene transfer into target cells. Vectors involved in the induction of apoptosis in leukemia cells can also be deduced from Walensky et al, 2004, "Activation of Apoptosisin Vivo by a Hydrocarbon-stabilized BH3 Helix (Activation of apoptosis in Vivo by Hydrocarbon-immobilized BH3 Helix)", Science, 305, p.1466-1470. The nucleic acid active ingredient may comprise, for example, a gene fragment of the human telomerase gene, in particular a promoter region. Suitable examples are the gene therapy vectors pGT62-codAupp described in WO 99/38964, or other vectors which can be deduced by the person skilled in the art from WO 99/38964. The nucleic acid active ingredient may comprise a tumor suppressor gene fragment, such as the p53 tumor suppressor gene or fragment thereof. US6,451,593B1 describes the principle of construction of expression vectors for gene therapy, which are suitable for preparing the active ingredients of nucleic acids in the sense of the invention.

Nanoparticles

The pharmaceutical dosage form comprises nanoparticles which can preferably have a size of from 20 to 1000, preferably from 50 to 250, particularly preferably from 80 to 220, in particular from 100 to 200, nm.

The nucleic acid present in the nanoparticle may preferably be present in the form of a complex with a cationic species.

The cationic species may be a cationic lipid, a cationic polypeptide, and/or a cationic polymer. Polyethyleneimine or derivatives may also be suitable.

The cationic lipid may be, for example, a commercially available mixture of N- [1- (2, 3-dioleyloxy) propyl ] -N, N, N-trimethylammonium chloride (DOTMA) and Dioleylphosphatidylethanolamine (DOPE). Also suitable are N- [1- (2, 3-dioleyloxy) propyl ] -N, N, N-trimethylammonium methylsulfate (DOTAP), Dioleylphosphatidylcholine (DOPC), dioctadecylamidoglycyl spermine (DOGS).

The cationic polypeptide is preferably a synthetically prepared homopolymer of amino acids having cationic side groups. Mention may be made of polylysine, polyarginine, polyornithine and polyhistidine. The chain length may be from several units to a large number of units, for example from 3 to 20, from 10 to 50, from 50 to 100 or up to 500 or up to 1000 amino acids. Naturally occurring proteins having predominantly cationic properties, such as histones, may also be used.

(meth) acrylate copolymers are preferred over other materials with relatively little pharmacological experience because they have been safe for use in oral pharmaceuticals for decades. The cationic polymer may therefore preferably be a (meth) acrylate copolymer, in particular a (meth) acrylate copolymer having tertiary or quaternary amino groups. Cationic polymerThe glass transition temperature (ISO 11357-2, fine mesh 3.3.3) of the (meth) acrylate copolymer is preferably from 40 to 60 ℃ and the molecular weight M_w(weight average) of 100000 to 200000 (molecular weight M)_wCan be determined, for example, by gel permeation chromatography or by scattered light methods (see, for example, h.f. mark et al, Encyclopedia of polymer Science and Engineering, second edition, volume 10, pages 1 and beyond, j.wiley, 1989). For improving excretion via the kidney or biliary tract, it is preferred to have a low molecular weight M_wE.g. M of 50000 or less, 5000 to 40000, 10000 to 30000 or 15000 to 25000_wThe cationic (meth) acrylate copolymer of (1).

Molecular weight M_w(weight average) can be determined, for example, by viscometry or gel exclusion chromatography (GPC). The viscosity measurement (intrinsic viscosity) can be determined in chloroform or in DMF (dimethylformamide) at 23 ℃ and should preferably be from 10 to 20, preferably from 11 to 15n_spez/c(cubic centimeter per gram). The viscosity number may be measured, for example, as specified in ISO 1628-6.

Particularly preferred are (meth) acrylate copolymers composed of radically polymerized units of 20 to 30% by weight of methyl methacrylate, 20 to 30% by weight of butyl methacrylate and 60 to 40% by weight of dimethylaminoethyl methacrylate. The (meth) acrylate copolymers can be used in particular in micronized form with an average particle size of 10 to 30 μm. A particularly suitable commercially available (meth) acrylate copolymer having a tertiary amino group is, for example, one composed of 25% by weight of methyl methacrylate, 25% by weight of butyl methacrylate and 50% by weight of dimethylaminoethyl methacrylate (bE100) In that respect Micronized form having a mean particle size of 10 to 20 microns (E PO, powder) is particularly preferred. This form can be processed particularly well into nucleic acid-containing nanoparticles. In this case, significant distinctions are obtainedFavorable complex formation with nucleic acid molecules, which can contribute to increased transfection rates.

Sodium comprising a nucleic acid active and a cationic and anionic (meth) acrylate copolymer Rice granules

The transfection rate of the respective nucleic acids for the target cell type can be further optimized by additionally adding an anionic (meth) acrylate copolymer in a proportion of 0.1 to 40 wt.%, in particular 1 to 30 wt.%, particularly preferably 2 to 25 wt.%, based on the nucleic acid active ingredient and the cationic (meth) acrylate copolymer, in the preparation of the nanoparticles comprising the nucleic acid active ingredient and the cationic (meth) acrylate copolymer. The transfection rate of the nanoparticles must then be measured in an in vitro assay with a cell culture of the cell type of interest (where available) or with at least similar or similarly reactive cell types. In this way, the appropriate balance between the binding force of the nucleic acid in the complex and its release from the complex into living cells can be adjusted. If the binding caused by the cationic (meth) acrylate copolymer alone is initially too strong, such that the transfection rate of the nucleic acid is unsatisfactorily low, the addition of the anionic (meth) acrylate copolymer weakens the binding until the transfection rate reaches an optimum value specific for the nucleic acid used and for the target cell type. Formulations of this type have the advantage that both cationic and anionic (meth) acrylate copolymers are pharmacologically acceptable, so that only little or no side effects are to be expected.

Suitable anionic (meth) acrylate copolymers are preferably of the same type which can also be used for the outer coating, i.e.a (meth) acrylate copolymer having a content of anionic group-containing monomers of from 5 to 60% by weight (meth) acrylate copolymerTypes L, S, L100-55, FS). In many cases, a surprising increase in transfection rate can be achieved when using an anionic (meth) acrylate copolymer constructed as follows:

20 to 33 wt% of methacrylic acid and/or acrylic acid

5 to 30% by weight of methyl acrylate and

20 to 40% by weight of ethyl acrylate and

from above 10 to 30% by weight of butyl methacrylate and

when appropriate, the first and second electrodes are,

0 to 10% by weight of other monomers capable of vinyl copolymerization,

wherein the proportions of the monomers add up to 100% by weight,

with the proviso that the copolymer has a glass transition temperature (midpoint temperature T) in accordance with ISO 11357-2, fine 3.3.3_mg) Is from 55 to 70 ℃.

The above copolymers are composed in particular of free-radically polymerized units formed from:

20 to 33, preferably 25 to 32, particularly preferably 28 to 31,% by weight of methacrylic acid or acrylic acid, preferably methacrylic acid,

5 to 30, preferably 10 to 28, particularly preferably 15 to 25,% by weight of methyl acrylate,

20 to 40, preferably 25 to 35, particularly preferably 18 to 22,% by weight of ethyl acrylate, and

from more than 10 to 30, preferably from 15 to 25, particularly preferably from 18 to 22,% by weight of butyl methacrylate,

wherein the monomer composition is selected such that the glass transition temperature of the copolymer is from 55 to 70 ℃, preferably from 59 to 66, particularly preferably from 60 to 65 ℃.

To improve excretion via the kidney or biliary tract, anionic (meth) acrylate copolymers having a low molecular weight are preferred, for example, having M_wThose of 50000 or less, 5000 to 40000, 10000 to 30000 or 15000 to 25000 are preferable.

Molecular weight M_w(weight average) can be determined, for example, by viscometry or gel exclusion chromatography (GPC). The viscosity measurement (intrinsic viscosity) can be determined in chloroform or in DMF at 23 ℃ and should preferably be from 10 to 20, preferably from 11 to 15n_spez/c(cubic centimeter per gram). The viscosity number may be measured, for example, as specified in ISO 1628-6.

Anionic (meth) acrylate copolymers with low molecular weight are pH sensitive polymers which have cytotoxic properties only at high concentrations, or no cytotoxic properties at all, in the pH7.0 or slightly higher range, but which already have haemolytic or membrane-lytic action at low concentrations in vivo below pH 6.5. The polymers can be used in nanoparticles as modulators of the binding strength between the nucleic acid active ingredient and the cationic (meth) acrylate copolymer and at the same time have a favorable effect on the transfection rate. The proportion of anionic (meth) acrylate copolymers having a low molecular weight in the nanoparticles can be particularly helpful for the intracellular release of the nucleic acid active ingredient after uptake into the endosomes by its subsequent destabilization or lysis.

Anionic (meth) acrylate copolymers with low molecular weight for nano-encapsulation

In a preferred embodiment, M is of low molecular weight, e.g.50000 or less, 5000 to 40000, 10000 to 30000 or 15000 to 25000_wIs applied as a shell by nano-encapsulation onto nanoparticles comprising a nucleic acid active and a cationic polymer, preferably a cationic (meth) acrylate copolymer. The proportion of anionic (meth) acrylate copolymers having a low molecular weight on the nanoparticle surface can in particular contribute to the intracellular release of the nucleic acid active ingredient after uptake into the endosomes by its subsequent destabilization or lysis. Furthermore, the nucleic acid active ingredient is better protected internally from nucleolytic degradation so that more active ingredient can reach the target site.

Ratio of active ingredients

The proportion of nanoparticles in the matrix layer is preferably up to 40, in particular from 0.001 to 15 or from 0.05 to 5,% by weight, based on the content of polymer having mucoadhesive action. The proportion of nucleic acid active ingredient in the nanoparticles can be, for example, from 1 to 50, preferably from 2 to 25,% by weight.

Preparation of nanoparticles

The preparation of nanoparticles is known. Known methods are agglomeration, complex formation, emulsion precipitation, evaporation of the organic solvent fraction from a water-in-oil emulsion, thereby producing nanoparticles in the aqueous phase, and evaporation of the organic solvent fraction from an oil-in-water emulsion, thereby producing nanoparticles in the aqueous phase. Leong et al (1998) described the preparation of nanoparticles in Journal of Controlled Release 53, page 183-193 "DNA-polynucleotides as non-viral gene delivery vehicles (DNA polycationic nanospheres as non-viral gene delivery vehicles)". Roy et al (1999) described the preparation of nanoparticles in Nature Medicine, Vol.5, No.4, p.387 391, "organic gene delivery with nanoparticles-DNAnanoparticules protection in the membrane model of the peer alarm".

Nanocapsulation refers to boundary layer polymerization (see, e.g., Chouinard F. et al, Pharm Res., 1994, 11.6: 869-. Nanocapsules can be made by dispersing nanoparticles as insoluble complexes in an aqueous medium and emulsifying the dispersion in an organic solvent. The dispersion in an organic solvent comprises, for example, a (meth) acrylate copolymer. Upon evaporation of the organic solvent, the (meth) acrylate copolymer precipitates and forms a shell around the nanoparticles. Encapsulation of the nanoparticles is advantageous in that an additional protection of the complexed nucleic acid active ingredient is ensured during absorption by the intestinal cells and the liver.

Polymers with mucoadhesive effect

The matrix layer further comprises a polymer having mucoadhesive properties.Suitable polymers having mucoadhesive action are in particular chitosan (chitosan and derivatives, chitosan varieties), copolymers of (meth) acrylates consisting of 20 to 45% by weight of methyl methacrylate and 55 to 80% by weight of methacrylic acid, cellulose, especially methylcellulose, for example sodium carboxymethylcellulose (for example sodium carboxymethylcellulose)Or). (meth) acrylate copolymers are preferred over other materials with relatively little pharmacological experience because they have been safe for use in oral pharmaceuticals for decades.

The polymer having mucoadhesive action is selected such that it exhibits a water absorption of 10 to 750%, preferably 10 to 250%, particularly preferably 10 to 160% within 15 minutes based on the pH value at the beginning of dissolution of the outer coating ± 0.5, preferably ± 0.3 pH unit.

Measurement of mucoadhesive Properties

Suitable measurement methods for characterizing mucoadhesive properties are found in Hassan and Gallo (1990) (see Hassan E.E. and Gallo J.M., "A Simple Rheological method for the in Vitro evaluation of the Mucin-Polymer Bioadhesive bond Strength", Pharma Res.7(5), 491 (1990)). The method is based on the following assumptions: the viscosity (η, dynamic viscosity or viscosity coefficient) of the mixture of polymer and mucin is different from the sum of the viscosities of the components. Application relation eta_{Mixtures of polymers and mucins}＝η_Mucins+η_{Polymer and method of making same}+η_bWherein eta_bRepresenting the difference. Eta_bThe higher the mucoadhesive properties. The viscosities of the individual components were first measured using a rotational viscometer. An aqueous solution of mucoadhesive polymer at a concentration of 0.5% (w/w) and a solution of porcine gastric mucin at a concentration of 15% were used. Is composed ofMeasurement of the mucoadhesive Property eta_bMucin and polymer are measured separately and as a mixture thereof at the concentrations.

The polymer having a mucoadhesive effect is selected so that it exhibits a viscosity η of 150 to 1000, preferably 150 to 600 mPas as a range of. + -. 0.5, preferably. + -. 0.3 pH units based on the pH at which the outer coating starts to dissolve_bForm the mucoadhesion measured.

Hydration and water absorption

The hydration of the polymer is based on the affinity of the polymer to absorb water. The polymer swells due to this water absorption. This is related to the imbalance between the chemical potentials of water in the polymer and water in the surrounding medium. Due to the osmotic pressure of the polymer, water is absorbed until equilibrium has been reached between the internal and external phases. The polymer was then 100% hydrated. For polymers with low average molecular weight, this is then in the form of a solution. For polymers with higher molecular weights or crosslinked polymers, gels are produced. The water uptake until equilibrium has been established can, for example, be up to 10 times the intrinsic weight, corresponding to 1000% by weight of the polymer.

Measurement of percent Water absorption

The measurement of the percent water absorption is familiar to the person skilled in the art. For example, in Lehrbuchner pharmazeutischen technology (textbook for pharmaceutical technology)/Rudolf Voigt, Basel: verlag Chemie (chemical Press), 5 th full revision, 1984, page 151, 7.7.6 "Suitable methods are described in the section (absorbency) ". The method utilizes a so-called Enslin device in which a glass suction filter is in communication with a graduated pipette through a hose. The pipette is mounted precisely horizontally so that it is at the same height as the frit filter plate. 100% water absorption in the present case is defined as per 1 gram of toolThe mucoadhesive polymer absorbed 1 ml of water within 15 minutes.

The rapid protection of the active ingredient and the direct binding to the intestinal mucosa at the moment the outer coating starts to dissolve is ensured by the relatively rapid water absorption or hydration and the high degree of hydration. The binding of the active ingredient in the mucoadhesive matrix should be only low so that the active ingredient can be transferred directly from the intestinal mucosa into the body.

Control of substrate pH

For many mucoadhesive polymers, mucoadhesion is pH dependent. The pH in the matrix can be controlled specifically by the addition of acids, bases or buffer systems. The inner matrix may comprise, for example, chitosan as a mucoadhesive polymer used together with acetate buffer systems. For example, an acetate/sodium acetate buffer adjusted to a pH of 5.0 to 5.5 may be present as an additive in the matrix or applied to the core to which the matrix is applied. In this way, chitosan can also be used in combination with a film-forming coating which starts to dissolve at a higher pH, for example at pH6.0 to 8.0. The pH remains low in the microenvironment of the matrix despite the high ambient pH. The mucoadhesive properties of the polymer can thus be exploited in a pH range where there would otherwise be no mucoadhesion or no mucoadhesion to that extent. This has the following advantages: it is possible to achieve a certain protection against nucleases with a pH optimum in a higher pH range. The pH of the matrix can also be raised by adding a base and combined with a film-forming coating that dissolves at lower pH, applying the same principle in the opposite way.

Examples of suitable mucoadhesive polymer choices

The selection of suitable mucoadhesive polymers is based on their mucoadhesive properties and their water absorption capacity. The polymers should have at least eta in the respective pH ranges_bMucoadhesive action of 150 to 1000 mPas and water absorption of 10 to 750% in 15 minutesAnd (6) harvesting. The following table gives a list as an example.

Chitosan is suitable, for example, for the ambient pH region at pH5.5 (duodenum) or for another ambient pH region (ileum or colon), provided that the pH range of the matrix has been adjusted, for example by means of a buffer system, to a region of approximately pH 5.5.

The (meth) acrylate copolymers listed in the table are more suitable for the pH region of pH7.2 than for the pH region of about pH 5.5.

Sodium alginate is suitable for a pH region of about pH5.5, but not for pH 7.2.

Sodium carboxymethylcellulose and crosslinked polyacrylic acid are suitable over a wide pH range of 5.5 to 7.2.

Mucoadhesive polymers	Mucoadhesive action eta_b[mPa·s]At pH5.5	Mucoadhesive action eta_b[mPa·s]At pH7.2	H₂O absorption [%, in 15 min]At pH5.5	H₂O absorption [%, in 15 min]At pH6.0	H₂O absorption [%, in 15 min]At pH7.2
						Chitosan	220	0	140	320	320
(meth) acrylate ester copolymer^*	150	480	170	50	125
						Sodium alginate	580	0	40	50	50
Sodium carboxymethylcellulose	300	250	55	50	50
						Polyacrylic acids, crosslinked	350	340	50	25	25

^*Composed of 30% by weight of methyl methacrylate and 70% by weight of methyl(meth) acrylate copolymer of acrylic acid

External coating made of anionic (meth) acrylate copolymer

The outer coating, which is composed of an anionic polymer or copolymer, acts as a gastric resistant coating to protect the inner matrix layer from gastric fluid. The outer coating also serves to protect the active ingredient from the nucleolytic enzyme until the point at which the coating reaches the section of the intestine (duodenum, jejunum, ileum or colon) where it begins to dissolve. The outer coating is in this case particularly used for so-called "gastrointestinal targeting", i.e. the targeted release of the inner matrix layer at the intestinal segment determined by the pH value at which it prevails. In order not to hinder the transport of the inner matrix layer, the outer coated (meth) acrylate copolymer should show a possibly small or only low interaction with the active ingredient or mucoadhesive polymer of the inner matrix layer.

Suitable anionic polymers or copolymers are cellulose glycolates () Cellulose acetate phthalate (CAP, Cellulosi acetas, PhEur, cellulose acetate phthalate, NF, Ph,) Cellulose Acetate Succinate (CAS), cellulose acetate trimellitate (trimeliat) Cellulose (CAT), hydroxypropylmethylcellulose phthalate (HPMCP, HP50, HP55), hydroxypropylmethylcellulose acetate succinate (HPMCAS-LF, -MF, -HF), polyvinyl acetate phthalate (PVAP,) Vinyl acetate-vinylpyrrolidone copolymer (PVAc,VA64), vinyl acetate crotonic acid 9: 1 copolymer (VAC: CRA,VAC) and/or shellac. The polymers or copolymers can in many cases be formulated in a completely satisfactory manner in order to achieve pH-specific dissolution.

The outer film-forming coating particularly preferably consists essentially of a (meth) acrylate copolymer having a content of monomers containing anionic groups of from 5 to 60% by weight, which copolymer is optionally formulated with pharmaceutically customary auxiliaries, in particular plasticizers. The anionic (meth) acrylate copolymers mentioned within the scope of the invention lead, in comparison with the polymers mentioned at the outset, to a pH-specific setting which in many cases makes it possible to adjust a more precise and more reproducible dissolution pH. The operation and application are also generally considered to be less complex.

The externally coated (meth) acrylate copolymers preferably comprise 40 to 95 wt.%, preferably 45 to 90 wt.%, in particular 30 to wt.%, of free-radically polymerized acrylic or methacrylic C₁-to C₄-an alkyl ester and may comprise from 5 to 60 wt%, preferably from 8 to 40 wt%, especially from 20 to 35 wt% of an anionic group-containing (meth) acrylate monomer.

The proportions mentioned generally add up to 100% by weight. However, in addition, minor amounts of other monomers capable of vinyl copolymerization, such as hydroxyethyl methacrylate or hydroxyethyl acrylate, in the range of 0 to 10, such as 1 to 5 wt%, may be present without causing a loss or change in basic properties.

C of acrylic acid or methacrylic acid₁-to C₄Alkyl esters are, in particular, methyl methacrylate, ethyl methacrylate, butyl methacrylate, methyl acrylate, ethyl acrylate and butyl acrylate.

The anionic group-containing (meth) acrylate monomer may be, for example, acrylic acid, but is preferably methacrylic acid.

Also suitable are from 40 to 60% by weight of methacrylic acid and from 60 to 40% by weightAnionic (meth) acrylate copolymers of methyl methacrylate or of 60 to 40% by weight of ethyl acrylate (meth)L orL100-55 type). Glass transition temperatures (ISO 11357-2, fine 3.3.3) of this type are from 105 to 160 ℃ and molecular weights M_wFrom 100000 to 300000 (molecular weight M)_wCan be determined, for example, by gel permeation chromatography or by scattered light methods (see, for example, H.F. Mark et al, Encyclopedia of Polymer science and Engineering, second edition, volume 10, pages 1 and later, J.Wiley, 1989).

L is a copolymer of 50% by weight of methyl methacrylate and 50% by weight of methacrylic acid. Such (meth) acrylate copolymers are particularly suitable for dissolution in the pH range (jejunum) of about pH6.0 to 6.5.

L100-55 is a copolymer of 50% by weight of ethyl acrylate and 50% by weight of methacrylic acid.L30D-55 is an emulsion containing 30 wt%Dispersions of L100-55. Such (meth) acrylate copolymers are particularly suitable for dissolution in the pH range (duodenum) of about pH5.5 to 6.0.

Also suitable are anionic (meth) acrylates composed of 20 to 40% by weight of methacrylic acid and 80 to 60% by weight of methyl methacrylateCopolymer (A) and (B)Type S). Such (meth) acrylate copolymers are particularly suitable for dissolution in the pH range (jejunum or ileum) of about pH6.5 to 7.0. The glass transition temperature of this type is from 140 to 180 ℃ and the molecular weight M_w100000 to 150000.

Particularly suitable (meth) acrylate copolymers are those composed of 10 to 30% by weight of methyl methacrylate, 50 to 70% by weight of methyl acrylate and 5 to 15% by weight of methacrylic acid.

The FS type is a copolymer composed of 25 wt% of methyl methacrylate, 65 wt% of methyl acrylate and 10 wt% of methacrylic acid.FS 30D is a dispersion comprising 30 wt% FS type copolymer. Such (meth) acrylate copolymers are particularly suitable for dissolution in the pH range (ileum or colon) of about pH7.0 to 7.8.

Also suitable are copolymers composed of:

20 to 34 wt.% of methacrylic acid and/or acrylic acid

20 to 69% by weight of methyl acrylate and

0 to 40 wt.% of ethyl acrylate and/or, where appropriate

0 to 10% by weight of other monomers capable of vinyl copolymerization,

with the proviso that the copolymers according to ISO 11357-2, fine 3.3.3 have a glass transition temperature of at most 60 ℃. Such (meth) acrylate copolymers are particularly suitable for compressing pellets into tablets due to their good elongation at break properties.

Also suitable are copolymers composed of:

20 to 33 wt% of methacrylic acid and/or acrylic acid,

5 to 30% by weight of methyl acrylate and

20 to 40% by weight of ethyl acrylate and

from more than 10 to 30% by weight of butyl methacrylate and

when appropriate

0 to 10% by weight of other monomers capable of vinyl copolymerization,

wherein the proportions of the monomers add up to 100% by weight,

with the proviso that the glass transition temperature (midpoint temperature T) of the copolymers according to ISO 11357-2, fine 3.3.3_mg) Is from 55 to 70 ℃. Copolymers of this type are particularly suitable for compressing pellets into tablets because of their good mechanical properties.

The above copolymers are composed in particular of free-radically polymerized units of:

Mixtures of the mentioned copolymers can also be used to adjust a specific release profile or release site.

The glass transition temperature here is in particular the midpoint temperature T according to ISO 11357-2, fine 3.3.3_mg. The measurements were carried out without addition of plasticizer at a residual monomer content (REMO) of less than 100ppm, at a heating rate of 10 ℃/min and under a nitrogen atmosphere.

The copolymers are preferably composed substantially to completely, with 90, 95 or 99 to 100% by weight, of the monomers methacrylic acid, methyl acrylate, ethyl acrylate and butyl methacrylate in the abovementioned quantity ranges.

However, in case this does not necessarily lead to an impairment of the basic properties, small amounts of other monomers capable of vinyl copolymerization, such as methyl methacrylate, butyl acrylate, hydroxyethyl methacrylate, vinylpyrrolidone, vinylmalonic acid, styrene, vinyl alcohol, vinyl acetate and/or derivatives thereof, may also be present in the range from 0 to 10, for example from 1 to 5% by weight.

The copolymers are obtained in a manner known per se by free radical bulk polymerization, solution polymerization, bead polymerization or emulsion polymerization. They must be brought into the particle size range according to the invention by suitable grinding, drying or spraying processes before processing. This can be done by simple crushing, or thermal shock (abchlag), of the extruded and cooled granular strands.

The use of powders may be advantageous, especially in the case of mixing with other powders or liquids. Suitable apparatus for preparing powders: () Are familiar to the person skilled in the art, for example air jet mills, rod mills, fan mills. Where appropriate, corresponding sieving steps may be included. Mills suitable for industrial large scale are, for example, opposed jet mills operating at an overpressure of about 6 bar (Multi No. 4200).

Preparation of copolymers

All (meth) acrylate copolymers mentioned can be obtained by free-radical polymerization of monomers in the presence of polymerization initiators and molecular weight regulators by means of block, bead or emulsion polymerization and discharge of the polymerization product (see, for example, EP 0704207a2, EP 0704208a2 or WO 03/092732). The (meth) acrylate copolymers can be prepared in a manner known per se by free-radical emulsion polymerization in the aqueous phase in the presence of preferably anionic emulsifiers, for example as described in DE-C2135073. Other preparation methods which are suitable are in principle "group transfer polymerization" (GTP) or "atom transfer radical polymerization" (ATRP) (see, for example, Matyjaszews ki, K. et al, chem. Rev.2001, 101, 2921-. The resulting polymer structure is a random copolymer or a block copolymer.

Preference is given to emulsion polymerization in the presence of from 2 to 15% by weight of molecular weight regulator, from 0.1 to 2% by weight of emulsifier proportion, from 0.02 to 0.4% by weight of polymerization initiator amount and at temperatures of from 65 to 90 ℃. Preferred are emulsifier mixtures, preferably consisting of sodium lauryl sulfate (e.g. 0.1 to 0.5 wt%) and polyoxyethylene-20 sorbitan monooleate (e.g. 0.4 to 1.5 wt%). Particularly suitable initiators are sodium peroxodisulfate or ammonium peroxodisulfate. In this way, for example, dispersions having a solids content of from 20 to 40% by weight can be prepared and the copolymers can be obtained by spray drying or by coagulation of water in an extruder and extrusion under pressure. The polymer product is then dissolved, preferably in an organic solvent, purified by multiple dialysis against water, and preferably lyophilized.

Examples of polymerization initiators which may be mentioned are: azo compounds, such as 2, 2 '-azobis (isobutyronitrile) or 2, 2' -azobis (2, 4-dimethylvaleronitrile); redox systems, such as tertiary amines in combination with peroxides, or preferably peroxides (see for example h."Acryl-und Methacryl verbindungen", Springer, Heidelberg, 1967, or Kirk-Othmer, Encyclopedia oChemical Technology (encyclopedia of Chemical Technology), volume 1, page 386 and the latter pages, j.wiley, New York, 1978. Examples of suitable peroxide-type polymerization initiators are dilauroyl peroxide, tert-butyl peroctoate, tert-butyl perisononanoate, dicyclohexyl peroxydicarbonate, dibenzoyl peroxide or 2, 2-bis (tert-butylperoxy) butane.

The polymerization can also preferably be carried out with mixtures of various polymerization initiators having different half-lives, for example dilauroyl peroxide and 2, 2-bis (tert-butylperoxy) butane, in order to keep the radical flow constant during the course of the polymerization and at various polymerization temperatures. The polymerization initiators are generally used in amounts of from 0.01 up to 1% by weight, based on the monomer mixture.

The molecular weight M can be adjusted by polymerizing the monomer mixture in the presence of a molecular weight regulator_w. Suitable molecular weight regulators are, in particular, mercaptans, such as n-butyl mercaptan, n-dodecyl mercaptan, 2-mercaptoethanol or 2-ethylhexyl thioglycolate, where the molecular weight regulator is generally used in an amount of from 0.05 to 15% by weight, based on the monomer mixture, preferably in an amount of from 0.1 to 10% by weight, particularly preferably from 2 to 12% by weight, based on the monomer mixture (see, for example, H.Rauch-Puntigam, Th."Acryl-undMe thacrylverbindungen", Springer, Heidelberg, 1967; Houben-Weyl, Methoden der organischen Chemie (methods of organic chemistry), volume XIV/1, page 66, Georg Thieme, Heidelberg, 1961 or Kirk-Othmer, Encyclopedia of Chemical Technology, volume 1, pages 296 and beyond, J.Wiley, New York, 1978). The molecular weight regulators preferably used are n-dodecylmercaptan or 2-ethylhexyl thioglycolate. Ethylhexyl thioglycolate offers the advantage that the hydrophobicity of the (meth) acrylate copolymer can be influenced, since the modifier is introduced terminally into the molecule. Preferably, 5 to 15% by weight of dodecyl mercaptan or 2 to 10% by weight of 2-ethylhexyl thioglycolate are used.

Organic solution

The (meth) acrylate copolymers mentioned may be provided in the form of organic solutions, for example in concentrations of from 10 to 30% by weight. Useful solvents are, for example, acetone, isopropanol or ethanol or mixtures thereof, which, where appropriate, may contain water in proportions of up to about 10% by weight. However, aqueous dispersions are preferred.

Dispersion product

The (meth) acrylate copolymers mentioned can be prepared and used as emulsion polymers, preferably in the form of aqueous dispersions in concentrations of from 10 to 50% by weight, in particular from 20 to 40%. As a commercially available form, a solids content of 30% by weight is preferred. For processing, a partial neutralization of the methacrylic acid units is not necessary; however, it may be of the order of up to 5 or 10 mole percent, for example, if stabilization or thickening of the coating composition dispersion is desired. The latex particles generally have a weight average size of from 40 to 100 nm, preferably from 50 to 70 nm, ensuring a viscosity of less than 1000 mPas which is advantageous in terms of processing technology.

At higher degrees of neutralization, for example 10 to 50 mole%, or complete neutralization, the copolymer can be converted into the dissolved state.

To prepare solutions of anionic copolymers, it is generally necessary to partially or completely neutralize the acid groups. The anionic copolymer can be added to the water, for example, with gradual stirring at a final concentration of 1 to 40% by weight, and is partially or completely neutralized there by addition of basic substances, for example NaOH, KOH, ammonium hydroxide or organic bases, for example triethanolamine. It is also possible to use a powder of the copolymer, to which a base, for example NaOH, has been added during its preparation for the purpose of (partial) neutralization, so that the powder is an already (partially) neutralized polymer. The pH of the solution is typically above 4, for example from 4 to about 7.

The dispersions can also be spray-dried or lyophilized, for example in a manner known per se, and be provided in the form of redispersible powders (see, for example, EP-A0262326). Alternative methods are lyophilization or coagulation with water in an extruder and extrusion under pressure followed by granulation (see, for example, EP-a 0683028).

It has surprisingly been found that copolymer dispersions made from spray-dried or freeze-dried and redispersed powders exhibit improved shear stability. This is particularly advantageous in the case of spraying. This advantage manifests itself particularly strongly when the copolymer present in the dispersion is present in a partially neutralized form of from 2 to 10 mol%, based on the acid groups present in the copolymer. Partial neutralization by addition of NaOH is preferred for this purpose. The anionic emulsifier is preferably present in an amount of 0.1 to 2 wt%. Sodium lauryl sulfate is particularly preferred as emulsifier.

Layer thickness

The layer thickness of the outer coating is preferably from 20 to 200, preferably from 50 to 120 μm.

Preparation of multiparticulate pharmaceutical dosage forms

The invention also relates to a process for preparing a multiparticulate pharmaceutical dosage form in the following manner:

a) the nucleic acid active ingredient is formulated with the auxiliary materials in a manner known per se into nanoparticles,

b) the inner matrix layer is produced by spraying onto the core or by rotary agglomeration (roto-polymerization), precipitation or spraying in the absence of a core, and comprises the nucleic acid active ingredient in the form of nanoparticles and a polymer having mucoadhesive action, and, where appropriate, further pharmaceutically customary auxiliary substances, and is subsequently applied

c) Applying by spraying an external film-forming coating consisting essentially of an anionic polymer optionally formulated with usual pharmaceutical adjuvants, in particular plasticizers, to obtain coated pellets containing the active ingredient, and

d) the resulting pellets are processed with the aid of pharmaceutically customary auxiliaries and in a manner known per se into multiparticulate pharmaceutical dosage forms, in particular tablets, mini-tablets, capsules, sachets or powders for reconstitution containing pellets, which are formulated such that the contained pellets are released in the pH range of the stomach.

Preformed pellets and preparation of pellets

Granulation may be carried out on pellets without active ingredient (Nonpareilles), or coreless pellets may be prepared.

First, nanoparticles containing an active ingredient are prepared.

Subsequently, an inner matrix layer with or without a core is prepared. This not yet coated rounded layer may be referred to as a preformed pellet (pellet core).

The solution or suspension of mucoadhesive polymer containing nanoparticles of a nucleic acid-containing active ingredient can be applied by a fluidized bed process to a placebo pellet or other suitable carrier material where the solvent or suspending agent is evaporated off. This preparation process may be followed by a drying step.

The nucleic acid active ingredient is added to an organic solvent or to water in the form of nanoparticles containing a polymer having a mucoadhesive effect, and mixed. To ensure satisfactory sprayability of the mixture, it is generally necessary to formulate low-viscosity mixtures. It may be advantageous to use the polymers having mucoadhesive action at relatively low concentrations, for example from 1 up to 10, preferably from 2 to 5% by weight, for this purpose. Furthermore, it may be advantageous to add detergents, such as tween, at concentrations of 0.1 to 20, preferably 0.5 to 10 wt%, to reduce the surface tension.

In addition to the active ingredients, other pharmaceutical adjuvants may also be present: binders (e.g. cellulose and its derivatives), polyvinylpyrrolidone (PVP), humectants, disintegration-promoting agents, lubricants, disintegrants, (meth) acrylates, starches and their derivatives, sugars, solubilizers or others.

For example, by Bauer, Lehmann, Os terrald, Rothgang "Corresponding application methods are known from Arzneiformen (coated pharmaceutical dosage form) ", Wissenschaftliche Verlags-gesellschaft mbH Stuttgart, Chapter 7, p.165-196.

Further, the details are known to those skilled in the art from textbooks. See, for example:

-Voig t，R.(1984)：Lehrbuch der pharmazeutischenTechnologie；Verlag Chemie Weinheim-Beerfield Beach/Florida-Basel

-packer, h., Fuchs, p., Speiser, p.: pharmazeutische technology, Georg Thieme Verlag Stuttgart (1991), in particular chapters 15 and 16, pages 626-

Gennaro, A., R. (eds.), Remington's pharmaceutical sciences (Remington pharmaceutical sciences), Mack Publishing Co., Easton Pennsylvania (1985), Chapter 88, p. 1567-

List, P.H (1982): arzneiformenlehre (pharmaceutical dosage form guide), Wissenschaftliche Verlagsgesellschaft mbH, Stuttgart.

The inner matrix may also be prepared without the aid of an inert core (Nonpareilles). The composition of the inner matrix may in this case be rounded by methods such as rotary agglomeration, precipitation or spraying, especially ultrasonic fluidised spraying, into uncoated pellets (pre-formed pellets) of a specified size, for example 50 to 1000 microns. This has the advantage that the entire core volume is available for loading with active ingredient. The loading of the active ingredient can thus be further increased compared to embodiments with inert cores.

After the inner matrix cores (or pre-formed pellets) are made, they are again preferably provided with an outer coating in a spray process to obtain finished pellets. The pellets are prepared from an organic solution, or preferably from an aqueous dispersion, by spraying. In this case, it is decisive for the implementation to produce a uniform, non-porous coating.

Top coating

In addition, the pellets may be provided with a colored coating, but it must not affect the dissolution pH. Suitable examples are coatings consisting of coloured hydroxypropylmethylcellulose or other polymers which are soluble or rapidly disintegratable in water.

Common pharmaceutical auxiliary material

Usual adjuvants or additives can be added to the formulations of the invention during the preparation. Of course, in principle all substances used must be toxicologically unproblematic and, in particular, in pharmaceuticals, pose no risk to the patient.

The amounts and use of conventional additives in drug coatings or coatings are familiar to those skilled in the art. Possible examples of customary additives are plasticizers, release agents, pigments, stabilizers, antioxidants, pore formers, penetration enhancers, gloss agents, fragrances, detergents, lubricants or odorants. They act as processing aids and are intended to ensure a reliable and reproducible preparation process and good long-term storage stability, or they achieve additional advantageous properties in pharmaceutical dosage forms. They are added to the polymer formulation prior to processing and can affect the permeability of the coating, which can be used as an additional control parameter when appropriate.

● release agent:

the release agent is generally lipophilic in nature and is typically added to the spray suspension. They prevent the cores from agglomerating during the film coating process. Preference is given to using talc, magnesium or calcium stearate, ground silicon dioxide: () Kaolin or a non-ionic emulsifier having an HLB value of from 3 to 8. Release agents are generally used in the coating composition and binder of the invention in amounts of 0.5 to 100 wt.%, based on the copolymer.

● pigment:

pigments incompatible with the coating composition are in particular the following pigments: which, if added directly to the (meth) acrylate copolymer dispersion, for example by stirring, in the usual amounts of, for example, 20 to 400% by weight, based on the dry weight of the (meth) acrylate copolymer, can lead to destabilization of the dispersion, coagulation, segregation phenomena or similar undesirable effects. Furthermore, the pigments used are of course non-toxic and suitable for pharmaceutical purposes. See also, for example, Deutsche Forschungsgemeinschaft (german scientific research association), farbstofe fur lebensmitel (food dye), Harald Boldt verlagkgk, Boppard (1978); deutsche Lebensmittelrundschau 74, phase 4, page 156 (1978); arzneimitelfarbstoffverdnung AmFarbV, 25.8.1980.

Pigments incompatible with the coating composition may be, for example, alumina pigments. Examples of incompatible pigments are yellow orange, cochineal lake, coloured pigments based on alumina or azo dyes, sulphonic acid dyes, yellow orange S (E110, c.i.15985, FD & C yellow 6), indigo carmine (E132, c.i.73015, FD & C blue 2), tartrazine (E102, c.i.19140, FD & C yellow 5), ponceau 4R (E125, c.i.16255, FD & C cochineal a), quinoline yellow (E104, c.i.47005, FD & C yellow 10), erythrosine (E127, c.i.45430, FD & C red 3), azorubine (E122, c.i.14720, FD & C Carmoisine), amaranth (E123, c.i.85, FD & C red 2), brilliant acid green (E142, c.i.44090, FD & C green S).

The E numbers of the pigments given relate to the EU numbering. See also "Deutsche Forschungsgeseeinschaft, Farbstoffe fur Lebensmittel, Harald Boldt Verlag KG, Boppard (1978); deutsche lebensmittelrundschau 74, phase 4, page 156 (1978); arzneimitelfarbstoffverdnung AmFarbV, 25.8.1980. The FD & C number relates to food, drug and cosmetic approval given by the U.S. Food and Drug Administration (FDA), which is described in: the united states food and drug administration, food safety and application nutrition center, cosmetics and color office: code of Federal Regulations Association 21 Color Additive Regulations 82 Part, Listing of Certified Provisional Listed colors and Specifications (CFR 21 Part 82).

● plasticizer:

other additives may also be plasticizers. Customary amounts are from 0 to 50, preferably from 2 to 20, in particular from 5 to 10,% by weight.

Plasticizers can affect the functionality of the polymer layer depending on the type (lipophilic or hydrophilic) and the amount added. Plasticizers achieve a reduction in the glass transition temperature by physical interaction with the polymer and promote film formation depending on the amount added. Suitable substances generally have a molecular weight of from 100 to 20000 and contain one or more hydrophilic groups in the molecule, such as hydroxyl, ester or amino groups.

Examples of suitable plasticizers are alkyl citrates, glycerol esters, alkyl phthalates, alkyl sebacates, sucrose esters, sorbitan esters, diethyl sebacate, dibutyl sebacate and polyethylene glycols from 200 to 12000. Preferred plasticizers are triethyl citrate (TEC) and acetyl triethyl citrate (ATEC). Mention may also be made of esters which are normally liquid at room temperature, such as citrates, phthalates, sebacates or castor oil. Preferably, citric acid esters and sebacic acid esters are used.

The addition of the plasticizer to the formulation can be carried out in a known manner, directly in aqueous solution, or after thermal pretreatment of the mixture. Mixtures of plasticizers may also be used.

Preparation of multiparticulate pharmaceutical dosage forms

The coated pellets containing the active ingredient can be processed with the aid of customary pharmaceutical excipients in a manner known per se into multiparticulate pharmaceutical dosage forms, in particular into tablets, mini-tablets, capsules, sachets or powders for reconstitution containing the pellets, which can be formulated such that the contained pellets are released in the pH range of the stomach. The preparation as a multiparticulate pharmaceutical dosage form provides high dose reliability and provides the advantage of good distribution of the pellets in the intestinal lumen. The multiparticulate pharmaceutical dosage form of the invention may also comprise a variety of different pellet types having different active ingredients and/or different pellet structures.

Compressed tablet

The preparation of multiparticulate pharmaceutical dosage forms by compressing together pharmaceutically customary binders with particles containing the active ingredient is described, for example, in Beckert et al (1996), "Compression of enteric-coated pellets to discrete tablets", International Journal of pharmaceuticals 143, pages 13 to 23 and in WO 96/01624.

Film coatings of pellets containing the active ingredient are typically applied in a fluid bed apparatus. The film-forming agent is generally mixed with the plasticizer and release agent by suitable methods. In this case, the film former may be present as a solution or suspension. The auxiliary materials for film formation can likewise be dissolved or suspended. Organic or aqueous solvents or dispersants may be used. Stabilizers may additionally be used to stabilize the dispersion (e.g., Tween 80 or other suitable emulsifiers or stabilizers).

Examples of release agents are glycerol monostearate or other suitable fatty acid derivatives, silicic acid derivatives or talc. Examples of plasticizers are propylene glycol, phthalates, polyethylene glycol, sebacates or citrates, and also other substances mentioned in the literature.

A barrier layer may be applied between the active ingredient-containing layer and the enteric soluble copolymer layer, which serves to barrier the active ingredient from the coating material to prevent interaction. Such a layer may be composed of an inert film former (e.g. HPMC, HPC or (meth) acrylic copolymer) or e.g. talc or another suitable pharmaceutical substance. Combinations of film formers and talc or similar materials may also be used. It is also possible to apply a separating layer consisting of a partially or completely neutralized (meth) acrylate copolymer dispersion.

The separator layer may also be composed of the same or different mucoadhesive polymer as in the underlying matrix layer. Possible interactions or incompatibilities of the active ingredient or mucoadhesive polymer with the film-forming (meth) acrylate copolymer layer can be prevented in this way.

The mixture for preparing tablets consisting of coated granules is formulated by mixing the pellets with a binder suitable for tabletting, if necessary with a disintegration-promoting substance and if necessary with a lubricant. The mixing can be carried out in a suitable machine. Mixers which cause damage to the coated particles are not suitable, for example ploughshare mixers. In order to achieve a suitably short disintegration time, a specific order may be necessary when adding the auxiliary materials to the coated granules. By premixing the coated granules with the lubricant or the release agent magnesium stearate, the surfaces thereof can be hydrophobized and thus adhesion can be avoided.

Mixtures suitable for tableting typically comprise 3 to 15 wt% of a disintegration aid, such as KollidonCL, and for example 0.1 to 1 wt% of a lubricant and a release agent, such as magnesium stearate. The binder ratio is determined according to the desired ratio of the coated particles.

Typical binders are, for exampleMicrocrystalline cellulose, calcium phosphate,Lactose or other suitable sugars, calcium sulfate or starch derivatives. Low bulk density materials are preferred.

Typical disintegration aids (disintegrants) are cross-linked starch derivatives or cellulose derivatives, and cross-linked polyvinylpyrrolidone. Cellulose derivatives are likewise suitable. The use of a disintegration aid can be dispensed with by the choice of a suitable binder.

Typical lubricants and mold release agents are magnesium stearate or other suitable fatty acid salts or substances cited in the literature for this purpose (e.g. lauric acid, calcium stearate, talc, etc.). The use of lubricants and mold release agents in the mixture can be dispensed with by suitable machines (e.g. tablet presses with external lubrication) or suitable formulations.

If appropriate, auxiliaries for improving the flowability (e.g. highly disperse silicic acid derivatives, talc, etc.) may be added to the mixture.

The compression can be carried out on a conventional tablet press, an eccentric or rotary press, under a pressure of from 5 to 40kN, preferably from 10 to 20 kN. The tablet press may be equipped with a system for external lubrication. A special system for filling the mould is used when appropriate, which avoids the mould filling process by means of a stirring paddle.

Other multiparticulate pharmaceutical dosage forms

As an alternative to compressed tablets or mini-tablets, the coated pellets containing the active ingredient may also be processed into any other orally administrable multiparticulate pharmaceutical dosage form. The coated pellets may, for example, be filled into capsules, such as gelatin capsules, or formulated into sachets or powders for reconstitution.

Advantageous effects of the invention

The pharmaceutical dosage form of the invention is suitable for targeted and effective release of nucleic acid active ingredients. The pharmaceutical dosage forms exhibit high dose reliability and good distribution in the stomach and in the intestinal lumen. In this case, the contained nucleic acid active ingredients are largely protected against physical or nucleolytic inactivation and can be released at the site of action in such a way that a high proportion of the active ingredient can be absorbed by the body. The pharmaceutical dosage form thus also suffices to use small amounts of active ingredient, since only small amounts of active ingredient are lost. The risk of side effects is reduced overall by targeted delivery. The site of action can be variably adjusted depending on the purpose of treatment. Thus, the point in time of absorption of the active ingredient can be better controlled. Because it is an oral pharmaceutical dosage form, it is generally better accepted by patients (patient compliance) than other administration forms. Thus, a large number of nucleic acid active ingredients can be made available for oral use. The risk of administration is generally less than in the case of parenteral administration in particular. The cost of administration can also be kept low, since no skilled technicians are required for administration.

Lipophilic matrix

A particular aspect of the invention results when the active ingredient is embedded in the form of nanoparticles in a lipophilic matrix having a melting point above 37 ℃, preferably above 45 ℃, particularly preferably above 55 ℃ and the lipophilic matrix containing the active ingredient is embedded in a matrix consisting of a polymer having mucoadhesive action. The aim of the formulation in lipophilic matrices is to improve the solubility or bioavailability of active ingredients, preferably sparingly soluble or poorly soluble active ingredients (in the sense of DAB 10, 2003).

Lipophilic matrix means in the sense of the present invention a substance or a mixture of substances: the active ingredient may be dissolved, suspended or emulsified therein. The lipophilic matrix is different from conventional pharmaceutical adjuvants and polymers with mucoadhesive effect. The substance or substances of the lipophilic matrix preferably have hydrophobic or amphiphilic characteristics. Lipophilic matrices may also be referred to as amphiphilic matrices or lipid matrices.

The lipophilic matrix may be composed of a single substance (e.g., a lipid) or a mixture of substances (e.g., a lipid mixture). In the case of mixtures, the properties described below for the water solubility, partition coefficient and/or HLB value of DAB 10 are calculated in each case from the arithmetic mean of the parts by weight and the values of the substances in the mixture. The substances used must be non-toxic.

Lipophilic matrix/polymer with mucoadhesion effect

In a preferred embodiment, possible interactions of the lipophilic matrix with the polymer having mucoadhesive effect are considered. In order to avoid uncontrolled interactions, the substance or substances forming the lipophilic matrix and the polymer having mucoadhesive action should preferably have the same ionic properties, i.e. both should consistently have at least predominantly cationic character or consistently anionic character. In the selection of substances with counter-ionic properties, the polymers with mucoadhesive action should preferably be present in a neutralized form in a proportion of at least 50, particularly preferably 100%. Neutralization can be carried out by adding an acid or a base in a known manner.

One or more substances for formulating lipophilic matrices

The lipophilic base preferably comprises from 80 to 100, preferably from 90 to 100, particularly preferably 100,% by weight of a substance or substance mixture having an (average) HLB value of from 0 to 15, preferably from 2 to 10. The lipophilic base may contain from 0 to 20, preferably from 0 to 10,% by weight of customary pharmaceutical auxiliaries, in particular stabilizers, thickeners or adsorbents. It is particularly preferred that no pharmaceutically customary auxiliaries are present.

The lipophilic matrix-forming substance or substances may for example belong to the following group of substances: oils, fats, mono-, di-or triglycerides, fatty acids, fatty alcohols, especially C₆To C₂₀Fatty acid and/or C₆To C₂₀Alcohols, including their salts, ether, ester or amide derivatives, phospholipids, lecithins, emulsifiers, lipids, lipid-soluble vitamins or surfactants.

The lipophilic matrix may comprise, for example, one of the following lipid preparations: (Imwitor 308) glycerol monocaprylate with a monoester content of greater than 80%, glycerol monolaurate with a monoester content of greater than 90%, glycerol monostearate (stearate) (C) with a monoester content of greater than 90%₁₆+C₁₈) Ester, (Imwitor 900P) monoester content is 40-55% and C₁₈Glyceryl monostearate with content of 40-60%, monoester (Imwitor 900K) with content of 40-55%, and C₁₈Glyceryl monostearate with a content of 60-80%, medium chain length C with a monoester content of (Imwitor 742) 45-55%₈And C₁₀Glycerides, (Imwitor 928) contain predominantly C₁₂And saturated plant C with monoester content of 34-36%₁₀-C₁₈Partial glycerides of fatty acids, C₈And C₁₀Glyceryl esters, sodium caprylate or sodium caprate.

The lipophilic matrix may comprise, for example, one of the following lipid preparations:

fats, such as mono-, di-, tri-glycerides of saturated and unsaturated fatty acids and mixtures thereof. In particular glycerol stearate, glycerol palmitate, glycerol myristate, glycerol palmitate stearate, glycerol laurate, glycerol caprylate, glycerol oleate, examples of these esters being-308, -312, -491, -742, -900, -928, -988 and44/14, -50/13, Geleol, Compritol E ATO, Dynasan114, Softisan, Witepsol, Dynacet 212, coconut oil fat, oils such as castor oil, sesame oil, sunflower oil, cottonseed oil, corn oil, almond oil, peanut oil, olive oil, coconut oil, carrot oil, wheat germ oil, walnut oil, neutral oils such as isopropyl myristate, isopropyl palmitate, isopropyl stearate, medium chain triglycerides (r) (oil, oil)，

Short-chain aliphatic and aromatic carboxylic acid esters, such as dibutyl phthalate, diethyl sebacate, dibutyl sebacate, tributyl citrate, acetyltributyl citrate, triacetin,

waxes, such as carnauba wax, beeswax, wool wax,

glycerol is added to the mixture of (A) and (B),

fatty acid amides, such as stearamide, palmitamide, lauramide,

aliphatic long-chain carboxylic acids, such as stearic acid, palmitic acid, lauric acid, myristic acid, oleic acid, caprylic acid, linoleic acid, linolenic acid, and, for example, their Na, Al and Mg salts,

fatty alcohols, such as stearyl alcohol, lauryl alcohol, cetyl alcohol, myristyl alcohol, glycerol formal,

W/O emulsifiers, e.g. cholesterol, glycerol monostearate, ethylene glycol monostearate, sorbitan monooleate (S) ((S))80) Sorbitan monopalmitate (b)40) Sorbitan monolaurate (A)20) Sorbitan monostearate (C)60) Sorbitan trioleate (85) Sorbitan tristearate (C)65) Sorbitan sesquioleate (f)83) Calcium stearate, aluminum stearate, magnesium stearate, polyoxyethylene sorbitan tristearate: (65) Polyoxyethylene sorbitan trioleate (85)，

Non-ionic O/W emulsifiers, e.g. polyethylene glycol (Macrogol) stearate 400 (C:)A) Polyethylene glycol lauryl ether, polyethylene glycol-20-sorbitan-monolaurate, -monostearate, -monopalmitate, -monooleate, polyethylene glycol-1500-glycerol triricinoleate, polyethylene glycol glycerol hydroxystearate ((C))RH), polyethylene glycol-1000-glycerol-monolaurate, -monostearate, -monooleate, sucrose monostearate, Polysorbat 60: (60) Polyoxyethylene monostearate (Myrj49), Polysorbat 80 (I)80)、Polysorbat 40(40)、Polysorbat 20(20) Poloxamer (Poloxamer)407 (b)F127) Poloxamer 188 (b)F68) Polyoxyethylene ricinoleate (A), (B), (C) and (C)EL), polyoxyethylene-5-stearyl stearate,

ionic O/W emulsifiers, e.g.Cetyl stearyl sulfate salt (C: (C))E) Sodium lauryl sulfate: (Z), sodium glycocholate, hederagenin,

amphiphilic emulsifiers, such as egg phosphatidylcholine (egg lecithin), soy phosphatidylcholine (soy lecithin), betaine, sulfobetaine, ceramide (sphingomyelin),

vitamins, for example retinol (vitamin A), cholecalciferol (vitamin D), alpha-tocopherol and alpha-tocopherol acetate (vitamin E), phylloquinone (microorganism K),

other adjuvants are galactolipids, such as monogalactosyldiacylglycerol, trigalactosyldiacylglycerol, and perfume oils, such as anise oil, citronella oil, eucalyptus oil, fennel oil, chamomile oil, cardamom oil, pine needle oil, amaranth oil, dwarf pine oil, lavender oil, peppermint oil, nutmeg oil, clove oil, peppermint oil, rosemary oil, sage oil, and terpenes, such as menthol, linalool, 1, 4-cineole, pyrethrin, borneol, eucalyptol, phytol, dacryferol, Azadirachtin (Azadirachtin), Azadirachtin.

The content of the lipid matrix containing the active ingredient in the inner matrix layer a) may be 1 to 50, preferably 10 to 20 wt%.

The lipophilic base preferably comprises at least 50% by weight of glyceryl monocaprylate, up to 10% by weight of sodium cholate, up to 10% by weight of tocopherol succinate, with 1 to 5% by weight of efflux pump inhibitors, such as Solutol HS15, triglycerides, in particular tristearate, in the case where the active ingredient is a substrate for the PgP efflux pump, wherein these components add up to 100%. Such lipophilic matrix can be incorporated directly into the mucoadhesive polymer or in emulsified form in water. In the latter case, the aqueous phase may comprise a weak acid, such as citric acid.

Method of producing a composite material

The invention also relates to a method for producing a multiparticulate pharmaceutical dosage form having the following steps

a) Preparing a lipophilic matrix containing the active ingredient by suspending nanoparticles containing the nucleic acid active ingredient with the substance or substances forming the lipophilic matrix and, where appropriate, further pharmaceutically customary auxiliaries by intensive mixing or melting of the ingredients,

b) by spraying the mucoadhesive polymer mixed with a lipophilic matrix containing the active ingredient on the core or by rotary agglomeration, precipitation or spraying in the absence of a core, to prepare preformed pellets (cores for pellets),

c) preparing the pellets by spraying an anionic polymer or copolymer coating, which optionally may contain a blend of pharmaceutically customary auxiliaries, in particular a plasticizer and a release agent, from a dispersion or an organic solution on the preformed pellets from step b),

d) multiparticulate pharmaceutical dosage forms are prepared by filling or incorporating the pellets from step c), in a manner known per se, using, where appropriate, the usual excipients for pharmacy, in particular by processing into tablets, mini-tablets, capsules, sachets or powders for reconstitution containing the pellets.

Preferred method

The process steps a) and b) are preferably carried out as follows:

a) the inner matrix layer was prepared as follows: preparing an emulsion or suspension comprising nanoparticles of a nucleic acid active ingredient by vigorously mixing the ingredients in water, using the substance or substances of the lipophilic matrix and, where appropriate, further customary pharmaceutical adjuvants, and preparing an oil-in-water formulation having an average particle size of not more than 60, preferably not more than 20,. mu.m,

b) spraying the oil-in-water formulation from step a) onto mucoadhesive polymers, optionally containing other pharmaceutically usual adjuvant blends, thereby preparing pre-formed pellets, wherein these ingredients are present in the form of micronized powder produced by rotary agglomeration, extrusion or granulation, the powder for example having an average particle size of 10 to 100 microns.

Detailed Description

Examples

The examples illustrate exemplary embodiments of the invention.

Example 1

Preparation of nanoparticles comprising cationic (meth) acrylate copolymers

2 mg of DNA (nucleic acid active ingredient), for example, a gene therapy vector composed of a double-stranded plasmid DNA having, for example, 3000 to 10000 base pairs, which contains a gene to be expressed in human cells and having an intended therapeutic effect, was dissolved in 4 ml of a phosphate buffer solution having pH7.4, and mixed with 2 ml of a mouse monoclonal anti-human DNA IgM solution (1mg/ml) and incubated at 37 ℃ for 1 hour. Then 1 ml of Lipofectin was added^TMOr preferably 3 ml (1mg/ml) modificationE (a (meth) acrylate copolymer of 25% by weight of methyl methacrylate, 25% by weight of butyl methacrylate and 50% by weight of dimethylaminoethyl methacrylate, low molecular weight, kidney-permeable: ()M_w21000) and kept at 37 ℃ for about 30 minutes with slow stirring. After this time the pH was measured and adjusted to 7.4 with 0.001N HCl. After intensive mixing on, for example, a Vortex oscillator (Vortex), use is made ofWhile producing nanoparticles having an average diameter of about 250 nm, using a modificationE, nanoparticles with an average diameter of about 150nm are produced. The suspension of nanoparticles was purified by dialysis. The suspension may be further processed directly, or the nanoparticles may be isolated by lyophilization.

Example 2

Nanoparticles comprising cationic and anionic (meth) acrylate copolymers

In preliminary experiments with appropriate human cell cultures, it was found that when in cationE (modified) with addition of, for example, 10% of an anionic (meth) acrylate copolymerL (modified), an optimal transfection rate of the nucleic acid active ingredient can be achieved.

2 mg of DNA (nucleic acid active ingredient), for example, a gene therapy vector composed of a double-stranded plasmid DNA having, for example, 3000 to 10000 base pairs, which contains a gene to be expressed in human cells and has an intended therapeutic effect, is dissolved in 4 ml of a phosphate buffer solution of pH7.4, and mixed with 2 ml of a mouse monoclonal anti-human DNA IgM solution (1mg/ml) and cultured at 37 ℃ for 1 hour. Then 1.1 ml, 4 ml (1mg/ml) were modifiedE (a (meth) acrylate copolymer composed of 25% by weight of methyl methacrylate, 25% by weight of butyl methacrylate and 50% by weight of dimethylaminoethyl methacrylate,low molecular weight, kidney-passable M_wAbout 21000) and 0.4 ml (1mg/ml) modificationL (copolymer of 50% by weight of methyl methacrylate and 50% by weight of methacrylic acid, low molecular weight, kidney-permeable M)_w21000) and kept at 37 ℃ for about 30 minutes with slow stirring. After vigorous mixing on, for example, a vortex oscillator, nanoparticles with an average diameter of about 250 nm are produced. The suspension of nanoparticles was purified by dialysis. The suspension may be further processed directly, or the nanoparticles may be isolated by lyophilization.

Example 3

Surface-modified nanoparticles comprising a cationic (meth) acrylate copolymer and having an anion Nanoparticles of ionic (meth) acrylate copolymer shell

2 mg of DNA (nucleic acid active ingredient), for example, a gene therapy vector composed of a double-stranded plasmid DNA having, for example, 3000 to 10000 base pairs, which contains a gene to be expressed in human cells and has the intended therapeutic effect, was dissolved in 4 ml of Dulbecco's phosphate buffer solution of pH7.4, and mixed with 2 ml of a mouse monoclonal anti-human DNA IgM solution (1mg/ml) and cultured at 37 ℃ for 1 hour. Then 4 ml (1mg/ml) was modifiedE (a (meth) acrylate copolymer of 25% by weight of methyl methacrylate, 25% by weight of butyl methacrylate and 50% by weight of dimethylaminoethyl methacrylate, low molecular weight, kidney-passable M_w21000) and kept at 37 ℃ for about 30 minutes with slow stirring. After this time the pH was measured and adjusted to 7.4 with 0.001N HCl.

Mixing in by modificationL (a (meth) acrylate copolymer of 50% by weight of methyl methacrylate and 50% by weight of methacrylic acid, low molecular weight, kidney-permeable, M_wAbout 21000) in phosphate buffer (ph7.4, 0.5 mg/ml) and the resulting latex-like buffered dispersion was brought up to ph5.0 by adding 0.001M citric acid. The suspension of nanoparticles was purified by dialysis. The suspension may be further processed directly, or the coated nanoparticles may be isolated by lyophilization.

Example 4

By embedding the nanoparticles from example 1, 2 or 3 into chitosan and acid Adjusting to pH 5.0-5.5 to prepare mucoadhesive uncoated inner matrix layer Pill (prefabricated pill)

Preparation of mucoadhesive solution:

4 g of chitosan acetate was dissolved in 20 g of water. Then 2 g of citric acid monohydrate were added under rapid stirring. A pH value of 5.2 was set. Then, 0.4 g of sodium dodecanoate was added to the resulting clear, yellowish viscous solution. The suspension from example 1, 2 or 3 was mixed into this solution with slow stirring.

Preparation of preformed pellets

The mixed suspension was sprayed onto 40 g of neutral pellets having a diameter of about 400-600 μm at an inlet air temperature of 30 ℃ using a fluidized bed apparatus (Micro-Lab from Huttling) at a spray rate of 5-8 g/min/kg. The inlet air is in this case adjusted to 35-45 m/h. The yield in this case is 85-90%.

Example 5

Preparation of (coated) pellets

In the flowUsed in chemical bed methodL12.5 (a (meth) acrylate copolymer of 50% by weight of methyl methacrylate and 50% by weight of methacrylic acid, M_wAbout 200000, 12.5% strength organic solution in isopropanol/acetone 3: 2) the preformed pellets made according to example 4 were coated. The polymer coating amount was 40 wt% based on the weight of the core. The coating suspension consisted of:

L 12.55 3.3％

1.33 percent of triethyl citrate

Isopropanol 38.3%

2.0 percent of slip

5.0 percent of water

A uniformly coated gastric resistant pellet is obtained, the coating of which dissolves rapidly in the duodenum or jejunum above ph6.0 and releases the mucoadhesive preformed pellet.

Example 6

Preparation of multiparticulate pharmaceutical formulations in capsule form

The pellets prepared as in example 5 were directly filled into hard gelatin capsules, size 0 capsules, using a capsule filling apparatus to produce unit dosage forms having a fill weight of 550 milligrams. After oral administration, the capsules rapidly dissolve in the pH range of the stomach and release the pellets, which are already uniformly distributed in the stomach.

Example 7

Multiparticulate pharmaceutical in tablet formPreparation of pharmaceutical dosage forms

Pellets prepared as in example 5 were formulated with tableting aid, binder, disintegration promoter and lubricant. 550 grams of pellets were mixed with 390 grams of microcrystalline cellulose, 150 grams of sodium carboxymethyl starch, and 10 grams of magnesium stearate. The mixture was compressed in a tablet press to give small compacts weighing 1100 mg. After oral administration, the tablets disintegrate within the pH range of the stomach and release the pellets, which are already homogeneously distributed in the stomach.

Claims

1. An oral multiparticulate pharmaceutical dosage form comprising pellets having an average diameter of from 50 to 2500 microns, the pellets consisting essentially of:

a) an inner matrix layer comprising nanoparticles which contain a nucleic acid active ingredient and are embedded in a matrix consisting of a mucoadhesive polymer which is chitosan, a (meth) acrylate copolymer consisting of 20-40% by weight of methyl methacrylate and 60 to 80% by weight of methacrylic acid, and/or cellulose, crosslinked and/or uncrosslinked polyacrylic acid, lectin, sodium alginate and/or pectin, wherein the matrix optionally comprises further pharmaceutically customary auxiliaries,

b) an external film-forming coating consisting essentially of an anionic polymer or copolymer, optionally formulated with pharmaceutically customary auxiliaries, which is cellulose glycolate, cellulose acetate phthalate, cellulose acetate succinate, cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, vinyl acetate-vinylpyrrolidone copolymer, vinyl acetate-crotonic acid 9: 1 copolymer and/or shellac, or of a (meth) acrylate copolymer having a monomer content of anionic groups of from 5 to 60% by weight,

it is characterized in that the preparation method is characterized in that,

formulating the multiparticulate pharmaceutical dosage form such that the contained pellets are released in the pH range of the stomach, adjusting the outer coating by selection of the formulation of the anionic polymer or copolymer or with the auxiliary material and its layer thickness such that the coating dissolves in the intestine within 15 to 60 minutes at a pH range of 4.0 to 8.0, thereby exposing the mucoadhesive matrix layer containing the active ingredient, and which layer is capable of engaging the intestinal mucosa and releasing the active ingredient there, wherein the polymer having mucoadhesive action is selected such that it exhibits at least eta in a range of + -0.5 pH units based on the pH at which the outer coating starts to dissolve_bA mucoadhesive effect of 150 to 1000mPa · s and a water absorption of 10 to 750% within 15 minutes, and the active ingredient content of the nanoparticles in the matrix layer is at most 40 wt% of the content of the polymer having mucoadhesive effect.

2. Pharmaceutical dosage form according to claim 1, characterised in that the pharmaceutically usual auxiliary material in b) is a plasticiser.

3. Pharmaceutical dosage form according to claim 1, characterised in that the nanoparticles have a size of 20 to 1000 nm.

4. Pharmaceutical dosage form according to any one of claims 1 to 3, characterised in that the nucleic acid contained in the nanoparticles is present in the form of a complex with a cationic substance.

5. Pharmaceutical dosage form according to claim 4, characterised in that the cationic substance is a cationic lipid, a cationic polypeptide and/or a cationic polymer.

6. Pharmaceutical dosage form according to claim 5, characterised in that the cationic polymer is a (meth) acrylate copolymer having tertiary or quaternary amino groups.

7. Pharmaceutical dosage form according to claim 6, characterized in that the (meth) acrylate copolymer consists of free-radically polymerized units of 20 to 30% by weight of methyl methacrylate, 20 to 30% by weight of butyl methacrylate and 60 to 40% by weight of dimethylaminoethyl methacrylate.

8. Pharmaceutical dosage form according to claim 4, characterised in that the nucleic acid active ingredient contained in the nanoparticles is present in the form of a complex with a cationic and anionic (meth) acrylate copolymer.

9. Pharmaceutical dosage form according to claim 8, characterised in that the anionic (meth) acrylate copolymer contains 5 to 60 wt.% of anionic group-containing monomers.

10. Pharmaceutical dosage form according to claim 8, characterized in that an anionic (meth) acrylate copolymer is present, which is composed of:

20 to 33 wt% of methacrylic acid and/or acrylic acid,

5 to 30% by weight of methyl acrylate and

20 to 40% by weight of ethyl acrylate and

from more than 10 to 30% by weight of butyl methacrylate and

is unnecessary

0 to 10% by weight of other monomers capable of vinyl copolymerization,

wherein the proportion of monomers amounts to 100% by weight, with the proviso that the copolymer has a glass transition temperature according to ISO 11357-2, fine 3.3.3, i.e.a mid-point temperature T_mgAnd is from 55 to 70 ℃.

11. Pharmaceutical dosage form according to claim 4, characterised in that the nanoparticles comprise an average molecular weight M_wA cationic or anionic (meth) acrylate copolymer of 50000 or less.

12. Pharmaceutical dosage form according to any one of claims 1 to 3, characterised in that the nanoparticles have a mean molecular weight M_wEncapsulation of anionic (meth) acrylate copolymers of 50000 or less.

13. Pharmaceutical dosage form according to any one of claims 1 to 3, characterised in that the nucleic acid active ingredient is a single-or double-stranded DNA or RNA or DNA-RNA chimera in which naturally occurring and/or non-naturally occurring synthetic modified nucleotides may be present.

14. Pharmaceutical dosage form according to any one of claims 1 to 3, characterised in that the nucleic acid is present in the form of a complex with an antibody and a cationic substance, the antibody specifically binding to the nucleic acid.

15. Pharmaceutical dosage form according to any one of claims 1 to 3, characterised in that the layer thickness of the outer coating is from 20 to 200 μm.

16. Pharmaceutical dosage form according to any one of claims 1 to 3, characterised in that the inner matrix comprises C₁₀To C₂₀Fatty acid and/or C₁₀To C₂₀Alcohols, including theirSalt, ether, ester or amide derivatives, and/or lipids and/or lipid-soluble vitamins and/or penetration enhancers.

17. Pharmaceutical dosage form according to claim 16, characterised in that the lipid is a phospholipid.

18. Pharmaceutical dosage form according to any of claims 1 to 3, characterised in that the mucoadhesive polymer is sodium carboxymethylcellulose.

19. Pharmaceutical dosage form according to claim 1, characterised in that the inner matrix comprises chitosan as mucoadhesive polymer together with an acid or buffer system present in or on the matrix or in or on the core on which the matrix is coated.

20. Pharmaceutical dosage form according to claim 19, characterised in that the inner matrix layer comprises chitosan and is adjusted to a pH of 5.0 to 5.5 with an acid or buffer system and combined with an outer filmogenic coating which starts to dissolve in the range of pH6.0 to 8.0.

21. Pharmaceutical dosage form according to any of claims 1 to 3, characterised in that a separating layer is applied between the active ingredient-containing matrix layer and the outer film-forming coating layer.

22. A process for the preparation of a multiparticulate pharmaceutical dosage form according to any one of claims 1 to 21, by the steps of:

a) the nucleic acid active ingredient is formulated together with adjuvants into nanoparticles in a manner known per se,

b) preparing an inner matrix layer comprising the nucleic acid active ingredient in the form of nanoparticles and a mucoadhesive polymer, optionally together with other pharmaceutically customary auxiliaries, by spraying onto the core or, in the absence of the core, by rotary agglomeration, precipitation or spraying, to form a preformed pellet, and subsequently

c) Applying an outer film-forming coating onto the preformed pellets by spraying, wherein the coating consists essentially of an anionic polymer optionally formulated with pharmaceutically customary auxiliaries, to obtain coated pellets containing the active ingredient, and

d) the resulting pellets are processed with the aid of customary pharmaceutical excipients and in a manner known per se into multiparticulate pharmaceutical dosage forms which are formulated such that the contained pellets are released in the pH range of the stomach.

23. The method according to claim 22, wherein the pharmaceutically acceptable excipient in step c) is a plasticizer.

24. The method according to claim 22, wherein the multiparticulate pharmaceutical dosage form is a tablet comprising pellets, a sachet or a powder for reconstitution.

25. The method of claim 24, wherein the tablet is a miniature tablet.

26. The method of claim 22, wherein the multiparticulate pharmaceutical dosage form is a capsule containing pellets.