DE19839515A1 - Association of active agent with colloidal polymer, preferably new polymeric branched polyol ester, useful for controlled transmucosal administration of e.g. peptide, DNA construct or vaccine - Google Patents

Abstract

A pharmaceutical composition contains at least one colloidal polymer-active agent association (A). An Independent claim is included for novel polymers (I) which are branched polyol esters consisting of a central molecule (II) to which short-chain, biodegradable hydroxycarboxylic acid ester groups (III) are attached. The reaction parameters (i.e. natural and amount of (II) and catalyst system, nature and length of (III), reaction temperature and reaction time) are selected to optimize (I) for use as the polymer component of (A).

Description

The invention relates to novel pharmaceutical formulations gene, in colloidal form, consisting of at least one biocompatible and biodegradable polymer in combination with at least one active substance, its place of action, type of action and drug-adapted production, and their use, especially for mucosal drug delivery.  

[State of the art]

In modern pharmacotherapy, drug formulations are used genes and combinations of active substances that Bring the active ingredient (s) into an applicable form and thereby especially the drug stability, its biodistribution, Affect bioavailability and / or absorption. Through great advances in molecular biology, gene and Biotechnology is becoming an ever increasing number pharmacologically active substances, such as. B. proteins, Glycoproteins, peptides, oligonucleotides and DNA Kon structures and growth factors, accessible. However the human or veterinary use of these Substances without suitable carrier systems by numerous unwanted side effects aggravated or even partially not possible.

In particular for vaccines, such as. B. tetanus and diphtheria Toxoid, as well as viral antigens, are currently lacking suitable ones Carrier systems that are simple, cheap, for the patient more pleasant and especially in terms of accessibility from patients, e.g. B. in the 3rd world, reliable immunization enable.

Experiments described in the literature show that parenteral len biopolymer depot or retardation formulations in An implant or microparticle shape is a basic one Improvement is achievable, so far, however, remain clear Successes for these systems, not least due to the Lack of suitable biopolymer carriers can be attributed.

That is why in many places another, more targeted way became for the administration of pharmacologically relevant effects fabrics trod. It is already known from the literature that particulate systems when applied to mucous membranes from specific structures of the mucosal immune system (MALT:  mucosa associated lymphoid tissues) in the best case can be absorbed. So far, however, only came Systems for use in which the observed transport phase nomene indicates largely non-specific absorption interpret. However, this indicates that a particle depending on size.

First attempts described in the literature, this way too pharmacologically active, low molecular weight substances with such colloidal systems are associated in the body transport already showed promising results nits.

In contrast, macromolecular, mostly hydrophilic active substances not yet accessible to these invasion procedures, what especially due to the lack of suitable carrier polymers as well to the previously known, too "aggressive" particle manufacturers technologies.

Problems with the association of mostly hydrophilic, macromo lecular, pharmacologically active substances with polymers or particulate systems, on the one hand, often lie in theirs too little stability compared to the usual manufacturing have z. B. too aggressive polymerization processes or Dispersing processes such as high pressure homogenization or ultra sound dispersion, or prevail in these processes the conditions (temperature increase, contact with lipophilic Solvents, etc).

However, there are also further problems in particular so far insufficient compatibility and interaction between Carrier (polymer) and active ingredient. Biocompatible polymers have been developed already for use in slow release formulations gene (depot systems) proposed claims or execution examples in the sense of the present invention are missing so far. Especially the conscious adaptation of all components, thus in particular carrier polymer (s), active ingredient (s), applicate onsart (s) and location (s), etc., to the special needs Such applications have so far not been targeted.

OBJECT OF THE INVENTION

It is therefore an object of the present invention to provide pharmaceutical formulations which consist of at least one biocompatible and biodegradable polymer (component 1) and at least one active ingredient (component 2) and as colloidal systems (ie particulate in the micron and submicron size range ) are available, which are distinguished from known carrier systems by

• 1. the ability to be the most macromolecular, hydrophilic Active ingredients in biologically active form without prejudice to as sociate.
• 2. the ability to associate the ones mentioned under 1. if required, controllable to the therapeutically sensible goal place to lead.
• 3. the ability according to 1. and 2. that this destination of mucosal destination is.
• 4. Select a smaller number of necessary procedural steps manufacturing.
• 5. Biocompatibility and biodegradability of the carrier polymer.
• 6. a lower toxic effect on the surrounding tissue.
• 7. a sufficiently long dwell time, according to 3. on the mucosa len surfaces.
• 8. an active substance enrichment, according to 3. on mucosal surface surfaces.
• 9. A possibly required sufficient cellular Admission.
• 10. according to 3., the ability to induce an immune response, if the relevant drug is an antigen for immunization tion is.
• 11. According to 10., the ability to induce an immunant word if the relevant drug is a DNA construct.

It is therefore another object of the present invention gentle process for the production of such pharmaceutical  to provide preparations which meet the requirements of correspond to one or more of the sub-aspects 1 to 11, and for their production at least one carrier polymer (com component 1) and at least one relevant biologically active Active ingredient (component 2), alone or in physical or chemical form may be associated with colloidal Carrier system should be united.

To this end, it is a further object of the present invention necessary to provide suitable carrier polymers that correspond to the tasks / aspects mentioned above.  

Solution of the Invention

Finding suitable carrier polymers is according to the previous lying invention solved by branched polyol esters, which consists of a central molecule (charge modified and uncharged polyols), to the short-chain, biodegradable Hy droxycarboxylic acid ester groups are appended.

It has surprisingly been found that the adjustable amphiphilic character, i.e. the balance of hydro phobic, hydrophilic and possibly loaded stock share, thus also the carrier polymers according to the invention is unexpectedly suited to this with suffices associated quantities of relevant active ingredients, and these associates then in colloidal form z. B. to, on and to carry through cell barriers.

On the properties of the polymers, which surprisingly turned out to be in addition to the amphi among other things, their solubility. she can be used for the wide range from water solubility to adjust towards solubility in organic solvents, the preferred range according to the invention being toxicological contains harmless solvents: from water to Alcohols (such as ethanol) and esters (such as ethyl acetate) to ketones (such as acetone), as well as their combination nations.

The solubility in water was beyond that not only in the range from good to bad set soluble, but it became one, depending adjustable overall polymer composition separation temperature (LCST) found.

Carrier systems according to the invention consist of at least one biodegradable and biocompatible polyol ester and at least one relevant active ingredient (ie components 1 and 2). The properties of the carrier polymers (component 1) can be synthetically optimized both with regard to their transport sport behavior in vivo and with regard to their compatibility with or their interaction with active substances. Furthermore, these properties also allow the selection of the gentle manufacturing technology to be used for the carrier system according to the invention (according to the second task mentioned).

• A) In the case of defined solubility of the Polymers in water, is the manufacturing technology for pharmaceutical formulations according to part Task 2 in that the manufacture of the Invention contemporary carrier system by surprisingly found spontaneous in-situ training by him too colloidal polymer active ingredient according to the invention Complexation occurs when a solution A with component B is united. Solution A must be a fiction contain appropriate polymer and component B is little at least one relevant active ingredient or contains it in chemically bound or physically associated Shape. Component B can be a) constituent itself of solution A or b) be a second solution.
• B) Is, e.g. B. for reasons of compatibility with the active ingredient, a higher lipophilicity of the polymers is desired the solubility of the polymers is defined as soluble in non-toxic organic solvents or also solvent mixtures, such as. B. esters, ethers, Alcohols, ketones discontinued and then fiction as a modified manufacturing technology Preferred solvent deposition methodology, in which invent carrier systems according to the invention simultaneously (in-situ) or in a subsequent step adsorptive or che mix with at least one relevant active ingredient asso be adorned.

Component 1 Polymers and their production

The thus as a component of the carrier according to the invention serving branched polyol ester consisting of a Central molecule (modified and unmodified polyols), the through the targeted synthetic attachment of short-chain bioab Buildable hydroxycarboxylic acid ester groupings property-specific is differentiated and property-optimized are preferred produced by so-called melt grafting.

Melt grafting preferred according to the invention denotes here the reaction of one or more hydroxycarboxylic acid (s) dimeric or lactone form (hereinafter also referred to as monomers designated) above one of their melting points in the presence of the central molecule using a ring opening Polymerization suitable catalyst.

Catalysts can all be used for ring opening polymerization suitable substances, but non-toxic are preferred and by z. B. FDA approved catalysts such as tin and its salts, zinc and its salts and hydrides such as e.g. B. Calcium hydride.

Peculiarity of this reaction to the targeted setting of the Properties of the carrier polymers according to the invention is thereby on the one hand, that the central polyols are defined in sufficient dimensions with melt grafting preferred according to the invention must dissolve in the melt of the monomers. Then through suitable selection of the synthesis conditions (quantitative ratio se, reaction temperature and reaction time, choice of central polyols, etc.) the required properties of the central polyols specifically manipulated and tailored to the requirements of the Carrier system can be tailored.

Central polyols are therefore preferred:

• a) linear synthetic polymers with <6 to 500 hydroxyl groups, especially polyvinyl alcohols (PVA) with polyme degrees of risk from <6 to 500 (and degrees of saponification from  70 to 98%, if by hydrolysis of poly (vinyl acetate) receive);
• b) linear synthetic vinyl copolymers, especially the Polyvinyl alcohol, with <6 to 500 hydroxyl groups, esp especially with vinyl acetate, vinyl pyrrolidones, vinyl amines, Vinylimidazoles, vinylpyridines, vinylsulfonic acids, Vinyl phosphoric acids.
• c) linear, branched or cyclic, charged or ungela their polysaccharides, especially starches or stocks parts, glycogens, celluloses or components, dextrans, Tunicine, Inuline, Chitine, Alginate, Pectine, Mannane, Galactans, xylans and other polyoses, chondroitin sulfa te, heparin, hyaluronic acid and other glycosaminoglycans, Mureine, dextrins, cyclodextrins, chitosans, and esp especially their partially hydrophobicized derivatives, where Hydro phobization by methyl and / or ethyl ether, ester and / or urethane linkage is reached.
• d) In particular the polyols listed above, with the particularity that they additionally contain one or more carboxyl, sulfobutyl or sulfopropyl group (s) and / or butylamine, propylamine or ethylamine (primary, secondary, tertiary or quaternary Amine, where corresponding N substituents, in particular —H, —CH ₃ and / or —C ₂ H ₅ - represent group (s)) group (s) which are in the form of ether, ester or urethane Link to the polyol are bound.

In the case of polyols, the charged and / or protonatable and / or deprotonable groups, including their alkali metal or halogen salts, in particular their sodium or Chloride salts.

Because of the preferred central polyols according to the invention if polysaccharides belong, a must sufficient solubility and stability in the melt Monomers can be guaranteed. For non-cargo modified  Polysaccharides this z. B. ge known from the literature targeted partial acylation achieved.

In the case of charge-modified polysaccharides, the additionally introduced hydrophil is overcompensated by suitable aliphatic lipophilic spacer chain lengths, owing to the charge-bearing and / or (de) protonatable group (s). This is e.g. B. as already explained above by sulfobutyl or sulfopropyl ether, ester or urethane groups and / or butylamine or propylamine (primary, secondary, tertiary and / or quaternary amine, preference being given to corresponding N substituents - H, -CH ₃ and / or -C ₂ H ₅ group represent) ether, ester or urethane groups reached.

Only if this solubility of the central polyols is guaranteed is, z. B. about synthesis amounts, choice and amount of the catalyst (s), reaction temperature (s) and time (s) the properties of the invention required Carrier polymers (component 1) are adequately adjusted, e.g. B. over the length of the hydroxycarboxylic acid groups. On Criterion that z. B. with known spectroscopic Analysis methods - such as NMR spectroscopy - slightly above can be checked.

Peculiarity of this reaction to the targeted setting of the Properties of the carrier polymers according to the invention is white terhin that only the combination of a suitable central polyol or its properties and the choice of the monomer (s), as well as the reaction conditions with regard to later Active ingredient (s) whose association with the polymer Carrier and its / their gentle production and the sen / their in vivo transport behavior to a success speaking carrier system leads.

Those preferred for the property adaptation of the central polyols Hydroxycarboxylic ester groups are preferred by  Reaction products of dimers / anhydrides / lactones from Hy droxycarboxylic acids (monomers). Everyone comes here those in question, their polymerization products as biological apply safely. According to the invention, L- Lactide, D, L-Lactid and Glycolid as these easily increase in vivo Lactic and glycolic acid can be metabolized.

It has surprisingly turned out, for example, that in particular ratios of polyol-OH group to acid building unit in the range from 0.6: 1 to 6: 1, in particular in the range from 1: 1 to 3: 1 to form water-soluble carrier po lymeren performs the cases outlined in the examples also a lower critical separation temperature in the Own watery.

Furthermore, it has been found that - if the hydroxy carboxylic acid groups on the central polyol each from 1 to 100, and in particular 5 to 50 building blocks are - a solubility in e.g. B. acetone results.

In the literature, polyol esters are based on a Polyols with poly (hydroxycarboxylic acids) side chains for bio medical applications proposed. Targeted studies on synthetic adjustment of the polymer properties and particular the adjustment of the polymer properties to the there are no special needs for the claims according to the invention but completely.

An example of this literature finding should therefore be given called this point: EP 0 407 617 describes reaction products from poly (hydroxycarboxylic acid esters) and saccharides, DE 34 30 852 A1 and DE 40 21 517 A1 likewise describe polymers Poly (hydroxycarboxylic acid esters) and polyols, claims or Exemplary embodiments in the sense of the colloid according to the invention len carrier systems are completely missing. WO 95/23175 describes Poly (hydroxycarboxylic acid ester) polyols and their use  as a depot matrix system for the retardation of pharmakolo gisch active substances. Claims or application examples in the sense of the colloidal carrier system according to the invention, especially for mucosal drug delivery. Furthermore, the targeted control of the polymer properties not described, as well as the general Possibility to doubt the examples given there. USP 4745160 describes biodegradable amphipathic copolymers, USP 5210108 degradable foam materials, according to the invention Exemplary embodiments or claims are completely absent. USP 5612412 and USP 5712334 describe the synthesis of lac clay-modified polyvinyl alcohols (PVA), the present Claims and exemplary embodiments corresponding to the invention but are completely missing. USP 5331045 describes the synthesis of poly (hydroxycarboxylic acid ester) modified polyvinyl alcohol get (PVA) by polycondensation of PVA with monomers Hydroxy carboxylic acids. A procedure that according to the current State of the art Impurities in the form of poly (hydroxy carboxylic acid ester) homopolymers, what such a manufacture for the invention colloidal carrier systems appear to be undesirable leaves.

Component 2 Active ingredients

It was surprisingly found that all relevant and esp special hydrophilic active ingredients used according to the invention can be. All are therefore preferred according to the invention Active substances that are selected from the group: active Peptides, proteins, enzymes, enzyme inhibitors, antigens, cyto static, anti-inflammatory, antibiotic, DNA constructs and Growth factors.

However, carrier systems according to the invention also serve in particular - but not limited to it - acc. Part aspects 3 and 8 to 11 of the first task as colloidal carriers for mucosal or transmucosal administration  Chung relevant hydrophilic active ingredients, such as. B. Pro stones, peptides, oligonucleotides and DNA constructs as well also lipophilic active ingredients, such as. B. Corticosteroids and steroid sex hormones.

Pharmaceutical formulations according to the invention for mucosal Applications can be administered in different ways be enough, e.g. B. sublingual, buccal, oral, nasal, pulmo nal, vaginal, ocular or gastrointestinal.

Such formulations are pharma rele for all vanten, biologically active substances of interest if a Drug transport to, in or through the mucosa is desirable is. They are particularly advantageous for active ingredients without the use of a colloidal carrier system according to the invention for sublingual, buccal, oral, nasal, pulmonary, vaginal, ocular or gastrointestinal administration destroyed or inactivated, or for which a high local mucosal concentration is desired.

Active substances (WS) preferred according to the invention for mucosal administration are accordingly:

• 1st group of hydrophilic macromolecular pharmacologically relevant WS:
  • 1.1. Vaccines that are selected from the group: live vaccines, dead vaccines, toxoid vaccines, DNA constructs and their adjuvanted preparation and are directed against pathogenic germs or their pathogenic metabolites, of which the mucosal penetration pathway is known or known from the literature which colonize predominantly on mucosal surfaces, such as B. Helicobacter pylori, typhiform Salmonella, Vibrio cholerae, Neisserien (N. gonorrhoee, N. meningitidis), Enta moeba histolytica, Haemophilus influenzae, Bordetella pertus sis, hepatitis A viruses, colon and vagina-colonizing streptococcal types, streptococcal types & B, glue points (K. pneu moniae, K. oxytoca, K. rhinoscleromatis), Shigella, Yersinia, vibrions, Pseudomonas aeroginosa, Legionella, Korynebacterium diphtheriae, Bacillus anthracis, Mycobacteria (M. tuberculosis, M. leprai-), Treponema palli dum, My coplasma pneumoniae, chlamydia (C. trachomatis, C. pneumo niae), polio viruses, rhino viruses, myxoviruses (measles V., Mumps- V., Parainfluenza V.), rubella virus, rotaviruses, Adeno viruses, influenza viruses, herpes viruses (varicella zoster virus, cyto megalievirus, herpes simplex virus, EBV), HAV, HEV
  • 2. 1.2 Vaccines that are selected from the group: live vaccines, dead vaccines, toxoid vaccines, DNA constructs and their adjuvanted preparation and are directed against pathogenic germs or their pathogenic metabolites, which are known to have a parenteral route of infection, but a mucosal application causes systemic, specific immunoglobulin, such as B. Clostridium tetani, Clostri dium botulinum, Yersinia pestis, Borrelia burgdorferi, rabies virus, HIV-1 & HIV-2, HBV, HCV, HDV
• 2nd group of lipophilic pharmacologically relevant active ingredient fe, such as B. corticosteroids, steroidal sex hormones, Cyclosporin A, Nystatin, Amphotericin B, Clotrimazole, Econa zol, miconazole, fluconazole, itraconazole, bifonazole, and Antibiotics

Production of the colloidal associates [I] Polymer-drug complexes (Solution task 2 - method 1)

The invention relates to pharmaceutical formulations which in Form of polymer-drug complexes in colloidal form at least one polymer and at least one active ingredient consist. The active ingredient can itself be a partner in the Be complex or bound to a partner of the complex his. It was surprisingly found that the invention Polymers with relevant active substances spontaneously self-assemble training associates, here also called complexes, and  that their properties via simple parameter variations could be manipulated and optimized.

It is already known from the literature that charged macromolecules with ions of opposite charge form ionic compounds which, depending on the charge distribution, the molecular weight and the hydrophobicity of the end product, often precipitate out of aqueous solutions. Low molecular weight ions of the same charge are displaced by the higher molecular weight compound, phenomena which are summarized under the generic term "polyelectrolyte effect". State of the art includes the formation of gels (combining alginate solutions and Ca ²⁺ ) or protein precipitations. Such polyelectrolyte complexes can in principle either consist of a macromolecular, multiply charged component of one polarity and many low molecular weight ions of the other polarity, or of two macromolecular, each multiply charged partners of different polarity. Hollow capsules made from such polyelectrolyte complexes are described, for example, in BE-A 9 01 704, EP-A 01 27 989, EP-A 01 88 309, EP-A 01 27 713, DE-A 32 09 127, EP -A 01 52 898, US 44 87 758, DE-A 32 09 098, US 44 09 331. It is also known from the literature, e.g. B. described in DE 00 040 13 110 A1 that polyelectrolyte complexes can be produced in microparticulate form and thereby enable an improvement in the therapeutic applicability and breadth of an active ingredient.

Surprisingly, however, it has now been found that she also invented biodegradable according to the invention, thus toxicologically completely unbe conceivable polymers, even without clear polyelectrolyte genes complexes with pharmacologically relevant active ingredients open spontaneously, due to the hydrophilic-hydrophobic interaction, form. Furthermore, the simple tax is surprising Ability of this interaction through targeted carrier polymer manipulation from hydrophilic-hydrophobic interaction to  towards interionic interaction. So they show Polymers to a particular extent also as carriers Properties that not only meet the requirement profile biokompa tibler and biodegradable polymer systems correspond, but which is also due to the slight variation in production special requirements of a therapeutic, in particular also slightly adapted for mucosal drug administration can be.

Surprisingly, even for the unmodified, thus have no clear polyelectrolyte properties the high active ingredient association according to the colloids according to the invention (Loads) found that are not actually expected would have been, furthermore the degree of loading targeted selection of the carrier polymer (component 1) manipulation change and optimize.

It was also surprising that the manufacturing process such complexes fine particles with controllable size of nm down to the µm range and not - as actually to was expected - just a clumped mass.

Particle sizes are deliberate in the range of less than 100 nm held down to a few µm, and particle size / distribution easily adjustable, for example via the choice of Carrier polymers, the concentrations used, salt concentrations trations, ionic strengths, pH values, temperatures, etc. The required parameters can be found in corresponding Preliminary tests easily determined and to the desired requirements Therapeutic agents, especially mucosal ones Drug administration can be adjusted.

It was also surprising that with the help of such complex like associate high local drug concentrations relevant cell tissues can be generated.

According to the invention, such self-assembling can be carried out colloidal systems particularly well by inventive  Produce polymer-drug complexes. About external influences, such as solution equilibria, charge interactions, pH Changes, etc. may continue, provided that therapeutic View required, slow decomplexing in vivo, So drug release can be achieved. This release condition like the consistency properties the complex composition can be controlled.

The advantages of such spontaneously arise from aqueous solutions training systems are obvious, u. a. are not special manufacturing technologies are necessary, they are not imperative to reduce the properties of the therapeutic Auxiliaries such as B. organic solvents, emulsifiers etc. necessary, whereby a faster, repro according to the invention Ducible and inexpensive way was found, efficient To receive therapeutic agents, especially with regard to on mucosal drug delivery.

[II] Production and loading of colloidal according to the invention Carrier systems using modified solvent deposition (Solution task 2 - method 2, for more lipophilic polymers)

Since, as already stated, most of the manufacturing techniques are particularly unsuitable for sensitive active ingredients Production of the carrier system according to the invention continues an adapted and optimized so-called solvent deposition Methodology preferred, with the colloids according to the invention easily and can be produced reproducibly, provided that Use of a more lipophilic, therefore no longer water-soluble Chen carrier polymer is desired or required.

This manufacturing methodology, which differs from that of Fessi introduced nanoparticle production method [USP 5118528, USP 5049322] based on controlled precipitation a dissolved polymer in a non-solvent and thus allows the production of particulate systems in the Micron and submicron size range without upstream emul  sification or dispersion steps. It was invented Carrier systems according to the invention, in particular also for mucosal Active substance administration through selection of the suitable polymer / solution medium / non-solvent / (emulsifier) system optimized.

Here, too, particle sizes became conscious according to the invention in a range from less than 100 nm to a few µm poses. The width of the particle size distribution is also easily adjustable, for example via the concentration of the polymer solution used, the rate of addition, Emul gator share and by suitable selection of one defined in Proportion of added cosolvency. These parameters can also easily identified in appropriate screenings and to the desired requirements can be adapted, in particular can They thereby made for the mucosal active ingredient according to the invention administration can be optimized.

It was found that the size range is below 500 nm diameter according to the invention is particularly good for mucosal suitable application, there is an increased active ingredient concentration set on relevant cells, as well as in vivo appropriate immune response.

Such polyol esters can be used Produce colloidal carrier systems particularly well. Because of the specifically manipulated properties of the carrier polymers was further surprisingly found that the Use of additional emulsifiers for stabilization completely or largely, which could not be done only the simultaneous preservation of the targeted set Surface properties of the carrier system according to the invention enabled, but also the number of compos to be used nent / process steps reduced and thus the manufac ling simplified and cheaper.  

This system can be associated with the active ingredient (s) in at least five ways:

• 1. by adsorption of the active ingredient from a solution with which the polymer colloids already produced by solution deprivation of funds come into contact
• 2. by in-situ adsorption of the active ingredient from a solution, with which the polymer colloids just produced before Lö detoxification come into contact
• 3. by in-situ inclusion of the active ingredient in the polymer trix during particle formation and subsequent loosening deprivation of food
• 4. by covalent bonding, the active substance being chemically attached at least one functional grouping of the used Polymer is bound.
• 5. by covalent binding to the particle surface, where the active ingredient chemically to at least one functional Grouping of the particles is bound.

Because of the one hand widely variable, on the other hand very speci fish adjustable properties show inventive Polymer colloid-active ingredient formulations property profiles, which is especially useful for mucosal application predestine.

It is summarized for both colloid production methods [(I) and (II)] a peculiarity of the invention that the respective biocompatible and biodegradable carrier polymer (I) consciously from metabolizable and thus non-toxic construction stones is produced and targeted (2) to the particular their requirements of the active substance (s), (3) to the targeted place of action or absorption and mechanism of action and (4) the possibility of using a gentle parti manufacturing process could be adapted.

ad (1)

For the synthesis of the carrier polymer (component 1) come at least one biocompatible component (polyo  le) and attached polyester fragments, which by simple hydrolysis in the body to ver in the citrate cycle metabolizable fragments disintegrate, for use.

ad (2) and (3)

Through suitable and targeted selection or Modification of the polyols (e.g. type, molecular weight, Number of esterifiable hydroxyl groups, number and type of charge-bearing groups, distance of the charge-bearing groups Groups of polyol backbones, etc.) specified properties of the polyol systematically by specifically linking polyester building blocks to the Requirements of the colloidal system according to the invention customized. Examples include: solubility, Molecular weight, structure, charge density, particles surface charge etc.

Is to the desired drug-carrier polymer interaction (such as active ingredient stabilization, degree of loading, etc.) a hydrophilic-hydrophobic interaction is desired, this is due to the targeted synthetic attitude the polymer amphiphile achieved is an interionic Interaction desired, this is through the selection and / or modification of a corresponding electrolyte Polyols reached.

ad (4)

[II] If a stable particulate carrier in more lipophilic active ingredients during manufacture or for more hydrophilic active ingredients after the production of the carrier system according to the invention associated with active ingredients The solubility becomes of the carrier polymer deliberately set so that a modi Defined and optimized gentle so-called solvent depo sition Particle generation process for use com men can.

[I] If on this technological manufacturing step can or should be dispensed with the carrier polymer is deliberately made water-soluble, so that when combining active ingredient and carrier polymer  spontaneously defined colloidal polymer-drug complexes form in aqueous solution and their properties, such as B. size, degree of loading, etc. also targeted can be adjusted.

With the help of this new colloidal, bioab buildable polymeric carrier system side that only by combination of all of the above points, their particular effectiveness are completely new therapeutic applications possible, but also conventional ones Therapy concepts are optimized, such as B. by the increases the therapeutic breadth and efficiency of an active ingredient fes.

Other advantages that result from the invention are the more comfortable type of application for the patient because not necessarily parenterally, i.e. via an injection, appli must be decorated, and the associated increased "Patien ten "compliance. This allows, compared to previous vaccination procedure, a vaccination z. B. by taking one Tablet or solution, similar to poliomyelitis vaccination (Polio). The resulting low The degree of infection in the population does not have one insignificant economic benefits that are the same if by using systems according to the invention in the Veterinary medicine results.

Examples

The invention is, but is not limited to, the following to be closer, based on a few selected examples explained.

Polymer synthesis Central polyol modification

The exemplary modification of an uncharged Zeritral polymers, here poly (vinyl alcohol), was comparable to  [F. Dolle, J. Le Moigne and P. Gramain, Eur. Polym. J. 6 (1970) 1227-1232; P. Gramain and J. Le Moigne, Eur. Polym. J. 8 (1972) 703-709] via etherification of its activated Hydroxyl groups with butane sultone or with diethyaminoe thyl chloride.

Table 1

Fig. 1 shows an example of FT-IR spectroscopy (Nicolet 510 P FT-IR spectrometer) of the introduction of the sulfobutyl groups into the originally uncharged central polymer and the increase in the intensities of the corresponding SO-related vibrations at 1190, 1040 and 610 cm ^-1 .

Fig. 2 shows the corresponding modifications by NMR spectroscopy (Jeol GX400 Delta N FT-NMR. Spectrometer), z. B. by reducing the signal intensity of -CH ₂ -CH-OH (~ 1.7 ppm) and -CH ₂ -CH-OH (~ 4.1 ppm), increasing the signal intensities -CH ₂ -CH-OC and - CH-O-CH ₂ - (~ 1.9 ppm) and the sulfobutyl group itself at ~ 3.08 and ~ 3.7 ppm.

Melt grafting for the production of carrier polymers (component 1) by appending hydroxycarboxylic acid groups to the Central polyols

Exemplary for the lactones of lactic and glycolic acid: D, L-lactide (dimeric anhydride of lactic acids) and glycolide (dimeric anhydride of glycolic acid) are defined in molar amounts, e.g. B. 1: 1, in the presence of defined amounts the central polyol, e.g. B. 1 wt .-% PVA at z. B. 130 ° C for z. B. three hours under inert, anhydrous conditions defined molar amounts of catalyst (e.g. 0.2 mmol of tinoc toat) implemented. Then the reaction product in taken up in a suitable solvent and in a 10: 1 Excess of a polymer nonsolvent miscible with it precipitated for purification and until constant weight in Vacuum dried. For this example, the Crude product in dichloromethane (DCM) and precipitation in etha nol. Depending on the solubility, combinations also resulted Acetone in water or inverse water in acetone. Further Details see Table 2.

Table 2

Structure and solubility of the polyol esters (component 1)

Fig. 3 shows the structural change in the polymers as a function of the synthesis conditions by NMR spectroscopy. Here the hydroxycarboxylic acid group lengths on the central polymer were systematically shortened, in particular to demonstrate the adjustability of solubility. While in PVAPLG10 the hydroxycarboxylic acid groups, which were attached to the OH groups of the central polymer, each consisted of about 10 acid units, e.g. B. on the ratio of the lactide signal intensities -CH ₃ chain: -CH ₃ - end group, ie at ~ 1.5 ppm to ~ 1.3 ppm, the groupings at PVAPLG50 are already shortened in such a way that this carrier polymer dissolves well in cold water, but - surprisingly found - can be precipitated again by increasing the temperature and therefore has a lower crystalline separation temperature.

Fig. 4 uses a combination of size exclusion chromatography and static light scattering (Merck-Hitachi system with RI 71 detector in combination with MiniDawn Light Scattering detector) to show how this structural change also compares to the molecular weight of the carrier polymers an unmodified central polyol.

Fig. 5 shows by means of photometric turbidity measurement (Shimadzu UV-VIS spectrophotometer UV 160) as an example for uncharged carrier polymers that the surprisingly found lower crystalline segregation temperatures could also be set as a function of the (hydroxycarboxylic acid grouping) chains.

Fig. 6 shows by means of photometric turbidity measurement, for example, a further possibility of manipulating the position of the lower crystalline separation temperature via the concentration of the polymer in the aqueous for a charge-modified carrier polymer.

Fig. 7 shows by means of photon correlation spectroscopy [PCS, Malvern Zetasizer 4], for example here for a charge-modified carrier polymer, the size of the pure carrier polymer associates depending on the temperature, which demonstrates one possibility, the properties of the polymer-drug complexes to control. Particularly for very sensitive active substances (growth factors are mentioned as an example), the active substance can be offered a particulate carrier polymer associate in water (comparable to a micellar system) even at low temperatures, on the surface of which the active substance itself is then associated.

Spontaneously trained polymer-active ingredient complexes in water

General procedure for the following examples: A defined amount of an active ingredient stock solution is presented, diluted with a defined amount of buffer, then with a Defined amount of polymer stock solution added, so that if not explicitly stated otherwise, a total volume of 1 ml results.

Fig. 8 shows the possibility of controlling the size of such polymer-drug complexes via the polymer concentration used (drug: fluorescent-labeled model protein FITC-BSA (bovine serum albumin) 640 µg; polymer PVAPLG50; isotonic phosphate buffer pH 6.0 (PBS 6.0), size measurement via PCS).

Fig. 9 shows an example of the easy adjustability of the polymer-drug complex properties via "procedural" parameters such as pH and / or ionic strength of the aqueous solutions (drug: tetanus toxoid, amount for this facto rial design varies within limits 332 µg ... 949 µg / 200 ml, polymer: PVAPLG57.1 1670 µg / 200 ml, size measurement by PCS, zeta potential measurement with Malvern Zetasizer 4).

Fig. 10 shows by way of example that - if desired - the addition of an emulsifier is also a possibility to influence the polymer-drug complex properties (drug: tetanus toxoid 3.367 mg; polymer: PVAPLG57.1 3.32 mg; in a total of 1.220 ml PBS 6.0, emulsifier Pluronik F68 BASF).

Fig. 11 shows an example of how the selection of the carrier polymer and its amount can be used to optimize the overall system, i.e. polymer-drug complex, for one drug. If an uncharged central polyol is synthetically provided with hydroxycarboxylic acid groups, 90% of the model active ingredient used (FITC-BSA, 450 µg, total liquid volume: 1 ml PBS 6.0) is bound in the form of the polymer-active ingredient complexes according to the invention if the carrier polymer has a sufficiently high concentration in water However, if a central polyol with only 10% charge-carrying groups is used for the synthesis of component 1, the entire amount of the protein used is associated. Once the desired polymer has been selected in this way, any amount of the protein used can also be associated in the form of the polymer-active substance complexes via the amount used.

Fig. 12 shows that - if therapeutically meaningful - decomplexing / releasing active substances, for example tetanus toxoid, can also be achieved from the polymer-active substance complexes. Releases that were controlled here, for example, by the selection of the carrier polymer. Furthermore, this example shows that, particularly when these systems are used according to the invention, no significant amounts of protein are released within the first hours, so that the mucosal destination can be reached, and only then does the active ingredient be released (photometric content determination at 280 nm, amount of components 1 (carrier polymer) each 10 mg, amount of component 2 (tetanus toxoid) each 1992 µg, each in a total of 1000 µl PBS 6.0, after spontaneous formation of the polymer-drug complexes, these became at specified times Centrifuged 44 ° C, the supernatant was examined photometrically, then the complexes were further incubated in fresh PBS 6.0 at 37 ° C).

Fig. 13 shows, using the example of a complex between a charge-carrying carrier polymer and the model protein FITC-BSA, the massive accumulation of the complexes on mucosal-like cell tissue (Caco 2 cell culture), a phenomenon that does not occur when free active substances are used alone and thus clearly demonstrates the surprisingly found effectiveness of the new carrier system according to the invention (640 µg protein, 10 mg polymer, in together 1000 µl PBS 6.0)

Colloids

If the use of a carrier polymer is desired, its hydrophilic-hydrophobic balance more on the side of the Lipophilia is postponed, e.g. B. for compatibility reasons to the active ingredient or when the relevant active ingredient emerges finally defined on the surface of the colloidal If there is a carrier, the total polymer can be corresponding composition then an acetone-soluble carrier polymer provides and in accordance with modified solvent deposition methodology the colloidal carrier according to the invention are transferred.  Exemplary manufacturing description (manufacturing parameters can deviate from this to optimize properties): 50 mg Polymer in 5 ml acetone (may contain cosolvent) solved. The solution is added at a constant rate in 50 ml PBS given (may contain some emulsifier). The first The suspension is continuously dispersed. After The organic solvent is added for 8 hours with stirring evaporated.

Fig. 14 uses the example of an uncharged biodegradable polymer to demonstrate a possibility to vary the size of such particles produced by controlled precipitation, here by adding a proportionally toxicologically harmless cosolvent (ethyl acetate) in the carrier polymer acetone solution.

Fig. 15 illustrates, here using the example of a charged carrier polymer SB50PVAPLG10, the morphology and size of such colloids (transmission electron microscopic (TEM) photography).

Fig. 16 shows an example of the possibility, due to the amphiphilic nature of the carrier polymers, that the solvent deposition method can not only be used without the addition of an emulsifier (emulsifier here PVA (18.88), Hoechst), but also the possible preservation of the surface properties of the colloids .

Fig. 17 shows, by way of example, that the colloid surface properties can be set as a function of the carrier polymers, more precisely the choice of the central polyol used for melt grafting and its properties in combination with suitable hydroxy carboxylic acid grouping modification - listed here by way of example: surface charge of the colloids (zeta potential measurement and particle Charge Detection (PCD) titration) and their hydrophobicity (Bengal adsorption method).

Fig. 18 demonstrates that the loading degree of such colloids with relevant active ingredient is a direct function of the carrier polymers, more precisely the choice of the central polyol used for melt grafting and its properties in combination with suitable hydroxycarboxylic acid grouping modification.

Fig. 19 shows the high effectiveness of the colloids according to the invention, here, for example, for Nile-red-marked SB30PVAPLG10 colloids in a cell culture model (confocal laser microscopy), not only on (apical), but also in and even basally, a high fluorescent association (optical cell section planes) is found in Fig. 19 from left to right about 0 µm, 10 µm, 20 µm).

Fig. 20 underpins this finding, since the cell-adsorbed and cell-absorbed amount of colloid is again a function of the carrier polymers, more precisely the choice of the central polyol used for melt grafting and its properties in combination with a suitable hydroxycarboxylic acid grouping modification.

Toxicity and drug stability

Fig. 21 demonstrates the toxicological safety of colloids according to the invention, according to the MTT assay a cell viability of 100% is maintained, ie no cell death by the colloids is recognizable, and the colloids are also neutral with regard to the release of LDH. In the propidium iodide test, this finding is also supported, less than 0.2% of the cells died on incubation with the colloids, a value which corresponds to that of pure aqueous buffer medium.

Fig. 22 shows the result of gel electrophoresis (GE) tests (native PAGE and SDS PAGE), which demonstrate the unchanged nature of the active ingredient, both in the polymer-active ingredient complex and in the desorbed active ingredient after 24 hours, show no changes in comparison to free untreated active ingredient (1 = free untreated model protein FITC-BSA; 2 = polymer-active ingredient complex: SB10PVAPLG50-FITC-BSA complex; 3 = FITC-BSA desorbed from 2 after 24 h).

General method description of cell tests Determination of cytoadhesion and cytotoxicity

Prerequisite for the application of colloidal medication carriers, in particular also mucosal application in addition to only a slight toxic effect on the surrounding Tissue (epithelial cells), also a sufficiently long dwell time on mucosal surfaces (cytoadhesion) and a Adequate cellular uptake may be required. For Interaction with mucosal cells has been studied the Caco 2 cell culture model was used. This in vitro The intestinal mucosa model has been used in many studies its suitability for studying intestinal absorption, Cytoadhesion and toxicity of drugs and drugs can show fabric support systems.

Cell culture

Caco 2 cells from passages 42 to 45 were cultivated in Dulbecco Modified Eagles medium with a glucose content of 4.5 g / l with the addition of 10% fetal calf serum, 2 mM L-glutamine and 1% non-essential amino acids in 10% CO ₂ atmosphere sphere at 95% RH and 37 ° C.

Toxicity studies

Caco 2 cells were sown in a cell density of 6.5 × 10 ⁴ cells / cm ² on polystyrene multiwell plates. Medium change took place every 2 days. On day 21 after sowing, the cells developed a differentiated monocell layer and can be used for in vitro investigations.

For this purpose, cells were washed twice with isotonic phosphate buffered saline (PBS, pH 7.2) and corresponding colloid suspensions according to the invention (0.25, 2.5 mg / ml) applied the cell surface. After several hours of incubation (Time intervals according to the figures) were the following mentioned toxicity tests.

1. Release of lactate dehydrogenase (LDH)

Principle: As a cytoplasmic enzyme, LDH occurs in one Damage to the plasma membrane from the inside of the cell to the outside medium (buffer) and can be determined using kinetic determinations methods against a standard UV photometric quantification be decorated. Triton X serves as the toxic reference standard 100 (0.1% in PBS), whose LDH release corresponds to 70%.

2. Transformation of MTT (3- (4,5-dimethylthiazol-2-yl) -2,5- diphenyl tetrazolium bromide)

Principle: The tetrazole compound MTT is used in mitochondria more viable cells to a blue-colored formazan dye reduced enzymatically. The blue color is there proportional to the viability of the cells and becomes photometric recorded, PBS serves as a non-toxic reference.

3. Propidium iodide staining

Principle: Propidium iodide penetrates as a hydrophilic substance only damaged cell membranes and binds to nucleic acids of the Nucleus, which when turned into UV leads to a red core fluo comes resence that is microscopically detectable. The evaluation is carried out using fluorescence microscopy with a wavelength filter (380/550) against a non-toxic PBS standard.

Cytoadhesion and cellular uptake

Caco 2 cells were sown in a cell density of 6.5 × 10 ⁴ cells / cm ² on polycarbonate membranes (Costar-Transwell). Medium change took place every 2 days. On day 21 after sowing, the cells developed a differentiated monolayer and can be used for adhesion and absorption studies.

Fluorescent-labeled colloids and polymer-active substance complexes (active substance: bovine serum albumin (FITC-BSA) labeled with fluorescein isothiocyanate) (NIT red (1%, w / w)) were applied to the apical surface of the cells and for 120 min at 37 ° C., 95% RH, 10% CO ₂ incubated. The colloid concentration was 250 µg / ml in both cases.

After incubation, the cell monolayers were washed 3 times with PBS washed and then with formalin solution (4% in PBS) Fixed for 60 minutes. The filter membranes were by means of Cut out the scalpel and embedded in glycerol gelatin tet. The preparations thus produced were confocal Laser Scanning Microscopy (CLSM) on adhesive and in tracytoplasmic fluorescence examined.

In controls only with active ingredient without colloids or complexes no fluorescence was detectable on or in the cells.

Claims (30)

1. Pharmaceutical preparation containing at least one colloidal polymer drug association. 2. Pharmaceutical preparation according to claim 1, in particular also for mucosal drug delivery. 3. Pharmaceutical preparation according to one or more of the Claims 1 to 2, characterized in that at least one component is a biocompatible and biodegradable poly mer (component 1). 4. Colloidal association according to one or more of the claims che 1 to 3, characterized in that it is a Polymer-drug complex deals. 5. Colloidal association according to one or more of the claims che 1 to 4, characterized in that spontaneous in-situ Formation of colloidal polymer-drug complexes follows when an aqueous solution A combines with component B. Is nigt, wherein solution A contains at least component 1 and component 8 must have at least one active ingredient (compo nente 2) or this (s) in chemically bound or contains physically associated form and component B thereby a) component of solution A or b) one second solution can be. 6. Colloidal association according to one or more of the An Proverbs 1 to 5, characterized in that the manufacturer development especially about the choice of component 1, as well Process parameters: concentrations, pH values, ionic strength ken and / or temperature agent, mode of action and place of action is optimized or can be optimized. 7. Colloidal association according to one or more of the claims che 1 to 3, characterized in that the polymeric Trä through controlled precipitation into colloidal form leads. 8. Colloidal association according to claim 6, characterized in that the association with active substances takes place:

• a) by adsorbing the active ingredient from a solution with which the polymer colloids already produced come into contact after solvent removal, and / or
• b) by in-situ adsorption of the active ingredient from a solution with which the polymer colloids just produced come into contact before solvent removal, and / or
• c) by in-situ inclusion of the active ingredient in the polymer matrix during particle formation and subsequent solvent removal, and / or
• d) by covalent bonding, the active substance being chemically bound to at least one functional group of the polymer used, and / or
• e) by covalent binding to the particle surface, the active substance being chemically bound to at least one functional grouping of the particles.

9. Colloidal association according to one or more of the claims che 7 to 8, characterized in that the manufacture especially about the choice of component 1, and procedure rens parameters: concentrations, pH values, ionic strengths, Precipitation rate, stirring speed, cosolvents and / or temperature, agent, mode of action and site of action is optimized or can be optimized. 10. Pharmaceutical preparation according to one or more of claims 1 to 9, characterized in that the Active substance (s) is selected from the group: active Peptides, proteins, enzymes, enzyme inhibitors, antigens, Cytostatics, anti-inflammatory drugs, antibiotics, DNA con structures and growth factors. 11. Pharmaceutical preparation according to one or more of claims 1 to 10, characterized in that col loidally an average particle size range of <1 mm designated. 12. Pharmaceutical preparation according to one or more of claims 1 to 11, characterized in that col loidally an average particle size range of <10 µm designated.   13. Pharmaceutical preparation according to one or more of claims 1 to 12, characterized in that col loidally an average particle size range of <1 µm designated. 14. Pharmaceutical preparation according to one or more of claims 1 to 13, characterized in that kolloi dal be an average size range of 10 to 10,000 nm records, and in particular their use for mucosal Drug administration, with mucosal drug administration in particular sublingual, buccal, oral, nasal, pulmonary, vaginal, ocular and / or gastrointestinal Administration referred. 15. Use of the pharmaceutical preparation according to one or more of claims 1 to 14 as a vaccine. 16. Polymers, characterized in that it is branched Polyol esters are the best of a central molecule hen, to the short-chain, biodegradable hydroxycarboxylic acids groups are attached, with the peculiarity that by reaction parameters: choice and amounts of Zen tralpolyol and catalyst (system), choice and length of Hydroxycarboxylic acid ester groups, and the reaction temperature and response time, for use as a com component 1 according to one or more of claims 1 to 15 are optimized or can be optimized. 17. Polymers according to claim 16, characterized in that modify the central molecules necessary for their production adorned or unmodified polyols. 18. Polymers according to one or more of claims 16 to 17, characterized in that for their manufacture necessary central molecules are selected from the group: linear synthetic polymers with <6 to 500 hydroxyl groups pen, especially polyvinyl alcohols (PVA) with Polymerisa degrees of saturation from <6 to 500 (and degrees of saponification from 70 up to 98%, if by hydrolysis of poly (vinyl acetate) hold).   19. Polymers according to one or more of claims 16 to 17, characterized in that for their manufacture necessary central molecules are selected from the group: linear synthetic vinyl copolymers, especially of the Po lyvinyl alcohol, with <6 to 500 hydroxyl groups, in particular others with vinyl acetate, vinyl pyrrolidones, vinyl amines, Vinyl imidazoles, vinyl pyridines, vinyl sulfonic acids, vinyl phosphoric acids. 20. Polymers according to one or more of claims 16 to 17, characterized in that for their manufacture necessary central molecules are selected from the group: linear, branched or cyclic, charged or ungela dene polysaccharides, especially starches or ingredients le, glycogens, celluloses or components, dextrans, Tu nicine, inuline, chitine, alginates, pectins, mannans, Ga lactans, xylans and other polyoses, chondroitin sulfates, Heparin, hyaluronic acid and other glycosaminoglycans, Mureine, dextrins, cyclodextrins, chitosans, and esp especially their partially hydrophobicized derivatives, where Hydro phobization by methyl and / or ethyl (ether), (ester), (-Urethane) link is reached. 21. Polymers according to one or more of claims 16 to 17, characterized in that the central molecules necessary for their preparation additionally one or more car boxyl, sulfobutyl or sulfopropyl group (s) and / or butylamine, propylamine or ethylamine - (Primary, secondary, tertiary or quaternary amine, where corresponding N-substituents are in particular -H, -CH ₃ and / or -C ₂ H ₅ - group (s)) - Group (s) may contain, in the form of ether, ester or urethane linkage are bound to the polymer. 22. Polymers according to one or more of claims 16 to 21, characterized in that for their manufacture necessary central molecules also in the form of alkali metal or halogen salts, especially sodium or chloride salts available.   23. Polymers according to one or more of claims 16 to 22, characterized in that the hydroxycarboxylic acid ester groups from biologically harmless Units, especially L, D milk and / or glycol acid, best in any molar mixing ratio hen. 24. Polymers according to one or more of claims 16 to 22, characterized in that the hydroxycarboxylic acid ester groups reaction products of dimers of bio are logically unobjectionable hydroxycarboxylic acids; Dimers are in particular L-lactide, D-lactide, D, L-lactide and / or glycolide in any molar composition. 25. Polymers according to one or more of claims 16 to 24, characterized in that the introduction of Hy droxycarboxylic acid ester groups to form water soluble polyol esters. 26. Polymers according to one or more of claims 16 to 25, characterized in that it is an aqueous solution Have lower critical segregation temperature, esp especially in the temperature range from 0 ° C to 100 ° C. 27. Polymers according to one or more of claims 25 to 26, characterized in that for the production of molar Ratios of polyol-OH group to acid unit in the area from 0.6: 1 to 6: 1, especially in the range of 1: 1 up to 3: 1. 28. Polymers according to one or more of claims 16 to 24, characterized in that the introduction of Hy droxycarboxylic acid ester groups to form non- leads to water-soluble polyol esters, but a sol in non-toxic organic solvents: Esters, ethers, alcohols, ketones, especially acetone, Ethyl acetate, ethanol and especially their mixtures gene, as well as in particular their mixtures with proportions Water. 29. Polymers according to claim 28, characterized in that the hydroxycarboxylic acid ester groups on the central polyol  each from 1 to 100, and in particular 5 to 50, hydroxy carboxylic acid building blocks are composed. 30. Use of the polymers according to one or more of the Claims 16 to 29 as component 1 according to one or more reren of claims 1 to 15.