DE10118312A1 - Solid particles for transporting hydrophobic active agents, e.g. drugs or nucleic acids, obtained from organic solvent solution of active agent and water-insoluble and amphiphilic polymers by ultrasonication and dialysis - Google Patents

Abstract

New solid particles (I), for transporting hydrophobic or hydrophobized active agents (A), are obtained by: (a) dissolving (A), a water-insoluble organic polymer (P1), an amphiphilic organic polymer (P2) and optionally supplementary components in organic solvent(s); (b) ultrasonicating the solution; (c) dialyzing the solution against water; and (d) separating the obtained particles (I) from the aqueous solution. Independent claims are included for: (i) new solid particles (I') for transporting (A), comprising a core of (P1) and an outer layer of (P2) (not covalently bonded to the core), where (P2) is polyvinyl alcohol (PVA) or its ester; (ii) the preparation of (I') by the procedure described above for (I); (iii) particles for transporting drugs (A), which (a) have linker molecules (LM), containing amino-reactive and/or thiol-reactive groups, covalently bonded to free amino or thiol groups present on the surface of the particles; or (b) have a mixture of two types of LM's covalently bonded to reactive groups on the particle surface, one type being a bifunctional LM having a further reactive group on the other end of the molecule and the other being a monofunctional LM with no further reactive groups; and (iv) the use of pure bifunctional polyethylene glycol (PEG) molecules (preferably N-hydroxysuccinimide (NHS) ester/vinyl sulfone PEG), optionally mixed with monofunctional PEG molecules (preferably NHS ester-PEG) for the preparation of surface-modified solid particles for transporting drugs.

Description

The invention relates to solid particles for the transport of pharmaceutical Active substances, process for their preparation, containing pharmaceuticals these particles as well as the use of these particles in different selected indications.

A primary goal of pharmaceutical research is to: to enhance the desired effects of known active ingredients and the minimize systemic side effects, which is particularly true in Substances with high intrinsic and therefore inevitable Toxicity (e.g. cytostatics) is of great importance. This can both about reducing the therapeutic effect required total dose as well as accumulation of the effectors on desired place of action can be achieved, both of which by the controlled, spatially specific release of effector molecules in the broadest sense (proteins, peptides, nucleic acids or low molecular weight substances) in the desired target tissue can be accomplished. Among them is the specific one Transfer of therapeutically or diagnostically usable substances into defined biological targets ("drug delivery", "drug targeting") understand that is an important goal of current pharmaceutical Research is.

The antibody technology available today allows generation of high affinity binding partners for almost any  biological structure; Furthermore, there are numerous natural ligands for cellular receptors have been characterized and cloned so that it no longer a problem, molecules with high and specific Generate affinity for the desired goals. In many cases low molecular weight ligands (e.g. glycosides) are also known which imitated chemically and thus used for target control can be. However, these "finder molecules" practice whether micro or macromolecular, generally not pharmaceutical itself usable function while the effectors themselves are not are target-specific. A main focus must therefore be on to bridge this separation and the therapeutic potential of available effectors with the target specificity of the searcher molecules connect.

The most efficient approach to achieving this goal known to date is to use support structures in the colloidal (ie submicron) size range, on the surfaces of which appropriate targeting molecules can be bound. In this way, both the optimal ratio of target seeker to effector molecules as well as maximum flexibility are achieved: While only a few (<10) effector molecules can be bound by direct covalent coupling to a single antibody or other ligand molecule (which with a molecular weight of an antibody of around 150 kDa means that over 90% of the mass of the conjugate is accounted for by the antibody part) and conjugates with low molecular weight finders usually have a molar ratio of 1: 1, an effector: target seeker ratio of 10 ³ - 10 ⁴ can be achieved with colloidal systems. Furthermore, no chemical change to the effector is required, which is advantageous in every respect.

An efficient way to do this is to use the Incorporate substances in colloidal carrier particles, which with Antibodies against or natural ligands for characteristic Molecular structures of the target linked and at the same time by inert Coating their surface protected against the immune system become.

A widely used method for the colloidal packaging of pharmaceuticals is to enclose the effectors in lipid membrane-enveloped vesicles (liposomes). By using appropriate membrane components, it is possible, on the one hand, to bind the desired targeting molecules to the liposome membranes, on the other hand, to coat the carriers with anti-immunogenic coatings (e.g. polyethylene glycol) and thereby protect them against non-specific removal from the blood stream by the reticuloendothelial system , The advantages of this system are offset by the following serious disadvantages:

• - The thermal and temporal stability of a single Lipid bilayer existing vesicles is limited, as is the Tightness. The permeability of the membranes for hydrophilic substances can be reduced in principle, but the required modified (e.g. fluorinated) lipids not biologically harmless.  In addition, a single "hit" of the complement system is sufficient to Leakage of a complete vesicle.
• - The lack of stability of the membrane vesicles in turn limits the possible variability in surface design and limited thereby the potential applications.
• - Only hydrophilic substances can in the aqueous inner phase Vesicles are transported in sufficient concentration.
• - The liposomes are loaded (from a few special cases apart from by simply including part of the aqueous phase and is accordingly inefficient: typically <0.5% the effector substance enclosed in the vesicles. Here, the Substantial thermal and chemical loads exposed (the working temperature must be above the critical phase transition temperature of the lipid mixture lie, and the reactive groups required for the covalent modification only survive this at a very low pH value).
• - Half of the membrane-based reactive groups responsible for Linkage with proteinaceous search molecules is needed after the vesicle formation on the inside and is not available Binding is available, however, after the resolution of the Liposomes are released in the organism and can become unpredictable Lead reactions.
• - The chemical coupling of proteins to liposomal membranes (e.g. directly or via a polyethylene glycol arm with lipids  linked SPDP) leads to the formation of highly immunogenic structures, that have already been successfully used for vaccination on the However, the field of "drug targeting" can be regarded as unusable because they lead to an immune reaction against the particles.

Solid colloid particles ("nanoparticles") are available as an alternative Available. In principle, nanoparticles are known. Particles in Micrometer and submicrometer range made of hydrophobic polymers can in principle by fine dispersion of the in a non-polar Solvent-absorbed polymer can be produced: By Removal of the solvent drops the polymer in the form of Particles whose diameter is below that of the droplets; a Loading with hydrophobic substances (in which category the most drugs fall) by simply adding the substance to the non-polar solvent: After removal of the solvent is almost 100% active substance associated with the polymer and remains when the particles are in a aqueous phase are introduced by Van der Waals forces and steric "entrapment" not covalent, but stable over the long term Particle matrix bound. It is essential here that a subsequent Coagulation of the hydrophobic particles (their large contact area with the hydrophilic medium is energetically unfavorable) is prevented, for example by producing the particles in the presence an amphiphilic substance that is between hydrophobic Mediated particle matrix and hydrophilic medium, can happen.  

Conventional nanoparticles and their production and Possibilities of a surface variation are from WO 96/20698 known, the system here in particular for the intravascular Use, especially in the case of restenosis has been optimized. The Patent application describes polymolecular nanoparticles natural or synthetic polycondensates with a predominantly generally described property-modifying Natural or synthetic surface covering Macromolecules.

The following documents may also be mentioned as examples.

DE 198 10 965 A1 describes polymolecular nanoparticles a polyelectrolyte complex of polycations and polyanions, which is treated with a crosslinking agent.

DE 198 39 515 A1 describes colloidal polymer Active substance associates with a property-optimized branched Polyol esters especially for use on mucosal tissues described.

US 6,117,454 describes in particular polymolecular Nanoparticles, which are coated with fatty acid derivatives Penetration of the blood-brain barrier are suitable.

In US 5,840,674 are firmly covalent via a "linker" bound complexes of active ingredient and microparticles in particular described for use against microorganisms.  

US 5,641,515 describes polymolecular nanoparticles Polycyanoacrylate containing insulin, which is the complex bound Release insulin in a controlled manner.

The previously known nanoparticles and methods for their Manufacturing often exhibit problems in stability and Producibility as well as in the degree and extent of loading on and can only be used to a very limited extent. In particular, that is also Problem of targeted application of the active ingredient to the desired one The place of action and the stability of the nanoparticles after administration up to Reaching the site of action, "drug targeting" insufficient solved. A central problem is the surface modification of nanoparticles. As already mentioned above, conventional ones solid nanoparticulate systems (in contrast to liposomes) due to their extremely high specific interphase between hydro phober particle matrix and hydrophilic medium an energetic unfavorable system that in the absence of stabilizers is unstable: The aggregation of particles makes the minimized energetically unfavorable contact area, a precipitation the result is the hydrophobic particle material.

For this reason, conventional nanoparticles need one Coating, for example, from amphiphilic molecules that the Reduce interfacial energy and in this way the particles stabilize. This coating completely covers the particle matrix and thus deprives them of access to modifying agents. A Modification of the amphiphilic molecules leads on the other hand  to a drastic change in physical properties and thus destabilizing the coating. For this reason it is hardly possible to modify conventional nanoparticles in such a way that binding of ligands and thus an application in the field of "drug targeting "is possible. Those previously offered in the prior art Solutions either only deal with surface modification generally or so specifically directed to a problem that a more general application is not possible or the presented solution is disadvantageous in itself.

The object of the invention was therefore to provide nanoparticles places that do not have the disadvantages described and in particular are able to produce a specific release on To reach the site of action.

This problem is solved by solid particles for the transport of hydrophobic or hydrophobized pharmaceutical active substances, which can be produced by a process with the following steps:

• a) Preparation of a solution in or a mixture of organic solvent (s) containing at least one hydrophobic or hydrophobized pharmaceutical active ingredient, water-insoluble organic polymer material and amphiphilic organic polymer material and optionally Supplements,  
• b) treatment of the solution with ultrasound,
• c) Dialysis of the solution against H ₂ O
• d) separation of the resulting particles from the resulting aqueous solution.

The particles according to the invention are particularly suitable forms with which to increase the impact and minimize the Side effects controlled and / or spatially specific release of the effector molecule is achieved. As a general rule are the particles encompassed by the invention preferably solid, colloidal and / or lipid-free Particle systems.

Hydrophobized active substances are originally meant more hydrophilic active ingredients by chemical modification have become more hydrophobic. One example is hydrophobic Absorption ester of hydrophilic active substances.

When producing the above nonoparticles according to the invention, it is particularly preferred if the organic solvent or solvents in water in the ratio solvent: water between 1:10 and 1:50, preferably between 1:20 and 1:40, in particular between 1:20 and 1: 30 solve and / or are preferably selected from:
Methylene chloride or benzyl alcohol, preferably benzyl alcohol.

The choice of a suitable solvent is by no means an issue Restricted methylene chloride or benzyl alcohol.

It is further preferred if the particles according to the invention the water-insoluble organic Polymer material together with the or the hydrophobic pharmaceutical active ingredient (s) dissolved and the amphiphilic organic Polymer material first separated from it, if necessary with the or the supplement (s) is dissolved and the solutions for Preparation of the solution after step (a) can only be mixed.

Another preferred form of production of the invention Particle contains the separately dissolved amphiphilic organic Polymer material and / or an optionally co-dissolved Supplement before mixing with the non-polar Polymer material in a concentration of between 10 and 45% (w / v), preferably between 20 and 40% (w / v), in particular of 35% (w / v) is present.

It is equally preferred if the water-insoluble organic Polymer material before step (b) in a concentration of between 3 and 0.1% (w / v), preferably between 2 and 0.5% (w / v), especially 1.7% (w / v).

It is further preferred if the particles according to the invention follow a process be made; where the solution in step (b) between 1 h and 15 h, preferably 4 h or 5 h to 10 h,  especially 5 h to 6 h, is treated with ultrasound. Doing the Ultrasound treatment is preferred at maximum power.

It is equally preferred if the concentration ratio in% (w / v) between the water-insoluble organic polymer material from step (a) and the amphiphilic polymer material from step (b) after completion of step (b) between 1: 2 and 1:32, preferably between 1: 4 and 1: 28, in particular between 1: 8 and 1: 20, is preferably between 1:12 and 1:16, in particular 1:14.

When producing the above particles according to the invention, it is particularly preferred if the water-insoluble, organic polymer material from step (a) is selected from
Polyesters, preferably polylactic acid (polylactide), polyglycolide or polylactide / polyglycolide copolymers (PLGA), in particular pure polylactide, pure polypropylene glycol, or polylactide / polyglycolide copolymer (for example in a ratio of 3: 1).

However, the particles according to the invention can also be produced on other biocompatible, degradable synthetic polymers such. B. corresponding polyanhydrides, polyamino acids, polycyanoacrylates, Polyacrylamides or polyurethanes can be used.

When producing the above particles according to the invention, it is preferred if the amphiphilic organic polymer material from step (a) is selected from
Polyvinyl alcohol or derivatives of polyvinyl alcohol, preferably pure polyvinyl alcohol, esters of polyvinyl alcohol and a hydrophobic carboxylic acid (preferably fatty acid) or coesters, in which each molecule of the polyvinyl alcohol is esterified with at least one hydrophobic carboxylic acid (preferably fatty acid) and a second, different carboxylic acid.

When producing the above particles according to the invention, it is particularly preferred if the supplement (s) is / are selected from
Alkali or alkaline earth metal salts of organic acids, especially magnesium salts, preferably magnesium acetate.

It is also preferred if the process steps (a) to (c) in physiological temperatures, preferably between 35 and 40 ° C, take place especially at 37 ° C.

Another object of the invention that fulfills the object of the invention is solid particles for the transport of hydrophobic or hydrophobized pharmaceutical active substances, which contain a core of organic, water-insoluble polymer material and an outer layer of amphiphilic polymer material which is non-covalently bound to the molecules of the core and in which the amphiphilic polymer material is selected from
Polyvinyl alcohol or esters of polyvinyl alcohol.

It is particularly preferred that the particles are noncovalent bound, hydrophobic or hydrophobized pharmaceutical Contain active ingredient.

It is also preferred if the amphiphilic polymer material is selected from among these particles according to the invention
pure polyvinyl alcohol, esters of polyvinyl alcohol and a hydrophobic carboxylic acid (preferably fatty acid) or coesters, in which each molecule of the polyvinyl alcohol is esterified with at least one hydrophobic carboxylic acid (preferably fatty acid) and a second, different carboxylic acid.

It is a selected embodiment for all of the abovementioned particles according to the invention if the amphiphilic organic polymer material used is selected from coesters of polyvinyl alcohol in which each molecule of the polyvinyl alcohol is esterified with at least one hydrophobic carboxylic acid (preferably fatty acid) and a second, different carboxylic acid,
wherein the hydrophobic carboxylic acid is preferably selected from fatty acids with a length between 10 and 24 carbon atoms, which are preferably not or not substituted with COOH, OH, SH or NH ₂ , in particular not with COOH or OH, preferably selected from C ₁₀ - C ₁₆ fatty acids, especially lauric acid,
the second, different carboxylic acid being selected from carboxylic acids which are preferably not substituted with COOH or OH and preferably with SH or NH ₂ , preferably NH ₂ , in particular amino acids, preferably alanine,
is preferably selected from coesters in which the polyvinyl alcohol is esterified with at least one fatty acid and at least one amino acid, in particular polyvinyl laurate-co-β-alanate, preferably polyvinyl laurate (25%) - co-β-alanate (7%).

It is for all of the inventive particles described above a preferred embodiment of this invention when the particles Nanoparticles are and / or accordingly in at least two Dimensions a length of between 10 and 500 nm, preferably <150 nm, in particular 50 to 100 nm.

Nanoparticles in the narrower sense of this invention are understood to mean particles which have a length of less than 1 μm in each dimension and, in a broader sense, particles which have a length of less than 1 μm in at least two dimensions. The narrower definition also includes in particular all particles which have a volume of less than 1 μm ³ , preferably 0.01 μm ³ , in particular 0.0001 μm ³ . Nanoparticles are solid colloidal particles.

Surface-modified particles in particular are a central subject of this invention and achieve the object of the invention in an outstanding manner. The invention therefore furthermore relates to particles for transporting pharmaceutical active substances to which linker molecules which have an amino- and / or thiol-reactive, preferably an amino-reactive, group are covalently above (ie before modification by linking the particle to the linkers) free NH ₂ or SH groups, preferably NH ₂ groups, are bound on the surface of the particle.

Linker molecules are understood to be polymers, in particular unbranched polymers, the properties, especially the Change surface properties of the particle, but especially for the sterically favorable connection of other bioactive Connections to the particles are used or, if necessary, the Protect particles sterically from degradation.

The "reactive group" includes, in particular, the prior art to understand known groups that are easy on amino, thiol or Bind hydroxyl groups, as well as epoxy or vinyl groups.

It is particularly preferred if the linker molecules are bifunctional and in addition to an amino- or thiol-reactive, preferably amino reactive, grouping at another end of the molecule also another, differently reactive functional Grouping, preferably a thiol-reactive grouping, exhibit.  

It is also preferred if the linker molecules are a mixture bifunctional molecules - as described above - and monofunctional molecules that are only either the amino reactive or the thiol-reactive, preferably the amino-reactive, grouping wear, are.

Another preferred object of the invention are particles for Transport of active pharmaceutical ingredients in which on the surface the particle is a mixture of two types of linker molecules is present that is reactive at one end of the linker molecule Grouping are covalently bound to the surface of the particle, where the one type of linker molecules (bifunctional) at least another reactive group at another end of the molecule carries while the other type of linker molecules (monofunctional) no more reactive at any other end of the molecule Groupings.

Both of the aforementioned groups of particles are very cheap because of that bulky residues such as antibodies on the bifunctional residues can be freely stored while the monofunctional linkers sterically prevent degradation. This Particles are also known as acanthus.

It is again particularly preferred if the surface of the both of the aforementioned particle types (acanthospheres) significantly more, preferably at least 100% more, monofunctional than bifunctional molecules are covalently bound.  

It is particularly preferred if the linker molecules Polyglycolides are, preferably polyethylene glycol derivatives, especially NHS ester polyethylene glycol or NHS Ester / vinylsulfone polyethylene glycol.

It is also preferred if those provided with linkers particles according to the invention are nanoparticles.

It is further preferred if the particles have hitherto been in the previous sections without mentioning linkers described particles according to the invention.

It is preferred if attached to the bifunctional linker molecules bioactive macromolecules or "finder" molecules selected from Peptides, proteins; preferably antibodies, Antibody fragments or antibody derivatives with target binding Properties such as "single-chain" antibodies; Hormones, sugars, preferably glycosides; synthetic or natural receptor Ligands; Proteins or peptides with a free cysteine group or thio sugars, are coupled, are coupled or in front of the Surface modification were coupled.

Under the term "finder" molecule in the sense of this invention is generally understood to mean the particles according to the invention connectable connections that are able with high affinity the biological goals of the active ingredients, as if there were proteins, Peptides, polysaccharides, oligosaccharides, lipoproteins, Glycoproteins or other biological molecules that are either in  healthy tissue (physiological) or in or near the sick Tissue (pathological) expressed to bind. "Viewfinder" - Molecules can, for example, peptides, proteins, for example Antibodies, antibody fragments or antibody derivatives with target-binding properties such as "single-chain" antibodies; Hormones, sugars, for example glycosides; synthetic or be natural receptor ligands. Are particularly preferred Antibodies, derivatives, fragments and glycosides.

It is further preferred if attached to the bifunctional linker molecules bioactive macromolecules or more generally "finder" molecules, preferably antibodies, antibody fragments or Antibody derivatives with target binding properties such as B. "Single chain "antibodies, especially those with a free cysteine group, are coupled, are coupled or before the Surface modification were coupled. This is especially true for particles whose coating is significantly more monofunctional than contains bifunctional molecules.

It is also preferred if the bifunctional linker molecules bioactive micromolecules or "finder" molecules, preferably Sugar, in particular thio sugar, hormones or proteins, in particular with a free cysteine group, are coupled are or were coupled before the surface modification. This applies in particular to particles whose coating is predominant or consists entirely of bifunctional molecules.  

It is further preferred if the particles according to the invention after binding the bioactive micromolecules or "finder" molecules free reactive groups are still saturated, preferably with Cysteine.

In general, a subsequent cleaning or if necessary Isolation preferably via dialysis, preferably with selective exclusion membranes.

For all particles according to the invention it is preferred if the transporting active pharmaceutical ingredient a synthetic or natural active ingredient, a protein, peptide, lipid, or sugar Nucleic acid or a low molecular weight organic or high molecular weight organic active ingredient, for example a hormone, an antineoplastic substance, an antibiotic, antifungal, Paraside, virustatic or antihelmintic, a cardiovascular active substance; a central active substance, especially a Analgesic, antidepressant or antiepileptic; is.

In general, it is a preferred embodiment of all particles according to the invention if the particle is linked directly or via a linker, preferably via bifunctional polyethylene glycol molecules, to a "viewfinder" molecule selected from:
Peptides, proteins; preferably antibodies, antibody fragments or antibody derivatives with target-binding properties such as "single-chain"antibodies; Hormones, sugars, preferably glycosides; synthetic or natural receptor ligands; Proteins or peptides with a free cysteine group or thio sugars.

Especially the use of the bifunctional polyethylene glycol Molecules for surface modification of particles for transport Active pharmaceutical ingredients is an important part of this invention. Therefore, another object of this invention is the use of pure bifunctional polyethylene glycol molecules, preferably NHS ester / vinyl sulfone polyethylene glycol; or Mixtures of bifunctional and monofunctional Polyethylene glycol molecules, preferably NHS esters "- Polyethylene glycol with NHS ester / vinyl sulfone polyethylene glycol; to Production of surface-substituted solid particles for Transport of active pharmaceutical ingredients.

It is particularly preferred if "seeker" molecules are selected from the bifunctional polyethylene glycol molecules
Peptides, proteins; preferably antibodies, antibody fragments or antibody derivatives with target-binding properties such as. B. single chain antibodies; Hormones, sugars, preferably glycosides; synthetic or natural receptor ligands; Proteins or peptides with a free cysteine group or thio sugars are bound to the particles, preferably when using pure bifunctional polyethylene glycol molecules gylycosides, especially thio sugar; when using mixtures of bifunctional and monofunctional polyethylene glycol molecules antibodies, antibody fragments or antibody derivatives with target-binding properties such as. B. single-chain antibodies, preferably antibodies with a free cysteine group.

The processes for producing particles according to the invention are also an important part of the invention. Another object of the invention is therefore a method for producing a particle according to the invention, in particular the second type of particle described with PLGA (polylactide / polyglycolide); does the following:

• a) Preparation of a solution in or in a mixture of organic solvent (s) containing at least one hydrophobic active pharmaceutical ingredient, water-insoluble, organic polymer material and amphiphilic organic Polymer material,
• b) (b treatment of the solution with ultrasound,
• c) Dialysis of the solution against H ₂ O
• d) separation of the resulting particles from the resulting aqueous solution.

The particles according to the invention are particularly suitable forms for Enhancement of the desired effects of known active ingredients and for Minimizing systemic side effects through controlled and / or spatially specific release of the effector molecule is achieved. This makes them suitable and intended in therapeutics to be used in various ways. Another item The invention is therefore pharmaceuticals, the particles of the invention and, if appropriate, suitable additives and / or auxiliaries contain.

In principle, the medicaments according to the invention can be used as liquid Dosage forms in the form of aerosols, injection solutions, drops or juices or as semi-solid dosage forms in the form of granules, Tablets, pellets or capsules can be administered.

Suitable additives and / or auxiliary substances are e.g. B. solution or Diluents, stabilizers, suspending agents, Buffer substances, preservatives, as well as dyes, fillers, and / or binders. The selection of excipients as well as the the amounts to be used depends on whether the Medicines e.g. B. inhalative, oral, oral, parenteral, intravascular, intravenously, intraperitoneally, rectally, subcutaneously or intramuscularly should be applied. Are suitable for oral applications Preparations in the form of tablets, dragees, capsules, granules or suspensions such as drops, juices and syrups, for others Applications suspensions as well as easily reconstitutable Dry formulations.  

As stated above, the particles according to the invention are special suitable forms with which to increase the effect and Minimization of side effects through controlled and / or spatially specific release of the effector molecule is achieved, so that a general usability of these particles for production of therapeutic agents and of course they are generally for one unlimited number of indications. Without the use of wanting to limit the particles of the invention to this offers their use for special indications. Another The invention therefore relates to the use of Particles of the invention for the manufacture of a medicament for Cancer treatment, to treat infectious diseases and Parasitosis, used to treat diseases and symptoms central nervous cause, for use in gene therapy or for genomic targeting. Another example is the use of Targeting cytostatics on tumor cells when transporting therapeutically usable substances through the blood-brain barrier and in the treatment of serious infections (especially through Eukaryotes) also preferred.

Other possible uses include e.g. B. the transfer of vegetable alkaloids with microbicidal activity in trypanosomes and of antioxidants and anti-inflammatory compounds [Vitamin E, gallic acid, N-acetyl-L-cysteine, 2,6-bis (tert-butyl) -4- Mercaptophenol, ibuprofen and gentisic acid] in (degenerative) Brain diseases, the transfer of substances in hepatocytes, primarily for the treatment of neoplasms, also increasing the  Effect of Primaquin on those that persist in the liver cells Plasmodium Hypnozoiten.

Among the possible applications for the particles according to the invention, the following should be mentioned in particular:

• - Transport of otherwise non-brain-accessible pharmaceuticals (e.g. Cytostatics, psychotropic drugs, pain relievers, M 'Alzheimer's Therapeutics) with antibody-conjugated carriers through the blood Brain barrier
• - Targeted introduction of pharmaceuticals (e.g. antivirals, Cytostatics, plasmodicides) with glycoside-conjugated carriers in hepatocytes
• - Oral administration of otherwise only parenterally available Pharmaceuticals by targeting intestinal epithelia
• - Increase the effect of anti-parasitic therapeutic agents Targeting parasite-specific surface molecules.

Another object of the process is treatment of a human or animal who needs this treatment, with or using the particles according to the invention. This treatment is particularly suitable for the aforementioned Indications and types of use.

In the following section, the invention is further illustrated by examples explained without restricting them to it.  

Examples and illustrations pictures

Fig. 1 shows schematically the general structure and shape of particles according to the invention, simple particles, simple stealth particles, target-seeking actinospheres and target-seeking acanthospheres.

Fig. 2 shows the uniformity in the size distribution of particles produced according to the invention according to Example 1.

Examples General remarks

The examples set out below describe particles encompassed by the invention, in particular nanoparticles, in which the possibility is realized of incorporating active substances into colloidal carrier particles. These can optionally be linked, for example, with antibodies to or natural ligands for characteristic molecular structures of the target or other “searcher molecules”. Optionally, the nanoparticles can be protected against the immune system at the same time, for example, by inert coating of their surface. In general, the particles encompassed by the invention described here by way of example are colloidal, lipid-free particle systems. Some of the particles described here and included in the invention are referred to below with the general terms actinospheres and acanthospheres (see Fig. 1). Because they denote structural concepts and non-steric basic forms, these designations are retained regardless of the actual geometry.

example 1 General nanoparticles

These are particles in the size range between 10 nm and 500 nm with a compact inner part made of organic water-insoluble polymer material, which with a layer of a amphiphilic organic polymer coated due to its Properties non-covalent, but firm to the water-insoluble Particle matrix is bound and the particles both against the aqueous environment stabilized as well before sticking together and the protects the resulting flocculation. Furthermore, this is amphiphilic organic polymer with reactive functional groups provided that make it possible to chemically add other components to it to dock.

As material for the production of the nanoparticles of this example polylactic acid (polylactide, PLA) was chosen. Existing Literature on the production of particles in the size range of 1-10 µm (used for other purposes) confirms the suitability of these  Substance that has the advantage over most other polymers has biodegradability. It is also possible to use PLA to provide more hydrophilic end groups, which then during production at the interface with the aqueous medium orient and either as a low-molecular target seeker act or used as an anchor point for protein targeters can be.

The manufacture of nanoparticles was based on the substance RG752 (from Boehringer Ingelheim), a block copolymer made from 75% Polylactide and 25% polyglycolide with a MW of on average 12 kDA, established and based on the pure polylactide R202H (Boehringer) and polypropylene glycol 26000 (Sigma; at Room temperature liquid) successfully applied. The respective Polymer is - possibly in the presence of those to be transported Substance - dissolved in benzyl alcohol (final concentration 5%) and this Solution with twice the volume of a highly osmotic Protective colloids (35% polyvinyl alcohol and 35% magnesium acetate) mixed. With ultrasound treatment lasting several hours, this becomes dissolved polymer finely dispersed. By dialysis of the emerging milky product against a large excess of water become magnesium acetate and benzyl alcohol (soluble in water 1:25) as well as excess polyvinyl alcohol removed, so that an aqueous Suspension of polyvinyl alcohol coated nanoparticles remains.  

The manufacturing process was evaluated according to the principle of "quasi-elastic light refraction" (QELS) in a Zetasizer device (Zetasizer 3000 HS, Malvern). It could be shown that after simply mixing dissolved polymer and Protective colloid (stirrer, vortex) still liquid particles in the Micrometer range are present that are only under the ultrasound treatment transition into nanoparticles. In accordance with the model, after which the polyvinyl alcohol stabilizes the interfaces is the Polyvinyl alcohol concentration is the decisive factor for the Diameter of the resulting nanoparticles: 10% polyvinyl alcohol led to the formation of particles in the range of 300-400 nm, 35% Particles of 50-100 nm. (The critical size for colloidal Systems is ~ 150 nm; Particles below this size can in principle d. H. when linked to suitable search molecules, the Cross the blood-brain barrier.) The temperature was at all Try largely in the physiological area, or between 25 ° and 45 ° C kept.

The duration of the ultrasound treatment has no discernible influence on the particle size, but probably on the yield of usable Particles: After a treatment of 15 minutes, around 90% of the counted particles, but together only ~ 1% of the total Make up polylactide mass, in the desired size range, while the rest consists of microparticles; after 1 h of sonication comprises the nanoparticle fraction ∼10% of the mass; after 5-6 hours between 70% and 99% of the total polylactide in nanoparticles implemented. The rest are in two largely discrete ones  Microparticle fractions before, a smaller one with a diameter from half to whole and a larger one Diameter of several micrometers, which is a two-phase Could reflect the course of the dispersion process. After about Decomposition (recognizable by the discoloration) and began at 15-18 h Clumping until finally the whole after 24 hours Reaction mixture had degenerated to a waxy mass.

A secondary peak in the range of occurred in some measurements 250-300 nm; through dilution experiments, however, was shown be that these are only aggregates of the relative acted hydrophobic particles. Lead to higher particle densities moreover, with all tested substances to an apparent Diameter of the individual particles, which is 20-30% above the real one Diameter lies because apparently interactions of the particles as Inhibit the basis for the movement used for the QUELS measurement. As expected, both manifestations were these Aggregation tendency for the more hydrophobic substances R202H and polypropylene glycol at both high and low Thinning significantly more pronounced than in the corresponding samples of nanoparticles from the Glycolide-containing copolymer RG752.

Below are the results of a typical measurement (1 vol. 5% RG752 in benzyl alcohol with 2 vol. 35% magnesium acetate / 35% Polyvinyl alcohol for 6 hours at maximum performance and 42 ° C sonicated, then dialyzed against 600 vol. water for 48 hours  

[Exclusion size of the membrane 25-30 kDa, after 24 hours of renewing the dialysis water]; Samples diluted 1:36 with water before measurement):

The size distribution observed after filtration is one good preparation of liposomes comparable, the advantage lies in the incomparably higher packaging efficiency.

By linking the used as a protective colloid Polyvinyl alcohol with various organic acids could Surface properties of the particles are changed. in this connection Hydrophobization of the polyvinyl alcohol, its Water solubility at the upper limit for one Coating substance is essential as the exclusive Introduction of reactive groups to the hydrophilicity of polyvinyl alcohol increased to such an extent that it simply went into solution and there was no more particle formation. This could be done through partial Esterification with fatty acids via the acid chlorides can be achieved. The polyvinyl laurate (20%) thus obtained showed Particle binding properties that those of the original Polyvinyl alcohol were superior: For comparable quantities Protective colloids were made much shorter with polyvinyl laurate Sonication time and somewhat smaller particles with higher yield educated.

In the next step, polyvinyl alcohol was reacted simultaneously with different amounts of lauryl chloride and β-alanyl chloride, so that polyvinyl laurate (25%) - co-β-alanate (7%) was formed. This substance was comparable to polyvinyl laurate in its particle formation properties, but provided large amounts of covalently bonded primary amino groups. Due to the loose surface structure, which allows the charges of NH ₄ ⁺ groups to be balanced by intermediate acetate, no surface potential was observed, but the existence of the amino groups bound to supermolecular structures could be clearly demonstrated by chemical means.

Example 2 Production of the actinospheres and acanthospheres a) General comments

They exist in both actinospheres and acanthospheres other components from a bifunctional Polyethylene glycol molecule that has an amino reactive at one end Group carries, at the other end a different group different reactivity. This bifunctional polyethylene glycol on the one hand, the particles become steric by blocking the surface with an inert molecule removed from the access of the immune system (Similar things were done in connection with liposomes under the "Stealth technology" tested) and beyond stabilized, on the other hand the possibility is offered, further Molecules that convey the actual target specificity in spatial convenient and flexible position to attach to the particles. With these Molecules can be micromolecules, e.g. B. sugar, in which case an exclusive use of the bifunctional Polyethylene glycol is possible (actinospheres), or around  Macromolecules such as antibodies (acanthospheres) in which If it is advisable for steric reasons, only a small part of the functional groups on the particle surface with bifunctional Implement polyethylene glycol and the rest monofunctional polyethylene glycol, which is then the only serves physical and immunological stabilization of the particles, saturate.

It is also recommended that after the nanoparticle PEG conjugates with the "searcher" molecules may still be free Coupling groups at the distal ends of the PEG spines a high molar excess of a suitable reactant saturate, in the case of vinyl sulfone z. B. Cysteine. In this way it is prevented that these coupling groups in the organism with the body's own molecules (e.g. serum proteins) react and thus the target specificity is falsified.

b) concrete implementation following example 1

Following the production of the nanoparticles according to Example 1, these were modified with polyethylene glycol on the surface. By reaction of the particles coated with polyvinyl laurate (25%) - co-β-alanate (7%) with NHS ester / fluorescin-polyethylene glycol in a neutral to weakly basic environment, followed by removal of the unbound polyethylene glycol by dialysis of the particle suspension against water "Proto-actinospheres" were obtained, the properties of which could be investigated on the basis of the fluorescence and the physical properties of the polyethylene glycol molecules. It could be shown that a covalently bound layer of fluorescence-labeled polyethylene glycol covers the surface of the nanoparticles in high molecular density ("self-quenching" of the fluorescence) and with a thickness of 10-15 nm (hydrated), as in the schematic drawing for " Actinospheres "( Fig. 1). The particle formation properties of polyvinyl laurate-co-β-alanate were clearly superior to those of unmodified polyvinyl alcohol.

By reacting with polyvinyl laurate (25%) - co-β-alanate (7%) coated particles with NHS ester / vinyl sulfone polyethylene glycol in a weakly basic environment and subsequent purification by Accordingly, dialysis against water can receive particles be at the distal, largely freely movable end of the Polyethylene glycol covalently bonded to the particle surface "Spines" wear thiol-reactive groups.

When using the slow-reacting, relatively water-stable Vinyl sulfone group as the distal reaction group of the PEG spines it is possible to use these thiophilic particles for all applications in one to produce standardized standard procedures and only after the To combine purification with the appropriate "finder molecules", which can be of any chemical nature and only one Must have sulfhydryl group, so that actinospheres and  Acanthospheres can be made. With acanthospheres a simple in-process control of the last linking step by adding fluorescence-labeled or self-fluorescent Proteins (GFP) or by Western blot of samples, for actinospheres by detection in the Thin layer chromatography.

Example 3 Efficacies and properties of nanoparticles produced according to example 1 or 2

The loading of the nanoparticles was determined using the relative hydrophobic, usable for staining cell membranes Fluorescent dye 4-Di-10ASP from the group of Dialkylaminostyrole (Molecular Probes Inc.) tested and corresponded to those based on the measured size distributions Expectations: Around 90% of the fluorescent dye used were properly packaged in nanoparticles. (In watery Solution, uncharacterized physical effects interfered in inverse dependence on the degree of dilution with the fluorescence, the 4-Di-10ASP content could therefore only be resolved after the particles had dissolved in an excess of a methanol-chloroform mixture can be determined fluorometrically.)

The structural stability of the particles turned out to be remarkable high: without the addition of preserving substances or the like, after a  eight-week storage at room temperature without further addition stabilizing substances no significant change in Size distribution or 4-Di-10ASP loading can be determined.

With the functional surface groups covalently with Linked "spikes" made of polyethylene glycol (MW 3400) Nanoparticles, at the distal ends of which are antibodies or other molecules that can be used for targeting are added may have been a reduction in unwanted intake of the Particles through the reticuloendothelial system on the rat model shown.

Example 4 Efficacy of acanthospheres

Particles for the transport of active pharmaceutical ingredients with a volume of <1 µm ³ loaded with tritium-labeled daunomycin were linked via their surface amino groups either to monofunctional polyethylene glycol (PEG) or via bifunctional (NHS-ester / vinylsulfone) PEG with an average molecular weight of 3400 Da Cysteine residues of different "seeker" proteins coupled:

• Human transferrin in various concentrations,
• - bovine serum albumin and  
• - single-chain antibodies against the transferrin receptor.

Finally, there were free coupling groups with cysteine saturated. There was also a fraction of the particles with no "seeker" - Molecule made.

The acanthospheres were parasitic-type Unicellular Trypanosoma brucei brucei incubated and binding as well Cytotoxicity determined.

The results of the binding and cytotoxicity studies show one Correlation between cytotoxicity and binding. And the one with Acanthospheres provided with "viewfinder" proteins reduced the Cell density of the parasites clearly compared to controls, i.e. i.e. it a pronounced cytotoxic effect was observed. In Absence of "seeker" proteins, however, were the same Daunomycin concentration neither binding nor cytotoxicity observe.

Claims (27)

1. Solid particles for the transport of hydrophobic or hydrophobized pharmaceutical active substances, producible by a method with the following steps:

• a) preparation of a solution in one or a mixture of organic solvents containing at least one hydrophobic or hydrophobized pharmaceutical active substance, water-insoluble organic polymer material and amphiphilic organic polymer material and, if appropriate, supplements,
• b) treatment of the solution with ultrasound,
• c) Dialysis of the solution against H ₂ O
• d) separation of the resulting particles from the resulting aqueous solution.

2. Particles according to claim 1, characterized in that the organic solvent or solvents in water in the ratio solvent: water between 1:10 and 1:50, preferably between 1:20 and 1:40, in particular between 1:20 and 1 : 30 solve, and / or are preferably selected from:
Methylene chloride or benzyl alcohol, preferably benzyl alcohol,
and or
that the water-insoluble organic polymer material before step (b) in a concentration of between 3 and 0.1% (w / v), preferably between 2 and 0.5% (w / v), in particular of 1.7% (w / v) is present,
and or
that the solution in step (b) is treated with ultrasound between 1 h and 15 h, preferably 4 h or 5 h to 10 h, in particular 5 h to 6 h,
and or
that the concentration ratio in% (w / v) between the water-insoluble organic polymer material from step (a) and the amphiphilic polymer material from step (b) after completion of step (b) is between 1: 2 and 1:32, preferably between 1: 4 and 1:28, in particular between 1: 8 and 1:20, preferably between 1:12 and 1:16, in particular 1:14,
and or
that process steps (a) to (c) take place at physiological temperatures, preferably between 35 and 40 ° C, in particular at 37 ° C,
and or
that the water-insoluble organic polymer material is dissolved together with the hydrophobic pharmaceutical active substance (s) and the amphiphilic organic polymer material is first separated therefrom, optionally with the supplementary substance (s), and the solutions for preparing the solution after step (a) only then be mixed, it being preferred
that the separately dissolved amphiphilic organic polymer material and / or an optionally added supplement before mixing with the non-polar polymer material in a concentration of between 10 and 45% (w / v), preferably between 20 and 40% (w / v), in particular of 35% (w / v) is present. 3. Particle according to one of claims 1 or 2, characterized in that the water-insoluble, organic polymer material from step (a) is selected from
Polyesters, preferably polylactic acid (polylactide), polyglycolide or polylactide / polyglycolide copolymers (PLGA), in particular pure polylactide, pure polypropylene glycol, or polylactide / polyglycolide copolymer (preferably in a ratio of 3: 1)
and or
that the amphiphilic organic polymer material from step (a) is selected from
Polyvinyl alcohol or derivatives of polyvinyl alcohol, preferably pure polyvinyl alcohol, esters of polyvinyl alcohol and a hydrophobic carboxylic acid (preferably fatty acid) or coesters in which each molecule of the polyvinyl alcohol is esterified with at least one hydrophobic carboxylic acid (preferably fatty acid) and a second, different carboxylic acid,
and or
that the supplement (s) is / are selected from
Alkali or alkaline earth metal salts of organic acids, especially magnesium salts, preferably magnesium acetate. 4. Solid particles for the transport of hydrophobic or hydrophobized pharmaceutical active substances, containing a core of organic water-insoluble polymer material and an outer layer of non-covalently bound to the molecules of the core, amphiphilic polymer material, characterized in that the amphiphilic polymer material is selected from
Polyvinyl alcohol or esters of polyvinyl alcohol. 5. Particles according to claim 4, characterized in that the Particles non-covalently bound, hydrophobic or contain hydrophobized pharmaceutical active ingredient. 6. Particles according to one of claims 4 or 5, characterized in that the amphiphilic polymer material is selected from
pure polyvinyl alcohol, esters of polyvinyl alcohol and a hydrophobic carboxylic acid (preferably fatty acid) or coesters, in which each molecule of the polyvinyl alcohol is esterified with at least one hydrophobic carboxylic acid (preferably fatty acid) and a second, different carboxylic acid. 7. Particles according to one of claims 1 to 6, characterized in that the amphiphilic polymer material
is selected from coesters of polyvinyl alcohol in which each molecule of the polyvinyl alcohol is esterified with at least one hydrophobic carboxylic acid (preferably fatty acid) and a second, different carboxylic acid,
wherein the hydrophobic carboxylic acid is preferably selected from fatty acids with a length between 10 and 24 carbon atoms, which are preferably not or not substituted with COOH, OH, SH or NH ₂ , in particular not with COOH or OH, preferably selected from C ₁₀ - C ₁₆ fatty acids, especially lauric acid, and / or
the second, different carboxylic acid being selected from carboxylic acids which are preferably not substituted with COOH or OH and preferably with SH or NH ₂ , in particular NH ₂ , in particular amino acids, preferably alanine,
is preferably selected from coesters in which the polyvinyl alcohol is esterified with at least one fatty acid and at least one amino acid, in particular polyvinyl laurate-co-β-alanate, preferably polyvinyl laurate (25%) - co-β-alanate (7%). 8. Particles according to one of claims 1 to 7, characterized characterized in that the particles are nanoparticles and / or in at least two dimensions a length of between 10 and  500 nm, preferably <150 nm, in particular 50 to 100 nm exhibit. 9. Particles for the transport of active pharmaceutical ingredients, characterized in that linker molecules which have an amino- and / or thiol-reactive, preferably an amino-reactive, grouping, covalently via NH ₂ or SH groups which are previously free on the surface of the particle, preferably NH ₂ groups to which particles are bound. 10. Particles according to claim 9, characterized in that the Linker molecules are bifunctional and in addition to an amino or thiol-reactive, preferably amino-reactive, grouping at another end of the molecule also another, different reactive functional grouping, preferably one thiol-reactive grouping. 11. Particles according to one of claims 9 or 10, characterized characterized in that the linker molecules are a mixture bifunctional molecules according to claim 10 and monofunctional molecules that only either amino reactive or the thiol reactive, preferably the are amino reactive, carry grouping. 12. Particles for the transport of active pharmaceutical ingredients, thereby characterized in that on the surface of the particle Mixture of two types of linker molecules is present  one end of the linker molecule via a reactive one Grouping covalently bound to the surface of the particle are, the one type of linker molecules (bifunctional) at least one other end of the molecule another reactive grouping while the other type of linker Molecules (monofunctional) at no other end of the Molecule carries further reactive groups. 13. Particle according to one of claims 11 or 12, characterized characterized that clearly on the surface of the particles more, preferably at least 100% more, monofunctional are covalently bound as bifunctional linker molecules. 14. Particles according to one of claims 9 to 13, characterized characterized in that the linker molecules are polyglycolides, preferably polyethylene glycol derivatives, especially NHS Ester polyethylene glycol or NHS ester / vinyl sulfone Polyethylene glycol. 15. Particles according to one of claims 9 to 14, characterized characterized in that the particles are nanoparticles. 16. Particles according to one of claims 9 to 15, characterized characterized in that the particles are one of claims 1 to 8 correspond. 17. Particles according to one of claims 9 to 14, characterized characterized in that the bifunctional linker molecules bioactive macromolecules or "finder" molecules  from peptides, proteins; preferably antibodies, Antibody fragments or antibody derivatives with target-binding properties such as "single-chain" antibodies; Hormones, sugars, preferably glycosides; synthetic or natural receptor ligands; Proteins or peptides with a free cysteine group or thio sugars are coupled or before the surface modification were coupled. 18. Particle according to one of claims 11 to 13, characterized characterized in that the bifunctional linker molecules bioactive macromolecules or "finder" molecules, preferably antibodies, antibody fragments or Antibody derivatives with target-binding properties such as "Single-chain" antibodies, especially those with free ones Cysteine group, are coupled, are coupled or in front the surface modification were coupled. 19. Particles according to claim 10, characterized in that the bifunctional molecules of the bioactive coating Micromolecules or "seeker" molecules, preferably sugar, especially thio sugar; Peptides or hormones, in particular with a free cysteine group, are coupled or were coupled before the surface modification. 20. Particle according to one of claims 17 to 19, characterized characterized in that after binding the bioactive  Micromolecules or "searcher" molecules are still reactive Groups are saturated, preferably with cysteine. 21. Particle according to one of claims 1 to 20, characterized characterized in that the active ingredient to be transported synthetic or natural active ingredient, a protein, peptide, Lipid, sugar or nucleic acid or a low molecular weight organic or high molecular weight organic active ingredient, for example a hormone, an antineoplastic substance Antibiotic, antifungal, parasiticide, virustatic or Antihelmintic, a cardiovascular active substance; a central active substance, especially an analgesic, Antidepressant or antiepileptic; is. 22. Particle according to one of claims 1 to 8, characterized in that it is directly or via a linker; preferably via bifunctional polyethylene glycol molecules; is linked to a "viewfinder" molecule selected from:
Peptides, proteins; preferably antibodies, antibody fragments or antibody derivatives with target-binding properties such as "single-chain"antibodies; Hormones, sugars, preferably glycosides; synthetic or natural receptor ligands; Proteins or peptides with a free cysteine group or thio sugars. 23. Use of pure bifunctional polyethylene glycol molecules; preferably NHS ester / vinyl sulfone Polyethylene glycol; or mixtures of bifunctional and monofunctional polyethylene glycol molecules; preferably NHS ester polyethylene glycol with NHS Ester / vinylsulfone polyethylene glycol; for production of surface-modified solid particles for transport active pharmaceutical ingredients. 24. Use according to claim 23, characterized in that "seeker" molecules selected from the bifunctional polyethylene glycol molecules
Peptides, proteins; preferably antibodies, antibody fragments or antibody derivatives with target-binding properties such as "single-chain"antibodies; Hormones, sugars, preferably glycosides; synthetic or natural receptor ligands; Proteins or peptides with a free cysteine group or thio sugars
are bound to the particles, preferably when using pure bifunctional polyethylene glycol molecules glycosides, in particular thiose sugar; when using mixtures of bifunctional and monofunctional polyethylene glycol molecules, antibodies, antibody fragments or antibody derivatives with target-binding properties such as "single-chain" antibodies, preferably those with a free cysteine group. 25. A method for producing a particle according to one of claims 4 to 6, characterized by the following steps:

• a) preparation of a solution in or in a mixture of organic solvents containing at least one hydrophobic pharmaceutical active substance, water-insoluble, organic polymer material and amphiphilic organic polymer material,
• b) treatment of the solution with ultrasound,
• c) Dialysis of the solution against H ₂ O
• d) separation of the resulting particles from the resulting aqueous solution.

26. Medicament containing particles according to one of claims 1 to 22 and, if appropriate, suitable additional and / or Excipients. 27. Use of particles according to one of claims 1 to 22 for the manufacture of a medicament for cancer treatment, for Treatment of infectious diseases and parsitosis, for Treatment of diseases and symptoms with central nervous cause, for use in gene therapy or for genomic targeting.