EP1978937A1

EP1978937A1 - Use of amphiphilic self-assembling proteins for formulating poorly water-soluble effect substances

Info

Publication number: EP1978937A1
Application number: EP07704011A
Authority: EP
Inventors: Burghard Liebmann; Marcus Fehr; Mario Brands; Thomas Scheibel; Arne Ptock; Daniel HÜMMERLICH; Ingrid Martin
Original assignee: BASF SE
Current assignee: Technische Universitaet Muenchen
Priority date: 2006-01-20
Filing date: 2007-01-19
Publication date: 2008-10-15
Also published as: US8288512B2; CN101370556A; US20100278882A1; CN101370481A; JP2009523766A; CN104856962A; CA2637065C; US20100278883A1; CA2637065A1; CA2638870A1; JP2009523767A; JP5236498B2; WO2007082923A3; WO2007082936A1; WO2007082923A2; EP1979055A2

Abstract

The invention relates to the use of amphiphilic self-assembling proteins for formulating poorly water-soluble effect substances.

Description

Use of amphiphilic, self-assembling proteins for the formulation of poorly water-soluble effect substances

The present invention relates to the use of amphiphilic, self-assembling proteins for the formulation of sparingly water-soluble effect substances

State of the art

DE 10059213A1 describes a process for preparing solid preparations of water-insoluble or sparingly water-soluble active substances by dispersing the active ingredients in a protein-containing protective colloid, flocculating and separating the active ingredient coated with the protective colloid and converting into a dry powder. Preferred protective colloids include casein and beef, pork and fish gelatin.

DE 102004057587A1 describes aqueous dispersions of a mixture of sparingly water-soluble active ingredients and single-cell protein material and dry powders prepared therefrom.

task

The hitherto known processes for the formulation of water-insoluble or poorly water-soluble active substances and effect substances do not meet all the requirements which are imposed on an active ingredient formulated in particular for cosmetic and pharmaceutical use, such as temperature, oxidation and light stability, mechanical stability, toxic safety.

It was therefore an object to provide a method that allows the formulation of poorly water-soluble drugs while the o.g. Meets criteria better than the known from the prior art method.

Description of the invention

The present invention relates in a first embodiment to the use of amphiphilic self-assembling proteins for the formulation of sparingly water-soluble effect substances. Amphiphilic, self-assembling proteins are suitable as formulation auxiliaries for poorly water-soluble, hydrophobic active ingredients. Due to their amphiphilic molecular character, these proteins can stabilize hydrophobic drugs in aqueous solutions. Their self-assembling properties enable these proteins to adopt higher molecular structures and thus permanently encapsulate hydrophobic active ingredients.

Another object of the invention is a process for the preparation of Effektstoffformulierungen, wherein (i) the sparingly water-soluble effect substance mixed together with the amphiphilic self-assembling protein in a common disperse phase, (ii) then a phase separation in a protein and effect-rich phase and a protein and low-phase phase performs.

The protein- and effect-rich phase can later be cured and separated as mechanically stable effect-containing protein microbeads and optionally dried.

(i) Amphiphilic, self-assembling proteins

Amphiphilic, self-assembling proteins consist of polypeptides composed of amino acids, in particular of the 20 naturally occurring amino acids. The amino acids may also be modified, for example, acetylated, glycosylated, farnesylated.

Suitable amphiphilic, self-assembling proteins for the formulation of sparingly water-soluble effect substances are those proteins which can form protein microbeads. Protein microbeads have a globular shape with an average particle diameter of 0.1 to 100, in particular from 0.5 to 20, preferably from 1 to 5 and particularly preferably from 2 to 4 microns.

Protein microbeads can preferably be prepared by the process described below:

The protein is dissolved in a first solvent. As the solvent, for example, aqueous salt solutions can be used. In particular, are high concentrated salt solutions having a concentration greater than 2, in particular greater than 4 and particularly preferably greater than 5 molar, whose ions have more pronounced chaotropic properties than sodium and chloride ions. An example of such a saline solution is 6 M guanidinium thiocyanate or 9 M lithium bromide. Furthermore, organic solvents can be used to dissolve the proteins. In particular, fluorinated alcohols or cyclic hydrocarbons or organic acids are suitable. Examples are hexafluoroisopropanol, cyclohexane and formic acid. The preparation of the protein microbeads can be carried out in the solvents described. Alternatively, this solvent can be replaced by another solvent, for example, low-concentration salt solutions (c <0.5 M) by dialysis or dilution. The final concentration of the dissolved protein should be between 0.1-100 mg / ml. The temperature at which the process is carried out is usually 0-80, preferably 5-50, and more preferably 10 40 ° C.

If aqueous solutions are used, they may also be mixed with a buffer, preferably in the range of pH 4-10, particularly preferably 5-9, very particularly preferably 6-8.5.

Addition of an additive induces phase separation. This results in a protein-rich phase emulsified in the mixture of solvent and additive. Due to surface effects, emulsified protein-rich droplets take on a round shape. By choosing the solvent, the additive and the protein concentration, the average diameter of the protein microbeads can be set to values between 0.1 μm and 100 μm.

As an additive, it is possible to use all substances which, on the one hand, are miscible with the first solvent and, on the other hand, induce the formation of a protein-rich phase. If the microbead formation is carried out in organic solvents, organic substances which have a lower polarity than the solvent, for example toluene, are suitable for this purpose. Salts can be used as an additive in aqueous solutions whose ions have more pronounced cosmotropic properties than sodium and chloride ions (eg ammonium sulfate, potassium phosphate). The final concentration of the additive should be between 1% and 50% by weight, based on the protein solution, depending on the type of additive. The protein-rich droplets are fixed by curing, whereby the round shape is retained. The fixation is based on the formation of strong intermolecular interactions. The nature of the interactions may be non-covalent, eg by the formation of intermolecular β-sheet crystals or covalent, eg by chemical cross-linking. The curing can be carried out by the additive and / or by the addition of another suitable substance. The curing takes place at temperatures between 0 and 80 ° C, preferably between 5 and 60 ° C.

This further substance can be a chemical cross-linker. A chemical cross-linker is understood to mean a molecule in which at least two chemically reactive groups are linked to one another via a linker. Examples of these are sulfhydryl-reactive groups (for example maleimides, pydridyl disulfides, α-haloacetyls, vinyl sulfones, sulfatoalkyl sulfones (preferably sulfatoethyl sulfones)), amine-reactive groups (for example succinimidyl esters, carbodiimde, hydroxymethyl phosphine, imido esters, PFP esters, aldehydes, isothiocyanates, etc .), Carboxy-reactive groups (eg, amines, etc.), hydroxyl-reactive groups (eg, isocyanates, etc.), unselective groups (eg, arylazides, etc.), and photoactivatable groups (eg, perfluorophenyl azide, etc.). These reactive groups can form covalent linkages with amine, thiol, carboxyl or hydroxyl groups present in proteins.

The stabilized microbeads are washed with a suitable further solvent, e.g. Water and then dried by methods known to those skilled in the art, e.g. by lyophilization, contact drying or spray drying. The success of the sphere formation is checked by scanning electron microscopy.

Proteins that are predominantly intrinsically unfolded in aqueous solution are suitable for the production of protein microbeads. For example, this condition may be calculated using an algorithm that underlies the lUpred program (http://iupred.enzim.hu/index.html; The Pairwise Energy Content Estimated from Amino Acid Composition Discriminates between Folded and Intrinsically Unstructured Proteins; Zsuzsanna Dosztanyi Veronika Csizmόk, Peter Tompa and Istvan Simon, J. Mol. Biol. (2005) 347, 827-839). A predominantly intrinsically unfolded state is assumed when a value> 0.5 is calculated for over 50% of the amino acid residues according to this algorithm (prediction type: long disorder). Other suitable proteins for the formulation of poorly water-soluble effect substances are silk proteins. By this we mean hereinafter those proteins which contain highly repetitive amino acid sequences and which are stored in the animal in a liquid form and whose secretion by shearing or spinning results in fibers (Craig, CL (1997) Evolution of arthropod silks. Annu. Rev. Entomol. 42: 231-67).

Particularly suitable proteins for the formulation of sparingly water-soluble effect substances are spider silk proteins, which could be isolated in their original form from spiders.

Especially suitable proteins are silk proteins which could be isolated from the "major ampullate" gland of spiders.

Preferred silk proteins are ADF3 and ADF4 from the "major ampullate" gland of Araneus diadematus (Guerette et al., Science 272, 5258: 12-5 (1996)).

Equally suitable proteins for the formulation of sparingly water-soluble effect substances are natural or synthetic proteins which are derived from natural silk proteins and which have been produced heterologously in prokaryotic or eukaryotic expression systems using genetic engineering working methods. Non-limiting examples of prokaryotic expression organisms are Escherichia coli, Bacillus subtilis, Bacillus megaterium, Corynebacterium glutamicum etc. Nonlimiting examples of eukaryotic expression organisms are yeasts such as Saccharomyces cerevisiae, Pichia pastoris and others, filamentous fungi such as Aspergillus niger, Aspergillus oryzae and Aspergillus nidulans. Trichoderma reesei, Acremonium chrysogenum and others, mammalian cells, such as Heia cells, COS cells, CHO cells and others, insect cells, such as Sf9 cells, MEL cells and others.

Particularly preferred for the formulation of sparingly water-soluble effect substances are synthetic proteins which are based on repeat units of natural silk proteins. In addition to the synthetic repetitive silk protein sequences, these may additionally contain one or more natural non-repetitive silk protein sequences (Winkler and Kaplan, J Biotechnol 74: 85-93 (2000)).

Among the synthetic silk proteins preferred for the formulation of poorly water-soluble effect substances are synthetic spider silk proteins, which Repeat units of natural spider silk proteins based. In addition to the synthetic repetitive spider silk protein sequences, these may additionally contain one or more natural non-repetitive spider silk protein sequences.

Among the synthetic spider silk proteins, preference is given to the so-called C16 protein (Huemmerich et al., Biochemistry, 43 (42): 13604-13612 (2004)). This protein has the polypeptide sequence shown in SEQ ID NO: 1. In addition to the polypeptide sequence shown in SEQ ID NO: 1, particularly functional equivalents, functional derivatives and salts of this sequence are also preferred.

According to the invention, "functional equivalents" are understood in particular to also be mutants which, in at least one sequence position of the abovementioned amino acid sequences, have a different amino acid than the one specifically mentioned but nevertheless have the property of packaging sparingly water-soluble effect substances.

"Functional equivalents" thus include the mutants obtainable by one or more amino acid additions, substitutions, deletions, and / or inversions, which changes may occur in any sequence position as long as they result in a mutant having the property profile of the invention Equivalence is especially given when the reactivity patterns between mutant and unchanged polypeptide match qualitatively.

"Functional equivalents" in the above sense are also "precursors" of the described polypeptides as well as "functional derivatives" and "salts" of the polypeptides.

"Precursors" are natural or synthetic precursors of the polypeptides with or without the desired biological activity.

Examples of suitable amino acid substitutions are given in the following table:

Original rest Examples of substitution

AIa Ser

Arg Lys

Asn GIn; His

Asp Glu Cys Ser

GIn Asn

Giu Asp

GIy Pro

His Asn; Gin

Me Leu; Val

Leu Me; Val

Lys Arg; GIn; Glu

Met Leu; He

Phe Met; Leu; Tyr

Ser Thr

Thr Ser

Trp Tyr

Tyr Trp; Phe

VaI Me; Leu

Salts are understood as meaning both salts of carboxyl groups and acid addition salts of amino groups of the protein molecules of the invention Salts of carboxyl groups can be prepared in a manner known per se and include inorganic salts such as, for example, sodium, calcium, ammonium, iron and zinc salts, as well as salts with organic bases such as amines such as triethanolamine, arginine, lysine, piperidine and the like, acid addition salts such as salts with mineral acids such as hydrochloric acid or sulfuric acid and salts with organic acids such as acetic acid and oxalic acid also the subject of the invention.

"Functional derivatives" of polypeptides of the invention may also be produced at functional amino acid side groups or at their N- or C-terminal end by known techniques Such derivatives include, for example, aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups obtainable by reaction with ammonia or with a primary or secondary amine; N-acyl derivatives of free amino groups prepared by reaction with acyl groups; or O-acyl derivatives of free hydroxy groups prepared by reacting with acyl groups. (ii) poorly water-soluble effect substances

In the following, the terms heavy water-soluble effect substances and hydrophobic effect substances and hydrophobic agents and effector molecules are used interchangeably. As sparingly water-soluble effect substances are referred to below those compounds whose water solubility at 20 ° C <5 wt .-%, preferably <1 wt .-%, more preferably <0.5 wt .-%, most preferably <0.1 Wt .-% is.

Suitable sparingly water-soluble effect substances are dyes, in particular those mentioned in the following table:

Particularly advantageous dyes are the oil-soluble or oil-dispersible compounds mentioned in the following list. The Color Index Numbers (CIN) are taken from the Rowe Color Index, 3rd Edition, Society of Dyers and Colourists, Bradford, England, 1971.

Further preferred effector molecules are fatty acids, in particular saturated fatty acids which carry an alkyl branching, particularly preferably branched eicosanoic acids, such as 18-methyl-eicosanoic acid.

Further preferred effector molecules are carotenoids. Carotenoids according to the invention are the following compounds and their esterified or glycosylated derivatives β-carotene, lycopene, lutein, astaxanthin, zeaxanthin, cryptoxanthin, citranaxanthin, canthaxanthin, bixin, β-apo-4-carotenal, β-apo-8-carotenal, β-apo-8-carotenoic acid ester, neurospores, Echinone, adonirubin, violaxanthin, torules, torularyhodine, singly or as a mixture. Preferably used carotenoids are β-carotene, lycopene, lutein, astaxanthin, zeaxanthin, citranaxanthin and canthaxanthin.

Further preferred effector molecules are vitamins, especially retinoids and their esters.

By retinoids in the context of the present invention is meant vitamin A alcohol (retinol) and its derivatives such as vitamin A aldehyde (retinal), vitamin A acid (retinoic acid) and vitamin A esters (e.g., retinyl acetate, retinyl propionate and retinyl palmitate). The term retinoic acid encompasses both all-trans retinoic acid and 13-cis retinoic acid. The terms retinol and retinal preferably comprise the all-trans compounds. The preferred retinoid used for the formulations according to the invention is all-trans-retinol, referred to below as retinol.

Further preferred effector molecules are vitamins, provitamins and vitamin precursors from groups A, C, E and F, in particular 3,4-didehydroretinol, .beta.-carotene (provitamin of vitamin A), palmitic acid ester of ascorbic acid, tocopherols, especially .alpha.-tocopherol and its esters, eg the acetate, nicotinate, phosphate and succinate; also vitamin F, which is understood as meaning essential fatty acids, especially linoleic acid, linolenic acid and arachidonic acid.

Further preferred effector molecules are lipophilic, oil-soluble antioxidants from the group vitamin E, i. Tocopherol and its derivatives, gallic acid esters, flavonoids and carotenoids, and butylhydroxytoluene / anisole.

Another preferred effector molecule is lipoic acid and suitable derivatives (salts, esters, sugars, nucleotides, nucleosides, peptides and lipids).

Further preferred effector molecules are UV light protection filters. These are understood to mean organic substances which are able to absorb ultraviolet rays and to release the absorbed energy in the form of longer-wave radiation, eg heat. As oil-soluble UV-B filters, for example, the following substances can be used:

3-benzylidene camphor and its derivatives, e.g. 3- (4-methylbenzylidene) camphor;

4-aminobenzoic acid derivatives, preferably 2-ethylhexyl 4- (dimethylamino) benzoate, 2-octyl 4- (dimethylamino) benzoate and 4- (dimethylamino) benzoic acid ester;

Esters of cinnamic acid, preferably 2-ethylhexyl 4-methoxycinnamate, 4-propyl methoxy cinnamate, isoamyl 4-methoxycinnamate, 4-isoacetyl methoxycinnamate, 2-cyano-3-phenylcinnamic acid 2-ethylhexyl ester (octocrylene); Esters of salicylic acid, preferably 2-ethylhexyl salicylate, 4-isopropylbenzyl salicylate, homomenthyl salicylate;

Derivatives of benzophenone, preferably 2-hydroxy-4-methoxybenzophenone, 2-

Hydroxy-4-methoxy-4'-methylbenzophenone, 2,2'-dihydroxy-4-methoxybenzophenone;

Esters of benzalmalonic acid, preferably di-2-ethylhexyl 4-methoxybenzmalonate;

Triazine derivatives, such as 2,4,6-trianilino- (p-carbo-2'-ethyl-1 ^'-hexyloxy) -1, 3,5-triazine (octyl tyltriazone) and Dioctyl Butamido Triazone (Uvasorb® HEB):

Propane-1,3-diones, e.g. 1- (4-tert-butylphenyl) -3- (4'-methoxyphenyl) propane-1,3-dione. Particularly preferred is the use of esters of cinnamic acid, preferably 4-

2-ethylhexyl methoxycinnamate, isopentyl 4-methoxycinnamate, 2-cyano-3-phenylcinnamic acid 2-ethylhexyl ester (octocrylene).

Furthermore, the use of derivatives of benzophenone, in particular 2-

Hydroxy-4-methoxybenzophenone, 2-hydroxy-4-methoxy-4 ^" -methylbenzophenone, 2,2'-dihydroxy-4-methoxybenzophenone and the use of propane-1, 3-diones, such as

1- (4-tert-butylphenyl) -3- (4-methoxy-phenyl) -propane-1,3-dione is preferred.

Typical UV-A filters are:

Derivatives of benzoylmethane, such as 1- (4'-tert-butylphenyl) -3- (4'-methoxyphenyl) propane-1,3-dione, 4-tert. Butyl 4'-methoxydibenzoylmethane or 1-phenyl-3- (4'-isopropylphenyl) -propane-1,3-dione;

Amino-hydroxy-substituted derivatives of benzophenones such as N, N-diethylamino hydroxybenzoyl-n-hexyl benzoate. Of course, the UV-A and UV-B filters can also be used in mixtures.

Suitable UV filter substances are mentioned in the following table.

In addition to the two aforementioned groups of primary light stabilizers, it is also possible to use secondary light stabilizers of the antioxidant type which interrupt the photochemical reaction chain which is triggered when UV radiation penetrates into the skin. Typical examples are tocopherols (vitamin E) and oil-soluble ascorbic acid derivatives (vitamin C).

According to the invention, suitable derivatives (salts, esters, sugars, nucleotides, nucleosides, peptides and lipids) of said compounds can be used as effector molecules. Further preferred are so-called peroxide decomposers, ie compounds which are able to decompose peroxides, particularly preferably lipid peroxides. These include organic substances, such as 5-pyrimidinol and 3-pyridinol derivatives and probucol.

Furthermore, the abovementioned peroxide decomposers are preferably those described in the patent applications WO / 0207698 and WO / 03059312, the contents of which are hereby incorporated by reference, preferably the boron-containing or nitrogen-containing compounds described there, the peroxides or hydroperoxides can reduce to the corresponding alcohols without radical radical education. Furthermore, sterically hindered amines can be used for this purpose.

Another group are anti-irritants, which have an anti-inflammatory effect on UV-damaged skin. Such substances are, for example, bisabolol, phytol and phytantriol.

Another group of poorly water-soluble effector substances are active substances that can be used in crop protection, for example herbicides, insecticides and fungicides.

Also suitable as sparingly water-soluble effect substances are active substances for pharmaceutical use, in particular those for oral administration. The inventive method is in principle applicable regardless of the medical indication on a variety of drugs.

Examples of suitable sparingly water-soluble pharmaceutical active substances are mentioned in the following table.

(iii) formulation of hydrophobic drugs

Formulations of poorly water-soluble drugs can be prepared in a variety of ways using amphiphilic self-assembling proteins. Sparingly water-soluble, hydrophobic drugs can be packaged in protein microbeads or colloidally dispersed by protein coating, e.g. can be achieved in Mikronisierungsansätzen be stabilized. The formulation of hydrophobic drugs can be done by inclusion in microbeads. This process involves two steps. In the first step, the hydrophobic drug and the amphiphilic self-assembling protein are in a common

Phase solved. For this purpose, the active ingredient and the protein can be brought into solution directly by a solvent or a solvent mixture. Alternatively, the active substance and the protein can first be dissolved in different solvents and the solutions subsequently mixed with one another, so that in turn a common phase is formed. The common phase may be a molecular disperse phase or a colloidally disperse phase.

The dissolution of the hydrophobic active ingredient and the protein in various solvents and the subsequent mixing of the two solutions is particularly advantageous if the hydrophobic active ingredient and the protein can not be dissolved in a common solvent or solvent mixture. In this way, colloidal-disperse solutions of hydrophobic active ingredients can be prepared by this procedure by the active ingredient dissolved in a suitable solvent is diluted into another solvent in which this active ingredient is insoluble. Since proteins are generally readily water-soluble, preference is given to working with aqueous solutions and mixtures of water and water-miscible organic solvents. Examples of suitable, water-miscible solvents are alcohols such as methanol, ethanol and isopropanol, fluorinated alcohols such as hexafluoroisopropanol and trifluoroethanol, alkanones such as acetone or sulfoxides such as dimethyl sulfoxide or formamides such as dimethylformamide or other organic solvents such as tetrahydrofuran and acetonitrile or N-methyl-2 pyrrolidone. In general, it is possible to work with all solvents and solvent mixtures in which the proteins can be dissolved. Examples of suitable solvents are fluorinated alcohols. Ie such as hexafluoroisopropanol or trifluoroethanol, ionic liquids such as EMIM acetate, aqueous solutions of chaotropic salts such as urea, guanidinium hydrochloride and guanidinium thiocyanate or organic acids such as formic acid and mixtures of these solvents with other organic solvents. Examples of solvents which can be mixed with the solvents for the protein include alcohols such as methanol, ethanol and isopropanol, alkanones such as acetone, sulfoxides such as dimethyl sulfoxide, formamides such as dimethylformamide, haloalkanes such as methylene chloride or other organic solvents such as tetrahydrofuran , The second step in the formulation of hydrophobic drugs in microbeads is a phase separation into a low-protein and low-drug phase and into a protein- and drug-rich phase, which subsequently hardens. The hydrophobic agent is included in the assembly form of the protein. Due to surface effects during the phase separation, preferably round protein structures, so-called microbeads, are formed.

The phase separation is preferably induced by addition of aqueous solutions of lyotropic salts to the mixtures of proteins and hydrophobic active ingredients. Suitable lyotropic salts are described by the Hofmeister ^'sche row. Particularly suitable are ammonium sulfate and potassium phosphate. The addition of these solutions can be done by simple mixing, dropwise or by dialysis. The interactions between the hydrophobic drug and the protein are based essentially on their hydrophobic properties, although hydrogen bonds, ionic interactions and van der Waals interactions may also be involved. The hydrophobic drug may be bound to the surface, be included in the microbeads, or be associated with the microbeads in both ways. The binding of the hydrophobic drug to the microbeads can be determined by the depletion of the drug-active assembly set-up. The concentration of the active ingredient can be measured by a quantitative analysis of its properties. For example, the binding of light-absorbing active substances can be analyzed by photometric methods. For this purpose, for example, the color of the microbeads or the decolorization of the protein- and drug-poor phase of the formulation batch are determined by measuring the absorption of a colored active substance. These methods can also be used to determine the level of active ingredient in the microbeads. The release of the active compounds from the microbeads can be effected by desorption into suitable solvents, by the degradation of the microbeads by proteases or by dissolution of the microbeads by suitable solvents. Suitable solvents for the desorption are all solvents or solvent mixtures in which the active ingredient can be dissolved. Suitable proteases can be added as technical proteases to a suspension of protein microbeads or occur naturally at the desired site of action of the effector molecules, such as skin proteases, proteases of the digestive tract, for example gastric or intestinal proteases or proteases released by microorganisms. Solvents that can dissolve the microbeads are, for example, fluorinated alcohols such as hexafluoroisopropanol or trifluoroethanol, ionic liquids such as EMIM acetate, aqueous solutions of chaotropic salts such as urea, guanidinium hydrochloride and guanidinium thiocyanate or organic acids such as formic acid and mixtures of these solvents with others organic solvents. The speed and kinetics of the release of the effector molecules can be controlled, for example, by the loading density with active substances and the size of the microbeads or their ratio of volume to the surface.

The formulation of poorly water-soluble hydrophobic drug may also be stabilized by stabilizing its colloidally disperse solution, e.g. by micronization.

Another object of the invention is the use of the protein microbeads prepared using the described amphiphilic self-assembling proteins or the e.g. micronization-produced colloidal-disperse protein formulations for the storage, transport or release of active ingredients in pharmaceutical products, cosmetic products, crop protection products, food and feed. In this case, e.g. the protein microbeads continue to protect the packaged active ingredients from environmental influences, e.g. oxidative processes or UV radiation, or destruction by reaction with other constituents of the products or degradation by certain proteases. The active substance can be released from the protein microbeads or colloidally disperse protein formulations by desorption, proteolytic degradation, targeted release or slow release or combination of these mechanisms.

Preference is given to protein microbeads and active ingredients formulated in pharmaceutical products for oral ingestion. In this case, the stability of the active ingredients in gastric passage can be increased because under the prevailing conditions there is no proteolytic degradation of the protein microbeads. The release of the active Substances from the orally absorbed drug-containing microbeads then takes place in the intestine. Also, topical applications of protein microbeads and embedded therein pharmaceutical agents are possible. The degradation of the protein microbeads and the resulting release of the active ingredients is then controlled by proteases contained on the skin or in the upper layers of the skin.

In pharmaceutical products, food and feed or crop protection products, formulation of active ingredients with the described amphiphilic self-assembling proteins can furthermore lead to increased bioavailability of the active ingredients. The packaging of active pharmaceutical ingredients in protein microbeads or the colloidally disperse formulation of active ingredients using the described amphiphilic self-assembling proteins can further lead to improved blood-brain barrier overcome the drug or improved uptake via the intestinal mucosa. Crop protection products can be protected from being washed out by encapsulation or embedding in protein microbeads. Certain drug particle sizes which are better incorporated or resorbed or more bioavailable may be prepared by packaging in protein microbeads or by colloidally dispersed formulation, e.g. by micronization approaches using amphiphilic self-assembling proteins.

By varying the amino acid sequence of the described amphiphilic self-assembling proteins or fusing with additional protein or peptide sequences, it is possible to generate structures which have certain surfaces, e.g. Skin, hair, leaves, roots or intestinal or blood vessel surfaces, specifically recognize or be recognized and bound by these surfaces or the receptors contained.

This makes it possible to more effectively bring the active ingredients formulated with the described amphiphilic self-assembling proteins to the desired site of action or to improve the absorption of active substance. The bioavailability of active pharmaceutical ingredients in food and feed can be increased if they are packaged in protein microbeads, which are additionally fused or associated with proteins that bind to certain surface markers (eg receptors) of cells of the intestinal tract (eg mucosal cells). Such proteins are, for example, the MapA protein or the collagen-binding protein CnBP from Lactobacillus. lus reuteri (Miyoshi et al., 2006, Biosci Biotechnol., Biochem 70: 1622-1628) or functionally comparable proteins from other microorganisms, especially the natural gastrointestinal flora. The described binding proteins mediate attachment of the microorganisms to cell surfaces. By coupling or fusing the binding proteins to the described amphiphilic self-assembling proteins, resulting protein-containing protein microbeads would be more selectively directed to appropriate sites or dwelling at these sites for longer, resulting in prolonged and improved drug release and uptake , Furthermore, by varying the amino acid sequence of the amphiphilic self-assembling proteins described for the active ingredient formulation or fusion with additional protein or peptide sequences, it is possible to target active ingredients to desired sites of action in order, for example, to achieve higher specificity, lower drug consumption or drug dose, faster or to achieve greater impact.

Experimental part

example 1

Packaging of β-carotene from THF and THF / isopropanol in microbeads and release by proteolysis Preparation of β-carotene solutions

A stock solution was prepared by dissolving 80 mg of β-carotene and 16 mg of tocopherol in 10 g of THF. Following this, solutions 1-4 were prepared from this stock solution by dilution according to Table 1.1. All solutions were prepared shortly before use and processed immediately after dilution.

Tab. 1 .1: ß-carotene solutions

Packaging of β-Carotene in Microbeads by Direct Addition of Potassium Phosphate In order to package β-carotene into the C16 protein microbeads, a common phase of C16 protein and β-carotene was first prepared. For this purpose, in each case 500 μl of a solution of 10 mg / ml C16 protein in 5 mM potassium phosphate, pH 8, were mixed with 50 μl or 100 μl of the β-carotene solutions (solutions 1-4) (Table 1.2). In the case of the β-carotene from THF / isopropanol (mixtures 1-4), orange dispersions were obtained, in the case of β-carotene mixtures of THF (mixtures 5-8), yellow dispersions. To induce C16 protein microbead formation by phase separation, 1000 μl of a 1 M potassium phosphate solution, pH 8.0, was added to each of the batches (Table 1.2). After 15 min incubation at room temperature, the mixtures were separated by centrifugation into a distinctly colored pellet of C16 protein microbeads with β-carotene and colorless supernatant. The ß-carotene used went so completely in the phase separation in the microbeads. The colorless supernatant was removed. The pellets were then washed twice with distilled water and redispersed.

After redispersion of the C16 protein microbeads, in the case of the batches with the β-carotene solutions of THF / isopropanol (batches 1-4), orange dispersions were obtained, in the case of batches with the β-carotene solutions of THF (batches 5-8). yellow dispersions.

Tab. 1 .2: Packaging of β-carotene from THF and THF / isopropanol in C16 protein microbeads by adding 1 M potassium phosphate solution

Packaging β-carotene in C16 protein microbeads by dialysis against potassium phosphate

As an alternative to the direct addition of potassium phosphate to the common phase of β-carotene and C16 protein, the phase separation can also be carried out by dialysis against 1 M potassium phosphate. Since the dialysis was carried out in dialysis tubing, the 10-fold batch volume was in each case pipetted in comparison to the direct addition of the potassium phosphate solution (Table 1.3).

Tab. 1 .3: Packaging of β-carotene from THF and THF / isopropanol in C16 protein microbeads by dialysis against 1 M potassium phosphate solution

For this purpose, the C16 solution (10 mg / ml in 5 mM potassium phosphate, pH 8.0) was mixed with the respective β-carotene solution and the mixture was immediately added to the dialysis tubing and dialyzed against 1 M potassium phosphate solution. After overnight dialysis, the microbead dispersion was removed from the slides and separated by centrifugation into a colorless supernatant and into a colored pellet. As in the case of the direct addition of potassium phosphate to the common phase of C16 protein and β-carotene, the β-carotene was bound quantitatively by the C16 protein in the form of the protein microbeads. The colorless supernatant was removed. men. The pellets were then washed twice with distilled water and then redispersed.

After redispersion of the protein microbeads, in the case of mixtures with the β-carotene solutions of THF / isopropanol (mixtures D1-D4), orange dispersions were obtained, in the case of mixtures with the β-carotene solutions of THF (mixtures D5-D8) yellow dispersions (Fig. 1).

FIG. 1: Dispersions of the C16 protein microbeads with β-carotene from THF and THF / isopropanol in water. From left to right: batches D1-D4 (THF / isopropanol) and batches D5-D8 (THF). The proportion of β-carotene in the C16 protein microbeads is given as a percentage by weight based on the weight of the C16 protein microbeads.

In order to reproduce the different staining of the C16 protein microbeads with β-carotene from THF and THF / isopropanol, the mixtures D4 and D5 were repeated on a larger scale (Table 1.4). Again, yellow C16 protein microbeads were obtained with β-carotene from THF and orange C16 protein microbeads with β-carotene from THF / isopropanol (FIG. 2).

Tab. 1 .4: Packaging of β-carotene from THF and THF / isopropanol in C16 protein microbeads by dialysis against 1 M potassium phosphate solution

In order to reproduce the different staining of the C16 protein microbeads with β-carotene from THF and THF / isopropanol, the mixtures D4 and D5 were repeated on a larger scale (Table 1.4). Again, β-carotene from THF gave yellow C16 protein microbeads and with β-carotene from THF / isopropanol orange C16 protein microbeads (FIG. 2).

FIG. 2: Dispersions of the C16 protein microbeads with β-carotene from THF / isopropanol (0.9 percent by weight β-carotene, mixture G1, left) and THF (0.3 percent by weight β-carotene, mixture G2, right).

Release of β-carotene from C16 protein microbeads by digestion with proteinase K. In order to show that the β-carotene in the C16 protein microbeads can be released by proteolysis, 200 μl of the microbead dispersions G1 and G2 were dissolved in water at 500 5 mM potassium phosphate pH 8.0. Subsequently, 5 μl proteinase K (Roche, 19.45 mg / ml) were added and incubated overnight at room temperature. In each case a batch of C16 protein microbead dispersions without proteinase K served as control. After overnight incubation, centrifugation was carried out. In the presence of the protease, the C16 protein microbeads were digested and the beta-carotene was released. After centrifugation, no pellet was visible. The supernatant was clearly colored. Without protease, the intact C16 protein microbeads could be centrifuged off. A distinctly colored pellet was observed. The supernatant was colorless.

Fig. 3 Digestion of the C16 protein microbead dispersions by proteinase K. A) C16 protein microbeads with β-carotene from THF / isopropanol (0.9 percent by weight β-carotene, mixture G1) without protease; B) C16 protein microbeads with β-carotene from THF / isopropanol (0.9 percent by weight β-carotene, mixture G1) with protease; C) C16 protein microbeads with β-carotene from THF (0.3% by weight β-carotene, batch G2) without protease; D) C16 protein microbeads with β-carotene from THF (0.3% by weight β-carotene, mixture G2) with protease. Example 2

Stability of β-carotene-containing microbeads and release of β-carotene from microbeads by proteolytic digestion

By treating β-carotene-containing C16 protein microbeads with different proteases, which are active in the human stomach or intestine, the suitability of protein microbeads as a storage, transport or "delivery" system for pharmacological Effect substances are detected.

To prepare β-carotene-containing C16 protein microbeads, 80 mg of β-carotene and 16 mg of vitamin E were dissolved in 10 ml of THF and then diluted with 90 ml of isopropanol. A portion of this solution was then mixed with 10 volumes of C16 protein solution (10 mg / ml in 5 mM potassium phosphate buffer pH 8). Subsequently, the batch was mixed with 2 volumes of 1 M potassium phosphate buffer pH 8. The resulting β-carotene-containing C16 protein microbeads were centrifuged off and excess free, free β-carotene was removed by washing the sediment with water. 20 mg of β-carotene-containing C16 protein microbeads were mixed with 2 ml of artificial gastric juice (6.4 mg pepsin, 80 mM HCl, 4 mg NaCl) or 2 ml of artificial intestinal juice I (20 mg pancreatin, 0.45 M sodium phosphate pH 7.5, 0.9 mM sodium taurocholate) or 2 ml of artificial intestinal juice II (20 mg pancreatin, 0.45 M sodium phosphate pH 7.5, 6 mM sodium taurocholate) and resuspended for 0, 1, 2, 6, 24 and 48 h with shaking (140 rpm) at 37 ° C incubated. Non-proteolytically degraded C16 protein microbeads were determined by scattering the suspension at 600 nm (Figure 4). Intact C16 protein microbeads were then centrifuged off and the supernatant analyzed for the β-carotene content by determining the absorbance at 445 nm (FIG. 5). By treatment with pepsin-containing artificial gastric juice, C16 protein microbeads could hardly be degraded even after 48 hours (Figure 4) and thus β-carotene was released (Figure 5). In contrast, when treated with pancreatin-containing artificial intestinal juice I and II, C16 protein microbeads were almost completely degraded within 6 h (Fig. 4) and the contained ß-carotene was released (Fig. 5). Accordingly, C16 protein microbeads would survive the human gastric passage without substantial degradation and only release the bound effector substances by proteolytic degradation in the intestinal tract.

Fig. 4: Determination of intact C16 protein microbeads by photometric measurement of the absorption at 600 nm. Fig. 5: Determination of β-carotene released from C16 protein microbeads by photometric absorption measurement at 445 nm.

Example 3 Micronization with C16 spider silk protein

2 g of crystalline lycopene and 0.4 g of alpha-tocopherol were dissolved in 500 g of THF. The drug solution was continuously stirred at room temperature and a flow rate of 2.42 g / min with an aqueous solution consisting of 0.2 g / l C16 protein in 5 mM potassium phosphate buffer (pH 8), and a flow rate of 25.4 g / min mixed. The drug particles formed during the mixing had a particle size of 103 nm in the THF / water mixture. After 2 hours, clear dispersion stabilization of the sample treated with C16 protein (FIG. 6B) was shown in comparison with the untreated sample (FIG. 6A). Even after several days, the lycopene dispersion with C16 protein appeared stable, while the untreated lycopene dispersion was highly flocculent (Figure 7). A portion of the C16 protein-stabilized lycopene dispersion was concentrated to a solids content of 0.28%. Lycopene dried in this state would be poorly redispersible. Alternatively, a C16 protein-stabilized lycopene dispersion was treated with 330 mM potassium phosphate (final concentration in the mixture) and dried. The resulting lycopene powder was readily redispersible.

Fig. 6: Formulation of lycopene with C16 spider silk protein. Absorption of untreated lycopene sample (A) and C16 protein-treated lycopene sample (B) immediately after mixing (black graph) and 2 hours after mixing (red graph).

Fig. 7: Formulation of lycopene with C16 spider silk protein. Comparison of untreated lycopene dispersion (left) with a C16 protein-stabilized lycopene dispersion (right) about 30 days after mixing.

Example 4

Packaging of metazachlor from isopropanol in microbeads and release by proteolysis

Poorly water-soluble plant compounds can be packaged in protein microbeads, which are made from amphiphilic self-assembling proteins and then release from it. For this purpose, the herbicide active substance metazachlor was selected as a non-limiting example.

500 μl of a 10 mg / ml C16 protein-containing potassium phosphate solution (5 mM, pH 8.0) were mixed with 100 μl of a metazachlor solution (50 mg / ml in isopropanol). C16 protein microbead formation was induced by the addition of 1 ml of 1 M potassium phosphate buffer (pH 8.0). The mixture was incubated for 1 h at room temperature and then centrifuged for 10 min at 20,000 x g. The pellet was redistilled twice with 5 ml H2O. washed and then lyophilized. As a control, a similar batch was carried out without C16 protein.

After precipitation with potassium phosphate, microbeads formed in the batch with C16 protein and metazachlor. These were morphologically comparable to those of a standard mixture with C16 protein but no active ingredient. In the C16-metazachlor approach, no metazachlor crystals were visible. In the approach metazachlor without C16 protein, however, formed large drug crystals. This illustrates that the C16 protein has a significant inhibiting effect on the crystallization of metazachlor in the presence of aqueous potassium phosphate buffer.

The determination of the metazachlor concentration in the supernatant after C16 protein precipitation or C16 microbead formation showed that about 90% of the active ingredient was packed in or associated with the protein microbeads. The wash supernatants each contained about 20% of the active ingredient.

The lyophilized metazachlor-containing C16 protein microbeads were resuspended in 1 ml of 10 mM Tris buffer; 0.1% SDS; 100 μg proteinase K proteolytically digested at 37 ° C for 1 h. After ten minutes of centrifugation (20,000 x g) remaining from this approach drug crystals were dissolved in 500 ul of isopropanol. In the supernatant of the protease digestion about 1 1% of the amount of metazachlor used was detected. The redissolved in isopropanol drug crystals accounted for about 35% of the amount of metazachlor used.

Analysis of drug distribution (photometric determination at λ = 215 nm)

Example 5

Packaging and stabilization of retinol in protein microbeads

Substances which are difficult or not water-soluble and which are labile to influences such as oxygen radicals, UV, etc., can be packaged in protein microbeads which are produced from amphiphilic self-assembling proteins. Then they can be released from it again. In addition, the active ingredients are protected by formulation or packaging in protein microbeads from the damaging effects and the resulting degradation. To show this, the active substance retinol was selected as a non-limiting example, which was packed in C16 protein microbeads and stirred under air aeration and homogeneous mixing for several hours. Samples were taken at various times and the remaining retinol quantitated after THF extraction. The approaches presented in Table 5.1 were examined. First, the retinol-THF solution was diluted in isopropanol, then treated with the aqueous C16 protein solution and then in the case of approach 1, the C16 protein microbead formation induced by the addition of 1 M potassium phosphate solution. Since the presence of cations, eg. For example, by potassium phosphate in the C16 protein packaging approach, in principle contributes to increase the oxidation of freely dissolved or particulate occurring retinol was in control batches with and without C16 spider silk protein, but in which no C16 protein microbead formation should be induced 154 mM sodium chloride solution (see Fisher et al., 1972, Biochem J. 132: 259-270). The batches were incubated in plastic containers with closed glass vessels with stirring on a magnetic stirrer and continuous gassing via a cannula for up to 7 hours. When taking samples, 4 x 300 .mu.l taken, in which mathematically a maximum of 9.38 ug Retinol should be included. After removal, the C16 microbeads of the packaging batch were centrifuged off and the retinol contained was extracted with 1.5 ml of THF and quantitated by absorption photometry at 325 nm. For the batches without C16 microbeads, directly 1.5 ml of THF were added to the 300 μl sample, and the sample was mixed and centrifuged to produce a phase separation. The retinol then contained in the upper THF phase was also quantified by absorption photometry at 325 nm.

Approach 1: Packaging batch 0.9 ml of isopropanol (Sigma, crystalline)

0.1 ml retinol 5 mg / ml in THF

5 ml C16 solution 10 mg / ml in 5 mM K ₂ HPO ₄ buffer

10 ml of 1 MK ₂ HPO ₄ buffer

Approach 2: Stabilization mixture 0.9 ml of isopropanol (from Sigma, crystalline)

"Coating" 0.1 ml Retinol 5 mg / ml in THF

5 ml C16 solution 10 mg / ml in 5 mM K ₂ HPO ₄ buffer

10 ml of 154 mM NaCl solution

Batch 3: Control batch without 0.9 ml of isopropanol (from Sigma, crystalline)

C16 protein 0.1 ml retinol 5 mg / ml in THF

5 ml of 5 mM K ₂ HPO ₄ buffer

10 ml of 154 mM NaCl solution

Table 5.1: Different approaches to quantify the C16 spider silk protein mediated oxidation stability of retinol.

In the course of the mixtures with C16 spider silk protein (batch 1 packaging in C16 microbeads, batch 2-soluble C16), a clear stabilization of retinol under atmospheric oxygen is to be observed in comparison to the control without C16 protein (Table 5.2, Fig. 8) While in approach 2, the amount of retinol but also significantly reduced from 5-7 h, in approach 1, in which the drug was packaged in the C16 microbeads, even after 7 h even more than 70% intact retinol detectable (Tab 5.2, Fig. 8). Thus, the packaging of retinol in C16 protein microbeads appears to be a convenient method by which stabilization against oxygen radical induced degradation can be achieved. Proteolytic degradation of the retinol-loaded C16 microbeads with proteinase K (2.25 U) in 1 ml of 5 mM potassium phosphate buffer pH 8 allowed the drug to be released.

Table 5.2: Determination of retinol stability in C16 formulation approaches.

Fig. 8: Determination of retinol stability in C16 formulations depending on the incubation period.

In order to determine the maximum loading density of the C16 microbeads with active ingredient, various amounts of retinol were used in packaging approaches (see approach 1). The solvent used for the active ingredient was exclusively THF. Subsequently, C16 protein microbead formation was induced by the addition of 1 M potassium phosphate buffer (pH 8.0). The mixture was incubated for 1 h at 10 ° C and then centrifuged for 10 min at 20,000 x g. The pellet was washed twice with distilled water. Thereafter, the active ingredient was removed by washing the C16 protein microbeads with 2 ml of THF and quantified by absorption photometry at 325 nm (see Table 5.3). It was found that the maximum loading density for retinol in this experiment is about 1.9 mg per 5 mg C16 protein used (Table 5.3). In quantitative precipitation to C16 microbeads, the retinol active substance concentration or loading density is therefore about 38%.

Tab. 5.3: Quantification of the retinol packed in 5 mg C16 microbeads and released therefrom.

Example 6

Packaging of ibuprofen from THF in microbeads and release by proteolysis

Hardly or not water-soluble pharmacologically active substances can be packaged in protein microbeads, which are produced from amphiphilic self-assembling proteins. Then they can be released from it again. In addition, these agents can be protected from the deleterious effects, e.g. by packaging in protein microbeads, e.g. certain proteases or strongly acidic pHs and resulting degradation. Certain drug particle sizes or drug structures that are better absorbed or better bioavailable can be adjusted by packaging in protein microbeads or by micronization approaches using amphiphilic self-assembling proteins. To demonstrate this, the active substance ibuprofen [(RS) -2- (4-isobutylphenyl) propionic acid] was selected as a non-limiting example. 500 μl of a 10 mg / ml C16 protein-containing potassium phosphate solution (5 mM, pH 8.0) were mixed with 100 μl of an ibuprofen solution (5 mg / ml in isopropanol). C16 protein microbead formation was induced by the addition of 1 ml of 1 M potassium phosphate buffer (pH 8.0). The mixture was incubated for 1 h at room temperature and then centrifuged for 10 min at 20,000 x g. The pellet was redistilled twice with 5 ml H2O. washed. After precipitation with potassium phosphate, microbeads formed in the batch with C16 protein and ibuprofen. These were morphologically comparable to those of a standard mixture with C16 protein but no active ingredient. The packaging of ibuprofen in the C16 protein microbeads was quantitative in this approach, which is why in the supernatant after induction of microbead formation no ibuprofen could be detected by absorption photometry. Non-specific proteolytic degradation of the I-buprofen-loaded C16 microbeads with proteinase K (2.25 U) in 1 ml of 5 mM potassium phosphate buffer pH 8 enabled the release of the active substance.

A proteolytic digestion of the ibuprofen-loaded C16 protein microbeads in a pepsin-containing batch (analogous to Example 2) did not lead to the release of the active ingredient. The treatment of the ibuprofen-loaded C16 protein microbeads in pancreatin-containing mixtures (analogously to Example 2) led to the release of the active ingredient. Accordingly, the C16 microbeads can provide protection against gastric protease as well as the very acidic pH values prevailing in the stomach. A release under intestinal conditions is possible. C16 microbeads are therefore suitable, among other things, for the packaging and formulation of orally administered active substances which are absorbed or act in the intestine and which are to be protected in the case of gastric passage.

Claims

claims

1. Use of amphiphilic self-assembling proteins for the formulation of sparingly water-soluble effect substances.

2. Use according to claim 1, characterized in that the amphiphilic self-assembling proteins are microbead-forming proteins.

3. Use according to claim 1, characterized in that the amphiphilic self-assembling proteins are intrinsically unfolded proteins.

4. Use according to claim 1, characterized in that the amphiphilic self-assembling proteins are silk proteins.

5. Use according to claim 1, characterized in that the amphiphilic self-assembling proteins are spider silk proteins.

Use according to claim 1, characterized in that the amphiphilic self-assembling proteins are the C16 spider silk protein.

7. Use according to claim 1 to 6, characterized in that pharmaceutically active substances are used as effect substances.

8. Use according to claim 1 to 6, characterized in that are used as effect substances crop protection agents.

9. Use according to claim 1 to 6, characterized in that are used as effect substances active ingredients for the skin and hair cosmetics.

10. A process for the preparation of Effektstoffformulierungen, wherein

(i) mixing the sparingly water-soluble effect substance together with the amphiphilic self-assembling protein in a common disperse phase and

(ii) subsequently carrying out a phase separation into a protein- and effect-rich phase and a phase which is low in protein and effect.

1 1. A method according to claim 10, characterized in that one causes the phase separation (ii) by lyotropic salts.

12. The method according to claim 10, characterized in that one operates at a temperature between 5 and 50 ° C.

13. The method according to claim 10-12, characterized in that the protein- and effect-rich phase hardens and separated as mechanically stable effect-containing protein microbeads and possibly dried.

14. Cosmetic preparations containing a sparingly water-soluble effect substance which has been formulated with at least one amphiphilic self-assembling protein, together with further cosmetic adjuvants.

15. Pharmaceutical preparations containing a sparingly water-soluble

Effect substance that has been formulated with at least one amphiphilic self-assembling protein, together with other pharmaceutical excipients.

16. Agrochemical preparations containing a sparingly water-soluble effect substance which has been formulated with at least one amphiphilic self-assembling protein, together with other agrochemical auxiliaries.