DE19732774A1 - Covalent modification of compounds in liquid phase used as e.g. injectable immunogens, contact lenses or catheters - Google Patents

Abstract

Covalent modification of chemically defined compounds which contain functional groups and which are present in the liquid phase, comprises: (a) bringing the compounds into intimate contact with other compounds or solid bodies which contain at least two silicon-bound vinyl or butenyl groups per molecule (or on the surface of the solid body); and (b) subjecting the mixture to conditions such that the functional groups of the first compounds are added to the vinyl/butenyl groups of the other compounds or solid bodies.

Description

This invention relates to a method for the covalent modification of in liquid Phase present, functional group-containing compounds and the Use of the process products.

In the following, the term “functional groups” is understood to mean chemical groups which can be added to CC double bonds in a known manner. Examples include -OH, -NH ₂ , -CHO, -CH ₂ -Cl, -SH etc., see also Jerry March: Advanced Organic Chemistry, John Wiley & Sons, 3 edition, pp. 657-779, New York 1985.

Examples of compounds containing such functional groups are wise monosaccharides, such as. B. glucose, fructose, mannose, etc. or disaccha ride, such as B. sucrose, lactose, etc. or polysaccharides, such as. B. starch, Amylose, agarose, dextran, agar-agar, arabinose, heparin, alginate, cellulose, Pectin, etc. or amino acids such as e.g. B. glycine, lysine, arginine, cysteine, etc. or (Oligo) peptides such as e.g. B. (genetically engineered) FSH, LH, ACTH, etc. or proteins, such as B. gelatin, collagen, microsomes, plasmids, enzymes, anti body, receptors, etc., as well as other chemically defined ones Connections such as B. pharmaceuticals or plastics, e.g. B. Polyvinyl alcohol (PVA).

O.g. Compounds are in liquid, in particular physiologically aqueous Phase before. Examples are a 5% by weight glucose solution in de-ionized Water, a 5% by weight albumin solution in isotonic saline solution, a 10% by weight dextran solution in isotonic saline (average molecular weight weight of dextran 40,000), a 20% by weight mannitol solution in de-ionized Water, a 3% by weight solution of a polymer degraded succinylated Gelatin (average molecular weight 35,000) in isotonic electrolyte solution, etc. called.  

The term "covalent modification" used here describes that the above. functional groups present in the liquid phase as examples containing compounds cross-linked covalently or but also be covalently bound to the surfaces of solids. The Networking of chemically defined compounds with each other or binding such connections to solid phases is in the field of biotechnology and the biomaterials known and common, see for example the PIERCE, USA-Rockford, Illinois catalogs. - By the covalent ver network connections with each other, get the above Connections for example as higher viscosities and / or higher sedimentation rates and / or altered immunogenic properties and / or altered (biological) degradation behavior. Through covalent bonding the above Connections Surfaces of solid particles get this new (surface) property the number of reactive groups available for further reactions stand, is increased and the (water) wetting behavior is changed, preferably improved so that this in turn is reflected in the sedimentation behavior and is reflected in the flow properties of particulate solids.

In the known methods for cross-linking connections or their binding to surfaces of dispersed solids Chemicals used, such as. B. CNBr, are toxic or that lead to bonds lead, with a half-life of approx. 18 hours, as with CDI, only limited are stable to hydrolysis or require the reaction conditions, e.g. B. epoxy where denature sensitive biologically active compounds.

The object of the invention is to provide chemically defined liquid phase compounds containing functional groups through the use of known, inexpensive, toxicologically harmless chemicals, in a simple way and to network with each other with great efficiency or on surfaces of To bind solids that the newly created covalent bonds are stable to hydrolysis and also by means of high-energy electromagnetic Radiation, e.g. B. UV, can be achieved and that the products in one Variety of training, such as B. as an immunogen, chromatography material or can be used as a medical device.  

The object is achieved by chemically defined, in the liquid phase present functional group connections with others Compounds or with solids containing at least two silicon-bonded vinyl or have butenyl groups per molecule or on the solid surface, bringing them into intimate contact and exposing them to reaction conditions, that the functional groups of the first-mentioned compounds are attached to the vinyl or Butenyl groups of the second-mentioned compounds or the solids added become.

Compounds that have at least two silicon-bonded vinyl or butenyl groups have per molecule are known. Examples include tetravinylsilane, 1,3-divinyl-tetramethyldisiloxane, methyltrivinylsilane, dimethyldivinylsilane, tetra methyltetravinylcyclotetrasiloxane; vinyl end-capped linear polydimethylsiloxanes; vinyl group-containing polymethylsiloxanes with terminal and pendant vinyl called groups etc.

Solids that have at least two silicon-bonded vinyl or butenyl groups the solid surface are also known or can easily be created. Examples include vinyl or butenyl groups surface-modified highly disperse silica (fumed silica), ground Glass, open-pore glass, e.g. B. Siran®, Bioran® or controlled pore glass (CPG), Diatomaceous earth, (Ouarz-) sand, molded particles made of silicone rubber or open-pore silicone foam as well as finished Molded body made of silicone rubber, such as. B. contact lenses, intra eyepiece lenses, (blood) catheters, (mamma) implants etc. These each with vinyl or butenyl group-modified particles or moldings are included easily obtainable, for example, by following Weethall, H.H .: Covalent Coupling Methods for Inorganic Support Materials in Methods in Enzymology (K. Mosbach, ed.), Vol. XLIV, pp 134-148 (1976), Academic Press, New York, the particle or shaped body surface, instead of with an amino group-containing silane, they now with a vinyl group-containing silane, such as. B. Vinyltriaethoxysilane, vinyltrimethoxysilane, vinytriacetoxysilane, vinyltripropenoxy silane, vinyl tris (2-methoxyethoxy) silane or the like. surface modified. Preferred Particles or moldings have a density of between 0.9 and 1.3 g / ml  have a particle between 5 nm and several millimeters diameters, are porous, are biologically inert and are covered with the above-mentioned vinyl or to modify butenyl group-containing silanes easily.

The crosslinking of hydroxyl-containing compounds according to the invention by means of divinyldimethylsilane can be described in terms of the formula:

The bond according to the invention of compounds containing amino groups to vinyl groups which are in the form of particles or mold surfaces can be described in terms of the formula:

The addition of functional groups, such as. B. -OH, -NH ₂ , -CHO, -COOH, -CH ₂ -Cl ,. . .-SH, ether, secondary amine, ester, alkyl obtained on CC double bonds. . . Compared to the prior art, sulfide bonds are considered to be extremely stable to hydrolysis.

The intimate contact of the functional liquid phase Group-containing compounds with compounds that have at least two contain silicon-bonded vinyl or butenyl groups per molecule takes place in the Usually by simply mixing the reactants together. Because vinyl or Silicon compounds containing butenyl groups are poorly water-soluble, one must look for solvent systems in which both hydrophilic and hydrophobic Connections are at least partially to be solved. The appropriate selection of such Systems are familiar to the person skilled in the art: for example, hydroxyl groups containing compounds with vinyl groups-containing silanes or vinyl group-containing  low molecular weight siloxanes in aqueous alcoholic solutions, e.g. B. 3 parts 2-propanol and 1 part water even without the use of surfactants Substances such as B. surfactants, sufficiently good in a homogenizer emulsify.

The intimate contact of the functional liquid phase Group-containing compounds with particles or moldings that at least two silicon-bonded vinyl or butenyl groups on the particle or have molded surface, is easily done by stirring in Particles in the liquid phase or by contacting the molded body with the liquid phase. The moistening of vinyl group-containing particles with a lower aliphatic alcohol to facilitate their dispersion.

The reaction conditions under which the functional groups are in liquid Phase present compounds on the vinyl or butenyl groups of the others Connections, particles or moldings are added to the person skilled in the art common. With regard to the intended use of the networked or connections to be bound to a solid surface, taking into account the nature of the respective functional group, its molecular weight or concentration related to the solid surface, the concentrations of the individual reactants, the chemical nature of the solvent (mixture), the pH of the reaction solution, the reaction temperature, type and concentration added catalysts and / or initiators and / or photosensitizers each experimentally determined and optimized.

As a rule of thumb: sensitive, functional, liquid phase should be used Group-containing compounds, such as. B. biologically active substances, e.g. B. native enzymes, receptors or antibodies cross-linked or on solid body surfaces are bound, so you choose the reaction conditions as mild as possible. "As mild as possible" means that the solvents and whose pH and temperature do not have a denaturing effect and that the additions reaction preferably by photosensitizers, such as. B. benzophenone or Acetophenone, and UV light is initiated. - However, become relatively insensitive Connections such as B. cross-linked saccharides or to a solid  bound to the surface, the addition reaction is advantageously carried out with increased Pressure and elevated temperature using polymerization catalysts, e.g. B. water-soluble peroxides.

The compounds covalently cross-linked by this method are used as an injectable immunogen, as an injectable (gene product) Therapeutic agent, as an injectable drug carrier, as a starting material for (affinity) chromatographic purposes etc. used. - Process products that by adding functional group-containing compounds to vinyl group-containing particle surfaces have been created are (injectable) Immunogen, as an (injectable) (gene product) therapeutic agent, as starting material for the immobilization of biologically active substances, as (basic) material for chromatographic purposes, etc. used. - Process products by Addition of functional group-containing compounds to vinyl groups Molded surfaces containing created are considered to be with blood, tears liquid, secretions, urine, cell-containing tissue, etc. contacting medicine products such as B. blood bags, contact lenses or (blood) catheters or as water-binding materials for soil loosening or as. . . used.

Examples Example 1: Covalent crosslinking of dextran, MW 40,000 using tetramethyl tetravinylcyclotetrasiloxane

50 ml Rheofusin® (from Kabi Pharmacia), which is acidified to pH 2 with 1 M HCl was with 2 ml of a 10 vol .-% solution of tetramethyltetravinyl cyclotetrasiloxane in methanol, which added a spatula tip of benzophenone was at room temperature for 48 hours and when irradiated with a Medium-pressure mercury lamps in a rotating round-bottom flask contacted. - There are clear differences in the behavior when solving each equal volumes of the product thus produced or of native Rheofusin® in Drops of 2-propanol.  

Example 2: Covalent crosslinking of human albumin using tetravinylsilane

50 ml of a 5% solution of albumin in an electrolyte solution (from Immuno) be with 5 ml of a 5 vol .-% tetravinylsilane emulsion in de-ionized Contacted water as described in Example 1. - The tetravinyl silane emulsion was by adding 5 vol% tetravinylsilane and 0.5 wt .-% Na dodecyl sulfate to boiling water and about 2 minutes of ultrasound treatment. - Differences in the solution behavior are evident when dropping the same volumes from uncrosslinked and crosslinked by means of tetravinylsilane to isotonic Saline solution, which contains 2.5 vol .-% 96% ethanol.

Example 3: Addition of sucrose to various silicon-bonded vinyl groups having solid phase particles

Equipping the surfaces of the particles with silicon-bonded vinyl groups was carried out by contacting open-pore glass (Siran®) for several days, open porous silicone foam (manufactured according to DE-OS 195 42 687), diatomaceous earth (DIAMOL GM®, Franz Bertram GmbH, Hamburg), highly disperse silica (HDK T 30, Wacker-Chemie GmbH, Burghausen) and silicone particles, the by grinding silicone moldings using liquid Nitrogen to a grain size of <800 microns were obtained with a 10 vol .-% Vinyltnmethoxysilan solution in methanol, pH 2, then Drying the particles at approx. 50 ° C for several hours and then washing them with one consisting of 1 part methanol and 1 part de-ionized water Wash solution, the pH of which was adjusted to about 2 with HCl. - Approx. 1 part by volume of respective, now vinyl group-containing solid phase particles were then in washing liquid-moist state in approx. 10 parts by volume of a 5% by weight aqueous sucrose solution, pH 2, dispersed. About the respective dispersions about 3% by weight of ammonium peroxodisulfate were then added before the Dispersions were then autoclaved for 45 min at 121 ° C and 1.2 bar. After that the dispersions were washed several times with deionized water and in stored in a moist state. - All surfaces with sucrose modified solid phase particles sediment much faster in water than that  corresponding untreated particles. - To these so covalently bound Hydroxyl groups of these particles can now in a second step biologically active compounds, e.g. B. antibodies or enzymes, by means of established Methods such as B. CNBr, CDI, sulfonyl chloride or FMP.

Example: 4: Addition of Polymin P® to various silicon-bonded vinyl groups having solid phase particles

Essentially the same procedure as described in Example 3: instead of one 5% by weight sucrose solution becomes a 2% by weight ethyleneimine polymer solution (from Fluka, Neu-Ulm), in de-ionized water, pH 11, used. - All solid phase modified with Polymin POR particles sediment much faster in water than the corresponding ones untreated particles, they also bind anionic dissolved in water Dyes, e.g. B. "Phenol red water soluble" (Fa. Fluka, Neu-Ulm).

Example: 5: Addition of sorbitol to shaped bodies designed as contact lenses Silicone rubber

Contact lenses made of silicone rubber (from Wöhlk, Schönkirchen near Kiel) were made for exposed to gaseous vinyltrimethoxysilane for about 8 days, namely that: at 30 ° C in a polypropylene net above approx. 10 ml vinyl trimethoxysilane were positioned in a desiccator. The contact lenses were then subjected to an approximately two-hour heat treatment at approximately 70 ° C. Then they were placed in quartz cuvettes with a saturated sorbitol solution Methanol, which was added about 0.5 wt .-% benzophenone, transferred. The The whole was then irradiated with a medium pressure mercury lamp for 48 hours. - After that, the contact lenses were equilibrated in de-ionized water. The so treated contact lens surface is now hydrophilic, which is inherent shaft is also extremely resistant to contact lens care products.

Claims (2)

1. Process for the covalent modification of chemically defined compounds containing functional groups in the liquid phase, characterized in that these compounds are combined with other compounds or solids which in turn have at least two silicon-bonded vinyl or butenyl groups per molecule or on the solid surface , brings into intimate contact with one another and exposes reaction conditions such that the functional groups of the former compounds are added to the vinyl or butenyl groups of the latter compounds or solids. 2. Use of the produced by the method of claim 1 Process products as (injectable) immunogen, as (injectable) (Gene product) therapeutic agent, as (starting) material for the immobilization of biologically active substances, as (basic) material for chromatographic Purposes as with blood, tear fluid, secretions, urine, cell tissue, etc. medical devices coming into contact, such as B. blood bags, contact lenses or (Blood) catheter, as a water-binding material for loosening the soil or as . . .