NO770394L - PROCEDURES FOR IMMOBILIZING ANTIMICROBIAL AGENTS. - Google Patents

Description

Procedure for the immobilization of antimicrobial agents

The present invention relates to antimicrobial compounds and, more specifically, antimicrobial compounds which are covalently bound to polymeric carriers with a relatively large particle size.

Many potentially useful antimicrobial compounds cannot be used for "therapeutic purposes" in humans or animals because these compounds become toxic when observed through the skin, mucous membranes or the gastrointestinal system. Until recently, e.g. hexachlorophene has routinely been used as a disinfectant and antimicrobial agent in soaps and detergents. However, hexachlorophene has now been banned for routine use due to the fact that it can be absorbed through the skin and exhibit neurological toxicity. In a similar vein, pharmaceutical research programs have led to the isolation and characterization of a large number of natural products that exhibit antimicrobial activity but cannot be used because of their toxic effects. In order to reduce absorption and additionally limit the antimicrobial agent, the antibiotic agent is frequently applied topically in immobilizing ointments. For example, Neosporin contains bacitracin, polymyxin and neomycin in an ointment base. These three antibiotics are rarely used internally due to secondary toxic effects associated with their use. In the case of Neosporin, the ointment primarily serves to limit the treatment area. However, there are many antimicrobial agents that cannot be<p>applied even in an ointment base to limited areas because they will be absorbed and be toxic. Chloramphenicol and novobicin are extremely toxic antimicrobial agents that are rapidly absorbed from the gastrointestinal tract. The use of these is only recommended against such infections that cannot be effectively treated with less toxic agents.

In certain areas, efforts have been made to permanently bind smaller molecules to much larger polymeric carrier molecules which, because of their size, prevent passage of the small chemicals through the skin or mucous membrane.

This molecular immobilization has been done with food additives, and a discussion of this particular area of application can be found in Food Engineering, for January and June 1974, The Manufacturing Confectioner, Volume 54 - No. 2, February 1974, and in an article in the Wall Street Journal, June 18, 1973. It appears that dyes, antioxidants and other food additives will pass through the gastrointestinal system without being absorbed when they are chemically bound to polymeric molecules.

While a food coloring or antioxidant, when immobilized by chemical binding to a polymeric carrier molecule, may retain its color or oxidation-inhibiting properties prior to ingestion, a pharmaceutical agent, such as an antimicrobial agent, cannot be expected to remain active when the corresponding immobilization. Furthermore, while the colorants and antioxidants exhibit their effect in food before ingestion, by improving the appearance or preserving the taste, there is no need for such chemicals to retain their desired properties after ingestion. In contrast, an antimicrobial must retain its biological activity against microbes in all environments. Furthermore, there are reasons to believe that when such pharmaceutical agents are permanently attached to polymeric carrier molecules through stable chemical bonds, their biological activity will be lost.

For example, cellulose fibers have been developed with antibacterial cations or organomercury or organotin residues. Such treated fibers are stated to exhibit antibiotic activity only as long as the active molecules are released by the breakdown of their chemical bond to the carrier fibers, when completely stable chains are used, the treated fiber has no antibiotic activity. (Z.A. ogovin and A.D. Virnik in "High Polymers", (N, M. Bikales and L. Segal, eds.) Volume V, Part V, Wiley-Interscience, 1971, pp. 1333-1336).

A number of patents describe the temporary immobilization of active pharmaceutical agents by adsorption or dispersion in oils or pectin (H. Welch, 2491537, 20 September 1949, H. Welch, 2518510, 15 August 1950, BS.

Whittingham, 2481805, Sept. 13, B.S. Whittingham, 2481804, September 13, 1949), by ionic bonds to polymeric acids or bases (P.C. Wirth, 3832340, August 27, 1974, L. Loewe, 2656298, October 20, 1953), or by unstable chemical bonds (G.F. Collins and L.J. Daher 3320236, 16 May 1967). similar preparations affect the duration of action of the therapeutic agent by enabling slow release, but it is clear that only the free therapeutic agent after release from the complex is active, and not the immobilized drug itself:

Thus, no method has been devised to provide a solution to the problem of cross-linking an antimicrobial agent which will be permanently immobilized by coupling through stable bonds to a polymeric carrier molecule, but which will still remain effective for its originally intended purpose, what than the purpose may be.

While the main obstacle overcome by the present invention is the need to preserve biological activity of immobilized antimicrobial agents, there are other aspects of the problems that need to be solved for a practical solution to be achieved: identification of polymeric carriers that will be appropriate in terms of size, shape and physical and chemical properties to provide the necessary immobilization with respect to passage through skin or entry into wounds therein or through mucous membranes, resistance to phagocytosis, or retention at the site of application, choice of size and nature of the chemical link between the antimicrobial agent and the carrier molecule, determination of the molecular site on the antimicrobial agent to which the immobilizing chemical

ke coupling can be attached. It is in reality these two sides that are critical to ensure that the antimicrobial agent retains

where its biological activity in an immobilized state.

An aim of the present invention is therefore to provide immobilized antimicrobially active pharmaceutical preparations which can either be administered internally through the gastrointestinal system or applied topically, without the risk of absorption and subsequent distribution through the circulatory system.

It is a further object of the invention to provide immobilized antimicrobially active pharmaceutical compositions which can either be administered internally through the gastrointestinal system or applied topically, without loss or dilution of the antimicrobial compound from the site of application due to the passage of the compound through the skin or wound therein or through mucous membranes.

A further aim of the invention is to provide ■ immobilized antimicrobially active pharmaceutical preparations which, when administered internally or applied topically, avoid

undergo secondary toxic effects, are resistant to phagocytosis and avoid immunological sensitization.

Another aim of the invention is to provide a method for producing such an immobilized antimicrobial active pharmaceutical preparation.

A further aim of the invention is to provide

provide a method for preventing secondary toxic effects, phagocytosis and immunological sensitization when administering antimicrobial compounds when administered internally

tes or application topically of only immobilized-active pharmaceutical<p>preparations.

Another aim of the invention is to achieve immobilisation

ring of an active pharmaceutical preparation by covalent chaining of the preparation to a relatively large polymeric carrier molecule while significant pharmaceutical activity of the preparation is retained.

Other goals and benefits will emerge from the following description and requirements.

According to the above, the invention relates to active antimicrobial compounds or preparations which are covalently bound to the polymer, e.g. polysaccharide-based carriers of natural or synthetic origin to immobilize the preparations. Such immobilization prevents dissolution, absorption and transport of the antimicrobial compounds into or through the skin or wound therein or mucous membranes, thereby limiting their action to specific areas and eliminating secondary toxic effects. Covalent binding of antimicrobial compounds to polymeric carriers according to the invention has led to this antimicrobial compound which retains antimicrobial activity, and in which the covalent bond between the antimicrobial compound and the polymeric carrier is stable in all environments, including the environment in the gastrointestinal system with low pH.

There are countless areas of application for immobilized antimicrobial compounds. For example, antibiotics that are too easily absorbed when used topically can now be used topically when these are immobilized by covalent binding to polymeric carriers. Antibiotics which when taken orally would pass through the gastrointestinal system can now be administered orally for the treatment of infections of the gut and stomach when immobilized by covalent binding to <p>olymeric carriers. The size and shape of the polymeric carrier can be tailored to suit the particular case in which the antimicrobial agent is to be used. In cases where open wounds or cuts are included, the shape of the polymeric carrier must be such that it is ensured against uptake of the antimicrobial compound through the open wound or cut into the vascular system. For such applications, a good example of a polymeric carrier for the antimicrobial agent would be cellulose fibers in the form of a bandage. In the case of antimicrobial agents in soaps or detergents. the polymeric carrier will preferably be a water-soluble compound.

Specific examples of the wide range of applications for immobilized antibiotics are exemplified by the two antibiotics vancomycin and bacitracin. Both of these antibiotics exhibit serious adverse effects when they enter the circulatory system. This can occur when these antibiotics are administered topically to open wounds. The adverse effects of vancomycin may include hypersensitivity reactions (skin rash and anaphylaxis), chills and fever, ototoxicity, nephrotoxicity, deafness and kidney damage. Thus vancomycin is currently not used very therapeutically. The adverse effects of the bacitracin peptides may include hypersensitivity reactions, nausea, vomiting, diarrhoea, cylinduria, proteinuria, hematuria and severe kidney damage (oliguria and anuria).

It is of course impossible to list all conceivable areas of application for immobilized antimicrobial compounds. In general, they appear to be applicable in all human or animal treatments (with the exception of parenteral administration, i.e., except for introduction into the cardiovascular system) where the antimicrobial compound without chaining to a carrier would be absorbed through the skin or mucous membranes and cause undesirable secondary effects toxic effects. Other main advantages are that immobilized antimicrobial compounds can be more effectively retained at the site of application and that they are relatively inert to chemical or enzymatic modification.

The purpose of the invention is to use any of the chemical reactions that lead to stable covalent binding of antimicrobial compounds in biologically and pharmaceutically active forms to relatively large molecular units, such as solid (e.g. glass) or polymeric (e.g. .eg cellulose) carriers to prevent absorption of the antimicrobial compound through the skin or mucous membranes, to prevent dilution or loss thereof, and to prevent the antimicrobial derivative from being phagocytized. This purpose is achieved by using e.g. coupling reactions with activated polysaccharides, aminotriazines, reactions capable of forming amine, sulfonamide, carbonate, carbamate, isourea and other imino-carbonate bonds, reactions capable of forming ester, amine, sulfide or ether bonds, reactions involving formation and reduction of Schiff bases, and reactions involving aldol, dehydrated aldol, alkyl and acyl bonds.

As used herein, the term "antimicrobial" includes

all types of antibiotic, antibacterial, antiviral, anti-parasitic, antifungal and similar anti-infective agents.'

There are countless antimicrobial compounds that are useful in combating different types of infection in humans and animals and the present invention is not limited to any particular group, and individual antimicrobial compounds or any molecular weight classes. As the invention is extremely applicable to the antimicrobial agents which have secondary toxic effects when applied to humans or animals, these compounds are natural and important preparations for use according to the present invention. However, it should be understood that even if an antimicrobial compound has no known secondary toxic effects, it can be of great advantage to bind it to a regulatory molecule to prevent inactivation or loss or dilution of the agent from the site of application. Furthermore, such immobilization ensures against immunological sensitization to the antimicrobial agent and thus enables repeated use. Examples of such compounds are given below.

Antimicrobial-antibiotic compounds

a. Aliphatic diamines with the general structure indicated below in which n = 2-18:

b. Polymyxins B^, B2 , , and whose structures are shown below:

In the preceding structural formula, R, X, Y and Z have the following chemical meanings:

MeOct - 6-methyloctanoic acid, MeHep = 6-methyl-heptanoic acid, DAB = L-a, γ-diaminobutyric acid, Thr = threonine, Phe = phenylalanine, Ser = serine, Leu = Leucine

c. Bacitracin A whose structure is shown below:

d. Penicillins, penicillin derivatives and cephalosporins as illustrated by the following representative examples: Benzylpenicillin 6-aminopenicillanic acid

Cephalosporin C derivative

e. Aminophenols and substituted aminophenols of the general structure shown below:

where A, B, C and D can be different functions including H, F, Cl, Br, NO2, N<H>2, COOH and OH. f. Polyene antibiotics as exemplified by the following representative examples: g. Organomercury compounds such as the three representative examples set forth below: h. The vancomycin group of antibiotics, as the •partially determined structure of Ristomycin A shown below:

R^- R^ represents known bonds

Partial structure of ristomycin A<1>" Befenium with the following structure:

j. Dichlorophene with the following structure.: k. Hexylresorcinol with the following structure:

The preferred antimicrobial compounds to be bound to polymeric carriers are antibiotics and specifically, those compounds which exhibit their pharmaceutical or antimicrobial function by acting on or in the cell walls or membranes. The most significant among these are the polymyxins, bacitricin, and the polyene antibiotics.

Just as there are too many antimicrobial compounds to list, the possible polymeric carriers that can be chosen are likewise too numerous to list fully. The following list of suitable carriers selected from natural and modified polysaccharides, glass and synthetic polymers illustrates the wide range of possible carrier materials. Most preferred among these are the polysaccharides, specifically dextran and cellulose.

Solid and/or polymeric carriers

Natural carriers

1. Cellulose. Cellulose is a linear polymer of D-glucopyranose units chained 3(1/4). The structure is shown below. 2. Dextrans. Dextrans are chained polymers of D-glucopyranose units, the backbone chain is chained a(l,6), and the branches are chained cx(1,2, a(l,3) and cx(l,4). The structure of the backbone chain is shown below.

Dextran chains can be cross-linked with epichlorohydrin, 2-hydroxypropyl chains are chained 1,3 to the free hydroxyl groups in the dextran chains. Dextrans with various degrees of crosslinking are available commercially in the form of beads under the trade name Sephadex from Pharmacia Corp.

3. Agarose. Agarose is a linear polysaccharide with alternating residues of D-galactopyranose and 3,6-anhydro-L-galacto-pyranose. The structure is shown below. Beads and cross-linked forms of agarose are commercially available under the trade names Sepharose 2B, 4B, 6B and CL2B, CL4B and CL6B from Pharmacia Corp. 4. Starch: For example a-amylose which is an unbranched polymer of D-glucopyranose chain a(1,4) and amylopectin which is a branched polymer with an a-amylose backbone and branches chain a(1,6). 5. ' Inulin is a complex copolymer of D-glucopyranose and D-fructofuranose units. 6. Xylans are linear polymers of D-xylopyranose units chained 3(1,4). 7. Mannans are polymers of the D-mannopyranose chain ■0(1,4), 8. Glucomannans are copolymers of D-glucose and D-mannose chain 3(1,4). 9. Galactans and arabinogalactans are highly branched polymers of galactose and galactose and arabinose units respectively. chain 1.6 and 1.3.

10. Gum arabic and other plant gums.

11. Collagen and other naturally occurring or synthetic polypeptides and their derivatives.

3

Modified polysaccharide carrier materials

1. Treatment of polysaccharides with periodic acid "under mild conditions leads to the splitting of some of the 1,2-, diol chains with the formation of dialdehyde groups. Extensive splitting of the polymer chain takes place. The aldehyde functions can be used as further modification sites. 2. Natural functional groups in the polysaccharide carriers are primary and secondary hydroxyl groups, and in certain cases carboxylic acids (e.g. gum arabic and xylans). Other functional groups that can be derived from these are shown below.

The natural functional group in glass is a Si-OH group. Examples of other functions that can be linked are:

Synthetic polymers

1. Polyacrylamides

The structure of polyacrylamide is (shown below. The structure is sometimes crosslinked with methylenebisacrylamide or methylenediacrylate units. These materials are available commercially in the form of beads, with varying degrees of crosslinking, under the trade names Bioels from Bio-Rad Laboratories.."

The natural function of acrylamides is carboxyamide groups. These can be converted into other groups suitable for binding drugs as shown below.

Several of these derivatives are commercially available from Bio-Rad Laboratories, e.g. hydrazide (Hydrazid Bio-Gel P-2 and P-150), aminoethyl (Affi-Gel 701 and Aminoethyl Bio-Gel P-2 and P-150), and carboxyl (.Affi-Gel 702 and Bio-Gel CM-2 ).

2. Polyacrylates The structure of the polyacrylates is shown below:

The natural function in these carrier materials is carboxylic acid or the ester group. Functional group modification reactions of importance for drug binding are shown below:

3. Polystyrenes

The structure of polystyrene is shown below. Different polystyrenes are commercially available, some in the form of beads containing a low percentage of 1,4-divinylbenzene as a cross-linking agent. •

Certain reactions that introduce functional groups into polystyrene that are suitable for binding drugs are shown in the following reaction core:

Chloromethylated cross-linked polystyrene beads are commercially available as Bio-beads S-Xl and S-X2 from Bio-Rad Laboratories.

4. Polyvinyl alcohol

This carrier material is produced by hydrolysis of polyvinyl acetate. The structure is shown below:

The natural function in this material is the hydroxyl group. Therefore, all functionalization reactions described in connection with the polysaccharide carriers, with the exception of periodate splitting, are applicable.

5. Polyethylene maleic anhydride copolymer

The material contains reactive succinic anhydride units and can be used to bind medicines directly. 6. Other possible carrier materials include condensation polymers such as polyesters and polyamides (nylon) and polyvinylpyrrolidone.

As the size of the carrier must be sufficiently large to prevent phagocytosis or penetration through biological membranes and typical phagocytic cell diameters in micron are as follows: Monocyt.es (14 - 20), macrophages (20 - 40), neutrophils (12), langhans- cells (40 - 50), it is assumed that the carrier molecules according to the invention must have a diameter above approx. 50 microns.

The antimicrobial compound and the solid carriers can be chained in many ways, which will be discussed in detail later, with the result that the antimicrobial compounds remain pharmaceutically active. One form of chaining contemplates the insertion of a molecular arm between the carrier and the antimicrobial compound to allow sufficient cellular penetration of the target cell by the therapeutically active agent while the carrier molecule restricts the movement of the therapeutic agent. To exemplify and demonstrate that chained drugs can retain their pharmaceutical activity, polymyxin B was covalently bound to activated agarose beads with a 10 Å hydrocarbon arm between them and the retained antimicrobial activity of polymyxin B was shown.

Example 1

Polymyxin B, a known naturally occurring antimicrobial agent, was covalently bound to agarose beads by reacting free polymyxin B with activated agarose beads (commercially available as Affi-Gel 10 from Bio-Rad Laboratories). The structure of activated agarose (Affi-Gel 10) is shown below:

The activated side arm is covalently bound to agarose through a stable ether bond. Polymyxin-B was coupled to Affi-gel 10 as shown below:

The conditions for connection were as follows:

1. 2.5 g of polymyxin B (1.8 mmol) sulfate was dissolved in

25 ml, of 0.1 M f osf atpuf f er , pH 7 , 0 .

2. The above-mentioned solution was added to 1 g of dehydrated Affi-Gel 10, forming 15 ml of swollen Affi-Gel containing approx. 0.18 mmol activated side arms.

This mixture was then reacted for 8 hours at 4° C. with stirring. The final derivative was washed with 1M NaCl until the absorbance of the eluate at 260 nanometers was zero.

As can be seen, derivatives of this type can be reacted with the free amino groups in polymyxin with the formation of amide chains between the agarose beads and the polymyxin. The hydrocarbon arm was approx. 10 Å long and the hydrated agarose beads were approx. 74 to 149 microns in diameter. This bead size is sufficiently large to prevent phagocytosis or penetration through biological membranes.

It is approx. 8 to 12 micromol/ml of the activated arm groups in Affi-Gel 10 when it swells. Therefore, a tenfold molar excess of polymyxin B is used in relation to the active arm groups, to prevent multiple amide chaining to polymyxin B, which has four free amino groups. Chemical modification of one of the amino groups does not destroy the antibiotic activity, but modification of two or more does. Accordingly, the coupling conditions were chosen so that the average polymyxin molecule was bound to the agarose beads only through an amide bond.

After covalent binding of polymyxin B to agarose beads, 1 ml of this derivative was washed thoroughly with ethanol and water to remove all free polymyxin. The washing procedure consisted of successive washings with 200 ml of 10% ethanol in water, 200 ml of 5% ethanol in water and 200 ml of water. The effectiveness of the washing procedure was determined by demonstrating that the successive washes completely eliminated unbound polymyxin B as determined by antimicrobial assays and ultraviolet absorption. The washing procedure is not critical as long as it removes all traces of unbound polymyxin. It is important that the final wash water does not contain any organic solvent, as ethanol residues could affect the results obtained.

The antimicrobial activity of polymyxim B bound to agarose beads was determined as follows.

Sterile culture medium was introduced into the inside (50 ml) and outside (100 ml) of a dialysis bag suspended in an Erlenmeyer flask. The polymyxin-agarose beads were placed on the inside of the dialysis bag after which both the inside and outside were inoculated with different numbers of viable Escherichia coli (ATCC #25922) ranging from 1 x 10 6 to 1 x 10 8 cells. When 0.1 mg of the chained polymyxin-agarose was placed on the inside of the dialysis bag, no bacterial growth was detected on the inside of the bag when the inoculum consisted of 1 x 10 cells or less (Table I). Likewise, when the chained polymyxin-agarose was placed on the outside of the bag, no bacterial growth was detected on the outside of the bag (Table I). The bacteria always grew on the outside when the polymyxin-agarose was placed on the inside of the bag and on the inside when the polymyxin-agarose was placed on the outside of the bag. In control experiments, free (unbound) polyoxyxin B was added to the inside of the dialysis bag. No bacterial growth was detected either on the inside or outside of the bag. In other control experiments, it was shown that agarose beads in the absence of covalently bound polymyxin B did not prevent bacterial growth.

This attempt establishes the following points:

1. Polymyxin bound to agarose beads retains its antimicrobial activity. 2. The polymyxin is covalently bound to the agarose beads on due to polymyxin. which-*was: chemically reacted with agarose beads was not-: ice tooth.--to pass again nom' the dialysis bag while free polymyxin passed through the dialysis bag. 3. Polymyxin in active form that can pass through the dialysis bag was not released from the beads by reaction with E. coli.

Example 2

The effectiveness of an aliphatic primary amine, an aliphatic carboxylic acid and an aminophenol is shown in the following experiments. The compounds shown below were covalently bound to agarose using Affi-Gel 10 as described for polymyxin B under Example 1:

Agarose derivatives of these compounds linked to agarose beads through their amine groups were investigated for antimicrobial activity against E. coli, S. typhimurium. B; subtilis and S. aureus in the following experiments. Inoculum was added to 9.5 ml of sterile culture medium. The inoculum consisted of 9.5 ml of stationary phase cells. In the test tubes, 0.5 ml of the immobilized antimicrobial agent was added. Control tubes contained either 0.5 mg of untreated agarose or no agarose. The samples were thoroughly mixed and then 1 to 10 serial dilutions of the original inoculated tube were made and the cultures were incubated at 37°C for 12 hours. Serial dilutions of the original culture were made to a final dilution of 1 x 10 ^ of the original sample. After 12 hours, the samples were examined for bacterial growth. In Table II below, the highest dilution at which bacterial growth was observed is indicated. These data support the following conclusions: 1. The untreated beads had no antimicrobial activity. 2. The aliphatic primary amine (ff 1) showed activity against E. coli, S. typhimurium and S. aureus. 3. The aliphatic carboxylic acid (^2) showed activity against E. coli. 4. C^q normal alkane ( ff3) showed no activity against any of the strains. 5. The phenol [ ffA) showed activity against S. typhimurium and B. subtilis.

These data indicate that the primary aliphatic amine and the phenol bound to agarose exhibit significant activity against at least one of the gram-positive and at least one of the gram-negative organisms tested.

Example. 3

Aminophenol was covalently bound to cellulose using carboxymethylcellulose and a carbodiimide reagent by the procedure outlined below to form the structure:

Phenolic cellulose I: (From carboxymethylcellulose and p-aminophenol): 1 g of carboxymethylcellulose (Sigma, medium, 0.7 milliequivalents per gram) was suspended in 10 ml of water and "5 ml of 5% hydrochloric acid was added. After stirring for 5 minutes, the slurry was filtered on a Buchner funnel and washed with 200 ml of water, 50 ml of ethanol and then 50 ml of ether. powder was transferred to a 125 mL Erlenmeyer flask with a magnetic stirrer, and 5 mL of dimethylformamide (DMF) was added, followed by 0.445 g (2.15 mmol) of dicyclohexylcarbodiimide (DCC, Aldrich), in 7 mL of DMF, and 0.545 g (5 mmol) p-aminophenol (Eastman) in 5 ml DMF. The sides of the flasks were washed with an additional 3 mL of DMF and the slurry was gently stirred at room temperature for 36 hours. At this point, 100 ml of water was added and the suspended solid was collected on a Buchner funnel and washed thoroughly with 200 ml each of water, ethanol and ether and then air dried to a light brown powder. The above derivative and unmodified cellulose were then tested for antimicrobial activity against E. coli using different amounts of the cellulose/phenol derivative and different inoculum sizes. The samples were cultured for 24 hours and examined for bakery growth. These data are listed in the following table III (+ = growth, - = growth). The data in Table III show that, unlike untreated cellulose, aminophenol coupled to cellulose has significant antibacterial activity. Aminophenol was also linked to agarose as described in example I. The last-mentioned derivative showed activity against S. typhimurium and B. subtilis but not against E. coli (Table II). These results indicate that the choice of polymer support and the covalent arm can have a significant effect on the antimicrobial activity of the compound bound to an immobilized support.

Other phenol-cellulose derivatives (II - V) and their di-chloro and di-iodo derivatives were prepared as described below: Phenol cellulose II: (From aminoethyl cellulose and 3-p-hydroxy-phenyl)-propionic acid): 3 g aminoethyl cellulose (Sigma, ca. 0 .7 milliequivalents per gram) was suspended in 20 ml of dimethylformamide (DMF), and 2.10 g (12.6 mmol) of 3-(p-hydroxyphenyl)-propionic acid (Aldrich) dissolved in a minimal volume of DMF was added, followed by 1.15 g (6.3 mmol) of dicyclohexylcarbodiimide (DCC, Aldrich) in 3 mL of DMF. This mixture was stirred at room temperature for 200 hours. After treatment with 20 ml of water, the slurry was transferred to a Buchner funnel and washed thoroughly with 200 ml of ethanol. The filter cake was resuspended in a beaker with 100 ml of ethanol, and then transferred back to a Buchner funnel and washed thoroughly with 100 ml each of ethanol, acetone and ether. After air drying, the product was a faint off-white solid.

The dichloro derivative of phenol cellulose II:

A 0.7 g portion of phenol cellulose II was slurried in 5 mL of water and 3 mL (3 mmol) of 1N sodium hypochlorite solution (Clorox) was added. The slurry was then stirred for 1 hour at 0°C, during which time the reaction mixture changed color from light yellow-green•to white. The reaction mixture was diluted with 20 ml of water and excess oxidant was destroyed (negative on starch-iodide paper) by the addition of 3 ml of saturated aqueous solution of sodium thiosulphate. The solid material was collected on a Buchner funnel and washed thoroughly with 200 ml each of water, 95% ethanol, acetone and ether and then air dried to form a white powder.

The diiodo derivative of phenol cellulose II:

Using the above-mentioned procedure, 0.7 g of phenol cellulose II was iodinated with 2.5 ml of an iodizing solution (15 g of potassium iodide and 25 g of iodine in 200 ml of 0.5 M water). After processing, washing and drying as described above, a light brown powder was obtained.

Phenolic cellulose III: (From aminoethyl cellulose and p-hydroxyphenylacetic acid)

3 g of aminoethylcellulose (Sigma, approx. 0.7 milliequivalents per gram) was suspended in .20 ml of dimethylformamide (DMF), and 1.96 g (12.6 mmol) of p-hydroxyphenylacetic acid (Aldrich) dissolved in a minimal volume of DMF was added, followed by 1.15 g

(6.3 mmol) of dicyclohexylcarbodiimide (DCC, Aldrich) in 3 mL of DMF. This mixture was stirred at room temperature for 48 hours. After treatment with 100 ml of acetone, the suspended solid was collected on a Buchner funnel and washed with 200 ml each of ethanol, water, acetone and ether and then air-dried to form an off-white powder.

The dichloro derivative of phenol cellulose III:

A 0.7 g portion of phenol cellulose III was slurried in 5 mL of water and 3 mL (3 mmol) of 1N sodium hypochlorite solution (Clorox) was added. The slurry was then stirred for 1 hour at 0°C, during which time the reaction mixture slowly changed color from light yellow-green to white. The reaction mixture was diluted with 20 ml of water, and excess oxidizing agent was destroyed (negative on starch-iodine paper) by the addition of 3 ml of a saturated aqueous solution of sodium thiosulfate. The solid was collected on a Buchner funnel and washed thoroughly with 200 ml each of water, 95% ethanol, acetone and ether and then air dried to give a white powder.

The diiodo derivative of phenol cellulose III:

Using the above procedure, 0.7 g of phenolic cellulose was iodinated with 2.5 ml of an iodizing solution (15 g of potassium iodide and 25 g of iodine in 200 ml of 0.5 M water). After processing, washing and drying as indicated above, a light brown powder was obtained. Phenolic cellulose IV: (From aminoethyl cellulose and p-hydroxybenzoic acid) 3 g of aminoethyl cellulose (Sigma approx. 0.7 milliequivalents per gram) were suspended in 20 ml of dimethylformamide (DMF), and 1.74 g (12.6 mmol) of -hydroxybenzoic acid (Eastman) dissolved in a minimal volume of DMF was added, followed by 1.15 g (6.3 mmol) of dicyclohexylcarbodiimide (DCC, Aldrich) in 3 mL of DMF. This mixture was stirred at room temperature for 200 hours. After treatment with 100 mL acetone, the suspended solids were collected on a Buchner funnel and washed with 200 mL each of ethanol, water, acetone and ether and then air-dried to form an off-white powder.

The diiodo derivative of phenol cellulose IV:

Using the above procedure, 0.7 g of phenol cellulose IV was iodinated with 2.5 ml of an iodizing solution (15 g of potassium iodide and 25 g of iodine in 200 ml of 0.5 M water). After processing, washing and drying as indicated above, a light brown powder was obtained.

Phenolic cellulose V: ( From carboxymethylcellulose and 2-( p-hydroxy-phenyl)-ethylamine: 3 g of carboxymethylcellulose (Sigma, Medium, 0.7 milliequivalents per gram) was suspended in 40 ml of dimethylformamide (DMF) and 1.72 g (12.6 mmol) of 2-(p-hydroxyphenyl)-ethylamine dissolved in a minimal volume of DMF was added, followed by 1.15 g (6.3 mmol) of dicyclohexylcarbodiimide (DCC, Aldrich) in 3 mL of DMF.

This mixture was stirred at room temperature for 48 hours during which time it changed color to brown.

After treatment with 100 mL of acetone, the suspended solids were collected on a Buchner funnel and washed with 200 mL each of ethanol, water, acetone, and ether, and then air-dried to form an off-white powder.

The dichloro derivative of phenol cellulose V:

A 0.7 g portion of phenol cellulose V was slurried in 5 mL of water and 3 mL (3 mmol) of 1N sodium hypochlorite solution (Clorox) was added. The slurry was then stirred for 1 hour at 0°C, during which time the reaction mixture changed color to light brown. The reaction mixture was diluted with 20 ml of water and excess oxidant was destroyed (negative on starch-iodine paper) by the addition of 3 ml of a saturated aqueous solution of sodium thiosulphate. The solid material remained collected on a Buchner funnel and washed thoroughly with 200 ml each of water, 95% ethanol, acetone and ether and then air dried to form a white powder.

The diiodo derivative of phenol cellulose V:

By applying the above-mentioned procedure, 0.7 g of phenol cellulose V was iodinated with 2.5 ml of an iodizing solution (15 g of potassium iodide and 25 g of iodine in 200 ml of water 9.5 M). The chocolate brown reaction mixture was worked up, washed and dried as above to form a light brown powder.

In light of the countless suitable antimicrobial compounds, the many suitable carrier materials and the various suitable chemical bonds between the antimicrobial compound and the carriers, it is impossible to exemplify every possible combination. For illustrative purposes, the following examples are intended to illustrate a typical chemical coupling of representative antimicrobial compounds to cellulose through the insert arms covalently bonded to the cellulose.

Example IV

Polymyxin B-, can be linked to cellulose as indicated below:

Example V

Mono-dinitrophenyl-Bacitracin A (derived from the amino group of ornithine) retains antibiotic activity. Therefore, Bacitracin A or its derivatives can be chained to cellulose through this amino group as follows:

. Example VI

6-Aminopenicillanic acid can be linked to cellulose as indicated below:

Example VII

The polyenes, illustrated here with Filipin, can be covalently attached to cellulose through an ester bond as shown below:

Example VIII

Derivatives of organic mercury compounds, such as phenylmercuric nitrate, can be covalently bonded to cellulose as shown below:

<->i

Example IX

Derivatives of bephenium can be attached to cellulose as shown below:'

Example X

Derivatives of dichlorophene can be attached to cellulose as shown below:

Example XI

Hexylresorcinol derivatives can be attached to cellulose through amide chains as shown below:

As already pointed out, there are many chemical reactions for chaining the antimicrobial compounds to the carrier materials. A brief description of illustrative reaction mechanisms is given in points A-H.

A. Derivatives of cyanogen bromide-activated agarose

Activation of agarose with cyanogen bromide is carried out by treating the agarose with excess finely divided cyanogen bromide at strongly basic pH and temperatures that do not exceed 20° C. Further derivation is carried out by reacting the activated agarose with a suitable pharmaceutical agent in a buffer or suitable organic solvent for several hours at room temperature or lower. Thus, the reaction with amines yields cyclic iminocarbonates substituted on the nitrogen and/or urethanes or carbamates, but principally N-substituted isoureas. Other nucleophilic compounds such as phenols can also be used in this reaction.

As the monovalent N-substituted isourea derivatives of ligands are not completely stable, it was decided to prepare stable polyvalent agarose derivatives. For example, cyanogen bromide-activated agarose will be linked to poly-D-lysine, bovine serum albumin or other large molecules containing polyamino groups. The polyvalent insertion arm (spacer) will then be further derived by standard procedures so that the insertion arm can in turn be chained to the active pharmaceutical agent by stable covalent bonds (peptide, ether etc.). The use of poly-D-lysine as one insert arm is particularly suitable, as it will be resistant to proteolysis.

B. Derivation of cyanuric acid-activated agarose

Several carriers, e.g. agarose, has been activated for further coupling by reaction with 2-amino-4,6-dichloro-s-triazine. Reaction of the activated support with nucleophilic reagents, including amines, thiols and alcohols, gives unsymmetrical triazine. The final derivation takes place in weakly basic buffers at room temperature for several hours.

C. Formation of amide bonds

Carboxylic acids can be converted into highly reactive acylating reagents by treatment with certain reagents. The resulting active esters, which is a general term for the products, can be of two types, those that develop in situ for immediate further reaction and those that are sufficiently stable for isolation and characterization. 1. Active esters developed in situ. The principal method for in situ development of acylating agents is by treating the carboxylic acid with a suitable carbodiimide, the choice of carbodiimide being determined by the solvent for the reaction. the tion. Typically, the carboxylic acid is dissolved in a suitable solvent where the pH is adjusted to 4.5 - 6.0. Treatment of the solution with the carbodiimide reagent provides the active acylating agent which further reacts with primary amines to form the amides. Typical reaction times are from 8 to 16 hours at room temperature. Alternatively, ethoxyacetylene can be used as an activating reagent. 2. Stable or isolable activated esters Carboxylic acids or active derivatives thereof can also be used to acylate p-nitrophenol or N-hydrdoxysuccinimide to form the corresponding esters. These materials are very active as acylating agents. While the typical procedure requires activation of the carboxylic acid with a carbodiimide reagent, it would seem that the utility of these active esters lies in their high stability which allows isolation and purification prior to coupling to the support. 3. Use of mixed anhydrides as acylating reagents

Carboxylic acids can be activated with respect to the acylerene by forming a mixed anhydride with a suitable carboxylic acid or carboxylic acid derivative. Preferred reagents include the isovaleryl or trimethylacetyl anhydrides formed in organic solvents by treatment of the carboxylic acid with the appropriate acid halide in the presence of the calculated amount of a tertiary amine base. Formation of the mixed anhydride is rapid even at low temperatures, i.e. -10° C, and the derivatives can be reacted with an appropriate nucleophilic compound within a very short time. The anhydrides are not normally isolated before further reaction.

Another type of mixed anhydride is the carboxylic acid-carboxylic anhydride, of which the isobutyl carbonates are the most preferred. These reagents are formed by reaction with isobutyl chloroformate and the carboxylic acid in the manner indicated above. The advantage of mixed carboxylic acid-carboxylic acid anhydrides in relation to mixed carboxylic acid anhydrides is that in the first-mentioned case the by-products are very volatile alcohols and carbon dioxide.

D. Formation of carboxylic acid esters

Formation of carboxylic acid esters can proceed by acylation of a suitable alcohol with one of the activated forms of a carboxylic acid described above.

E. Acylation on carbon

The active esters described in section C. 2 regarding stable or isolable activated esters are suitable electrophilic compounds for reaction with nucleophilic carbon centers, e.g. enols and certain electron-rich arene systems. Three cases can be taken into account, as described below.

1. Enolate nucleophilic compounds. Materials containing active hydrogen atoms can be converted either, reversibly or irreversibly, to their enolate anions by treatment with suitable agents. In the case of irreversible formation of the enolate, the active hydrogen component in the system will be boiled

o

during reflux with e.g. sodium metal or potassium hydride to form the metalled derivatives. The chosen solvent for this reaction will be from ethers, e.g. tetrahydrofuran or diglyme. Acylation of the metal compounds would be assumed to proceed rapidly at room temperature under these conditions.

Alternatively, the enolate can be developed reversibly by treatment with such reagents as sodium alkoxides in the appropriate alcohol. However, it should be noted in this case that reagents themselves must be non-nucleophilic and that the choice of solvents is thus limited to those that are strongly hindered. In this procedure, the alkoxide, the material to be acylated, and the acylating agent are boiled under reflux until the absence of acylating agent indicates completion of the reaction.

2. Enol nucleophilic compounds. Acylation of enolizable compounds can be carried out by treating a mixture of the acylating agent and the active hydrogen compound with a

suitable catalyst, e.g. boron trifluoride or boron trifluoride etherate, at temperatures close to 0° C. This method is best suited for the preparation of (3-diketones. 3. Aromatic nucleophilic compounds. Aromatic systems can be acylated in reactions catalyzed by Lewis acids, e.g. aluminum trichloride, boron trifluoride and stannous or zinc chloride, under anhydrous conditions.Suitable acylating agents include simple anhydrides and acid chlorides.

F. Alkylation of carbon nucleophilic compounds

1. Enolates as described in section E.l are suitable nucleophilic compounds for displacement of halide or sulfonate anion from primary or secondary alkyl groups. The reactions usually take place in ether or alcohol solvents, depending on the base used when forming the enolate. Epoxides can also be used as the electrophilic compound, and the alkylation gives rise to secondary or tertiary alcohols. With aldehydes or ketones as electrophilic components, the reaction products can be (3-hydroxy or α, (3-unsaturated carbonyl compounds (aldol condensation)). 2. Acylation of aromatic nucleophilic compounds (Friedel-Crafts alkylation) takes place by reacting alkyl halides and sulfonates with arenes in the presence of Lewis acid catalysts, usually aluminum trichloride.The use of reactive aromatic systems is thought to moderate the often vigorous reaction states.

A related reaction is the "hydroxyalkylation" of aldehydes and ketones. This reaction with phenols is particularly useful, as it gives rise to biphenylated methylene by reaction with aldehydes in alkaline media.

G. Alkylation of oxygen nucleophilic compounds

Reaction of alkyl or aryl alkoxides with suitable electrophilic compounds gives rise to ethers with good yield. Substrates for the reaction include alkyl halides, sulfonates, sulfates, and epoxides. The reaction gives the best yields and fewer side reactions if the alkylation involves displacement from primary carbon centers in alkoxides of primary or secondary alcohols. Under forced conditions, alcohols would react with aryl diazonium salts to form aryl ethers.

H. Alkylation of amines

1. Amines can be reacted with alkyl halides, sulfates or sulfonates to form an alkylated amine. The reaction can be controlled only with difficulty and addition of more than one alkyl group to the nitrogen is common, so that the method is of particular applicability in the preparation of tertiary amines.

The reaction of amines with epoxides to form the amino alcohol is a general reaction, but is most successful with epoxides with a primary center of attack. Under forced conditions, amines will react with aryldiazonium salts to form arylamines. 2. Reductive alkylation. Primary amines react with aldehydes and ketones in alcohol solution to form Schiff's base (imine). These intermediates can be reduced without isolation with a variety of reagents, including sodium borohydride, formic acid, zinc and hydrochloric acid, and by catalytic reduction over Raney nickel.

It should be noted that the reductive alkylation of the second nitrogen function is possible, if the functional group is unstable to reduction by a reagent as indicated above, e.g. nitro, nitroso or azo groups...

3. Formation of Mannich bases. Aliphatic amines are reacted with compounds containing active hydrogen atoms, including aldehydes, ketones, carboxylic acids and esters, nitriles, nitro compounds and certain phenols and benzyl compounds, in the presence of formaldehyde or other low molecular weight aldehydes with the formation of Mannich bases, i.e. active hydrogen compounds which has been aminomethylated at the site of the active hydrogen. The reaction usually takes place by refluxing in aqueous or alcoholic solution and can be either acid or base catalyzed.

I. Alkylation of sulphur-nucleophilic compounds

Mercaptans or alkali mercaptides react well with alkyl halides, sulphonates, aryldiazonium salts and epoxides. during formation of the sulfide (thioether). Conditions for the reaction are largely the same as for the formation of ethers (see section G) or amines (see section H.l) from these electrophilic compounds.

The covalent bond between the carrier and the therapeutically active agent formed by the reaction of the functional group of component 1 (activated component; electrophilic compound) with the functional group of component 2 (nucleophilic). The therapeutically active agent or carrier may be any of the components. In each case, the key groups in the two components involved in chain formation and the final chain structure are indicated and identified in the following Table IV. In general, amide, ether and amine chains are preferred for stability in different environments.

In certain cases, as previously indicated, it may be preferred to insert a molecular arm between the carrier material and the therapeutically active agent. The arm can be on either component 1 or component 2. After the reaction, the arm is thus located between the carrier and the biologically active material. The arms exemplified below can be constructed by the same reactions used to connect the carrier and the therapeutically active agent:

where m, n and o are at least 1.

Claims (51)

1. - Pharmaceutically active medicinal composition, characterized in that it comprises a pharmaceutically active antimicrobial material covalently bound to the molecules in a carrier, which carrier molecules have a sufficiently large dimension to prevent passage through the skin or mucous membranes, or entry through wounds in the skin. 2. Composition according to claim 1, characterized in that the active antimicrobial material is selected from antimicrobial compounds with sufficiently small molecules to allow passage through the skin, mucous membranes or wounds in the skin, and which cause secondary toxic effects when distributed in the circulatory system. 3. Composition according to claim 1, characterized in that the active antimicrobial material is selected from the group consisting of aliphatic diamines, polymyxins, bacitracin, penicillins, penicillin derivatives, cephalosporins, aminophenols, substituted phenols, polyene antibiotics, organic mercury compounds, vancomycin antibiotics. befenium, dichlorophene and hexylresorcinol. 4. Composition according to claim 3, characterized in that the active antimicrobial material is a compound that acts on or in the cell walls or membranes of the organism against which its activity is directed. 5. Composition according to claim 4, characterized in that the antimicrobial material is selected from the group consisting of polymyxins, bacitracin and polyene antibiotics. 6. Composition according to claim 1, characterized in that the carrier molecules are selected from natural polysaccharides, modified polysaccharides, natural polypeptides, synthetic polypeptides, glass, glass with active functional groups bound thereto, and synthetic polymers. 7. Composition according to claim 6, characterized in that the carrier molecules are polysaccharides selected from the group consisting of cellulose, dextrans, agarose, starch, inulin, xylans, mannans, glucomannans, galactans, arabinogalactans, gum arabic, plant gums and derived and periodized oxidation-modified forms of any of the foregoing compounds. 8. Composition according to claim 6, characterized ' . in that the carrier molecules are polymers selected from the group consisting of polyacrylamides, polyacrylates, polystyrenes with functional groups attached thereto, polyvinyl alcohol, polyethylene-maleic anhydride copolymer, polyvinylpyrrolidone and condensation polymers such as polyesters and polyamides. 9. Composition according to claim 7, characterized in that the carrier molecules are selected from dextran and cellulose. 10. Composition according to claim 1, characterized in that the covalent bond between the antimicrobial material and the carrier is stable in all the intended use environments. 11. Composition according to claim 10, characterized in that the bond is a covalent bond selected from the group consisting of amide, carbonate, carbamate, iminocarbonate, isourea, triazine, acyl and alkyl bonds, Mannich, base, sulfide, sulfonamide, ester, amine and ether linkages. 12. Composition according to claim 11, characterized in that the bond is a covalent bond selected from amide, amine and ether bond. 13. Composition according to claim 1, characterized in that a molecular arm is inserted between the active antimicrobial material and the carrier. 14. Composition according to claim 13, characterized in that the molecular arm is selected from the group consisting of those under points (a) - (e) in the following and combinations thereof, in which n, m and o are at least 1: 15. Composition according to claim 5, characterized in that the carrier molecules are selected from polysaccharides and that the covalent bond between the antimicrobial material and the carrier is selected from the group consisting of amide, amine and ether bond. 16. Composition according to claim 15, characterized in that the diameter of the carrier molecule is over 50 microns. 17. Process for the production of a pharmaceutically active medical composition that cannot pass through skin, mucous membranes or wounds in the skin into the circulatory system, characterized in that a pharmaceutically active antimicrobial agent is reacted with a carrier material with molecules that are sufficiently large to prevent passage through the skin, mucous membranes or wounds in the skin, forming a covalent bond between these, which bond is stable in all the intended use environments. 18. Method according to claim 17, characterized in that the covalent bond formed by the reaction is selected from the group consisting of amide, carbonate, carbamate, iminocarbonate, isourea, triazine, acyl and alkyl bonds, Mannich base, sulfide, sulfonamide , ester, amine and ether linkages. 19. Method according to claim 18, characterized in that a molecular arm is inserted between the active antimicrobial material and the carrier. 20. Method according to claim 19, characterized in that the molecular arm is selected from the group consisting of those specified under points (a) - (e) and combinations thereof, in which n, m and o are at least 1: 21. Method according to claim 18, characterized in that the carrier molecules are selected from natural polysaccharides, modified polysaccharides, natural polypeptides, synthetic polypeptides, glass, glass with active functional groups bound thereto, and synthetic polymers. 22. Method according to claim 21, characterized in that the diameter of the carrier molecules is over 50 microns. 23. Method according to claim 21, characterized in that the carrier molecules are collagen or polysaccharides, selected from the group consisting of cellulose, dextrans, agarose, starch, inulin, xylans, mannans, glucomannans, galactans, arabinogalactans, gum arabic, plant gums and derivatives and periodatoxidation- modified forms of any of those previously stated. 24. Method according to claim 21, characterized in that the carrier molecules are polymers selected from the group consisting of polyacrylamides, polyacrylates, polystyrenes with functional groups bound thereto, polyvinyl alcohol, polyethylene maleic anhydride copolymer, polyvinyl pyrrolidone and condensation polymers such as polyesters and polyamides. 25'. Method according to claim 18, characterized in that the active antimicrobial material is selected from antimicrobial compounds with sufficiently small molecules to allow passage through the skin, mucous membranes or wounds in the skin and which cause secondary toxic effects when distributed in the circulatory system. 26. Method according to claim 25, characterized in that the active antimicrobial material is selected from the group consisting of aliphatic diamines, polymyxins, bacitracin, penicillins, penicillin derivatives, cephalospirins, aminophenols, substituted phenols, polyene antibiotics, organic mercury compounds, vancomycin antibiotics. biphenium, dichlorophen and hexylresorcinol. 27. Method according to claim 26, characterized in that the active antimicrobial material is a compound that acts on or in the cell walls or membranes of the organism against which its activity is directed. 28. Method according to claim 27, characterized in that the antimicrobial material is selected from the group consisting of polymyxins, bacitracin and polyene antibiotics. 29«. Method according to claim 18, characterized in that the carrier molecules are natural polysaccharides selected from dextran and cellulose. 30. Method according to claim 28, characterized in that the carrier molecules are selected from polysaccharides and that the covalent bond between the antimicrobial material and the carrier is selected from the group consisting of amide, amine and ether bonds. 31. Method for preventing secondary toxic effects when administering antimicrobial compounds due to the passage of these compounds through the skin, mucous membranes or wounds in the skin into the circulatory system, characterized in that a pharmaceutically active medicinal composition is administered comprising a pharmaceutically active microbial material that is covalently bound to molecules. ler in a carrier, which carrier molecules have sufficiently large dimensions to prevent passage through the skin or wounds of the skin when the composition is applied topically, and through mucous membranes when administered internally. 32. Method according to claim 31, characterized in that the active antimicrobial material is selected from antimicrobial compounds with sufficiently small molecules to allow passage through the skin, mucous membranes or wounds in the skin and which cause secondary toxic effects when distributed within the circulatory system. 33. Method according to claim 31, characterized in that the antimicrobial material is selected from the group consisting of aliphatic diamines, polymyxins, bacitracin, penicillins, penicillin derivatives, cephalosporins, aminophenols, substituted phenols, polyene antibiotics. organomercury compounds, vancomycin antibiotics, befenium, dichlorophene and hexylresorcinol. 34. Method according to claim 33, characterized in that the active antimicrobial material is a compound that acts on or in the cell walls or membranes of the organism against which its activity is directed. 35. Method according to claim 34, characterized in that the active antimicrobial material is selected from the group consisting of polymyxins, bacitracin and polyene antibiotics. 36. Method according to claim 31, characterized in that the carrier molecules are selected from natural polysaccharides, modified polysaccharides, natural polypeptides, synthetic polypeptides, glass, glass with active functional groups bound thereto and synthetic polymers. 37. Method according to claim 36, characterized in that the carrier molecules are polysaccharides selected from the group consisting of cellulose, dextrans, agarose, starch, inulin, xylans, mannans, glucomannans, galactans, arabino-galactans, gum arabic, plant gums and derivatives and periodate oxidation -modified forms of any of the foregoing compounds. 38. Method according to claim 36, characterized in that the carrier molecules are polymers selected from the group consisting of polyacrylamides, polyacrylates, polystyrenes with functional groups bound thereto, polyvinyl alcohol, polyethylene maleic anhydride copolymer, polyvinyl pyrrolidone and condensation polymers such as polyesters and polyamides. 39. Method according to claim 37, characterized in that the carrier molecules are selected from dextran and cellulose. 40. Method according to claim 31, ^ characterized in that the covalent bond between the antimicrobial material and the carrier is stable in all intended use environments. 41. Method according to claim 40, characterized in that the bond is a covalent bond selected from the group consisting of amide, carbonate, carbamate, iminocarbonate, isourea, triazine, acyl and alkyl bonds, Mannich base, sulphide, sulfonamide, ester, amine and ether. 42. Method according to claim 41, characterized in that the bond is a covalent bond selected from amide, amine and ether bonds. 43. Method according to claim 31, characterized in that the molecular arm is inserted between the active antimicrobial material and the carrier. 44. Method according to claim 43, characterized in that the molecular arm is selected from the group consisting of those specified under points (a) - (e) and combinations thereof, in which ri, m and o are at least 1: 45. Method according to claim 35, characterized in that the carrier molecules are selected from polysaccharides and that the covalent bond between the antimicrobial material and the carrier is selected from the group consisting of amide, amine and ether bonds. 46. Method according to claim 45, characterized in that the diameter of the carrier molecules. is over 50 microns. 47. Method according to claim 46, characterized in that the carrier molecules are selected from dextran and cellulose. 48. Composition according to claim 1, characterized in that the antimicrobial material is selected from the group consisting of polymyxin, bacitracin and polyene antibiotics and that the covalent bond is a covalent bond that is stable in all intended use environments and is selected from the group consisting of amide, amine and ether bonds, and that the carrier molecules are selected from dextran and cellulose. 49. Method of preventing loss or dilution of antimicrobial compounds from the site of application due to passage of the compounds through the skin, mucous membranes or wounds in the skin into the circulatory system, characterized in that a pharmaceutically active medical composition is administered comprising a pharmaceutically active antimicrobial material which is covalently bound to molecules in a carrier, which carrier molecules have sufficiently large dimensions to prevent loss or dilution of the composition from the site of application due to passage of the composition through the skin or wounds therein when the compositions are applied topically and through mucous membranes when administered internally'. 50. Method for preventing immunological sensitization of antimicrobial compounds, characterized in that a pharmaceutically active antimicrobial material is administered which is covalently bound to molecules in a carrier with such a physical character that immunological sensitization of the antimicrobial component is prevented. 51. Method for preventing phagocytosis when administering antimicrobial compounds, characterized in that a. pharmaceutical active antimicrobial ingredient that is covalently bound to molecules in a carrier with. sufficiently large dimensions to prevent penetration <4> through the biological membranes.