COPOLYMERS HAVING BACTERICIDAL ACTIVITY, PROCESS FOR THE PREPARATION THEREOF AND PHARMACEUTICAL COMPOSITIONS THEREFOR

Abstract: Copolymers of formula I are described in which A is the monomeric unit of a natural or synthetic polymer, m is the degree of polymerization; m and n are different one from the other, and m is a number between about 10 and about 10,000 and n is a number between about 100 and about 250; R is a quaternary ammonium radical; x⁻ is an anion. A may be the monomeric unit of starch or the monomeric unit of hydroxyethylcellulose.

Description: Copolymers having bactericidal activity, process for the preparation thereof and pharmaceutical compositions therefrom 'rhe present invention relates to copolymers and more specifically to copolymers of formula (I), EMI1.1 in which A is the monomer unit of a natural. or svnthe- tic polymer with degree of polymerization m; m and n which, in general are different one from the other, are numbers between about 10 and about 101000 in the case of m and between about 100 and about 250 in the case of n; R is a quaternary ammonium radical, preferably one of the following formulae hereinbelow; EMI1.2 in which R', R", R"', which may be the same or different one from the other, are alkyl or benzyl radicals and p is an integral number between 0 and 10; x is the anion from an inorganic or organic acid, preferably C1 , Br , I , F , HSO4 Compounds of formula I unexpectedly have exhibited hi qh bactericidal activity accompanied by a very low toxicity. It is well known that disinfectants and antiseptics ordinarilycalled surface active agents which contain quaternary ammonium groups exhibit high bactericidal activity against both gram-positive and gram-negative organisms, as well as against funguses and viruses The toxicity of these compounds varies from one product to the other: however, it is certain that the administra- tion by the oral, rectal or vaginal route may cause serious damages particularly if the administration had tween made erroneously. Particularly, when these substances are administered morally, they cause nausea and vomiting, and after they are absorbed by the body, they may produce muscular paralysis because of the possibilaity of blockage of the respiratory muscles with the result that asphyxia follnws. The lethal dose of the most common quaternary surface active agents such as henzethonium chloride, benzalko nium chloride, methylbenzethonium chloride, cetylpyridinium chloride, has been estimated to be about 1-3 grams. The symptoms of poisoning are gastrointestinal irritation, dyspnea, cyanosis, collapse, convulsions, a state of dizziness, muscular weakness and finally death due to paralysis of the respiratory muscles. In. the concentrations used on the human skin, the so Ructions of the quaternary ammonium bases do not product ce irritation but in some patients which are hypersensitive and after protracted use, they < .may produce phenomena of irritation and'or allergic reactions. It has now been found surprisingly that the compounds of formula I in addition to exhibiting a very high activity, are also substantially devoid to toxicity both Thy the oral route and by application onto the skin as well as mucosae. The synthesis of the products of the present application is carried out by means of a reaction of copolymerization which may be either ionic or of the free radical type by means of suitable initiators. The reaction of copolymerization may be carried out using as starting materials different monomeric units, thus obtaining copolymers which may be block copolymers or with an alternating or sequential arrangement. It is also possible to use as a starting material a pre-prepared polymer and then attach to it polymeric chain of different molecular weight of another functional mono mer The monomeric unit which imparts to the product the chain - EMI3.1 may also be in the form of a quaternary salt and the quaternization may be carried out prior to the reaction of copolymerization or after the reaction of copolymerization. One object of the present application is to provide the novel copolymers of formula I hereinabove. Another object is to provide a process for the prepa ration of the copolymers of formula I which involves grafting nitrogenous monomeric units, preferably 4 vinylpyridine onto oligosaccharides, polysaccharides, cellulosic derivatives, polyvinyl alcohol, etc. The reaction of grafting is carried out by means of initiators for the purpose of forming suitable free radicals at a temperature lower than 40 C. For this reason, it is preferable to use redox initiator systems among which it is possible to mention the systems Fe++/H202; R-sWH202; Na2S2%/H202; Ce4+ in solution of sulfuric or nitric acid, metallic salts of transi 5+ tion metals such as V and other similar salts. Obviously, the products which are obtained depend upon the conditions used in the copolymerization reaction and specifically depend upon the quantity of the initiator used, the quantity of the monomer, the temperature and the reaction medium. It is, therefore, possible to obtain different quantities of synthetic polymers produced by an addition reaction with different molecular weight of the vinylpyridine chains which are grafted onto them. In general, the quantity of the polymer which is grafted varies between 10 and 60%, while the molecular weight is between 10,000 and 200,000. The quantity of the polymer which is grafted may be determined on the basis of the increase in weight of a known amount of oligosaccharide and poiysaccharide after the reaction of copolymerization as soon as the homopolymer such as polyvinylpyridine has been eliminated by means of a selective extraction with a suitable solvent or by means-f elemental analysis of the nitrogen which is contained in a known quantity in the copolymer. The reaction of quaternization of the copo].ymer may he carried out using different alkylating agents such as ethyl. bromide, -propyl bromide, butyl bromide, etc., cetyl chloride, benzyl chloride, etc., in a variety of solvents. The polyvinylpyridine, either as such or copolymerized, is difficult to quaternize and the rate of quaternization dicreases as the reaction proceeds. The choice of the reaction medium in which the reaction is carried out and the choice of the alkylating agent determine the extent of quaternization which may be achieved. Solvents having a high dielectric constant such as ni trobenzene, dimethylformamidet tetrahydrofuran, sili fo- lane, propylene carbonate have been found to be very suitable. The quaternization reaction may be followed spectrophotometrically by infrared analysis during the course ol reaction. Polyvinylpyridine exhibits a clear absorpli- -1 on band at 1600 cm As soon as the reaction of quaternization proceeds, a -1 new tand at 1640 cm appears. The degree of quaterni-- zdt is calculated on the basis of the results of elemental analysis and the amount of halogen ions and other negative ions. The final products exhibit a degree of quaternization between 60 and 95%. The following non-limitative examples are given by way of illustration of the invention. EXAMPLE 1 a) Natural maize starch, in the amount of 5 grams is clissolved in 50 ml of boiling water. To the solution, there is added concentrated nitric acid in such a manner as to obtain a concentration of 1.5 N, with respect to the acid. The mixture is deaerated to remove. air for 10-20 minutes by passing nitrogen or argon through; then 10 ml of 4-vinylpyridine are added and subsequently a solution of cerium ammonium nitrate in nitric acid, so that the reaction system has a 0.01 M concentration with respect to Ce4+ The reaction mixture is stirred for 12 hours at room temperature; then 400 ml of a mixture of 10% sodium hydroxide and 1,2-propandiol,in the ratio of 3:1 are added. The precipitate which is formed is centrifuged and washed with distilled water until the starch disappears in the washing water. Subsequently, the material is washed with methanol in order to eliminate the 4-polyvinylpyridie which has not been grafted onto the starch matrix. The product after filtering exhibits an infrared spectrum in KBr with the following characteristics bands: 1600 cm-. 1220 -1 -1 -1 cm , 1070 cm and 820 cm . The elemental analysis has given the following values: C = 52.78%; H = 4,82%; N - 7.34%. b) The copolyifler obtained as described in Example 1 in the amount of 7 grams, is suspended in 50 ml of methanol and treated with 10 grams of ethyl bromide. The reaction mixture is warmed under reflux for 120 hours, then the solvent and the excessive agent are evaporated up to a volume of about 15-20 ml. The material is then cooled to 0 C and the resulting quaternary salt which is formed is filtered. I.k. KBr: 1600 cm band has disappeared and the 1640 cm 1 band is now present. This band is characteristic of quaternized pyridine. Analysis: Br = 39.80%. The product which is obtained is designated hereinbµ- low for short with the symbol FCB 1010. EXAMPLE 2 a) Twenty grams of hydroxyethylcellulose of average molecular weight 40,000 are dissolved in 350 ml of water. To the solution there are added 20 grams of concentrated nitric acid and then after deaeration by means of nitrogen, 30 ml of 4-vinylpyridine followed by a solution of cerium ammonium nitrate in nitric acid in such a quantity to achieve a concentration of Ce4 of 0.01 M. The reaction mixture is maintained under stirring for a period of 24 hours at 250 C and then 1000 ml of 10% sodium hydroxide are added. The precipitate which is formed, is filtered, washed with water and then with methanol. I.R. KBr: 1600 cm-Ú. 1200 cm-Ú, 1045 cm-Ú, 830 cm-Ú Analysis: C - 42.46%; H = 5.76%; N = 3.05%. b) The copolymer described in Example 2a, in the amount of 15 grams, is dissolved in 75 grams o± nitrobenzene and quaternized in 25 grams of ethyl bromide at 100 C for a period of 48 hours. After the reaction is completed, the material is diluted with pentane and the quaternary salt which is formed is filtered and washed with a small amount of methanol. T.R.: The 1600 cm band has disappeared and the 1640 cm band is now present. cm Analysis: Br = 16.54%. The material which is obtained will bè referred to.he- reinbelow with the symbol FCB 1011. Microbiological Testing of the Antibacterial Activity of the Compounds FOB 1010 and FCB 1011 1) Antibacterial activity as a function of the concen- tration of the products. The products in the form of a whitish powder are dissolved in saline phosphate buffer prepared according to Sorensen of pH 7.2 in a concentration of 10% weight/volume which corresponds to 100 mg/ml. For the purpose of testing the antibacterial activity of the products, 6 bacteral stocks purified by plaque donation derived from human pathogenic germs maintai- ned in the phase of logarithmic growth are used. In particular, there. are used the following organisms: 1) Sram - : Escherichia Coli (E.Coli) Pseudomonas Aeruginosa (Ps.Aer.) Proteus Vulgaris (Pr.Yulg.) 2) Gram + : Micrococcus Luteus (M.Lut.) Streptococcus Fecalis (S.Fec.) Staphylococcus Aureus (S.Aur.) A liquid medium with ample spectrum of capacity of growth is used as the culture medium, that is Bactotryptic Soy Broth (Difco). The activity of the products is determined using the concentrations of 0.1, 0.01% and 0.001% in a liquid culture medium both òr FCB 1010 and FCB 1011. For corn parison purposes, there is used oL-(p-tolyl)dodecyl- trimethylammonium methoxysulfate in the same concen- rations. For each dilution of the product there are utilized four test tubes each containing 20 ml of the liquid medium. The antibacterial activity is determined by the turbidom.etric test after 24 hours of incubation at 370 C. The test consists of determining the values of reduction of the light transmitted from the culture media which contain the micro-organisms under test and the products in different concentrations using as a control the same media together with the pharmaceuti cal substance being tested in the same concentration. The values are expressed in percentage of the cultures without addition of any antibacterial substance. For each test, there are used 7.5 x 105 live gram-negative bacteria and 9.2 x 105 live gram-positive bac terio. RESULTS Jn Table 1 hereinbelow are reported the effects of FCB 1010 in different concentrations with respect to the organism under test. It is possible to observe that the product FCB 1010 in the concentration of 0.1% and 0.01% inhibits totally the growth of all bacteria tested. In the concentration of 0.001%, the substance exhibits an antibacterial activity, particularly with respect to Escherichia Coli, Pseudomonas Aeruginosa and Micrococcus Luteus. In Table 2 hereinbelow, the antibacterial activity ex hibited by the substance FCB 1011 is shown. Also, this substance prevents the antibacterial growth with all the organisms which have been studied up to a concentration of 0.01%, while in the concentration oP 0.0010, the activity decreases substantially, but the inhibitory effect of 50-60% is maintained. The e±èct of &alpha;-(p-tolyl)-dodecyltrimethylammonium methoxysulfate (Desogen), are tabulated in Table 3. They are substantially inferior with respect to the two preceding products. In fact, in the concentration of 0.01%. this substance does not prevent bacterial growth with any of the organisms tested. TABLE 1 Antibacterial activity of the Product FCB 1010 at different concentrations with respect to several bacteria under test Test Conc.of No. the E.Coli Ps.Aer. Pr.Vul. M.Lut. S.Fec. S.Aur. Substance 1 ------- 100% 100% 100% 100% 100% 100% 2 0.1% 0% 0% 0% 0% 0% 0% 3 0.01% 0% 0% 0% 0% 0% 0% 4 0.001% 51% 42.2% 76.31% 55% 63% 64% TABLE 2 Antibacterial activity of the Product FCB 1011 at different concentrations with respect to several bacteria under test Test Conc. of No. the E.Coli Ps.Aer. Pr.Vul. M.Lut. S.Fec. S.Aur. Substance 1 -------- 100% 100% 100% 100% 100% 100% 2 0.1% 0% 0% 0% 0% 0% 0% 3 0.01% 0% 0% 0% 0% 0% 0% 4 0.001% 59% 44.3% 66.4% 61.3% 58% 61% TABLE 3 Activity of the Product Desogen at different concentrations with respect to the bacteria under test Test Conc. of No. the E.Coli Ps.Aer. Pr.Vul. M.Lut. S.Fec. S.Aur. Substance 1 -------- 100% 100% 100% 100% 100% 100% 2 0.1% 0% 0% 0% 0% 0% 0% 3 0.01% 18% 27% 39% 16% 26% 21% 4 0.001% 78% 66% 72% 86% 84% 83% 2) Antibacterial activity as a function of the concen- trations of the products and their contact times with the bacteria. The antibacterial activity as a function of the con,- tact time of the surface active agents with the bacteria has also been investigated. The bacteria are suspen- ded in water; FCB 1010, FCB 1011 and &alpha;-(p-tolyl)-dodecyl trimethylammonium methoxysulfate have been used in concentrations of 0.1% and 0,01. After 2, 5, 15 minutes of contact time, Example of 0.1 ml has been placed into a medium of liquid culture as described hereinabove for a period. of 48 hours. Each test according to the different times and different concentrations has been carried out four times. The mediwn for the evaluation of the bacterial growth was the same as the method previously described. However, the results are expressed in a semi-quantitative manner in order to provide a better evaluation of the effective antibacterial activity (turbidimetric values of 1-30%+++; 3060%++; 60-90%+). The results so obtained are reported in Trifle 4 hereinbelow using different contact times of ta, product with respect to the organisms with the two concentrations which have been studied. It is evident that the products FCB 1010 and 1011 are active as antibacterial agents against all the organisms tested in the concentrations of 0.1% and 0.01% e ven after only two minutes of contact time. The product used as a comparison exhibits an activity in the same order of magnitude against all bacteria. except Pseudomonas Aeruginosa because in the case of this organism, the substance at the concentration of 0,01 does not exhibit a total bactericidal action. TABLE 4 EMI15.1 Substances <tb> Tested <SEP> C.% <SEP> T.C. <SEP> E.Coli <SEP> Ps.Aer. <SEP> Pr.Vulg. <SEP> M.Lut. <SEP> S.Fec. <SEP> S.Aur. <tb> FCB <tb> 1010 <SEP> 0.1 <SEP> 2 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> 5 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> 15 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> 0.01 <SEP> 2 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> 5 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> FCB <tb> 1011 <SEP> 0.1 <SEP> 2 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> 5 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> 15 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> 0.01 <SEP> 2 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> 5 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> 15 <SEP> - <SEP> + <SEP> - <SEP> - <SEP> - <SEP> DESOGEN0.1 <SEP> 2 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> 5 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> 15 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> 0.01 <SEP> 2 <SEP> - <SEP> + <SEP> - <SEP> - <SEP> - <SEP> + <tb> 5 <SEP> - <SEP> + <SEP> - <SEP> - <SEP> - <SEP> 15 <SEP> - <SEP> + <SEP> - <SEP> - <SEP> - <SEP> Disinfectant and antimycotic activity in "ViVO" a) The disinfectant activity in "vivo" has been studied in 40 patients affected by dermatosis of infec- tive nature, generally pyogenic such as pyodermia; streptodermia, staphylodermia, acne, and in general inflamed prominent elevations of the skin. FCB 1010 and FCB 1011 have been applied on the skin of 20 patients both in the concentration of 1%, two or three times a day up to an improvement of the clinical condition. The therapeutic effect has been found to be op times in 80% of the patients treated both in the case of FCF 1010 and FCB 1011. In addition, none of the treated patients exhibited any reactions of cutaneous intnlerance nor did any of the patients exhibit any reactions of allergic nature. h) The antimycotic activity in "vivo" was determined wi th 30 patients affected by superficial cutaneous mycosis (epidermofizie) or different types both from the morphologic-clinical point of view as well as Sta ge, caused by a variety of different funguses. Also, in this case, FCB 1010 and FCB 1011 were applied onto the skin in a concentration of 1%, two or three times d day to 15 patients up to an improvement of the clinical condition. This improvement was achieved very substantially in 86 of the individuals with FCB 1010 and 80% with FCB 1011. During treatment, no secondary reactions, either at the cutaneous level or in general, were noted. Allergological Tests The allergological tests have been carried out with 60 individuals who had an allergic dermatosis and who had, therefore, high cutaneous activity, by application onto the skin. FCB 1010 and FCB 1011 have been applied three times a day in different concentrations on an equal number of sections of the skin -for a period of 30 days. The concentrations of the products selected for the test have been 4/, 2ib and 1%. None of the patients manifested any symptoms of cutaneous intolerance connected with the treatment. Toxicity Tests FCB 1010 and FCB 1011 have been administered orally to Swiss rats and Sprague-Dawley rats in order to determine the acute toxicity, that is DL50. The DL50 has been found in both species to be greater than 3 g/kg. Another test has been carried out to determine the toxicity over an extended period of time with Beagle dogs For a period of four months. The tests have shown that with a daily dose of 1 or 2 g/kg, no change attri.butable to the products FCB 1010 and FCB 1011 has been found. Another object of the invention is the preparation of pharmaceutical compositions for topical use, such as creams, ointments, lotions, etc., having antibacterial and antimycotic activity which contain as the active principle at least one of the copolymers of formula I, together with carriers and excipients conventionally used in the formulations of pharmaceutical compositions. Still another object of the invention resides in the method of treatment of patients affected by dermatosis of infective nature, such as pyodermia, streptodermia, staphylodermia, acne and cutaneous mycosis by means o the compounds of formula I. Clearly, the in vention covers all the operations required for the preparation of the copolymers, their purification, their formulation in pharmaceutical forms suitable for the administration and/or the final confection in containers suitable for the administration.

Claims: Claims: 1. A copolymer of formula I EMI19.1 wherein A is the monomeric unit of a natural or synthetic polymer, m is the degree of polymerization; m and n are different one from the other, and m is a number between about 10 and about 10,000 and n is a number between about 100 and about 250; It is a quaternary ammonium radical. 2. A copolymer according to claim 1 wherein R is a quaternary ammonium radical which is a member selected from the group consi.sti ng of EMI19.2 wherein R', R'', are the same or different and are arkya. or benzyl and p is an integral number between O and 10; x is the anion of an inorganic or organic acid. 3. A copolymer according to claim 1 wherein x is Cl Br, I, F, or HSO4. 4. A copolymer according to claim 1 wherein A is the monomeric unit of starch. 5. A copolymer according to claim 1 wherein A is the monomeric unit of hydroxyethylcelulose. 6. A copolymer according to claim 2 wherein R is EMI20.1 wherein R' has the same meanings as in claim 2. 7. A copolymer according to claim 5 wherein R is EMI20.2 and X is Cl, Br or I . 8. A pharmaceutical composition for topical use which contains as the active ingredient at least one compo und of formula I EMI21.1 wherein A is the monomeric unit of a natural or synthetic polymer, m is the degree of polymerization: rn and n are different one from the other, and m is c number between about 10 and about 10,000 and n is a number between about 100 and about 250; R is a quaternary ammonium radical; and at least one inert carrier and inert excipient. 9. The method of treatment of a patient affected by dermatosis of infective character of the type of pyodermia, streptodermia, staphylodermia, acne, cutaneo Us mycosis which consists of treating said patient with an effective amount of a compound according t claim 1 Claims: 1. Process for the preparation of copolymers having bactericidal activity, of the formula I EMI22.1 wherein A is the monomeric unit of a natural or synthetic polymer, m is the degree of polymerization; m and n are different one from the other, and m is a number between about 10 and about 10,000 and n is a number between about 100 and about 250; R is a quaternary ammonium radical; comprising the grafting of monomeric units of the formula EMI22.2 wherein R1 is a basic group which can be converted in onto a polymeric chain of the formula EMI22.3 (wherein A and m have the above mentioned meanings), the reaction of grafting being carried out by means of ini tiators capable of forming free radicals at a temperature lower than 400 C; and the quaternization of the basic groups R1 in the grafted copolymers so obtained. 2. Process according to claim 1, wherein the initiators are redox systems selected from the group consisting of Fe++/H2O2; thiols/H202; Na2S2O3/H2O2; Ce+ in solution of sulfuric or nitric acid; salts of transition metals. 3. Process according to claims 1-2, wherein the quantity of the grafted chains of formula EMI23.1 varies between 10 and 60% of the copolymer of formula (I), and their molecular weight is between 10,000 and 200,000* 4. Process according to claims 1-3, wherein R is a quaternary ammonium radical which is a member selected from the group consisting of EMI23.2 wherein R', R", R"' are the same or different and are alkyl or benzyl and p is an integral number between 0 and 10; X is the anion of an inorganic or organic acid. 5. Process according to claims 1-4, wherein K is Cl Br, I, F or HSO4. 6. Process according to claims 1-5, wherein A is the monomeric unit of starch. 7. Process according to claims 1-5, wherein A is the monomeric unit of hydroxyethylcellulose. 8. Process according to claims 1-7, wherein R is EMI24.1 and X is Cl-, Br or I-.

Inventors: MALTZ, JAVIER ESTEBAN
Applicants: TEXCONTOR-ANSTALT
Application year: 1981
Main classification: A61K31
IPC codes: C08F251, C08F8, A61P31, C08F291, A61K31