IL87829A - Compositions containing quaternary ammonium fluoride salts for inhibiting or destroying unicellular living organisms and a process for preparing such salts. - Google Patents

Description

&>>n ο»>κη-*τη DmmviiK tntam ι Compositions containing quaternary ammonium fluoride salts for Inhibiting or destroying unl eel lul ar riving organisms and a process for preparing such salts ATLANTIC PHARMACEUTICAL PRODUCTS LIMITED C. 75746 87829/2 •COMPOSITION ΤΗΛ.Τ INH BITS Ofl DESTROYS M LtftOr ONE UMICCLLULnn LIVING ORGANISM CONTAINING ft *jUft TERHRRY _-|[ν1ΜΠΜ ΓΙ IM Fl linRrnF ΔΝΠ Δ PRMf!FSS FHP ΡΡΓίΛΡΓ^ Γ,, ^ ^ F.

The invention relates to the inhibition or des ruction of unicellular living organisms such as protozoa, microbes bacteria, gametes, fungi, yeasts or the like, and viruses. It relates, more especially, to the technical fields of local contraception, of antibiotic therapy, of antisepsis, of combating sexually transmitted diseases including mycoses, and of combating viral conditions, in the context of pharmacy for human or veterinary use or of cosmetic The invention may also be advantageously ap lied in the field of the disinfection of surfaces or alternatively in agriculture, for example in combating fungi and bactei.ia.

Many substances that inhibit or destroy unicellular living organisms are already known, and these include surfactant agents such as quaternary ammonium salts. Γη particular, it is known that quaternary ammonium h-ilides such as benzalkonium chloride (or dimeth lben*y lammnnium chloride), alone or in combination with other active principles, are advantageous in these applications (sen, for example, British Patent 1,5S4,615. French Patents 2,431,859, 2,483,177, 2,37 . S08, 2,3^4, ?.457.64 I, 2,573,624, 2,418,221, 2,562,888, US Patent 4,321 77, European Patents 0,243,713, 0,175,338, 0,132,963, 0.127.J3I , Q .0'-»4. . 0,076,136. 0,068,399, 0,037,593, unpublished Furnpean Patent Application No. 86/402,716.4, filed on n.|?.]«BA. and international applications WO 84/00,877 and WO It is also known, moreover, that the quaternary ammonium ha1 ides which are the easiest to manufacture and the best known, for these applications and in other-known applications, are the chlorides, iodides, bromides and chloroiodi tes (see patents mentioned above, and also French Patents 2,002,945, 2.1.85,71.7, ?,¾7,0'^, . 66 , 260. 2,366,261, 2,391,991, 2,41.8,220, 2,41 ,024, 2,482,090, 2,505,325, 2,517,672, 2 , 562 , 79 and ur pean P tent 0, 191 ,236) .

Moreover, the prior art has already prov ded many processes for manuf c uri g those quate nary ammonium salts, but which are directly applicabl , among quaternary ammonium halides, only to the chlorites and/or iodides and/or bromides and/or chloroiod tes (see above patents and also French Patents 2,472,558, 2,033,044 and European Patents 0,094,552 and 0,012,296).

Ammonium fluoride, and processes for preparing and purifying it, are also known (see, for example, French Patents 2,244,713, 2,253,710 and European Patent 0,002,016), as are perf luorinated or polyf luor i nated quaternary ammonium salts (for example, Trench Patents 2,038,421, 2,051,095, 2,153,489 and European Patents 0,073,760, 0,100,478, 0,100,477, 0,149,172), Ionic fluorine is, moreover, well known for its anti caries properties in dental applications (for example. US Patent 4,473,547; VIDAL dictionary, L985, page 582, O.V.P. , Paris, "fluor monal"), optionally combined with a catiomc quaternary ammonium compound (see French Patents 1,486,676 and 1,297,708). In these latter documents, the advantages demonstrated of combining, in one and the same compound, on the one hand fluorine, well known for its anti caries properties, and on the other hand surfactant qu tern y ammonium salts, known for their bactericide L proper ies. However, no genuinely synergistic activity is demonstrated in these applications for dentifrices, with the quaternary ammonium fluorides tested, either with respect to anti caries properties or with respect to bactericidal proper ties .

French Patent 1,297,708 describes, in particular, laurylbenzyldimethylammonium fluoride, a process for preparing this fluoride and its application in a toothpaste. This patent teaches the bactericidal effect of the quaternary ammonium compound and the anti naries effect-of the fluorine. However, no synergy is demonstrated between the fluorine and the quaternary ammonium compound. Finally, Italian Patent L,.153, 530 describes a process for preparing quaternary ammonium carbonates and then, rom these carbonates, quaternary ammonium ha'l ides by anion exchange with the corresponding acid, which is stronger than carbonic acid. However, this patent does not mention how this process can be applied to the preparation of fluorides. Moreover, while the compounds obtained by meuis of this process are suitable for application in the disinfection of surfaces and in industry, they cannot be used directly on human beings or animals, for example in the pharmaceutical compositions in which the compounds mu .t be of high purity.

With respect to this general state of the art, the invention raises the problem of providing a composition that completely inhibits or destroys unicellular living organisms and which is simultaneously appLicable to living organisms, human, animal or plant, without thereby causing harmful side effects. In effect, it is difficult, or even 87829/2 impossible, to prepare such a composition at the present time using quaternary ammonium ha 1 ides withou being reconciled either to producing side effects ( ir itations, allergies, red blotches, etc. ) or to being insufficiently active. The probLem arises espFciail with known spermicidal compositions based on benz lkonium chloride, or in combating viruses or retroviruses such ,τ?. herpes or the LAV, responsible for AIDS .

The invention relates,, in particular, to improving the invention which is the subject of European Patent No. 0,253,037.

The inventors have demonstrated that, among known quaternary .ammonium halides, the benzalkonium fluor de of general formula: OH3 where P. is an alkyL radical which can vary between C8H17 and C13H37. provides a markedly better and unexpected activity in the field of the destruction or inhibition of unicellular living" organisms .

In particular, the results are markedly better than could have been expected on the basis of the known respective properties of ionic fluorine and of the benzalkonium cation, in particular thos_e__stated in European Patent No. 0.253,037 or in French Patent 1,297,708.

The invention reLates to a composition that inhibits or destroys at least one unicellular living organism, protozoan, microbe, fungus, yeast, virus or the like, and more especially an antibiotic composition, a virucidal composi ion, a fungicidal compos ion, a bactericidal pharmaceutical composition, a composition intended for application on the genital organs in order to combat sexually transmitted diseases, a local contraceptive composition, in particular a spermicidal composi on, a cosmetic composition or composition for body hygiene, nr a composition for the local disinfection of the human body or of surfaces, characterized in that it contains at least one benzalkonium fluoride as defined above. Preferably, the radical R is an alkyl radical between C12H25 and Ci^Hrg. More especially, the invention relates to a galenical composition, characterized in that it contains between 0.05% and 7% by weight, in particular of the order of L'¾ , of a benzalkonium fluoride of this kind, sufficiently purified so as to be usable for human beings or animals.

A composition according to the invention can contain another active principle, chosen in accordance with the purpose for which the composition is intended - in particular an antibiotic - in combination with the abovementioned benzalkonium fluoride.

Similarly, a composition according to the invention advantageously contains, in addition to the abovementioned benzalkonium fluoride, at least one metal fluoride. The proportions of benzalkonium fluoride and of metal fluoride to be used enable the proportion of ionic fluorine lr_ to be varied relative to the proportion of benzalkonium. A composition according to the invention preferably contains lithium fluoride, which has proved to have a surprising additional synergistic effect.

The invention relates to such a composition characterized Ln that it contains one and onLy one quaternary ammonium halide which formula cor responds to the above mentioned formula.

More generally, the invention relates to medicinal product, characterized in that it contains at least one benzalkoniurn fluoride as mentioned above. The radical R is preferably an alkyl radical between _?H_>s and Onllj-v, and the benzalkoniurn fluoride is preferably present in a proportion varying between 0.2% and 1¾ by w ight.

The invention also relates to the application of at least one quaternary ammonium fluoride of general formula Ri R2 R3 RN+ F- for obtaining a composition according to the invention, and more especially a spermicidal composition, an antibiotic pharmaceu ical composi ion that can be administered systemically, an antibiotic topical composition, a bactericidal pharmaceutical composition, a topical composition intended for combating viral conditions, in particular v ral conditions of the skin and the genital organs, a pharmaceutical compo i ion that can be administered systemically or parenteral, ly intended for combating viruses or retrov ruses, in particular herpes or. the LAV (or HIV) responsible for AIDS, or a composition intended for combating fungal conditions. Preferably, the radical Rl is composed of a benzyl radical, R? and Rj are composed of a methyl radical and R is an alkyl radical between CeH and CisHs?, so that the quaternary ammonium fluoride referred to is a benzalkoniurn fluoride as mentioned above. More especially, R is preferably an alkyl radical varying between C12H25 and C14H29.

The invention also relates to a process for preparing a quaternary ammonium fluoride in the form of ··« solid o pasty salt that is suff cien ly pure to he inclu ed in a composition intended for human beings or .mimals, characterized in that a quaternary ammonium methyl carbonate is reacted in toichiom tric amount.-.?, with hydrof uoric acid solubilized in an alcohol, until the gaseous evolution of carbon dioxide has ceased., and the remaining alcohol is then removed by evapora ion under vacuum. Preferably, the quaternary ammonium methyl carbonate is purified beforehand by washing it in the heated state in petroleum ether until its amber yellow colour has disappeared, and the quaternary ammonium methyl, carbonate is then separated off after settl ng has taken place. The hydrofluoric acid solubi ized in an alcohol is obtained by the displacement of gaseous hydrofluoric acid with a stream of air from a 40% strength aqueous solution, and solubilization in the alcohol at 0°C. Preferably, t tv*. hydrofluoric acid is solubili ed in methanol. The evaporation of the alcohol under vacuum is preferably carried out in two stages, in particular- a stage under a pressure of 10 mm of mercury (i.333224 x lOJPa) followed by a stage under a pressure of 0,01 mm of mercury (1.33224 Pa). The quaternary ammonium fluoride obtained is subsequently purified by washing in the heated state in petroleum ether, followed by separation by centrifugation and drying under vacuum. It is also possible to purify thf quaternary ammonium fluoride obtained by dissolution in the heated state in acetone, followed by slow cooling, separation of the pasty residue and drying under vacuum.

The preparation process of the invention is advantageously applicable to the preparation of a benzalkonium fluoride as mentioned above. It e bl ? an extremely pure salt to be obtained, which is usable in a composition intended for human beings or anim ls, in particular a pharmaceutic l compos tion. The benza 1 kon ii.irn methyl carbonate used together with the hydrofluoric acid may be obtained as taught in Italian Patent .1 , 1 ,Ζ>Λ , o0 , from the corresponding tertiary amine and dime hyl carbonate.

Three kilogrammes of 012 dimethy Ibensy lammoni urn methyl carbonate are treated with 6.3 1 of 1.25 N HF solubi I i/.ed in methanol.

A strong evolution of carbon dioxide takes place. At the end of the reaction, the pH is between 5 and 6. After evaporation of the methanol under vacuum, an amber yellow translucent pasty mass is obtained.

The purification of this pasty mass is performed by dissolution in the heated state of the tran lucent mass in acetone in the heated state. After slow cooling, the pasty residue is separated off and then freed from the remaining solvent under vacuum.

The purity of the benza] konium fluoride obtained is monitored by an assay of the F ions with a specific electrode, and an HPLC (high pressure liquid chromatography) assay of the quaternary ammonium. In this example, the purity of the product obtained was 99%. rAni BiJ lf ica.tiar .

One kilogram of Ci2 dimethyIbenzy Lammoni urn methyl carbonate brought to its melting point is washed with petroleum ether and dried under vacuum.

It is then attacked in the molten state with 2.76 l of 1.5 N HF in methanol. A strong evolution o f carbon dioxide takes place. At the end of the reaction, the pH is between 5 and 6. After evaporation of the methanol under vacuum, an amber yellow translucent pasty mass i s obtained. I he purification is performed by dissolution in the heated state of the translucent mass in acetone i n the heated state. After slow cooling, the pasty residue i s separated off, and then freed of the remaining solvent under vacuum. The purity of the product, monitored according to the same method as in the previous example, was 9*}%.

The same processes of manufacture as in the 2 previous examples were used, substituting C14 dimethy Ibenzy l ammon urn methyl carbonate for C 1 2 dimethy Lben.Tyl amrnon i. urn methyl carbonate .

The benzalkonium fluoride obtained a lso takes the form of an amber yellow pasty mass.

After purification, the pu ity was and '^Γ·?¾, respectively.

The benzalkonium fluorides prepared as described above were used in the trials described below, enabling their activity to be measured.

Except where otherwise stated, the methodologies employed were those already described in European Patent Application 86/402,416.4, filed on 8.12.1^ 6. The teaching of this European patent application is considered to be known in the description below.

Y...1JLLQ..L The SANDERS CRAMER tests, according to IPPF ( International 1.0 PLanned Parenthood Federation) standards, were carried out:, in order to determine the minimal, inhibitory concentration of benzalkonium f luoride. A "minimal i nhib tory concentration (MIC) of Ci 2 benzalkonium fluoride of 20 mg/i, equivalent to 0.0C2¾, was thereby obtained. This value is to be compared with the MIC of benza konium chloride alone, which is 0.063%, and with the MIC of benzalkonium chloride in the presence of F~ anions at a concentration of 1 |jg/ml , which is 0.003%. As is hence observed, benzalkonium fluoride procures a higher activity than ^benzalkonium chloride alone, and also a higher activity than the combination of benzalkonium chloride with fluoride anions F~ , f o r example in the form of sodium f luoride .

Under the same conditions, benzalkonium fluoride in the presence of sodium fluoride at a concentra ion of J. ing/ 1 has an MIC of 18 mg/i, equivalent to 0.0018%. This MIC falls to 15 mg/l, equivalent to .0O15%, in the presence of lithium fluoride at a concentration of 1 mg/l.

Trial No. 2: Activi ies of spermicidal galenical. p reiaa ai..:^ This in vitro trial was carried out on galenical preparations containing, as active principle, either benzalkonium chloride alone, or benz lkonium chloride wi h 0.45% by weight of fluoride anions F- in the form of sodium fluoride, or benzalkonium fluoride. The MIC values are determined after in vitro simulation of the true in vivo conditions (extraction, solubilization, etc.). The fi-ANDE S CRAMER test of the IPPF is then carried out in vitro. The results obtained are summarized in the following tables: GALENICAL Proportion of benza konium M I C ( % by w t . ) FORM chloride alone (by weight) of benzalkonium chloride in the galecinal form Pessary 1.2 % 0.00fc>3 '¾ Cream i .2 % 0.0083 '¾ Tablet 1.2 % 0.0075 * Soluble film 3.12 % 0.0095 % Jelly 1.2 % 0.0080 % GALENICAL ■ Proportion of benzalkonium MIC ( % by wt . ) FORM chloride (by weight) of benzalkonium chloride in the galecinal form with 0.45% of F- anions Pessary 1.2 % 0.0023 % Cream 1.2 % 0.0030 % Pad 1.2 % 0.0025 % Tablet 3.12 % 0.0025 % Soluble film 1.2 % 0.00L7 % Jelly 1.2 % 0.0033 % GALENICAL Proportion of benzalkonium MIC (¾ by wt. ) FORM fluoride alone (by weight) of benzalkonium fluoride in the galecinal form Pessary 1 % O. OL9 ¾ Cream 1 % 0.0022 % Pad 1 % 0.0020 % Tablet 2.5 % 0.0020 % Soluble film 1 % 0.0O12 % Jelly 1 % 0.0026 % It is hence observed that benza Lkonium fluoride has a minimal inhibitory concentration which is very much lower than that of benzalkonium chloride, and even than that of benzalkonium chloride in the presence of F~ anions, A surprising synergistic effect is hence due to the benzalkonium fluoride.

.Lrj,«¾l_..Mo .....3jL. EJing-LcJ-d-aJ^ The fungicidal activities were tested on three strain:-, of mould according to AFNOR Standard T72-201. The thre strains of mould were as follows: Aspergillus versicolor CNCM 1187-79, Cladosporium cladospor oides CNCM 11.85-79, Penicillium virrucosum variety cylopium CNCM )18fe-79.

The trial was also carried out on a strain of yeast: Candida albicans (ATCC 2091) CNCM 1180-79.

The trials were carried out with a contact time of J min. at 20°C.

Two types of trials were carried ou : a fi st tr al comparing the fungicidal activity of benzalkonium chloride alone with benzalkonium fluoride alone, and a second trial comparing the fungicidal activity of benzalkonium chloride combined with lithium fluoride and the preservative KATHON (registered trademark) with the fungicidal activity of -benzalkonium fluoride combined with lithium fluoride and the same preservative KATHON (registered trademark).

KATHON (registered trademark) is a known preservative marketed by RHOM AND HASS COMPANY (U.S.A. ), consisting of mixture of two isothiazo Lines : 5-ch Loro-2 -methy L The results are summarized in the fol lowing table's: It is hence observed that the combination ofbenzalkoniurn fluoride with lithium fluoride and the preservative KATHON l registered trademark) i , in some cases, better than the combination of benzalkoniurn chloride with lithium fluoride and the same preservative KATHON (registered trademark).

The activity of benzalkoniurn chloride sn benza Ikon i i.im fluoride, alone or in the presence of lithium fluoride .¾fc ·¾ concentr tion of 0.9 The results are as follows: Fungicide concentration in mg/ 1.

Benzalkonium chloride alone 150 Benzalkonium chloride XX~ih 1 135 + LiF at 0.96 mg/1 Trichomonas, Chlamydia, Gardnerell and Pucrey's bacillus The results obtained are summarized in the following t¾blp. ttinimal n jb_ic-£Y. Minimal inhibitory Minimal inhibitory STRAIN concentration concentration concentr t on LMICJL. LM ICi X L. ..LQ.1.

Benzalkonium Benzalkonium" en:-:a 1. kon um chloride alone chloride + F- 1 f luo i de m crocirain / ml Gonoccus 1.15 mg/litre 0.t>0 mg/l tre 0.44 mg/ 1 i Trichomonas 1.3 mg/litre 0,90 mg/litre 0.. '/ mg/ litre Chlamydia 100 mg/litre 85 mg/litre f9 mg/ li re Gardnerella 50 mg/litre 4 mg/litre 32.4 mg/litre Ducrey 's 75 mg/litre 62 mg/litre 50.5 mg/li re bacillus A very strong decrease in the minimal inhibitory concentration is hence observed for benzaLkonium fluori Ltii.aI..„Jil.Q..,._5.

This trial was performed according to At-'NOR Standard NFT 72-150, March 198L. The neutralizing agent used was follows: 3% of Tween 80 (V/V) and 0.3% of lecithin ( M/V ) THe pH of the medium was 7.2. The results obtained are summarized in the following table: Minimal inhibitorv Minimal i nhibi tor v i im i nh bi to v STRAIN concentration concent ra OA. cnnne r ati on ( MIC J m.i.01 fyfof Benzalkonium Benzalkonium / Benja Ikoniurn chloride chloride + F- 1 f luor de m crogram / ml Pseudomonas 31.25 mg/ litre 18 mg/ l re 14.2 mg I Tit r & aeruginosa CNCM A 22 Escherichia coli 6.57 mg/ litre 3 mg 1 i tre 2. ¾ mg 1 i i CNCM 54 127 Staphy loccocus 1.56 mg/' litre 1.1 mg/ 1. i re 1.1 mg/ I. \ t « aureus Oxford strain CNCM 53 154 Staphyloccocus 4 mg/ litre 3.6 mg/ litre 2.9 mg/ l re faecalis CNCM 5 855 Mycobacterium 30 mg/ litre 26 mg/ l tre •21. ■ mg 1.1 i smegmatis CNCM 7 326 — % In this case also, a marked improvement is observed for benzalkonium fluoride.

IdfllJliL-i..

The activity of ^benzalkonium chloride and benzalkonium fluoride, alone or in the presence of lithium fluoride at concentration of 0.96 mg/1, or in the presence of lithium fluoride at a concentration of 0.96 mg/ L and KATHON (registered trademark) preservative at a concen ration of 0.47 mg/1, was tested on 80 wild- type strains of multi- resistant Escherichia coli producing plasmid 8-lactamase.

The MIC was dtermined in accordance with the methodology described by Profssors F . CAT LAN , P. SEDMAOUl A. MILOVANOVIC at al . , Institut A. FOU NIER. Paris.

The results are as follows: The resistance of Pseudomonas aeruginosa to various reputedly bactericidal active principles or commercial antiseptics was studied in terms of the hardness of the water and the presence or absence of proteins. The same trial was carried out on pure benzalkonium fluoride.

The methodology employed is that defined by AFMOR.

Standard T72-150, using the products diluted in accordance with the manufacturers' recommendations as regards the concentrations and conditions of use. · Mercryl Lauryle is a foaming solution marketed by Laboratoires LA8AZ (Paris, France).

Cresol is the name given to three isomeric phenols, ortho, meta and para, CTHQQ, and immediate homologue of phenol, HO-CeH-i -CH3.

Solubacter is a registered trademark denoting a foaming solution marketed by Laboratoire INNOTHERA (Aroueil, France ) .

Hibisprint is an alcohol solution marketed by rci PHARMA (Cergy, France).

Cetavlon is an antiseptic solution marketed by LCI PHARMA (Cergy, France).

Hexomedine is a registered trademark denotinj a nnn foaming solution marketed by THERAPLIX S.A. (Paris, France") .

In the following table, R denotes resistance (a growth of colonies was observed), S denotes sensitive (no significant growth of colonies was observed), P+ means that proteins were added to the solvent, and "P- means that the solvent does not contain proteins.

ANTISEPTIC 0.2 ml of innoculum 0.4 ml of innoculum 0.t> ml of innocul' PRODUCTS TESTED Contact time Contact time Contact time 10 min 20 min 30 min Dist lied Hard Distilled Hard Disti lied Hard water water wa er water wa er water P+ P- p+ P- P+ P- P+ P- p+ P- Crude cresol S S S S R S S R R R R Mercryl R S R R R R R R R R R Lauryle Solubacter R S S R S R R R R R R R Formaldehyde S S S S S S S S S S S Hexomedine R S S S S R S R R R R R Hibisprint S S S S S S R R R R R Cetavlon S S R S 5 5 S S S S S Zinc sulphate R R R R R R R R R R R Benzalkonium fluoride S S S S S S s s S S XJIJSJSLI bliaL. —8JL .J__ .!JL.C_iji3j,_ a,.C_tLlLiOi_.t. ..»..

This trial was carried out according to the method of gelf iltration on SEPHADEX LH 20.

The antiseptics used were C 1 4 benzalkonium chloride as powder, C12 benzalkonium fluoride as crystals and lithium *— fluoride as powder. Dilution series in a geometric ratio of 2 were prepared in sterile double distilled water, and the trials were performed with 10, 30, 50, 80, 100, 200 and 500 mg/litre of benzalkonium chloride and benzalkonium —= . —I fluoride, with or without the addition of lithium fluoride at a concentration of 1 or L.70 mg/ Litre, The cell cuLtures employed are VER0 cells (strains AI'CC CCL81) , maintained on DMEM medium (. EUROBIO), to which 5% or foetal calf serum and antibiotics (streptomycin at 50 microgramnie per mxllilitre and penicillin at 200,000 ) were added.

The test virus was 5ABIN type L polio virus. It w ^. cultured on VERO cells in RQUX dishes. The determination of the infectious titre was carried out according to the method of REED and MUENCH, using the conversion table of WYSHAK and DETRE.

The trial was carried out in three stages with, in the first place, the determination of the threshold of cytotoxicity of the antiseptics, then the study of the capacity of the cells treated with the antiseptics to develop the infection, and finally the determ nation of the virucidal effects of these antiseptics. 1 - Determination of the threshold of cytotoxicity of the antiseptics : Before any investigation' of the virucidal effects, it is necessary to investigate the dilution of antiseptics responsible for a cytotoxic activity, before and after filtration.

Before filtration, the VERO cells are cuLtured on 96-well microf 1 tration plates. The antiseptics are then arranged in an increasing dilution at a geometric ratio on the basis of 0.1 ml per depression and 8 depressions per dilution on the cell lawns. The cells and the dilutions of virucidal substances are left in contact for one hour at 37°C, and the antiseptic substances are then removed and replaced by the culture medium without serum. I is then possible to calculate the LDO (dilution of antiseptics not adversely affecting the cultured cells) af er a 48- hour incubation. 30 ml of the suspension of SEPHADEX LH 20 gel are introduced into the filtration apparatus. This operation is followed by a centrifugatio , and the solution of antiseptic is then deposited on the basis of 2 ml on the SEPHADEX column. A further centr fugation is carried out before the determination of the LOo . 2 - Capacity of the cells treated with antiseptic to develop the viral infection: VERO cells are cultured on 96-well mic oplates. 1 he antiseptic is employed at its LDo and left in contact for one hour, and is then replaced by increasing 10-fo).d dilutions of polio virus. After one hour's contact, the? viral suspension is removed and replaced by a culture medium without serum. The measurement of the cytopathic effect is made after a 48-hour incubation at 37°C. 3 - Determination of the virucidal activity of the antiseptics: All the doses of the antiseptics used are, obviously. Less than the threshold of cytotoxicity recorded above. From the mixtures containing an equal volume of viral suspension and antiseptic, 1 ml is withdrawn at 30, 60, 120 and 180 minutes. The sarnpLes are then deposi ted on SEPHADEX columns, after which they are centrifuged. In parallel, a control viral suspension without antiseptic is processed under the same cond tions.

The results are given in the following tabLes: BKC: Benzalkonium chloride BKF: Benzalkonium fluoride LiF: Lithium fluoride PFU = plaqueforming unit (PFU/ml) 3 The virucidal activity in , terms of the virus/ antiseptic contact time hence shows that, for all the concentrations employed, the result obtained, that is to say the viral inhibition, is equal to or better than that required by the Env ronmental Protection Agency (see SS. BLOCK, "Disinfection, sterilization and preservation" Editions Lea and Febiger, Philadelphia U.S.. A., 1977 ).

Benzalkonium chloride is active at a concentration of 0.01% in 30 minutes' contact. The same applies to benzalkonium fluoride. Howver, with lower concentrations, such as 0.0085% and 0.0082%, viral inhibition is noted from 120 minutes onwards with benzalkonium fluoride, whereas it is necessary to wait at least 180 minutes in the case of benzalkonium chloride.

Ixlal No. 9; The virucidal activity of benzalkonium luoride was determined following AFN0R Standard 172-180. The following results were obtained: 1/ QjlJkyj¾_LJ!L Inhibi tory Ce 11 concentration Toxici ty (% by weight) Benzalkonium chloride 0.01% 0.05% Benzalkonium fluoride 0.0075% 0.08% THE PREFERRED GALENICAL FORMS OF THE INVENTION FOR ITS APPLICATIONS IN LOCAL CONTRACEPTION ARE AS FOLLOWS : .

E£.SSj¾fiJjEJa,: Benzalkonium fluoride L.O ¾ Excipients: semi-synthetic glycerides or cocoa butter, or gelatin, glycerin and purified water, antioxidants, antiseptics .

. CBEfiLHS: Benzalkonium fluoride 1.00¾ Excipients: distilled or purified water, humectants, gelling agents, emulsifier, stabilizer, antioxidant, antiseptic. (To be distributed in variable proportions according to the viscosity.) pH between 4.5 and 6.5 (preferably citric acid).

Qm.i £tiis_.aw.s._saL.y-.E.s.: Benzalkonium fluoride 1 00'5; Excipients: distilled or purified water, emu Is if ie , excipients of the fatty type (vaseline, lanolin, lanovaseline , stearovaseline ) , stabilizer, antioxidant, antiseptic. (To be distributed in variable proportions according to the viscosity.) pH between 4.5 and 6,5 (preferably citric acid).

Benzalkonium fluoride 1 00% Excipients: soluble derivatives of cellulose that are compatible with cationic surfactant detergents, distilled or purified water, glycerin, sorbitol, antioxidant, antiseptic. (To be distributed in variable proportions according to the viscosity.) pH between 4.5 and 6.5 (preferably citric acid. ) Benzalkonium fluoride 1 00% Excipients: polyvinyl alcohol, glycerin, sorbitol, propylene glycol, distilled or purified water, antioxidant. pH between 4.5 and 6.5 (preferably citric acid). lASJLrJEJ-S.* Senzalkonium fluoride 20 nig per ta let Excipients: lactose, magnesium s te ate , cellulose, starch citric acid, sodium bicarbonate.

Benzalkonium fluoride 2% Excipients: foaming and wetting products that are compatible with quaternary ammonium compounds (for exam le amphoteric surfactant of the betaine or aminobetaine typo) emollients, stabilizer, antioxidant, antiseptic. pH of between 4.5 and 6.5 (preferably citric acid).

REftPY-TQ-USE SQU T IQtlS..

Benzalkonium fluoride 0.40% Excipients: distilled or purified water, ethanol, antioxidant, glycerin, sorbitol, antiseptic. pH between 4.5 and 6.5 (preferably citric acid).

Benzalkonium fluoride 10% Excipients: distilled or purified water, ethanol. , antioxidant, glycerin, sorbitol, antiseptic, pH between 4.5 and 6.5 (preferably citric acid).

The preferred galenical forms combining benzalkonium fluoride with lithium fluoride are as follows: - CREAM: benzalkonium fluoride 0.80% L F 0.55¾ Excipients: emulsive agent, preservative such .=is ΓΗϋΜ (registered trademark), purified water, perfume qs. 100¾.

- JELLY : same formulations as the cream Excipients: soluble derivatives of cellulose, glycerin, preservative such as ATHQN (registered trademark), purified water, perfume qs. 100 %.

- PAP : impregnated with the cream defined above. benzalkonium fluoride 0.016 g LiF 0.010 g Excipients: sodium bicarbonate, citric acid, colloidal silica, cellulose, magnesium stearate, lactose qs. 1 tablet.

- SOLUBLE SHEET: same formulations as the cream Excipients: polyvinyl alcohol, glycerin, preservative such as KATHON (registered trademark), purified water qs . 100%.

- EE.SS.aa.IES.: benzalkonium fluoride 0.0.15 g LiF 0.0L0 g Excipients: semi-synthetic giycerides, preservative such as KATHON (registered trademark) qs. 1 pessary.

APPLICATION OF THE INVENTION IN THE FIELD OF AN Γ If]EPS I.f.i , ANTIBIOTIC THERAPY, etc., ANO, IN PARTICULAR, IN COMBA [MG STD's: The preferred galenical forms of the invention, for example, for its application in dermatology and venereology by way of an antiseptic are as follows: CREAMS Benzalkonium fluoride 1 00% Excipients: distilled or purified water, humectants, , emulsifier, stabilizer, antioxidant, antiseptic. (To be distributed in variable proportions according to the viscosity.) pH from 4.5 to 6.5 (preferably citric acid).

Benzalkonium fluoride L 00% Excipients: distilled or purified water, emulsif ie , excipients of the fatty type (vaseline, lanolin, lanovaseline, stearovaseline) , stabilizer, antioxidant, antiseptic. (To be distributed in variable proportions according to the viscosity.) pH from 4.5 to 6.5 (prefera ly citric acid).

. SYNTHETIC SOftPS IN BAR OR PASTE FORM: Benzalkonium fluoride L.8 to 2.0¾ Excipients: foaming and wetting products that are compatible with quaternary ammonium compounds (for example, amphoteric surfactant of the betaine or aminobetaine type), emollients, stabilizer, antioxidant, antiseptic, citric acid pH regulator.

- SOLUTIONS: Benzalkonium fluoride 1 00% Excipients: distilled or purified water, ethanol, antioxidant, glycerin, sorbitol, antiseptic. pH from 4.5 to 6.5 (preferably cvtric acid).

The following galenical forms may be (for example, and without implied limitation) used by way of a dermatologica I local antiseptic (skin, mucosa, etc.): - EQfiClIUS-fiaB.: Benzalkonium fluoride Excipients: foaming synthetic base, preservative such a KATHON (registered trademark), purified water, perfume 100%.

- FORMING PASTE: same formulations as for the foaming b. except for the excipient formulated as a paste.

- MOISTURIZING C EAM: Benzalkonium fluoride 0.18% LiF 0.04% NaF 0.8% Excipients: emulsive agent, moisturizing principle, wheatgerm oil, sweet almond oil, liquid paraffin, preservative such as KATHON (registered trademark), purified water qs. 100%.

The preferred galenical forms of the inventicn, fcr example for its application in dermatology by way of a local antibiotic, are as follows: Erythromycin base 4% Benzalkonium fluoride f rom 0.2% to O .8% Excipients: ethyl alcohol, propylene glycol, distilled water .

Q.EJLS.: Erythromycin base 4% Benzalkonium fluoride rom 0.2% 0.8% Excipients: ethyl alcohol, hydroxypropylcel lulose , distilled water, glycerin.

Neomycin base 0.35% Benzalkonium fluoride from 0.2% to 0.8% or Bacitracin 50,000 IU% Benzalkonium fluoride from 0.2% to 0.8% or Oxytetracycixne hydrochloride Benzalkonium fluoride from 0.2% to 0.8% or Aureomycin hydrochloride Benzalkonium fluoride from 0.2% to 0.8% Excipients: vaseline, liquid paraffin, lanolin Soframycin sulphate 2.5% Benzalkonium fluoride om 0.2% to 0.8% Excipients: propylene glycol, polyox ethylene glycol, distilled water.

The preferred embodiments of the invention, for example for its application in otorhinolaryngology, by way of a local antibiotic, are as follows: Aureomycin L -o Benzalkonium fluoride from 0.2% to 0.8% or Oxytetracycl ine 0.50% Benzalkonium fluoride from 0.2% 0.5% Excipients: vaseline, liquid paraffin, lanolin.

Soframycin sulphate I. 25% Benzalkonium fluoride f rom 0.50% ho L% Excipients: distilled water, citric acid, sodium chloride.

APPLICATION OF THE INVENTION IN THE FIELD OF DISINFECTION This field relates to the treatment of floors, surfaces, instruments, etc., with contact bactericidal products .

The preferred embodiments of the invention for this application are as follows: USE BENZALKONIUM EXCIPIENTS FLUORIDE Hands 0.05% to 0.*1% purified water or alchohol Epidermis qs. 1.00% Instruments to 0.08% to 1.0% purified water be sterilized or to be disinfected qs. 100% Texti les 0.05% purif ed water qs. 100% Instruments of 10% ethan l thermometer 0.08% to 1.0% purified water type qs. 100% Washing of surpu if ed water faces (rooms, 0.1% boarding, floors) qs. J 00% APPLICATION OF THE INVENTION IN COSMETOLOGY: The preferred embodiments of the invention in cosmetology are described below.

The following galenical forms may be presented in cosmetology: creams, milks, salves, solutions, foam baths, synthetic soaps, shampoos, personal hygiene lotions and disinfectant lotions.

The formulae of the excipients are the same as for the pharmaceutical presentations, but the concentr ions of benzalkonium fluoride will be different.

These concentrations are as follows: 1..8¾ to 2% for rinsed products (synthetic soap type) 0.1% to 0.2% for non rinsed products.

Active principles, chemical or of naturaL origin, can also participate in these formulations, at concentrations and doses permitted in cosmetology.

The creams and milks can be in an aqueous continuous phase (oil-in-water or water-in-oil emulsion), or pasty in the heated state, to be d luted in water.

The various excipients mentioned by way of example correspond, as a guide and without implied limitation, to the following products: . Humectants: glycerol, propylene glycol, diethylene glycol, polyoxyethylene glycol.

. Emulsifiers: sodium stearate, beeswax, sorbitol ester, polyoxyethylene glycol ester, fatty alcohol, triethanolamine-lanoLin , Tween, glycol, stearate and polyglycols.

. Stabilizers: glycol stearate, cetyl alcohol alginate, pectin, gum, polyol fatty esters, soluble esters of 87829/2 - 35 - cellulose.

. Antioxidants: tartaric, citric and ascorbic acids.

..Antiseptics: boric acid, benzoic acid, parabenzoic acid and their methyl or propyl esters, in the form of sodium salts or otherwise. . pH: all these formulations are especially effective at pH values between 4.5 and t>.5. To obtain this range, citric acid is mainly used.

Claims (1)

1. CLAIMS 1. Composition tha,t inhibits or destroys at least one unicellular living organism such as a protozoan, microbe, bacterium, gamete, fungus, yeast, yirus characterised in that i contains at least one quaternary ammonium s.ilt of formula: CH3 where R is an alkyl radical which can vary between CeH and Ci Θ-Η37 , 2. Antibiotic composition, characterized in th t it contains at least one quaternary ammonium salt of formula where R is an alky I radical which can vary between C9H17 87829/2 and Ci SH 7. 3. Virucidal composition, characterized in that it contains at Least one ansternary ammonium salt of formula: CH3 where R is an alkyl radical which can vary between C0H.7 and Ci 8H37 - . 4. Fungicidal composition, characterized in that it . contains at least one quaternary ammonium salt of formula: CH3 inhere R is an alkyl radical which can vary between O13H17 and Ci 8H 7 · 5. Bactericidal pharmaceutical composition, ch ra cterized in that it contains at least one quaternary anunon ium salt of formula: CH3 CH where R is an alkyl radical which can vary between OgHi? and Ci 8H37. 6. Composition intended for. appl i cation on the genital organs in order to combat sexua ly ransmi ted diseases, 87829/2 37 characterized in that it contains at least one (quaternary ammonium salt of formula: CH3 where is an alkyl radical which can vary between C13H.7 and Ci 8H37. 7. Local contraceptive composition, in particular a spermicidal composition, characterized in that t contains at least one quaternary ammonium salt of formula: CH3 where R is an alkyl radical which can vary between C0H17 and Ci 8H37. 8. Composition for the local disinfection of the human body or of surfaces, characterized in that it contain? at least one quaternary ammonium ¾.* I 1; of formuLa: CHs CH3 where R is an alkyl radical which can vary between C13H17 and Ci sHs7. 9. Composition according to any one of Claims 1 to S, characterized in that R is between CizHzs and C14H29. 87829/3. 10. Galenical composition according to any one of Claims to 9, characterized in that it contains between/ C.05% and 7% by weight, in particular of the order of L "'·. , of a quaternary ammonium salt of this kind, sufficiently purified so as to be sable for human beings or anim ls. 1. 11. Composition according to any one of Claims I. to 10. characterized in that it contains another active principle, chosen in accordance with the purpose for which the composition is intended - in particular an antibiotic - , in combination with the quaternary ammonium fluoride. 12. Composition according to any one of Claims 1 to li , characterized in that it contains, in addition, at J east one metal fluoride. 13. Composition according to Claim 12. charar ter zed in that it contains lithium fluoride. 14. Composition according to either of Claims 12 and 13, characterized in that it contains, in addition, a preservative such as KATHON ( registered trade mark ) . 15. Pharmaceutical composition characterized in that it contains at least one quaternary ammonium salt of formula: where R is an alky I radical which can vary between Cell.7 and Ci 8H3 . 16. Pharmaceutical composition according to Claim 15, characterized in that R is between C12H2S and C.4H29. 17. Pharmaceutical composition according to either of Claims 15 and 16, characterized in- that it contains between 0.2% and 1¾ by weight of quaternary ammonium fluoride. - 39 - 87829/3 18. Use of a quaternary ammonium fluoride of general formul in the manufacture of a medicament having spermicidal activity, substantially as described in the specification. 19. A quaternary ammonium fluoride of general formula : CH3 for use as an antibiotic. 20. A quartemary ammonium fluoride of general formula: for use as a topical antibiotic medicament. 21. a quaternary ammonium fluoride of general formula CH- CH- - 40 87829/3 for use as a medicament having a bactericidal activity. 22. A quarternary ammonium fluoride of general formula: for use as a medicament having an activity for topically combating viral conditions, in particular viral conditions of the skin and the genital organs. 23. A quarternary ammonium fluoride of general formula: for use as a medicament having an activity for combating viruses or retroviruses, in particular herpes and the LVA (or HIV) responsible for AIDS for systemically or parentarally administration. 24. A quaternary ammonium fluoride of general formula: for use as a medicament having an activity to combating fungal conditions. - 41 - 87829/3 A quartemary ammonium fluoride of general formula for use as a medicament having an activity for combating sexually transmitted diseases. 26. A compound of general formula: CH- characterized in that R is an alkyl radical between C8H17 for use according to any one of claims 18 to 25. 27. A compound of the general formula: CH2 i " CH- characterized in that R is between C12H2s and Ci4H29 for use according to claim 26. 28. Process for preparing a quaternary ammonium fluoi id in the form of a solid or pasty salt- that is sufficient! pure to be included in a composition intended for human beings or animals, characterized in that a qua ernary ammonium methyl carbonate is reacted in stoichiometri amounts with hydrofluoric acid soluh Lized in an alcohol until the gaseous evolution of carbon dioxide has ceased and the remaining alcohol is then removed by v.?.poi a ion under vacuum. 29. Process according to Claim 2*3, characterized in that the quaternary ammonium methyl carbonate is purified beforehand by washing it in the heated state in petroleum ether until its yellow colour has disappeared, and the quaternary ammonium methyl carbonate is then separated off after settling has taken place. 30. Process according to either of Claims 28 and 29, characterized in that the hydrofluoric acid solubilized in an alcohol is obtained by the displacement of gaseous hydrofluoric acid with a stream of air from a 40% strength aqueous solution, and solubilization in the alcohol at 0°C. 31. Process according to any one of Claims 28 to 30, characterized in that the hydrofluoric acid is solubilized in methanol. 32. Process according to any one of Claims 28 to 31, characterized in that the evaporation of the alcohol under vacuum is carried out in two stages, in particular a stage at 10 mm of mercury (1.316 x 104 Pa) followed by a stage at 0.01 mm of mercury (13.16 Pa). 33. Process according to any one of Claims 28 to 32, characterized in that the quaternary ammonium fluoride obtained is subsequently purified by washing in the heated state in petroleum ether, followed by separation by centrifugation and drying under vacuum. 34. Process according to any one of Claims 28 to 33, characterized in that the quaternary ammonium fluoride obtained is subsequently purified by dissolution in the heated state in acetone, followed by slow cooling, separation of the pasty residue and drying under vacuum. For