CARBOHYDRATE DERIVATIVES FOR INHIBITING BACTERIAL ADHERENCE

Abstract: Pharmaceutical preparations containing globotetraosylceramide, globotriaosylceramide or globotetraos, and the use of said preparations or compounds for treatment, prophylaxis or diagnosis of bacterial infections in the urinary tract. The compounds have the ability to inhibit the adherence of bacteria to human urinary tract epithelial cells.

Description: Carbohydrate Derivatives for Inhibiting Bacterial Adherence DESCRIPTION Technical_Field The present invention relates to compounds for use in medicine, and novel pharmaceutical preparations containing as active ingredient these compounds which are known per se. The pharmaceutical preparations are especially useful for inhibiting the adherence of bacteria to human urinary tract epithelial cells, and can be used for treatment, prophylaxis and diagnosis of urinary tract infections. §§9! $ g?9 Qd_Ar _ Bacteria tend to grow attached to surfaces. Besides the general "stickiness" enabling bacteria to bind to almost any surface, host and tissue specific attachment of bacteria is known. Such specific attachment guides the distribution on mucous membranes, both of bacteria constituting the indigenous flora and of potential pathogens and is believed to be a prerequisite for susceptibility to infection of the host. For Escherichia coli isolated from patiens with urinary tract infection the severity of infection produced in vi o is strongly related to the capacity to adhere tc human urinary tract eoithelial cells in vitro. The carbohydrate chains of glycosphingolipids are extremely variable, and are known to be involved in self- not self recognition as blood group antigens and receptors for bac¬ terial toxins. Furthermore, the glycolipid pattern is species specific and differs between epithelial and non epithelial tissue. Carbohydrates at the cell surface have been implicated as possible receptors or a ac ng ac er a u no epithelial cell component interacting with bacteria has been identified. In connection with a recent observation (Svanborg Eden C, Leffler H. Scaπd. J. Infect. Pis. 1980, Suppl 24:144-147) that a fraction of glycolipids, isolated from human urinary tract epithelial cells, inhibited attachment of E.coli to cells from the same donor, a rale for glycolipids as receptors for the attaching bacteria was suggested but no specific glycolipid had been identified before the date of our invention. Disclosure of Invention According to the present invention it has been found that the compounds globotetraosylceramide, globotriaosylceramide, and globotetraos have the ability to inhibit the adherence of bacteria to human urinary tract epithelial cells. Bio¬ logical tests have indicated that this unexpected action is due to the fact that these compounds have the ability to attach to the cell walls of bacteria and thereby block some ligaπd function which is responsible for the adherence of bacteria to human urinary tract " epithelial cells. Urinary tract infections are commonly caused by an invasion of pathogenic bacteria, either from bacteria in th.e bowels or by bacteria invading through the urethra oper.ing. The present invention provides pharmaceutical compositions having the ability to prevent aαherence cf potential pathogenic bacteria to human urinary tract epithelial cells, and thereby curing or preventing urinary tract infections. .'lore speci ically, the invention relates to pharmaceutical preparations for inhibiting bacterial adherence to human urinary tract epithelial cells, nich certain as active iπ- gredient an effective amount of at least ne of the compounds globotetraosylceramide of the formula Ga lfJAcS ' -- 3 Ga l l } 4 Ga l£ l ■ 7 4G l c 5 l -> ICΞRAΠICΞ ( I J globotriaosylceramide of the formula Galαl 4GalSl Glcgl >1CERAMIDE (II) and globotetraos of the formula GalNAcSl - 3Galαl ->4Gal2l -4Glc (III) in association with a pharmaceutically acceptable carrier. In a preferred embodiment the invention also relates to pharmaceutical preparations which contains solely globo¬ tetraosylceramide and/or globotriaosylceramide as an active ingredient. These preparations may as an additional in- grεdieπt contain an effective amount of the compound lactosylceramide of the formula. Galβl GlcSl ^lCERAMIDE " (IV) in proportions of 1:10 to 10:1 per weight of the ceramides of the formulas I and . II. Lactos lceramide does not in itself possess any ability to inhibit bacterial adherence. However, most surprisingly it has been found to strongly potentiate the inhibitory effects of globotetraosylceramide and globotriaosylceramide. The active ingredients glccctetraosylceramide, globotriaosyl¬ ceramide, globotetraos and lactosylceramice are compounds known per se, which can be prepared according to known methcds. These compounds have not previously beer, suggested to possess any medical use. The active ingredients may according to the invention be formulated for use in human medicine for therapeutic p rophylactic or diagnostic .se. In clinical practice the active ingredients will nor-.≤IIy be administered tcoically, orally, or by rectal administration, in the form of ≤ pharmaceu ical preparation comprising the active ingredients, m association with a pharmaceutically acceptable carrier which may be a solid, semi-solid or liquid diluent or an ingestible capsule, and such preparations comprise a further aspect of the invention. The compounds may also be used without carrier material. As examples of pharmaceutical preparations may be mentioned tablets, drops, suppositories, preparations for topical application such as ointments, jellies, creams, powders, drops and suspensions. Usually the active substance will comprise from 0.05 to 99 % by weight of the preparation, for example from 0.1 to 50 % for preparations intended for oral adminiitratiαn and from 0.5 to 80 % for preparations intended for topic'a ' l administration. For topical application, especially for application to the urethra opening, the preparations are suitably in the form of an ointment, gel, suspension, or cream. The amount of active substance may vary, for example from 0.5 - 80 % by weight of the active substance. Such preparations for topical application may be prepared in known manner by mixing the active substance with known carrier materials such as isopropanol, glycarol, paraffin, stearyl alcohol, and polyethylene glycol. To produce pharmaceutical preparations in the form of dosage units for oral application containing a compound of the invention, the active ingredients may be mixed with a solid, pulverulent carrier, for example lactose, saccharose, sorbitol, mannitol, a starch such ' as potato starch, corn starch, amylαpectin, laminaria powder or citrus pulp powder, a cellulose derivative or gelatine and also may include lubricants such as magnesium or calcium stearate or a Carbowax or other polyethylene glycol waxes compressed to form tablets or cores for drageεs. If dragees are required, the cores may be coated for example with concentrated sugar solutions which may contain gum arable, talc and/or titanium cioxide, or alternatively with a film forming agent dissolved in easily volatile organic solvents or mixtures of organic solvents. Dyestuffs can be added to these coatings, for example, to distinguish between different contents of active substance. For the preparation of soft gelatine capsules consisting of gelatine and,for example, glycerol as a plasticizsr, or similar closed capsules, the active substance may be admixed with a Carbowax or a suitable oil as e.g. sesame oil, olive oil, cr arachis oil. -Hard gelatine capsules may contain granulates of the active substance with solid, pulverulent carriers such as lactose, saccharose, sorbitol, mannitol, starches (for e'xample potato starch, corn starch or amylopectin), cellulose derivatives or gelatine, and may also include magnesium stearate or stsaric acid as lubricants. By using several layers of the active α-rug, separated by slowly dissolving coatings sustained release. ablets are obtained. Another way of preparing sustained release tablets is to divide the dose of the active drug into granules with coatings of different thicknesses and compress the granules into tablets together with the carrier substance. The active substance can also be incorporated in slowly dissolving tablets mads for instance of fat and wax substances or evenly distributed in a tablet of an insoluble substance such as a physiologically inert plastic substance. In order to obtain dosage units of oral preparations - tablets and capsules - which are designed so as to prevent release of and pαssiole decomposition of the active substance in the gastric juice, the taclets and dragees may be enteric-coated, that is provided with a layer of a gastric juice-resistant enteric film or coating having such properties that it is not dissolved at the acidic pH in the gastric juice. Thus, the active substance will not be released until the preparation reaches the intestines. As examples of such known enteric coatings may be mentioned cellulose acetate phthalate, hydroxypropyimethylceilulose p thalates such as those sold under the trade names r.P 55 and HP 50, and Eudragit L and EudraΞit S. scer-t powders -.ay be prepared by mixing the active ent .-.ith non-tcxic carbonates or hydrogen carbcnates soci'jm, potassium or calcium, such as calcium - __. j. . te, cotassium carbonate and potassium hydrogen te, and solid, non-toxic acids such as tartaric acic. , , . Liquid preparations for oral application may be in the form of elixirs, syrups or suspensions, for example solutions containing from .0..1 to 20% by weight of. active substance, sugar and a mixture or ethanol, water, glycerol, propylene glycol and optionally aroma, saccharine and/or carboxymethyl- cellulose as a dispersing agent. The ~ dosage at which the active ingredients are administered may vary -within a wide range and will depend on various factors such as for example the severity of the infection, the age and weight of the patient, and may have to be individually adjusted. As a possible range for the amount of active ^ ingredients which may be administered per day may be mentioned from 0.1 mg to 2000 mg or from 1 mg to 2000 mg. The pharmaceutical compositions containing the active ingredients may suitably be formulated so that they provide doses within these ranges either as single dosage units or as multiple dosage units. Thus, it has been found according to the present invention that globotetraosylceramide, globotriaocylseramide and globotetraos have the ability to inhibit the adherence of bacteria to human urinary tract epithelial cells. Since such an adherence in many cases is an essential prerequisite for susceptibility to urinary tract pathogenic bacteria, the above active ingredients and corresponding pharmaceutical preparations are useful in therapeutic and/or prophylactic treatment cf urinary tract infections. 5§§i . _ i l-2ds_c£_Carrying_αu _the_Iπyention The preferred aspect cf the invention is the use of globo¬ tetraosylceramide in the treatment of urinary tract infections caused by £.coli . ^Q§sigπ_inhibitign_bγ_gly_cqligids ι The in vitro test system for bacterial adhesion to urinary tract epithelial cells has been described in Infect. I muπ. 18, 767-774 (1977). The cells were collected from the sediment of freshly voided urine from one healthy donor, and consisted of about 80 per cent squamous and 20 per cent transitional epithelial cells. An E■coli 3669 strain (02 nontypable), isolated from the urine of a girl with acute pyelonephritis was used. The strain attached to human but not to rat 10 urinary tract epithelial cells and agglutinated human but not guinea pig erythrσcytes. For testing, bacteria were cultivated in Luria broth for 16 h,sedimented by centri- fugation and rεsuspended in phosphate-buffered saline, PBS (pK 7.1, 300 mOsm per 1). To 10 8 bacteria were added 105 15 urinary tract epithelial cells. After incubation for 60 min at 37 C the mixture was washed free cf unattached bacteria, and the number of bacteria on the epithelial cells counted by light microscopy. Adhesion is given as the mean number of bacteria attached to 40 epithelial cells. 20 For inhibition experiments samples of the different glyco¬ lipids to be tested were taken from solutions in chlorofαrm- ethanol 2:1. The solvent was removed by evaporation in a stream of nitrogen followed by suction in an exsiccator eve: -> c πi cn • t.< a d: of 0.5 or 1.0 ml of 0.85 σεr cent c* N'aCl or PBS the samples were sonicated in a water bath for 20-50 seconds to give a glycolipid suspension. Bacteria were preincubated with glycolipid suspension (10 c bacteria in 100 μ l and 0.5 ml of glycolipid) for 30 min ter ≤cdi ■ti.cn of i -i 5 urinary sediment epithelial cells adhesion testing was continued as described above. The glycolipids to be tested were prepared in the laboratory cf -A Karlssαn, Institute of f-ledical Biochemistry, Gotebcrg, i5 S ' .'.ΞGsn. The known structures (L≤ine, R.A., Steliπsr, . end * i, S.-i. (1374) in: Methecs 1". ."- ' smbra e Siclogy (Kern, E.D. ed) vol. 2, pp. 205-244, Plenum Press, Mew York) were confirmed by mass spectromεtry , NFIR spectrosccpy and degradative methods. 5 Thus, lactosy lceramide was purified from human brain and globotriaosylceramide and globotetraosylceramide from human erythrocytes . For the preparation of lactoneotetrao- sylceramide, the major gangliαside of human erythrocytes, N-Acetylneuraminosyllactoneotεtraosylceramide, was iso- 10 lated and degraded by Vibrio cholera neuraminidase Calbiochem. (San Diego, USA). The results from the tests are depicted in Tables 1 and 2 below.. -- The globotetraosylceramide was found to be the most effective inhibitor of the bacterial attachment while globotriaosylceramide also had some inhibitory activity 20 (Table 1 and 2). The other glycolipids tested were not inhibitory (Table 1). Also, some combinations of pure glyco¬ lipids wsre tested. Thus, it was found that lower amounts of globotetraosylceramide were needed for inhibition of adhesion when a glycosphingolipid in itself not affecting 25 attachment viz._ lactosylceramide in an amount of 100 μg. was added together with globotetraosylceramide (Table 2). Table 1 30 Preincubated ith μg in 0.5 ml P.c. bacteria adhε * ring per cell, % cf contra1 Lactasylcerernide 100 13- Globotriaosylceramide 150 35 Globotetraosylceramide 100 Lactoneotetraosylcsramide 10C i ab l e 2 . Inhibition of bacterial attachment by globotetraosyl¬ ceramide and globotriaosylceramide presented in differεnt forms. Average number of bacteria adhering per cell, % of control. With addition of lacto¬ sylceramide Amount Globotetrao- Globotriao¬ Globotetrao- Globotriao¬ ( μg in 0.5 ml) sylceramide sylceramide sylceramide sylceramidε 400 S 61 2 24 200 4 54 11 75 100 20 12 40 51 67 12 59 20 85 41 10 99 66 a) 200 μg of lactosylcsramidε wεre added to the 400 μg sample of globotstra-glαbotriaosylceramide end 100 μg was added to the other samples. The inhibitory affects of globotetraosylceramide has been confirmed by tests with other E.coli strains attaching to human urogenital epithelial cells. In a still further test it has been shown that 90 % out of a large sam le of pyelonephritis E.coli strains which attach to human urogenital epithelial cells also do bind to glcbotetraosyl ceramide. In a further series of tests it has been shown that globotetraos, viz. the free tetrasaccharide residue of . globotetraosylcεramids also can inhibit the adhesion of pyelonefritis E.coli to uroepithelial cells. The test were made in accordance with the general procedure described g above, using the strain E.coli 36692. Bacteria (10 /ml) were mixed with globotetraos ( * -" ■ -150 μg/ l) or PBS and then incubated for 30 min at 37 C. Epithelial cells (human and mouse) were then added and after incubation the number of bacteria on the cells were counted as described above. The results are depicted in Table 3 below. The globotetraos was obtained by αzonolysis according to .'iegandt and Baschang (1965) using globotstraosylceramide obtained from human erythrocytes. Table Bacteria prεincubatεd with Adhesion (' saline control) Human cells Mouse cells Phαsphatebuffer, PBS 1 π•__ * - ICO Globotetraos 27 15 ? 9t § 9i-: 0_ S iD§5_.§§9§!_! iDa__ . The strain £. - ____ 36592, original isolated from urine αf ' a patient with pyelonephritis, was used for testing. The strain was acnssive to human uric.ary tract epithelical cells by recognizing glcbctstraosylceramide as receptor (Leffler,H., and Svanborg Ξcs.n, C. 1980. Chemical identification of a glycosp ngolipid receptor for Escherichia coli attaching to human urinary tract epithelial cells and agglutinating human erythrocytes. FΞ.'.S ."icrobio1. Lett. 3:127) It has now been shown that this strain also adheres to uroepithelial cells of mice and that it gives experimental t elonεphritis (bacteria in the kidney) wr.en injected in i ~ s ciaddεr of mice. n ;ne tests :o OQO5 were oreincuDa e; w11n iG r≤os or phcsphatebuffer for 30 min at 37 C. The adhesion tc mouse bladder epithelial cells of the two samples was compared in vitro, before injection of 0.05 ml into the bladdεr of mice. Ten animals were used in each group. After sacrifice of the animals two hours after injection cultures were performed on kidneys, bladders and urine. Thε results are .given,in Table 4 below. In the first (A) series of test the injected bacterial suspension contained 10 bacteria/ml, and the preincubatioπ was made with a solution containing 200 jjg/ml of globotetraos. In the second (3 ) g- series of test thε bacterial suspension containad 2 ■ 10 bacteria/ml, and the prεihcubatiαn was made with a solution containing about 150 ug/ml of globotetraos. Table 4 Bacteria No. of positive cultures Mo . dead pretreatεd with Kidney Bladder Urine phosphatεbuffer (A) 10 o 0 globqtetrags ( _0_ 10 0 phosphatabuffer ( ** :) 8 7 5 globotetraos (3) 2 3 0 It is seen from the results in the table t.-.at, firstly, with a low dose (A) of prstrsatεd osctεria the tendency for pyelonephritis (bacteria in the kidney) is eliminated. Secondly, with a higher bacterial doss (B) the survival of mouse is increased from 5 tc 10 whe using globotetraos pretreated bacteria. These c≡-.εfici≤ results b≡en confirmed in tests with ether bacteria and with a larger number of animal tests. Discussion In a previous st'_dy (Svaπbcr- Ede , C . , Haissor,, L . A . , jodel, U., Lindc≡rg, U., and Ξohl A -- = r 1 u π , A. 1 = 76. a ia le adnesion to normal urinarv t: • act soithelial cells urinary tract infection. Lancet 11:490) it has been shown that at least 70 % α- E.coli strains isolated from pyelonephritis -patients are adhesive to urinary tract 5 epithelial cells. This indicates a general therapeutic usefulness of agents interfering with this adhesion, such as thε compounds tested above. The therapeutic effect of interference with adhesion is 0 dependent on whether adhesion is a descisive event in the infectious process or not. Two lines of evidence indicate that this is the case. Firstly, adhesive capability is common among pyeloπεphritis E.coli and rare among normal faecal E.coli (Lancet, 11:490 (1976). Secondly, people with 5 a decreasεd capacity to synthesize glycolipid receptors (as measured by blocdgroup) has been shown to run a lower risk of attracting urinary tract infεction (Lancet, 1981). DIAGNOSTIC USE 0 The characterist i cs of a bacteri a l strai n to recogn ize a receptor, such as g lobotstraosylceramide (g lobos ide ) , globo¬ triaosylceramide , or . globotetraos may be dεmo nstratεd by us i ng part i cles or surfaces wi th adf ixed receptor . 5 The particles may be erythrocytes coated with receptor as in Leffler, H. and Svanborg Edan.C, (1980) FEMS Microbiol. Letters _8, 127-134. Also possibly useful are synthetic particles or surfaces with bound glycolipid, for example 0 covalsntly bound to agarose or glass beads. CThe Journal of Biological Chemistry, Vol. 249, \io . 14, 4460-4466 (1974)1. polystyrene tubes, micrαtiter plates and similar. Linkage to or coating cf other materials such as latex is also possible. 5 The bacterial property is demonstratsd by agglutination of particles after addition cf bacteria, or visualization cf bacterial binding to a surface. If agglutination occurs for particles with receptor but not tarticles without rεceptor C recognition cf the rεceptor by bacteria is demonstratsd .^ __ Pharmaceutical- p re oa rations The following Examples illustrate the preoaration α- pharmacεutical prεparations of thε invention. Example 1. Ointment I Globotetraosylceramide 2-5 g Cetyltrimethy lammonium bromide 0.6 g Stearyl alcohol 2.25 g Cetanol 6.75 g Liquid paraffin 17.0 g Glycerol 12.0- g Hydrochloric acid to pH 6.5 Destilled water aά 100.0 Example 2. Ointment II Globotriaosylceramide 2.5 Polyethylεne glycol 1500 50 Polyethylene glycol 4000 15 Propylε-ne glycol ac ICQ Examole 3. Ointment III Globotetraosylceramide 2.0 g Lactosy lceramide 1.0 g Scrbitan onoleate 5.0 z Petro latum Examole 4. Ointment IV Globotetraos 2.5 c- Polythylene glycol 1500 50 e Polyethylene glycol 4000 13 Propylεne glycol .00 xam e . e v G αbαtεtradsylceramide 4.0 g ethαcεl 4.0 g Methyl paraoxybεn∑oatε D.I2 g Propyl paraoxybenzoate 0.05 g Sodium hydroxide and hydrochloric acid to pH 6.7 Distilled water ad 100.0 ml Example * 6. Solution for topical use Globotetraosylceramide 2.00 g Isopropanol 3c.0 g Glycerol 12.6 e Hydrochloric acid to pH 5.0-7.0 Purified water ad uu Example 7. Drops Globotetraosylceramide 2.00 Ascorbic acid 1.00 g Sodium cyrosulfite c.io g Disodiu εdatatε 0.10 g Liquid glucose Ξ-o.cα g Absoluts alcohol 10. CO g Purified water GO.O ml Example Drops Glctcts raαεylceremi Lactos lceramide 00 Citric acid 00 Discdium adetate 10 Liquid glucose Ethano1 5 % ,co Sodium hydroxide a. d hydrochloric acid Purifiec "water ml Examp le 9. - Tab l ets Each tab let contai ns Globotstraosylcerami de 20 .0 mg Maize starch 25 . 0 mg Lactose 190 .0 mg Gelati n 1 . 5 mg laic f 12.0 mg Magnesi um stearate 1. 5 mg 250.0 mg ExamplelO. Gastric.juice-resistant tablets _» Tablets according to Example 6 are coated with an enteric coating solution with the following composition: Cellulose acetate phtalate 120.0 g Propylεne glycol 30.0 g Sorbitan monσleate 10.0 g Ethanol 95 % 450.0 ml Acetone q.s. ad 1000.0 ml Thε coating is carried out by a pouring procedure in a conventional coating pan or by spraying the tablets in a pan spray tablet coater. Examole 11 ' . Suppositories Each suppository contains: Globotetraosylceramide 20.0 mg Asccrbyl palmitate 1.0 mg Suppository base (Imhausεn H cr l-.'itaosol H) ad 2000.0 g ... -._.-i u example 12. Syrup Globotetraosylceramide 0.200 Liquid glucose 30-, 0 Sucrose 50.0 g Ascorbic acid 0.1 σ — Sodium pyrosulfite 0.01 g Oisodium edetate 0.01 g Orange essence 0.025 σ Certified colour 0.015 σ __. Purified water ad 100.0 σ Example 13. Syrup Globotriaosylceramide 0. .200 g Liquid glucose 30, .0 g Sucrose = 0. .0 σ Ascorbic acid 0.1 Oisodium edetate 0.01 Orange essence with solubilizer 0.25 Hydrochloric acid to pH 6.0-6.5 Purified water ad i •-•-. p

Claims: CLAIMS 1. A compound selected from globotetraosylceramide, globo¬ triaosylceramide and globotetraos for use in ths trεatment, prophylaxis or diagnosis of bacterial infections • in the urinary tract. 2. A compound according to claim 1 for use as an inhibitor of bacterial adherencε to human urinary tract epithelial cells. 3. A pharmaceutical preparation containing as active " ' ingredient an effectivε amount of a compound according to claim 1 in association with a pharmaceutically acceptable carrier. 4. A pharmaceutical preparation according to claim 3 con- taining as active ingredient an effεctive amount of at least one cf the compounds globotetraosylceramide and globotriaosylceramide. 5. A pharmaceutical preparation according to claim 4 containing as additional ingrediεnt thε compound lactosyl- cεramica in proportions cf 1:10 to 10:1 per weight of the globotetraosylceramide or globotriaosylceramide. 5. A pharmacεutical preparation according to claim 3 designεc fcr topical application and being in tne form of drops, a suspension, a powder, a jelly, an ointment or a cream. 7. A pharmaceutical preparation according to claims 3, 4, 5 or 6 ir sterilized form. 5. A p-armaceutical preparation according to claim 7 in dcsage ur.it form. . in— hod for the treatment or prophylaxis cf urinary tract i factions in humans comprising administering to a human 1 c need cf such treatment an effectve amtun compou-. ■z according to claim 1. - u • -

Inventors: LEFFLER H, SVANBORG E
Applicants: LEFFLER H, SVANBORG E
Application year: 1981
Main classification: C07H15
IPC codes: A61K31, A61K35, A61K38, C07H3, C07H15, G01N33