A COMPOUND AND A COMPOSITION FOR THERAPEUTIC OR DIAGNOSTIC USE AND THE USE OF SUCH COMPOUND AND COMPOSITION FOR THERAPEUTIC TREATMENT AND ISOLATION OF SHIGATOXINE

Abstract: A compound for use in the treatment of profylaxis of or diagnosis on disorders caused by the toxine Shigella dysenteriae having formula (I), wherein R1 and R2 independently are hydrogen, lower alkyl or acyl, with the proviso that R1 is not hydrogen when R2 is acetyl, or R1 and R2 together with the nitrogen atom to which they are connected form a cyclic imide, for example phtalamide, and wherein R3 is hydrogen, an organic residue, such as a carbohydrate residue, or the residue of a natural or synthetic glycoconjugate; composition including such compound in combination with a pharmaceutically acceptable carrier or diluent; method for treatment determining the presence of the toxine; method for identification or quantification of such compound; and method for isolating the toxine.

Description: A compound and a composition for therapeutic or diagnostic use and the use of such compound and composition for the.-, rapeutic treatment and isolation of Shiσatoxine. The present invention relates to compounds and com¬ positions which are useful for therapeutic treatment of dis¬ orders caused by the toxine of Shiσella dvβenteriae as well as profylaxiβ and diagnosis in connection herewith. The in¬ vention also relates to a method of therapeutic treatment of mammals including man and a process for isolating the toxine in question. Necrosis of the epithelium of the colon iβ part of the pathogenesiβ of bacillary dyββnteria after infection with Shiσella dvsenteriae (see Davis, B.D., Dulbecco, R., Eisen, H.N., and Ginsberg, N.S. <edβ> Microbiology Harper and Row, Publishers, Philadelphia, third edition, 1980). The necrosis is due to an invasion of the bacterium into the epithelium to produce therein a toxine which appears to inhibit protein synthesis (see Brown, J .E. , . Rothman, S.W. and Doctor, B.P. (1980) Infect . Immun. 29, 98-107). The purified toxine has a molecular weight of about 70 000 (see Brown, J.E., Griffin, D.E., Rothman, S.W. and Doctor, B.P. (1982) Infect . Immun. 36, 996-1005) and is composed of one heavy and four to five light βubunits similar to the case for cholera toxine (see Olβneβ, S. and Eiklid, K. <1980) J.Biol.Chem. 255, 284-289). The to¬ xine has recently been purified on a milligram scale (see Brown, J.E., Griffin, D.E., Rothman, S.W. and Doctor, B.P. <1982) Infect . Immun. 36., 996-1005), which has enabled more detailed studies of its mechanism of action. The present invention has for its main object to pro¬ vide a compound or a composition which can be used thera- peutically for the treatment of disorders due to infection with Shiσella dvsenteriae and which can also be used for diagnosing the toxine generated by the just mentioned bac- terium. Another object of the invention iβ to provide a method for therapeutic treatment of mammals including man. Still another object of the invention iβ to provide a process for isolating the toxine of Shiαella dvsenteriae. In connection with extensive research and experimen¬ tation it has now been found that the active receptor sub¬ stance vis-a-vis the toxine of Shiαella dvββntβriae haβ the formula CD: R-, wherein ^ and & independently are hydrogen, lower alJcyl or acy , with the proviso that R^ iβ not hydrogen at the same time aβ R2 iβ acetyl, or R^ and R2 together with the nitrogen atom to which they are connected form a cyclic imide, for example " phthalimide, and wherein R3 iβ hydrogen, an organic reβidue, βuch aβ a carbohydrate residue, or the residue of a natural or synthetic glycoco ugate. A preferred compound according to the invention fal- ling within formula (I) above has the formula (II): wherein j, and R have the above meaning and wherein R4 iβ hydrogen, an organic reβidue, βuch aβ a carbohydrate reβidue, or the reβidue of a natural or βynthetic glycoconjugate. I In the compounds given' above it is preferred that R^ iβ hydrogen or lower alkyl and R2 is hydrogen, lower alkyl or acyl . A preferred proviso iβ that R^ and R2 are not simul¬ taneously hydrogen. In a particularly preferred embodiment of the inven¬ tion, in the above formula, R^ is hydrogen, whereas R2 has the formula < 111> : 0 v R ς CUD X / - C - C— R 6 \ R 7 wherein R5, R5 and R7 idependently are hydrogen, lower alkyl or halogen. It is particularly preferred that R5, Rg and R7 all are fluoro or chloro, particularly fluoro. With regard to compound <II) it is preferred that -OR3 is in β-configura- tion. The expression "lower" used in the present disclosure refers to a group containing 1-6 carbon atoms, particularly 1-4 carbon atoms and especially 1 or 2 carbon atoms. The active substance constituted by the compound of formular (I) can be used as βuch or in combination with a pharmaceutically acceptable carrier. The active substances according to the present inven¬ tion can be formulated for use in human or veterinary medi¬ cine for therapeutic, profylactic or diagnostic uses. In clinical practice the active constituents are normally ad¬ ministered orally or rectally or by injection in the form of a pharmaceutical preparation containing the active consti¬ tuents in combination with a pharmaceutically acceptable carrier, which may be solid, βemiβolid or liquid, or as a capsule, and βuch compositions constitute a further aspect of the invention. The compounds may also be used as such without carrier and in a form of an aqueous solution for injection. As examples of pharmaceutical preparations there may be men¬ tioned tablets, drops, solutions and suppositories. The ac- tive substance usually conβtituteβ from 0.05 to 99% by weight of the preparation, for example from 0.1 to 50X for prepara- tionβ intended for oral administration. To manufacture pharmaceutical preparations in the form of dose unite for oral application containing a compound ac¬ cording to the invention the active constituents can be ad¬ mixed with a solid pulverulent or other carrier, for example lactose, saccharose, sorbitol, mannitol, starch, βuch as potatoe starch, corn starch, amylopectin, a cellulose de- rivative or gelatin and may also include lubricants, βuch aβ magnesium or calcium stearate, or polyethylene glγcol waxes compressed to form tablets or cause for drageeβ . By using several layers of the active drug separated by slowly dissolving layers tablets of delayed release are ob¬ tained. Liquid preparations for oral application can be in the form of elixires, syrups or suspensionβ, for example solu- . tionβ containing from 0.1 to 20% by weight of active βub- stance, sugar and a mixture of ethanol, water, glycerol, propylene, glycol and optionally other additives of a con¬ ventional character. The dose by which the active constituents are admi¬ nistered may vary within wide limits and depend on different factors, such aβ the severity of the disorder, the age and the weight of the patient and can be individually adjusted. As a conceivable range for the quantity of active consti¬ tuents that may be administered per day there may be mentio¬ ned from 0.1 to 2000 mg or from 1 g to 2000 mg. The present invention has also for an object to provide a method for therapeutic treatment of mammals including man, and in this treatment a therapeutically active amount of a substance or a composition in accordance with the invention iβ administered. The present invention has furhter ore for an object to provide use of the composition or the substance according to the invention for therapeutic treatment or diagnosis. The composition or compound according to the present invention is thus uβeful in treatment of profylaxiβ or dia- gnoβiβ of diβorders caused by the toxine of Shiqella dvsen¬ teriae . As iβ clear from the definition of compound <I) given above R3 of formula (I) can designate the reβidue of a natu¬ ral or βynthetic glycoconjugate. Substituent R3 may also be constituted by a macromolβcular carrier to which the active part of the compound according to the invention iβ coupled. The coupling can suitably be provided by a covalent coupling obtained via a coupling arm. Aβ a macromolβcular carrier there may be used a synthetically or naturally occurring po- lypeptide, polysaccharide, other polymer or particle. In actual practice the coupling arm between the βtruc- tura element and macromolecular carrier suitably consists of : - NH-(CH 2 ) n -CH 2 (θ N-N- - NH-(CH 2 ) n -C0NH- - NH- According to another aspect of the invention the com¬ pound or composition according to the invention can be used to determine the presence of the toxine of Shiqella dvsen¬ teriae in a sample taken from a mammal including man. In this context one determines the degree of interaction between the toxine in question and the compound or composition in ques¬ tion. This interaction can be determined by inhibition or induction of adhesion of the toxine to cells or to other sur¬ faces . " According to yet another aspect of the invention the compound which constitutes active receptor vis-a-vis the Shi- gatoxine can be identified or quantified in native biological material, the procedure using antibodieβ, the generation of which haβ been induced by the uβe of a compound or a composi¬ tion according to the invention. Moreover, the invention can be used for isolation of the toxine of Shiαella dvsenteriae from a culture containing βuch toxine, a compound according to the invention being as¬ sociated with a macromolecular or particular carrier and the culture being brought into contact with the carrier to bind the toxine thereto, the toxine being then released from the carrier and recovered. The toxine can suitably be released by providing in a column containing βuch carrier a change in pH. The toxine may alβo be relβaβed by displacement while utili¬ zing a compound according to the invention in free form. Al¬ ternatively, the toxine may be released by treatment with a borate buffer or by treatment with βo called chaiotropic ions. Regarding the meaning of βubβtituent R 3 and R 4 in the compound of formula (I) or formula (II) aβ given above it can be of any type aβ long aβ it doeβ not negatively effect the conditionβ in connection with the practical application of the invention. Thuβ, the βaid βubβtituent may be hydrogen, lower alkyl or lower acyl, but R^ can also be constituted by the reβidue of a natural or synthetic glycoconjugate, whereby the compound according to the invention thuβ iβ a glycocon- jugate, for example a glycolipid. The invention will in the following be further describ¬ ed in relation to non-limiting examples. EXAMP ES 1. Syntheβiβ of β -Ji-G INTF^-<l-3>-α-Ji-βalja-<l-O-g-J: • H CO-CF j SIobotetraosylceramide (from human red cell membranes) was treated with trifluoroacetic acid/tri luoroacetic acidanhydride (TFA/TFAA) (1:100; v/v; 20 ml) at 100°C for 46 hours. After cooling the reaction mixture was evaporated to dryness. The residue was dissolved in 10 ml glacial acetic acid, 10 ml distilled water being then added. The mixture was heated at 100°C for 4 hours and then evaporated into dryness. 10 ml water and 10 ml chloroform/methanol (2:1; v/v) were added and the mixture was shaken. The organic phase was sepa¬ rated and another 10 ml chloroform/methanol were added. After shaking the organic phase was separated and the aqueous phase was evaporated into dryness. The crude product was gelcromatographed on Sephadex S15 (2x100 cm) using water as an eluent. The fractions containing tetrasaccharide were combined and freezedried, 43 mg of pro- duct being obtained. The product was analyzed using FAB-techniques (V6 ZAB) in a masspectrometer, and (M+l)+τn/e 7ό2 was obtained. When adding NaCl there was obtained (M+Na)+m/e 784. iHNMR, 500 MHz (D 2 0), TSP, 70°C: Spectroscopic data for TF 1HNMR, 500 MHz (D 2 0, TSP, 70°C) δ 5,23 (d, 0,38H, y-3,7Hz, H-1x) δ 4,95 (d, 1H, Y- 3,7Hz, H-1'») 6 4,80 (d, 1H, γ«8,2Hz, H-1"*) δ 4,66 (d, 0,64H, γ*7,9Hz, H-1β) δ 4,52 (d, < 1H, γ-7,8Hz, H-1') δ 4,52 (d, <1H, γ-7,8Hz, H-Tβ) δ 4,31 (1 (bred), 1H, Yl c?Y 2 c?6,SHz, H-5") δ 4,24 (m, 1H, H-4") δ 3,28 (t, 0,62 H, γ 1 βγ 2 • 7,9Hz, H-28) *C; SO, 3 MH 2 (D 2 0, TSP, 21°C): II 162,59 (q, -γ37,5Hz, Q - CF j ) δ 118.49 (q,γ - 286,4H 2 , - CF, δ 106,14 (C - 1') δ 105,40 (C - 1"') δ 103,32 (C - 1") δ 98,53 C-0.6C, C - Ifl) δ 94,61 (<*Q,4C, C - 1 ) δ 56,23 (C - 21 " ) The identity of IH- and 13 C-resonances was confirmed by homo- and heteronuclear 20-correlatiαn spectra. Analysis of the product using gascromatography after reduction with NaBDz, (1 mg/ml , 1 hour, 20°C) and permethylation showed a pique on SE 30 glasscapi11ary column (25 mm) at 330°C. Masspectrum (E.I.) of reduced and permethylated product showed the expected result according to what is shown * below: CHDOMe 2. Binding of Shigella dysenteriae type 1 toxine to HeLa- nd Vero-oells. Two HeLa-cel lines originally coming from Walter Reed Army Institute of Research, Washington D.C. were used. They are called "sensitive" and "resistent" based on the 10 000 fold difference for the cytotoxic effects of the toxine. TheVero-cel 1ine came from the Governe ental Bacteriological Laboratory, Stockholm. The cells were cultivated in Eagle's minimal essential medium having added thereto a fetal calf serum (Sibco), final concentration 5%; O M NaHCOs; mM glutamin; and 100 lU/ml strepto ycine and penicillin (Gibco). The cellcultures were incubated in 3% CO2 at 37°C for 3 days (HeLa-cel Is) or for 7 days (Vero-oells). Trypsinized cells for experimental use were suspended in medium, centrifuged and resuspended either in phosphate-buffered physiological saline (PBS) (0.05 M, pH7.2) for the binding studies or in a medium for toxicity studies. The cells were counted under microscope and imme¬ diately used. Toxine (18/ug protein in 10 / ul PBS) were labelled with 250 uCi 125 I using Bolton Hunter reagent (2200 Ci mmol; New England Nuclear). Separation of the toxine from unbound iso¬ tope was obtained using gel cromatography with Sephadex G25 column (10x0.6 cm) (Pharmacia Fine Chemicals) and PBS as an eluting liquid. Labelled toxine was stored at + °C until used. Isotope-labelled toxine was dialuted to a concentrat ion of 1.25 ug protein/ml with PBS having added thereto bovine serum albumin (BSA, Sigma Chemical) 1 mg/ml . Dialuted toxine was preincubated with antigen, compound (I) according to Example 1 above dialuted in PBS or only with PBS for 1 hour at +25°C. Then cells were added suspended in PBS and the mixture was stirred for 1 hour at +25°C. Controls containing toxine but no cells were incubated in parallel. The total volume of each experiment was 0.3 ml. Each reaction mixture was then stored on top of a discontinuous Percoll gradient (Pharmacia Fine Chemicals). The gradient was prepared by adding 1 ml of 100% Percoll (9 ml concentrate + 1 ml 15% NaCl) to a polycarbonate tube, 4 ml of 20% Percoll in PBS and at last 1 ml PBS being added on top. The gradient was centrifuged for 15 minutes at 80Qxg +4°C. The cells form bands between 20% and 100% Percoll. The top layers containing toxine not bound to the cells were transferred with a Pasteur pipette to another polycarbonate tube. Both fractions are counted in a ga macounter (Packard Instruments). On the appended drawings 1-3 the results of the expe¬ riments as carried out are presented in the form of diagrams. In Fig. 1 the binding of the toxine to cells as a function of the quantity of labelled toxine is shown. The binding is shown in relation to Vero-cells (• ) , to HeLa-cells (+), to resistent HeLa-cells (o) and no cells (A). In Fig. 2 there is a corresponding diagram concerning ' 125j-_ι aDe . led toxine binding to Vero-cells after competition of labelled toxine (100 ng) with unlabelled toxine. In Fig. 3 there is shown a diagram over the inhibition of the binding of 12 ^J-label led Shigatoxine to Vero-cells. Toxine (100 ng) was in the experiments preincubated with galabiose (+), globotetraose (Δ) , 1 , 1 ' , 1 ' '-triacetylcito- triose (□) and the compound according to Example 1 above (o), respectively, before the addition of 2 x 10^ Vero-cells. Each testpoint in the diagram represents the average of two ex¬ periments. As is clear from Fig. 3 there is obtained 50% inhibi¬ tion at a concentration of the active compound of about 1.2 mg/ml .

Claims: 1. A compound having the formula (I): R R, 1. wherein Rj. and R2 independently are hydrogen, lower alkyl or acyl, with the proviβo that R^ iβ not hydrogen when R2 iβ acetyl, or Rj_ and R2 together with the nitrogen atom to which they are connected form a cyclic imide, for example phtalamide, and wherein R 3 ia hydrogen, an organic reβidue, βuch aβ a carbohydrate reβidue, or the residue of a natural or βynthetic glycoconjugate. 2. A compound according to claim 1 having the formula <II) : wherein j_ and Rj have the meaning given in claim 1 and wherein R4 iβ hydrogen, an organic reβidue, βuch aβ a carbohydrate residue, or the reβidue of a natural or βynthetic glycoco jugate. 3. A compound according to claim 1 ' or 2, characterized thereby, that Rj iβ hydrogen or lower alkyl and R2 iβ hydrogen, lower alkyl or acyl 4. A compound according to claimj 1 or 2, characterized thereby that R^ ^ and R 2 are not simultaneously hydrogen. 5. A compound according to claim 1 or 2, characterized thereby that R 1 iβ hydrogen and R2 has the formula ( III ) : - wherein R5 , R Q and R7 independently are hydrogen, ower alkyl or halogen. 6. A compound according to claim 5, wherein R5, R5 and R7 all are fluoro or chloro. 7. A compound according to claim 6, wherein R5, R5 and R7 all are fluoro. 8. A compound according to claim 2, wherein -OR3 is in -configuration * . 9. A composition for use in treatment of profylaxis or diagnosis of disorders caused by the toxine of Shiqella dvsenteriae. comprising a compound according to any of the preceding claims in combination with a pharmaceutically acceptable carrier or diluent . 10. A composition according to claim 9, characterized thereby that it contains a residue of the compound coupled to a macromolecular carrier. 11. A composition according to claim 10, characterized thereby that it contains said residue covalently coupled to a macromolecular carrier via a coupling arm. 12. A composition according to claims 10 or 11, characterized by using as a macromolecular carrier a synthetically or naturally occurring polypeptide, polysaccharide, other polymer or particle. 13. A composition according to any of claims 10-12, characterized thereby that the coupling arm between the structural element and macromolelcular carrier consists of : - NH- (CH 2 ) n -CH 2 (θ) N =N- - NH- (CH- _.) n -CONH- - NH- whβrein R is an alkyl- or an arylrβsidue and wherein n may vary from 1 to 20. 14. A compound according to any of claims 1-8 or a composition according to any of claims 9-13 for use as a receptor to the toxine Shi ella dvsenteriae. 15. A method for therapeutic treatment of mammals including man, characterized by administering a therapeutically active amount of a compound according to any of claims 1-8 or a composition according to any of claims 9- 14. 16. A method for determining the presence of the toxine of Shiqella dvsenteriae in a sample taken from a mammal including man, characterized by determining the degree of interaction between the toxine in the sample and the compound according to any of claims 1-8 or the composition according to claims 9-15. 17. A method according to claim 16, characterized by determining the interaction by inhibition or induction of the adhesion of the toxine to cells or to other surfaces. 18. A method for identi ication or quantification of the compound according to any of claims 1-8 in native biological material from mammal including man, characterized in using antibodies the generation of which has been induced by the use of the compound according to any of claims 1-8 or the composition according to any of claims 9-15. 19. A method of isolating the toxine of Shiqella dvsenteriae from a culture containing said toxine, characterized by associating a compound according to any of claims 1-8 with a macromolecular or particular carrier, bringing the culture into contact with the carrier, binding the toxine to said carrier, the toxine being then released from the carrier and recovered. 20. A method according to claim 19, characterized by releasing the toxine by providing in a column a change in pH, 21. A method according to claim 20, characterized by releasing the toxine by displacement while utilizing a compound according to any of claims 1-8 in free form. 22. A method according to claim 19, characterized by releasing the toxine by treatment with a borate buffer. 23. A method according to claim 19, characterized by releasing the toxine by treatment with chaiotropic ions.

Inventors: LINDBERG ALF, SVENSSON SIGFRID, LINDBERG, ALF, SVENSSON, SIGFRID
Applicants: BIOCARB AB
Application year: 1985
Main classification: C07H3
IPC codes: A61K39, C07H3, C07H21, C07K14, C12Q1, G01N33