WO1993023031A9 - Substances mimetiques polyvalentes et substances mimetiques peptidiques destinees a bloquer l'interaction cellulaire dependant de l'hydrate de carbone et a provoquer la reponse anti-hydrate de carbone des lymphocytes t - Google Patents

Substances mimetiques polyvalentes et substances mimetiques peptidiques destinees a bloquer l'interaction cellulaire dependant de l'hydrate de carbone et a provoquer la reponse anti-hydrate de carbone des lymphocytes t

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WO1993023031A9
WO1993023031A9 PCT/US1993/004163 US9304163W WO9323031A9 WO 1993023031 A9 WO1993023031 A9 WO 1993023031A9 US 9304163 W US9304163 W US 9304163W WO 9323031 A9 WO9323031 A9 WO 9323031A9
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naturally occurring
carbohydrate
sialosyl
carbohydrate epitope
corresponding naturally
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PCT/US1993/004163
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  • Multivalent mimetics and peptide mimetics for blocking carbohydrate-dependent cellul interaction and for eliciting anticarbohydrate T-cell response
  • the instant invention relates to stable multivalent carbohydrate epitope ⁇ and mimetics of carbohydrate epitopes and their uses.
  • the stable carbohydrate epitope ⁇ are prepared by chemically modifying the structure of known carbohydrate epitopes using, for example, 6-trifluoromethylfucose, carbocyclic fucose, N-trifluoroacetyl or N-carbamylneuraminic acid, or S-glycoside ⁇ of sialic acid and fucose.
  • the peptide mimetics complementary to carbohydrates can be based on amino acid sequences of complementarity-determining regions (CDR) 1, 2 or 3 of the variable heavy or variable light regions of anti-carbohydrate idiotype antibodies which mimic carbohydrate structure.
  • CDR complementarity-determining regions
  • the chemically modified carbohydrate epitopes and the carbohydrate mimetics are useful for inhibiting carbohydrate-mediated cell adhesion.
  • the peptide mimetics complementary to carbohydrates also are useful to induce
  • sialosyl-Le* (SLe x ) (1) .
  • sialosyl-Le a (SLe a ) (2), Le x (3) , Le a (4) , Le y (5) , Le b (7) , GM3 (8) , GD3 (9) , GD2 (10) , Gg3Cer (11) , Tn (13) , sialosyl-Tn (14) , T (15) and sialosyl-T (16) (see Table I) are important epitopes recognized as tumor-associated carbohydrate antigens (TACA' ⁇ ) .
  • SLe and SLe a have been identified as the epitope ⁇ recognized by selectin ⁇ .
  • Expression of the antigens listed above may be instrumental in the ability of tumor cells to invade surrounding tissues, and metastasize in vivo, based on the following types of observations: i. Strong correlation between expression of TACA in primary tumors and grade of subsequent tumor progression. ii. Identification of some of those antigens as adhesion molecules recognized by glycosphingolipids or other glycoconjugates expressed on a particular type of cell (e.g., microvascular endothelial cell).
  • SLea or analogue thereof by selectins expressed on activated platelets or endothelial cells SLea or analogue thereof by selectins expressed on activated platelets or endothelial cells.
  • Active immunization with carbohydrates or derivatives thereof is designed primarily to elicit humoral immune responses (mainly IgM or IgG 3 ) (which may not be sufficient to eliminate tumor cells) or to block activity of leukocytes and monocytes recruited at sites of inflammation.
  • Active immunization with Le x glycolipid may reduce inflammatory myelocytic response at the inflammatory lesion of rheumatoid arthritis (Ochi et al., J. Rheumatol. , 15:1609-1615, 1988).
  • Carbohydrate epitopes designed to block carbohydrate-carbohydrate interaction or selectin-dependent adhesion should be stable and not destroyed in vivo. It should also be designed to gain high affinity to carbohydrates or to lectin domains of selectin. Tritiated galactosyl ⁇ l->4 glucose (lactose) has a half-life of only 3-5 min (degraded and recovered as 3 H-labeled Gal) when injected into mice. Similarly, if sialosyl or fucosyl carbohydrate derivatives (such as SLe a or SLe x , the epitopes recognized by selectin) are injected, they are degraded rapidly. Other ⁇ tudie ⁇ by the instant inventors also have shown that bivalent sialosyl-Le x or bivalent Le x had higher binding affinity to selectin, which indicates that stable carbohydrate epitopes should be designed in a multivalent structure.
  • important objects of the instant invention are: (i) preparation of stable, conformationally-restrictedcarbohydrateoligosaccharide epitopes which has high affinity to carbohydrate ⁇ or to selectin and can efficiently block carbohydrate-dependent cell adhesion (i.e., based on carbohydrate-carbohydrate or carbohydrate-selectin interaction) ; (ii) preparation of oligosaccharide analogues which negatively effect the normal expression of carbohydrates that mediate intercellular adhesion; (iii) bivalent or multivalent structures of carbohydrate mimetics as described hereinabove; and (iv) preparation of peptide mimetics having a peptide conformational surface structure the same as specific carbohydrate antigens.
  • Such peptide/non-carbohydrate mimetics are useful not only for blocking carbohydrate-dependent cell adhesion but also for inducing a T cell response against carbohydrate antigens since many non-peptide/ non-carbohydrate epitopes are known to elicit a T cell response quite well (Kochibe et al., Proc. Natl. Acad. Sci. USA. 72:4582-4587, 1975; Handa et al. , J. Immunol.. 135:1564, 1985).
  • the present invention provides a stabilized carbohydrate epitope having more resistance to metabolic degradation than a corresponding naturally occurring carbohydrate epitope and having high affinity to block cell adhesion based on carbohydrate-carbohydrate interaction and carbohydrate-selectin interaction.
  • a high affinity structure could be based on multimeric mimetics.
  • the instant invention provides a preparation of oligosaccharide analogues which negatively effect ⁇ the normal expression of carbohydrates that mediate intercellular adhesion.
  • the present invention also provides a mimetic of a peptide epitope, wherein the mimetic has a structure such that the mimetic has about the same antibody-binding or selectin-binding activities, immunogenicity and antigenicity as that of a corresponding naturally occurring carbohydrate epitope.
  • the present invention further provides a process for preparing the above-described mimetic of a peptide epitope having the same surface structure as carbohydrate, the process comprising:
  • the present invention further provides a medicament for inhibiting metastasis of tumor cells, inhibiting inflammatory processes and inhibiting microbial infection caused by carbohydrate-mediated cell adhesion, the medicament comprising:
  • the present invention further provides a method for inhibiting carbohydrate-mediated cell adhesion including metastasis of tumor cells, inflammatory processes and microbial infection, the method comprising contacting cells with an inhibitory amount of a stabilized carbohydrate epitope having more resistance to metabolic degradation than a corresponding naturally occurring carbohydrate epitope or with a mimetic of a carbohydrate epitope wherein the mimetic has a structure such that the mimetic has about the same antibody-binding or selectin-binding activities, immunogenicity and antigenicity as that of a naturally occurring carbohydrate epitope.
  • the present invention additionally provides a vaccine for induction of an anti-carbohydrate T cell immune response, the vaccine comprising:
  • the present invention also provides a method of vaccinating to induce an anti-carbohydrate T cell immune response, the method comprising vaccinating a host with an anti-carbohydrate T cell inducing amount of a vaccine comprising a mimetic of a carbohydrate antigen, wherein the mimetic has a structure such that the mimetic has about the same antibody-binding or selectin-binding activities, immunogenicity and antigenicity as that of. a corresponding naturally occurring carbohydrate epitope.
  • Figures IA and IB graphically depict inhibition of B16 melanoma cell adhesion of HUVEC's (Fig. IA) and LacCer (Fig. IB) by methyl- ⁇ -lactoside (Me- ⁇ -Lactoside) , mimetic 6-deoxy-6-fluoro-galactopyranosyl- ⁇ l->4- glucopyranosyl- ⁇ l-methylglycoside (compound (1')) and galactopyranosyl- ⁇ l->4-6-deoxy-6-deoxy-6-fluoro- glucopyranosyl- ⁇ l-methylglycoside (compound (2')).
  • the abscissa represents concentration of added compound and the ordinate represents cell binding expressed as radioactivity per well.
  • Figures 2A, 2B and 2C show various synthesis schemes involved in preparing compound (1') and compound (2 1 ) shown in Fig. IA.
  • Figures 4A and 4B are the synthetic schemes for compounds 1-6 shown in Fig. 3.
  • Figure 5 is the synthetic scheme for compounds 7 and 8 shown in Fig. 3.
  • Figures 6A and 6B show the synthetic scheme for compound 9 shown in Fig. 3.
  • Figure 7 is the synthetic scheme for compounds 10 and 11 shown in Fig. 3.
  • Figure 8 shows a structural analogue common to both the sialosyl-Le x and sialosyl-Le a structure ⁇ .
  • Figures 9A and 9B show the synthetic scheme for synthesizing the cyclohexanediol analogue (87) shown in Fig. 8.
  • Figure 10 depicts a synthetic scheme, beginning with fucose (3) , to make a carbocyclic derivative thereof.
  • (a) is DMSO, Ac 2 0, rt, overnight;
  • (b) is LiCH 2 P(0) (OMe) 2 , THF, N 2 , -77°C, 30 min;
  • (c) is NaBH 4 , THF, rt, overnight;
  • (d) is DMSO, TFAA, Et 3 N, CH 2 C1 2 , -77°C, 1.5 h;
  • (e) is NaH, diglyme, N 2 , 65°C, 1 h;
  • (f) is (Ph 3 PCuH) 6 , H 2 0, THF, N 2 , rt, 48 h;
  • (g) is NaBH 4 , CeCl 3 , MeOH, rt, 5 min;
  • (h) is NaBH 4 , EtOH, rt, 4 h; and
  • (i) is 10%
  • Figure 12 depicts a continuation of the scheme ⁇ set forth in Figures 10 and ll.
  • (a) is (BnO) 2 PN(i-Pr) 2 , lH-tetrazole, CH 2 C1 2 , rt, 2 h;
  • (b) is Li, liq. NH 3 , THF, 2h;
  • (c) is Dowex 50X8-400 (Et 3 HN + ) ;
  • (d) is m-CPBA, -40°C-+0°C, 45 min; and
  • (e) is GMP-morpholidate, pyridine, rt, 5 d.
  • HPLC separation comprised RP-18; 24:10.05M aq Et 3 HNHC0 3 -MeCN, isocratic and the remaining chromatographic treatment (the triethyl ammonium salt to the sodium salt) comprised Bio-Rad AG 50 -X2 (Na + ) . Percent values indicate yield. Numbers identify compounds.
  • Figure 13 depicts a scheme for obtaining an intermediate of a carbocyclic derivative of a selectin epitope.
  • Figure 14 (Scheme IV, Route 1) depicts a continued synthesis to yield carbocyclic derivative intermediates, Compounds (23) and (24) .
  • Figure 14 also shows continuous synthesis from Compounds (24) to (26) by extension of the supporting arm of Le x having carbocyclic fucose.
  • Figure 15 depicts another route for synthesis of a carbocyclic compound of Le x , having carbocyclic fucose (Compound (26) and Compounds (28) or (29)).
  • Figure 16 (Scheme VI, Plan 1) depicts synthesis of a carboxyl group linked at the 3 position of the terminal galactose of Le x having carbocyclic fucose with appropriate arms (Compound (32)).
  • Figure 17 (Scheme VII, Plan 2) shows another scheme for synthesis of Le x having carbocyclic fucose that has a carboxyl group at the 3 position (Compound (34)).
  • Figure 18 (Scheme VIII, Plan 3) shows another plan for synthesis of a sulfonated group at the terminal Gal of Le x having carbocyclic fucose (Compound (39)).
  • Figure 19 shows another plan for synthesis of Le x having carbocyclic fucose that has a sulfonyl group linked through an intermediate carbon (Compound (41) ) .
  • Figure 20 depicts a phosphono group at the galactose residue of Le x having carbocyclic fucose (Compound (43)).
  • Figure 21 depicts synthesis of any alkyl group with acidic functionalities at the 3 position of the galactose of Le x having carbocyclic fucose using an alkyl halide.
  • Figure 22 depicts Scheme XII for synthesis of a • trifunctional stabilized carbohydrate epitopes in which M represents any carbohydrate mimetic structure of SLe x , SLe a , HLe y , Le etc. The structures have arms with an amino group to make trivalent structures as depicted.
  • Figure 23 depicts possible synthesis of lipids which carry various stabilized carbohydrate mimetics (Compound (46) ) which can be incorporated readily into liposomes.
  • Figure 24 depicts a synthetic scheme for multimerization of carbohydrate mimetics (Compound (48) ) .
  • the phrase "having more resistance to metabolic degradation than a corresponding naturally occurring carbohydrate epitope (or carbohydrate antigen) means that the half-life of the structure, when tested by any art-recognized test for measuring a half-life of a metabolite, is more than the half-life of the corresponding naturally occurring carbohydrate epitope or carbohydrate antigen, the difference being statistically significant.
  • E-selectin The epitopes recognized by E-selectin have been identified as SLe (Phillips et al., Science. 250:1130-1132, 1990) and SLe a (Takeda et al., Biochem. Biophys. Res. Commun. , 179:713-719, 1991; Berg, E.L. et al., J. Biol. Chem.. 266:14869-14872, 1991). Those recognized by P-selectin also were shown to be SLe x (Polley et al., Proc. Natl. Acad. Sci. USA. 88:6224-6228, 1991) and SLe a (Handa et al., Biochem. Biophvs.
  • N-modified carbohydrates can be obtained readily by known methods (Hakomori et al. (1980) "Cell Biological and Immunological Significance of Ganglioside Changes Associated With Transformation” in Structure-Function of Gan ⁇ lioside ⁇ . (Svennerhol et al., Eds.) Plenum Publishing Corp., N.Y., pp. 247-261). (iii) Instead of O-glycosylation of sialic acid and fucose, replace with S-glycoside, which is resistant to sialidase or fucosidase. (iv) Use carbasugar derivatives, such as carbocyclic fucose.
  • stable Le y or Le x can be constructed using 6-trifluorofucose; stable STn can be constructed using lactones or lactams, or with N-substituted sialic acid.
  • Carbohydrates constructed in those ways are more stable and therefore better able to inhibit carbohydrate-dependent cell adhesion.
  • Six-trifluorofucose is capable of inhibiting H-hemagglutination induced by anti-H lectin. Therefore, the H structure or Lewis structure in which fucose is replaced by 6-trifluorofucose also is capable of binding to antibodies or lectins which bind to fucose.
  • the following procedure allows the artisan to estimate and predict mimetic structures presenting a conformational surface structure such that the mimetic has about the same, i.e., within experimental error, antibody-binding or lectin-binding activities, immunogenicity and antigenicity as that of the native carbohydrate epitopes.
  • a monoclonal antibody (mAb) directed against the carbohydrate epitope of interest (Abl) is made by known methods. In fact, that step already has been accomplished for all the epitopes shown in Table I.
  • Ab2 anti-idiotype mAb directed against the internal image structure of Abl is made by known methods. To confirm that the desired Ab2 has been made, it is essential to demonstrate inhibition by Ab2 of Abl binding to the carbohydrate epitope of interest, or inhibition by the carbohydrate of interest of Ab2 binding to Abl. 3. A specific peptide region
  • CDR complementarity-determining region
  • the conformational structure thereof can be determined by a minimum-energy modeling program (e.g., Sybyl 5.5, Tripos Associates) .
  • the conformational structure is compared to that of the original carbohydrate epitope. In general, more than two sequences at different CDR's of the V H or of the V L region will cooperate for complete satisfaction of complementarity. 5.
  • a r ig id , conformationally-restricted peptide mimetic is synthesized by cross-linking or by substitution of appropriate amino acids as described.
  • a large ⁇ -loop structure can be maintained by appropriate cross-linking.
  • the same side chains of the essential peptide structure as in the original should be maintained.
  • a peptide analogue which mimics the surface structure of the original carbohydrate epitope can be synthesized.
  • Such carbohydrate mimetics made from anti-carbohydrate idiotype CDR sequences should demonstrate the same antibody-binding or lectin-binding activities, as well as the same immunogenicity, as the original carbohydrate epitope. However, the mimetics are more stable than the original carbohydrate epitope, or the original CDR peptide of Ab2, in terms of hydrolyzability with glycosidases and peptidases.
  • Step 1 Obtain hybridoma 1 producing anti-CHO mAb
  • Step 2 Obtain hybridoma 2 producing Ab2 (anti-anti-CHO) immunization with Abl or hybridoma 1
  • Step 3 Sequence information for Ab2 sequence the V H and v L regions, focus on CDR 1, 2 and 3
  • Step 4 Comparison of conformational structures of peptides in CDR 1, 2 and 3, focusing on regions where hydroxylated amino acids (Ser, Thr and Tyr) are clustered, which may mimic the CHO Ag
  • the conformational structure of the original CHO epitope based on hard sphere exano eric calculation (a) is compared with the conformational structures of the hydroxy-amino acid cluster sequences of regions CDR 1 and 2 (b and c) .
  • the majority of the surface structure of (a) is shared with b (top to right-side portion as shown) .
  • a part of the surface structure of (a) is shared with (c) (lower part) . Since CDR 3 in the above case does not show a cluster of hydroxylated amino acids, no structure can be assigned. Similar conformational analysis can be applied to the V L region.
  • Step 5 Chemical synthesis of peptide mimetics based on a defined peptide sequence found in CDR 1 and 2 above, whose conformation mimics that of the original CHO epitope
  • Step 6 Determination of biological activity of peptide mimetics created in step 5
  • Peptide mimetics obtained as in step 5 should have the same biologic properties as the original CHO antigen. That is, the mimetics should: (i) bind the appropriate lectin or inhibit binding of lectin to the original antigen; (ii) (if the original epitope is SLe x or SLe a ) bind to E-selectin or P-selectin, or inhibit binding of E-selectin or P-selectin to SLe or SLe a ; (iii) bind to appropriate anti-CHO mAb's or inhibit binding of those mAb's to the original CHO epitope; (iv) induce antibody response when conjugated with macromolecular carriers and injected into the body. In some cases, T cell response should be observed, or even predominate over humoral immune response. Humoral antibody response, or T cell response after immunization, should be stimulated equally by the mim
  • Anti-idiotype mAb 87.92.6 which binds to mAb 9BG5 as well as to the cell-binding site of reovirus, subsequently was established and thus mimics the cell-surface receptor function. mAb 87.92.6 also down-regulates receptor function and inhibits DNA synthesis in cells.
  • the essential peptide sequence of the CDR region of mAb 87.92.6 was found in the V L region and has the sequence shown below (Williams et al., Proc. Natl. Acad. Sci. USA. 86:5537-5541, 1989). Lys-Pro-Gly-Ly ⁇ -Thr-A ⁇ n-Ly ⁇ -Leu-Leu-Ile-
  • Tyr-Ser-Gly-Ser-Thr-Leu-Gln (SEQ. ID NO. 3)
  • the pentapeptide Tyr-Ser-Gly-Ser-Thr (SEQ. ID NO. 4) shown in bold hereinabove is the essential site for binding to mAb 9BG5, and also binds to the cell receptor, down-regulates receptor function and inhibits DNA synthesis in cells and inhibits binding of reovirus to cells.
  • Those conclusion ⁇ were ba ⁇ ed on a number of inhibition studies using various peptides with altered sequences, substitutions and other modifications.
  • To fix the pentapeptide conformation a mimetic was synthesized by using a cross-linking molecule.
  • the mimetic inhibited binding of the long peptide representing the V L region to mAb 9BG5, strongly down-regulated expression of reovirus receptor at the surface and inhibited reovirus-induced cellular DNA synthesis.
  • the peptide mimetic was resistant completely to proteolysis (Saragovi et al., Science. 253:792-795, 1991) .
  • the present invention also provides a medicament for inhibiting metastasis of tumor cells, inflammatory processes and microbial infection caused by carbohydrate-mediated cell adhesion, the medicament comprising:
  • the present invention also provides methods for inhibiting carbohydrate-mediated cell adhesion, which include inhibiting metastasis of tumor cells, inhibiting inflammatory processes and inhibiting microbial infection caused by carbohydrate-mediated cell adhesion.
  • the method comprises contacting the cells with or administering to a host in need of treatment an inhibitory amount of a stabilized carbohydrate epitope having more resistance to metabolic degradation than a corresponding naturally occurring carbohydrate epitope or a mimetic of a carbohydrate epitope, wherein the mimetic has a structure such that the mimetic has about the same antibody-binding or selectin-binding activities, immunogenicity and antigenicity as that of a corresponding naturally carbohydrate.
  • a majority of relevant carbohydrates comprise one or more fucose residues.
  • fucosyltransferases catalyze transfer of L-fucopyranose from GDP-fucose at appropriate sites on putative glycoconjugates.
  • fuco ⁇ e occur ⁇ at nonreducing termini of glycoconjugate ⁇ . Accordingly, inhibitors of fucosylation, by affecting the proper expression of cell adhesion molecules, can affect profoundly intercellular interactions, such as cell adhesion.
  • One such inhibitor is based on the replacement of the glycosyl moiety of a sugar nucleotide by a more stable carba-sugar.
  • carbocyclic analogues of GDP-fucose serve as suitable inhibitors of fucosyltransferases.
  • the conversion of L-fucose to the carbocyclic analogues thereof can be achieved by intramolecular olefination.
  • a suitable reaction scheme is intramolecular Emmons-Horner-Wadsworth olefination (Paulsen et al., Liebigs Ann. Chem. 1987:125; Marquez et al., J. Or . Chem.
  • carbohydrates comprising a fucose, such as Le x , Le 8 , Le y , SLe x , SLe a and the like, can be manipulated using a carbocyclic fucose derivative.
  • the carbocyclic fucose can be attached to the carbohydrate chain through a variety of linkages but preferably is linked in the ⁇ configuration at any of the available sites on the sugars comprising the backbone.
  • bivalent Le x structure 17 in Table 1
  • bivalent SLe x structure 18 in Table 1
  • Le x linked to SLe x structure 19 of Table 1
  • Multivalent structures can be prepared conveniently by either use of a multifunctional molecule, liposome ⁇ or polymerization. Since the Le mimic (26) and the SLe x mimics (32), (34), (39), (41), (43) and (44) all possess a spacer arm with an amino group, derivatizations of those mimics are performed readily by reaction with a carboxyl group of another compound. For (43) , for convenience, the mimics are depicted by an "M" bonded to an NHBoc group or to a NH 2 group, thus, M-NHBoc or M-NH 2 .
  • a suitable lipid such as the commercially available 2-tetradecylhexadecanoic acid (D) (Wako Chemical Co., Japan) , is converted to the active ester (E) and coupled to M-NH 2 .
  • D 2-tetradecylhexadecanoic acid
  • E active ester
  • M-NH 2 M-NH 2
  • carbohydrates can be made comprising such carbocyclic analogues.
  • Replacement of the ring oxygen in a sugar pyranoside by a methylene group transforms a glycosidic linkage into an ether linkage, which is resistant to the glycohydrolase reaction. •
  • incorporation of such an analogue will increase the metabolic stability of the parent carbohydrates without altering the stereochemistry.
  • the stereochemistry of the carbohydrate is one of the most important factors for molecular recognition.
  • the carbocyclic analogue of Le x can be modified further to mimic SLe x by an incorporation of simple anionic functionalities.
  • the terminal sugar of the backbone of carbocyclic analogues may be derivatized to contain a negatively charged substituent, such as a carboxyl group, sulfono group, phosphono group and the like.
  • a specific use of the method of inhibiting tumor cell metastasis includes treatment of malignancies.
  • the method of inhibiting inflammation is applicable to any inflammation which is due to neutrophil motility and invasion into blood vessel walls.
  • Another important application of peptide mimetics of carbohydrate epitope ba ⁇ ed on anti-idiotype monoclonal antibodies against anti-carbohydrate monoclonal antibodies is for induction of an anti-carbohydrate T-cell immune re ⁇ ponse for suppression of tumor growth.
  • Most carbohydrate antigens induce humoral antibody response, i.e., a T cell-independent B cell response.
  • carbohydrates are not processed by the host immune machinery by regular antigen pathways.
  • the present invention also provides a- vaccine for inducing of an anti-carbohydrate T cell immune response, the vaccine comprising:
  • the present invention also provides a method of vaccinating to induce an anti-carbohydrate T cell immune response, the method comprising vaccinating a host with an anti-carbohydrate T cell inducing amount of a vaccine comprising the above-described mimetic of a carbohydrate antigen.
  • the inhibitory effective amount and the anti-carbohydrate T cell inducing amount of stabilized carbohydrate epitope or of the carbohydrate mimetic according to the present invention can be determined using art-recognized methods, such as by establishing dose response curves in suitable animal models and extrapolating to humans; extrapolating from in vitro data; or by determining effectiveness in clinical trials.
  • Suitable doses of the stabilized carbohydrate epitope or of the carbohydrate mimetic according to the instant invention depend on the particular medical application, i.e., inhibiting carbohydrate-mediated cell adhesion or inducing anti-carbohydrate T cells, the severity of the disease, the weight of the individual, the age of the individual, the half-life in circulation etc. , and can be determined readily by the skilled artisan.
  • the number of doses, daily dosage and course of treatment may vary from individual to individual.
  • the stabilized carbohydrate epitope or the carbohydrate mimetic can be administered in a variety of ways, such as orally, parenterally and topically.
  • Suitable pharmaceutically acceptable carriers, diluents or excipients which can be combined with the stabilized carbohydrate epitopes and the carbohydrate mimetics for administration depend on the particular medical use and can be determined readily by the skilled artisan.
  • the stabilized carbohydrate epitopes and the carbohydrate mimetics with or without carrier can take a variety of forms, such as tablets, capsules, bulk or unit dose powders or granules; may be contained with liposomes; or may be formulated into solutions, emulsions, suspensions, ointments, pastes, creams, jells, foams or jellies.
  • Parenteral dosage forms include solutions, suspensions and the like.
  • Such subsidiary ingredients include disintegrants, binders, lubricants, surfactants, e ulsifiers, buffers, moisturizer ⁇ , solubilizers and preservatives.
  • stabilized carbohydrate epitopes or carbohydrate mimetics can be administered in a suitable fashion to ensure effective local concentrations.
  • the stabilized carbohydrate epitopes or the carbohydrate mimetics may be injected in a depot or adjuvant, carried in a surgically situated implant or reservoir that slowly releases a fixed amount of the substance over a period of time or may be complexed to recognition molecules with the ability of binding to a site presenting with abnormal cell growth.
  • An example of such a contemplated scenario is a recognition molecule that is an antibody with binding specificity for a bone marrow specific antigen, wherein the bone marrow-specific antibody is complexed to the stabilized carbohydrate epitope or the carbohydrate mimetic, the complex being administered to a patient with leukemia.
  • Human umbilical vein endothelial cells (HUVEC' ⁇ ) were grown in 20-well plates. Radiolabeled B16 melanoma cells (2 x 10 /well) were added in the presence of various concentrations of methyl- ⁇ -lactoside, compound (1') or compound (2') (Fig. IA) and the cells bound by the compound were measured, in terms of radioactivity.
  • Figs. IA and IB graphically depict inhibition of B16 melanoma cell adhesion to HUVEC's (Fig. IA) and LacCer (Fig. IB) by methyl- ⁇ -lactoside (Me- ⁇ -Lactoside) , mimetic 6-deoxy-6-fluoro-galactopyranosyl- ⁇ l-+4- glucopyranosyl- ⁇ l-methylglycoside (compound (1')) and galactopyranosyl- ⁇ l->4-6-deoxy-6-deoxy-6-fluoro- glucopyranosyl- ⁇ l-methylglycoside (compound (2 1 )).
  • the abscissa represents concentration of added compound and the ordinate represents cell binding expressed as radioactivity per well.
  • Compound (l 1 ) therefore is considered to be a useful anti-adhesion reagent for suppression of tumor cell metastasis.
  • Flash column chro atography (Still et al., J _ Org. Chem. 1978, 43, 2923) was performed over Merck silica gel 60 (230-400 mesh ASTM) .
  • 2,4-Dimethylbenzoyl chloride (Ador & Meier, Ber. 1879, .12., 1970) was prepared by reaction of 2,4-dimethylbenzoic acid with thionylchloride by a standard methodology (Vogel et al., "Vogel's Textbook of Practical Organic Chemistry”; Longman: New York, 1978; p 498) .
  • a mixture of (6) (0.5 g, 1.17 mmol), 2,4, 6-trimethylbenzoic acid (mesitoic acid) (2.35 g, 14.3 mmol) and trifluoroacetic anhydride (2.3 mL, 16.3 mmol) in dry benzene (90 mL) was stirred at rt under dry N 2 for 2 h and then poured into a pre-cooled solution of sat. aq NaHC0 3 . The organic layer was separated, washed with H 2 0 and dried over anhyd Na 2 S0 4 .
  • TLC indicated that, in addition to the starting material (> 60%) , a mixture of several compounds was formed due to partial de-O-acylation.
  • reaction mixture was neutralized with Amberlite IR-120 (H + ) resin and purified by flash column chromatography (10:1 toluene/EtOAc) . The following compounds were obtained.
  • Methyl 6-Deoxy-6-fluoro- ⁇ -lactoside (29) (Compound (2') in Fig. IA) A mixture of 28 (118 mg, 0.19 mmol) in 0.01M methanolic NaOMe (6 mL) was left at 0°C overnight. The mixture was neutralized with Amberlite IR-120 (H * ) resin and passed through a column of Bio-Gel P-2 with H 2 0.
  • SLe substitutions of SLe" are shown as structures (1) through (11) in Fig. 3.
  • the synthetic schemes are presented in Figs. 4A and 4B (for structures (l)-(6)), Fig. 5 (structure ⁇ (7)-(8)), Fig ⁇ . 6A and 6B (structure (9)) and Fig. 7 (structure ⁇ (l ⁇ )-(ll)).
  • the compound ⁇ all are ⁇ table and are more effective a ⁇ inhibitors than are native SLe x and SLe a in view of the finding that the 6-fluoro-6-deoxy-galactopyranosyl substituted form of methyl- ⁇ -lactoside is much more effective than native methyl- ⁇ -lactoside for inhibition of cell adhesion.
  • thioglycoside-mediated glycosidation between (22)-(26) was employed along with the properly protected methyl 1-thio- ⁇ -L-fucopyranoside (31) , which is readily available from L-Fuc (27) ((27) ⁇ (29) ⁇ (31)).
  • the glycosidation was initiated by the addition of dimethyl(methylthio)sulfoniu triflate 7 • (DMTST) to construct the trisaccharides, (33) and (35)-(38).
  • the regioselectivity of the reaction depend ⁇ on the selective reaction of the equatorial hydroxyl group. Subsequent deprotection of the tetrasaccharides (49)-(54) yields the deoxy analogues (3) and (5) and the deoxyfluoro analogues (2), (4) and (6).
  • the methyl glycosides (49)-(54) can be converted to the glycosyl chlorides (55)-(60) by treatment 11 with dichloromethyl methyl ether and ZnCl.
  • Metabolically stable analogues of SLe x containing a thiofucopyranoside linkage or a carba-Fuc residue also were synthesized. Both analogues are expected to inhibit ⁇ -fucosidase activities.
  • the synthetic plan for the incorporation of a thioglycoside linkage involves inversion of the configuration at the glycosyl position of the acceptor by nucleophilic attack of a thiolate ion. Therefore it is necessary to epimerize the 3-OH group of lactoside.
  • the dibutylstannylene-mediated oxidation of 3' ,4•-isopropylidene lactoside (17) results in the selective oxidation of the 3-OH group ((17) -> (65) 13 ).
  • the thiosialoside analogue of SLe which is expected to resist sialidases also is synthe ⁇ ized.
  • the triflate derivative (84) is required as a thiosialosylation acceptor.
  • the 3*-0-allyl derivative (76) is prepared from (12) through stannylation.
  • the ⁇ -L-Fuc residue is introduced to the 3-position of (78) by use of benzyl-protected fucopyranosyl fluoride (79) as a glycosyl donor ((78) + (79) ⁇ (80)).
  • the fluoride (79) is prepared readily from (31) by treatment with NBS and HF-pyridine. 16 Isomerization of the allyl group and subsequent hydrolysis yield (81) . The hydroxyl group then is epimerized by an oxidation-reduction procedure ((81) -> (82) ⁇ (83)).
  • N-acetyl group on the NeuAc residue appears to be important for binding through hydrophobic interactions of the methyl group. Therefore, the acetyl group of the NHAc group is replaced by trifluoroacetyl and benzoyl groups, expecting that those hydrophobic groups increase binding activity.
  • De-N-acetylation is carried out by refluxing (1) under strong basic ccoonnddiittiioonnss (((11)) ⁇ ((8866))))) ..
  • cyclohexanediol analogue (87) is synthesized as shown in Figs. 9A and 9B.
  • Tri-0-Benzyl-L-fucono-l,5-lactone (4) A mixture of tri-O-benzyl-L-fucopyranose (Dejter-Juszynski & Flowers, Carbohydr. Res. 1971, 18, 219) (3) (3.85 g, 8.86 mmol), acetic anhydride (27 mL) and DMSO (40 mL) was stirred at rt overnight.
  • reaction mixture was warmed to 0°C, and poured into a chilled mixture of CH 2 C1 2 (260 mL) and 2M aq HC1 (220 mL) .
  • the organic layer was separated, washed with sat. aq NaHC0 3 (2 x 400 mL) and H 2 0 (2 x 400 mL) and dried over anhyd Na 2 S0 4 .
  • the mixture was diluted with CH 2 C1 2 (20 mL) , washed sequentially with 10% aq Na 2 S0 3 (2 x 20 mL) , sat. aq NaHC0 3 (2 x 20 mL) and H 2 0 (2 x 20 mL) and then dried over anhyd Na 2 S0 4 .
  • the dihydrogen pho ⁇ phate (17) wa ⁇ converted into the bis(triethylammonium) salt by pas ⁇ ing it ⁇ aqueous solution over a column of Dowex 50X8-400 (Et 3 HN+) .
  • the eluate was lyophilized and the resulting amorphou ⁇ ⁇ olid was dried over P 2 0 5 overnight prior to use.
  • the dihydrogen phosphate (19) was first converted into the bis(triethylammonium) salt as described above.
  • Colo205 cells (ATCC) were grown to confluency in RPMI 1640 medium containing 10% fetal calf ⁇ erum, were trypsinized, centrifuged, washed twice with PBS (pH 7.4) and counted using a hemacytometer. Cells (4 x 10 ) were injected subcutaneously into athymic (nude) mice. Tumors were excised after 2 weeks and stored frozen at -80°C. The tumors then were homogenized at 4°C in two volumes of 50 mM HEPES (pH 7.2), 0.5M sucrose and ImM EDTA.
  • the crude homogenate was centrifuged at 30,000 g for 30 min and the pellet wa ⁇ rehomogenized in the presence of the above buffer containing 0.2% Triton-XlOO.
  • the homogenate was centrifuged at 100,000 g for 1 h and the supernatant was concentrated to the original volume of the tumors by dialysis.
  • the enzyme preparation was stored at -80°C until needed.
  • the inhibition assay was performed at a 25 ⁇ L scale.
  • the mixture contained the following components: HEPES (pH 7.2; 0.625 ⁇ mol) , MnCl 2 (0.125 ⁇ mol) , GDP-[U- U C]Fuc (20,000 cpm/nmol; 2.5 nmol) , LNF 1 (50 nmol) , enzyme preparation (10 ⁇ L) and inhibitor (5 nmol, 10 nmol, 20 nmol and 40 nmol) .
  • the mixture was incubated at 37°C for 20 min and stopped by addition of ice-cold H 2 0 (1 mL) .
  • (17) is a useful precursor for the preparation of the 3-OH unprotected ⁇ -D-N-acetyllactosaminide (21) .
  • the 2-OH group in (18) was transformed into the 2-N 3 group (compound (20)) in good yield via the iodo derivative (19) .
  • the Le x -mimic (26) which contains an ⁇ -carba-fucose residue is constructed either by condensation reactions between (9) and (22) and between (12) , the Tf derivative of (10) , and (21) (Scheme IV, Route l, Figure 14) or by coupling of (13) and (21) followed by saturation of a double bond (Scheme V, Route 2, Figure 15).
  • the sialosyl Le x mimics are elaborated from (25) a ⁇ depicted in Schemes VI-XI as set forth in the Figures.
  • the reactions schemes are based on the regioselective alkylation at the 3-OH group in the galactose residue via the stannylene complex.

Abstract

Epitope d'hydrate de carbone stabilisé présentant une résistance plus grande à la dégradation métabolique qu'un épitope d'hydrate de carbone naturel correspondant, ou que ceux assemblés dans des structures polyvalentes. Substance mimétique relative à un épitope d'hydrate de carbone, cette substance présentant une structure telle qu'elle présente des activités de liaison d'anticorps ou de la sélectine et une immunogénicité et une antigénicité pratiquement égales ou supérieures à celles d'un épitope d'hydrate de carbone naturel correspondant. On décrit un procédé de préparation de la substance mimétique relative à un épitope d'hydrate de carbone mentionnée ci-dessus, ainsi que les médicaments et les procédés de blocage d'adhésion cellulaire induite par l'hydrate de carbone, et un vaccin et une méthode de vaccination pour induire une réponse anti-hydrate de carbone des lymphocytes T.
PCT/US1993/004163 1992-05-08 1993-05-10 Substances mimetiques polyvalentes et substances mimetiques peptidiques destinees a bloquer l'interaction cellulaire dependant de l'hydrate de carbone et a provoquer la reponse anti-hydrate de carbone des lymphocytes t WO1993023031A1 (fr)

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DE4402756A1 (de) * 1994-01-31 1995-08-03 Boehringer Mannheim Gmbh Spezifische Bindungssubstanzen für Antikörper und deren Verwendung für Immunoassays oder Vakzine
CA2224346A1 (fr) * 1995-06-29 1997-01-16 Novartis Ag 1,2-diols diglycosyles utilises comme agents mimetiques de sialyl-lewis x et de sialyl-lewis a
CA2227013A1 (fr) 1995-07-14 1997-02-06 Glycotech Corp. Composes et methodes de traitement de cancers lies au recepteur egf et purification du recepteur egf
SK15699A3 (en) * 1996-08-08 1999-07-12 Novartis Ag Modified oligosaccharides, process for their preparation and pharmaceutical composition containing them
WO1998011118A1 (fr) * 1996-09-13 1998-03-19 Daikin Industries, Ltd. Derives x de lewis
JP2023528400A (ja) * 2020-06-03 2023-07-04 サイモン フレイザー ユニヴァーシティー タンパク質フコシル化の阻害剤及びその用途

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