EP0247792A2

Immunoglobulin conjugates.

Abstract

Immunoglobulin conjugates formed by reaction of an antineoplastic indole-dihydroindole vinca alkaloid containing a hydrazide group attached at C-3 or C-4 with an oxidized glycoprotein containing aldehyde groups, are provided.

EP0247792A2, drawing sheet 1 — Sheet 1 of 24

Term

Term ended

Projected expiry passed 21 May 2007, 19.3 years ago.

14 claims: 7 independent, 7 dependent

1
A C-3 vinca hydrazone conjugate of formula (Ia):wherein R² is H, CH₃ or CHO;when R⁴ and R⁵ are taken singly, R⁵ is H, and one of R³ and R⁴ is ethyl and the other is H or OH;when R⁴ and R⁵ are taken together with the carbons to which they are attached, they form an oxirane ring in which case R³ is ethyl;R¹ is H, (C 1-3 alkyl)-CO, or chloro-substituted (C 1-3 alkyl)-CO;m is 1 to 25;and GP is an oxidized glycoprotein or a fragment thereof.
2
A C-4 vinca hydrazone conjugate of formula (Ib):wherein R² is H, CH₃ or CHO;when R⁴ and R⁵ are taken singly, R⁵ is H, and one of R³ and R⁴ is ethyl and the other is H or OH;when R⁴ and R⁵ are taken together with the carbons to which they are attached, they form an oxirane ring in which case R³ is ethyl;R is O(C 1-3 alkyl), NH₂, NH(C 1-3 alkyl), NH-CH₂CH₂-Y, 1-pyrrolidinyl or 1-piperidinyl, wherein n is 2-4 and Y is Cl, OCH₃ or SCH₃;X is C 1-4 straight chain alkylene, C 2-8 branched chain alkylene, C 2-4 alkenylene, C 3-4 alkynylene, C 3-6 cycloalkylene, phenylene, hydroxy-substituted C 1-4 alkylene, or a direct bond, m is 1 to 25;and GP is an oxidized glycoprotein, or a fragment thereof.
3
A conjugate according to Claim 1 wherein R¹ is H.
4
A conjugate according to any one of claims 1 to 3 in which the glycoprotein moiety is an immunoglobulin or a fragment thereof.
7
A conjugate as claimed in any one of claims 1 to 6 in which the glycoprotein is KS 1/4.
8
A conjugate as claimed in any one of claims 1 to 7 in which the average ratio of vinca residues per antibody is about 1 to 10.
10
A pharmaceutical formulation which comprises, as an active ingredient, a conjugate as defined in any one of claims 1 to 9, associated with a pharmaceutically-acceptable carrier or diluent therefore.
11
A process for preparing a hydrazone conjugate as defined in any one of claims 1 to 9, which comprises reacting, with an aldehyde group of an oxidized glycoprotein, a vinca hydrazide of formula I:wherein R² is H, CH₃ or CHO;when R⁴ and R⁵ are taken singly, R⁵ is H, and one of R³ and R⁴ is ethyl and the other is H or OH;when R⁴ and R⁵ are taken together with the carbons to which they are attached, they form an oxirane ring in which case R³ is ethyl;R is NHNH₂, O(C 1-3 alkyl), NH₂, NH(C 1-3 alkyl), NH-CH₂CH₂-Y, 1-pyrrolidinyl or 1-piperidinyl, wherein n is 2-4 and Y is Cl, OCH₃ or SCH₃;R¹ is H, (C 1-3 alkyl)-CO, chloro-substituted (C 1-3 alkyl)-CO or R⁶ wherein R⁶ is COXCONHNH₂ wherein X is C 1-4 straight chain alkylene, C 2-8 branched chain alkylene, C 2-4 alkenylene, C 3-4 alkynylene,, C 3-6 cycloalkylene, phenylene, hydroxy-substituted C 1-4 alkylene, or a direct bond, except that R cannot be NHNH₂ when R¹ is R⁶ and R¹ cannot be R⁶ when R is NHNH₂.
12
A process according to Claim 11 wherein the vinca hydrazide is 4-desacetyl-VLB-3-carboxhydrazide, 4-desacetyl-VLB-4-hemisuccinate hydrazide, 4-desacetyl-VLB-3-carboxhydrazide-N²-succinimide-4-hemisuccinate hydrazide, 4-desacetyl-VLB-3-carboxamide-4-hemisuccinate hydrazide, 4-desacetyl-VLB-4-hemiglutarate hydrazide, 4-desacetyl-VCR-4-hemi-succinate hydrazide, 4-desacetyl-4ʹ-epideoxy-VLB-4-hemisuccinate hydrazide, 4-desacetyl-VLB-3-carboxhydrazide-N²-glutarimide-4-hemisuccinate hydrazide, 4-desacetyl-VLB-3-carboxamide-4-hemisuccinate hydrazide, 4-desacetyl-VCR-3-carboxhydrazide, or 4-desacetyl-4ʹ-epideoxy-VLB-3-carboxhydrazide.
14
A vinca hydrazone conjugate, as claimed in any one of claims 1 to 9, for use as an antineoplastic agent.

Independent claims10

77 paragraphs, as filed

0001Immunoglobulins are glycoproteins, i.e., they are proteins with oligosaccharides attached at various sites. Vicinal diols of these oligosaccharides can be oxidized with periodate to yield dialdehydes, and the aldehyde groups produced can be reacted with various amines and hydrazines to form Schiff bases and hydrazones. For example, Willan etal., FEBSLetters, 80 133 (1977) oxidized an oligosaccharide attached to an asparagine residue at position 297 (using the numbering system of the human IgGl myeloma protein Eu) in the CH2 region of rabbit IgG. This oxidized material was, after purification, reacted with 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl free radical and the resulting imine (reaction of amino group with sugar aldehyde) reduced with NaBH₃CN to yield a spin labelled protein. Timofeev etal., FEBSLetters89 191 (1978) also used oxidized glycoprotein monoclonal antibodies to prepare spin-labeled material. Murayama etal., Immunochemistry, 15 523 (1978) labeled, with amino-containing compounds via Schiff base formation, periodate oxidized oligosaccharide-groups in an immunoglobulin (IgG). Aspartic acid and horseradish peroxidase were the "amines" employed. The aspartic acid-Schiff base was used to detect the antigenicity of human IgG by counter immunoelectrophoresis. O'Shannessy etal.,ImmunologyLetters, 8 273 (1984), coupled biotin hydrazide with aldehyde groups of oxidized immunoglobulins and suggested the use of the procedure for conjugation of fluorescent dyes to monoclonal antibodies.

0002Chua etal., BiochimicaandBiophysicaActa, 800 291 (1984), oxidized carbohydrate groups on the constant region of the heavy chain of IgM (which has a relatively high carbohydrate content) to yield aldehydes. These aldehyde groups were reacted with hydrazide groups linked to a liposomal membrane. The monoclonal IgM antibodies were specific to a protein carrying a 1-dimethylamino naphthalene-5-sulfonyl hapten.

0003Rothfus etal., J.B.C., 238 1402 (1963) used periodate oxidation followed by borohydride reduction to determine the identity of the sugar component of the glycopeptide from human gamma globulin.

0004The linking of cytotoxic agents to immunoglobulins is reviewed by Blair etal., J.Immun.Meth., 59 129 (1983) and by Ghose, Blair and Kulkarni, MethodsinEnzymology, 93 280 (1983). Blair etal. reveal, at page 130, the possibility of linking the cytotoxic agent to the oligosaccharide of an Ig. Oxidation to an aldehyde and coupling of the cytotoxic agent to the aldehyde is not mentioned. Ghose etal. mentions the periodate oxidation of rabbit anti-BSA IgG having attached oligosaccharides to form aldehyde groups and the reaction of these groups with ethylenediamine followed by borohydride reduction of the Schiff base to form new aminoethyl primary amine groups attached to the oligosaccharide portion of the glycoproteins.

0005EPO Application Publication 88695 broadly teaches processes such as those described in the above publications; that is, the reaction of an aldehyde derived by oxidation of a carbohydrate moiety with a hydrazine, hydrazide or amine linker group of a compound to form a conjugate. The application is apparently directed to linker groups which are conjugated to the aldehyde-antibody and the linker group itself attached to an insoluble support or a second compound. There are also claims to antibody conjugates comprising any compound attached to a carbohydrate moiety of an antibody via a covalent hydrazone, imine or enamine bond. More specific claims are to a peptide linker, an amino acid linker or a linker of the general formula W(CH₂)nQ where W is C₆H₅NH-CH₂- or -CH₂- and Q is an amino acid, peptide, chelator or chelator derivative. Drugs specifically disclosed for antibody-mediated delivery via this system include daunomycin, bleomycin, vinblastine, vincristine, and 5-fluorouracil. There is no teaching of how these drugs are to be linked and, specifically, no teaching of how VLB or vincristine are to be linked since neither contains a hydrazide or amine function. Coupling to Fab antibody fragments is emphasized. Specific disclosure is limited to coupling ALKERAN® (N-[Bis(β-chloroethyl)phenyl]alanine) and monoclonal antibodies against sheep red blood cells. No invivo data are provided.

0006Further, VLB, vincristine, and other antineoplastic drugs have been linked to immunoglobulins or other proteins.

0007U.K. Patent Application G.B. 2,090,837A discloses immunoglobulin conjugates covalently linked to a vinca moiety by amide formation. The amide bond itself is produced by the reaction of a vinca indole-dihydroindole alkaloid such as 4-desacetyl-VLB via a C-3 carboxazide group with a free amino group of an immunoglobulin or immunoglobulin fragment. The azide is prepared from 4-desacetyl-VLB-3-carboxhydrazide which is itself prepared by the action of hydrazine on VLB. The covalent bond formed between the vinca moiety and the immunoglobulin is not ordinarily reversible; i.e., the bond is not subject to hydrolytic (chemical) cleavage at physiological pH.

0008U.S. Patent 4,203,898 (Cullinan I) discloses and claims 3-carboxazides of vinca indole-dihydroindole alkaloids such as VLB, vincristine, etc. It is these azides which were used in preparing the covalently linked immunoglobulin conjugates of the previous reference. The azide is prepared from a 4-desacetyl-3-carboxhydrazide of an indole-dihydroindole alkaloid. Such hydrazides are also disclosed and claimed.

0009U.S. Patent 4,166,810 (Cullinan II) discloses a group of derivatives of 4-desacetyl-VLB-3-carboxhydrazide including N²-alkyl derivatives. The compounds are prepared by forming a hydrazone by reaction of the N² amine group with an aldehyde or ketone and then reducing the resulting enamine.

0010Neuss etal., TetrahedronLetters, 783 (1968), disclose leurosine hydrazide.

0011Barnett etal., J.Med. Chem., 21 88 (1978), also disclose the hydrazides of VLB and related indoledihydroindole derivatives.

0012Conrad etal., ibid, 22 391 (1979), discuss generally the amides of VLB and their activities as anti-cancer drugs. The same amides discussed above in connection with Cullinan I are disclosed. In addition, a bridged disulfide was prepared in which the carboxamide group was attached to an ethyldithioethylcarboxamide grouping. Also disclosed are radioimmune assays for VLB and vincristine. The radioimmune assay was developed by coupling 4-desacetyl-vinblastinoic acid azide (4-desacetyl-VLB-3-carboxazide) to BSA. This antigen was used to prepare antibodies in rabbits which antibody would then pick up the VLB moiety of the antigen. The amount of antigen was determined by using VLB labeled with tritium -- see also Root etal., F.A.C.S.S., 2nd National Meeting, October 6-10, 1975, Abstract 183.

0013Hargrove, U.S. Patents 3,392,173 and 3,387,001, disclose novel C-4 esters of VLB, vincristine, leurosidine, etc. Among these esters is a chloroacetyl ester, which derivative was employed in EPO 124,502 to couple with a protein. Hargrove II reacted this 4-chloroacetyl ester with amines to prepare, for example, an N,N-dimethylglycine ester -- vinglycinate.

0014Langone etal., Anal.Biochem., 95 214 (1979), developed radioimmune assays for vinblastine and vincristine. The antigens used were prepared by oxidizing VLB to a dicarboxylic acid and then coupling this product to a protein with a carbodiimide coupling reagent.

0015Teale etal., Brit.J.Clin.Pharm., 4 169 (1977), also developed radioimmune assays for vinblastine and vincristine. The vinca alkaloid is conjugated to the albumin by a Mannich reaction using an amine group in the protein (BSA), formaldehyde and vinblastine. The point of attachment of the vinca alkaloid, vinblastine or vincristine, is not specified.

0016Johnson etal., Brit.J.Can., 44 372 (1981), disclose the preparation of vindesine linked to anti-CEA immunoglobulin via an azide. It should be emphasized that the azide from desacetyl VLB and the azide congener of vindesine are identical since the compounds differ only as to the moiety on the C-3 carboxylic acid and this moiety (ester or amide) disappears after the coupling reaction. These conjugates were found to be cytotoxic for human cancer cells invitro. U.K. Patent application GB 2,090,837, inventors Rowland and Simmonds, covers the same subject matter.

0017Rowland etal., CancerImmunologyandImmunotherapy, 19 1 (1985), discuss the anti-tumor properties of vindesine-monoclonal antibody conjugates (4-desacetyl-VLB derivatives conjugated via the C-3 carboxazide group as in U.K. 2,090,837A and Johnson etal). Four conjugates were tested against human tumor xenografts in athymic mice, and all showed some anti-tumor activity. Vindesine was inactive at non-toxic levels with the same tumor.

0018EPO 124502 covers conjugates prepared from an immunoglobulin and a 4-desacetyl-VLB or vindesine or other 4-desacetyl-VLB-3-carboxamide linked via a 4-succinate; i.e., a bridging group or linker between the 4-hydroxy group and the protein. The bridging group of the structure can be CO-CH₂ (from a 4-chloroacetyl group) or CO(CH₂)n-CO (succinic or glutaric acid). The same 4-succinoyl derivatives are disclosed in United Kingdom Patent application GB 2,137,202, and conjugates formed therefrom are disclosed in GB 2,137,210.

0019Bumol etal., Proc. Am. Assn. Cancer Research, 25 356 Abstract 1410 (1984) (Bumol I), discuss the characteristics of a conjugate prepared according to GB 2,137,210; i.e., a 4-desacetyl-4-succinoyl-VLB conjugate with a monoclonal antibody to a human adenocarcinoma (KS1/4). Bumol etal., Federation Proceedings, 44 1864 Abstract 8484 (1985) (Bumol II) discuss a similar conjugate with a monoclonal antibody to a human melanoma. Bumol etal., J. Cellular Biochemistry, Supplement 9A Abstract 0124 (1985) (Bumol III), discuss further work on the 4-desacetyl-4-succinoyl-VLB KS1/4 conjugate (adenocarcinoma).

0020None of the above references disclose or suggest conjugating an amine or hydrazine derivative of an antineoplastic dimeric indole-dihydroindole alkaloid with an oxidized carbohydrate (oxidized to one or more aldehyde groups) on the surface of a monoclonal antibody which is also a glycoprotein.

0021This invention provides conjugates formed by reaction of an oxidized glycoprotein containing one or more aldehyde groups with a vinca hydrazide. The hydrazide group can be either a C-3 carboxhydrazide (COR in formula I below) or a C-4 hydrazide-containing ester linked (R⁶ as defined below) via a hydrocarbon chain.

0022The vinca portion of the conjugate is described by Formula I below. The conjugates of this invention are formed by reaction of an aldehyde group of an oxidized glycoprotein with a compound of structure I: <chemistry id="chem0001" num="0001"><img file="EP0247792A2_D0001.tif" /></chemistry> wherein R² is H, CH₃ or CHO; when R⁴ and R⁵ are taken singly, R⁵ is H, and one of R³ and R⁴ is ethyl and the other is H or OH; when R⁴ and R⁵ are taken together with the carbons to which they are attached, they form an oxirane ring in which case R³ is ethyl; R is -NHNH₂, <chemistry id="chem0002" num="0002"><img file="EP0247792A2_D0002.tif" /></chemistry> -O(C1-3 alkyl), NH₂, -NH(C1-3 alkyl), -NH-CH₂CH₂-Y, 1-pyrrolidinyl or 1-piperidinyl, wherein n is 2-4 and Y is Cl, -OCH₃ or -SCH₃; R¹ is H, (C1-3 alkyl)-CO, chloro-substituted (C1-3 alkyl)-CO or R⁶ wherein R⁶ is -COXCONHNH₂ wherein X is C1-4 straight chain alkylene, C2-8 branched chain alkylene, C2-4 alkenylene, C3-4 alkynylene, C3-6 cycloalkylene, phenylene, hydroxy-substituted C1-4 alkylene, or a direct bond, except that R cannot be NHNH₂ when R¹ is R⁶ and R¹ cannot be R⁶ when R is NHNH₂.

0023Groups illustrative of X in the above formulas include methylene, ethylene, propylene, butylene, vinyl, propylene, butenylene, butynylene, propynylene, hydroxyethylene, 1,2-dihydroxyethylene, 1,2-dimethylethylene, 1,2,3,4-tetrahydroxybutylene, 3,4-dimethylbutylene, 1,4-cyclohexylene, 1,4-phenylene, 1,2-phenylene, and the like.

0024Indole-dihydroindole alkaloids useful in preparing the intermediate hydrazides which form the conjugates of this invention, can be represented by formula II: <chemistry id="chem0003" num="0003"><img file="EP0247792A2_D0003.tif" /></chemistry> wherein R², R³, R⁴, and R⁵ have their previous meaning.

0025In formula II above, where R² is methyl, R³ is hydroxyl, R⁴ is ethyl and R⁵ is H, VLB (vinblastine) is represented; where R² is formyl, R³ is hydroxyl, R⁴ is ethyl and R⁵ is H, vincristine (VCR) is represented; where R² is methyl, R³ is ethyl, R⁴ is hydroxyl, and R⁵ is H, leurosidine is represented; where R² is methyl or formyl, R³ is ethyl and R⁴ and R⁵ taken together with the carbons to which they are attached form an alpha-epoxide ring, leurosine and leuroformine, respectively, are represented; where R² is methyl, R³ is ethyl, and R⁴ and R⁵ are H, deoxy VLB "B" (4ʹ-deoxyleurosidine or 4ʹ-epideoxy VLB) is represented; where R² is methyl, R⁴ is ethyl and R³ and R⁵ are H, deoxy VLB "A" or 4ʹ-deoxy VLB is represented; and where R² is CHO, R³ is ethyl, R⁴ and R⁵ are H, 4ʹ-epideoxyvincristine (1-formyl-1-desmethyl-4ʹ-deoxyleurosidine) is represented.

0026Literature references to the parent vinca alkaloids (II) are as follows: leurosine (U.S. Patent No. 3,370,057), VLB (U.S. Patent No. 3,097,137), leurosidine (vinrosidine) and leurocristine (to be referred to hereafter as vincristine) (both U.S. Patent No. 3,205,220), desmethyl VLB (U.S. Patent No. 3,354,163), 4ʹ-epivincristine (U.S. Patent No. 4,143,041), leuroformine, formylleurosine (U.S. Patent No. 4,279,816), and deoxy VLB "A" and "B" [TetrahedronLetters, 783 (1958)].

0027The hydrazides useful in forming the conjugates of this invention are prepared differently, depending on whether the hydrazide is attached at C-3 or C-4. The C-3 hydrazides are prepared by the procedure of U.S. Patent 4,203,898, col 12, line 65 etseq. and Example 3, col 18. In this procedure, anhydrous hydrazine is reacted with a vinca alkaloid of formula II in ethanol in a sealed tube at about 60°C. The product of this reaction is a 4-desacetyl 3-carboxhydrazide since the acetoxy group at C-4 is hydrolysed under the basic reaction conditions. If it is desirable to prepare an ester at C-4 (R¹ in I is (C1-3 alkyl)-CO or chloro (C1-3-alkyl)-CO), the C-3 carboxhydrazide according to I wherein R¹ is H is first protected by reaction with acetone to form an N²-propylidene derivative. With the acylable NH₂ group of the hydrazide effectively protected against acylation, this "protected" derivative can then be acylated in routine fashion with an acyl halide (chloroacetyl chloride for example) or an acyl anhydride (propionic anhydride for example). The protecting group can then be removed by treatment with acid. If acylation also occurs on the C-3 hydroxyl, as it usually does, this C-3 acyl group can be preferentially removed by treatment with wet silica gel--see Hargrove, U.S. Patent 3,392,173.

0028When the hydrazide group is part of a C-4 chain (i.e., part of R⁶ in I above), the 4-desacetyl starting materials are prepared as follows, depending on the nature of the C-3 group: where C-3 is an ester group, the 4-desacetyl derivative is prepared by the procedure of Hargrove, U.S. patent 3,392,173, Examples 1-5; where C-3 is an amide group, the 4-desacetyl is prepared by the procedure of Conrad etal., supra, or Cullinan, U.S. patent 4,203,898. This procedure involves preparation of a C-3 carboxhydrazide which is accompanied by hydrolysis at C-4. The hydrazide is then converted to the azide and the azide reacted with ammonia or a primary amine of the structure NH₂-(C1-3 alkyl), or with NH₂-CH₂-CH₂-S-CH₃, NH₂-CH₂-CH₂-O-CH₃, pyrrolidine, piperidine or NH₂-CH₂-CH₂-Cl. A preferable method of preparing this last compound, the chloroethylamide to yield the desired C-3 carboxamide C-4 hydroxyl derivative is by decomposition of a 3ʹʹ-(β-chloroethyl)-3-spiro-5ʹʹ-oxazolidine-2ʹʹ,4ʹʹ-dione of a vinca alkaloid according to formula II above. The preparation of these oxazolidinedione derivatives is set forth in Miller and Gutowski, U.S. Patent RE 30,560. The preparation of the β-chloroethylamide from the above oxazolidinedione is set forth in Example 1 of Miller and Gutowski U.S. patent 4,357,334. (Example 2 of the same patent prepares the same β-chloroethylamide using the classical azide process). The primary amide (R in formula I is NH₂) can also be prepared by direct ammonolysis or by reduction of the hydrazide with Raney Nickel -- see also U.S. patent 4,203,898. Finally, when R in I is <chemistry id="chem0004" num="0004"><img file="EP0247792A2_D0004.tif" /></chemistry> where n is 2-4; i.e., a cyclic succinimide, glutarimide, or adipimide, respectively, the 4-hydroxyl derivatives are prepared by the procedure set forth in the copending application of Cullinan, Serial No. 745,562, corresponding to European Published Application No. 86304522.5.

0029The following list illustrates some of the vinca derivatives of Formula I which may be employed in this invention and their common names as known in the art: 4-Desacetyl-VLB-3-carboxhydrazide (Formula I, R = NHNH₂; R¹ = R⁵ = hydrogen; R² = methyl; R³ = hydroxy; R⁴ = ethyl) 4-Desacetyl-VLB-4-hemisuccinate hydrazide (R = OCH₃; R¹ = COCH₂CH₂CONHNH₂; R² = methyl; R³ = hydroxy; R⁴ = ethyl; R⁵ = hydrogen) 4-Desacetyl-VLB-3-carboxhydrazide-N²-succinimide-4-hemisuccinate hydrazide <chemistry id="chem0005" num="0005"><img file="EP0247792A2_D0005.tif" /></chemistry> R₁ = COCH₂CH₂CONHNH₂; R² = methyl; R³ = hydroxy; R⁴ = ethyl; R⁵ = hydrogen) 4-Desacetyl-VLB-3-carboxamide-4-hemisuccinate hydrazide (R = NH₂; R¹ = COCH₂CH₂CONHNH₂; R² = methyl; R³ = hydroxy; R⁴ = ethyl; R⁵ = hydrogen) 4-Desacetyl-VLB-4-hemiglutarate hydrazide (R = OCH₃; R¹ = COCH₂CH₂CONHNH₂; R² = methyl; R³ = hydroxy; R⁴ = ethyl; R⁵ = hydrogen) 4-Desacetyl-VCR-4-hemisuccinate hydrazide R = OCH₃; R¹ = COCH₂CH₂CONHNH₂; R² = CHO; R³ = hydroxy; R⁴ = ethyl; R⁵ = hydrogen) 4-Desacetyl-4ʹ-epideoxy-VLB-4-hemisuccinate hydrazide (R = OCH₃; R¹ = COCH₂CH₂CONHNH₂; R² = methyl; R³ = ethyl; R⁴ = R⁵ = hydrogen) 4-Desacetyl-VLB-3-carboxhydrazide-N²-glutarimide-4-hemisuccinate hydrazide <chemistry id="chem0006" num="0006"><img file="EP0247792A2_D0006.tif" /></chemistry> R¹ = COCH₂CH₂CONHNH₂; R² = methyl; R³ = hydroxy; R⁴ = ethyl; R⁵ = hydrogen) 4-Desacetyl-VCR-3-carboxhydrazide (R = NHNH₂; R¹ = R⁵ = hydrogen; R² = CHO; R³ = hydroxy; R⁴ = ethyl) 4-Desacetyl-4ʹ-epideoxy-VLB-3-carboxhydrazide (R = NHNH₂; R¹ = R⁴ = R⁵ = hydrogen; R² = methyl; R³ = ethyl) 4-Desacetyl-VLB-3-ethylcarboxamide-4-hemisuccinate hydrazide (R = NHCH₂CH₃; R¹ = COCH₂CH₂CONHNH₂; R² = methyl; R³ = hydroxy; R⁴ = ethyl ; R⁵ = hydrogen) 4-Desacetyl-VLB-3-methoxyethylcarboxamide-4-hemisuccinate hydrazide (R = NHCH₂CH₂OCH₃; R¹ = COCH₂CH₂CONHNH₂; R² = methyl; R³ = hydroxy; R⁴ = ethyl; R⁵ = hydrogen) 4-Desacetyl-VLB-4-propionyl-3-carboxhydrazide (R = NHNH₂; R¹ = COCH₂CH₃; R² = methyl; R³ = hydroxy; R⁴ = ethyl; R⁵ = hydrogen).

0030The preparation of several cyclic amides and their salts from European application Serial 86304522.5, noted above, are as follows:

Preparation I

4ʹ-Deoxy-4-desacetylleurosidine-3-carboxhydrazide-N²-succinimide

0031A solution was prepared from 1320 mg of 4ʹ-deoxy-4-desacetylleurosidine-3-carboxhydrazide in 25 ml of pyridine. 175 mg of succinic anhydride were added and the reaction stirred at room temperature under nitrogen for about 24 hours. The volatile constituents were then removed from the reaction mixture invacuo and the resulting residue taken up in methylene dichloride plus sufficient methanol to solubilize the entire residue. The organic layer was twice washed with an equal volume of water and was then dried. Evaporation of the volatile constituents invacuo gave a residue comprising N²-succinoyl 4ʹ-deoxy-4-desacetylleurosidine-3-carboxhydrazide which residue was dissolved in 25 ml of pyridine to which 350 mg of acetic anhydride were added, thus forming a mixed anhydride of the succinic and acetic acids, which anhydride spontaneously cyclized to yield 4ʹ-deoxy-4-desacetylleurosidine-3-carboxhydrazide-N²-succinimide. The reaction mixture was evaporated to dryness invacuo and the residue was dissolved in methylene dichloride. The methylene dichloride extract was washed twice with an equal volume of water and then dried. Evaporation of the methylene dichloride yielded 4ʹ-deoxy-4-desacetylleurosidine-3-carboxhydrazide-N²-succinimide which was chromatographed over silica gel using ethyl acetate containing increasing amounts (0-50%) of methanol as the eluant. Fractions shown by TLC to contain the desired succinimide derivative were combined and the solid evaporated therefrom invacuo. 190 mg of 4ʹ-deoxy-4-desacetylleurosidine-3-carboxhydrazide-N²-succinimide were obtained having the following physical characteristics: Infrared spectrum (in chloroform): peaks at 3470, 1736, 1615, and 1572 cm⁻¹.

0032NMR (CDCl₃): δ at 10.90, 7.88, 7.49, 7.12, 6.48, 6.05, 5.80, 4.10, 3.76, 3.56, 2.82, 0.93, 0.85.

0033The sulfate salt was prepared by dissolving the free base in anhydrous ethanol at pH = ∼ 8.0. The pH was then adjusted to about 3.9 with a freshly prepared solution of 2% ethanolic sulfuric acid. Evaporation of the reaction mixture to dryness yielded 4ʹ-deoxy-4-desacetylleurosidine-3-carboxhydrazide-N²-succinimide sulfate.

0034Following the above procedure, 4-desacetyl VLB hydrazide (2.4 g) was dissolved in 125 ml of pyridine to which 385 mg of glutaric anhydride were added. N²-glutaroyl 4-desacetyl-VLB-3-carboxhydrazide thus formed was isolated by the above procedure and purified by chromatography over silica gel using the same solvent system as above. The glutaroyl compound was reacted with acetic anhydride to form the mixed anhydride which cyclized spontaneously to yield 4-desacetyl-VLB-3-carboxhydrazide-N²-glutarimide. The compound was purified by chromatography over silica gel using the above solvent system. A yield of 460 mg of the N²-glutarimide derivative were obtained having the following physical characteristics: Mass spectrum m/e = 864 (C₄₈H₆₀N₆O₉). Infrared spectrum (CHCl₃): peaks at 3475, 1714, and 1616 cm⁻¹.

0035pKa (66% aqueous DMF) = 5.1, 7.4, 12.9.

0036NMR (CDCl₃): δ at 9.96, 8.91, 8.04, 7.52, 7.14, 6.58, 6.08, 5.83, 4.15, 3.78, 3.69, 3.61, 2.86, 0.93, 0.89.

0037The sulfate salt was prepared by the above procedure; yield = 210 mg from 290 mg of starting material.

0038Following the above procedure but again substituting 4-desacetyl-VLB-3-carboxhydrazide for 4ʹ-deoxy-4-desacetylleurosidine-3-carboxhydrazide, and using acetyl chloride in place of acetic anhydride, there was prepared N²-succinoyl-4-desacetyl-VLB-3-carboxhydrazide. Chromatography of the succinimide product yielded two fractions, one of which was the expected 4-desacetyl-VLB-3-carboxhydrazide-N²-succinimide and the other the 4-acetyl derivative thereof which formed as a by-product during the cyclization procedure. 4-Desacetyl-VLB-3-carboxhydrazide-N²-succinimide had the following physical characteristics. Mass spectrum m/e = 850 (C₄₇H₅₀N₆O₉).

0039Infrared spectrum (KBr): peaks at 3480, 1734 and 1616 cm⁻¹.

0040NMR (CDCl₃): δ at 9.69, 9.47, 8.22, 7.49, 7.12, 6.53, 6.07, 5.82, 5.71, 4.01, 3.77, 3.63, 3.59, 2.84, 2.82, and 0.89.

0041The sulfate salt was prepared according to the above procedure; yield = 250 mg from 350 mg starting material.

0042The preparation of C-4 derivates of these dimeric indole-dihydroindole alkaloids variously substituted at C-3 (see R in formula I) is carried out by reacting compounds according to I where R¹ is H and R and R²-R⁵ have their assigned meaning in a multistep process as outlined in G.B. 2,137,202A (previously cited), or Cullinan U. S. Serial No. 745,562, mentioned earlier, (Example 4). In these procedures, a cyclic anhydride of the formula <chemistry id="chem0007" num="0007"><img file="EP0247792A2_D0007.tif" /></chemistry> wherein X has its previous meaning, is reacted with the compound of Formula I where R¹ is H. The product of this reaction is a half-acid ester of the formula <chemistry id="chem0008" num="0008"><img file="EP0247792A2_D0008.tif" /></chemistry> where V is the 4-desacetyl vinca alkaloid of Formula I attached through the C-4 hydroxyl. An acylating group then replaces the "OH" of the carboxylic acid (COOH). The acid chloride is a very useful acylating group. Alternatively a mixed anhydride produced by reacting, successively, N-methylmorpholine and an alkyl chloroformate with the free carboxylic acid group of the hemi-acid can be used. This "activated" carboxylic acid, <chemistry id="chem0009" num="0009"><img file="EP0247792A2_D0009.tif" /></chemistry> for example, is reacted with hydrazine to produce the desired hydrazide V-CO-X-CONHNH₂. A specific preparation illustrating this procedure follows.

Preparation II

0043A solution was prepared by dissolving 2.5 g of 4-desacetyl-VLB-4-hemisuccinate (4-succinoyl VLB) from Example 1 of GB 2,137,202A, in 50 ml of chloroform. 870 mg of N-methylmorpholine were added. After solution was complete, the reaction mixture was cooled in an ice bath and a stream of nitrogen introduced. 985 mg of isobutyl chloroformate were added and the reaction mixture stirred at 0°C for about 45 minutes. One ml of anhydrous hydrazine was added and the reaction mixture stirred for about 10 minutes. The reaction mixture was then extracted with water, the water extract separated and discarded, and the organic layer dried. Evaporation of the solvent yielded a residue containing 4-desacetyl-VLB-4-hemisuccinate hydrazide. A methylene dichloride solution of the residue was chromatographed over silica gel using ethyl acetate/methanol (100/0 to 50/50) as the eluant. The appropriate fractions provided 950 mg of purified 4-desacetyl-VLB-4-hemisuccinate hydrazide; m/e = 882.

0044The sulfate salt was prepared by dissolving the base in anhydrous ethanol (25 ml), adjusting the pH to approximately 4 with 2% ethanolic sulfuric acid, (24.5 g of anhydrous ethanol, 0.5 g of 18 M H₂SO₄) and removing the solvents invacuo.

0045Infrared spectrum; peaks at 3400, 3009, 1738, 1680, and 1616 cm⁻¹.

0046NMR CDCl₃ δ at 9.9, 8.35, 8.05, 7.55, 7.10, 6.85, 6.10, 5.85, 5.45, 5.30, 3.80, 3.70, 3.60, 2.70, 0.90, 0.85.

0047The novel conjugates of the invention comprise an oxidized glycoprotein, preferably an immunoglobulin and, of that class, preferably a monoclonal antibody (MoAb), which is a gamma-globulin such as an IgG or an IgM. Immunoglobulin (Ig) fragments containing carbohydrate, as in the parent immunoglobulin from which these fragments are derived, also can be used to form novel conjugates of the type disclosed in this specification.

0048The preferred class of glycoproteins, the immunoglobulins, are those which are reactive with or at least recognize antigens on the desired target cell; i.e., they have antigen recognizing properties. Particularly preferred are those glycoproteins which recognize antigens on the surface of the desired target cell.

0049Techniques for the production of such immunoglobulins from the serum of immunized animals or by culturing hybridomas secreting monoclonal products are well known. See, forexample, Kohler and Milstein, Nature256 495 (1975). The preferred type of antibody for use in the invention is an immunoglobulin which is a gamma-globulin. IgG, IgA, IgE, and IgM subclasses are particularly preferred. Some representative immunoglobulins are as follows: Mono- or polyclonal antibodies to <ul id="ul0001" list-style="none"><li>(i) human or animal tumor associated antigens;</li><li>(ii) human B- and T-cell antigens;</li><li>(iii) human Ia antigens;</li><li>(iv) viral, fungal and bacterial antigens; and</li><li>(v) cells involved in human inflammatory or allergic reactions.</li></ul>

0050Of the preferred antibodies to human or animal tumor associated antigens there may be mentioned: <ul id="ul0002" list-style="none"><li>(i) Ig from goats or sheep immunized with carcinoembryonic antigen;</li><li>(ii) Ig from rabbit antiacute lymphoblastic leukemia serum;</li><li>(iii) Ig from various primate antisera raised against acute lymphoblastic leukemia, acute myleoblastic leukemia, chronic lymphoblastic leukemia and chronic granulocytic leukemia;</li><li>(iv) Ig from goats or sheep immunized with lung carcinoma cells, or cellular fractions;</li><li>(v) monoclonal Ig from mouse hybridomas secreting anti-human colorectal carcinoma antibodies;</li><li>(vi) monoclonal Ig from mouse hybridomas secreting anti-human melanoma antibodies;</li><li>(vii) monoclonal Ig from mouse hybridomas that secrete antibodies reacting with human leukemia cells;</li><li>(viii) monoclonal Ig from mouse hybridomas secreting antibodies reacting with human neuroblastoma cells;</li><li>(ix) monoclonal Ig from mouse hybridomas secreting antibodies reacting with human breast cancer antigens;</li><li>(x) monoclonal Ig from mouse hybridomas secreting antibodies reacting with human ovarian carcinoma cells;</li><li>(xi) monoclonal Ig from mouse hybridomas secreting antibodies reacting with human osteosarcoma cells, with human pancreatic carcinoma cells, with human prostatic carcinoma cells etc.;</li><li>(xii) monoclonal Ig from mouse hybridomas secreting antibodies to adenocarcinomas including lung, renal, breast and pancreas;</li><li>(xiii) monoclonal Ig from mouse hybridomas secreting antibodies reacting with human squamous carcinoma cells;</li><li>(xiv) monoclonal Ig from human hybridomas (hybridomas which secrete antibodies to the human tumor-associated antigen including, but not limited to, those monoclonals above);</li><li>(xv) any antibody or fragment thereof that contains carbohydrate in either the light or heavy chain;</li><li>(xvi) Monoclonal Ig from rat, hamster, or other mammalian species not specifically mentioned above, from hybridomas which secrete antibodies to human tumor associated antigens including, but not limited to, those mentioned above.</li></ul>

0051As indicated above, the conjugate can also be made with immunoglobulin fragments Igʹ, referred to also as Fab, Fabʹ, F(abʹ)₂ and IgM monomer derived from an antibody by, for example, proteolytic enzyme digestion or reductive alkylation. Such materials and methods of preparation are well known. The preferred method for preparing such fragments is with proteolytic enzymes such as pepsin and papain. See generally Parham, J. Immunology, 131, 2895 (1983); Lamoyi et al., J. Immunological Methods, 56, 235 (1983); Parham, id., 53, 133 (1982); and Matthew et al., id., 50, 239 (1982).

0052Specific MoAbs exist that are reactive against various tumors; such immunoglobulins useful in the present invention which are associated with or recognize antigens on the surface of tumor cells, include but are not limited to the following: <tables id="tabl0001" num="0001"><img file="EP0247792A2_D0010.tif" /></tables><tables id="tabl0002" num="0002"><img file="EP0247792A2_D0011.tif" /></tables>

0053Preferred conjugates are those prepared from monoclonal antibodies, especially those which recognize human cancer cells such as adenocarcinoma, sguamous cell carcinoma, transitional cell carcinoma, melanoma, neuroblastoma, small cell carcinoma, leukemia, lymphoma, and sarcoma.

0054A glycoprotein (GP) of the type set forth above may be oxidized by periodate or other suitable oxidizing agent so that a bond between vicinal diols in a surface carbohydrate is ruptured to produce aldehyde groups on either side of the original bond. <chemistry id="chem0010" num="0010"><img file="EP0247792A2_D0012.tif" /></chemistry>

0055The number of dialdehyde units produced is a result of the following factors as will be appreciated by those skilled in the art: amount of periodate employed, general reaction conditions of oxidation (eg, time, temperature, solvent, concentration, etc.), number of vicinal diol carbohydrate units present on the protein, and their accessibility to the periodate reagent.

0056Alternatively, or in combination with the periodate oxidation above, one may employ enzymatic oxidation with, for example, galactose oxidase. This is a more selective and restrictive reagent catalyzing the conversion of the 6-CH₂OH position of galactose residues on the glycoprotein carbohydrate chains to an aldehyde functionality. This 6-CH₂OH group must be unsubstituted to allow for successful oxidation. Unmasking a blocked group, such as a group in which a sialic acid residue is attached, may be accomplished with the enzyme neuraminidase. As above, the number of aldehyde moieties so produced will be a function of many variables.

0057The aldehyde-containing glycoproteins, (OCH)m-GP, are then conjugated with the vinca hydrazide, Vʹ-3-CONHNH₂ or V-4-O-CO-X-CONHNH₂, to produce a hydrazone, (Vʹ-3-CONHN=CH)m-GP or (V-4-O-COX-CONHN=CH)m-GP, where Vʹ is the vinca radical defined in Formula I, less the -COR radical at the 3-position, and m is the number of aldehyde or vinca groups attached to the glycoprotein. These conjugates are the subject of this invention and are defined by formulae Ia and Ib, respectively: <chemistry id="chem0011" num="0011"><img file="EP0247792A2_D0013.tif" /></chemistry>

0058In general, the number represented by m is a function of the number of aldehyde groups arising from the oxidation procedures described above. Conjugates with up to about 25 vinca residues per antibody have been found. The average ratios (vinca residues per antibody) are about 1-10 (ie, m is 1-10), with ratios of about 4-10 preferred.

0059The conjugation of the vinca hydrazides with oxidized glycoproteins is accomplished by standard methods known in the art. In general, a solution of the vinca in a water-miscible solvent such as dimethylformamide is added to a chilled buffered aqueous solution of the oxidized glycoprotein. Temperatures of about 0-8°C are preferred and a 0.1N sodium acetate buffer is normally employed. The reaction is best carried out in the dark and under an inert atmosphere. The reaction is usually complete in about 10-24 hours and the resulting conjugate may be purified by standard methods, such as by chromatography over Sephadex.

0060The oxidation and conjugation of specific MoAbs follows.

Preparation III

Conjugates with X-63AG8-S1 MoAb.

0061A solution was prepared by dissolving 200 mg of X-63AG8-S1 (mw = approximately 150,000, 1.34 × 10⁻⁶ moles; cell line deposited and freely available as ATCC TIB9, American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852) in 20 ml of a 0.1M sodium acetate buffer pH 5.6 (29.3 g sodium acetate, 2.44 ml acetic acid plus sufficient sterilized water to make 4 L of buffer). The solution was stored at about 0°C overnight (about 10% of the protein had not dissolved). 685 mg of sodium metaperiodate were added in a single batch with rapid stirring.

0062The mixture was stirred for 21 minutes at about 0°C in the dark and was then quenched by the addition of a 5-fold excess (for the total periodate) with 1.28 ml of a 12.5M solution of ethylene glycol in sterile water. The new mixture was stirred at 0°C for 5 minutes in the dark and was then centrifuged to leave a clear supernatant and a white pellet. The supernatant was loaded onto a Sephadex G25 (medium mesh) gel column and the product eluted with the same sodium acetate buffer. The eluate was monitored with UV light at 280 nm. Any periodate was washed from the column and discarded. Concentration of the oxidized product was assessed in each eluate fraction at 279 nm; yield was 96%. A second run carried out identically gave a 96.5% yield.

0063The above eluate solutions containing 4.72 and 4.02 mg/ml of oxidized product were mixed and 0.1N sodium acetate buffer added to give a final protein concentration of 2.77 mg/ml (Total volume = 69 ml). The solution was cooled to about 0°C.

0064A solution of 4-desacetyl VLB 3-carboxhydrazide in DMF (5.6 ml of a 53.7 mg/ml solution) was added in dropwise fashion to the chilled buffered MoAb solution. The reaction vessel was flushed with nitrogen gas and then sealed. The reaction mixture was stirred in the cold and dark with magnetic stirring for 24 hours. The reaction vessel was then unsealed and the clear, pale yellow reaction mixture was centrifuged. The supernatant was chromatographed over Sephadex G25 gel preequilibrated with pH = 7.4 phosphate buffered saline (0.01M H₃P0₄, 0.15M NaCl) which was also used as the eluant. The conjugate (formed by hydrazone formation between the 3-carboxhydrazide group and an aldehyde group in a carbohydrate on the MoAb) was eluted first followed by unreacted 4-desacetyl VLB 3-carboxhydrazide. The yield of conjugate obtained (from 4 columns) was 173 mg in 224 ml (90% yield). The conjugate contained about 6 moles of 4-desacetyl-VLB-3-carboxhydrazone per mole of X-63AG8-S1 MoAb.

0065A second complete run was carried out using 200 mg of KS1/4 (a MoAb capable of recognizing surface antigens of human adenocarcinoma cells) in 20.0 ml of acetate buffer and using the same quantities of periodate and ethylene glycol as in Preparation 3. The run gave 176 mg of oxidized MoAb in 39.9 ml of buffer after chromatography (88% yield). Conjugation with 274 mg of 4-desacetyl-VLB-3-carboxhydrazide in pH = 5.6 acetate buffer gave 146 mg (83% yield) of conjugate containing about 7.5 moles of 4-desacetyl-VLB-3-carboxhydrazone per mole of KS1/4.

0066Following the above procedure, a conjugate was prepared by reacting 4-desacetyl-VLB-3-carboxhydrazide with aldehyde groups formed by oxidation of the surface carbohydrates of 9.2.27, a glycoprotein MoAb capable of recognizing antigenic sites on the surface of human melanoma cells. A run using 200 mg of the MoAb oxidized with 685 mg of sodium metaperiodate in pH = 5.6 acetate buffer (0.1 molar) gave an aldehyde-containing oxidized 9.2.27 MoAb in 92% yield (184 mg). This material was conjugated with 4-desacetyl-VLB-3-carboxhydrazide (279 mg). Final yield of the poly 4-desacetyl-VLB-hydrazone of oxidized, aldehyde group-containing 9.2.27 conjugate was 91% as a solution in phosphate buffered saline. The product contained about 4.9 moles of the starting vinca per mole of 9.2.27.

0067Following the procedures used in preparation III, several other antibody conjugates, using 4-desaetyl-VLB-3-carboxhydrazide as the vinca moiety, were prepared as outlined in Table II: <tables id="tabl0003" num="0003"><img file="EP0247792A2_D0014.tif" /></tables>

0068As indicated above, conjugates can also be made with immunoglobulin fragments Igʹ, IgM monomer, or other Ig monomers, containing carbohydrate derived from a parent antibody by, for example, proteolytic enzyme digestion or reductive alkylation. Such materials and methods of preparation are well known. Preferred proteolytic enzymes for preparing these fragments are pepsin and papain.

0069Evaluation of the conjugates of this invention can be carried out using well known techniques such as affinity chromatography. The efficacy of the conjugate can be estimated by counting the number of viable cells after treatment of a suspension of tumor cells with the conjugate, or from measurements of the uptake of tritiated uridine. Protein and drug concentrations are determined by measuring optical densities of conjugate solutions at two wavelengths, for example 270 and 279 nm, and relating the values obtained to those for the free drug and unconjugated immunoglobulin at the same two wavelengths. The conjugate can also be evaluated invivo against human tumor xenografts in athymic mice.

0070The novel conjugates of the invention are useful antineoplastic agents and as such are preferably prepared for use in formulations suitable for injection. Thus the invention includes a pharmaceutical formulation, for example an injectable preparation comprising a conjugate of the invention associated with a pharmaceutically-acceptable carrier or diluent such as are well known in the art. The formulation is preferably in unit dosage form, each dosage containing, for example, from 0.01 to 10 mg of the active ingredient (in terms of the vinca drug moiety). A "pharmaceutically acceptable carrier or diluent" is one useful in the chemotherapy of a warm-blooded animal.

0071The novel conjugates are effective over a wide dosage range and dosages per week. For example, for the treatment of adult humans suffering from cancer, dosages will normally fall within the range of 0.01 to 10 mg/kg (vinca drug moiety), more usually in the range of from 0.03 to 9 mg/kg. However it will be understood that the amount of conjugate actually administered will be determined by a physician in light of the relevant circumstances, such as the condition to be treated and the route of administration.

0072The following Tables show the efficacy of the described conjugates in treating neoplastic diseases. <tables id="tabl0004" num="0004"><img file="EP0247792A2_D0015.tif" /></tables><tables id="tabl0005" num="0005"><img file="EP0247792A2_D0016.tif" /></tables><tables id="tabl0006" num="0006"><img file="EP0247792A2_D0017.tif" /></tables>

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867183	United States of America	–	1986-05-27
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DK260687D0	Denmark	D0	1987-05-22
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IL82579A0	Israel	A0	1987-11-30
EP0247792A2This record	European Patent Office (EPO)	A2	1987-12-02
AU7334987A	Australia	A	1987-12-03
JPS62287000A	Japan	A	1987-12-12
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EP0247792A3	European Patent Office (EPO)	A3	1989-07-12
NZ220441A	New Zealand	A	1989-09-27
PT84932B	Portugal	B	1990-02-08
SU1561825A3	Soviet Union (until 1991)	A3	1990-04-30
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Immunoglobulin conjugates.

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