Drug – bio-affecting and body treating compositions – Conjugate or complex of monoclonal or polyclonal antibody,... – Conjugated via claimed linking group – bond – chelating agent,...
Reexamination Certificate
1994-10-19
2003-08-26
Celsa, Bennett (Department: 1639)
Drug, bio-affecting and body treating compositions
Conjugate or complex of monoclonal or polyclonal antibody,...
Conjugated via claimed linking group, bond, chelating agent,...
C424S178100, C514S002600, C514S004300, C514S023000, C536S001110
Reexamination Certificate
active
06610299
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to glycosyl-etoposide prodrugs, a process for the preparation thereof and the use thereof in combination with functionalized tumor-specific enzyme conjugates for treating cancers, and specifically relates to 4′-O-glycosyl-etoposides as prodrugs which can be cleaved by the action of tumor-specific enzyme conjugates to give cytotoxic active substances, the liberated active substance being suitable, by reason of its cytostatic activity, for treating cancers.
The present invention further relates to enzyme conjugates for prodrug activation, including fusion proteins of the general formula huTuMAb-L-&bgr;-Gluc, where huTuMAb is a humanized or human tumor-specific monoclonal antibody, a fragment or a derivative thereof, L is linker, and &bgr;-Gluc comprises human &bgr;-glucuronidase. These fusion proteins are prepared by genetic manipulation. huTuMAb ensures the specific localization of tumors, L connects the huTuMAb to &bgr;-Gluc in such a way that the specific properties of the two fusion partners are not impaired, and &bgr;-Gluc activates a suitable prodrug compound by elimination of glucuronic acid, where a virtually autologous system for use in humans is provided by the humanized or human fusion partners.
BACKGROUND OF THE INVENTION
The combination of prodrug and tumor-specific antibody-enzyme conjugates for use as therapeutic agents is described in the specialist literature. This has entailed injection of antibodies which are directed against a particular tissue and to which a prodrug-cleaving enzyme is covalently bonded into an animal which contains the transplanted tissue, and subsequently administering a prodrug compound which can be activated by the enzyme. The prodrug is converted by the action of the antibody-enzyme conjugate, which is anchored to the tissue, into the cytotoxin which exerts a cytotoxic effect on the transplanted tissue.
A therapeutic system which contains two components, an antibody-enzyme component and a prodrug component that can be activated by the enzyme is described in WO 88/07378. In this case, the use of non-mammalian enzymes is described for the preparation of the antibody-enzyme conjugates, and that of endogenous enzymes is ruled out because of non-specific liberation of active compound. Since the exogenous enzymes are recognized by the body as foreign antigens, the use thereof is associated with the disadvantage of an immune response to these non-endogenous substances, which can result in the enzyme immobilized on the antibody being inactivated, or possibly, the entire conjugate being eliminated. In addition, in this case p-bis-N-(2-chloroethyl) amino-benzylglutamic acid and derivatives thereof are used as prodrug, and the chemical half-life thereof is only 5.3 to 16.5 hours. It is a disadvantage for a prodrug to be chemically unstable because of the side effects to be expected.
A therapeutic system which contains two components and in which the antibody-enzyme conjugate located on the tumor tissue cleaves a prodrug compound to form a cytotoxic active compound is likewise described in EPA 0302473 A2. The combined use of etoposide 4′-phosphate and derivatives thereof as prodrug and of antibody-immobilized alkaline phosphatases for liberating the etoposides, which is described therein, inter alia, is disadvantageous because of the presence of large amounts of endogenous alkaline phosphatases in the serum. As described in DE 38265662 A1, the etoposide 4′-phosphates have already been used alone as therapeutic antitumor agents, with the phosphatases present in the serum liberating the etoposide from the prodrug.
DESCRIPTION OF THE INVENTION
It has emerged, surprisingly, that the synthetically prepared, hitherto unobtainable compound 4′-O-alpha-D-glucopyranosyl-etoposide can be cleaved in vitro into etoposide and D-glucose with the enzyme alpha-glucosidase as well as a tumor-specific antibody-glucosidase conjugate.
Based on this finding, and taking into account the disadvantages, described above, of prior art combinations of prodrugs and antibody-enzyme conjugates, the object of the present invention was to prepare synthetic, enzymatically cleavable 4′-O-glycosyl-etoposides as well as functionalized tumor-specific enzymes to cleave them, and to test the pharmacological utility of the combination of the two components in suitable mammalian test models. This object has been achieved by preparing 4′-O-glycosyl-etoposides of formula I and functionalized tumor-specific enzymes of formula II which, on combined use thereof, exhibit cytostatic activity.
The invention thus relates to 4′-O-glycosyl-etoposides of the formula I
in which
R
1
is a methyl, benzyl or 2-thienyl group,
R
2
is a hydrogen atom, an acyl or tri-C
1
-C
4
-alkylsilyl protective group,
R
3
is a hydroxl group, an acyl or tri-C
1
-C
4
-alkylsilyl protective group which is bonded via an oxygen atom, an amino, acetylamino, benzyloxycarbonylamino or dimethylamino group,
R
4
is a hydrogen atom or a methyl group,
R
5
is a hydrogen atom, a hydroxyl group, an acyl or tri-C
1
-C
4
-alkylsilyl protective group which is bonded via an oxygen atom, or an amino, benzyloxycarbonyl-amino, azido or acetylamino group,
R
6
is a hydroxyl group, an acyl or tri-C
1
-C
4
-alkylsilyl protective group which is bonded via an oxygen atom, or an amino, benzyloxycarbonylamino or azido group,
R
7
is a hydrogen atom, an acyl or tri-C
1
-C
4
-alkylsilyl protective group, and
R
8
is a methyl or hydroxymethyl group or an acyl protective group which is bonded via a methyleneoxy group, or a benzyloxycarbonyl group, where an acyl protective group means an acetyl, mono-, di- or trihalogenoacetyl group with halogen meaning fluorine or chlorine;
and functionalized tumor-specific enzymes of the formula II
A—Sp—E II
in which
A is an antibody or one of the fragments thereof, which have specificity for a tumor-associated antigen, or is a biomolecule which accumulates in a tumor, such as EGF (epidermal growth factor), TGF-&agr; (transforming growth factor a), PDGF (platelet derived growth factor), IGF I+II (insulin like growth factor I+II) or a+b FGF (acidic+basic fibroblast growth factor),
E is a glycosidase which is not immunogenic or is of low immunogenicity, preferably mammalian glycosidase, as &agr;-or &bgr;-glucosidase, &agr;-galactosidase, &agr;- or &bgr;-mannosidase, &agr;-fucosidase, N-acetyl-&agr;-galactosaminidase, N-acetyl-&bgr;-/N-acetyl-&agr;-glucosaminidase or &bgr;-glucuronidase,
Sp (spacer) is a polypeptide spacer or a bifunctional sulfide- or disulfide-containing group of the formula III or IV
X(S)
n
Y III
X(S)
n
IV
in which
X is —CO—R
9
—(N-succinimido)- or —C(═R)—CH
2
—CH
2
— with R
9
being —CH
2
—CH
2
—, 1,4-cyclohexylidene, 1,3- or 1,4-phenylene or methoxycarbonyl- or chloro-1,4-phenylene and R being O or NH,
Y is —C(═R
10
)—CH
2
—CH
2
—, where R
10
has the stated meaning, and
n is 1 or 2.
Preferred within the scope of the invention are compounds of the formula I in which the radicals
R
1
is a methyl, benzyl or 2-thienyl group,
R
2
is a hydrogen atom, an acetyl or chloroacetyl group or a tri-C
1
-C
4
-alkylsilyl protective group,
R
3
is a hydroxyl group, an acetyl, chloroacetyl or tri-C
1
-C
4
-alkylsilyl protective group which is bonded via an oxygen atom, or an amino, acetylamino, benzyloxycarbonylamino or dimethylamino group,
R
4
is a hydrogen atom or a methyl group,
R
5
is a hydrogen atom, a hydroxyl group, or an acetyl, chloroacetyl or tri-C
1
-C
4
-alkylsilyl protective group which is bonded via an oxygen atom, or an amino, benzyloxycarbonylamino, azido or acetylamino group,
R
6
is a hydroxyl group, an acetyl, chloroacetyl or tri-C
1
-C
4
-alkylsilyl protective group which is bonded via an oxygen atom, or an amino, benzyloxycarbonyl-amino or azido group,
R
7
is a hydrogen atom, an acetyl, chloroacetyl or tri-C
1
-C
4
-alkylsilyl protective group, and
R
8
is a methyl, hydroxymethyl, acetyloxy or chloro-acetyloxymethy
Bosslet Klaus
Czech Jörg
Hoffmann Dieter
Kolar Cenek
Sedlacek Hans-Harald
Aventis Pharma Deutschland GmbH
Celsa Bennett
Finnegan, Henderson Farabow, Garrett and Dunner L.L.P.
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