US 2016.0002298A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0002298 A1 MULLER et al. (43) Pub. Date: Jan. 7, 2016 (54) AMATOXIN DERIVATIVES (30) Foreign Application Priority Data (71) Applicant: HEIDELBERG PHARMA GMBH, Mar. 4, 2013 (EP) .................................. 13OO 1074.7 Ladenburg (DE) Publication Classification (72) Inventors: Christoph MULLER, Birkenau (DE); (51) Int. Cl Jan ANDERL, Modautal (DE); Werner cok /64 (2006.01) LUTZ, Weinheim (DE): Torsten C07K 16/32 (2006.01) HECHLER, Bensheim (DE) (52) U.S. C. CPC. C07K 7/64 (2013.01); C07K 16/32 (2013.01); A61K47/48569 (2013.01); A61K 47/48492 (73) Assignee: HEIDELBERG PHARMA GMBH, (2013.01) Ladenburg (DE) (57) ABSTRACT (21) Appl. No.: 14/769,970 The invention relates to tumour therapy. In one aspect, the 1-1. present invention relates to conjugates of an amatoxin and a (22) PCT Filed: Mar. 10, 2014 target-binding moiety, e.g. an antibody, connected by certain linkages, which are useful in the treatment of cancer and other (86). PCT No.: PCT/EP2014/OOO614 disorders and diseases. In a further aspect the invention S371 (c)(1), relates to pharmaceutical compositions comprising Such con (2) Date: Aug. 24, 2015 jugates. O R R2 R R O-amanitin OH OH NH2 OH 3-amanitin OH OH OH OH Y-amanitin H OH NH2 OH e-amanitin H OH OH OH amanin OH OH OH H amaninamide OH OH NH2 H amanulin H H NH2 OH amanullinic acid H H OH OH Patent Application Publication Jan. 7, 2016 Sheet 1 of 7 US 2016/0002298 A1 Fig. 1 O-amanitin OH NH2 OH 3-amanitin OH OH OH V-amanitin OH NH2 OH 8-amanitin OH OH OH amanin OH OH OH amaninamide OH OH NH2 amanullin NH2 OH amanullinic acid OH OH Patent Application Publication Jan. 7, 2016 Sheet 2 of 7 US 2016/0002298 A1 Fig. 2 SKOV-3 120 110 100 10-13 10-12 10-11 10-10 10-9 10-8 10-7 10-6 Concentration M Her-30.0643 (4.4 O Her-30.10333.9 v Her-30.1036 (4.0) Her-30.0643 (4.4). Her-30.1033 (3.9 Her-30.1036 (4.0) EC50 2.942e-O 11 2.748e-011 3.832e-O 11 Patent Application Publication Jan. 7, 2016 Sheet 3 of 7 US 2016/0002298 A1 Fig. 3 SKOV-3 120 110 100 10-13 10-12 10-11 10-10 10-9 10-8 10-7 10-6 Concentration M Her-30.0643 (4.3) O Her-30.1165 (5.0) Her-30.0643 (4.3) Her-30.1165 (5.0) EC50 2.713e-011 1,081 e-010 Patent Application Publication Jan. 7, 2016 Sheet 4 of 7 US 2016/0002298 A1 Fig. 4 SKBR-3 120 110 100 10-13 10-12 10-11 10-10 10-9 10-8 10-7 10-6 Concentration (M) Her-30.0643 (4.4) • Her-30.10333.9 v Her-30.1036 (4.0) Her-30.0643 (4.4). Her-30.1033 (3.9 Her-30.1036 (4.0) EC50 1997e-011 1258e-011 3.271e-011 Patent Application Publication Jan. 7, 2016 Sheet 5 of 7 US 2016/0002298 A1 Fig. 5 UIMT-1 120 110 100 i 10-13 10-12 10-11 10-10 10-9 10-8 10-7 10-6 Concentration M Her-30.0643 (4.4 o Her-30.1033 (3.9 v Her-30.1036 (4.0) Her-30.0643 (4.4). Her-30.1033 (3.9 Her-30.1036 (4.0) EC50 1004e-009 7.912e-010 2.1.03e-009 Patent Application Publication Jan. 7, 2016 Sheet 6 of 7 US 2016/0002298 A1 Fig. 6 400 3SOa. 300 25 O 2 O 150 10-is SO single dose i.v. w8- PBS. were 30pug/kg Her-30.0643 -0-150 g/kg Her-30.0643 co 30g/kg. Her-30.1036 -- 150 g/kg Her-30.1036 Patent Application Publication Jan. 7, 2016 Sheet 7 of 7 US 2016/0002298 A1 Fig. 7 day 9, all animals dead 0 1 2 3 4 5 S 8 9, 10 1 12 13 14 15 Day -- PBS -0-300g/kg her-30.0643 --300g/kg Her-30.1036 US 2016/0002298 A1 Jan. 7, 2016 AMATOXIN DERVATIVES of these conjugates on the proliferation of breast cancer cells (cell line MCF-7), pancreatic carcinoma (cell line Capan-1), FIELD OF THE INVENTION colon cancer (cell line Colo205), and cholangiocarcinoma 0001. The invention relates to tumour therapy. In one (cell line OZ) were shown. aspect, the present invention relates to conjugates of an ama 0006 Structure activity relationship of amatoxins is toxin and a target-binding moiety, e.g. an antibody, connected reviewed in by Wieland (T. Wieland, Peptides of Poisonous by certain linkages, which are useful in the treatment of Amanita Mushrooms, Springer series in molecular biology, cancer and other disorders and diseases. Inafurther aspect the Springer Verlag New York, 1986). The hydroxyl group of invention relates to pharmaceutical compositions comprising amino acid 2 (hydroxy proline) and the Y-hydroxy group of Such conjugates. amino acid3 (dihydroxy-isoleucine) are assumed as essential for activity, whereas the functionalities at amino acid 1 (as BACKGROUND OF THE INVENTION partate or asparagine), amino acid 4 (6-hydroxy-tryptophan) 0002 Amatoxins are cyclic peptides composed of 8 amino and the 8-hydroxy-group at amino acid 3 are more tolerant for acids. They can, for example, be isolated from Amanita phal chemical modifications. This indicates that the latter posi loides mushrooms or prepared synthetically. Amatoxins spe tions are the preferred sites for linker attachment, while modi cifically inhibit the DNA-dependent RNA polymerase II of fications of the first ones should be avoided. mammalian cells, and thereby also the transcription and pro 0007. It is known that amatoxins are relatively non-toxic tein biosynthesis of the affected cells. Inhibition of transcrip when coupled to large biomolecule carriers. Such as antibody tion in a cell causes stop of growth and proliferation. Though molecules, and that they exert their cytotoxic activity only not covalently bound, the complex between amanitin and after the biomolecule carrier is cleaved off. In light of the RNA polymerase II is very tight (K, 3 nM). Dissociation of toxicity of amatoxins, particularly for liver cells, it is of out amanitin from the enzyme is a very slow process, thus making most importance that amatoxin conjugates for targeted recovery of an affected cell unlikely. When the inhibition of tumour therapy remain highly stable after administration in transcription lasts too long, the cell will undergo programmed plasma, and that the release of the amatoxin occurs after internalization in the target cells. In this context, minor cell death (apoptosis). improvements of the conjugate stability may have drastic 0003. The use of amatoxins as cytotoxic moieties for tumour therapy had already been explored in 1981 by cou consequences for the therapeutic window and the safety of the pling an anti-Thy 1.2 antibody to C-amanitin using a linker amatoxin conjugates for therapeutic approaches. attached to the indole ring of Trp (amino acid 4: see FIG. 1) OBJECTS OF THE INVENTION via diazotation (Davis & Preston, Science 1981, 213, 1385 1388). Davis & Preston identified the site of attachment as 0008. It was an object of the present invention to provide position 7". Morris & Venton demonstrated as well that sub further target-binding moiety amatoxin conjugates that are stitution at position 7" results in a derivative, which maintains stable in plasma, so that harmful side effects to non-target cytotoxic activity (Morris & Venton, Int. J. Peptide Protein cells are minimized. Res 1983, 21419-430). 0004 Patent application EP 1859811A1 (published Nov. SUMMARY OF THE INVENTION 28, 2007) described conjugates, in which the Y C-atom of 0009. The present invention is based on the unexpected amatoxin amino acid 1 of B-amanitin was directly coupled, observation that a ring formation via the two oxygen atoms i.e. without a linker structure, to albumin or to monoclonal bound to they and the ÖC atoms of amatoxinamino acid3 can antibody HEA125, OKT3, or PA-1. Furthermore, the inhibi improve the tolerability of target-binding moiety amatoxin tory effect of these conjugates on the proliferation of breast conjugates without interfering with the interaction of Such cancer cells (MCF-7), Burkitt's lymphoma cells (Raji), and amatoxins with their target, the DNA-dependent RNA poly T-lymphoma cells (Jurkat) was shown. The use of linkers was merase II of mammalian cells. To date, the presence of the Suggested, including linkers comprising elements such as hydroxyl group bound to the YC atom of amatoxinamino acid amide, ester, ether, thioether, disulfide, urea, thiourea, hydro 3 has been considered essential for the efficacy of amatoxins carbon moieties and the like, but no such constructs were and amatoxin conjugates. Wieland (T. Wieland, Peptides of actually shown, and no more details, such as attachment sites Poisonous Amanita Mushrooms, Springer Series in Molecu on the Amatoxins, were provided. lar Biology, Springer Verlag New York, 1986: Wieland T. 0005 Patent applications WO 2010/115629 and WO Rempel D, Gebert U, Buku A, Boehringer H. Uber die 2010/115630 (both published Oct. 14, 2010) describe conju Inhaltsstoffe des grinen Knollenblatterpilzes. XXXII. Chro gates, where antibodies, such as anti-EpCAM antibodies Such matographische Auftrennung der Gesamtgifte und Isolierung as humanized huHEA125, are coupled to amatoxins via (i) der neuen Nebentoxine Amanin und Phallisin sowie des the YC-atom of amatoxin amino acid 1, (ii) the 6' C-atom of ungiftigen Amanullins. Liebigs Ann Chem.