Development of a novel strategy for cancer therapy by combining chimeric adaptertoxins and triterpenoids investigated on mammary gland tumors.
Hematology, Oncology
Final Report Abstract
In the last decade targeted chimeric toxins have received an increasing interest for the treatment of metastasizing and inoperable tumors. By introducing a multi-functional molecular adapter into conventional targeted toxins we drastically reduced side-effects and concomitantly enhanced efficacy in both cell culture and a mammary gland cancer model in BALB/c mice. Before we began with the here reported project we demonstrated in vitro that a combined application with the saponins from Saponinum album (Spn) enhances the specific cytotoxicity on tumor target cells up to 385 000-fold whereas non-target cells are affected less than 1000-fold thus broadening the therapeutic window. Our objective thus was to get insights into the mechanism of the synergistic effect and to develop and optimize the combined application in a mouse tumor model. The use of the 131I- labeled targeted toxin SA2E – a chimera consisting of saporin, a molecular adapter and epidermal growth factor (EGF) – revealed a rapid transport of the protein through the body of the healthy mice into the urine. Within 1 h after s.c injection the amount of labeled SA2E in the skin is reduced by about 50% and at the same time the activity in the urine reached its maximum. Spn distributes within 10 min throughout the entire body of the mice and a strong accumulation of Spn in the urine was detectable after 30 min. No strong prevalence for tumor tissue or other organs was observed. The Spn/SA2E combination therapy significantly (p = 0.001) reduced the mean tumor volume from 465 mm3 in the mock-treated control to 29 mm3 in the Spn/SA2E group, a remarkable 94% reduction of tumor volume. While Spn alone had no effect on tumor growth, 0.1 µg SA2E alone reduced the mean tumor volume slightly by 42% compared to the control, which is very close to significance (p = 0.051) and, as expected, less than obtained with 5 µg SA2E (71% reduction). The anti-tumorigenic efficacy was higher after 60 min preincubation of Spn in comparison to 10 min. To analyze any non-visual side effects, blood plasma samples were collected at different time points and the following parameters quantified: alanine transaminase, aspartate transaminase, ferritin, glutamate dehydrogenase and cholinesterase 2 as predominantly liver-specific parameters, creatinine as kidney-specific parameter and interleukin-6 as inflammation-specific parameter. The liver-specific values showed an increase of about 2 to 3-fold after the first treatment with Spn/SA2E, indicating moderate liver damage. The same parameters except serum ferritin increased after treatment with Spn alone while SA2E alone did not affect these parameters. Five days after the last treatment all these values returned to normal levels for all treatments. After the third application the levels of cholinesterase 2 and interleukin-6 increased for the Spn/SA2E combination and returned to normal levels at the end of the experiment. Further studies suggest that saporin-based EGFR-targeted toxins specifically bind to the target receptor and are then moderately internalized by the cell via clathrin-independent pathways with no need of an acidic environment or microtubule-dependent transport. After preincubation with specific saponins, cellular uptake massively increases and this enhancement is clathrin-dependent and requires an acidic endosomal environment and formation of microtubules.
Publications
- A cleavable molecular adapter reduces side effects and concomitantly enhances efficacy in tumor treatment by targeted toxins in mice. J. Control. Release 117(3):342–350 (2007)
H. Fuchs, C. Bachran, T. Li, I. Heisler, H. Dürkop, M. Sutherland
- Patents on immunotoxins and chimeric toxins for the treatment of cancer. Recent Patents Drug Deliv. Formulation 1(2):105–115 (2007)
C. Bachran, M. Sutherland, D. Bachran, H. Fuchs
- Quantification of diphtheria toxin-mediated ADP-ribosylation in a solid phase assay. Clin. Chem. 53(9):1676–1683 (2007)
C. Bachran, M. Sutherland, D. Bachran, H. Fuchs
- Chimeric toxins inhibit growth of primary oral squamous cell carcinoma cells. Cancer Biol. Ther. 7(2):234–239 (2008)
C. Bachran, I. Heisler, D. Bachran, K. Dassler, M. F. Melzig, J. Ervens, H. Fuchs
- Der Nachweis von Diphtherietoxinaktivität durch einen hoch sensitiven colorimetrischen Festphasenassay. Dtsch. Ges. Klin. Chem. Mitt. 39(4+5):106–114 (2008)
C. Bachran, R. Urban, D. Bachran, R. Tauber, H. Fuchs
- Enhancement of saporin toxicity against U937 cells by Gypsophila saponins. J. Immunotoxicol. 5(3):287–292 (2008)
A. Weng, M. F. Melzig, C. Bachran, H. Fuchs
- Saponins in tumor therapy Mini-Rev. Med. Chem. 8(6):575–584 (2008)
C. Bachran, S. Bachran, M. Sutherland, D. Bachran, H. Fuchs
- Small cleavable adapters enhance the specific cytotoxicity of a humanized immunotoxin against CD64-positive acute myeloid leukemia cells. J. Immunother. 31(4):370–376 (2008)
C. Hetzel, C. Bachran, R. Fischer, H. Fuchs, S. Barth, M. Stöcker
- Soapwort saponins trigger clathrin-mediated endocytosis of saporin, a type I ribosome-inactivating protein. Chem. Biol. Interact. 176(2-3):204–211 (2008)
A. Weng, C. Bachran, H. Fuchs, M. F. Melzig
- A simple method for isolation of Gypsophila saponins for the combined application of targeted toxins and saponins in tumor therapy Planta Med. 75 (epub ahead of print) (2009)
A. Weng, D. Bachran, C. Görick, C. Bachran, H. Fuchs, M. F. Melzig
- Enhancement of saporin cytotoxicity by Gypsophila saponins – More than stimulation of endocytosis. Chem. Biol. Interact. 181(3):424–429 (2009)
A. Weng, C. Bachran, H. Fuchs, E. Krause, H. Stephanowitz, M. F. Melzig
- Improved immunotoxins with novel functional elements. Curr. Pharm. Des. 15(23):2700–2711 (2009)
C. Hetzel, C. Bachran, M. K. Tur, H. Fuchs, M. Stöcker
- Inhibition of tumor growth by targeted toxins in mice is dramatically improved by Saponinum album in a synergistic way. J. Immunother. 32(7):713–725 (2009)
C. Bachran, H. Dürkop, M. Sutherland, D. Bachran, C. Müller, A. Weng, M. F. Melzig, H. Fuchs
- Saponins as tool for improved targeted tumor therapies. Curr. Drug Targets 10(2):140–151 (2009)
H. Fuchs, D. Bachran, H. Panjideh, N. Schellmann, A. Weng, M. F. Melzig, M. Sutherland, C. Bachran
- Targeted tumor therapies at a glance. Curr. Drug Targets 10(2):89–93 (2009)
H. Fuchs, C. Bachran
- The Fabisch-Symposium 2009 on targeted tumor therapies Current Drug Targets 10(2):88 (2009)
C. Bachran, H. Fuchs
