CytRx

Elastin-like polypeptides (ELPs) undergo a characteristic phase transition in response to ambient temperature. Therefore, it has been be used as a thermosensitive vector for the delivery of chemotherapy agents since it can be used to target hyperthermic tumors. This novel strategy introduces unprecedented options for treating cancer with fewer concerns about side effects. In this study, the ELP system was further modified with an enzyme-cleavable linker in order to release drugs within tumors. This system consists of an ELP, a matrix metalloproteinase (MMP) substrate, a cell-penetrating peptide (CPP), and a 6-maleimidocaproyl amide derivative of doxorubicin (Dox). This strategy shows up to a 4-fold increase in cell penetration and results in more death in breast cancer cells compared to ELP-Dox. Even in doxorubicin-resistant cells (NCI/ADR and MES-SA/Dx5), ELP-released cell-penetrating doxorubicin demonstrated better membrane penetration, leading to at least twice the killing of resistant cells compared to ELP-Dox and free Dox. MMP-digested CPP-Dox showed better membrane penetration and induced more cancer cell death in vitro. This CPP-complexed Dox released from the ELP killed even Dox-resistant cells more efficiently than both free doxorubicin and non-cleaved ELP-CPP-Dox.

Abstract Introduction and objectives: Maytansine and its analogs (DM1 and DM4) are potent microtubule-targeting compounds that inhibit proliferation of cells during mitosis.1 Unfortunately, their narrow therapeutic window prevents a clinical application of these molecules. So far only T-DM1, an antibody-maytansinoid conjugate targeting the HER2 receptor, has been approved for the treatment of resistant breast cancer. Previous work on maytansinoids showed that their potent cytotoxic activity is related to the nature of the substituent at the C3 acyloxy side chain. In order to harness the potential of compounds of the maytansinoid family while diminishing dose-limiting side effects, we synthesized a library of novel analogs which can be attached to serum albumin in vivo through an acid-sensitive linker ensuring release of the active maytansinoid at the tumor site. All novel maytansine analogs contain a keto moiety as an attachment point for the linker and differ in their substitution pattern at C3. The analogs we studied can be classified into those containing an amino acid spacer between C3 and the keto group (maytansine-type) and those obtained through direct esterification at the C3-OH-position with various carboxylic acids (ansamitocin-type). Methods: All the newly synthesized molecules were screened for their in vitro cytotoxicity against 11 cancer cell lines. In order to explore the influence of the maytansine functional groups at C3, we designed novel analogs with different amino acid spacers at this position. We evaluated the influence of the chain length, the degree of steric hindrance, the need of the chiral center and the influence of the N-methyl on the cytotoxicity as well as stability in murine and human blood plasma. Moreover, we studied the structure-activity relationship of the ansamitocin-type of analogs including the influence of the chain length, the degree of steric hindrance and the effect of introducing different heteroatoms in the alkyl chain. Result and conclusion: A total of 32 new maytansinoid analogs were synthesized, and seven of them were found to be more potent than the parent drug maytansine in inhibiting the growth of human cancer cells in vitro. Clear SARs were identified for both classes of compounds. Based on these studies, lead compounds have been selected for creating albumin-binding derivatives and their further in vivo evaluation.2 1) W. C. Widdison et al, J. Med. Chem., 49: 4392-4408 (2006); K. A. Poon et al, Toxicology and Applied Pharmacology, 273: 298-313 (2013); H. L. Perez et al, Drug Discovery Today, 19:869-881 (2014) 2) Abstract "In vivo efficacy of novel acid-sensitive albumin-binding Maytansinoid-based prodrugs in human cancer xenograft models in nude mice" Citation Format: Friederike I. Nollmann, Patricia Perez Galan, Javier Garcia Fernandez, Heidi K. Walter, Johannes P. Magnusson, Federico Medda, Felix Kratz, Stephan D. Koester, Khalid Abu Ajaj, Lara Pes, Serghei Chercheja, Anna Warnecke. Structure-activity relationship studies and biological evaluation of novel maytansinoids, a class of highly selective tubulin inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1657.

Abstract Introduction and objectives: Maytansine and its analogs (DM1 and DM4) are potent microtubule-targeting compounds that inhibit proliferation of cells during mitosis. Their potent anticancer activity made them attractive for drug development.1 Unfortunately, their narrow therapeutic window prevents the clinical application of these molecules. So far only T-DM1, an antibody-maytansinoid conjugate targeting the HER2 receptor, has been approved for the treatment of Herceptin®-resistant breast cancer. Previous work2 showed that by direct esterification at the C3-OH-position of maytansinol, in vitro cytotoxicity may be enhanced compared to the parent drug maytansine. To make these highly potent compounds available for cancer treatment, novel maytansinoids were attached to a water-solubilizing hydrazone linker which selectively binds to the Cys-34 position of human serum albumin upon i.v. injection. The active drug is designed to be liberated in the acidic environment of the tumor as well as in acidic cellular compartments of tumor cells.3 Based on stability and release data in vitro, albumin-binding maytansinoids were selected for further in vivo evaluation. Herein, we present the data from a head-to-head comparison of novel albumin-binding maytansinoids with their parent maytansine in different cell- and patient-derived human tumor xenograft models. Methods: Patient-derived human tumor xenografts (LXFE937 etc.) or tumor cells (A2780, MDA-MB 231, MDA-MB 468 etc.) were transplanted subcutaneously to female NMRI nu/nu mice (n=7-8 per group). Therapy with maytansine (0.4 or 0.5 mg/kg) and albumin-binding maytansinoids (2-3 mg/kg maytansine eq) was initiated after tumors reached a mean volume of 80-380 mm3. Animals were treated by i.v. injection once per week over 4 weeks. Result and conclusion: Treatment with novel albumin-binding maytansinoids was better tolerated than with maytansine and MTDs were 4-7 times higher showing no significant body weight loss (< 5%). The albumin-binding maytansinoids demonstrated a significantly higher antitumor activity compared to both the control group and the group treated with maytansine and induced long-term partial and complete tumor regressions in all experiments. 1) W. C. Widdison et al, J. Med. Chem., 49: 4392-4408 (2006); K. A. Poon et al, Toxicology and Applied Pharmacology, 273: 298-313 (2013); H. L. Perez et al, Drug Discovery Today, 19:869-881 (2014) 2) Abstract XXX (Structure-activity relationship studies and biological evaluation of novel maytansinoids, a class of highly selective tubulin inhibitors) 3) F. Kratz et al., ChemMedChem, 3:20-53 (2008); US 7, 387, 771; F. Kratz, J. Control. Release, 132:171-183 (2008), F. Kratz, U. Beyer, Drug Delivery, 5: 281-299 (1998). Citation Format: Friederike I. Nollmann, Patricia Perez Galan, Javier Garcia Fernandez, Heidi K. Walter, Johannes P. Magnusson, Federico Medda, Felix Kratz, Stephan D. Koester, Khalid Abu Ajaj, Lara Pes, Serghei Chercheja, Anna Warnecke. Novel albumin-binding maytansinoids inducing long-term partial and complete tumor regressions in several human cancer xenograft models in nude mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2661.

Abstract Auristatins are highly cytotoxic antimitotic tubulin-binding peptides. Of this family, only Adcetris®, an antibody drug conjugate (ADC) derived from monomethyl auristatin E (MMAE), is approved and marketed. Other auristatins such as dolastatin 10, dolastatin 15, and auristatin PE reached phase 1 and 2 clinical trials, but due to systemic toxicity and low antitumor activity they were discontinued.1 Because of the high cytotoxicity, it is important to deliver these drugs selectively to the tumor avoiding premature release in the blood circulation. We developed an acid-sensitive drug delivery system that uses circulating endogenous serum albumin as a macromolecular carrier. The drug is rapidly bound covalently to albumin upon i.v. injection and, following accumulation in the tumor due to enhanced uptake and retention mechanisms, the highly cytotoxic agent is released in a pH-dependent manner.2 Herein we present in-vivo data of the considerably improved efficacy of two auristatin E derived prodrugs, AE-Keto-Sulf07 and AE-Ester-Sulf07, compared to the parent compound auristatin E. The acid-sensitive hydrazone prodrugs were prepared from the respective carbonyl-containing auristatin E derivatives and the water-solubilizing maleimide-bearing linker Sulf07. Female NMRI nu/nu mice were inoculated subcutaneously with patient- or cell-derived human tumor xenografts (A375, A2780, RXF631, LXFA737) and randomized (n=7-8 per group) with starting tumor volumes in the range of 140-350 mm3. AE-Keto-Sulf07 showed excellent antitumor response over a wide dose range (3.0-6.5 mg/kg twice per week over 3-4 weeks), with optimal dosage at 4.5 mg/kg twice per week over 4 weeks. AE-Ester-Sulf07 was highly efficacious between 1.9 and 2.4 mg/kg dosed twice per week over 3-4 weeks or at 3.8 mg/kg dosed once per week over 4 weeks, but caused cumulative skin lesions due to scratching and biting. In contrast, auristatin E was dosed at 0.3 mg/kg twice per week over 3-4 weeks and was only marginally active. In summary, we have shown for the first time that auristatin E derivatives, namely AE-Keto-Sulf07 and AE-Ester-Sulf07, bound to circulating albumin demonstrate promising antitumor efficacy and induce statistically significant long-term partial or complete remission in a panel of human xenograft models in mice. Citation Format: Stephan D. Koester, Lara Pes, Johannes P. Magnusson, Serghei Chercheja, Federico Medda, Friederike I. Nollmann, Patricia Perez Galan, Javier Garcia Fernandez, Heidi-Kristin Walter, Khalid Abu Ajaj, Anna Warnecke, Felix Kratz. Superior efficacy of novel albumin-binding auristatin E-based prodrugs compared to auristatin E in a panel of human xenograft models in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3703.

Glioblastoma (GBM) ranks among the most common, aggressive, and least curable cancers due to a strong tendency for intracranial dissemination, high proliferation potential and inherent tumor resistance to radiation or chemotherapy. Current treatments face the critical challenge of adverse treatment effects in normal tissue within the brain itself and also when the agent cannot adequately penetrate the blood brain barrier (BBB) to reach the tumor microenvironment. We have developed a system to selectively deliver chemotherapeutic doxorubicin to GBM tumors. This carrier is based on elastin-like polypeptide (ELP), which is soluble at physiological temperatures but undergoes a phase transition and accumulates at tumor sites with externally applied, mild (40-41°C) hyperthermia. The CPP-ELP-Dox conjugate consists of a cell penetrating peptide (CPP), which facilitates transcytosis through the BBB and cell entry, and the 6-maleimidocaproyl hydrazone derivative of doxorubicin (Dox) at the C-terminus of ELP. The acid-sensitive hydrazone linker ensures release of Dox in the lysosomes/endosomes after cellular uptake of the drug conjugate. We have shown that CPP-ELP-Dox effectively inhibits cell proliferation in three GBM cell lines; GBM6, D54, and U251 with IC50 values of 250 nM, 60nM, and 30nM, respectively. Both the free drug and CPP-ELP-Dox conjugate exhibited similar in vitro cytotoxicity, although their subcellular localization was considerably different. The Dox conjugate was mainly dispersed in the cytoplasm, while free drug was largely dispersed in the cytoplasm but also had partial nuclear accumulation. The accumulation of free Dox in GBM cells was measured by flow cytometry to determine whether the cytotoxic activity of free Dox or CPP- ELP-Dox is related to intracellular Dox levels. Intracellular Dox concentration was increased in the CPP-ELP-Dox cells compared to that in the free Dox cells, which positively correlates with cytotoxic activity. Flow cytometry was also used to quantitatively investigate apoptosis of GBM cells. After incubation for 24 h, the total apoptosis percentage of free Dox was 1.5% in GBM6, 6% in D54, and 7% in U251 cells. However, the total apoptosis rates of CPP-ELP-Dox treated cells were 5%, 12% and 5% in GBM6, D54, and U251 cells respectively, which confirms increased apoptosis compared to free Dox treated cells. Cell cycle analysis of GBM cells treated with free Dox showed enhanced arrest of the cells in G1 phase, while there was increased percentage arrest of cells in G2 phase in cells treated with CPP-ELP-Dox.In summary, our findings demonstrate that CPP-ELP-Dox effectively kills GBM cells. Development of such a drug carrier has the potential to greatly improve current therapeutic approaches for GBM by increasing the specificity and efficacy of treatment and reducing cytotoxicity in normal tissues. Citation Format: Sonja Dragojevic, Rebecca B. Mackey, Felix Kratz, Drazen Raucher. Targeted delivery of doxorubicin to glioblastomas by thermally sensitive polypeptides [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3708.