Dragojevic, Sonja

The anticancer agent doxorubicin(dox) has been widely used in the treatment of a variety of hematological malignancies and solid tumors. Despite doxorubicin’s efficiency in killing tumor cells, severe damage to healthy tissues, along with cardiotoxicity, limits its clinical use. To overcome these adverse side effects, improve patient safety, and enhance therapeutic efficacy, we have designed a thermally responsive biopolymer doxorubicin carrier that can be specifically targeted to tumor tissue by locally applying mild hyperthermia (41 °C). The developed drug vehicle is composed of the following: a cell penetrating peptide (SynB1) to promote tumor and cellular uptake; thermally responsive Elastin-like polypeptide (ELP); and the (6-maleimidocaproyl) hydrazone derivative of doxorubicin (DOXO-EMCH) containing a pH-sensitive hydrazone linker that releases doxorubicin in the acidic tumor environment. We used the in vivo imaging system, IVIS, to determine biodistribution of doxorubicin-delivered ELP in MDA-MB-231 xenografts in nude mice. Tumor bearing mice were treated with a single IV injection of 10 mg/kg doxorubicin equivalent dose with free doxorubicin, thermally responsive SynB1 ELP 1-DOXO, and a thermally nonresponsive control biopolymer, SynB1 ELP 2-DOXO. Following a 2 h treatment with hyperthermia, tumors showed a 2-fold higher uptake when treated with SynB1 ELP 1-DOXO compared to free doxorubicin. Accumulation of the thermally non-responsive control SynB1 ELP2 –DOXO was comparable to free doxorubicin, indicating that an increase in dox accumulation with ELP is due to aggregation in response to thermal targeting. Higher levels of SynB1 ELP1–DOXO and SynB1 ELP2 –DOXO with respect to free doxorubicin were observed in kidneys. Fluorescence intensity from hearts of animals treated with SynB1 ELP1–DOXO show a 5-fold decrease in accumulation of doxorubicin than the same dose of free doxorubicin. SynB1-ELP1-DOXO biopolymers demonstrated a 6-fold increase in tumor/heart ratio in comparison to free doxorubicin, indicating preferential accumulation of the drug in tumors. These results demonstrate that thermally targeted polymers are a promising therapy to enhance tumor targeting and uptake of anticancer drugs and to minimize free drug toxicity in healthy tissues, representing a great potential for clinical application.

Although doxorubicin (dox), an anthracycline antibiotic, is widely used and effective in treating cancer, its treatment efficiency is limited by low blood plasma solubility, poor pharmacokinetics, and adverse side effects, including irreversible cardiotoxicity. Moreover, cancer cells often develop drug resistance over time, which decreases the efficacy of anti-cancer drugs, including dox. In this study, we examine a macromolecular drug delivery system for its ability to specifically deliver doxorubicin to cancer cells with and without drug resistance. This drug delivery system consists of a multi-part macromolecule, which includes the following: elastin-like polypeptide (ELP), cell penetrating peptide (CPP), a cleavable linker (releasing at low pH), and a derivative of doxorubicin. ELP is thermally responsive and improves drug solubility, while the CPP mediates cellular uptake of macromolecules. We compared cytotoxicity of two doxorubicin derivatives, where one is cleavable (DOXO) and contains a pH-sensitive linker and releases dox in an acidic environment, and the other is non-cleavable (ncDox) doxorubicin. Cytotoxicity, apoptosis, cell cycle distribution and mechanism of action of these constructs were tested and compared between dox-responsive MCF-7 and dox-resistant NCI/ADR cell lines. Dox delivered by the ELP construct is comparably toxic to both sensitive and drug resistant cell lines, compared to unconjugated doxorubicin, and given the pharmacokinetic and targeting benefits conveyed by conjugation to ELP, these biopolymers have potential to overcome dox resistance in vivo.

Abstract Radio-resistance mechanisms limit the benefit of radiation therapy (RT) for melanoma brain metastases. A key pathway for radiation-induced DNA double-strand break repair is non-homologous end joining, which is critically mediated by DNA-dependent protein kinase (DNA-PKcs). Here we evaluated radio-sensitizing effects of M3814, a selective oral inhibitor of DNA-PKcs, in patient-derived xenografts (PDXs) of melanoma brain metastases. In a clonogenic survival assay, M3841 augmented RT-induced killing of M12 cells at concentrations of ≥300 nM, and a minimum of 16 h exposure with ~300 nM M3814 was required for effective sensitization. M3814 inhibited RT-induced (5 Gy) auto-phosphorylation of serine-2056 of DNA-PKcs in primary cultures of M12, M15 and M27 PDX lines. Interestingly, inhibition of RT-induced DNA-PKcs by M3814 coincided with increased KAP1 phosphorylation, a DNA damage signaling regulated via ATM. Persistent γH2AX foci were observed in 28% M12 cells at 24 hours after co-treatment with M3814 and RT as compared to 12% cells following RT alone. In vivo pharmacokinetic analysis after single oral dose of 20 mg/kg M3814, showed reasonably short half-life (~2.44 hours) and poor brain distribution in wild-type FBV mice (Kpuu, 0.027). Consistent with an efflux liability, brain distribution of M3814 in triple knockout mice for BCRP/MDR1A/B was ~11 fold higher (Kpuu, 0.215). Compared to normal brain, much higher M3814 concentrations were detected in intracranially implanted M12 tumors (~23 fold and ~20 fold) 2 and 6 hours after a single oral dose of 50mg/kg respectively. The relative exclusion of M3814 from normal brain as compared to brain metastases suggests that this drug may have a favorable toxicity profile when combined with radiation for treatment of melanoma brain metastases, and this hypothesis is being tested in ongoing efficacy studies.

AbstractThe application of rationally designed therapeutic peptides (TP) may improve outcomes in cancer treatment. These peptides hold the potential to directly target proliferative pathways and stimulate cell arrest or death pathways. Elastin‐like polypeptide (ELP) is an elastin derived biopolymer that undergoes a thermally mediated phase transition. This study employs p50, a nuclear localization sequence derived peptide that inhibits the activation of NFκB and is implicated in cancer cell survival and metastasis. In order to effectively delivery p50, it is conjugated to SynB1‐ELP1, a thermally responsive macromolecular carrier. By applying an external heat source, mild hyperthermic conditions (41 °C) induce aggregation and therefore can be used to specifically target ELP to solid tumors in cancer therapy. The addition of a cell penetrating peptide (CPP) to the N‐terminus of the macromolecular carrier enhances the cellular uptake and directs the subcellular localization of the bioactive peptide. The novel TP, p50, inhibits proliferation and induces apoptosis of breast cancer cells by blocking the intranuclear import of NFκB. By expanding the repertoire of oncogenic targets, CPPs, and ELP carrier sizes, ELP‐based polypeptides may be modulated to optimize the delivery of these novel therapies and allow for the flexibility to create individualized cancer therapies.