Development of Novel Peptide-Drug Conjugates for Targeting Brain Tumors and Brain Metastases



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Primary brain tumors and brain metastasis remain as big challenges in oncology. Glioblastoma multiforme (GBM) is the most aggressive diffuse glioma with a median survival less than one year. Patients with human epidermal growth factor receptor 2 (HER-2) positive breast cancer and triple-negative breast cancer (TNBC) are at higher risk of developing brain metastases compared to patients with other subtypes of breast cancer. Treatment of brain tumors is challenging partially due to the blood-brain barrier (BBB) and the blood-brain-tumor barrier (BBTB) by preventing sufficient delivery of effective therapeutics to brain. Lapatinib is an orally administered HER-2 tyrosine kinase inhibitor and has shown the potential for improving the outcomes of HER2+ breast cancer brain metastases. Irinotecan is the prodrug of anticancer drug SN-38 which stabilizes the topoisomerase I-DNA complex and inhibits the enzyme leading to DNA damaging. It is used as a first-line treatment for the advanced colorectal cancer. Despite their clinical use, there are several limitations of lapatinib and irinotecan in the treatment of breast cancer brain metastasis or brain tumors. High dose of lapatinib is required to reach therapeutic level intracranially to exert its efficacy which generally associates with severe diarrhea and hepatotoxicity. Irinotecan has dose-limiting toxicities such as severe diarrhea and large interindividual pharmacokinetic variability due to the variation in carboxylesterase activity. The active metabolite SN-38 itself is poorly water-soluble and chemically unstable, which prevented the development of parenteral formulations. Additionally, both lapatinib and irinotecan are the substrates of efflux transporters that abundantly expressed at the BBB, thus greatly limit the brain drug delivery and clinical use of lapatinib or irinotecan for the treatment of brain metastasis or brain tumors. The goal of this dissertation research is to develop peptide/peptoid-lapatinib and peptide/peptoid-SN38 conjugates to improve the delivery of lapatinib or SN-38 to the brain, and demonstrate antitumor efficacy in the breast cancer brain metastasis and glioblastoma mouse models. In this dissertation, we first developed Angiopep-2 peptide-based first-generation peptide-lapatinib conjugate. Angiopep-2 is a 19-amino acid peptide that is known to transport across the BBB via receptor-mediated transcytosis mechanism after binding to the low-density lipoprotein receptor-related protein-1 (LRP-1) receptor, which is overexpressed in the brain capillary endothelial cells. We synthesized and characterized three lapatinib-Angiopep-2 conjugates via three linkers including succinic anhydride, SPDP, and N-maleoyl-β-alanine. We also synthesized four poly(ethylene glycol) (PEG)-based lapatinib-PEG conjugates with different length of PEG to enhance the water solubility and brain delivery. All lapatinib conjugates were characterized by NMR, MALDI-TOF MS, and reversed phase HPLC. We compared the solubility, stability in PBS and human plasma, and in vitro cytotoxicity of conjugates in HER2+ breast cancer BT-474 and SK-Br-3 cell lines using free lapatinib as positive control. The brain distribution of lapatinib-Angiopep-2 conjugates was investigated in CD-1 mice after intravenous (IV) injection in comparison to free lapatinib at 30 min post injection. Lapatinib-Angiopep-2 conjugates showed greatly enhanced solubility (>25 mg/ml) compared with free lapatinib (7 µg/ml). One lapatinib-Angiopep-2 conjugate, and lapatinib-PEG conjugates showed good stability in both PBS pH 7.4 and human plasma. The IC50 values of lapatinib-Angiopep-2 and lapatinib-PEG conjugates decreased when compared to that of free lapatinib in breast cancer cells, suggesting the prodrug properties of the conjugates. However, the in vivo biodistribution evaluation of the IV bolus injection of lapatinib-Angiopep-2 conjugate showed that mouse brain concentration of released lapatinib was significantly less than that of free lapatinib at 30 min post injection, suggesting slow conversion of the conjugates to active lapatinib. In the second-generation of peptide-drug conjugates, SN-38 was conjugated to three different peptides including glutathione, L/D-Angiopep-2, and a peptoid TRAN-6. Three different types of linkers were developed for peptide/peptoid-SN-38 conjugate: stable ester chain with bulky group, pH-sensitive linker, and a PEG linker. We firstly validated the brain permeability and brain distribution of Cy5.5-labeled L-Angiopep-2 and Cy5.5-labeled TRAN-6 using in vitro transwell assay and in vivo whole-body fluorescence imaging. The cytotoxicity of each peptide/peptoid-SN-38 conjugate was evaluated in MDA-MB-231, JIMT-1, SK-Br-3-Luc, HCT-116, H460-Luc and U87-Luc cell using sulforhodamine B (SRB) assay. The peptide/peptoid-SN-38 conjugates displayed a comparable and potent cytotoxicity against all cells tested similar to free SN-38. Two sensitive HPLC-fluorescence (FL-HPLC) methods were developed to detect and quantify the free SN-38 and intact prodrugs (irinotecan or conjugates) using a single injection in mouse/human plasma matrix, and tissues to support the pharmacokinetics studies. The stability results demonstrated that the irinotecan stability is species dependent, and all the SN-38 conjugates can release SN-38 in the mouse or human plasma. Among all the tested conjugates, TRAN6-BCH-SN38 and D-Angiopep-BCH-SN38 were identified to be the lead conjugates with improved water solubility and good stability and were used for subsequent in vivo evaluations. The brain distribution of lead conjugates and release of SN-38 were investigated in CD-1 mice after IV injection of conjugates. The mouse brain intact conjugates and SN-38 active drug were quantified by FL-HPLC method. TRAN6-BCH-SN38 and D-Angiopep-BCH-SN38 conjugates displayed approximate 10-fold higher liberated SN-38 concentration than the liberated SN-38 generated from irinotecan at 10 min after IV administration at an equivalent SN-38 dose of 8 μmol/kg. We tested the antitumor efficacy of TRAN6-BCH-SN38 and irinotecan at 30 mg/kg (IV injection) in MDA-MB-231-Luc intracranial breast cancer brain metastasis mouse model and U87-luc glioblastoma mouse model. The anticancer efficacy results in both models have shown comparable median survival time for both TRAN6-BCH-SN38 and irinotecan. The result is significant because only 1/3 molar dose of TRAN6-BCH-SN38 (30 mg/kg, 15.5 μmol SN-38/kg) compared to that of irinotecan (30 mg/kg, 44.3 μmol SN-38/kg) can achieve similar effective tumor growth delay with less toxicity in aggressive brain metastasis and primary brain tumor mouse models. Our in vivo efficacy results suggest that peptide/peptoid-drug conjugate approach is a feasible and effective strategy to enhance brain delivery of anticancer drugs in preclinical mouse models. The TRAN6-BCH-SN38 conjugate has potential as an effective brain penetrating anticancer drug therapeutic. Further validation of brain targeting potential of TRAN6-BCH-SN38 conjugate is needed to translate this novel peptoid-drug conjugate for future therapeutic application. In summary, current research has provided quantitative results to support the feasibility of utilizing peptide-drug conjugates to effectively improve brain delivery of anticancer agents and enhance the anticancer efficacy in brain tumor models.



Primary brain tumors, Brain metastases, Blood-brain barrier, Peptide-drug conjugates, Receptor-mediated transcytosis


Portions of this document appear in: Zhou, Xue, Quentin R. Smith, and Xinli Liu. "Brain penetrating peptides and peptide–drug conjugates to overcome the blood–brain barrier and target CNS diseases." Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology 13, no. 4 (2021): e1695.