• Polymer-dual drug conjugates targeted to HER2 overexpressing breast cancer

      Lee, Jun Hyoung; Nan, Anjan (2012)
      Current FDA approved treatments for Human epidermal growth factor receptor type 2 (HER2) overexpressing breast cancers include a humanized monoclonal antibody, Trastuzumab (TRZ) and small molecule tyrosine kinase inhibitors (TKIs). However, TRZ develops rapid drug resistance while TKIs and their metabolites cause hepatotoxicity due to nonselective distribution. To overcome these limitations of each agent it is essential to develop novel strategies to increase efficacy and reduce toxicity. This dissertation evaluates the potential of using conjugates of water soluble, N-(2-hydroxypropylmethacrylamide) (HPMA) copolymers with TRZ and TKI for targeted delivery of dual drugs to HER2 overexpressing tumors. Comb-like random and star-like semitelechelic (ST) HPMA copolymers (PHPMA) and their conjugates to TRZ were synthesized and characterized. Star-like conjugate system showed narrower size distribution, more potent and prolonged anticancer activity in HER2 overexpressing breast cancer cell lines (BT-474 and SK-BR-3) compared to comb-like conjugates. Therefore, the star-like conjugate system was selected to additionally conjugate a second anticancer agent for targeted combination drug delivery. Also fluorescence microscopy studies with Alexa488 labeled star-like conjugate demonstrated cellular internalization. The combination drug delivery system was successfully synthesized by covalently conjugating PKI166 (a model TKI) and TRZ to ST-PHPMA backbone. PKI166 release from the conjugate was observed in the presence of lysosomal enzyme cathepsin B. The polymer dual-drugs conjugate showed synergistic anticancer activity on HER2 overexpressing BT-474 and SK-BR-3 cells. The conjugate also exhibited prolonged anticancer activity up to 72 h by inhibiting the phosphorylation of MAPK and Akt when compared to free TRZ and free PKI166. The in vivo antitumor activity of the conjugates was evaluated in a pilot study in mice bearing SK-BR-3 tumor xenografts. The conjugates showed concentration dependent and synergistic antitumor activity over a 3 weeks treatment. The conjugates also showed no apparent indications of toxicity in non-tumor bearing mice. The results demonstrate the potential of HPMA copolymer based dual mode of action drugs conjugate as a novel combination drug delivery system that could i) prolong anticancer efficacy, ii) synergistically enhance anticancer effects of the drug combination and iii) reduce non-specific toxicity by way of cancer cell specific targeting and synergistic activity.
    • Repurposing Artemisinins for the Treatment of Acute Leukemias

      Fox, Jennifer Marie; Civin, Curt I.; 0000-0002-8651-2968 (2015)
      Artemisinins are endoperoxide-containing molecules derived from the natural compound artemisinin. Although best known as antimalarials, artemisinins also have potent antineoplastic activity. The purpose of this study was to characterize the in vitro anti-leukemic activity of artesunate (AS), a monomeric artemisinin derivative in worldwide use for malaria treatment, and ART-838, a novel artemisinin dimer. Both AS and ART-838 inhibited growth of all 23 tested acute leukemia cell lines, including several harboring mutations known to confer poor prognoses in patients (MLL rearrangement and FLT3 internal tandem duplication [FLT3/ITD]). ART-838 more potently induced cell cycle arrest, caspase-dependent apoptosis, and ROS generation, than did AS. Pretreatment with the antioxidant N-Acetyl-L-cysteine (NAC) or the superoxide scavenger Tiron abrogated AS-induced growth inhibition and apoptosis, demonstrating that ROS generation is an important component of the anti-leukemic mechanism of AS. While ART-838-induced apoptosis was reduced in the presence of the NAC, antioxidant pretreatment offered little to no protection against ART-838's antiproliferative effects, suggesting additional, ROS-independent, mechanisms. Though both ART-838 and AS synergized with current chemotherapy drugs to inhibit leukemia cell growth, ART-838 synergized with FLT3 inhibitors more effectively than did AS, further suggesting mechanistic differences between the monomer and the dimer. Despite over 20 years of study, the artemisinins' full antineoplastic mechanisms of action have yet to be elucidated. A synthetic lethal drug combination screen with AS/ART-838 and a library of targeted inhibitors was used to discover novel synergistic combinations and to identify novel anti-leukemic mechanisms of action of artemisinins, as well as potential mechanisms of resistance, including activation of compensatory signaling pathways. In the screen and in subsequent validation experiments AS or ART- 838 potently synergized with the BCL2 inhibitors ABT-199 and ABT-737, potentially due to MCL1/BCL2 downregulation and/or BAX upregulation by AS/ART-838. The potent anti-leukemic efficacy of AS and especially ART-838 supports further development of the artemisinins, alone and in combinations, to treat acute leukemias.