Abstract NG06: The Trojan Horse Strategy: Packaging chemotherapeutics can help alleviate toxicity (2015)

Abstract Most cancer drugs have a narrow therapeutic window where dose is dictated by toxicity and, in turn, causes decreased efficacy. Adverse side effects are the primary cause of early cessation of chemotherapy as well as high failure rates of new entities during clinical trials. This has led to intense research into various tumor-selective delivery modalities. We present here two novel strategies by which we reformulate promising cancer drug candidates and dramatically lower their toxic side effects. The first strategy involved development of a generalizable method for efficiently loading poorly soluble, non-ionizible organic molecules into stealth liposomes using a pH gradient. Using this new methodology we successfully encapsulated the PLK-1 inhibitor, BI-2536 and the MEK-1 inhibitor, PD-0325901, both of which had failed Phase II despite exceptional promise in preclinical and Phase I stages. In the second case we successfully packaged the potent anti-glycolytic alkylating agent, 3-bromopyruvate, previously used only under loco-regional therapy settings, for systemic therapy and demonstrated its exceptional promise in an orthotopic xenograft model of pancreatic cancer. Together, our data presented below demonstrate innovative strategies for opening up the therapeutic window of toxic cancer drugs. Our first approach uses drug-packaged stealth However, liposomal formulations have not gained widespread use because many drugs are highly hydrophobic and non-ionizable, and as such, cannot be efficiently loaded into liposomes through established passive or active techniques. To overcome these issues we designed a generalized strategy that have three important features: 1) pH gradient across liposome bilayer for directional loading of drugs, 2) use of β-cyclodextrins to create solubilize hydrophobic drugs regardless of the physico-chemical properties of the agents themselves, 3) synthetic analogs of β-cyclodextrins containing multiple weakly basic or weakly acidic functional groups on their solvent exposed surfaces to trap the cyclodextrin-drug complexes in liposomes, exploiting the ionizable groups on the cyclodextrins, rather than on the drugs themselves. First, we established that cyclodextrins labelled with a fluorescent moiety, but without a drug payload, accumulated in liposomes containing acidic citrate buffer with &amp;gt;90% loading efficiency compared to minimal accumulation in neutral liposomes at pH 7.4 without the pH gradient. These data suggests that the cyclodextrins can be transported across the lipid membrane and are trapped within the aqueous milieu. We next tested if the modified cyclodextrins could ferry and trap hydrophobic compounds within the liposome using common hydrophobic organic dyes (coumarins) to determine whether the modified cyclodextrins could ferry hydrophobic compounds across the liposome lipid bilayer. All cyclodextrin-coumarin complexes were incorporated into liposomes with high efficiency (&amp;gt;95%). In contrast, coumarins in the absence of cyclodextrins and unmodified cyclodextrin-coumarins complexes were poorly incorporated into liposomes under identical conditions. These results establish that ionizable cyclodextrins not only cross the lipid bilayer, they are able to carry a non-ionizable payload with them. We next investigated the ability of functionalized cyclodextrins to load into liposomes anti neoplastic drug candidates that had failed Phase II human trials. For this purpose we chose 1) BI-2536 (Boehringer Ingelheim), a highly selective inhibitor of PLK1, and 2) PD-0325901 (Pfizer), an allosteric MEK-1 inhibitor as our “rescue” candidates. BI-2536 was the subject of several clinical trials in patients with cancers of the lung, breast, ovaries, and uterus. Although BI-2536 showed evidence of efficacy in cancer patients, development was abandoned after Phase II trials revealed unacceptable toxicity (grade 4 neutropenia) at sub-therapeutic doses. Similarly, PD-0325901 was the subject of multiple clinical trials before being abandoned after Phase II trials due to retinal vein occlusion. We hypothesized that packing of these drugs in liposomes would reduce normal tissue exposure and mitigate the clinical toxicities observed. Using our new methodology we were able to reproducibly load both BI-2536-cyclodextrin and PD-0325901-cyclodextrin complexes into liposomes, achieving stable aqueous solutions containing 10 mg/ml and 5mg/ml of the drugs respectively. We, then, assessed their effects in nude mice bearing subcutaneous xenografts of colon cancer cells. The CYCL version of both drugs proved far superior to the corresponding free forms with respect to both toxicity and efficacy. While the free drugs exhibited both acute and delayed toxicity in our murine models, the CYCL-drugs did not ellicit any noticeable adverse reactions even at doses far greater than the MTD. In three different xenograft models, not only did equivalent doses of the CYCL forms exhibit better efficacy than the corresponding free drugs, but the CYCL forms resulted in partial regressions of tumors when administered at doses 2-4 times higher than the MTD of the free form. Moreover, no observable bone marrow toxicity resulted with single high dose of CYCL-BI-2536 while a single dose of free BI-2536 resulted in significant neutropenia in our xenograft model. Notably, this was one of the serious side-effects observed during the Phase II evaluation of this drug, leading to it eventual failure in Phase II. Our results demonstrate a novel, general strategy for loading hydrophobic drugs into liposomes. Importantly, the loaded liposomes exhibit substantially less toxicity and greater activity when tested in murine models of cancer. In our second approach, we explored the potential of selectively targeting the altered tumor metabolism in pancreatic cancers. We focused our attention on the well documented anti-glycolytic agent, 3-bromopyruvate, which can only be delivered by local injections or via tumor feeding arteries. The primary limitation in reaching the milestone of systemic deliverability is the reported toxicity due to its alkylating properties. In our effort to mitigate the toxicity we encapsulated 3-BrPA in anionic cyclodextrins alone and evaluated its efficacy in in vitro and in vivo models. Microencapsulation of 3 BrPA into cyclodextrins did not adversely affect the therapeutic efficacy of the agent in vitro against two different pancreatic cancer cells lines under hypoxic and normoxic conditions. 3D organotypic cell culture model using lucMiaPaCa-2 and Suit-2 cells demonstrated the ability of this novel complex to effectively penetrate the ECM rich environment, commonly observed in pancreatic cancers, and inhibit proliferation and induce apoptosis. In fact, treatment of the highly metastatic Suit-2 cells with CD-3BrPA showed visible reduction in cell protrusions. Encouraged by these promising in vitro results we attempted systemic delivery of these complexes. The median lethal dose of CD-3BrPA was determined to be double that of free 3BrPA. One-fourth of the LD50 was chosen as a safe dose for our orthotopic model of pancreatic cancer using lucMiaPaCa-2 cells surgically implanted at the tail of the pancreas. Microencapsulated 3BrPA demonstrated complete inhibition of tumor growth without eliciting any systemic and organ toxicities while the free form resulted in majority of animals succumbing to the therapy. In comparison, standard-of-care Gemcitabine resulted in only moderate deceleration of tumor growth. In summary, we demonstrated that packaging of 3-BrPA is a significant step forward in realizing systemically deliverable anti-glycolytic therapy with the reduction of toxicity paving the way for clinical trials in patients with pancreatic cancer and other malignancies. Because many of the most promising drugs developed today and in the past are relatively insoluble and exceedingly toxic, the approaches described here may be broadly applicable in mitigating those issues. These strategies therefore has the capacity to “rescue” drugs that fail at one of the last steps in the laborious and expensive process of drug development, allowing administration at higher doses and with less toxicity than otherwise obtainable. Citation Format: Surojit Sur, Julius Chapiro, Lynn J. Savic, Ganapathy S. Kaniappan, Juvenal Reyes, Raphael Duran, Sivarajan C. Thiruganasambandam, Cassandra R. Moats, Weibo Luo, Andrew J. Ewald, Shibin Zhou, Kenneth W. Kinzler, Jean-Francois Geschwind, Bert Vogelstein. The Trojan Horse Strategy: Packaging chemotherapeutics can help alleviate toxicity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr NG06. doi:10.1158/1538-7445.AM2015-NG06