ojit Sur

Genetically defined subsets of human pancreatic cancer show unique in vitro chemosensitivity

Purpose: Pancreatic cancer is the fourth cause of death from cancer in the western world. Majority of patients present with advanced unresectable disease responding poorly to most chemotherapeutic agents. Chemotherapy for pancreatic cancer might be improved by adjusting it to individual genetic profiles. We attempt to identify genetic predictors of chemosensitivity to broad classes of anticancer drugs. Experimental Design: Using a panel of genetically defined human pancreatic cancer cell lines, we tested gemcitabine (antimetabolite), docetaxel (antimicrotubule), mitomycin C (MMC; alkylating), irinotecan (topoisomerase I inhibitor), cisplatin (crosslinking), KU0058948 (Parp1 inhibitor), triptolide (terpenoid drug), and artemisinin (control). Results: All pancreatic cancer cell lines were sensitive to triptolide and docetaxel. Most pancreatic cancer cells were also sensitive to gemcitabine and MMC. The vast majority of pancreatic cancer cell lines were insensitive to cisplatin, irinotecan, and a Parp1 inhibitor. However, individual cell lines were often sensitive to these compounds in unique ways. We found that DPC4/SMAD4 inactivation sensitized pancreatic cancer cells to cisplatin and irinotecan by 2- to 4-fold, but they were modestly less sensitive to gemcitabine. Pancreatic cancer cells were all sensitive to triptolide and 18% were sensitive to the Parp1 inhibitor. P16/CDKN2A-inactivated pancreatic cancer cells were 3- to 4-fold less sensitive to gemcitabine and MMC. Conclusions: Chemosensitivity of pancreatic cancer cells correlated with some specific genetic profiles. These results support the hypothesis that genetic subsets of pancreatic cancer exist, and these genetic backgrounds may permit one to personalize the chemotherapy of pancreatic cancer in the future. Further work will need to confirm these responses and determine their magnitude in vivo. Clin Cancer Res; 18(23); 6519–30. ©2012 AACR.

Systemic Delivery of Microencapsulated 3-Bromopyruvate for the Therapy of Pancreatic Cancer

Purpose: This study characterized the therapeutic efficacy of a systemically administered formulation of 3-bromopyruvate (3-BrPA), microencapsulated in a complex with β-cyclodextrin (β-CD), using an orthotopic xenograft mouse model of pancreatic ductal adenocarcinoma (PDAC). Experimental Design: The presence of the β-CD–3-BrPA complex was confirmed using nuclear magnetic resonance spectroscopy. Monolayer as well as three-dimensional organotypic cell culture was used to determine the half-maximal inhibitory concentrations (IC50) of β-CD–3-BrPA, free 3-BrPA, β-CD (control), and gemcitabine in MiaPaCa-2 and Suit-2 cell lines, both in normoxia and hypoxia. Phase-contrast microscopy, bioluminescence imaging (BLI), as well as zymography and Matrigel assays were used to characterize the effects of the drug in vitro. An orthotopic lucMiaPaCa-2 xenograft tumor model was used to investigate the in vivo efficacy. Results: β-CD–3-BrPA and free 3-BrPA demonstrated an almost identical IC50 profile in both PDAC cell lines with higher sensitivity in hypoxia. Using the Matrigel invasion assay as well as zymography, 3-BrPA showed anti-invasive effects in sublethal drug concentrations. In vivo, animals treated with β-CD–3-BrPA demonstrated minimal or no tumor progression as evident by the BLI signal as opposed to animals treated with gemcitabine or the β-CD (60-fold and 140-fold signal increase, respectively). In contrast to animals treated with free 3-BrPA, no lethal toxicity was observed for β-CD–3-BrPA. Conclusion: The microencapsulation of 3-BrPA represents a promising step towards achieving the goal of systemically deliverable antiglycolytic tumor therapy. The strong anticancer effects of β-CD–3-BrPA combined with its favorable toxicity profile suggest that clinical trials, particularly in patients with PDAC, should be considered. Clin Cancer Res; 20(24); 6406–17. ©2014 AACR.

Deregulation of energy metabolism promotes antifibrotic effects in human hepatic stellate cells and prevents liver fibrosis in a mouse model

Liver fibrosis and cirrhosis result from uncontrolled secretion and accumulation of extracellular matrix (ECM) proteins by hepatic stellate cells (HSCs) that are activated by liver injury and inflammation. Despite the progress in understanding the biology liver fibrogenesis and the identification of potential targets for treating fibrosis, development of an effective therapy remains elusive. Since an uninterrupted supply of intracellular energy is critical for the activated-HSCs to maintain constant synthesis and secretion of ECM, we hypothesized that interfering with energy metabolism could affect ECM secretion. Here we report that a sublethal dose of the energy blocker, 3-bromopyruvate (3-BrPA) facilitates phenotypic alteration of activated LX-2 (a human hepatic stellate cell line), into a less-active form. This treatment-dependent reversal of activated-LX2 cells was evidenced by a reduction in α-smooth muscle actin (α-SMA) and collagen secretion, and an increase in activity of matrix metalloproteases. Mechanistically, 3-BrPA-dependent antifibrotic effects involved down-regulation of the mitochondrial metabolic enzyme, ATP5E, and up-regulation of glycolysis, as evident by elevated levels of lactate dehydrogenase, lactate production and its transporter, MCT4. Finally, the antifibrotic effects of 3-BrPA were validated inxa0vivo in a mouse model of carbon tetrachloride-induced liver fibrosis. Results from histopathology & histochemical staining for collagen and α-SMA substantiated that 3-BrPA promotes antifibrotic effects inxa0vivo. Taken together, our data indicate that sublethal, metronomic treatment with 3-BrPA blocks the progression of liver fibrosis suggesting its potential as a novel therapeutic for treating liver fibrosis.

Abstract 1341: PRINT: A protein bioconjugation method with exquisite N-terminal specificity

The use of proteins and peptides for therapeutic applications are often compromised by low biological stability, high renal clearance, and non-optimal biodistribution. Chemical attachment of poly-(ethylene glycol) (PEGylation) is often considered the most effective way to improve these pharmacologic properties by increasing circulation half-life as well as reduced renal clearance, immunogenicity and protease mediated degradation. However, random conjugation results in heterogeneous derivatives with undefined composition and can substantially lower the bioactivity of the modified protein, leading to unpredictable in vivo behavior. Site-specific modification of proteins is therefore an attractive approach to circumvent the non-specificity resulting from random conjugation. We have developed a novel technique named PRINT (PRotect, INcise Tag) for N-terminal specific bioconjugation of proteins and peptides. Conceptually, PRINT can be performed on any protein that has any N-terminal tag for purification and a protease cleavage site following the tag. The recombinant protein is first treated with an excess of citraconic anhydride to reversibly block all reactive primary amine sites. Proteolytic cleavage then exposes only a single amine (the primary amine at the N-terminus) for desired bioconjugation by amine-reactive NHS ester chemistry. Lowering of reaction pH results in removal of the citraconates, leaving N-terminal specific mono PEGylated protein molecules. We used Tumor Necrosis Factor-α (TNF-α) as a model protein as it suffers from inherent instability and short biological half-life, and exhibits toxic side effects at therapeutic concentrations in both small animals and human patients. We demonstrate that PRINT results in a single product with exquisite selectivity and specificity in contrast to conventional reaction using the same NHS reagent, which was further confirmed by mass spectrometric analyses. Subsequent de-blocking generated an N-terminal protected TNF-α molecule with enhanced serum stability, superior pharmacokinetic properties, and reduced systemic toxicity. Importantly, N-terminal protection by PRINT did not affect the bioactivity of TNF-α. Existing site-selective bioconjugation approaches are either specific to amino acid tags or involve substantial non-trivial chemical or biotechnological manipulations to synthesize a desired bioconjugate. In contrast, PRINT employs ubiquitously used recombinant DNA techniques and easily acquired commercial reagents to generate exquisite N-terminal selective protection. We show that PRINT is a robust, reproducible and mild strategy which is able to target the α -amine and provide N-terminal specific protection to proteins or peptides that suffer from similar issues. We believe that this approach is strongly orthogonal to current methods and will be applicable to many biotherapeutics and bioprobes that are currently being designed to treat cancer. Citation Format: Surojit Sur, Yuan Qiao, Anja C. Fries, Robert N. O’Meally, Robert N. Cole, Kenneth W. Kinzler, Bert Vogelstein, Shibin Zhou. PRINT: A protein bioconjugation method with exquisite N-terminal specificity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1341.

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

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAnnMost 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.nnOur 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.nnFirst, we established that cyclodextrins labelled with a fluorescent moiety, but without a drug payload, accumulated in liposomes containing acidic citrate buffer with >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.nnWe 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 (>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.nnWe 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.nnUsing 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.nnMicroencapsulation 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.nnEncouraged 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.nnBecause 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.nnCitation 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