Dose-limiting Gastrointestinal Toxicity Research Articles

Therapeutic resurgence of 6-diazo-5-oxo-l-norleucine (DON) through tissue-targeted prodrugs.

The recognition that rapidly proliferating cancer cells rely heavily on glutamine for their survival and growth has renewed interest in the development of glutamine antagonists for cancer therapy. Glutamine plays a pivotal role as a carbon source for synthesizing lipids and metabolites through the TCA cycle, as well as a nitrogen source for synthesis of amino acid and nucleotides. Numerous studies have explored the significance of glutamine metabolism in cancer, providing a robust rationale for targeting this metabolic pathway in cancer treatment. The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) has been explored as an anticancer therapeutic for nearly six decades. Initial investigations revealed remarkable efficacy in preclinical studies and promising outcomes in early clinical trials. However, further advancement of DON was hindered due to dose-limiting gastrointestinal (GI) toxicities as the GI system is highly dependent on glutamine for regulating growth and repair. In an effort to repurpose DON and mitigate gastrointestinal (GI) toxicity concerns, prodrug strategies were utilized. These strategies aimed to enhance the delivery of DON to specific target tissues, such as tumors and the central nervous system (CNS), while sparing DON delivery to normal tissues, particularly the GI tract. When administered at low daily doses, optimized for metabolic inhibition, these prodrugs exhibit remarkable effectiveness without inducing significant toxicity to normal tissues. This approach holds promise for overcoming past challenges associated with DON, offering an avenue for its successful utilization in cancer treatment.

Discovery of DRP-104, a tumor-targeted metabolic inhibitor prodrug.

6-Diazo-5-oxo-l-norleucine (DON) is a glutamine antagonist that suppresses cancer cell metabolism but concurrently enhances the metabolic fitness of tumor CD8+ T cells. DON showed promising efficacy in clinical trials; however, its development was halted by dose-limiting gastrointestinal (GI) toxicities. Given its clinical potential, we designed DON peptide prodrugs and found DRP-104 [isopropyl(S)-2-((S)-2-acetamido-3-(1H-indol-3-yl)-propanamido)-6-diazo-5-oxo-hexanoate] that was preferentially bioactivated to DON in tumor while bioinactivated to an inert metabolite in GI tissues. In drug distribution studies, DRP-104 delivered a prodigious 11-fold greater exposure of DON to tumor versus GI tissues. DRP-104 affected multiple metabolic pathways in tumor, including decreased glutamine flux into the TCA cycle. In efficacy studies, both DRP-104 and DON caused complete tumor regression; however, DRP-104 had a markedly improved tolerability profile. DRP-104's effect was CD8+ T cell dependent and resulted in robust immunologic memory. DRP-104 represents a first-in-class prodrug with differential metabolism in target versus toxicity tissue. DRP-104 is now in clinical trials under the FDA Fast Track designation.

Inflammatory-Dependent Bidirectional Effect of Bile Acids on NLRP3 Inflammasome and Its Role in Ameliorating CPT-11-Induced Colitis.

Irinotecan (CPT-11) in combination with 5-fluorouracil and leucovorin is a first-line chemotherapy regimen for the treatment of colorectal cancer; however, its clinical application is limited by the dose-limiting gastrointestinal toxicity of colitis. In our previous studies, several bile acids (BAs) were found significantly elevated in the colon of the CPT-11-induced rat colitis model. On the other hand, NLRP3 inflammasome has been reported to play important roles in mediating colitis. Interestingly, BA was stated to activate the NLRP3 inflammasome in some studies, while in some other reports, it showed an inhibitory effect. We assumed that the inflammatory status in different circumstances might have contributed to the controversial findings. In this study, we first discovered, under non-inflammatory conditions, that supplementing BA could activate the NLRP3 inflammasome in THP-1-differentiated macrophages and promote inflammation. In lipopolysaccharide (LPS)-induced inflammatory macrophages, however, BA inhibited the NLRP3 inflammasome and reduced inflammation. Further experiments demonstrated that Takeda G protein-coupled receptor 5 (TGR5) is essential in mediating the inhibitory effect of BA, while phospho-SP1 (p-SP1) is key to the activation. Furthermore, we applied the above findings to ameliorate CPT-11-caused colitis in rats by inhibiting SP1 with mithramycin A (MitA) or activating TGR5 using oleanolic acid (OA). Our findings may shed light on the discovery of effective interventions for reducing dose-limiting chemotherapy-induced colitis.

Quantitative Investigation of Irinotecan Metabolism, Transport, and Gut Microbiome Activation.

The anticancer drug irinotecan shows serious dose-limiting gastrointestinal toxicity regardless of intravenous dosing. Although enzymes and transporters involved in irinotecan disposition are known, quantitative contributions of these mechanisms in complex in vivo disposition of irinotecan are poorly understood. We explained intestinal disposition and toxicity of irinotecan by integrating 1) in vitro metabolism and transport data of irinotecan and its metabolites, 2) ex vivo gut microbial activation of the toxic metabolite SN-38, and 3) the tissue protein abundance data of enzymes and transporters relevant to irinotecan and its metabolites. Integration of in vitro kinetics data with the tissue enzyme and transporter abundance predicted that carboxylesterase (CES)-mediated hydrolysis of irinotecan is the rate-limiting process in the liver, where the toxic metabolite formed is rapidly deactivated by glucuronidation. In contrast, the poor SN-38 glucuronidation rate as compared with its efficient formation by CES2 in the enterocytes is the key mechanism of the intestinal accumulation of the toxic metabolite. The biliary efflux and organic anion transporting polypeptide-2B1-mediated enterocyte uptake can also synergize buildup of SN-38 in the enterocytes, whereas intestinal P-glycoprotein likely facilitates SN-38 detoxification in the enterocytes. The higher SN-38 concentration in the intestine can be further nourished by β-d-glucuronidases. Understanding the quantitative significance of the key metabolism and transport processes of irinotecan and its metabolites can be leveraged to alleviate its intestinal side effects. Further, the proteomics-informed quantitative approach to determine intracellular disposition can be extended to determine susceptibility of cancer cells over normal cells for precision irinotecan therapy. SIGNIFICANCE STATEMENT: This work provides a deeper insight into the quantitative relevance of irinotecan hydrolysis (activation), conjugation (deactivation), and deconjugation (reactivation) by human or gut microbial enzymes or transporters. The results of this study explain the characteristic intestinal exposure and toxicity of irinotecan. The quantitative tissue-specific in vitro to in vivo extrapolation approach presented in this study can be extended to cancer cells.

Notch Signaling in Breast Cancer: A Role in Drug Resistance.

Breast cancer is a heterogeneous disease that can be subdivided into unique molecular subtypes based on protein expression of the Estrogen Receptor, Progesterone Receptor, and/or the Human Epidermal Growth Factor Receptor 2. Therapeutic approaches are designed to inhibit these overexpressed receptors either by endocrine therapy, targeted therapies, or combinations with cytotoxic chemotherapy. However, a significant percentage of breast cancers are inherently resistant or acquire resistance to therapies, and mechanisms that promote resistance remain poorly understood. Notch signaling is an evolutionarily conserved signaling pathway that regulates cell fate, including survival and self-renewal of stem cells, proliferation, or differentiation. Deregulation of Notch signaling promotes resistance to targeted or cytotoxic therapies by enriching of a small population of resistant cells, referred to as breast cancer stem cells, within the bulk tumor; enhancing stem-like features during the process of de-differentiation of tumor cells; or promoting epithelial to mesenchymal transition. Preclinical studies have shown that targeting the Notch pathway can prevent or reverse resistance through reduction or elimination of breast cancer stem cells. However, Notch inhibitors have yet to be clinically approved for the treatment of breast cancer, mainly due to dose-limiting gastrointestinal toxicity. In this review, we discuss potential mechanisms of Notch-mediated resistance in breast cancer cells and breast cancer stem cells, and various methods of targeting Notch through γ-secretase inhibitors, Notch signaling biologics, or transcriptional inhibitors. We also discuss future plans for identification of novel Notch-targeted therapies, in order to reduce toxicity and improve outcomes for women with resistant breast cancer.

Preclinical toxicology profile of squalene epoxidase inhibitors

Small cell lung cancer (SCLC) is a particularly aggressive subset of lung cancer, and identification of new therapeutic options is of significant interest. We recently reported that SCLC cell lines display a specific vulnerability to inhibition of squalene epoxidase (SQLE), an enzyme in the cholesterol biosynthetic pathway that catalyzes the conversion of squalene to 2,3-oxidosqualene. Since it has been reported that SQLE inhibition can result in dermatitis in dogs, we conducted a series of experiments to determine if SQLE inhibitors would be tolerated at exposures predicted to drive maximal efficacy in SCLC tumors. Detailed profiling of the SQLE inhibitor NB-598 showed that dogs did not tolerate predicted efficacious exposures, with dose-limiting toxicity due to gastrointestinal clinical observations, although skin toxicities were also observed. To extend these studies, two SQLE inhibitors, NB-598 and Cmpd-4″, and their structurally inactive analogs, NB-598.ia and Cmpd-4″.ia, were profiled in monkeys. While both active SQLE inhibitors resulted in dose-limiting gastrointestinal toxicity, the structurally similar inactive analogs did not. Collectively, our data demonstrate that significant toxicities arise at exposures well below the predicted levels needed for anti-tumor activity. The on-target nature of the toxicities identified is likely to limit the potential therapeutic utility of SQLE inhibition for the treatment of SCLC.

Abstract NT-110: EFFICACY OF NOVEL TOPOISOMERASE 1 INHIBITORS AND THEIR SYNERGY WITH PARP INHIBITORS IN A MURINE MODEL FOR OVARIAN CANCER

Abstract Camptothecin derivatives topotecan and irinotecan are currently the only FDA-approved topoisomerase I (Top1) inhibitors. Despite their wide usage they have many drawbacks: chemical instability, short half-life, dose-limiting bone marrow and gastrointestinal toxicity, and they are drug efflux substrates. The novel non-camptothecin Top1 inhibitors, indenoisoquinolines (LMP400, LMP776 and LMP744), have been developed to overcome these limitations. We explored whether treatment with LMPs could induce “synthetic lethality” in cells with impaired homologous recombination (HR) repair mechanism and whether it could be enhanced by combining them with PARP inhibitor, olaparib. Using the DT40 cell line, we assessed in vitro the role of HR in the cellular responses to the LMPs. We found that BRCA1-, BRCA2- and PALB2-deficient cells are 3 to 5 times more sensitive to the LMP compounds than wild type cells. Moreover, combination treatments showed a significant synergy between each of the three LMPs and olaparib. To confirm the differential response of Brca1 wild type or Brca1 deficient cells to LMP treatment we assessed compound potency in primary ovarian cancer cell lines derived from a genetically engineered mouse (GEM) model for serous epithelial ovarian cancer (SEOC). As in DT40 cells, we observed that murine Brca1 deficient cells were more sensitive to all 3 LMP compounds than Brca1 wild type cells. Our results suggest that Brca1 deficiency renders cells more sensitive to treatment with LMPs or combination of LMPs and olaparib, potentially directing selection of patients with impaired HR repair mechanism in the phase 2 clinical trials that are in preparation. To confirm this observation in vivo, we are performing efficacy studies using LMP compounds alone or in combination with olaparib in orthotopic allograft mouse models for ovarian cancer derived from the GEM model. In Brca1 deficient tumors, treatment with each of the 3 LMP compounds led to significant increase of survival compared to vehicle treated group. LMP400 had the greatest beneficial impact on survival and tumor regression. To confirm synergy between LMPs and olaparib we selected LMP400 to represent the LMPs and repeated the efficacy study using Brca1 deficient tumor model. We observed that suboptimal doses of LMP400 or olaparib used as single treatments resulted in some increase in survival, however, when combined they significantly increased survival due to tumor regression. Our data demonstrate that the LMPs synergize with olaparib and provide a rationale for personalized treatment and Phase 2 clinical trials with the indenoisoquinolines in combination with PARP inhibitors in HR-deficient ovarian cancers. Citation Format: Ludmila Szabova, Melanie Gordon, Laetitia Matzi, Yves Pommier, Zoe Weaver Ohler. EFFICACY OF NOVEL TOPOISOMERASE 1 INHIBITORS AND THEIR SYNERGY WITH PARP INHIBITORS IN A MURINE MODEL FOR OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-110.

Abstract 365: Indotecan (LMP400), indimitecan (LMP776) and LMP744, a new class of non-camptothecin topoisomerase I inhibitors selective for schlafen11-positive and BRCA-deficient cells that synergize with olaparib

Abstract To overcome the limitations of topoisomerase inhibitors, irinotecan and topotecan (chemical instability, drug efflux substrates, short half-life, dose-limiting bone marrow and gastrointestinal toxicity), we have developed the indenoisoquinolines (LMP400, LMP776 and LMP744). To rationally select patients for phase 2 clinical trials, we have taken two approaches. First, we mined the cancer cell lines genomic databases with CellMinerCDB and found that a dominant response determinant to the indenoisoquinolines is Schlafen 11 (SLFN11), a recently identified executor of cells undergoing replication stress. We validated this finding in isogenic cell lines. Second, because the major lesions generated by the trapping of TOP1 are replication-induced DNA double-strand breaks that are repaired by homologous recombination (HR), we determined whether the indenoisoquinolines exhibit a “synthetic lethality” in cells presenting BRCA1, BRCA2 or PALB2 deficiency. In addition, we tested wether this selectivity could be enhanced when combined with the PARP inhibitor, olaparib. Survival and cell cycle alterations were tested after treatment with the indenoisoquinolines as single agents in isogenic DT40, DLD1 and OVCAR cell lines, with BRCA1, BRCA2 or PALB2 deficiencies, and organoids cultured from patient-derived xenografts with BRCA2 loss, as well as in combination with olaparib. We found that BRCA1-, BRCA2- and PALB2-deficient cells are 3 to 5 times more sensitive to the indenoisoquinolines compared to isogenic cell lines. Moreover, combination treatments showed high synergy between all three indenoisoquinolines and olaparib. We also established the synergy between LMP400 (Indotecan) and olaparib in orthotopic allograft models derived from genetically engineered mouse models for serous epithelial ovarian cancer harboring BRCA1 loss. Better efficacy was observed with the combination over single agent treatments of LMP400 or olaparib. Our results provide a rationale for Phase 2 indenoisoquinoline clinical trials with the indenoisoquinolines in HR-deficient cancers as single agents and in combination with PARP inhibitors, and for measuring Schlafen 11 (SLFN11) as a clinical response determinant. Citation Format: Laetitia Marzi, Ludmila Szabova, Zoe Weaver Ohler, Shyam Sharan, Mike Beshiri, Junko Murai, Kathy Kelly, Yves Pommier. Indotecan (LMP400), indimitecan (LMP776) and LMP744, a new class of non-camptothecin topoisomerase I inhibitors selective for schlafen11-positive and BRCA-deficient cells that synergize with olaparib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 365.

Preclinical Pharmacokinetics of Fosciclopirox, a Novel Treatment of Urothelial Cancers, in Rats and Dogs

Pharmacokinetic studies in rats and dogs were performed to characterize the in vivo performance of a novel prodrug, fosciclopirox. Ciclopirox olamine (CPX-O) is a marketed topical antifungal agent with demonstrated in vitro and in vivo preclinical anticancer activity in several solid tumor and hematologic malignancies. The oral route of administration for CPX-O is not feasible due to low bioavailability and dose-limiting gastrointestinal toxicities. To enable parenteral administration, the phosphoryl-oxymethyl ester of ciclopirox (CPX), fosciclopirox (CPX-POM), was synthesized and formulated as an injectable drug product. In rats and dogs, intravenous CPX-POM is rapidly and completely metabolized to its active metabolite, CPX. The bioavailability of the active metabolite is complete following CPX-POM administration. CPX and its inactive metabolite, ciclopirox glucuronide (CPX-G), are excreted in urine, resulting in delivery of drug to the entire urinary tract. The absolute bioavailability of CPX following subcutaneous administration of CPX-POM is excellent in rats and dogs, demonstrating the feasibility of this route of administration. These studies confirmed the oral bioavailability of CPX-O is quite low in rats and dogs compared with intravenous CPX-POM. Given its broad-spectrum anticancer activity in several solid tumor and hematologic cancers and renal elimination, CPX-POM is being developed for the treatment of urothelial cancer. The safety, dose tolerance, pharmacokinetics, and pharmacodynamics of intravenous CPX-POM are currently being characterized in a United States multicenter first-in-human Phase 1 clinical trial in patients with advanced solid tumors (NCT03348514).

Abstract 5882: Bench-to-bedside translation of ciclopirox prodrug for the treatment of non-muscle invasive and muscle-invasive bladder cancer

Abstract Ciclopirox (CPX) is contained in a number of FDA-approved topical antifungal drug products as the free acid and olamine salt. CPX possesses anticancer activity in a number of in vitro and in vivo preclinical models. Its clinical utility is limited as an oral anticancer agent, however. The oral bioavailability of CPX is quite low due to extensive first pass effect. The poor water solubility of CPX and its olamine salt prevent formulation as an injectable drug product. Thirdly, dose-limiting gastrointestinal toxicities were observed following four times daily oral dosing of CPX in patients with advanced hematologic malignancies. Ciclopirox Prodrug (CPX-POM), in contrast, has demonstrated excellent bioavailability via injectable routes of administration. Here we describe the preclinical characterization of CPX-POM, a novel anticancer agent being developed for the treatment of non-muscle invasive (NMIBC) and muscle invasive (MIBC) bladder cancer. Following IV, SQ and IP administration to mice, CPX-POM is rapidly and completely metabolized to CPX in blood via circulating phosphatases. CPX and its major, inactive glucuronide metabolite are extensively eliminated in urine. At well-tolerated doses, steady-state urine concentrations of CPX exceed in vitro IC50 values in mice by 15-30 fold. CPX inhibited cell proliferation, colony formation, and bladdosphere formation in vitro in T24 (NMIBC) and 253JBV (MIBC) human cell lines in both concentration- and time-dependent manners with IC50 values of 2-4 µM. CPX exposure increased the percentage of NMIBC and MIBC cells arrested at the S and G0/G1 phases, and induced cell death. CPX exposure significantly reduced expression of genes at the mRNA level involved in cancer stem cell signaling pathways including Notch, Wnt, and Hedgehog. CPX was shown to inhibit bladder cancer cell growth in vitro by inhibiting the Notch 1 signaling pathway. The validated N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) chemical carcinogen mouse model of bladder cancer was employed to establish in vivo preclinical proof of principle for CPX-POM. Over the once-daily IP dose range of 25-200 mg/kg, CPX-POM treatment resulted in significant decreases in bladder weight, a clear migration to lower stage tumors, dose-dependent reduction in Ki67 and PCNA staining, as well as a reduction in PCNA-expressing cells. All CPX-POM doses were well tolerated with no evidence of toxicity to the urinary tract based on blinded pathologic evaluation. There were also dose-dependent decreases in Notch 1, Presenilin 1, and Hey 1 in bladder cancer tissues obtained from CPX-POM treated animals. Tumor response was similar, in vivo, following once-daily and three-times weekly CPX-POM administration. CPX-POM has received FDA clearance to proceed to Phase I, and is currently being evaluated in a first-in-human trial in patients with advanced solid tumors. Citation Format: Scott J. Weir, Partha Ranjarajan, Robyn Wood, Karl Schorno, Prabhu Ramamoorthy, Lian Rajweski, Kathy Heppert, Michael J. McKenna, William McCulloch, Greg A. Reed, Amanda Brinker, Michael J. Baltezor, Roy A. Jensen, John A. Taylor, Shrikant Anant. Bench-to-bedside translation of ciclopirox prodrug for the treatment of non-muscle invasive and muscle-invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5882.

Abstract CT152: Phase I dose- and regimen-finding study of NVP-HDM201 in pts with advanced TP53 wt acute leukemias

Abstract Background: NVP-HDM201 is a selective inhibitor of the p53-HDM2 interaction and has demonstrated potent single-agent activity in various in vitro and in vivo tumor models, dependent on wild-type (wt) TP53. This study aims to determine the optimal dose and schedule of NVP-HDM201 for treating patients (pts) with TP53 wt tumors for further clinical study. Here we focus on pts with advanced, TP53 wt acute leukemias. Methods: In this multicenter, open-label, dose-finding, Phase I study, pts with advanced, TP53 wt tumors who had relapsed or refractory acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) were treated with single-agent oral NVP-HDM201. Four treatment regimens were explored: two high-dose intermittent regimens (reg), Reg 1A and 1B (1A: Day 1 of a 3-week [wk] cycle; 1B: Days 1 and 8 of a 4-wk cycle) and two low-dose extended regimens, Reg 2A and 2C (2A: once daily for the first 2 wks of a 4-wk cycle; 2C: once daily for the first wk of a 4-wk cycle). Results: As of Dec 07, 2016, a total of 37 pts, comprising 35 pts with AML and 2 pts with ALL, had been enrolled in the study (Reg 1A n=16; Reg 1B n=6; Reg 2A n=7; Reg 2C n=8); treatment is ongoing in 3 pts (2 in Reg 1B and 1 in Reg 2C). The most common Grade 3/4 adverse events (AEs) suspected to be treatment-related (occurring in ≥25% of pts; Reg 1A; Reg 1B; Reg 2A; Reg 2C) were thrombocytopenia (50%; 50%; 29%; 50%), tumor lysis syndrome (TLS; 44%; 0; 14%; 13%), neutropenia (38%; 17%; 0; 25%), anemia (25%; 33%; 29%; 38%), febrile neutropenia (25%; 33%; 29%; 38%), and decreased white blood cell count (0; 0; 14%; 25%). Six dose-limiting toxicities (DLTs) were observed in 4 pts at 400 mg in Reg 1A: G4 hypophosphatemia (n=2), G3 infection (n=1), G3 chronic graft versus host disease (n=1), G3 stomatitis (n=1), and G4 subarachnoid hemorrhage (n=1). One DLT each occurred in Reg 1B (G4 acute kidney injury at 150 mg) and Reg 2C (G4 TLS at 45 mg). Importantly, there were no dose-limiting gastrointestinal (GI) toxicities. NVP-HDM201 also showed approximate dose-proportional pharmacokinetics (PK) and pharmacodynamics. Investigator-assessed overall response rate (CR + CRi + PR) for all pts with AML who had ≥1 post-baseline assessment (n=34) was 20.6% (95% confidence interval: 8.7-37.9%). There were 3 CRs (2 in Reg 1A; 1 in Reg 2C) and 4 CRis (1 in Reg 1B; 3 in Reg 2C). CRs/CRis were observed in pts receiving a cumulative dose of 250 mg within the first wk of treatment. Conclusions: Across all regimens, the AEs reported were overall expected and manageable, with no dose-limiting GI toxicities. The recommended dose for expansion (RDE) was declared as 45 mg in Reg 2C, based on the manageable safety profile, therapeutically relevant exposures determined by PK modeling, and meaningful antitumor activity seen at this dose level of NVP-HDM201. RDE determination for Reg 1A and Reg 1B is ongoing. Preliminary anti-leukemic activity is promising in these pts and warrants further study of this agent in AML. Citation Format: Eytan Stein, Joerg Chromik, Daniel J. DeAngelo, Manik Chatterjee, Richard Noppeney, Filip de Vos, Hironobu Minami, Sébastien Jeay, Christophe Meille, Ensar Halilovic, Luisa Mariconti, Matthieu Klopfenstein, Nelson Guerreiro, Rajkumar Radhakrishnan, Emil T. Kuriakose, Cecilia Carpio. Phase I dose- and regimen-finding study of NVP-HDM201 in pts with advanced TP53 wt acute leukemias [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT152. doi:10.1158/1538-7445.AM2017-CT152

Cholinesterase Inhibitor Therapy in Alzheimer's Disease: The Limits and Tolerability of Irreversible CNS-Selective Acetylcholinesterase Inhibition in Primates.

Irreversible acetylcholinesterase (AChE) inhibition accumulates to high levels in the central nervous system (CNS) because AChE turnover in the brain is much slower than in peripheral tissues. As expected from this CNS selectivity, the irreversible AChE inhibitor methanesulfonyl fluoride (MSF) produces significant cognitive improvement in Alzheimer's disease patients without the gastrointestinal toxicity that plagues other AChE inhibitors. However, without dose-limiting gastrointestinal toxicity, one shortcoming of the prior human studies of MSF is that the upper limits of CNS AChE inhibition that might be tolerated could not be tested. Therefore, in this study, monkeys were treated with escalating intramuscular (IM) doses of MSF that culminated with several weeks of 1.5 mg/kg dosing, more than eight times the prior human clinical dose, still without signs of toxicity. Brain biopsies showed that ∼80% AChE inhibition had been produced and that the new synthesis of cortical AChE had a half-time (t1/2) of ∼12 days. A single IM dose of 1.5 mg/kg MSF produced ∼59% inhibition in cerebrospinal fluid (CSF) AChE as measured one day later. This corresponds to a peak of ∼80% inhibition in CSF AChE at the time of the injection, recovering with a t1/2 of 2.4 days. Computational analyses suggest that MSF at clinically relevant doses could theoretically produce a steady-state AChE inhibition between 65% and 85% in the CNS. These data suggest that the full therapeutic advantage of AChE inhibition therapy can be realized without interference from dose-limiting gastrointestinal toxicity if an irreversible inhibitor is employed.

Abstract 1895: Ciclopirox prodrug for the prevention and therapy of non-muscle invasive bladder cancer

Abstract Objective: Ciclopirox prodrug for the prevention of Non-Muscle Invasive Bladder Cancer (NMIBC) represents a potential breakthrough in the management of high-grade, NMIBC. If successful, this represents the first systemic approach to manage NMIBC. Ciclopirox olamine, the active pharmaceutical ingredient in several topical antifungal drug products, has demonstrated anticancer activity in blood and solid tumors. We developed a phosphoryloxymethyl prodrug of ciclopirox (CPX) for efficient intravenous delivery, thereby avoiding dose-limiting gastrointestinal toxicities and first-pass effect associated with oral administration. Methods: In vitro anticancer activity of CPX was performed in both NMIBC (T24) and MIBC (253-JBV) cell lines. Cell proliferation was determined by hexoseaminidase assay. Stemness was determined by the Spheroid assay. Flow cytometric analyses was performed for cell cycle analysis. RT-PCR array analysis was performed to identify signaling pathways. Real Time PCR, western blot and immunofluorescent studies were performed for determining gene expression. Results: Pharmacokinetic studies conducted in mice, rats and dogs demonstrated that the drug is rapidly and completely metabolized and eliminated via the urine. In vitro, CPX inhibited growth of T24 and 253-JBV cells at 4 and 2 μM, respectively coupled with S-phase cell cycle arrest. FITC conjugated Annexin V-coupled flow cytometry studies showed that CPX induces apoptotic cell death. CPX also suppressed bladdosphere formation, suggesting that stem cells are affected. Notch signaling pathway plays a significant role in stem cell behavior, and previous studies have suggested that the pathway may be a therapeutic target for bladder cancer. Notch receptor activation, which occurs following ligand binding, involves specific intracellular cleavage by the γ-secretase complex. First, RT-PCR array analyses suggested that CPX inhibits the Notch signaling pathway. Furthermore, CPX significantly inhibited the expression of the γ-secretase complex proteins Presenilin1, Nicastrin, APH-1 and PEN-2. In addition, there was a reduction in the expression of downstream targets of the pathway including Hes1 and Cyclin-D1. These data suggest that CPX significantly inhibits Notch intracellular signaling pathway proteins. Ectopic expression of the activated/cleaved Notch protein reversed CPX mediated inhibition of cell proliferation. Conclusion: CPX is a novel chemotherapeutic and preventive agent for bladder cancers, and this occurs by suppressing the stem cells. We are confident that there will be no failure of the compound as a result of unacceptable toxicity because drug safety profile of CPX has been previously characterized in animals and humans. These studies provide insight into understanding the role of CPX as a potent chemotherapeutic and preventive agent. Citation Format: Parthasarathy Rangarajan, Satish Ramalingam, Dharmalingam Subramaniam, Michael J. Baltezor, Robyn Wood, Shrikant Anant, Scott Weir. Ciclopirox prodrug for the prevention and therapy of non-muscle invasive bladder cancer. [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 1895. doi:10.1158/1538-7445.AM2015-1895

Abstract 2926: DR5-targeting sensitizes Lgr5+ stem cells to p53 and Chk2-dependent chemotherapy-induced cell death and produces dose-limiting gastrointestinal toxicity (GIT)

Abstract Current ongoing clinical trials are exploring combined targeting of the TRAIL-death receptor DR5 with radiochemotherapy. It is not clear mechanistically why targeting of the DR5 increasingly sensitizes cancer cells to cell death without affecting cells in normal tissues. Preclinical data suggests that integration of DR5 agonist antibodies into chemotherapy protocols has the promise to significantly improve cancer specific responses without negatively impacting dose-limiting toxicities. Here we show that targeting of DR5 in mice with the mouse-specific mAb MD5-1 in combination with chemotherapy triggers synergistic gastrointestinal toxicities (GIT) that is associated with the death of Lgr5+ crypt basal columnar (CBC) cells. This type of GIT is mediated by p53 and in the case of irinotecan, the cell cycle checkpoint kinase 2 (Chk2). Pharmacologic targeting of Chk2 sensitized syngeneic mouse colorectal cancer tumors lacking P53 to the irinotecan/MD5-1 combination while at the same time protecting the gastrointestinal tract from injury. Our data indicates that blockade of cell cycle checkpoint kinases can help prevent GIT triggered by a combination of DNA damaging chemotherapy and DR5 agonists. Pharmacologic targeting of Chk2 can improve the anti-tumor response of such drug combinations and subsequently provides a rationale on how to improve therapeutic indices of DR5-targeting in the context of chemotherapy usage. Citation Format: Niklas K. Finnberg, Prashanth Gokare, Arunasalam Navaraj, Krystle A. Lang Kuhs, George Cerniglia, Hideo Yagita, Kazuyoshi Takeda, Noboru Motoyama, Wafik S. El-Deiry. DR5-targeting sensitizes Lgr5+ stem cells to p53 and Chk2-dependent chemotherapy-induced cell death and produces dose-limiting gastrointestinal toxicity (GIT). [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 2926. doi:10.1158/1538-7445.AM2015-2926

Pharmacologically blocking p53-dependent apoptosis protects intestinal stem cells and mice from radiation.

Exposure to high levels of ionizing radiation (IR) leads to debilitating and dose-limiting gastrointestinal (GI) toxicity. Using three-dimensional mouse crypt culture, we demonstrated that p53 target PUMA mediates radiation-induced apoptosis via a cell-intrinsic mechanism, and identified the GSK-3 inhibitor CHIR99021 as a potent radioprotector. CHIR99021 treatment improved Lgr5+ cell survival and crypt regeneration after radiation in culture and mice. CHIR99021 treatment specifically blocked apoptosis and PUMA induction and K120 acetylation of p53 mediated by acetyl-transferase Tip60, while it had no effect on p53 stabilization, phosphorylation or p21 induction. CHIR99021 also protected human intestinal cultures from radiation by PUMA but not p21 suppression. These results demonstrate that p53 posttranslational modifications play a key role in the pathological and apoptotic response of the intestinal stem cells to radiation and can be targeted pharmacologically.

FHL1C induces apoptosis in Notch1-dependent T-ALL cells through an interaction with RBP-J.

BackgroundAberrantly activated Notch signaling has been found in more than 50% of patients with T-cell acute lymphoblastic leukemia (T-ALL). Current strategies that employ γ-secretase inhibitors (GSIs) to target Notch activation have not been successful. Many limitations, such as non-Notch specificity, dose-limiting gastrointestinal toxicity and GSI resistance, have prompted an urgent need for more effective Notch signaling inhibitors for T-ALL treatment. Human four-and-a-half LIM domain protein 1C (FHL1C) (KyoT2 in mice) has been demonstrated to suppress Notch activation in vitro, suggesting that FHL1C may be new candidate target in T-ALL therapy. However, the role of FHL1C in T-ALL cells remained unclear.MethodsUsing RT-PCR, we amplified full-length human FHL1C, and constructed full-length and various truncated forms of FHL1C. Using cell transfection, flow cytometry, transmission electron microscope, real-time RT-PCR, and Western blotting, we found that overexpression of FHL1C induced apoptosis of Jurkat cells. By using a reporter assay and Annexin-V staining, the minimal functional sequence of FHL1C inhibiting RBP-J-mediated Notch transactivation and inducing cell apoptosis was identified. Using real-time PCR and Western blotting, we explored the possible molecular mechanism of FHL1C-induced apoptosis. All data were statistically analyzed with the SPSS version 12.0 software.ResultsIn Jurkat cells derived from a Notch1-associated T-ALL cell line insensitive to GSI treatment, we observed that overexpression of FHL1C, which is down-regulated in T-ALL patients, strongly induced apoptosis. Furthermore, we verified that FHL1C-induced apoptosis depended on the RBP-J-binding motif at the C-terminus of FHL1C. Using various truncated forms of FHL1C, we found that the RBP-J-binding motif of FHL1C had almost the same effect as full-length FHL1C on the induction of apoptosis, suggesting that the minimal functional sequence in the RBP-J-binding motif of FHL1C might be a new drug candidate for T-ALL treatment. We also explored the molecular mechanism of FHL1C overexpression-induced apoptosis, which suppressed downstream target genes such as Hes1 and c-Myc and key signaling pathways such as PI3K/AKT and NF-κB of Notch signaling involved in T-ALL progression.ConclusionsOur study has revealed that FHL1C overexpression induces Jurkat cell apoptosis. This finding may provide new insights in designing new Notch inhibitors based on FHL1C to treat T-ALL.

Oral ciclopirox olamine displays biological activity in a phase I study in patients with advanced hematologic malignancies

The antimycotic ciclopirox olamine is an intracellular iron chelator that has anticancer activity in vitro and in vivo. We developed an oral formulation of ciclopirox olamine and conducted the first-in-human phase I study of this drug in patients with relapsed or refractory hematologic malignancies (Trial registration ID: NCT00990587). Patients were treated with 5-80 mg/m² oral ciclopirox olamine once daily for five days in 21-day treatment cycles. Pharmacokinetic and pharmacodynamic companion studies were performed in a subset of patients. Following definition of the half-life of ciclopirox olamine, an additional cohort was enrolled and treated with 80 mg/m² ciclopirox olamine four times daily. Adverse events and clinical response were monitored throughout the trial. Twenty-three patients received study treatment. Ciclopirox was rapidly absorbed and cleared with a short half-life. Plasma concentrations of an inactive ciclopirox glucuronide metabolite were greater than those of ciclopirox. Repression of survivin expression was observed in peripheral blood cells isolated from patients treated once daily with ciclopirox olamine at doses greater than 10 mg/m², demonstrating biological activity of the drug. Dose-limiting gastrointestinal toxicities were observed in patients receiving 80 mg/m² four times daily, and no dose limiting toxicity was observed at 40 mg/m² once daily. Hematologic improvement was observed in two patients. Once-daily dosing of oral ciclopirox olamine was well tolerated in patients with relapsed or refractory hematologic malignancies, and further optimization of dosing regimens is warranted in this patient population.

Abstract C67: Safety, efficacy, and pharmacokinetics of a new humanized anti-Trop-2 antibody-SN-38 conjugate (IMMU-132) for the treatment of diverse epithelial cancers: Phase I clinical experience.

Abstract Trop-2 (trophoblastic antigen-2; aka EGP-1, GA733-1) is a surface glycoprotein differentially expressed on a variety of malignant epithelial cancers, making it an interesting target for antibody directed therapeutics. Preclinical studies with an anti-Trop-2 IgG, designated RS7 (Stein et al. Cancer Res 1990;50:1330-36), showed targeting of human cancer xenografts and antibody internalization. An antibody-drug conjugate (IMMU-132) was prepared by conjugating humanized RS7 with SN-38 (6 moles SN-38/IgG), the highly potent topoisomerase-I-inhibitor metabolite of irinotecan. The linker allows SN-38 release from the conjugate at low pH, such as in the tumor microenvironment or lysosomes. IMMU-132 was effective against several human cancer xenograft models, with a high therapeutic window. Primate studies found dose-limiting neutropenia and gastrointestinal (GI) toxicity identical to irinotecan, but no selective damage to normal tissues expressing Trop-2 (Cardillo et al. Clin Cancer Res 2011;17:3157-69). A multicenter Phase I clinical trial was initiated in a diverse, metastatic, solid tumor population, who had failed ≥ 1 prior standard therapeutic regimen for their tumor type. In a standard 3+3 trial design, eligible patients (pts) received multiple 21-day cycles of IMMU-132 with weekly dosing in the first 2 weeks followed by 1 week rest. Dose delay and reduction were permitted for treatment-related toxicities occurring on scheduled treatment days. Doses started at 8 mg/kg with 3 pts tolerating multiple cycles with no toxicity. Escalation continued to 12 and 18 mg/kg, when further escalation was stopped due to dose-limiting neutropenia in 2/3 pts at 18 mg/kg. In addition, grade 1-2 GI toxicities were reported in several pts, and grade 1/2 rash in 2 pts. The maximum tolerated dose was 12 mg/kg (0.19 mg/kg SN-38 equivalents), with no pts experiencing a dose-limiting event (i.e., grade 4 neutropenia >5 days) in the first cycle. However, there were frequent interruptions in the planned dosing at this level, which also required dose reductions. To establish a maximum acceptable dose, an intermediate dose level of 10 mg/kg is under evaluation with 5 pts currently in the early stages of treatment without any need for dose reduction or delay. Twenty-four pts have been enrolled to date at all dose levels, with 14 having at least 1 CT assessment and 10 not due yet for the first assessment. Objective partial responses by RECIST 1.0 were observed in 3 pts, one each with colon cancer (CEA also decreased from 781 to 39 ng/mL), triple-negative breast cancer, and small cell lung cancer at the 8 and 12 mg/kg, with lesser degrees of tumor shrinkage (stabilization) in several other pts. Clearance of the intact conjugate and IgG monitored by ELISA showed the intact conjugate clearing more quickly than the IgG, reflecting the expected release of SN-38 from the conjugate. SN-38 concentrations were estimated to be nearly 30-times higher in the serum than that reported with irinotecan therapy. Accrual is completed and therapy continuing in diverse cancers at the 8 and 10 mg/kg dose levels. Thus, IMMU-132 represents a promising, novel antibody-drug conjugate with acceptable toxicity and significant activity in this refractory, advanced, cancer population with difficult-to-treat tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C67. Citation Format: Alexander N. Starodub, Allyson J. Ocean, Michael J. Guarino, Ellen Chuang, Manish A. Shah, Scott Tagawa, Vincent J. Picozzi, Sajeve S. Thomas, Pius P. Maliakal, William A. Wegener, Robert M. Sharkey, Serengulam V. Govindan, David M. Goldenberg. Safety, efficacy, and pharmacokinetics of a new humanized anti-Trop-2 antibody-SN-38 conjugate (IMMU-132) for the treatment of diverse epithelial cancers: Phase I clinical experience. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C67.

Abstract 4448: Notch1 inactivation contributes to PI3Ki resistance in T-ALL.

Abstract Tyrosine kinases inhibitors induce genotype-specific regression in advanced human cancers. However, these responses are almost invariably transient and patients ultimately relapse due to outgrowth of drug-resistant clones. Thus, increased knowledge of the mechanisms underlying de novo and acquired resistance is essential for enhancing clinical efficacy. T cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer for which current therapies are toxic and frequently ineffective. Mutations in genes encoding components of the Notch1 and Ras/PI3K/PTEN/Akt signaling networks are prevalent in T-ALL and frequently coexist in individual patients. Thus, inhibiting these pathways alone and in combination is a rational therapeutic strategy. Notch1 inhibition with a γ-secretase inhibitor (GSI) was recently evaluated in refractory T-ALL, with disappointing results that may have been due to dose-limiting gastrointestinal toxicity. We are not aware of clinical trials of Ras/PI3K/Akt/mTOR pathway inhibitors in T-ALL. Retroviral insertional mutagenesis (RIM) in mice is a robust strategy for modeling both the clonal heterogeneity that exists within individual tumors and the genetic diversity found across a population of patients with the same cancer. We generated transplantable T-ALLs by performing RIM in mice expressing oncogenic KrasG12D from its endogenous genetic locus (Mx1-Cre, KrasG12D) and in wild-type (WT) littermates. These aggressive multi-step cancers are characterized by recurring viral integrations in known oncogenes such as Ikzf1 and late occurring acquired Notch1 PEST domain mutations. We have extensively investigated patterns of response and resistance to targeted inhibitors of PI3K (PI3Ki) and MEK (MEKi) in this system. Whereas KrasWT T-ALLs respond to either PI3Ki or to a combination regimen that also include a MEKi, KrasG12D T-ALLs are largely refractory to PI3Ki, but are sensitive to the MEKi + PI3Ki combination. T-ALLs invariably relapsed despite ongoing treatment, and we verified phenotypic drug resistance in relapse leukemias by transplanting and retreating them in vivo. We unexpectedly identified multiple independent PI3Ki resistant T-ALLs with markedly reduced levels of activated Notch1, including some in which Notch1 mutations that were present at enrollment were absent at relapse. Expression of activated Notch1 increases the sensitivity of mouse and human T-ALL cells to a PI3Ki, and loss of activated Notch promotes resistance and enhances PI3K signaling. Thus, oncogenic Notch1 mutations that promote outgrowth during transformation unexpectedly “switch” to become deleterious during in vivo treatment with a PI3Ki. These data suggest that aberrant Ras/PI3K signaling plays a dominant role in T lineage leukemogenesis and also raise the unexpected possibility that administering Notch1 and PI3K inhibitors sequentially or in combination might inadvertently promote resistance. Citation Format: Monique Dail, Jason Wong, Daniel O'Connor, Joy Nakitandwe, Shann-Ching Chen, Jessica Lawrence, James R. Downing, Deepak Sampath, Kevin Shannon. Notch1 inactivation contributes to PI3Ki resistance in T-ALL. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4448. doi:10.1158/1538-7445.AM2013-4448

Involvement of Multidrug Resistance-Associated Protein 1 in Intestinal Toxicity of Methotrexate

Methotrexate (MTX) causes dose-limiting gastrointestinal toxicity due to exposure of intestinal tissues, and is a substrate of the multidrug resistance-associated protein (MRP) 1. Here we examine the involvement of MRP1, which is reported to be highly expressed in the proliferative crypt compartment of the small intestine, in the gastrointestinal toxicity of MTX. MTX was intraperitoneally administered to mrp1 gene knockout (mrp1 ((-/-))) and wild-type (mrp1 ((+/+))) mice. Body weight, food and water intake were monitored, intestinal histological studies and pharmacokinetics of MTX were examined. mrp1 ((-/-)) mice more severely decreased body weight, food and water intake than mrp1 ((+/+)) mice. Almost complete loss of villi throughout the small intestine in mrp1 ((-/-)) mice was observed, whereas the damage was only partial in mrp1 ((+/+)) mice. Plasma concentration and biliary excretion profiles of MTX were similar in mrp1 ((-/-)) and mrp1 ((+/+)) mice, though accumulation of MTX in immature proliferative cells isolated from mrp1 ((-/-)) mice was much higher compared to mrp1 ((+/+)) mice. Immunostaining revealed localization of Mrp1 in plasma membrane of the intestinal crypt compartment in mrp1 ((+/+)) mice, but not in mrp1 ((-/-)) mice. Mrp1 determines the exposure of proliferative cells in the small intestine to MTX, followed by gastrointestinal toxicity.