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 ß-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.

Nanoliposomal irinotecan plus fluorouracil and folinic acid as a second-line treatment option in patients with metastatic pancreatic ductal adenocarcinoma: a retrospective cohort study.

BACKGROUND: According to the NAPOLI-1 trial, nanoliposomal irinotecan (nal-IRI) plus fluorouracil/folinic acid (5-FU/LV) showed improved overall survival compared to fluorouracil alone for patients with metastatic pancreatic cancer who were previously treated with gemcitabine-based therapy. In that trial, Asian patients had frequent dose modification due to haematological toxicity. There has been limited information on the clinical benefits and toxicity of this regimen in real-world settings. In this study, we assessed real-world experience of nal-IRI plus 5-FU/LV in patients with advanced pancreatic cancer after gemcitabine failure. METHODS: We conducted a single institution, retrospective analysis of response, survival and safety in patients who had been treated with nal-IRI with 5-FU/LV. Patients with metastatic pancreatic ductal adenocarcinoma previously treated with gemcitabine-based therapy received nal-IRI (80 mg/m2) with 5-FU/LV every 2 weeks. Kaplan-Meier analysis was performed to obtain median progression free survival and median overall survival. The hazard ratio and 95% confidence interval (CI) were estimated using a stratified Cox regression model. A multivariate Cox proportional hazards regression model was used to identify the effects of clinical factors. RESULTS: Fifty-one patients received nal-IRI plus 5-FU/LV between January 2015 and December 2020. The median age was 67 years, and males were 58.8%. A total of 40 (78.4%) and 11 (21.6%) patients had received one and two lines of prior chemotherapy before enrollment, respectively. Median progression-free survival was 2.8 months (95% CI 1.8-3.7) and median overall survival was 7.0 months (95% CI 6.0-7.9). Chemotherapy doses were reduced or delayed in 33 (64.7%) patients during the first 6 weeks and median relative dose intensity was 0.87. Thirty-six (70.6%) patients experienced grade 3 or 4 adverse events, most commonly neutropenia (58.8%). Most non-haematologic adverse events were under grade 2. Since the start of first-line chemotherapy, median overall survival was 16.3 months (95% CI 14.1-18.4). CONCLUSIONS: Nal-IRI plus 5-FU/LV seems to be effective, with manageable toxicities, following gemcitabine-based treatment in patients with metastatic pancreatic ductal adenocarcinoma. Nal-IRI plus 5-FU/LV following gemcitabine with nab-paclitaxel is a feasible sequential treatment option in patients with metastatic pancreatic cancer. TRIAL REGISTRATION: Retrospectively registered.

Novel Nontoxic 5,9-Disubstituted SN38 Derivatives: Characterization of Their Pharmacological Properties and Interactions with DNA Oligomers.

Novel nontoxic derivatives of SN38 with favorable antineoplastic properties were characterized in water solution using NMR. The phenomena observed by NMR were linked to basic pharmacological properties, such as solubility, bioavailability, chemical and stereochemical stability, and binding to natural DNA oligomers through the terminal G-C base pair, which is commonly considered a biological target of Topo I inhibitors. Compound 1, with bulky substituents at both C5(R) and C20(S) on the same side of a camptothecin core, manifests self-association, whereas diastereomers 2, with bulky C5(S) and C20(S) substituents are mostly monomeric in solution. The stereogenic center at C5 is stable in water solution at pH 5-6. The compound with an (N-azetidinyl)methyl substituent at C9 can undergo the retro Mannich reaction after a prolonged time in water solution. Both diastereomers exhibit different abilities in terms of binding to DNA oligomers: compound 1 is strongly bound, whereas the binding of compound 2 is rather weak. Molecular modeling produced results consistent with NMR experiments. These complementary data allow linking of the observed phenomena in NMR experiments to basic preliminary information on the pharmacodynamic character of compounds and are essential for planning further development research.

Antagonistic Effects of CAPE (a Component of Propolis) on the Cytotoxicity and Genotoxicity of Irinotecan and SN38 in Human Gastrointestinal Cancer Cells In Vitro.

The incidence of gastrointestinal cancers is increasing every year. Irinotecan (CPT-11), a drug used in the treatment of colorectal cancer and gastric cancer, is metabolized by carboxylesterases to an active metabolite, SN-38, which is more cytotoxic. CAPE (caffeic acid phenethyl ester) is an active component of propolis, which has a high antibacterial, antiviral, and antineoplastic potential. This study analyses the impact of CAPE on the cytotoxic (MTT assay), genotoxic (comet assay) and proapoptotic (caspase-3/7 activity) potential of irinotecan and its metabolite SN-38 in cultures of gastrointestinal neoplastic cells (HCT116, HT29, AGS). Cytotoxicity and genotoxicity activities of these compounds were carried out in comparison with human peripheral blood lymphocytes (PBLs) in vitro. The antioxidant potential of CAPE was investigated in relation H2O2-induced oxidative stress in the both neoplastic cells and PBLs. CAPE expressed cytotoxic, genotoxic, and pro-apoptotic activity against AGS, HCT116, and HT29 tumor cells. CAPE, in the presence of different concentrations of irinotecan or SN38, decreased the cytotoxicity, genotoxicity, and pro-apoptotic activity in these cell lines, but it has no such action on normal human peripheral blood lymphocytes.

Irinotecan and its metabolite SN38 inhibits procollagen I production of dermal fibroblasts from Systemic Sclerosis patients.

Systemic sclerosis (SSc) is a rare autoimmune connective tissue disease characterized by a microangiopathy and fibrosis of the skin and internal organs. No treatment has been proved to be efficient in case of early or advanced SSc to prevent or reduce fibrosis. There are strong arguments for a key role of topo-I in the pathogenesis of diffuse SSc. Irinotecan, a semisynthetic derivative of Camptothecin, specifically target topo-I. This study was undertaken to evaluate the effects of noncytotoxic doses of irinotecan or its active metabolite SN38 on collagen production in SSc fibroblasts. Dermal fibroblasts from 4 patients with SSc and 2 healthy donors were cultured in the presence or absence of irinotecan or SN38. Procollagen I release was determined by ELISA and expression of a panel of genes involved in fibrosis was evaluated by qRT-PCR. Subcytotoxic doses of irinotecan and SN38 caused a significant and dose-dependent decrease of the procollagen I production in dermal fibroblasts from SSc patients, respectively - 48 ± 3%, p < 0.0001 and - 37 ± 6.2%, p = 0.0097. Both irinotecan and SN38 led to a global downregulation of genes involved in fibrosis such as COL1A1, COL1A2, MMP1 and ACTA2 in dermal fibroblasts from SSc patients (respectively - 27; - 20.5; - 30.2 and - 30% for irinotecan and - 61; - 55; - 50 and - 54% for SN38). SN38 increased significantly CCL2 mRNA level (+ 163%). The inhibitory effect of irinotecan and its active metabolite SN38 on collagen production by SSc fibroblasts, which occurs through regulating the levels of expression of genes mRNA, suggests that topoisomerase I inhibitors may be effective in limiting fibrosis in such patients.

Salinomycin-Loaded Small-Molecule Nanoprodrugs Enhance Anticancer Activity in Hepatocellular Carcinoma.

BACKGROUND: There is currently no effective treatment for advanced hepatocellular carcinoma (HCC), and chemotherapy has little effect on long-term survival of HCC patients, largely due to the cancer stem cell (CSC) chemoresistance of HCC. METHODS: We constructed a small-molecule nanometer-sized prodrug (nanoprodrug) loaded with salinomycin (SAL) for the treatment of HCC. SAL was encapsulated by the prodrug LA-SN38 (linoleic acid modified 7-ethyl-10-hydroxycamptothecin) to construct a self-assembled nanoprodrug further PEGylated with DSPE-PEG2000. We characterized this codelivered nanoprodrug and its antitumor activity both in vitro in human HCC cell lines and in vivo in mice. RESULTS: Delivery of the SAL- and LA-SN38-based nanoprodrugs effectively promoted apoptosis of HCC cells, exerted inhibition of HCC tumor-sphere formation as well as HCC cell motility and invasion, and reduced the proportion of CD133+ HCC-CSC cells. In nude mice, the nanoprodrug suppressed growth of tumor xenografts derived from human cell lines and patient. CONCLUSION: Our results show that SAL-based nanoprodrugs are a promising platform for treating patients with HCC and a novel strategy for combination therapy of cancers.

Hydrophobized SN38 to redox-hypersensitive nanorods for cancer therapy.

The clinical application of SN38 (7-ethyl-10-hydroxy-camptothecin) is severely restricted by its extremely low water solubility. Nanoaggregates formed by amphiphilic SN38 prodrugs have been widely used for the delivery of SN38. In this study, we used a hydrophobized SN38 prodrug, rather than a typical SN38 amphiphile, to construct rod-shaped nanoaggregates for efficient SN38 delivery. The hydrophobized SN38 was synthesized by conjugating SN38 with oleic acid using disulfanyl-ethyl carbonate as the linker. Interestingly, the resulting prodrug self-assembled into nanorods with high drug loading capacity (45%) and colloidal stability. Moreover, these nanorods displayed an impressively high redox-sensitivity to release 100% SN38 within 1 h in 10 mM DTT, versus 1% in phosphate buffer (pH 7.4). The efficient drug release resulted in an uncompromised in vitro cytotoxicity, which was comparable to free SN38 and nearly 93-fold more potent than CPT-11. Most importantly, these novel prodrug nanoaggregates exhibited potent antitumor activity in the CT26 colorectal cancer xenograft. The nanoaggregates of such redox-hypersensitive hydrophobized SN38 represent an effective alternative strategy for developing novel SN38 nanomedicines.

Azo-based small molecular hypoxia responsive theranostic for tumor-specific imaging and therapy.

We report herein, an azo-derivative (AzP1) of FDA approved antineoplastic drug SN-38 (irinotecan analogue) as a theranostic agent with a potential for both tumor hypoxia-specific activation and therapy. The theranostic AzP1 was found to be stable within a biologically relevant pH scale and was chemically inert towards other competitive biological analytes. However, upon treatment with rat-liver microsomes, AzP1 showed a self-calibrated fluorescence enhancement at λem = 560 nm. The cytotoxicity profile of AzP1 was tested in various cancer lines. Under hypoxic conditions, prodrug AzP1 exhibited activation to release the parent drug (SN-38) and enhanced cytotoxicity in cancer cells with concomitant fluorescence enhancement at 560 nm, which served to monitor both the drug activation and tracing purposes. The therapeutic potential of AzP1 for both tumor-specific activation and suppression of tumor weights was validated in xenograft mouse model. Collectively, the synthetic ease and hypoxia-sensitive activation along with promising therapeutic properties highlight the potential of theranostic AzPI in future cancer treatment programs.