Active Metabolite SN-38 Research Articles

Abstract 3972: Activity of MM-398, nanoliposomal irinotecan (nal-IRI), in Ewings family tumor xenografts is associated with high exposure of tumor to drug and high SLFN11 expression

Abstract Introduction: Pharmacokinetic properties of irinotecan limit tumor exposure to the active metabolite SN-38. MM-398, a nanoliposomal irinotecan (nal-IRI) developed to attempt to enhance delivery of irinotecan and SN-38 to tumor cells is being tested in clinical trials. We assessed the pharmacokinetics and anti-tumor activity of nal-IRI in comparison to the current clinical formulation of irinotecan (free irinotecan) in preclinical models of pediatric cancer in relationship to expression of the putative DNA/RNA helicase SLFN11. Experimental Design: Plasma and tissue samples were analyzed by liquid chromatography-tandem mass spectrometry for irinotecan and SN-38. In vivo activity of free irinotecan and nal-IRI was compared in subcutaneous xenograft models (3 each) of Ewing's sarcoma family of tumors (EFT), neuroblastoma (NB), and rhabdomyosarcoma (RMS). SLFN11 mRNA expression (real-time RT-PCR) was correlated to in vitro cytotoxicity of SN-38 (by DIMSCAN) in 20 EWS, 13 RMS, and 20 NB cell lines. SLFN11 mRNA expression in primary tumors (10 EFT, 10 NB, and 39 RMS) was assessed by Affymetrix HuEx microarrays. Results: Plasma and tumor concentrations of irinotecan and SN-38 were ∼ 10-fold higher for nal-IRI than for free irinotecan at 4h, 6h, and 24h post-intravenous injection (p<0.01). In 7 of 9 xenograft models, nal-IRI significantly extended event-free survival (EFS) compared to controls, and for 6 of 9 models compared to irinotecan. Combining the 3 EFT models tested, 23 of 25 nal-IRI treated mice achieved complete responses for > 100 days (88% EFS on day 120). EFS for mice with RMS (median 39 days) and NB (median 81 days) was significantly shorter (P<0.001) than for EFT. SN-38 exposure in tumors from nal-IRI may be due to tumor-associated macrophage accumulation, but neither tumor infiltration of macrophages nor tumor SN-38 levels showed significant differences between EFT and RMS or NB xenografts. SLFN11 expression has been reported to correlate with sensitivity to DNA damaging agents and the median SLFN11 mRNA expression was > 100-fold higher in EFT cell lines and > 10-fold in EFT primary tumors compared with NB or RMS cell lines or primary tumors (P<0.0001). Relative cytotoxic/inhibitory concentrations (rIC50) of SN-38 showed a significant inverse correlation with SLFN11 expression in 20 EWS cell lines (r=-0.63, P=0.004). Conclusions: Nal-IRI demonstrated higher anti-tumor activity than free irinotecan in pediatric solid tumor xenografts, presumably due to increased systemic and tumor exposures to the SN-38 active metabolite. The high SLFN11 expression reported in EFT cell lines and the strong nal-IRI activity we have observed in EFT xenografts warrants testing in correlative clinical studies to determine if SLFN11 can reliably be used as a biomarker to predict nal-IRI activity in patients. Citation Format: Min H. Kang, Michael M. Song, Monish Makena, Joo-Sang Lee, Connor P. Hall, Ashly Hindle, Winford Ko, Nancy Paz, Jonathan Fitzgerald, Daryl C. Drummond, Timothy J. Triche, C. Patrick Reynolds. Activity of MM-398, nanoliposomal irinotecan (nal-IRI), in Ewings family tumor xenografts is associated with high exposure of tumor to drug and high SLFN11 expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3972. doi:10.1158/1538-7445.AM2014-3972

Abstract CT224: Pilot study in patients with advanced solid tumors to evaluate feasibility of ferumoxytol (FMX) as tumor imaging agent prior to MM-398, a nanoliposomal irinotecan (nal-IRI)

Abstract Introduction MM-398, a stable nanoliposomal irinotecan (nal-IRI), is designed to exploit leaky tumor vasculature for enhanced drug delivery to tumors. Tumor deposition of nal-IRI and subsequent irinotecan conversion by CES enzymes in both neoplastic cells and tumor associated macrophages (TAM) may positively correlate with activity. Predictive biomarkers to measure tumor deposition could identify patients likely to benefit from nal-IRI. FMX is a 30nm iron-oxide, superparamagnetic nanoparticle with MRI contrast properties. The particle size, its propensity for uptake by TAMs and similar distribution patterns to nal-IRI in preclinical models led to a clinical study evaluating the feasibility of correlating FMX-based MRI (Fe-MRI) acquisition with tissue drug metabolite levels and other biomarkers to estimate drug delivery to tumor. Patients and methods Eligible patients (n=12) with refractory solid tumors with at least two metastatic lesions >2cm accessible for a percutaneous biopsy were enrolled from one institution. Fe-MRI scans were performed on a 1.5T MRI using T2* iron sensitive sequences prior to and following FMX infusion (0.5h, 24h, 72h). MR images were used to direct biopsies at 72h to FMX high or low regions, permitting intra- and inter-patient comparisons of FMX and nal-IRI tumor levels. Patients continued on nal-IRI at 80mg/m2 q2w until progression. Tissue iron and TAM distribution were assessed by IHC, tissue-bound metabolite levels by mass-spectrometry. T2* signal was used to calculate FMX levels in total lesions along with FMX estimates on biopsy images derived from fused MRI-CT biopsy images. The first 9 patients (2M 7F; median age 57 years, range 28-71 years) are reported. Results There were no safety-related or other potential interactions with nal-IRI and FMX. Adverse events of nal-IRI were consistent with previous studies. FMX levels, quantified in 36 tumor lesions from the first 9 subjects, showed mean FMX accumulation of 37.9 mcg/mL [3.3-101.2 mcg/mL] and 13.2 mcg/mL [0.1-41.0 mcg/mL] at 24h and 72h, respectively. Lesions were localized mostly in liver (67%) and lymph nodes/peritoneal sites (25%). A mechanistic PK model indicated that tissue permeability to FMX contributed to Fe-MRI signals at 24h, while FMX binding contributed at 72h. Levels of irinotecan and SN-38 were 3.59mcg/g [2.29-4.89mcg/g] and 11.43ng/g [4.04-18.8ng/g], respectively, at 72h in biopsies from the first 6 patients. Conclusions This study is one of the first to measure active metabolite SN-38 levels in patient tumors. FMX can be used as a tumor contrast agent prior to nal-IRI treatment. T2* MRI sequences allowed for quantitation of FMX concentrations in tumor and reference tissue. A mechanistic model provided an estimation of FMX tumor tissue permeability and binding that may be useful as a predictive biomarker of nanotherapeutics such as nal-IRI. Citation Format: Ramesh K. Ramanathan, Ronald L. Korn, Jasgit C. Sachdev, Gerald J. Fetterly, Katie Marceau, Vickie Marsh, John M. Neil, Ronald G. Newbold, Natarajan Raghunand, Joshua Prey, Stephan G. Klinz, Eliel Bayever, Jonathan B. Fitzgerald. Pilot study in patients with advanced solid tumors to evaluate feasibility of ferumoxytol (FMX) as tumor imaging agent prior to MM-398, a nanoliposomal irinotecan (nal-IRI). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT224. doi:10.1158/1538-7445.AM2014-CT224

Cetuximab increases concentrations of irinotecan and of its active metabolite SN-38 in plasma and tumour of human colorectal carcinoma-bearing mice

In a previous study, we showed that cetuximab, a monoclonal antibody directed towards epidermal growth factor receptor, could inhibit P-glycoprotein (P-gp), an efflux protein of ATP-binding cassette family, and lead to an increased P-gp substrate intracellular concentration. Cetuximab is given with irinotecan to patients with metastasis colorectal cancer who did not respond to irinotecan-based therapy. The mechanism of this successful clinical reversion remains unknown. As irinotecan is a P-gp substrate, we tested here whether cetuximab could modify irinotecan concentration in mice. Therefore, concentrations of irinotecan and of its active metabolite SN-38 were measured by HPLC in plasma and tumour of mice bearing a human colorectal carcinoma xenograft when irinotecan is given orally alone or after a pretreatment with cetuximab. Pharmacokinetic analysis showed no significant modification of irinotecan concentrations but a significant increase (1.7-fold) in SN-38 AUCs in plasma and in tumour after a pretreatment with cetuximab. Those results suggest that cetuximab influence irinotecan distribution into tissues probably due to inhibition of P-gp. As SN-38 is 200-fold more potent than irinotecan, cetuximab could reverse irinotecan resistance by an effect on its active metabolite. Inhibiting SN-38 efflux by P-gp drug transporters in biliary system and tumour can lead to pharmacokinetic modification and a higher anticancer efficacy.

Clinical observations on associations between the UGT1A1 genotype and severe toxicity of irinotecan.

Severe toxicity is commonly observed in cancer patients receiving irinotecan (CPT-11). UDP glucuronosyltransferase1A1 (UGT1A1) catalyzes the glucuronidation of the active metabolite SN-38 but the relationship between UGT1A1 and severe toxicity remains unclear. Our study aimed to assess this point to guide clinical use of CPT-11. 89 cancer patients with advanced disease received CPT-11-based chemotherapy for at least two cycles. Toxicity, including GI and hematologic toxicity was recorded in detail and UGT1A1 variants were genotyped. Regression analysis was used to analyse relationships between these variables and tumor response. The prevalence of grade III-IV diarrhea was 10.1%, this being more common in patients with the TA 6/7 genotype (5 of 22 patients, 22.7%) (p<0.05). The prevalence of grade III-IV neutropenia was 13.4%and also highest in patients with the TA 6/7 genotype (4 of 22 patients; 18.2%) but without significance (p>0.05). The retreatment total bilirubin levels were significantly higher in TA6/7 patients (mean, 12.75μmol/L) with compared to TA6/6 (mean, 9.92 μmol/L) with p<0.05. Our study support the conclusion that patients with a UGT1A1*28 allele (s) will suffer an increased risk of severe irinotecan-induced diarrhea, whether with mid-or low-dosage. However, the UGT1A1*28 allele (s) did not increase severe neutropenia. Higher serum total bilirubin is an indication that patients UGT1A1 genotype is not wild-type, with significance for clinic usage of CPT-11.

Macromolecular Prodrug That Provides the Irinotecan (CPT-11) Active-Metabolite SN-38 with Ultralong Half-Life, Low Cmax , and Low Glucuronide Formation

We have recently reported a chemical approach for half-life extension that utilizes β-eliminative linkers to attach amine-containing drugs or prodrugs to macromolecules. The linkers release free drug or prodrug over periods ranging from a few hours to over 1 year. We adapted these linkers for use with phenol-containing drugs. Here, we prepared PEG conjugates of the irinotecan (CPT-11) active metabolite SN-38 via a phenyl ether that release the drug with predictable long half-lives. Pharmacokinetic studies in the rat indicate that, in contrast to other SN-38 prodrugs, the slowly released SN-38 shows a very low C(max), is kept above target concentrations for extended periods, and forms very little SN-38 glucuronide (the precursor of enterotoxic SN-38). The low SN-38 glucuronide is attributed to low hepatic uptake of SN-38. These macromolecular prodrugs have unique pharmacokinetic profiles that may translate to less intestinal toxicity and interpatient variability than the SN-38 prodrugs thus far studied.

Abstract OT3-1-08: The PROCEED trial KCSG BR11-01: Phase III multicenter randomized open label study of irinotecan plus capecitabine versus capecitabine in patients previously treated with anthracycline and taxane for HER2 negative metastatic breast cancer

Abstract Background: Most patients with metastatic breast cancer (MBC) experience disease progression after being treated with an anthracycline or taxane. Irinotecan, a semisynthetic agent derived from the natural alkaloid camptothecin is metabolized to the active metabolite SN-38 which targets topoisomerase I leading to single and double strand DNA breaks. Irinotecan as a single agent demonstrated tumor activity with an objective response rate ranging from 5 to 23% in patients with MBC refractory to taxane and anthracycline. Irinotecan increased the activity of 5-FU, the active metabolite of capecitabine, and overcomes the negative effect of thymidylate synthase overexpression, which is the main target of an active metabolite of 5-FU. A phase II study that evaluated the efficacy and safety of irinotecan and capecitabin combination (IX) showed that the median progression free survival (PFS) was 7.6 months (95% CI, 5.0-10.2months), and the median OS was 22.6 months (95% CI, 15.4 – 29.8 months) with good tolerability in anthracycline and taxane pretreated MBC patients. Based on these results, we planned to conduct a multicenter, randomized phase III study which assesses the efficacy of irinotecan and capecitabine combination therapy compared with capecitabine alone in patients with anthracycline and taxane resistant MBC. Methods: In this trial, patients with HER2 normal tumor who previously received anthracycline and taxane based chemotherapies are enrolled. Eligible patients are randomly assigned in a 1:1 ratio to receive irinotecan plus capecitabine or capecitabine alone. The primary end point of this trial is PFS and a total number of accrual patients will be 222. Randomization is done using a random block size permutation method and stratified by hormone receptor status (negative vs. positive), first line vs. ≥second lines, visceral metastasis (negative vs. positive). Patients receive irinotecan at 80 mg/m2 on day 1 and 8 every 3 weeks and capecitabine 1000mg/m2 bid from day 1 to day 14 every 3 weeks. In control arm, patients receive capecitabine 1250mg/m2 bid from day 1 to day 14 every 3 weeks. Response will be assessed using RECIST1.1 criteria and toxicity will be graded according to NCI-CTCAE 4.0 criteria. Study Status: A total of 107 patients consented for the study since June 2011, and accrual is ongoing. Clinical trial information: NCT01501669. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr OT3-1-08.

In vitro cytotoxicity, pharmacokinetics and tissue distribution in rats of MXN-004, a novel conjugate of polyethylene glycol and SN38

1. MXN-004 is a water-soluble PEGylated 7-ethyl-10-hydroxy-camptothecin (SN38). The aim of this study was to evaluate the in vitro cytotoxicity of MXN-004 and investigate pharmacokinetics and tissue distribution of MXN-004 and its active metabolite SN38 in rats.2. In vitro cytotoxicity of MXN-004 was tested in A549, HepG2 and Caco-2 cancer cell lines by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and compared with irinotecan. The pharmacokinetics and tissue distribution of MXN-004, irinotecan and their identical active metabolite SN38 were investigated after intravenous administration of MXN-004 and irinotecan at a same dose level of 16 μmol/kg in rats.3. In vitro cytotoxicity study showed that MXN-004 was more potent in comparison with irinotecan. In rats, MXN-004 exhibited a longer half-life (sixfold) and much greater Vss as compared with irinotecan. The AUC0–∞, T1/2 and Cmax of SN38 after intravenous administration of MXN-004 were higher than those of irinotecan (3.5-, 1.92- and 10.6-fold, respectively). In addition, the concentrations of SN38 released from MXN-004 were significantly higher in all tissues than those from irinotecan, especially in the lung.4. These results suggested that MXN-004 might be a more potential water-soluble antitumor agent with prolonged half-life of SN38 compared to irinotecan.

Neural Stem Cell-Mediated Delivery of Irinotecan-Activating Carboxylesterases to Glioma: Implications for Clinical Use

CPT-11 (irinotecan) has been investigated as a treatment for malignant brain tumors. However, limitations of CPT-11 therapy include low levels of the drug entering brain tumor sites and systemic toxicities associated with higher doses. Neural stem cells (NSCs) offer a novel way to overcome these obstacles because of their inherent tumor tropism and ability to cross the blood-brain barrier, which enables them to selectively target brain tumor sites. Carboxylesterases (CEs) are enzymes that can convert the prodrug CPT-11 (irinotecan) to its active metabolite SN-38, a potent topoisomerase I inhibitor. We have adenovirally transduced an established clonal human NSC line (HB1.F3.CD) to express a rabbit carboxylesterase (rCE) or a modified human CE (hCE1m6), which are more effective at converting CPT-11 to SN-38 than endogenous human CE. We hypothesized that NSC-mediated CE/CPT-11 therapy would allow tumor-localized production of SN-38 and significantly increase the therapeutic efficacy of irinotecan. Here, we report that transduced NSCs transiently expressed high levels of active CE enzymes, retained their tumor-tropic properties, and mediated an increase in the cytotoxicity of CPT-11 toward glioma cells. CE-expressing NSCs (NSC.CEs), whether administered intracranially or intravenously, delivered CE to orthotopic human glioma xenografts in mice. NSC-delivered CE catalyzed conversion of CPT-11 to SN-38 locally at tumor sites. These studies demonstrate the feasibility of NSC-mediated delivery of CE to glioma and lay the foundation for translational studies of this therapeutic paradigm to improve clinical outcome and quality of life in patients with malignant brain tumors.

Population pharmacokinetics of PEGylated liposomal CPT-11 (IHL-305) in patients with advanced solid tumors

To investigate pharmacokinetics (PK) of encapsulated CPT-11, released CPT-11 and the active metabolite SN-38 following administration of IHL-305 and to identify factors that may influence IHL-305 PK. Plasma samples from 39 patients with solid tumors were collected in a phase I study. IHL-305 was administered as a 1 h IV infusion with doses ranging from 3.5 to 210 mg/m(2). Plasma concentrations of encapsulated CPT-11, released CPT-11 and SN-38 were used to develop a population PK model using NONMEM®. PK of encapsulated CPT-11 was described by 1-compartment model with nonlinear clearance and PK of released CPT-11 was described by a 1-compartment model with linear clearance for all patients. PK of the active metabolite SN-38 was described by a 2-compartment model with linear clearance for all patients. Covariate analysis revealed that gender was a significant covariate for volume of distribution of encapsulated CPT-11. Vencap in male patients is 1.5-fold higher compared with female patients. The developed population PK modeling approach is useful to predict PK exposures of encapsulated and released drug and can be applied to the more than 300 other nanoparticle formulations of anticancer agents that are currently in development. The effect of gender on PK of IHL-305 needs to be further evaluated.

Abstract 975: Sorafenib overcomes irinotecan resistance in colorectal cancer by inhibiting the ABCG2 drug-efflux pump..

Abstract Background: Despite recent advances in the treatment of colorectal cancer, tumor resistance is a frequent cause of chemotherapy failure. Thus, new treatment options are needed to improve survival of patients with irinotecan-refractory colorectal cancers, particularly those bearing KRAS mutations that preclude the use of anti-EGFR therapies. In this study, we investigated whether sorafenib could reverse irinotecan resistance, thereby enhancing the therapeutic efficacy of routinely used irinotecan-based chemotherapy. Materials and methods: We used both in vitro (the HCT116, SW48, SW620 and HT29 colon adenocarcinoma cell lines and four SN-38 resistant HCT-116 and SW48 clones) and in vivo models (nude mice xenografted with SN-38 resistant HCT116 cells) to test the efficacy of sorafenib alone or in combination with irinotecan, or its active metabolite SN-38. Results: Sorafenib improved the anti-tumoral activity of irinotecan in vitro, in both parental and SN-38 resistant colon adenocarcinoma cell lines independently of their KRAS status, as well as in vivo, in xenografted mice. By inhibiting the drug-efflux pump ABCG2, sorafenib favors irinotecan intracellular accumulation and enhances its toxicity. Conclusion: Our results show that sorafenib can suppress resistance to irinotecan and suggest that sorafenib could be used to overcome resistance to irinotecan-based chemotherapies in colorectal cancer, particularly in KRAS mutated tumors. Citation Format: Thibault Mazard, Annick Causse, Joelle Simony, Wilhem Leconet, Marta Jarlier, Marc Ychou, Maguy Del Rio, Pierre Martineau, Bruno Robert, Céline Gongora. Sorafenib overcomes irinotecan resistance in colorectal cancer by inhibiting the ABCG2 drug-efflux pump. [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 975. doi:10.1158/1538-7445.AM2013-975

Abstract 225: A c-Met inhibitor increases the chemosensitivity of cancer stem cells to the irinotecan active metabolite SN38 in gastric carcinoma.

Abstract Purpose: Gastric cancer remains a major global health threat and most patients with advanced stage disease require chemotherapy. The development of drug resistance is a major obstacle in the treatment of gastric cancer and only few effective therapies for combating chemoresistance are currently available. It has been demonstrated that a small subset of cancer cells with stem cell properties, referred to as “cancer stem cells” (CSCs), survive intensive anticancer therapies better than proliferating progenitor cells or differentiated tumor cells. CSCs have been reported to be postulated mediators of chemoresistance, so it might be important to comprehend the drug resistance mechanisms of CSCs to develop a promising therapy to combat chemoresistance. The stemness signal might be associated with the chemosensitivity of CSCs. In the present study, we analyzed the effect of c-Met inhibitors on the chemosensitivity of stem-like cancer cells in gastric cancer. We demonstrated that a c-Met inhibitor synergistically increased the antitumor activity of SN38 in CSCs. To determine mechanisms underlying this observed synergism, we observed that a c-Met inhibitor combined with SN38 also led to a significant increase in UGT1A1 and its subsequent interaction with apoptosis-related genes, i.e., bcl-2 and caspase-6. Experimental Design: We used three gastric cancer cell lines and three side population (SP)-enriched cell lines. We used three signal inhibitors, c-Met inhibitor SU11274, GSK3β inhibitor AR-A014418, and mTOR inhibitor rapamycin, and five anticancer drugs. We examined the combined effects of signal inhibitors and anticancer drugs on proliferation, mRNA expression, and cell cycle. Results: The IC50 of irinotecan, oxaliplatin, taxol, and gemcitabine in SP cells were 10.5, 2.0, 2.8, and 2.0 times higher than their parent cells, respectively. In contrast, the IC50 of 5-fluorouracil did not differ between the two cell lines. There was a synergistic anti-proliferative effect with a combination of c-Met inhibitor and SN38 in SP cells. In contrast, the GSK3β inhibitor and mTOR inhibitor had no synergistic effects in combination with any anticancer drugs. The SP cell lines had higher expression levels of UGT1A1, ABCG2, and ABCB1 than their parent cell lines, while the c-Met inhibitor significantly decreased the expression of UGT1A1, but not ABCG2 and ABCB1. G0-phase of SP cells was higher than their parent cells. c-Met inhibition induced S-phase arrest in SP cells. Conclusions: CSCs are associated with multiresistance during chemotherapy. c-Met inhibitors could be a novel strategy to overcome chemoresistance. c-Met inhibitor may be a promising target molecule for irinotecan-based chemotherapy of gastric cancer. A UGT1A1 alteration might be involved in the irinotecan refractory process in CSCs. Citation Format: Masakazu Yashiro, Tatsunari Fukuoka, Haruhito Kinoshita, Takafumi Nishii, Tsuyoshi Hasegawa, Taro Matsuzaki, Tamami Morisaki, Kosei Hirakawa. A c-Met inhibitor increases the chemosensitivity of cancer stem cells to the irinotecan active metabolite SN38 in gastric carcinoma. [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 225. doi:10.1158/1538-7445.AM2013-225

Abstract 3353: Effect of erlotinib on the pharmacokinetics of irinotecan and its metabolites: A drug-drug interaction study.

Abstract Purpose: Since irinotecan and erlotinib are used in treatment for non-small cell lung cancer (NSCLC), co-administration of these drugs might be an effective treatment for NSCLC. However, drug-drug interaction should be concerned because these drugs are metabolized by CYP3A4 and excreted into bile by ABC transporters. To clarify the drug-drug interaction when these drugs are co-administered, we examined the effect of this co-administration on the pharmacokinetics of irinotecan, its active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38), SN-38 glucuronide (SN-38G) in animal model. Methods: Cytotoxicity of irinotecan and SN-38 with erlotinib against NSCLC cell line (A549) was assessed. To elucidate the effect of drug-drug interaction between irinotecan and erlotinib, male Sprague-Dawley rats were treated with 10 mg/kg irinotecan and 25 mg/kg erlotinib. The concentrations of irinotecan, SN-38, SN-38G and erlotinib in plasma, bile, and tissues were analyzed by high-performance liquid chromatography. Pharmacokinetic parameters were calculated by non-compartmental model methods. Results: Co-exposured erlotinib enhanced the cytotoxicity of SN-38 in a dose-dependent manner, however it did not significantly affect the cytotoxicity of irinotecan against A549 that is resistant to erlotinib. Additionally, co-administration of erlotinib resulted in a significant increase of plasma concentration of irinotecan, SN-38 and SN-38G compared to irinotecan alone, but decrease in the biliary excretion. Erlotinib significantly increased the concentration of irinotecan and SN-38G in kidney and that of SN-38 in lung and jejunum. On the other hand, co-administration of irinotecan intended to decrease the plasma concentration of erlotinib but significant differences of pharmacokinetic parameters were not observed. Also, levels of erlotinib in tissues were not affected by co-administration of irinotecan. Conclusions: We proposed the possibility of combination therapy of irinotecan with erlotinib because the plasma concentration of irinotecan and its active metabolite SN-38 significantly increased in vivo and the cytotoxicity of SN-38 against NSCLC cell line enhanced. New effective salvage chemotherapy regimens are needed in patients with NSCLC. We conducted a phase I/II clinical study of combination chemotherapy with irinotecan and erlotinib for recurrent NSCLC because our data have shown synergistic/additive effects between irinotecan and epidermal growth factor receptor-tyrosine kinase inhibitor. Citation Format: Emiko Sanematsu, Issei Toyooka, Yuki Takashima, Tamio Okimoto, Yukari Tsubata, Hideyuki Saito, Takeshi Isobe, Akinobu Hamada. Effect of erlotinib on the pharmacokinetics of irinotecan and its metabolites: A drug-drug interaction study. [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 3353. doi:10.1158/1538-7445.AM2013-3353

Gimatecan and other camptothecin derivatives poison Leishmania DNA-topoisomerase IB leading to a strong leishmanicidal effect

The aim of this work is the in vitro and ex vivo assessment of the leishmanicidal activity of camptothecin and three analogues used in cancer therapy: topotecan (Hycantim®), gimatecan (ST1481) and the pro-drug irinotecan (Camptosar®) as well as its active metabolite SN-38 against Leishmania infantum. The activity of camptothecin and its derivatives was studied on extracellular L. infantum infrared-emitting promastigotes and on an ex vivo murine model of infected splenocytes with L. infantum fluorescent amastigotes. In situ formation of SDS/KCl precipitable DNA–protein complexes in Leishmania promastigotes indicated that these drugs are DNA topoisomerase IB poisons. The inhibitory potency of camptothecin derivatives on recombinant L. infantum topoisomerase IB was assessed in vitro showing that gimatecan is the most active compound preventing the relaxation of supercoiled DNA at submicromolar concentrations. Cleavage equilibrium assays in Leishmania topoisomerase IB show that gimatecan changes the equilibrium towards cleavage at much lower concentrations than the other camptothecin derivatives and that this effect persists over time. Gimatecan and camptothecin were the most powerful compounds preventing cell growth of free-living L. infantum promastigotes within the same concentration range. All these compounds killed L. infantum splenocyte-infecting amastigotes within the nanomolar range. The amastigote form showed higher sensitivity to topoisomerase IB poisons (with high therapeutic selectivity indexes) than free-living promastigotes. All the compounds assayed poisoned L. infantum DNA topoisomerase IB leading to a strong leishmanicidal effect. Camptothecin derivatives are suitable for reducing the parasitic burden of ex vivo infected splenocytes. The selectivity index of gimatecan makes it a promising drug against this neglected disease.

Irinotecan Resistance is Accompanied by Upregulation of EGFR and Src Signaling in Human Cancer Models

Irinotecan is a major drug for treatment of metastatic colorectal cancer and a promising agent for other applications like gastric cancer. Its clinical activity is currently limited by both intrinsic (natural) and acquired drug resistance. A better understanding of the underlying resistance mechanisms is needed to develop novel therapeutic strategies. Exposure of tumor cells to irinotecan or its active metabolite SN-38 is accompanied by EGFR activation, either by stimulation of EGFR autophosphorylation or by Src-mediated phosphorylation. Accordingly, combinations of irinotecan and EGFR inhibitors have been associated with supra-additive activity. We now show that acquired resistance to SN-38 is accompanied by increased expression of EGFR, HER2, HER3 and Src proteins in two colorectal cancer cell models as well as by Src activation. One SN-38 resistant model (HT-29) showed increased sensitivity to erlotinib, an EGFR inhibitor, and afatinib, a dual EGFR/HER2 inhibitor, while the other SN-38 resistant model (HCT-116) showed increased resistance to erlotinib but unchanged or increased sensitivity to afatinib. Unexpectedly, both models showed increased or unaltered resistance to the Src inhibitor dasatinib. Therefore, tyrosine kinase upregulation is not necessarily accompanied by increased sensitivity to targeted agents. Taken together, our findings demonstrate that prolonged exposure to topoisomerase I inhibitors is accompanied by upregulation of different signal transduction pathways which can alter tumor sensitivity to molecular targeted agents. These results suggest that chemotherapy exposure may lead to creation of novel targets which could be exploited therapeutically.

Prediction of pathologic response of colorectal liver metastases (CRLM) to transcatheter hepatic therapy with irinotecan-eluting beads by hepatic activation of irinotecan.

438 Background: For patients with CRLM, good pathologic response to neoadjuvant chemotherapy is associated with improved overall survival. However, systemic therapy can increase postoperative morbidity and mortality. DEBIRI gives sustained delivery of drug directly to tumor, maximising response and reducing systemic exposure. Predictive markers of response would allow selection of patients most likely to benefit. This study assessed drug metabolism within normal hepatic parenchyma as a predictor of pathologic response of CRLM to DEBIRI. Methods: Patients with resectable CRLM received single targeted DEBIRI (up to 200mg) 1 month pre-hepatectomy. Hepatic parenchyma was harvested at resection. Parenchymal drug metabolism and protein expression were characterised with a novel LC-MS/MS assay and western blot, and correlated with pathologic tumour response. Results: Ten patients assessed. Median residual tumour 30% (range 10-50%), necrosis 40% (range 40-60%) and fibrosis 20% (range 10-40%). Microsomal formation of SN-38 was 0.52-2.19 nmol/mg/min (mean 1.02), SN-38G 0.30-0.59 nmol/mg/min (mean 0.43), APC 0.1-0.24 nmol/mg/min (mean 0.18). There was a negative correlation between SN-38 production and degree of SN-38 glucuronidation (r2=0.42, p=0.04), with no difference between heterozygous/wild-type UGT1A1*28 patients. SN-38 production demonstrated a strong correlation with CES-2 expression (r2=0.96, p&lt;0.001), with a strong correlation between the glucuronidation of SN-38 and UGT1A1 expression (r2=0.76, p=0.01). A moderate correlation between microsomal SN-38 production and degree of tumour necrosis was obtained (r2=0.30, p=0.07). A highly significant correlation between microsomal SN-38 production and degree of tumour fibrosis was observed (r2=0.54, p=0.01). Conclusions: Metabolism of irinotecan varies widely. Production of the active metabolite SN-38 is directly mediated by CES-2, and hepatic production of SN38 predicts degree of tumour fibrosis after DEBIRI. Metabolic profiling may offer a novel predictor of response to DEBIRI.

Irinotecan Resistance is Accompanied by Upregulation of EGFR and Src Signaling in Human Cancer Models

Irinotecan is a major drug for treatment of metastatic colorectal cancer and a promising agent for other applications like gastric cancer. Its clinical activity is currently limited by both intrinsic (natural) and acquired drug resistance. A better understanding of the underlying resistance mechanisms is needed to develop novel therapeutic strategies. Exposure of tumor cells to irinotecan or its active metabolite SN-38 is accompanied by EGFR activation, either by stimulation of EGFR autophosphorylation or by Src-mediated phosphorylation. Accordingly, combinations of irinotecan and EGFR inhibitors have been associated with supra-additive activity. We now show that acquired resistance to SN-38 is accompanied by increased expression of EGFR, HER2, HER3 and Src proteins in two colorectal cancer cell models as well as by Src activation. One SN-38 resistant model (HT-29) showed increased sensitivity to erlotinib, an EGFR inhibitor, and afatinib, a dual EGFR/HER2 inhibitor, while the other SN-38 resistant model (HCT-116) showed increased resistance to erlotinib but unchanged or increased sensitivity to afatinib. Unexpectedly, both models showed increased or unaltered resistance to the Src inhibitor dasatinib. Therefore, tyrosine kinase upregulation is not necessarily accompanied by increased sensitivity to targeted agents. Taken together, our findings demonstrate that prolonged exposure to topoisomerase I inhibitors is accompanied by upregulation of different signal transduction pathways which can alter tumor sensitivity to molecular targeted agents. These results suggest that chemotherapy exposure may lead to creation of novel targets which could be exploited therapeutically. Keywords: Topoisomerase I, EGFR, Src, irinotecan, SN-38, erlotinib, afatinib, dasatinib, colorectal cancer, autophosphorylation

Irinotecan Resistance is Accompanied by Upregulation of EGFR and Src Signaling in Human Cancer Models

Irinotecan is a major drug for treatment of metastatic colorectal cancer and a promising agent for other applications like gastric cancer. Its clinical activity is currently limited by both intrinsic (natural) and acquired drug resistance. A better understanding of the underlying resistance mechanisms is needed to develop novel therapeutic strategies. Exposure of tumor cells to irinotecan or its active metabolite SN-38 is accompanied by EGFR activation, either by stimulation of EGFR autophosphorylation or by Src-mediated phosphorylation. Accordingly, combinations of irinotecan and EGFR inhibitors have been associated with supra-additive activity. We now show that acquired resistance to SN-38 is accompanied by increased expression of EGFR, HER2, HER3 and Src proteins in two colorectal cancer cell models as well as by Src activation. One SN-38 resistant model (HT-29) showed increased sensitivity to erlotinib, an EGFR inhibitor, and afatinib, a dual EGFR/HER2 inhibitor, while the other SN-38 resistant model (HCT-116) showed increased resistance to erlotinib but unchanged or increased sensitivity to afatinib. Unexpectedly, both models showed increased or unaltered resistance to the Src inhibitor dasatinib. Therefore, tyrosine kinase upregulation is not necessarily accompanied by increased sensitivity to targeted agents. Taken together, our findings demonstrate that prolonged exposure to topoisomerase I inhibitors is accompanied by upregulation of different signal transduction pathways which can alter tumor sensitivity to molecular targeted agents. These results suggest that chemotherapy exposure may lead to creation of novel targets which could be exploited therapeutically.

Determination of the Camptothecin Derivatives CPT-11 and SN-38 in Urine and Saliva by Micellar Electrokinectic Chromatography

The anti-cancer synthetic drug irinotecan (CPT-11) and its active metabolite SN-38 have been determined by micellar electrokinetic capillary chromatography (MEKC). The detection of the analytes was made at 368 nm and their separation took less than 7 min using a borate buffer (pH 8.8 at 25 mmol L−1) solution containing sodium dodecyl sulfate (45 mmol L−1) and acetonitrile (13.5% v/v). On-line analyte concentration (normal stacking mode) and the use of a highly sensitive cell (Z shaped cell) improved detection limits (at the 10−8 mol L−1 level). Recovery in fortified human saliva was 108 ± 5%, in agreement with the result achieved with the reference HPLC method. For the analysis of urine from rats submitted to a single dose of CPT-11 and SN-38, camptothecin was used as internal standard enabling recoveries close to 100% when compared to the results achieved using HPLC.

A template model for studying anticancer drug efflux transporter inhibitors in vitro

Efflux transporters play an important role in drug absorption and also in multidrug resistance. ABCG2 (BCRP) is an efflux transporter conferring cross-resistance to mitoxantrone (Mit), irinotecan (CPT11), and its active metabolite SN38. MBLI87, a new ABCG2 inhibitor has proven its efficacy against ABCG2-mediated efflux in vitro and in vivo. This work aimed at modeling and quantifying the cellular interaction between MBLI87 and different substrates using a mechanistic template model. An in vitro competition experiment study was carried out with HEK293 cells overexpressing ABCG2 exposed to fixed concentrations of substrates (Mit, CPT11, SN38) and to MBLI87 at several concentration levels. A nonlinear mixed-effects transport inhibition model was developed to fit intracellular drug concentrations. In this model, drugs cross the cell membrane through passive diffusion, active drug efflux is ABCG2 mediated, interaction between substrates and inhibitor occurs within the transporter. The interaction was found to be noncompetitive. The MBLI87 Ki was estimated to 141 nm for Mit, 289 nm for CPT11, and 1160 nm for SN38. The ratio of intrinsic transport clearance divided by diffusion clearance was estimated to 2.5 for Mit, 1.01 for CPT11, and 5.4 for SN38. The maximal increase in the intracellular substrate concentration that is possible to achieve by inhibition of the transporter was estimated to 1.5 for Mit, 0.1 for CPT11, and 4.4 for SN38. This mechanistic template model describes both drug accumulation and cellular transport, and the mixed-effects approach allows an estimation of intra- and interassay variability. This model is of great interest to study cytotoxic cellular pharmacokinetics.

An investigator-initiated monocentric phase I dose escalation trial of temsirolimus and irinotecan (TEMIR) in patients with relapsing glioblastoma multiforme (reGBM).

2086 Background: Deregulation of the PI3K/AKT pathway has been preclinically involved in the pathophysiology of GBM. The mammalian target of rapamycin (mTOR) is an important downstream mediator of PI3K/AKT signalling. The mTOR-inhibitor temsirolimus (Tem) achieves significant drug levels in brain. Thus, Tem may be effectively combined with irinotecan (Iri) which has shown promising results in combination with bevacizumab in patients (pts) with reGBM refractory to temozolomide (Tmz) in phase II. Exposure to Tem as well as Iri is substantially reduced in pts receiving CYP3A4 enzyme-inducing anticonvulsants (CYP3A4-Ind). Methods: TEMIR is a phase I trial of escalating doses of Tem in combination with Iri in pts with reGBM refractory to Tmz depending on the cotreatment with CYP3A4-Ind. In pts without CYP3A4-Ind dose escalation of Tem was performed according to the “3+3 protocol” from 15 via 20 to 25 mg iv wkly together with 85 mg/m2 Iri iv wkly. In pts with CYP3A4-Ind the doses of Tem were 30, 40 and 50 mg iv wkly together with 170 mg/m2 Iri iv wkly, respectively. The determination of the pharmacokinetics (PK) of Tem and its influence on the PK of Iri and its active metabolite SN38 was performed by validated RP-HPLC methods with fluorescence detection. Results: Up to now, 14 pts [4 female and 10 male, median age 47 yrs (range 41-65 yrs), 9 without and 5 with CYP3A4-Ind] have been treated. All 9 pts without CYP3A4-Ind (3 pts on 15, 20 and 25 mg Tem each) are evaluable for safety with only mild toxicity [highest CTC-grade ≥2 (gr) per pt]: diarrhea 4 (gr2) and 1 (gr3), neutropenia 1 (gr3), thrombopenia 1 (gr2), anemia 1 (gr2), pneumonia 2 (gr2), rash 1 (gr2). PK parameters of Iri and SN38 were in agreement with previously published studies and there was no significant effect of 15 to 25 mg Tem combined with 85 mg/m2 Iri on the PK of Iri and SN38. In 7 pts evaluable for efficacy there was no remission (RECIST) and median time to symptomatic and/or MRT progression was 10 wks (7, 9+, 10, 10+, 11, 15, 20 wks). Conclusions: The combination of Tem in standard dose of 25 mg iv wkly with 85 mg/m2 Iri wkly is safe in pts with reGBM without CYP3A4-Ind. Tem has no significant effect on the PK of Iri and SN38.