Water-soluble Camptothecin Analog Research Articles

Dissociation of NSC606985 induces atypical ER-stress and cell death in prostate cancer cells.

Castration-resistant prostate cancer (CRPC) is a major cause of prostate cancer (Pca) death. Chemotherapy is able to improve the survival of CRPC patients. We previously found that NSC606985 (NSC), a highly water-soluble camptothecin analog, induced cell death in Pca cells via interaction with topoisomerase 1 and activation of the mitochondrial apoptotic pathway. To further elucidate the role of NSC, we studied the effect of NSC on ER-stress and its association with NSC-induced cell death in Pca cells. NSC produced a time- and dose-dependent induction of GRP78, CHOP and XBP1s mRNA, and CHOP protein expression in Pca cells including DU145, indicating an activation of ER-stress. However, unlike conventional ER-stress in which GRP78 protein is increased, NSC produced a time- and dose-dependent U-shape change in GRP78 protein in DU145 cells. The NSC-induced decrease in GRP78 protein was blocked by protease inhibitors, N-acetyl-L-leucyl-L-leucylnorleucinal (ALLN), a lysosomal protease inhibitor, and epoxomicin (EPO), a ubiquitin-protease inhibitor. ALLN, but not EPO, also partially inhibited NSC-induced cell death. However, both 4-PBA and TUDCA, two chemical chaperons that effectively reduced tunicamycin-induced ER-stress, failed to attenuate NSC-induced GRP78, CHOP and XBP1s mRNA expression and cell death. Moreover, knockdown of NSC induction of CHOP expression using a specific siRNA had no effect on NSC-induced cytochromec release and NSC-induced cell death. These results suggest that NSC produced an atypical ER-stress that is dissociated from NSC-induced activation of the mitochondrial apoptotic pathway and NSC-induced cell death in DU145 prostate cancer cells.

Biophysical and molecular docking insight into interaction mechanism and thermal stability of human serum albumin isoforms with a semi-synthetic water-soluble camptothecin analog irinotecan hydrochloride

In the present work, we have examined the binding parameters, thermodynamics, and stability of human serum albumin (HSA) isoforms at pH 7.4 and 9.0, using spectroscopic, calorimetric, and molecular docking methods in the presence of water-soluble camptothecin analog irinotecan hydrochloride (CPT-11). We observed that CPT-11 binds to HSA through a static quenching procedure of ground-state complex formation with N-isoform and B-isoform. Hydrogen bond and hydrophobic interactions are the major governing forces that participating in the formation of protein–drug complex. To determine the binding site of CPT-11 within HSA molecules, we also have performed molecular docking experiments. We explored the CPT-11-mediated stability and modulation of HSA by performing dynamic light scattering (DLS) and differential scanning calorimetry (DSC) experiments. DLS and DSC techniques are used to determine the size and the melting point (Tm) of HSA, which was decreased in the presence of CPT-11. Therefore, CPT-11 plays an important role in HSA stability and protein–ligand interactions. The present study provides valuable information in the field of pharmacokinetics, pharmaco-dynamics, and drug discovery.

Abstract 5377: Assessing the chemotherapeutic response of nanoliposomal topotecan in conjunction with radiotherapy on tumor and its microenvironment

Abstract Topotecan (TPT), a highly active, water soluble analogue of camptothecin, is currently being used in the treatment of various localized cancers from different tissue origin including the breast. Topotecan is classically known for its ability to inhibit topoisomerase I activity as well as accumulation of HIF-1α to block upregulation of pro-angiogenic factors. Despite its known anticancer and antiangiogenic properties the clinical use of TPT is limited owing to its rapid clearance and hematological toxicity. Liposomal encapsulation of TPT can potentially increase its efficacy by shielding it from systemic clearance, allowing greater uptake and extended tissue exposure in solid tumors. We hypothesize that tumor cell biology and treatment response would be more informative when done in the presence of stromal components, like endothelial cells. To that end we have developed a 3D in-vitro/ in-vivo murine model of Triple Negative Breast Cancer where 4T1-GFP tumor cells are co-cultured with their stromal component (2H11 murine endothelial cells) in ‘hanging drops’ of medium. In the present study we have determined the effective dose of radiation and TPT for 4T1 tumor and 2H11 endothelial cells by cell viability and clonogenic assays. Based on the observed toxicity treatment response of actively loaded liposomal topotecan (ALLTs) and free drug with or without radiation exposure (3Gy) in tumor-endothelial spheroids was determined by monitoring the spheroid size and quantitation of apoptotic changes observed. 100nm DSPC and m-PEG DSPE (95:5 mol: mol) nanoliposomes were prepared in ammonium besylate and then TPT was actively loaded by pH gradient method. Tumor-endothelial spheroids grown in hanging drops, were subsequently transferred to cell repellent plates for high throughput in-vitro analysis. Our studies indicate that endothelial cells are more sensitive to TPT treatment (IC50 of 0.13µM) than tumor cells (IC50=63µM). Combinatorial treatment with topotecan loaded nanoliposome (ALLT) and radiation of tumor endothelial spheroid reveals a decrease in spheroid size and severe disruption of the peripheral layers with time. ALLTs based chemotherapy in conjunction with radiation was thus observed to be more efficacious in disrupting the integrity, more specifically the extracellular matrix of tumor-endothelial spheroids. Unlike the free drug which gets hydrolyzed into its relatively inactive carboxylate form, we expect that the ability of ALLTs for extended release of active TPT (more than 24 hours) in a metronomic fashion, may contribute to the therapeutic efficacy of its combined treatment with radiation. Efforts are underway to implant these spheroids in mammary fat pad and dorsal skin fold window chamber and study the effect of ALLTs in combination with radiation on tumor growth and neovascularization and metastasis. Supported by NCI grants R25CA153954 and CA173609 Citation Format: Amar Jyoti, Pallavi Sethi, Kyle Fugit, Ulrich Langner, William St. Clair, Ronald C. McGarry, Bradley D. Anderson, Meenakshi Upreti. Assessing the chemotherapeutic response of nanoliposomal topotecan in conjunction with radiotherapy on tumor and its microenvironment. [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 5377. doi:10.1158/1538-7445.AM2014-5377

The Anti-Proliferative and Pro-apoptotic Effect of Topotecan in Combination With Thymoquinone in Acute Myelogenous Leukemia

Topotecan (a novel topoisomerase I inhibitor and a water-soluble camptothecin analog) is active against numerous human tumor cell lines, and has shown promising antineoplastic activity in solid tumors and acute leukemia. Due to the primary dose-limiting toxicity of topotecan which is myelosuppression, it is necessary to identify other chemotherapeutic agents that can work synergistically with topotecan, potentially increasing its efficacy while limiting its toxicity. Many studies showed synergism upon combination of topotecan with other chemotherapeutic agents like Gemcitabine, bortezomin and CP-4055. Other studies report the increase in growth inhibition of gemcitabine or oxaliplatin when cells are pre-exposed to naturally occurring drugs such as thymoquinone.

Practical Synthesis of (20S)‐10‐(3‐Aminopropyloxy)‐7‐ethylcamptothecin, a Water‐Soluble Analogue of Camptothecin

A robust, practical synthesis of (20S)-10-(3-aminopropyloxy)-7-ethylcamptothecin (T-2513, 5), which is a water-soluble analogue of camptothecin, has been developed. The key step in this synthesis is a highly diastereoselective ethylation at the C20 position by using N-arylsulfonyl-(R)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid ester as a chiral auxiliary, which affords the key intermediate ethyl-(S)-2-acyloxy-2-(6-cyano-5-oxo-1,2,3,5-tetrahydroindolizin-7-yl)butanoate (8k) in 93% yield and 87% de. Optically pure compound 8k was obtained by a single recrystallization from acetone and its further elaboration through Friedlander condensation afforded compound 5. This synthesis does not require any chromatographic purification steps and can provide compound 5 on a multi-gram scale in 6.3% overall yield (16 steps).

Preclinical evaluation of the novel 7-substituted camptothecin Namitecan (ST1968) in paediatric tumour models

The present study aimed to evaluate the new water soluble camptothecin analogue Namitecan (ST1968) in preclinical paediatric tumour models of the nervous system comprehensive of neuroblastoma, primitive neuroectodermal tumours/PNET and medulloblastoma where the drug was compared to Irinotecan. Cellular sensitivity to the drug was assessed by MTT and clonogenic assays. Propidium iodide staining was used for cell cycle perturbation studies. The genotoxic effects were quantified by Comet assay, whereas apoptosis was assessed by PARP cleavage and sub-G1 accumulation. Tumour response was investigated in xenograft models in nude mice. The cellular response to Namitecan was heterogeneous with IC(50) (2 h) ranging between 0.14 and 13.26 μM, whereas SN38 (the active metabolite of Irinotecan) appeared more effective (IC(50): 0.03-11.7 μM). Interestingly, prolonged drug incubation times up to 72 h enhanced Namitecan cytotoxicity, with similar colony inhibition curves between the two analogues (IC(50), nM-SN38: 0.9 ± 0.2; Namitecan: 0.7 ± 0.4). DNA damage, accumulation in late-S/G2 phases and induction of apoptosis appeared important players of Namitecan cytotoxicity in our models. In vivo, Namitecan was superior to Irinotecan in three out of five xenograft models, with reversible weight loss (10 %). In the sensitive SK-N-AS xenograft, Namitecan showed a high retention in tumours consistently with: high antitumour response, rapid drug-mediated DNA damage (60 % mean TailDNA after 1 h from drug inoculation), persistent cell cycle perturbation (60-40 % G2 accumulation after 48-72 h, respectively) and apoptosis. Studies with Namitecan and platinum agents in this model showed a significant enhancement of antitumour activity of the drugs combination versus single agents. Our preclinical data strongly support the interest of further investigations on the well-tolerated Namitecan either as a single agent or in combination in paediatric oncology.

Abstract C205: Persistent cell cycle perturbations and apoptosis mediate the antitumor activity of ST1968 in pediatric models.

Abstract Introduction. ST1968 (Namitecan) is a new water-soluble camptothecin analog belonging to the 7-oxyiminomethyl series that has been shown to have activity against a wide spectrum of adult tumor xenografts. Based on its therapeutic efficacy, Namitecan was selected for clinical development. Methods. We investigated the cellular pathways underlying the antitumor activity of ST1968 in the following pediatric tumor models: neuroblastoma (SK-N-DZ, SK-N-AS, SK-N-BE(2)c, HTLA and SHEP-1); primitive neuroectodermal tumors/PNET (PFSK) and medulloblastoma (DAOY). Cell growth inhibitory effect was evaluated with the 5-day MTT assay. The genotoxic potential of the camptothecin analog was evaluated in vitro and in vivo with the alkaline Comet assay. Cell cycle perturbation were studied by conventional propidium iodide staining whereas apoptosis was assayed by hypodiploid cell population and PARP cleavage. For in vivo studies, tumor xenografts were treated with the compounds at the following schedule: i.v. q4d×4, at the concentration of 30mg/kg for ST1968 and 50mg/kg for CPT-11. The in vivo tumor distribution of Namitecan was studied in the responsive neuroblastoma model SK-N-AS that was selected also for preliminary evaluations of drugs combination with platinum agents. Results. In the initial growth inhibition screening ST1968 appeared between 2 and 22-fold less effective than SN38, the active metabolite of irinotecan. However, the cellular response to the novel analog increased with prolonged drug incubation (24 and 72h) in line with the reported evidence that ST1968 uptake is poor and would therefore be favoured by longer exposure times. Overall, we observed a heterogeneous cellular response to ST1968. Cell cycle studies suggested that the enhanced cellular sensitivity to Namitecan in our models appeared mediated by more persistent cell cycle perturbations (late-S/G2 accumulation up to 72h) and increased sub-G1 peak indicative of apoptosis commitment. These effects were likely a consequence of the strong DNA damaging properties of the drug as shown by the rapid kinetic of DNA breaks induction. Interestingly this toxic cellular pathways appeared p53-independent as indicated by the lack of correlation between cellular sensitivity and p53 status of the cell lines from our panel. The antitumor potency of Namitecan was superior to CPT-11 in almost all our xenografts models where the drug was well tolerated. In SK-N-AS xenografts the pharmacokinetic study showed prolonged ST1968 retention in tumors consistently with a rapid and persistent DNA damage induction, G2-block (48–72h) and apoptosis. Combination studies with platinum agents showed a significant enhancement of antitumor activity of the drugs combination vs single agents. Particularly impressive was the observation of a very high rate of complete regression with the ST1868-carboplatin combination that persisted for 25–35 days after the cessation of treatment. Conclusions. Our data confirm the superior activity of Namitecan as compared to irinotecan that is currently utilized in pediatric oncology, strongly supporting further investigations of the novel camptothecin derivative as a single agent or in combination. Supported by FOP. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C205.

Preclinical Results of Camptothecin-Polymer Conjugate (IT-101) in Multiple Human Lymphoma Xenograft Models

Camptothecin (CPT) has potent broad-spectrum antitumor activity by inhibiting type I DNA topoisomerase (DNA topo I). It has not been used clinically because it is water-insoluble and highly toxic. As a result, irinotecan (CPT-11), a water-soluble analogue of CPT, has been developed and used as salvage chemotherapy in patients with relapsed/refractory lymphoma, but with only modest activity. Recently, we have developed a cyclodextrin-based polymer conjugate of 20-(S)-CPT (IT-101). In this study, we evaluated the preclinical antilymphoma efficacy of IT-101 as compared with CPT-11. We determined an in vitro cytotoxicity of IT-101, CPT-11, and their metabolites against multiple human lymphoma cell lines. In human lymphoma xenografts, the pharmacokinetics, inhibitions of tumor DNA topo I catalytic activity, and antilymphoma activities of these compounds were evaluated. IT-101 and CPT had very high in vitro cytotoxicity against all lymphoma cell lines tested. As compared with CPT-11 and SN-38, IT-101 and CPT had longer release kinetics and significantly inhibit higher tumor DNA topo I catalytic activities. Furthermore, IT-101 showed significantly prolonged the survival of animals bearing s.c. and disseminated human xenografts when compared with CPT-11 at its maximum tolerated dose in mice. The promising present results provide the basis for a phase I clinical trial in patients with relapsed/refractory lymphoma.

Clinical development of namitecan (ST1968), a novel camptothecin derivative with high antitumor activity: Phase I clinical data

2570 Background: Namitecan is a new water-soluble camptothecin analogue which showed high antitumor activity in preclinical models. Aim of this trial was to determine safety, PK profile and activity in adult patients with advanced solid tumors. Methods: The dose escalation started at 2.5 mg i.v. on days 1 and 8 of a 21 day cycle (D1, D8 Q21D) and increased according to 3+3 cohort design depending on the observed toxicity. Dose limiting toxicity (DLT) definitions were: ANC <0.5x109/L for >5 days; PLT ≥ Grade 3 (CTC V3); grade ≥2 liver/renal toxicity not recovered by D22; any non-hematologic toxicity ≥ Grade 3; D8 dose skipping due to toxicity. Maximum tolerated dose (MTD) and recommended dose (RD) were the primary end-points. Blood and urine samples were collected at cycle 1 for PK evaluation. Results: 31 pts (11 endometrial ca., 5 CRC, 5 ovarian ca., 2 NSCLC, 8 other) have been included, with 6 dose levels evaluated (2.5; 5; 10; 15; 17.5 and 20 mg). 17.5 mg was introduced later when 2/7 DLTs at 20 mg were observed (ANC G4>5days, one with D8 skipping). At 17.5mg 2/4 pts experienced DLTs (ANC G4; D8 skipped). Uncomplicated neutropenia and thrombocytopenia were the most relevant G3/4 hematological toxicities. Other toxicities were mild or moderate asthenia, fatigue and alopecia. The MTD was defined at 17.5 mg and the RD was 15 mg. Stable disease ≥ 6 cycles was recorded in 6 pts (2 stable diseases ≥ 10 cycles). PK was linear and data suggest an entero-hepatic recirculation. No metabolites were found in plasma and the product resulted poorly excreted into urine. Conclusions: The MTD and RD of D1, D8 Q21D schedule have been identified. The study will continue with the evaluation of MTD and RD of a single administration per cycle (D1 Q21D), to optimize the schedule of treatment. [Table: see text]

NSC606985, a novel camptothecin analog, induces apoptosis and growth arrest in prostate tumor cells

Prostate cancer is a major cause of cancer mortality in American males. Once prostate cancer has metastasized, there is currently no curative therapy available. The development of effective agents is therefore a continuing effort to combat this disease. In the present study, the effects and potential mechanisms of NSC606985 (NSC), a water-soluble camptothecin analog, in prostate cancer cells were investigated. Prostatic tumor cells, DU-145, LNCaP and PC-3, were used for the study. Cell proliferation, cell cycle, cell apoptosis and caspase 3/7 activity were determined in the presence or absence of NSC. The levels of Bax and Bak, and the release of cytochrome c from mitochondria were analyzed by Western blot. Treatment with NSC at nanomolar concentrations produced a time- and dose-dependent decrease in viable cell numbers of multiple prostate cancer cells. In DU-145 cells, NSC produced a time-and dose-dependent induction of cell apoptosis and cell cycle arrest as evidenced by cell morphological changes, increases in S-phase and sub-G1 cell fractions, an elevation of caspase 3/7 activity, DNA fragmentation and apoptotic cells. NSC increased the levels of apoptotic proteins, Bax and Bak, and induced a release of cytochrome c from mitochondria to cytosol in DU-145 cells. Co-administration of Z-VAD-FMK, a pan-caspase inhibitor, blocked NSC-induced caspase 3/7 activity and cell apoptosis without affecting NSC-induced cell cycle arrest. In contrast, co-administration of a PKCdelta inhibitor, rottlerin, had no significant effect on NSC induction of caspase activity, and slightly potentiated NSC-induced cell death. Furthermore, like camptothecin, a mutation of topoisomerase 1 that prevents the binding of camptothecin to the enzyme completely abolished the NSC effect in DU-145 cells. The data obtained suggest that NSC is able to decrease cell growth, induce cell apoptosis and cause growth arrest in prostatic tumor cells, which may involve an interaction with topoisomerase 1 and an activation of mitochondrial apoptotic pathway.

CPT21, a novel compound with anti-proliferative effect against gastric cancer cell SGC7901

7-[(3-piperidyl)-1-propinyl]-camptothecin (CPT21) is a novel semi-synthetic water-soluble analogue of camptothecin. In this context, we assessed the anti-tumor activity of CPT21 both in vivo and in vitro and explored its molecular mechanism. We found that CPT21 presented a broad anti-tumor spectrum against ten cancer cell lines in vitro, and the IC(50) values ranged from 0.1 to 12.0 microM. CPT21 was also capable to interrupt the DNA topoisemerase I activity and caused DNA double strand breaks during DNA replication. Proportion of apoptotic SGC7901 cells induced by CPT21 showed a time- and concentration-dependent increase accompanied with the decrease in mitochondria membrane potential (DeltaPsim). We also observed that CPT21 up-regulated the protein expression of p53, phospho-p53, p21, BAX, phospho-c-Jun NH2-terminal protein kinase (JNK), meanwhile down-regulating the protein expression of Bcl-2, procaspase-9, XIAP, and phospho-ERK1/2. In the study of SGC7901 xenograft model, the results suggested that both 5.0 mg/kg and 10.0 mg/kg CPT21 achieved high anti-tumor activity, and the tumor inhibition rates were 42.5% and 75.1% respectively. Taken together, our study demonstrates that CPT21 displays an extensive anti-tumor spectrum and CPT21 can induce the apoptosis of SGC7901 cells via activating the caspases cascade followed by disrupting mitochondrion function.

Preclinical Results of the Camptothecin-Polymer Conjugate IT-101 in Multiple Human Lymphoma Xenografts.

Camptothecin (CPT), a natural alkaloid isolated from Camptotheca acuminata, has potent broad spectrum antitumor activity by inhibiting type I DNA topoisomerase. It has not been used clinically because it is water-insoluble and highly toxic. As a result, irinotecan (CPT-11), a water-soluble analogue of CPT, has been developed and used as salvage chemotherapy in patients with refractory/relapsed lymphoma, but with only modest activity. For intravenous administration, we have developed IT-101, a ca. 40 nm diameter nanoparticle that is an assembly of cyclodextrin-based polymer conjugates of 20-(S)-CPT. The purpose of this study is to compare the preclinical efficacy of IT-101 with CPT-11 both in vitro and in vivo. Fluorescence microscopy studies demonstrated that incubation of a human lymphoma cell line with IT-101 resulted in the uptake and intracellular accumulation of CPT. Based on the demonstration of in vitro cytotoxity of IT-101 against multiple human lymphoma cell lines, we initiated experiments in xenograft models of lymphoma. In subcutaneous human xenograft models, a single cycle of three weekly doses of intravenous IT-101 at 10 mg/kg (CPT equivalents) showed significantly potent antitumor activity against Daudi, Karpas 299, and L540 cell lines compared to three weekly doses of intraperitoneal CPT-11 at its maximum tolerated dose in mouse of 100 mg/kg (P < 0.0001, P = 0.0072, and P < 0.0001, respectively). In the same animal models, IT-101 led to pathologically complete remissions in 78%, 44%, and 78% of animals inoculated with Daudi, Karpas 299, and L540 cell lines, respectively. In disseminated human xenograft models, IT-101, administered using the same dosing schedule, significantly prolonged the survival of animals intravenously injected with either Daudi or Karpas 299 cell lines when compared to CPT-11 (P < 0.0001 and P = 0.0049, respectively). The promising present results in a variety of lymphomas provide the basis for a phase I/II clinical trial in patients with refractory/relapsed lymphoma.

655. Development of a Human Carboxylesterase That Can Efficiently Metabolize CPT-11

CPT-11 is a water-soluble camptothecin analogue that has shown remarkable antitumor activity against a variety of tumors in xenograft models and in clinical trials. It is currently approved for the treatment of advanced colorectal cancers, and is likely to be approved for use against a variety of other solid tumors. CPT-11 is a prodrug that is metabolized in vivo by carboxylesterases (CEs) to generate the potent topoisomeraseI inhibitor, SN-38. Among the highly homologous mammalian CEs, a rabbit liver carboxylesterase (rCE) is the most efficient at CPT-11 activation.

Synthesis of 14-Azacamptothecin, a Water-Soluble Topoisomerase I Poison

14-Azacamptothecin, a potent, water-soluble analogue of the antitumor agent camptothecin, has been prepared by a convergent synthesis. The key condensation of the AB and DE rings with concomitant formation of ring C of 14-aza CPT was carried out in two stages, the latter of which involved a radical cyclization strategy. [structure: see text]

On the role of E-ring oxygen atoms in the binding of camptothecin to the topoisomerase I–DNA covalent binary complex

A recent X-ray crystallographic analysis of the binding of a water soluble camptothecin analogue to the human topoisomerase I–DNA covalent binary complex has suggested the existence of some novel features in the way that camptothecin is bound to the binary complex. Four additional models based on chemical and biochemical data have also been proposed. Presently we describe S-containing analogues of camptothecin prepared on the basis of these models, and report their ability to form stable ternary complexes with human topoisomerase I, and to mediate cytotoxicity at the locus of topoisomerase I. The results indicate that replacement of the 20-OH group of CPT with a SH functionality results in diminution of the potency of CPT as a topoisomerase I poison, while replacement of the O atoms at positions 20 and 21 with S atoms results in essentially complete loss of topoisomerase I inhibitory activity.

Phase II study of exatecan mesylate (DX-8951f) as first line therapy for advanced non-small cell lung cancer

Background: Exatecan mesylate (DX-8951f) is a water soluble analogue of camptothecin that inhibits topoisomerase I. This multi-centre phase II study evaluated the activity of single agent exatecan in previously untreated patients with advanced non-small cell lung cancer (NSCLC). Patients and methods: Patients with histologically or cytologically proven stage IIIb or IV NSCLC were treated with exatecan 0.5 mg/m 2 per day by 30 min intra-venous (i.v.) infusion for 5 days every 3 weeks to a maximum of six cycles. Measurable disease was documented prior to study entry and patients were re-staged every two cycles. Pharmacokinetic (PK) sampling was performed during cycle one. Results: 39 patients (32 patients ECOG performance status 0 or 1; 29 male and ten female; mean age 63 years) were entered into the study. Thirty-three completed at least two cycles of exatecan and 11 completed six cycles. Two patients (5.1%, 95% C.I. 0.3–21.3%) had a partial response, 7 (18.0%) minor response and 8 (20.5%) stable disease. Median time to tumour progression (TTP) was 88 days and median overall survival 262 days. The main toxicity was reversible neutropenia. PK analysis of exatecan demonstrated a mean clearance of 2.28 l/h per m 2, volume of distribution 18.2 l/m 2 and mean elimination half-life of 7.9 h. Conclusions: Exatecan mesylate has limited activity in advanced NSCLC and is not recommended for further evaluation as a single agent in this tumour type. PK data from this trial supports results established in phase I studies.

Topotecan inhibits VEGF- and bFGF-induced vascular endothelial cell migration via downregulation of the PI3K-Akt signaling pathway.

Angiogenesis plays a crucial role in tumor growth and metastases. The extent of angiogenesis correlates with the increased invasion and metastasis in a variety of human neoplasms. Vascular endothelial cell proliferation and migration are critical steps in angiogenesis and are regulated by various growth factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). The topoisomerase I inhibitor topotecan (10-hydroxy-9-dimethylaminomethyl-(S)-camptothecin) is a water-soluble camptothecin analogue and possesses an indirect in vivo antitumor effect mediated through the inhibition of angiogenesis. We found that topotecan inhibited VEGF- and bFGF-induced migration of human umbilical vein endothelial cells (HUVECs) in vitro. The migration of HUVECs was also inhibited by a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002. Thus, we investigated the possibility that topotecan's antiangiogenic property might be mediated by its inhibitory effect on VEGF- and bFGF-induced activation of the PI3K-Akt signaling pathway. We found that topotecan treatment decreased the amount of the phosphorylated (activated) form of Akt, but not the amount of Akt protein, in HUVECs. Moreover, transient transfection of constitutive active akt cDNA into HUVECs reversed the topotecan-mediated decrease in HUVEC migration. These results suggest that the antiangiogenic activity of topotecan is mediated in part by downregulating the PI3K-Akt signaling pathway.

Successful therapy of subcutaneously growing human hepatoblastoma xenografts with topotecan.

Human hepatoblastoma is an infrequent liver tumor in children. Although many hepatoblastomas can be treated adequately with well-defined treatment regimens, problems still persist with advanced and non-resectable tumors; in these cases, an effective chemotherapy is necessary to improve the patients' prognosis. This underlines the need for alternative anti-tumor agents in the treatment of human hepatoblastoma. The aim of this study was to investigate the therapeutic effects of topotecan, a water-soluble camptothecin analog (topoisomerase-I-antagonist), in an in vivo model of three human hepatoblastomas xenografted subcutaneously into nude mice. Hepatoblastoma cell suspensions from three children were transplanted subcutaneously into nude mice NMRI (nu/nu). Treatment with topotecan was initiated when the tumors reached a volume between 50 and 80 mm(3). A dose of 6.6 mg/kg of topotecan were given intraperitoneally every 4 days on four occasions. The tumor volume development and alpha-fetoprotein alterations were measured and statistically analyzed. After the treatment, the tumors were investigated histologically and by immunohistochemistry. There was a significant reduction of tumor growth in all treated tumor xenografts vs. untreated control groups (mean relative volume 3.1 vs. 47.4; P = 0,0015-0,0079). Serum alpha-fetoprotein levels were reduced in all three cell lines, in two of them significantly (mean 44,535 kU/l vs. 228,883 kU/l; P = 0.005-0.246). Histologically, the tumor necrosis rates were higher and immunohistochemistry showed lower proliferation activities in the treated tumor xenografts vs. the control groups. The data show that topotecan is an effective agent in the treatment of human hepatoblastoma xenografts. From these results, treatment with topotecan appears to be a promising alternative in the pre- and postoperative therapy of patients suffering from human hepatoblastoma

Activity and toxicity of GI147211 in breast, colorectal and non-small-cell lung cancer patients: An EORTC—ECSG phase II clinical study

Background: GI147211 is a water-soluble synthetic analogue of camptothecin showing promising in vivo and in vitro antitumor activity and an acceptable toxicity profile.Patients and methods: Between April 1995 and November 1996, 67 eligible patients with pretreated breast cancer (25 patients) and chemo-naïve colorectal (19 patients) and non-small-cell lung cancer (23 patients) were entered into three multicentric, non-randomized phase II trials. Treatment schedule consisted of intravenous GI147211 administered at a dose of 1.2 mg/m2/day for five consecutive days every three weeks.Results: Hematological toxicity was common with grade 3–4 neutropenia in 54% of patients and neutropenic fever together or not associated with infection in 14.5% of patients. Grade 3–4 thrombocytopenia and grade 2–4 anemia were observed in 20% and in 68% of patients, respectively. Non-hematological toxicity was generally mild to moderate and consisted mainly of gastrointestinal toxicity, asthenia and alopecia. A dose-escalation to 1.5 mg/m2/d was feasible in 17 (25%) patients. The antitumor activity of GI147211 was moderate in breast cancer patients (3 partial responses (PRs), response rate (RR) 13%) and minimal in non-small cell lung cancer patients (2 PRs, RR 9%). No objective responses were obtained in colorectal patients.Conclusions: GI147211, at the dose and schedule employed in this study, showed an acceptable safety profile but a modest antitumor activity in the examined tumor types.

Antitumor activity of 7-[2-(N-isopropylamino)ethyl]-(20S)-camptothecin, CKD602, as a potent DNA topoisomerase I inhibitor

We developed a novel water-soluble camptothecin analogue, CKD602, and evaluated the inhibition of topoisomerase I and the antitumor activities against mammalian tumor cells and human tumor xenografts. CKD602 was a nanomolar inhibitor of the topoisomerase I enzyme in the cleavable complex assay. CKD602 was found to be 3 times and slightly more potent than topotecan and camptothecin as inhibitors of topoisomerase, respectively. In tumor cell cytotoxicity, CKD602 was more potent than topotecan in 14 out of 26 human cancer cell lines tested, while it was comparable to camptothecin. CKD602 was tested for the in vivo antitumor activity against the human tumor xenograft models. CKD602 was able to induce regression of established HT-29, WIDR and CX-1 colon tumors, LX-1 lung tumor, MX-1 breast tumor and SKOV-3 ovarian tumor as much as 80, 94, 76, 67, 87% and 88%, respectively, with comparable body weight changes to those of topotecan. Also the therapeutic margin (R/Emax: maximum tolerance dose/ED58) of CKD602 was significantly higher than that of topotecan by 4 times. Efficacy was determined at the maximal tolerated dose levels using schedule dependent i.p. administration in mice bearing L1210 leukemia. On a Q4dx4 (every 4 day for 4 doses) schedule, the maximum tolerated dose (MTD) was 25 mg/kg per administration, which caused great weight loss and lethality in < 5% tumor bearing mouse. This schedule brought significant increase in life span (ILS), 212%, with 33% of long-term survivals. The ex vivo antitumor activity of CKD602 was compared with that of topotecan and the mean antitumor index (ATI) values recorded for CKD602 were significantly higher than that noted for topotecan. From these results, CKD602 warrants further clinical investigations as a potent inhibitor of topoisomerase I.