Inhibitory effect of combined administration with CPT-11 and 5-fluorouracil in vitro and in vivo

Combined antitumor activity of CPT-11 and 5-fluorouracil (5-FU) was evaluated in a human cultured cell line derived from lung cancer. After 24 h culture with SN-38 followed by 5-FU 24 h, synergistic effect was observed in the cell line. In addition, the antitumor effect of this combination was studied in in vivo experiments using Donryu rat with Yoshida sarcoma cells. CPT-11 and 5-FU synergistically inhibited tumor growth. There was no significant increase of toxicity as assessed by the body weights. These results might support for the combination with 5-FU and CPT-11 in a chemotherapy for cancer.     

The Study of Polyethyleneglycol-Coated Liposomes Containing CPT-11

Abstract

Polyethyleneglycol (PEG) -coated liposomal CPT-11 (PEG-LCPT(11)) was prepared and its pharmaceutical usefulness was examined. These liposomes, plain liposomal CPT-11 (PLCPT(11)) and PEG-LCPT(11), were composed of dimyristoylphosphatidylcholine, cholesterol, and dimyristoylphosphatidylglycerol (10 : 10 : 6, mol/mol) with or without PEG. The mean particle diameters were both about 1 60 nm. The trapping efficiencies were approximately 90%. In a distribution study, CDFl mice were injected with CPT-11 solution (CPT(11)sol), PLCPT(11) and PEG-LCPT(11) at a dose of 10 mg/kg (i.v.). Concentrations in each tissue of CPT-11 and SN-38, the active metabolite of CPT-11, were determined. After the administration, CPT-11 and SN-38 concentrations in the blood increased by liposomal encapsulation (liposomalization), and the circulation time in the blood was prolonged further by PEG-modification of the liposomes (PEGylation). In the liver, PLCPT(11) was rapidly taken up by the reticuloendothelial system (RES), and the uptake was avoided by PEGylation. Tumor accumulations of CPT-11 and SN-38 were accompanied by an increase in antitumor activity of CPT-11 by liposomalization. Thus, the prolongation of the circulation time in the blood by liposomalization and the avoidance of the RES uptake by PEGylation caused passive targeting of the tumor, with a resulting increase in the antitumor activity of CPT-11.     

Impediments to Enhancement of CPT-11 Anticancer Activity by E. coli Directed Beta-Glucuronidase Therapy

CPT-11 is a camptothecin analog used for the clinical treatment of colorectal adenocarcinoma. CPT-11 is converted into the therapeutic anti-cancer agent SN-38 by liver enzymes and can be further metabolized to a non-toxic glucuronide SN-38G, resulting in low SN-38 but high SN-38G concentrations in the circulation. We previously demonstrated that adenoviral expression of membrane-anchored beta-glucuronidase could promote conversion of SN-38G to SN-38 in tumors and increase the anticancer activity of CPT-11. Here, we identified impediments to effective tumor therapy with E. coli that were engineered to constitutively express highly active E. coli beta-glucuronidase intracellularly to enhance the anticancer activity of CPT-11. The engineered bacteria, E. coli (lux/βG), could hydrolyze SN-38G to SN-38, increased the sensitivity of cultured tumor cells to SN-38G by about 100 fold and selectively accumulated in tumors. However, E. coli (lux/βG) did not more effectively increase CPT-11 anticancer activity in human tumor xenografts as compared to non-engineered E. coli. SN-38G conversion to SN-38 by E. coli (lux/βG) appeared to be limited by slow uptake into bacteria as well as by segregation of E. coli in necrotic regions of tumors that may be relatively inaccessible to systemically-administered drug molecules. Studies using a fluorescent glucuronide probe showed that significantly greater glucuronide hydrolysis could be achieved in mice pretreated with E. coli (lux/βG) by direct intratumoral injection of the glucuronide probe or by intratumoral lysis of bacteria to release intracellular beta-glucuronidase. Our study suggests that the distribution of beta-glucuronidase, and possibly other therapeutic proteins, in the tumor microenvironment might be an important barrier for effective bacterial-based tumor therapy. Expression of secreted therapeutic proteins or induction of therapeutic protein release from bacteria might therefore be a promising strategy to enhance anti-tumor activity.     

Cell kinetics-dependent antitumor effect of irinotecan hydrochloride induced by the synchronizing effect of hydroxyurea: cell kinetics and dosing time

Influence of hydroxyurea (HU) on the antitumor effect of irinotecan hydrochloride (CPT-11) was investigated in ICR male mice transplanted with sarcoma 180 cells (S-180). A single dose of CPT-11 (100 mg/kg) was injected at various times after a single dose of HU (300 mg/kg). The relative tumor weight varied significantly depending on the timing of CPT-11 injection after HU injection (P < 0.01). The higher antitumor effect of CPT-11 was observed when DNA synthesis of S-180 cells increased (20 hr), and the lower effect was observed when the DNA synthesis decreased (0 hr). The loss of body weight also varied significantly depending on the timing of CPT-11 injection after HU injection (P < 0.01). The toxicity of CPT-11 was higher when the inhibitory effect of HU on DNA synthesis of bone marrow cells was stronger (15 hr), and the lower toxicity was observed when the inhibitory effect was not observed (0 hr). The plasma SN-38 concentration at 2 hr after CPT-11 injection was higher at 20 hr after HU injection than at 0 hr after HU injection. The difference in plasma esterase activity between 0 hr and 20 hr after HU injection was regarded as the mechanism underlying the dosing time-dependent difference of the SN-38 concentration. These experiments suggest that HU can produce a different phase of cell cycle between tumor cells and normal cells. This leads to increase the antitumor effect of CPT-11 without increasing the adverse effect of the drug. It is essential to consider the dosing time in the two-drug combination therapy.     

Identification and activities of human carboxylesterases for the activation of CPT-11, a clinically approved anticancer drug.

CPT-11 is a clinically approved anticancer drug used for the treatment of advanced colorectal cancer. Upon administration, the carbamate side chain of the drug is hydrolyzed, resulting in the release of SN-38, an agent that has approximately 1000-fold increased cytotoxic activity. Since only a very small percentage of the injected dose of CPT-11 is converted to SN-38, there is a significant opportunity to improve its therapeutic efficacy and to diminish its systemic toxicity by selectively activating the drug within tumor sites. We envisioned that a mAb-human enzyme conjugate for CPT-11 activation would be of interest, particularly since the conjugate would likely be minimally immunogenic, and the prodrug is clinically approved. Toward this end, it was necessary to identify the most active human enzyme that could convert CPT-11 to SN-38. We isolated enzymes from human liver microsomes based on their abilities to effect the conversion and identified human carboxylesterase 2 (hCE-2) as having the greatest specific activity. hCE-2 was 26-fold more active than human carboxylesterase 1 and was 65% as active as rabbit liver carboxylesterase, the most active CPT-11 hydrolyzing enzyme known. The anti-p97 mAb 96.5 was linked to hCE-2, forming a conjugate that could bind to antigen-positive cancer cells and convert CPT-11 to SN-38. Cytotoxicity assays established that the conjugate led to the generation of active drug, but the kinetics of prodrug activation (48 pmol x min(-1) x mg(-1) was insufficient for immunologically specific prodrug activation. These results confirm the importance of hCE-2 for CPT-11 activation and underscore the importance of enzyme kinetics for selective prodrug activation.     

The role of topoisomerase I inhibitor in cisplatin-resistant ovarian cancer.

We discussed the role of DNA topoisomerase I (topo I) inhibitor, which is now widely used in clinical practice, in cisplatin-resistant ovarian cancer. Our study showed the synergistic actions between cisplatin and 7-ethyl-10-hydroxycamptothecin (SN-38), an active metabolite of 7-ethyl-10-[4-(1-pyperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11), in two cisplatin-resistant cancer cell lines, HeLa/CDDP and KFr cells, but not in each parent cell line, HeLa and KF cells. Furthermore, HeLa/CDDP cells had a collateral sensitivity to SN-38. The levels of topo I protein in the cisplatin-resistant cells did not differ from those of their parent cell lines and were unaffected by exposure to cisplatin. In contrast, topo I enzymatic activity was 2-4 fold higher in the cisplatin-resistant cell lines compared with their respective parent cell lines. A significant correlation between the sensitivity for SN-38 and topo I activity human clear cell carcinoma cell lines, which are known as intrinsically ciasplatin-resistant cancer, was observed. Next, we examined the relationship between topo I activity and sensitivity to second-line chemotherapy consisting of cisplatin and CPT-11. A total of 30 patients with ovarian cancer who had initially undergone chemotherapy consisting of cisplatin, doxorubicin, and cyclophosphamide (CAP) and exhibited measurable lesions were entered in the study. Tumor samples were obtained in the period between the initial and the second-line chemotherapy. Of those 30 patients, 18 responded to second-line chemotherapy and 12 did not. Topo I activity in tumor samples of responder was significantly greater than that of in nonresponders. In 8 cases whose samples could be obtained before and after CAP, topo I activity significantly increased after CAP therapy. Consequently, the combination therapy with cisplatin and CPT-11 may be effective for patients with cisplatin-resistant ovarian cancer. In addition, topo I enzymatic activity may be a predictor of the sensitivity for topo I inhibitor.     

Intracellular dynamics of topoisomerase I inhibitor,CPT-11, by slit-scanning confocal Raman microscopy

Most molecular imaging technologies require exogenous probes and may have some influence on the intracellular dynamics of target molecules. In contrast, Raman scattering light measurement can identify biomolecules in their innate state without application of staining methods. Our aim was to analyze intracellular dynamics of topoisomerase I inhibitor, CPT-11, by using slit-scanning confocal Raman microscopy, which can take Raman images with high temporal and spatial resolution. We could acquire images of the intracellular distribution of CPT-11 and its metabolite SN-38 within several minutes without use of any exogenous tags. Change of subcellular drug localization after treatment could be assessed by Raman imaging. We also showed intracellular conversion from CPT-11 to SN-38 using Raman spectra. The study shows the feasibility of using slit-scanning confocal Raman microscopy for the non-labeling evaluation of the intracellular dynamics of CPT-11 with high temporal and spatial resolution. We conclude that Raman spectromicroscopic imaging is useful for pharmacokinetic studies of anticancer drugs in living cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of Cellular Location of Human Carboxylesterase 2 on CPT-11 Hydrolysis and Anticancer Activity

CPT-11 is an anticancer prodrug that is clinically used for the treatment of metastatic colorectal cancer. Hydrolysis of CPT-11 by human carboxylesterase 2 (CE2) generates SN-38, a topoisomerase I inhibitor that is the active anti-tumor agent. Expression of CE2 in cancer cells is under investigation for the tumor-localized activation of CPT-11. CE2 is normally expressed in the endoplasmic reticulum of cells but can be engineered to direct expression of active enzyme on the plasma membrane or as a secreted form. Although previous studies have investigated different locations of CE2 expression in cancer cells, it remains unclear if CE2 cellular location affects CPT-11 anticancer activity. In the present study, we directly compared the influence of CE2 cellular location on substrate hydrolysis and CPT-11 cytotoxicity. We linked expression of CE2 and enhanced green fluorescence protein (eGFP) via a foot-and-mouth disease virus 2A (F2A) peptide to facilitate fluorescence-activated cell sorting to achieve similar expression levels of ER-located, secreted or membrane-anchored CE2. Soluble CE2 was detected in the medium of cells that expressed secreted and membrane-anchored CE2, but not in cells that expressed ER-retained CE2. Cancer cells that expressed all three forms of CE2 were more sensitive to CPT-11 as compared to unmodified cancer cells, but the membrane-anchored and ER-retained forms of CE2 were consistently more effective than secreted CE2. We conclude that expression of CE2 in the ER or on the membrane of cancer cells is suitable for enhancing CPT-11 anticancer activity.     

Molecular modeling of CPT-11 metabolism by carboxylesterases (CEs): use of pnb CE as a model

CPT-11 is a prodrug that is converted in vivo to the topoisomerase I poison SN-38 by carboxylesterases (CEs). Among the CEs studied thus far, a rabbit liver CE (rCE) converts CPT-11 to SN-38 most efficiently. Despite extensive sequence homology, however, the human homologues of this protein, hCE1 and hiCE, metabolize CPT-11 with significantly lower efficiencies. To understand these differences in drug metabolism, we wanted to generate mutations at individual amino acid residues to assess the effects of these mutations on CPT-11 conversion. We identified a Bacillus subtilis protein (pnb CE) that could be used as a model for the mammalian CEs. We demonstrated that pnb CE, when expressed in Escherichia coli, metabolizes both the small esterase substrate o-NPA and the bulky prodrug CPT-11. Furthermore, we found that the pnb CE and rCE crystal structures show an only 2.4 A rmsd variation over 400 residues of the alpha-carbon trace. Using the pnb CE model, we demonstrated that the "side-door" residues, S218 and L362, and the corresponding residues in rCE, L252 and L424, were important in CPT-11 metabolism. Furthermore, we found that at position 218 or 252 the size of the residue, and at position 362 or 424 the hydrophobicity and charge of the residue, were the predominant factors in influencing drug activation. The most significant change in CPT-11 metabolism was observed with the L424R variant rCE that converted 10-fold less CPT-11 than the wild-type protein. As a result, COS-7 cells expressing this mutant were 3-fold less sensitive to CPT-11 than COS-7 cells expressing the wild-type protein.     

Simultaneous determination of irinotecan (CPT-11) and SN-38 in tissue culture media and cancer cells by high performance liquid chromatography: application to cellular metabolism and accumulation studies

A simple and sensitive HPLC method was developed to simultaneously determine CPT-11 and its major metabolite SN-38 in culture media and cell lysates. Camptothecin (CPT) was used as internal standard (I.S.). Compounds were eluted with acetonitrile-50 mM disodium hydrogen phosphate buffer containing 10 mM sodium 1-heptane-sulfonate, with the pH adjusted to 3.0 using 85% (w/v) orthophosphoric acid (27/73, v/v) by a Hyperclon ODS (C18) column (200 mm x 4.6 mm i.d.), with detection at excitation and emission wavelengths of 380 and 540 nm, respectively. The average extraction efficiencies were 96.9-108.3% for CPT-11 in culture media and 94.3-107.2% for CPT-11 in cell lysates; and 87.7-106.8% for SN-38 in culture media and 90.1-105.6% for SN-38 in cell lysates. Within- and between-day precision and accuracy varied from 0.1 to 10.3%. The limit of quantitation (precision and accuracy <20%) was 5.0 and 2.0 ng/ml for CPT-11 and 1.0 and 0.5 ng/ml for SN-38 in culture media and cell lysates, respectively. This method was successfully applied to quantitate the cellular accumulation and metabolism of CPT-11 and SN-38 in H4-II-E, a rat hepatoma cell line.     

Structural insights into CPT-11 activation by mammalian carboxylesterases

Mammalian carboxylesterases cleave the anticancer prodrug CPT-11 (Irinotecan) into SN-38, a potent topoisomerase I poison, and 4-piperidino-piperidine (4PP). We present the 2.5 A crystal structure of rabbit liver carboxylesterase (rCE), the most efficient enzyme known to activate CPT-11 in this manner, in complex with the leaving group 4PP. 4PP is observed bound adjacent to a high-mannose Asn-linked glycosylation site on the surface of rCE. This product-binding site is separated from the catalytic gorge by a thin wall of amino acid side chains, suggesting that 4PP may be released through this secondary product exit pore. The crystallographic observation of a leaving group bound on the surface of rCE supports the 'back door' product exit site proposed for the acetylcholinesterases. These results may facilitate the design of improved anticancer drugs or enzymes for use in viral-directed cancer cotherapies.     

Effects of quercetin combined with anticancer drugs on metastasis-associated factors of gastric cancer cells: in vitro and in vivo studies

Chemotherapy is essential to most patients with gastric cancer and the anticancer drug, irinotecan (CPT-11), and its metabolite, SN-38, an inhibitor of DNA topoisomerase I, are first-line chemotherapies for gastric cancer. Quercetin, a flavonoid that is widely found in various vegetables and fruits, has the ability to potentiate the efficacy of anticancer drugs. The purpose of this study was to investigate the therapeutic effect of quercetin combined with irinotecan/SN-38 in the AGS human gastric cancer cell line in vitro and in vivo. The in vitro study evaluated the efficacy of high-dose SN-38 and quercetin combined with low-dose SN-38 on cell viability, apoptosis, and β-catenin expression. Results showed that cell viability and the percentage of apoptosis in combined treatments with quercetin and SN-38 were comparable to treatment with high-dose SN-38 alone. AGS cells treated with a high dose of SN-38 exhibited up-regulation of β-catenin protein expression, whereas quercetin-treated cells (either quercetin alone or combined with low-dose SN-38) exhibited lower protein levels of β-catenin. In the AGS xenograft mouse model, gene expression of cyclooxygenase-2 and epithelial-mesenchymal transition-related markers, such as Twist1 and ITGβ6, were lower in combined treatments with quercetin and low-dose irinotecan than high-dose irinotecan alone. Furthermore, the concentration of angiogenesis-associated factors (vascular endothelial growth factor (VEGF)-A and VEGF-receptor 2) and percentage of Tie2-expressing monocytes was significantly down-regulated in combined treatments with quercetin and irinotecan. These results suggest that quercetin may enhance the efficacy of irinotecan/SN-38 in the human AGS cell line.     

Crystal structure of the Geobacillus stearothermophilus carboxylesterase Est55 and its activation of prodrug CPT-11

Several mammalian carboxylesterases were shown to activate the prodrug irinotecan (CPT-11) to produce 7-ethyl-10-hydroxycamptothecin (SN-38), a topoisomerase inhibitor used in cancer therapy. However, the potential use of bacterial carboxylesterases, which have the advantage of high stability, has not been explored. We present the crystal structure of the carboxyesterase Est55 from Geobacillus stearothermophilus and evaluation of its enzyme activity on CPT-11. Crystal structures were determined at pH 6.2 and pH 6.8 and resolution of 2.0 A and 1.58 A, respectively. Est55 folds into three domains, a catalytic domain, an alpha/beta domain and a regulatory domain. The structure is in an inactive form; the side-chain of His409, one of the catalytic triad residues, is directed away from the other catalytic residues Ser194 and Glu310. Moreover, the adjacent Cys408 is triply oxidized and lies in the oxyanion hole, which would block the binding of substrate, suggesting a regulatory role. However, Cys408 is not essential for enzyme activity. Mutation of Cys408 showed that hydrophobic side-chains were favorable, while polar serine was unfavorable for enzyme activity. Est55 was shown to hydrolyze CPT-11 into the active form SN-38. The mutant C408V provided a more stable enzyme for activation of CPT-11. Therefore, engineered thermostable Est55 is a candidate for use with irinotecan in enzyme-prodrug cancer therapy.     

Simultaneous determination of the lactone and carboxylate forms of the camptothecin derivative CPT-11 and its metabolite SN-38 in plasma by high-performance liquid chromatography

CPT-11 (irinotecan) and mainly its metabolite SN-38 are potent antitumor derivatives of camptothecin. As the active lactone forms of both CPT-11 and SN-38 exist in pH-dependent equilibrium with their respective less potent open-ring hydroxy acid species, the simultaneous monitoring of both forms of both compounds is relevant. CPT-11 and SN-38 derivatives have quite different fluorescence responses. In order to avoid any compromise on the wavelength setting, we developed chromatographic conditions allowing simple automated wavelength setting changes which have been prevented using existing methods involving conventional C₁₈ columns. This was achieved by means of a Symmetry C₁₈ column combined to a gradient elution program using acetonitrile and 75 mM ammonium acetate plus 7.5 mM tetrabutylammonium bromide at pH 6.4. The developed conditions allowed an elution order suitable for a simple automated wavelength change in respect to reliable peak integration. CPT-11 and SN-38 derivatives were detected at λ_ex=362 nm/λ_em=425 nm and λ_ex=375 nm/λ_em=560 nm, respectively. The developed method allowed the detection of amounts less than 3 pg of each derivative injected on column. The method was successfully applied to pharmacokinetic and toxicokinetic studies in rat and dog.     

Synthesis of new camptothecin analogs with improved antitumor activities

Novel hexacyclic camptothecin analogs containing cyclic amidine, urea, or thiourea moiety were designed and synthesized based on the proposed 3D-structure of the topoisomerase I (Topo I)/DNA/camptothecin ternary complex. The analogs were prepared from 9-nitrocamptothecin via 7,9-diaminocamptothecin derivatives as a key intermediate. Among them, 7c exhibited in vivo antitumor activities superior to CPT-11 in human cancer xenograft models in mice at their maximum tolerated doses though its in vitro antiproliferative activity was comparable to SN-38 against corresponding cell lines.     

Simultaneous determination of the lactone and carboxylate forms of irinotecan (CPT-11) and its active metabolite SN-38 by high-performance liquid chromatography: application to plasma pharmacokinetic studies in the rat

Irinotecan (CPT-11) and its main metabolite SN-38 are potent anticancer derivatives of camptothecin (CPT), with active lactone and inactive carboxylate forms coexisting. A simple and sensitive HPLC method using the ion-pairing reagent tetrabutylammonium hydrogen sulfate (TBAHS) was developed to simultaneously determine all four analytes in rat plasma samples. Camptothecin (CPT) was used as internal standard. The mobile phase was 0.1M potassium dihydrogen phosphate containing 0.01 M TBAHS (pH 6.4)-acetonitrile (75:25, v/v). Separation of the compounds was carried out on a Hypersil C18 column, monitored at 540 nm (excitation wavelength at 380 nm). All four compounds gave linear response as a function of concentration over 0.01-10 microM. The limit of quantitation in rat plasma was 0.01, 0.008, 0.005 and 0.005 microM for CPT-11 lactone, CPT-11 carboxylate, SN-38 lactone and SN-38 carboxylate, respectively. The method was successfully used in the study on the effect of coadministered thalidomide on the plasma pharmacokinetics of CPT-11 and SN-38 in rats. Coadministered thalidomide (100mg/kg body weight by intraperitoneal injection) significantly increased the AUC(0-10h) values of CPT-11 lactone and CPT-11 carboxylate by 32.6% and 30.3 %, respectively, (P < 0.01), but decreased the values by 19.2% and 32.4% for SN-38 lactone and carboxylate, respectively, (P < 0.05). Accordingly, the value of total body clearance (CL) of CPT-11 lactone was significantly lower in combination group compared to the control (1.329 versus 1.837 L/h/kg, P = 0.0002). Plasma t(1/2beta) values for SN-38 lactone and carboxylate were significantly (P < 0.01) smaller in rats with coadministered thalidomide, as compared to rats receiving CPT-11 alone. Further studies are needed to explore the underlying mechanisms for the observed kinetic interaction between CPT-11 and thalidomide.     

Hypoxia-targeted over-expression of carboxylesterase as a means of increasing tumour sensitivity to irinotecan (CPT-11)

The induced expression of carboxylesterase (CE) enzymes, which convert the prodrug irinotecan (CPT-11) into its active cytotoxic metabolite SN-38, constitutes a promising strategy for cancer gene therapy. By incorporating hypoxia-responsive elements (HREs) in conjunction with the transgene, expression can be targeted specifically to hypoxic tissues (such as solid tumours), expressing the hypoxia-inducible factor 1 (HIF-1). We have constructed a recombinant adenoviral vector, AdHRE-rCE, encoding the cDNA for the highly efficient rabbit liver CE (rCE), under the control of a HRE derived from the human phosphoglycerate kinase 1 (PGK-1) gene in conjunction with a minimal SV40 promoter. In vitro, HT1080 fibrosarcoma and SW480 colon carcinoma cells demonstrated an approximately 10-fold hypoxia-dependent induction in CE expression following pre-infection with AdHRE-rCE, which led to a15-30-fold increased sensitivity to CPT-11. Furthermore, in vivo, SW480 tumour xenografts infected with AdHRE-rCE demonstrated a 2-fold decrease in tumour doubling time, when combined with 7 days of CPT-11 treatment, in comparison to mock-infected controls, with rCE expression shown to be limited to hypoxic regions only. As the cytotoxicity of CPT-11 is reduced under hypoxic conditions, over-expression of a highly efficient CE such as rCE under hypoxia control within these hypoxic cells could reverse this effect and, therefore, form the basis for future clinical treatment strategies.     

Differential expression of DNA topoisomerase I gene between CPT-11 acquired- and native-resistant human pancreatic tumor cell lines: detected by RNA/PCR-based quantitation assay.

RNA/PCR quantitation method was developed to determine DNA Topoisomerase I(Topo I)-specific mRNA in order to study its gene expression in CPT-11 sensitive, acquired- or native-resistant human pancreatic tumor cell lines. The results were supported by Northern blotting and Western blotting analyses. Acquired-resistant cells have shown decreased levels of Topo I mRNA, compared with their parental cells. On the contrary, in the wild type cells no correlation was shown between sensitivity and gene expression. On the other, specific Topo I activity of the native resistant cell lines was fairly lower than that of sensitive cell lines, suggesting that immunoreactive Topo I protein contains low levels of active form enzyme which could be targets of CPT-11 in these native-resistant ones. Finally, the different mechanisms might be operative between acquired- and native-resistant tumor cells.     

Simple non-ion-paired high-performance liquid chromatographic method for simultaneous quantitation of carboxylate and lactone forms of 14 new camptothecin derivatives

SN-38 (7-ethyl-10-hydroxycamptothecin) is an active metabolite derived from the semi-synthetic compound camptothecin (CPT) named Irinotecan (CPT-11). The antitumor activity of SN-38 is 1000-fold more potent than the parent CPT-11. Fourteen new derivatives of camptothecin have recently been developed by Yakult Honsha (Tokyo, Japan). Here we describe a simple and cost-effective high-performance liquid chromatography (HPLC) method without an ion-pairing agent, which allows the simultaneous determination of both lactone and carboxylate forms of SN-38 and other camptothecin derivatives. A weak linear relationship between the HPLC retention factors (ln k') and the cellular concentrations of these compounds was observed. These results suggest that low-polarity compounds easily accumulate in cancer cells and may circumvent drug resistance. The HPLC analysis herein described is expected to greatly assist in derivative synthesis and chemical modification of camptothecin-based antitumor drugs.