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Background: The inactivation of tumor suppressor genes (TSGs) by aberrant DNA methylation plays an important role in the development of malignancy. Since this epigenetic change is reversible, it is a potential target for chemotherapeutic intervention using an inhibitor of DNA methylation, such as 5-aza-2'-deoxycytidine (DAC). Although clinical studies show that DAC has activity against hematological malignancies, the optimal dose-schedule of this epigenetic agent still needs to be established.Methods: Clonogenic assays were performed on leukemic and tumor cell lines to evaluate the in vitro antineoplastic activity of DAC. The reactivation of TSGs and inhibition of DNA methylation by DAC were investigated by reverse transcriptase-PCR and Line-1 assays. The in vivo antineoplastic activity of DAC administered as an i.v. infusion was evaluated in mice with murine L1210 leukemia by measurement of survival time, and in mice bearing murine EMT6 mammary tumor by excision of tumor after chemotherapy for an in vitro clonogenic assay.Results: Increasing the DAC concentration and duration of exposure produced a greater loss of clonogenicity for both human leukemic and tumor cell lines. The reactivation of the TSGs (p57KIP2 in HL-60 leukemic cells and p16CDKN2A in Calu-6 lung carcinoma cells) and the inhibition of global DNA methylation in HL-60 leukemic cells increased with DAC concentration. In mice with L1210 leukemia and in mice bearing EMT6 tumors, the antineoplastic action of DAC also increased with the dose. The plasma level of DAC that produced a very potent antineoplastic effect in mice with leukemia or solid tumors was > 200 ng/ml (> 1 microM).Conclusion: We have shown that intensification of the DAC dose markedly increased its antineoplastic activity in mouse models of cancer. Our data also show that there is a good correlation between the concentrations of DAC that reduce in vitro clonogenicity, reactivate TSGs and inhibit DNA methylation. These results suggest that the antineoplastic action of DAC is related to its epigenetic action. Our observations provide a strong rationale to perform clinical trials using dose intensification of DAC to maximize the chemotherapeutic potential of this epigenetic agent in patients with cancer. [ABSTRACT FROM AUTHOR]

Flavone-8-acetic acid (FAA) is a potent antivascular agent in mice but not in humans. Assuming that FAA was bioactivated in mice, we previously demonstrated that 6-OH-FAA was formed from FAA by mouse microsomes but not by human microsomes; its antivascular activity was 2.1- to 15.9-fold stronger than that of FAA, and its antivascular activity was mediated through the Ras homolog gene family (Rho) protein kinase A (RhoA) pathway. The present work aimed to study FAA metabolism in order to verify if 6-OH-FAA is formed in mice. Using synthesized standards and high-performance liquid chromatography (HPLC) coupled with ultraviolet (UV) detection and mass spectrometry (MS) analysis, we herein demonstrated, for the first time, that in vitro FAA and its monohydroxylated derivatives could directly undergo phase II metabolism forming glucuronides, and two FAA epoxides were mostly scavenged by NAC and GSH forming corresponding adducts. FAA was metabolized in mice. Several metabolites were formed, in particular 6-OHFAA. The antitumor activity of 6-OH-FAA in vivo is worthy of investigation., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Flavone-8-acetic acid (FAA) has been proved to be a potent vascular-disrupting agent in mice. Unfortunately, FAA did not produce any anticancer activity in clinical trials. Previously, we had reported that FAA is metabolized by mouse microsomes into six metabolites, whereas it was poorly metabolized by human microsomes, with fewer metabolites formed in lesser amounts. Especially, 6-OH-FAA was not formed by human microsomes. In this work, two major available metabolites, 4'-OH-FAA and 6-OH-FAA, were tested and compared with the parent compound FAA for their potential antivascular activities in vitro. The ability of the products to induce morphological changes, disrupt preformed capillaries of EA.hy926 endothelial cells and inhibit tubulin polymerization in vitro was assessed. The action mechanism was determined using the RhoA and Rac1 inhibitors. At 25 µg/ml, 6-OH-FAA induced morphological changes and membrane blebbing, whereas 300 µg/ml of FAA and 4'-OH-FAA slightly changed the morphology without inducing membrane blebbing. At 300 µg/ml, 6-OH-FAA produced morphological changes that were 2.1-6.9-fold greater than that produced by FAA and 4'-OH-FAA, an effect that was consistent with its much greater inhibitory effect on tubulin polymerization compared with FAA and 4'-OH-FAA. 6-OH-FAA significantly disrupted the EA.hy926 cell capillaries. 6-OH-FAA activities were prevented in EA.hy926 cells pretreated with RhoA, but not Rac1, inhibitor. In this short communication we report for the first time that, in vitro, 6-OH-FAA, a mouse-specific FAA metabolite, exhibits significantly stronger antivascular activities compared with FAA and 4'-OH-FAA, which are mediated through the RhoA kinase pathway.

A series of 16 flavonoids were isolated and prepared from bud exudate of Gardenia urvillei and Gardenia oudiepe, endemic to New Caledonia. Most of them are rare polymethoxylated flavones. Some of these compounds showed noticeable activity against Leishmania (Leishmania) amazonensis, Plasmodium falciparum and Trypanosoma brucei gambiense, in addition to tubulin polymerization inhibition at low micromolar concentration. We also provide a full set of NMR data as some of the flavones were incompletely described., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Objective of this study was to assess the various steps leading to spherulite obtention by means of optical and cryoelectron microscopy. The formulation, resting and hydration steps were optimised. Green-based process and organic-based process were compared. It was found that spherulites could be obtained only when two key steps were followed: a prior resting phase of excipients and the shearing stress of the hydrated excipients. Moreover, the new formulation under study formed spherulites in the 100-200 nm range, which is smaller than previously reported spherulites. Such laboratory scale optimised process led the integration of spherulites in a larger number of prospective studies. Indeed, we finally showed that the encapsulated payload of a hydrophobic compound, such as the anti-angiogenic agent fisetin, was increased to a much higher degree than with a liposomal encapsulation., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Dietary polyphenols, including flavonoids, have long been recognized as a source of important molecules involved in the prevention of several diseases, including cancer. However, because of their poor bioavailability, polyphenols remain difficult to be employed clinically. Over the past few years, a renewed interest has been devoted to the use of liposomes as carriers aimed at increasing the bioavailability and, hence, the therapeutic benefits of polyphenols. In this paper, we review the causes of the poor bioavailability of polyphenols and concentrate on their liposomal formulations, which offer a means of improving their pharmacokinetics and pharmacodynamics. The problems linked to their development and their potential therapeutic advantages are reviewed. Future directions for liposomal polyphenol development are suggested.

To evaluate the influence of stereochemistry on biological activities of cis-cyclopropyl combretastatin A4 (CA4) analogues, we have prepared several cyclopropyl compounds in their pure enantiomeric forms. The key reactions in our synthesis are the cyclopropanation of a (Z)-alkenylboron compound bearing a chiral auxiliary, and the cross-coupling of both enantiomeric cyclopropyl trifluoroborate salts with aryl and olefinic halides. Three pairs of cis-cyclopropyl CA4 analogues were evaluated for their potential antivascular activities. The diarylcyclopropyl compounds with SR-configuration (-)-1b, (-)-2b and the cyclopropylvinyl enantiomer (+)-3a with RR-configuration were the most potent tubulin polymerization inhibitors. A correlation was noted between anti-tubulin activity and rounding up activity of endothelial cells. The cytotoxic activity on B16 melanoma cells was in the submicromolar range for most compounds, but unlike the anti-tubulin activity, there was no difference in cytotoxic activity between racemic and enantiomerically pure forms for the three series of compounds. Molecular docking studies within the colchicine binding site of tubulin were in good agreement with the tubulin polymerization inhibitory data and confirmed the importance of the configuration of the synthesized cis-cyclopropyl CA4 analogues for potential antivascular activities., (Copyright © 2013. Published by Elsevier Ltd.)

The natural flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) has shown antiangiogenic and anticancer properties. Because of fisetin limited water solubility, we designed a liposomal formulation and evaluated its biological properties in vitro and in Lewis lung carcinoma (LLC) bearing mice. A liposomal formulation was developed with DOPC and DODA-PEG2000, possessing a diameter in the nanometer range (173.5±2.4nm), a high homogeneity (polydispersity index 0.181±0.016) and high fisetin encapsulation (58%). Liposomal fisetin incubated with LLC cells were internalized, induced a typical fisetin morphological effect and increased the sub-G1 cell distribution. In vivo, liposomal fisetin allowed a 47-fold increase in relative bioavailability compared to free fisetin. The effect of liposomal fisetin on LLC tumor growth in mice at low dose (21mg/kg) allowed a higher tumor growth delay (3.3 days) compared to free fisetin at the same dose (1.6 day). Optimization of liposomal fisetin therapy was attempted by co-treatment with cyclophosphamide which led to a significant improvement in tumor growth delay (7.2 days) compared to cyclophosphamide with control liposomes (4.2 days). In conclusion, fisetin liposomes markedly improved fisetin bioavailability and anticancer efficacy in mice and this formulation could facilitate the administration of this flavonoid in the clinical setting., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In order to find new molecules with cytotoxic activity against cancer cells, we prepared bis-akyne amides derived from propiolic acid. The bis-alkynes were then transformed in their mono-1,2,3-triazole analogs onto the amide side, due to its greater reactivity, using a catalyst-free Huisgen's reaction. The mono-triazoles were then subjected to the copper (I)-catalyzed version of the previous reaction (CuAAC), using a supported catalyst, to produce bis-triazoles. All products were obtained pure after simple trituration or filtration procedures. All synthetic compounds were tested in vitro for their cytotoxic activity using B16 melanoma cells. Four compounds (7, 23, 25 and 33) showed activities in the micromolar range (<21 μM) whereas three compounds (3, 22 and 38) presented activity at low micromolar concentrations (<10 μM), and two analogs (2 and 13) were active at nanomolar levels (<1 μM)., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Background and Objectives. To determine the most appropriate technique for tumour followup in experimental therapeutics, we compared ultrasound (US) and magnetic resonance imaging (MRI) to characterize ectopic and orthotopic colon carcinoma models. Methods. CT26 tumours were implanted subcutaneously (s.c.) in Balb/c mice for the ectopic model or into the caecum for the orthotopic model. Tumours were evaluated by histology, spectrofluorescence, MRI, and US. Results. Histology of CT26 tumour showed homogeneously dispersed cancer cells and blood vessels. The visualization of the vascular network using labelled albumin showed that CT26 tumours were highly vascularized and disorganized. MRI allowed high-resolution and accurate 3D tumour measurements and provided additional anatomical and functional information. Noninvasive US imaging allowed good delineation of tumours despite an hypoechogenic signal. Monitoring of tumour growth with US could be accomplished as early as 5 days after implantation with a shorter acquisition time (<5 min) compared to MRI. Conclusion. MRI and US afforded excellent noninvasive imaging techniques to accurately follow tumour growth of ectopic and orthotopic CT26 tumours. These two techniques can be appropriately used for tumour treatment followup, with a preference for US imaging, due to its short acquisition time and simplicity of use.

The endothelial cell adhesion molecules, including the integrin alpha v beta 3 (αvβ3) and E-selectin, are involved in the process of angiogenesis required for tumour growth, cell migration and metastasis. The purpose of this study was to assess and compare widely used tumour models to select the ones most suitable for angiogenesis research. Fifteen murine tumours were selected including melanoma (B16), colon (C26, C38, C51), mammary (MA13, MA16, MA16/Adr, MA17, MA17/Adr, MA25, MA44), pancreatic (PO2, PO3), Glasgow osteogenic sarcoma (GOS) and Lewis lung carcinoma (LLC). The tumour vascular density, assessed using the platelet endothelial cell adhesion molecule 1 (PECAM-1; CD31) immunostaining, revealed that B16 melanoma was poorly vascularized (<5 %), whereas the colon and mammary tumours were well vascularized (5-15 %). The most vascularized tumours (>15 %) were the pancreatic tumours (PO2 and PO3), the sarcoma (GOS) and the lung tumour (LLC). The integrin αvβ3 and E-selectin, evaluated by immunohistology, showed that 7/15 tumours expressed the αvβ3 integrin which was homogeneously distributed on all tumour sections (B16, C26, MA17/Adr, MA25, MA44, PO2, LLC). E-selectin was expressed in 4/15 tumours and its expression was restricted to the tumour periphery. Only 2/15 tumours (B16 and C26) were shown to express both integrin αvβ3 and E-selectin. In conclusion, these data not only contribute to a better understanding of the tumour biology of murine tumours but can also guide the choice of appropriate models for antiangiogenic therapy, for selective drug delivery to tumours and the validation of tumour imaging modalities targeting these endothelial cell adhesion molecules.

Purpose: To investigate the potential value of magnetic resonance (MR) elastography and diffusion-weighted (DW) MR imaging in the detection of microstructural changes of murine colon tumors during growth and antivascular treatment., Materials and Methods: The study was approved by the regional ethics committee for animal care. Sixty Balb-C mice, bearing ectopic and orthotopic colon tumors, were monitored for 3 weeks with high-resolution T2-weighted MR imaging, three-dimensional steady-state MR elastography, and DW MR imaging at 7 T. The same imaging protocol was performed 24 hours after injection of combretastatin A4 phosphate (CA4P) in 12 mice. The absolute value of the complex shear modulus (|G*|) and the apparent diffusion coefficient (ADC) were measured in the viable zones of tumors and compared with microvessel density (MVD), cellularity, and micronecrosis by using the Pearson correlation coefficient., Results: During tumor growth, |G*| increase was correlated with MVD (r = 0.70 [P = .08] and r = 0.78 [P = .002], for both the ectopic and orthotopic models, respectively). Moreover, the ectopic tumors displayed decreased ADC, which correlated with increased cellularity (r = 0.77, P = .04), whereas no changes in ADC and cellularity were observed in orthotopic tumors. After CA4P administration, |G*| decreased in the ectopic model (P < .0001), similar to the MVD evolution (P = .03), whereas no significant changes in |G*| (P = .7) and MVD (P = .6) were observed in the orthotopic model. ADC increased in both models (P = .047 and P = .01 for the ectopic and the orthotopic models, respectively) in relation to increased micronecrosis., Conclusion: Imaging of mechanical properties and diffusivity provide complementary information during tumor growth and regression that are respectively linked to vascularity and tumor cell alterations, including cellularity and micronecrosis.

To find new and better antivascular agents for cancer therapy, a series of combretastatin A4 (CA4) analogs were prepared from 1,3-diaryl-2-nitroprop-1-enes (6-12) obtained in a two-step synthesis from appropriate arylaldehydes and 2-aryl-1-nitroethanes (4 or 5). Treatment of these 1,3-diaryl-2-nitroprop-1-enes 6-12 by sodium azide in DMSO yielded the targeted compounds. The synthesized 1,2,3-triazoles disubstituted in 4- and 5-positions by one benzyl group and one aryl nucleus have also been tested for biological activities involved in antivascular action. It was found that several new compounds exhibited interesting biological activities in the nanomolar or low micromolar range, in terms of rounding up of endothelial cells, inhibition of tubulin polymerization, and cytotoxicity on B16 melanoma cancer cells. In silico docking studies of 11 and 19 within the active site of tubulin were also carried out in order to rationalize the inhibitory properties of these compounds and further understand their inhibition mechanism. In vivo evaluation of compounds 11 and 19 in mice bearing colon 26 carcinoma indicated modest anticancer activity., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

The natural flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) has shown antitumour activity but its administration is complicated by its low water solubility. Our aim was to incorporate fisetin into a nanoemulsion to improve its pharmacokinetics and therapeutic efficacy. Solubility and emulsification tests allowed to develop an optimal nanoemulsion composed of Miglyol 812N/Labrasol/Tween 80/Lipoid E80/water (10%/10%/2.5%/1.2%/76.3%). The nanoemulsion had an oil droplet diameter of 153 ± 2 nm, a negative zeta potential (-28.4 ± 0.6 mV) and a polydispersity index of 0.129. The nanoemulsion was stable at 4 °C for 30 days, but phase separation occurred at 20 °C. Pharmacokinetic studies in mice revealed that the fisetin nanoemulsion injected intravenously (13 mg/kg) showed no significant difference in systemic exposure compared to free fisetin. However, when the fisetin nanoemulsion was administered intraperitoneally, a 24-fold increase in fisetin relative bioavailability was noted, compared to free fisetin. Additionally, the antitumour activity of the fisetin nanoemulsion in Lewis lung carcinoma bearing mice occurred at lower doses (36.6 mg/kg) compared to free fisetin (223 mg/kg). In conclusion, we have developed a stable nanoemulsion of fisetin and have shown that it could improve its relative bioavailability and antitumour activity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The natural flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) has been shown to possess antiangiogenic and anticancer properties. Because of the limited water solubility of fisetin, our aim was to design and optimize a liposomal formulation that could facilitate its in vivo administration, taking into account the availability and cost of the various components. Several methods were evaluated such as probe sonication, homogeneization, film hydration and lipid cake formation. A selection of lipid and lipid-PEG was also performed via their incorporation in different formulations based on the size of the liposomes, their polydispersity index (PDI) and the fisetin encapsulation yield. An optimal liposomal formulation was developed with P90G and DODA-GLY-PEG2000, possessing a diameter in the nanometer scale (175nm), a high homogeneity (PDI 0.12) and a high fisetin encapsulation (73%). Fisetin liposomes were stable over 59 days for their particle diameter and still retained 80% of their original fisetin content on day 32. Moreover, liposomal fisetin retained the cytotoxicity and typical morphological effect of free fisetin in different tumour and endothelial cell lines. In conclusion, based on its physico-chemical properties and retention of fisetin biological effects, the developed liposomal fisetin preparation is therefore suitable for in vivo administration., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Although the natural flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) has been recently identified as an anticancer agent with antiangiogenic properties in mice, its in vivo pharmacokinetics and metabolism are presently not characterized. Our purpose was to determine the pharmacokinetics and metabolism of fisetin in mice and determine the biological activity of a detected fisetin metabolite. After fisetin administration of an efficacious dose of 223 mg/kg i.p. in mice, the maximum fisetin concentration reached 2.5 μg/ml at 15 min and the plasma concentration declined biphasically with a rapid half-life of 0.09 h and a terminal half-life of 3.1h. Three metabolites were detected, one of which was a glucuronide of fisetin (M1), whereas another glucuronide (M2) was a glucuronide of a previously unknown fisetin metabolite (M3). HPLC-MS/MS analysis indicated that M3 was a methoxylated metabolite of fisetin (MW=300 Da). The UV spectrum of M3 was identical to that of fisetin and standard 3,4',7-trihydroxy-3'-methoxyflavone (geraldol). In addition, because M3 co-eluted with standard geraldol in 4 different chromatographic ternary gradient conditions, M3 was therefore assigned to geraldol. Of interest, this metabolite was shown to achieve higher concentrations than fisetin in Lewis lung tumors. We also compared the cytotoxic and antiangiogenic activities of fisetin and geraldol in vitro and it was found that the latter was more cytotoxic than the parent compound toward tumor cells, and that it could also inhibit endothelial cells migration and proliferation. In conclusion, these results suggest that fisetin metabolism plays an important role in its in vivo anticancer activities., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We report here the discovery of a potent series of HIV-1 integrase (IN) inhibitors based on the ferrocenyl chalcone difluoridoborate structure. Ten new compounds have been synthesized and were generally found to have similar inhibitory activities against the IN 3' processing and strand transfer (ST) processes. IC(50) values were found to be in the low micromolar range, and significantly lower than those found for the non-coordinated ferrocenyl chalcones and other ferrocene molecules. The ferrocenyl chalcone difluoridoborates furthermore exhibited low cytotoxicity against cancer cells and low morphological activity against epithelial cells., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A series of combretastatin A4 (CA4) analogues with a lactam or lactone ring fused to the trimethoxyphenyl or the B-phenyl moiety were synthesized in an efficient and stereoselective manner by using a domino Heck-Suzuki-Miyaura coupling reaction. The vascular-disrupting potential of these conformationally restricted CA4 analogues was assessed by various in vitro assays: inhibition of tubulin polymerization, modification of endothelial cell morphology, and disruption of endothelial cell cords. Compounds were also evaluated for their growth inhibitory effects against murine and human tumor cells. B-ring-constrained derivatives that contain an oxindole ring (in contrast to compounds with a benzofuranone ring) as well as analogues bearing a six-membered lactone core fused to the trimethoxyphenyl ring are endowed with significant biological activity. The most potent compound of this series (oxindole 9 b) is of particular interest, as it combines chemical stability and a biological activity profile characteristic of a vascular-disrupting agent., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: The natural flavonoid fisetin was recently identified as a lead compound that stabilizes endothelial cell microtubules. In this study, we investigated the antiproliferative and antiangiogenic properties of fisetin in vitro and in vivo., Methods: Fisetin cytotoxicity was evaluated using Lewis lung carcinoma cells (LLC), endothelial cells and NIH 3T3 cells. Endothelial cell (EC) migration and capillary-like structure formation were evaluated using EAhy 926 cells. In vivo tumour growth inhibition studies were performed using LLC-bearing mice treated with fisetin and/or cyclophosphamide (CPA)., Results: The fisetin IC(50) was 59 μM for LLC and 77 μM for EC cells, compared to 210 μM for normal NIH 3T3 cells (24 h). Fisetin inhibited EC migration and capillary-like structure formation at non-cytotoxic concentrations (22-44 μM). In mice, fisetin inhibited angiogenesis assessed using the Matrigel plug assay. In LLC-bearing mice, fisetin produced a 67% tumour growth inhibition (223 mg/kg, intraperitoneal), similar to the 66% produced by low-dose CPA (30 mg/kg, subcutaneous). When fisetin and CPA were combined, however, a marked improvement in antitumour activity was observed (92% tumour growth inhibition), with low systemic toxicity. Tumour histology showed decreased microvessel density with either fisetin or CPA alone, and a dramatic decrease after the fisetin/CPA combination., Conclusions: We have shown that fisetin not only displays in vitro and in vivo antiangiogenic properties, but also can markedly improve the in vivo antitumour effect of CPA. We propose that this drug combination associating a non-toxic dietary flavonoid with a cytotoxic agent could advantageously be used in the treatment of solid tumours.

The metabolism of flavone-8-acetic acid (FAA) has been hypothesized to be partly responsible for its potent anticancer activity in mice. The purpose of this study was to identify the mouse enzymes involved in FAA Phase I metabolism and evaluate their possible induction in vivo by FAA. Mouse microsomes metabolized FAA into 6 metabolites: 3',4'-dihydrodiol-FAA, 5,6-epoxy-FAA, 4'-OH-FAA, 3'-OH-FAA, 3',4'-epoxy-FAA and 6-OH-FAA. Using Cyp-specific inhibitors (furafylline, Cyp1a2; α-naphthoflavone, Cyp1b1; tranylcypromine, Cyp2b9; quercetin, Cyp2c29; quinidine, 2d9; diethyldithiocarbamate, Cyp2e1; ketoconazole, Cyp3a11), the formation of 5,6-epoxy-FAA was mainly attributed to Cyps 1a2, 1b1, 2b9, 2c29 and 2e1, whereas the 3',4'-epoxy-FAA was formed by Cyps 2b9 and 3a11. The 4'-OH-FAA was generated by Cyps 1a2, 1b1, 2b9 and 2e1, and the 6-OH-FAA was formed by Cyps 1b1 and 2c9. Using the epoxide scavenger N-acetyl cysteine, 4'-OH-FAA, 3'-OH-FAA and 6-OH-FAA were shown to derive partly from non enzymatic isomerisation of their corresponding epoxides. The specific epoxide hydrolase inhibitor elaidamide allowed the confirmation that 3',4'-dihydrodiol-FAA was formed via the epoxide hydrolase. FAA treatment in vivo in mice led to a significant increase in the hepatic expression of Cyp1a2 (1.9-fold), 2e1 (2.1-fold), 2b10 (3.2-fold), 2d9 (2.3-fold) and 3a11 (2.2-fold), as evaluated by qRT-PCR. In conclusion, several Cyps were shown to be involved in FAA metabolism, particularly Cyps 3a11 and 2b9 which were responsible for the formation of the principal metabolites (5,6-epoxy-FAA, 3',4'-epoxy-FAA), and that FAA could induce the expression of several Cyps after in vivo administration. The possible implication of these enzymes in the in vivo anticancer activity of FAA in mice is discussed., (©2011 Bentham Science Publishers Ltd.)

Two series of 2-aroyltrimethoxyindoles were designed to investigate the effects of the replacement of the trimethoxyphenyl ring of phenstatin with a trimethoxyindole moiety. These compounds were efficiently prepared through a domino palladium-catalyzed sequence from 2-gem-dibromovinylanilines substituted by three methoxy groups and arylboronic acids under carbon monoxide atmosphere. These novel heterocyclic combretastatin A4 analogues were evaluated for their cell growth inhibitory properties and their ability to inhibit the tubulin polymerization., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

We designed and synthesized two novel series of azapodophyllotoxin analogues as potential antivascular agents. A linker was inserted between the trimethoxyphenyl ring E and the tetracyclic ABCD moiety of the 4-aza-1,2-didehydropodophyllotoxins. In the first series, the linker enables free rotation between the two moieties; in the second series, conformational restriction of the E nucleus was considered. We have identified several new compounds with inhibitory activity toward tubulin polymerization similar to that of CA-4 and colchicine, while displaying low cytotoxic activity against normal and/or cancer cells. An aminologue and a methylenic analogue were shown to disrupt endothelial cell cords on Matrigel at subtoxic concentrations, and an original assay of drug washout allowed us to demonstrate the rapid reversibility of this effect. These two new analogues are promising leads for the development of vascular-disrupting agents in the podophyllotoxin series.

A series of 5-(3',4',5'-trimethoxyphenyl)pyrrolo[3,4-a]carbazole-1,3(2H,10H)-diones was designed as cis-restricted analogues of 3-aroylindoles, arylthioindoles and 3-benzylidoneindolin-2-ones derived from combretastatin A4 (CA-4). Starting from various indoles, compounds were synthesized by means of a convenient two-step procedure involving a one-pot multicomponent reaction as key step. Intermediate tetrahydro[3,4-a]carbazoles and their corresponding carbazoles were submitted to biological screening tests involved in antivascular action, including the cytotoxicity against murine B16 melanoma cells, the rounding up of endothelial cells (EA.hy 926) and the inhibition of tubulin polymerization. Of the 31 compounds screened, those bearing a methoxy group at the 8-position endowed significant biological activities. A carbazole compound 30 was identified as a promising candidate for further development of novel vascular targeting agents., (2010 Elsevier Masson SAS. All rights reserved.)

The oxidation of the ferrocenyl group of 2'-hydroxyferrocenyl chalcones activates the beta-position of the unsaturated ketone to nucleophilic attack to yield the first examples of ferrocenyl flavones. These compounds are significantly more cytotoxic than their organic analogs on B16 melanoma cells, with IC(50) values in the low micromolar range.

Flavonoids are common components of the human diet and appear to be of interest in cancer prevention or therapy, but their structure-activity relationships (SAR) remain poorly defined. In this study, were compared 24 flavonoids for their cytotoxicity on cancer cells (B16 and Lewis lung) and their morphological effect on endothelial cells (EC) that could predict antiangiogenic activity. Ten flavonoids presented inhibitory concentrations for 50% of cancer cells (IC50, 48 h) below 50 microM: rhamnetin, 3',4'-dihydroxyflavone, luteolin, 3-hydroxyflavone, acacetin, apigenin, quercetin, baicalein, fisetin, and galangin. Important SAR for cytotoxicity included the C2-C3 double bond and 3',4'-dihydroxylation. Concerning the morphological effects on EC, only fisetin, quercetin, kaempferol, apigenin, and morin could induce the formation of cell extensions and filopodias at noncytotoxic concentrations. The SAR for morphologic activity differed from cytotoxicity and involved hydroxylation at C-7 and C-4'. Fisetin, the most active agent, presented cell morphology that was distinct compared to colchicine, combretastatin A-4, docetaxel, and cytochalasin D. Resistance to cold depolymerization and a 2.4-fold increase in acetylated alpha-tubulin demonstrated that fisetin was a microtubule stabilizer. In conclusion, this study disclosed several SAR that could guide the choice or the rational synthesis of improved flavonoids for cancer prevention or therapy.

6-Methoxy-3-(3',4',5'-trimethoxybenzoyl)-1H-indole (BPR0L075) (1) is a potent inhibitor of tubulin polymerization which exhibits both in vitro and in vivo activities against a broad spectrum of solid tumors. This compound was designed as a heterocyclic analogue of combretastatin A4 (CA-4), a natural stilbene derivative that disrupts the tumor vasculature and causes tumor regression. In the present work, we describe the design and synthesis of several new disubstituted analogues of 1, along with their biological evaluation as potential antivascular agents. Compound 13, bearing a hydroxyl group at the 7-position of the indole nucleus that mimics the hydroxyl group at the 3-position of the B-ring of CA-4, was identified as a potent inhibitor of tubulin polymerization and also as a cytotoxic agent against B16 melanoma cells at sub-micromolar concentration. In addition, compound 13 displayed marked morphological activity (rounding up) at nanomolar concentrations on endothelial cells (EA.hy 926 cells), which is indicative of potential antivascular activity. Interestingly, the corresponding 7-O-mesylate derivative 28 (an intermediate in the synthesis of 13), was also found active in cellular assays, although it was moderately active in the tubulin polymerization inhibition test. Finally, in order to better understand the SAR of disubstituted analogues of 1, two other position isomers (10 and 14), were synthesized and evaluated for their biological activities. It was noted that the 7-hydroxysubstituted analogue 13 was more potent than the 5-hydroxysubstituted analogue 10. In conclusion, this work has allowed the identification of biologically potent CA-4 analogues (13 and 28) and also contributes to a better understanding of the SAR of 1.

Flavone-8-acetic acid (FAA) is a potent anticancer agent in mouse but has not shown activity in humans. Because FAA metabolism could play a role in this interspecies difference, our aim was to identify the metabolites formed in vitro using mouse microsomes compared with those in human microsomes. Mouse microsomes produced six metabolites as detected by reversed-phase high-performance liquid chromatography-mass spectrometry (MS). Three metabolites were identified as the 3'-, 4'-, or 6-hydroxy-FAA, by comparison with retention times and UV and MS spectra of standards. Two metabolites presented a molecular weight of 296 (FAA = 280) indicating the presence of one oxygen but did not correspond to any monohydroxylated FAA derivative. These two metabolites were identified as epoxides because they were sensitive to epoxide hydrolase. The position of the oxygen was determined by the formation of the corresponding phenols under soft acidic conditions: one epoxide yielded the 3'- and 4'-hydroxy-FAA, thus corresponding to the 3',4'-epoxy-FAA, whereas the other epoxide yielded 5- and 6-hydroxy-FAA, thus identifying the 5,6-epoxy-FAA. The last metabolite was assigned to the 3',4'-dihydrodiol-FAA because of its molecular weight (314) and sulfuric acid dehydration that indicated that the 3'- and 4'-positions were involved. Compared with mouse microsomes, human microsomes (2 pools and 15 individual microsomes) were unable to metabolize FAA to a significant extent. In conclusion, we have identified six new FAA metabolites formed by mouse microsomes, whereas human microsomes could not metabolize this flavonoid to a significant extent. The biological importance of the new metabolites identified herein remains to be evaluated.

Interest in elucidating the mechanisms of action of various classes of anticancer agents and exploring the pathways of the induced-nitric oxide (NO) release provides an impetus to conceive a better designed approach to locally detect NO in tumors, in vivo. We report here on the first use of an electrochemical sensor that allows the in vivo detection of NO in tumor-bearing mice. In a first step, we performed the electrochemical characterization of a stable electroactive probe, K4Fe(CN)6, directly injected into the liquid microenvironment especially created around the electrode in the tumor. Second, the ability of the inserted electrode system to detect the presence of NO itself in the tumoral tissue was achieved by using the chemically modified Pt/Ir electrode as NO sensor and two NO donor molecules: diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium 1,2-diolate (DEA-NONOate) and (Z)-1-[N-(2-aminopropyl)-N-(2-ammonio propyl)amino]diazen-1-ium 1,2-diolate (PAPA-NONOate). These two NO donor molecules allowed proving the electrochemical detection of (i) directly injected exogenous NO phosphate buffer solution into the tumor (decomposed DEA-NONOate) and (ii) biomimetically induced endogeneous release of NO in the tumoral tissue, upon injection of PAPA-NONOate into the tumor. This approach could be applied to the in vivo study of candidate anticancer drugs acting on the NO pathways.

The experimental anticancer agent flavone-8-acetic acid (FAA) is metabolized into several monohydroxylated derivatives using mouse microsomes. Because these metabolites could be involved in the biological effects of FAA, the aim of this study was to characterize all its possible monohydroxylated derivatives. To do so, we have developed a methodology using reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with ultraviolet (UV) detection and mass spectrometry (MS) to analyze and identify FAA derivatives hydroxylated at the 2', 3', 4', 3, 5, 6, or 7 position. In RP-HPLC, 4'-, 3'-, 2'-, 6-, and 7-OH-FAA eluted before FAA, whereas 3- and 5-OH-FAA eluted after FAA. UV spectra showed a bathochromic shift of band I for all derivatives and of band II for 5- and 6-OH-FAA. In addition, the position of the OH group could be determined by the presence of certain product ions in MS. Ions at m/z 133 and 151 were specific for 2'-, 3'-, 4'-, and 3-OH-FAA, whereas the ion at m/z 177 was specific for 3-OH-FAA only. The ions m/z 133, 151 and 167 were specific for 2'-OH-FAA. Ions at m/z 149 were specific for the presence of the OH group on cycle A only (i.e., 5-, 6- or 7-OH-FAA). The presence of both product ions m/z 149 and 179 were specific for 7-OH-FAA. Finally, ions at m/z 149 and several product ions of even m/z values were specific for 5-OH-FAA. In conclusion, the methodology described can be used to identify all possible monohydroxylated FAA derivatives., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

Combretastatin A-4 (CSA-4), a stilbene derivative, is a potent vascular disrupting agent (VDA) with the structural requirement of a cis-configuration to maintain a molecular geometry and a correct orientation of both phenyl groups. A series of indolic analogues of CSA-4 was synthesized by means of an efficient strategy. Six compounds (20b, 25b-27b, 32b, and 35b) were identified as potent inhibitors of tubulin polymerization and also displayed cytotoxic activities on B16 melanoma cells at a nanomolar level. Both activities were well correlated with the ability to induce morphological changes of EA.hy 926 endothelial cells. In conclusion, the cis-stilbene skeleton of CSA-4 could conveniently be replaced by the 3-aroylindolic moiety, thus avoiding any isomerization leading to inactive trans compounds.

All-trans retinoic acid (ATRA) can induce complete remission in acute promyelocytic leukemia (APL), but resistance to this treatment develops rapidly partly due to increased ATRA metabolism. Among the cytochrome P450s (CYPs) involved in ATRA metabolism, the ATRA-inducible cytochrome P450 26A1 (CYP26A1) is particularly active although the molecular mechanisms involved in its regulation are not well defined in the target leukemia cells. To study CYP26A1 expression and regulation in APL cells, we used the NB4 promyelocytic leukemia cell line. CYP26A1 constitutive expression was barely detectable in NB4 cells, but ATRA could induce high levels of CYP26A1 expression, which reached a maximum at 72h. To further define CYP26A1 induction mechanisms in the NB4 leukemia cells, we used RARs and RXR selective agonists. The RARalpha agonist BMS753 could elicit maturation, as expected, but not CYP26A1 expression. Treatment with the RARbeta agonist BMS641, or the RARbeta/gamma agonist BMS961, could not elicit maturation, as expected, nor induce CYP26A1 expression. Because CYP26A1 expression could not be induced by RAR ligands alone, NB4 cells were then co-treated with the RXR agonist BMS649. The RXR agonist alone could not induce CYP26A1 expression, nor in combination with either the RARbeta agonist or the RARbeta/gamma agonist. However, the combination of the RXR agonist and the RARalpha agonist could elicit a marked induction of CYP26A1 expression. In conclusion, we have shown that CYP26A1 induction is not essential for the granulocytic maturation of NB4 leukemia cells, and that CYP26A1 induction requires the activation of both RARalpha and RXR in these cells.

Retinoids are vitamin A (retinol) derivatives essential for normal embryo development and epithelial differentiation. These compounds are also involved in chemoprevention and differentiation therapy of some cancers, with particularly impressive results in the management of acute promyelocytic leukemia (APL). Although highly effective in APL therapy, resistance to retinoic acid (RA) develops rapidly. The causes of this resistance are not completely understood and the following factors have been involved: increased metabolism, increased expression of RA binding proteins, P-glycoprotein expression, and mutations in the ligand binding domain of RARalpha. RA exerts its molecular actions mainly through RAR and RXR nuclear receptors. In addition to the nuclear receptor based mechanism of RA action, covalent binding of RA to cell macromolecules has been reported. RA derives from retinol by oxidation through retinol and retinal dehydrogenases, and several cytochrome p450s (CYPs). RA is thereafter oxidized to several metabolites by a panel of CYPs that differs for the different RA isomers. Phase II metabolism, mainly glucuronidation, is also observed. The role RA metabolism plays in the expression of its biological actions is not completely understood: in several systems, metabolism decreases RA activity, whereas in other systems metabolism appears involved in its action. In addition, several RA metabolites have shown activity and cannot be classified as only catabolites. Therapy of cancer with retinoids is still in its infancy, but the use of new analogues with improved pharmacological properties, along with combination with other drugs, could undoubtedly improve the management of several cancers in the future.

We have shown that four metabolites of all-trans-retinoic acid (ATRA) (4-oxo-, 4-OH-, 18-OH-, and 5,6-epoxy-RA) can induce maturation of NB4 promyelocytic leukemia cells (Idres, N., Benoit, G., Flexor, M. A., Lanotte, M., and Chabot, G. G. (2001) Cancer Res. 61, 700-705). To better understand the mechanism of action of ATRA metabolites and isomers, we assessed their binding to retinoic acid receptors (RARs) and activation of RAR-mediated transcription via a retinoic acid response element (RARE). Competition binding experiments with tritiated ATRA showed that all metabolites could bind to RARs with variable affinity. For transactivation studies, COS-7 cells were cotransfected with RAR alpha, beta, or gamma expression vectors and the reporter plasmid RARE-tk-Luc, and the retinoid concentrations for half-maximal luciferase activity (EC(50)) were determined. All retinoids tested could activate the three RAR isotypes. The lowest EC(50) value for RAR alpha was with 9-cis-RA (13 nM), followed by 4-oxo-RA (33 nM), 5,6-epoxy-RA (77 nM), 13-cis-RA (124 nM), 18-OH-RA (162 nM), ATRA (169 nM), and 4-OH-RA (791 nM). For RAR beta, the EC(50) values increased as follows: 4-oxo-RA (8 nM), ATRA (9 nM), 18-OH-RA (14 nM), 5,6-epoxy-RA (35 nM), 13-cis-RA (47 nM), 4-OH-RA (64 nM), and 9-cis-RA (173 nM). For RAR gamma the EC(50) values were: ATRA (2 nM), 5,6-epoxy-RA (4 nM), 18-OH-RA (14 nM), 13-cis-RA (36 nM), 9-cis-RA (58 nM), 4-oxo-RA (89 nM), and 4-OH-RA (94 nM). By comparing the -fold induction of luciferase activity, all retinoids tested were equipotent at transactivating RARE-tk-Luc whatever the RAR considered. However, the best induction of the transcription was obtained for RAR alpha, which was 5-fold higher than for RAR beta and 10-fold higher than for RAR gamma. In conclusion, these data show that ATRA metabolites can bind to and activate the three RARs with variable relative affinity but with similar efficacy. These results suggest that ATRA metabolites may activate several signaling pathways and probably play an important role in cellular physiology and cancer therapy.

The purpose of this work was to identify the principal human cytochrome P450s (CYPs) involved in the metabolism of the retinoic acid (RA) isomers, 9-cis- and 13-cis-RA, by using a combination of techniques including human liver microsomes (correlation of activity and inhibition), and lymphoblast microsomes expressing a single CYP. Concerning the 9-cis-RA, 4-OH- and 4-oxo-9-cis-RA were formed with human liver microsomes, and their formation correlated with activities linked to CYPs 3A4/5, 2B6, 2C8, 2A6, and 2C9. The use of lymphoblast microsomes expressing a single human CYP identified CYPs 2C9>2C8>3A7 as the most active in the formation of 4-OH-9-cis-RA. With regard to 13-cis-RA, specific P450 activities linked to CYPs 2B6, 2C8, 3A4/5, and 2A6 were correlated with the formation of 4-OH- and 4-oxo-13-cis-RA. Microsomes expressing a single CYP identified CYPs 3A7, 2C8, 4A11, 1B1, 2B6, 2C9, 2C19, 3A4 (decreasing activity) in the formation of 4-OH-13-cis-RA. The use of CYP-specific inhibitors in human liver microsomes disclosed that the formation of the 4-OH-9-cis-RA was best inhibited by sulfaphenazole (72%) and quercetin (66%), whereas ketoconazole and troleandomycin inhibited its formation by 55 and 38%, respectively; the formation of 4-OH-13-cis-RA was best inhibited by troleandomycin (54%) and ketoconazole (46%), whereas quercetin was a weak inhibitor (14%). In conclusion, adult human CYPs 2C9, 2C8, 3A4 have been identified as active in the 9-cis-RA metabolism, whereas CYPs 3A4 and 2C8 were active in 13-cis-RA metabolism. The fetal form CYP3A7 was also identified as very active in either 9-cis- or 13-cis-RA metabolism. The role of these human CYPs in the biological response or resistance to RA isomers remains to be determined.

The metabolism of all-trans retinoic acid (ATRA) has been reported to be partly responsible for the in vivo resistance to ATRA seen in the treatment of human acute promyelocytic leukemia (APL). However, ATRA metabolism appears to be involved in the growth inhibition of several cancer cell lines in vitro. The purpose of this study was to evaluate the in vitro activity of the principal metabolites of ATRA [4-hydroxy-retinoic acid (4-OH-RA), 18-hydroxy-retinoic acid (18-OH-RA), 4-oxo-retinoic acid (4-oxo-RA), and 5,6-epoxy-retinoic acid (5,6-epoxy-RA)] in NB4, a human promyelocytic leukemia cell line that exhibits the APL diagnostic t(15;17) chromosomal translocation and expresses the PML-RAR alpha fusion protein. We established that the four ATRA metabolites were indeed formed by the NB4 cells in vitro. NB4 cell growth was inhibited (69-78% at 120 h) and cell cycle progression in the G1 phase (82-85% at 120 h) was blocked by ATRA and all of the metabolites at 1 microM concentration. ATRA and its metabolites could induce NB4 cells differentiation with similar activity, as evaluated by cell morphology, by the nitroblue tetrazolium reduction test (82-88% at 120 h) or by the expression of the maturation specific cell surface marker CD11c. In addition, nuclear body reorganization to macropunctated structures, as well as the degradation of PML-RAR alpha, was found to be similar for ATRA and all of its metabolites. Comparison of the relative potency of the retinoids using the nitroblue tetrazolium reduction test showed effective concentrations required to differentiate 50% of cells in 72 h as follows: ATRA, 15.8 +/- 1.7 nM; 4-oxo-RA, 38.3 +/- 1.3 nM; 18-OH-RA, 55.5 +/- 1.8 nM; 4-OH-RA, 79.8 +/- 1.8 nM; and 5,6-epoxy-RA, 99.5 +/- 1.5 nM. The ATRA metabolites were found to exert their differentiation effects via the RAR alpha nuclear receptors, because the RAR alpha-specific antagonist BMS614 blocked metabolite-induced CD11c expression in NB4 cells. These data demonstrate that the principal ATRA Phase 1 metabolites can elicit leukemia cell growth inhibition and differentiation in vitro through the RAR alpha signaling pathway, and they suggest that these metabolites may play a role in ATRA antileukemic activity in vivo.

Cytochrome P450 (P450)-dependent metabolism of all-trans-retinoic acid (atRA) is important for the expression of its biological activity. Because the human P450s involved in the formation of the principal atRA metabolites have been only partially identified, the purpose of this study was to identify the human P450s involved in atRA metabolism. The use of phenotyped human liver microsomes (n = 16) allowed the identification of the following P450s: 2B6, 2C8, 3A4/5, and 2A6 were involved in the formation of 4-OH-RA and 4-oxo-RA; 2B6, 2C8, and 2A6 correlated with the formation of 18-OH-RA; and 2A6, 2B6, and 3A4/5 activities correlated with 5, 6-epoxy-RA formation (30-min incubation, 10 microM atRA, HPLC separation, UV detection 340 nm). The use of 15 cDNA-expressed human P450s from lymphoblast microsomes, showed the formation of 4-OH-RA by CYP3A7 > CYP3A5 > CYP2C18 > CYP2C8 > CYP3A4 > CYP2C9, whereas the 18-OH-RA formation involved CYPs 4A11 > 3A7 > 1A1 > 2C9 > 2C8 > 3A5 > 3A4 >2C18. Kinetic studies identified 3A7 as the most active P450 in the formation of three of the metabolites: for 4-OH-retinoic acid, 3A7 showed a V(max)/K(m) of 127.7, followed by 3A5 (V(max)/K(m) = 25.6), 2C8 (V(max)/K(m) = 24.5), 2C18 (V(max)/K(m) = 15.8), 3A4 (V(max)/K(m) = 5.7), 1A1 (V(max)/K(m) = 5.0), and 4A11 (V(max)/K(m) = 1.9); for 4-oxo-RA, 3A7 showed a V(max)/K(m) of 13.4, followed by a 10-fold lower activity for both 2C18 and 4A11 (V(max)/K(m) = 1.2); and for 18-OH-RA, 3A7 showed a V(max)/K(m) of 10.5 compared with a V(max)/K(m) of 2.1 for 4A11 and 2.0 for 2C8. 5,6-Epoxy-RA was only detected at high substrate concentrations in this system (>10 microM), and P450s 2C8, 2C9, and 1A1 were the most active in its formation. The use of embryonic kidney cells (293) stably transfected with human P450 cDNA confirmed the major involvement of P450s 3A7, 1A1, and 2C8 in the oxidation of atRA, and to a lesser extent, 1A2, 2C9, and 3A4. In conclusion, several human P450s involved in atRA metabolism have been identified, the expression of which was shown to direct atRA metabolism toward the formation of specific metabolites. The role of these human P450s in the biological and anticancer effects of atRA remains to be elucidated.

The clinical pharmacokinetics of irinotecan (CPT11) can be described by a 2 or 3 compartment model, a mean terminal half-life of 12 hours, a volume of distribution at steady state of 168 l/m2 and a total body clearance of 15 l/m2/h. Irinotecan is 65% bound to plasma proteins. The areas under the plasma concentration-time curve (AUC) of both irinotecan and active metabolite SN38 increase proportionally to the administered dose, although interpatient variability is important. SN38 levels achieved in humans are about 100-fold lower than corresponding irinotecan levels, but these concentrations are important since SN38 is 100- to 1,000-fold more cytotoxic than the parent compound. SN38 is 95% bound to plasma proteins. SN38 plasma decay follows closely that of the parent compound. Irinotecan is extensively metabolized in the liver. The bipiperidinocarbonylxy group of irinotecan is first removed by a carboxyesterase to yield the corresponding carboxylic acid and SN38. This metabolite can be converted into SN38 glucuronide by UDP-glucuronyltransferase (1.1 isoform). A recently identified metabolite is the 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]-carbonyloxy-camptothecin (APC), which is formed by the action of cytochrome P450 3A4. Numerous other unidentified metabolites are detected in bile and urine. The mean 24 h irinotecan urinary excretion represents 17-25% of the administered dose, whereas SN38 and its glucuronide recovery in urine is minimal (0.5 and 6%, respectively). Irinotecan and SN38 pharmacokinetics are not influenced by prior exposure to the parent drug. Irinotecan and SN38 AUCs correlate significantly with leuko-neutropenia and sometimes with the intensity of diarrhea. Increased bilirubin levels appear to influence irinotecan total body clearance. The observation that most tumor responses were seen at the highest doses administered in phase I trials suggest a dose-response relationship with this drug. These pharmacokinetic-pharmacodynamic relationships may prove useful for a better clinical management of this drug aimed at a better control of toxicities and a better prediction of tumor response for the benefit of the individual patient.

This article reviews the clinical pharmacokinetics of a water-soluble analogue of camptothecin, irinotecan [CPT-11 or 7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxy-camptoth eci n]. Irinotecan, and its more potent metabolite SN-38 (7- ethyl-10-hydroxy-camptothecin), interfere with mammalian DNA topoisomerase I and cancer cell death appears to result from DNA strand breaks caused by the formation of cleavable complexes. The main clinical adverse effects of irinotecan therapy are neutropenia and diarrhoea. Irinotecan has shown activity in leukaemia, lymphoma and the following cancer sites: colorectum, lung, ovary, cervix, pancreas, stomach and breast. Following the intravenous administration of irinotecan at 100 to 350 mg/m2, mean maximum irinotecan plasma concentrations are within the 1 to 10 mg/L range. Plasma concentrations can be described using a 2- or 3-compartment model with a mean terminal half-life ranging from 5 to 27 hours. The volume of distribution at steady-state (Vss) ranges from 136 to 255 L/m2, and the total body clearance is 8 to 21 L/h/m2. Irinotecan is 65% bound to plasma proteins. The areas under the plasma concentration-time curve (AUC) of both irinotecan and SN-38 increase proportionally to the administered dose, although interpatient variability is important. SN-38 levels achieved in humans are about 100-fold lower than corresponding irinotecan concentrations, but these concentrations are potentially important as SN-38 is 100- to 1000-fold more cytotoxic than the parent compound. SN-38 is 95% bound to plasma proteins. Maximum concentrations of SN-38 are reached about 1 hour after the beginning of a short intravenous infusion. SN-38 plasma decay follows closely that of the parent compound with an apparent terminal half-life ranging from 6 to 30 hours. In human plasma at equilibrium, the irinotecan lactone form accounts for 25 to 30% of the total and SN-38 lactone for 50 to 64%. Irinotecan is extensively metabolised in the liver. The bipiperidinocarbonylxy group of irinotecan is first removed by hydrolysis to yield the corresponding carboxylic acid and SN-38 by carboxyesterase. SN-38 can be converted into SN-38 glucuronide by hepatic UDP-glucuronyltransferase. Another recently identified metabolite is 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]-carbonyloxy-camptothecin (APC). This metabolite is a weak inhibitor of KB cell growth and a poor inducer of topoisomerase I DNA-cleavable complexes (100-fold less potent than SN-38). Numerous other unidentified metabolites have been detected in bile and urine. The mean 24-hour irinotecan urinary excretion represents 17 to 25% of the administered dose. Recovery of SN-38 and its glucuronide in urine is low and represents 1 to 3% of the irinotecan dose. Cumulative biliary excretion is 25% for irinotecan, 2% for SN-38 glucuronide and about 1% for SN-38. The pharmacokinetics of irinotecan and SN-38 are not influenced by prior exposure to the parent drug. The AUC of irinotecan and SN-38 correlate significantly with leuco-neutropenia and sometimes with the intensity of diarrhoea. Certain hepatic function parameters have been correlated negatively with irinotecan total body clearance. It was noted that most tumour responses were observed at the highest doses administered in phase I trials, which indicates a dose-response relationship with this drug. In the future, these pharmacokinetic-pharmacodynamic relationships will undoubtedly prove useful in minimising the toxicity and maximise the likelihood of tumour response in patients.

Quantitative reverse-transcriptase polymerase chain reaction was used to determine the content of mRNA derived from four CYP3A genes (CYP3A2, CYP3A9, CYP3A18, and CYP3A23) in rat liver. CYP3A2 and CYP3A9 gene expression was age- and sex-dependent, whereas CYP3A18 and CYP3A23 mRNA were observed before and after puberty at fairly constant levels that were about 20% higher in males than in females. CYP3A9 mRNA was detected only in adult rats, with a nearly twofold higher expression in females. CYP3A9 induction was different from other CYP3A isoenzymes, since phenobarbital was a more effective inducer than dexamethasone and clotrimazole. The results presented for CYP3A23 are those anticipated for CYP3A1, which may be an allelic variant of CYP3A23 not detected in these experiments. These data show that rat CYP3A genes are variably expressed depending on age, sex, or inducer type.

Purpose: The purpose of this study was to determine the bioavailability (F) of etoposide (E;VP-16) after oral administration of the water-soluble prodrug etoposide phosphate (EP;BMY-40481) during a phase I trial in cancer patients., Patients and Methods: Twenty-nine patients received oral EP (capsules, 50 to 150 mg/m2/d of E equivalent) for 5 days in week 1 (course 1), followed every 3 weeks thereafter by a daily intravenous (i.v.) infusion for 5 days of E (80 mg/m2, 1-hour i.v. infusion; course 2); in three patients, the i.v. E course was given before oral EP. Plasma and urine E pharmacokinetics (high-performance liquid chromatography [HPLC]) were performed on the first day of oral EP administration and on the first day of i.v. E., Results: Twenty-six of 29 patients completed two courses or more, whereas three patients received only one course due to toxicity. Myelosuppression was dose-dependent and dose-limiting, with grade 4 leukoneutropenia in four of 15 patients at 125 mg/m2 and in five of seven patients at 150 mg/m2. One patient died of meningeal hemorrhage related to grade 4 thrombocytopenia. Other toxicities were infrequent and/or manageable. No objective response was observed. The maximum-tolerated dose (MTD) is therefore 150 mg/m2, and the recommended oral dose of EP for phase II trials in this poor-risk patient population is 125 mg/m2. Twenty-six patients had pharmacokinetic data for both oral EP and i.v. E, whereas three had pharmacokinetic data on the i.v. E course only. After oral administration of EP, the pharmacokinetics of E were as follows: mean absorption rate constant (Ka), 1.7 +/- 1.7 h-1 (mean +/- SD); lag time, 0.3 +/- 0.2 hours; time of maximum concentration (t(max)), 1.6 +/- 0.8 hours; and mean half-lives (t1/2), 1.6 +/- 0.2 (first) and 10.3 +/- 5.8 hours (terminal); the increase in the area under the plasma concentration-versus-time curve (AUC) of E was proportional to the EP dose. After the 1-hour i.v. infusion of E, maximum concentration (C(max)) was 15 +/- 3 micrograms/mL; mean AUC, 88.0 +/- 22.0 micrograms.h/mL; mean total-body clearance (CL), 0.97 +/- 0.24 L/h/m2 (16.2 mL/min/m2); and mean t1/2, 0.9 +/- 0.6 (first) and 8.1 +/- 4.1 hours (terminal). The 24-hour urinary excretion of E after i.v. E was significantly higher (33%) compared with that of oral EP (17%) (P < .001). Significant correlation was observed between the neutropenia at nadir and the AUC of E after oral EP administration (r = .58, P < .01, sigmoid maximum effect [E(max)] model). The mean F of E after oral administration of EP in 26 patients was 68.0 +/- 17.9% (coefficient of variation [CV], 26.3%; F range, 35.5% to 111.8%). In this study, tumor type, as well as EP dose, did not significantly influence the F in E. There was no difference in F of E, whether oral EP was administered before or after i.v. E. Compared with literature data on oral E, the percent F in E after oral prodrug EP administration was 19% higher at either low ( < or = 100 mg/m2) or high ( > 100 mg/m2) doses., Conclusion: Similarly to E, the main toxicity of the prodrug EP is dose-dependent leukoneutropenia, which is dose-limiting at the oral MTD of 150 mg/m2/d for 5 days. The recommended oral dose of EP is 125 mg/m2/d for 5 days every 3 weeks in poor-risk patients. Compared with literature data, oral EP has a 19% higher F value compared with oral E either at low or high doses. This higher F in E from oral prodrug EP appears to be a pharmacologic advantage that could be of potential pharmacodynamic importance for this drug.

Although the induction of cytochromes P450 3A (CYP3A) is relatively well characterized in liver, its inducibility in an easily available tissue such as the peripheral leukocytes is not known. The purpose of this study was, therefore, to determine if CYP3A is inducible in vivo in peripheral leukocytes. Microsomes from rat leukocytes and liver were examined for CYP3A protein expression using Western blotting with a rabbit polyclonal antibody against rat CYP3A. Although CYP3A was not detected in control leukocytes, in vivo treatment with known CYP3A inducers (dexamethasone, clotrimazole, phenobarbital, pregnenolone-16 alpha-carbonitrile) resulted in CYP3A leukocyte levels of 0.2-0.8 pmol/mg protein. This leukocyte induction was approximately 1000-fold lower than in induced liver. Interestingly, there was an apparent linear relationship between leukocyte and liver CYP3A contents (r2 = 0.748, n = 29). These results not only demonstrate for the first time that CYP3A is inducible in rat leukocytes after in vivo treatment with various CYP3A inducers, but also suggest that peripheral leukocytes could be used to assess induction in vivo.

Irinotecan (CPT-11) is a semi-synthetic derivative of camptothecin currently in clinical trials. In vitro, CPT-11 presented preferential cytotoxicity toward some solid tumor cells (mouse colon 38 and pancreas 03; human pancreas MIA PaCa-2) as compared to leukemia cells (L1210), whereas SN-38, a metabolite of CPT-11, was not solid tumor selective. In vivo, schedule of administration studies in P388 leukemia and mammary adenocarcinoma 16/C (MA16/C) showed that CPT-11 was not markedly schedule dependent. In order to determine its spectrum of anticancer activity, CPT-11 was evaluated against a variety of mouse and human tumors. The end points used were total log cell kill (Lck) for solid tumors and increase in life span (% ILS) for leukemia. Intravenous CPT-11 was found highly active against both early and advanced stage pancreatic ductal adenocarcinoma 03 (P03), with 60% long-term survivors and 100% complete regressions, respectively. Other responsive tumors included: colon adenocarcinomas 38 and 51 (both 1.0 Lck); MA16/C (3.4 Lck); MA13/C (1.0 Lck); human Calc18 breast adenocarcinoma (2.8 Lck); Glasgow osteogenic sarcoma (1.8 Lck); Lewis lung carcinoma (1.4 Lck); B16 melanoma (1.4 Lck); P388 leukemia (170% ILS) and L1210 leukemia (64% ILS). Of interest, CPT-11 was active against tumors with acquired resistance to vincristine (P388/Vcr), to doxorubicin (P388/Dox) and to docetaxel (Calc18/TXT). CPT-11 was also found highly active after oral administration in mice bearing P03 and MA16/C tumors. Pharmacokinetic evaluations performed i.v. at the highest non-toxic dosage in mice bearing P03 tumors revealed CPT-11 peak plasma concentrations (Cmax) of 8.9 micrograms/ml and a terminal half-life of 0.6 h. The metabolite SN-38 plasma concentrations presented a Cmax of 1.6 micrograms/ml and a terminal half-life of 7.4 h. Although the CPT-11 tumor levels were similar to the plasma concentrations for early time points, drug levels decreased more slowly in the tumor compared to plasma (half-life, 5.0 h). SN-38 tumor levels reached concentrations in the range of 0.32-0.34 micrograms/g and decayed with a half-life of 6.9 h. No significant difference in plasma or tumor pharmacokinetics of either CPT-11 or SN-38 were noted after one or five daily i.v. injections. Overall, these data show that CPT-11 has good activity in experimental models, when administered both by the i.v. and the oral routes. Compared to humans, a similar schedule of administration independence was observed and similar CPT-11 levels could be reached at efficacious dosages although metabolite SN-38 levels were found higher in mice.

Intoplicine (RP 60475F; NSC 645008) is a novel 7H-benzo[e]pyrido[4,3-b]indole derivative which interacts with both topoisomerases I and II. Because of its high activity in preclinical cancer models, original mechanism of action and acceptable toxicity profile, intoplicine was further evaluated in a phase I and pharmacology study. Thirty-three (33) patients (24 men and nine women) meeting standard phase I eligibility criteria were included: median age was 56 years, performance status 0-1 in 28 patients and 2 in five patients. Tumor primary sites were head and neck (9), colon (6), lung (3) and various other sites (15). Thirty-one patients had received prior radiotherapy and/or chemotherapy. Sixty-nine coursed of intoplicine were administered as a 1 h i.v. infusion at dose levels ranging from 12 to 360 mg/m2. Dose-dependent and reproducible hepatotoxicity was dose limiting in three out of four patients at 360 mg/m2: this toxicity was reversible in two of three patients, but fatal in one. Two sudden deaths occurred in this study at 12 and 48 mg/m2, and the drug implication could not be excluded. No myelosuppression was noted. Hepatotoxicity is therefore dose limiting at 360 mg/m2, and the phase II recommended dose is 270 mg/m2 every 3 weeks with close monitoring of hepatic and cardiac functions. Intoplicine pharmacokinetics was determined in plasma (23 patients) and whole blood (18 patients) at doses ranging from 12 to 360 mg/m2. Intoplicine plasma concentration decay was either bi- or triphasic with the following pharmacokinetic values (mean +/- SEM): half-life alpha, 0.04 +/- 0.004 h; half-life beta, 0.61 +/- 0.13 h; terminal half-life, 19.4 +/- 4.0 h; mean residence-time (MRT), 11.3 +/- 2.4; total plasma clearance (CL), 74 +/- 5 l/h; volume of distribution beta (V beta), 1982 +/- 477 l: volume of distribution at steady state (Vss): 802 +/- 188 l. both the area under the plasma concentration versus time curves (AUC) and the maximum plasma concentrations (Cmax) increased linearly with the intoplicine dose, indicating linear pharmacokinetics (AUC: r = 0.937; slope = 0.01305; p < 0.001; Cmax: r = 0.847; slop = 0.01115; p < 0.001). Plasma AUC was also predicted very accurately by the Cmax values (r = 0.909; slope = 1.0701; p < 0.001). Other plasma pharmacokinetic parameter values increased significantly with dose, e.g. the terminal half-life (r = 0.748, p < 0.001) the MRT (r = 0.728, p < 0.001), the V beta (r = 0.809, p < 0.001), and the Vss (r = 0.804, p < 0.001). This was probably due to a longer detectability of the drug in plasma at higher doses. Blood pharmacokinetics was also evaluated in 18 patients since it was found that red blood cells represented a significant drug reservoir for intoplicine. Blood intoplicine disposition curves were either bi- or triphasic with the following pharmacokinetic parameter values (mean +/- SEM): half-life alpha, 0.04 +/- 0.01 h; half-life beta, 0.94 +/- 0.22 h; terminal half-life, 57.1 +/- 6.6 h; MRT, 82.2 +/- 9.9 h; CL, 18 +/- 3 l/h; V beta, 1188 +/- 147 I; Vss 1163 +/- 138 I. Blood pharmacokinetics was linear, since AUC and Cmax increased linearly with dose (AUC: r = 0.879; slop = 0.06884; p < 0.001; Cmax: r = 0.835, slop = 0.01223; p < 0.001). Blood AUC values could also be determined by the blood Cmax (r = 0.768; slop = 5.0206; p < 0.001). Other blood pharmacokinetic parameter values presented a dose dependence, e.g. the terminal half-life (r = 0.626, p = 0.005), the V beta (r = 0.682, p = 0.002) and the Vss (r = 0.555, p = 0.017). The plasma or blood intoplicine concentrations achieved in vivo in humans are potentially cytotoxic levels based on preclinical in vivo and in vitro data. In conclusion, the phase II recommended dose of intoplicine is 270 mg/m2 administered as a 1 h i.v. infusion every 3 weeks. Plasma and blood pharmacokinetics were linear within the dose range studied. Potentially cytotoxic concentrations were reached at clinically achievable doses.