Giorgio Grosa

In vitro metabolism study of combretastatin A-4 in rat and human liver microsomes.

The phase I biotransformation of combretastatin A-4 (CA-4) 1, a potent tubulin polymerization inhibitor with antivascular and antitumoral properties, was studied using rat and human liver subcellular fractions. The metabolites were separated by high-performance liquid chromatography and detected with simultaneous UV and electrospray ionization (ESI) mass spectrometry. The assignment of metabolite structures was based on ESI-tandem mass spectrometry experiments, and it was confirmed by comparison with reference samples obtained by synthesis. O-Demethylation and aromatic hydroxylation are the two major phase I biotransformation pathways, the latter being regioselective for phenyl ring B of 1. Indeed, incubation with rat and human microsomal fractions led to the formation of a number of metabolites, eight of which were identified. The regioselectivity of microsomal oxidation was also demonstrated by the lack of metabolites arising from stilbenic double bond epoxidation. Alongside the oxidative metabolism, Z-E isomerization during in vitro study was also observed, contributing to the complexity of the metabolite pattern. Moreover, when 1 was incubated with a cytosolic fraction, metabolites were not observed. Aromatic hydroxylation at the C-6′ of phenyl ring B and isomerization led to the formation of M1 and M2 metabolites, which were further oxidized to the corresponding para-quinone (M7 and M8) species whose role in pharmacodynamic activity is unknown. Metabolites M4 and M5, arising from O-demethylation of phenyl ring B, did not form the ortho-quinones. O-Demethylation of phenyl ring A formed the metabolite M3 with a complete isomerization of the stilbenic double bond.

IDENTIFICATION OF THE HUMAN UDP-GLUCURONOSYLTRANSFERASES INVOLVED IN THE GLUCURONIDATION OF COMBRETASTATIN A-4.

The stilbenic compound (Z)-combretastatin A-4 (CA-4) has been described as a potent tubulin polymerization inhibitor. In vivo, CA-4 binds to tubulin and inhibits microtubule depolymerization, which results in morphological changes in proliferating endothelial cells. Combretastatin A-4 prodrug phosphate is a leading vascular disrupting agent and is currently being evaluated in multiple clinical trials as a treatment for solid tumors. The aim of this study was to identify and characterize the UDP-glucuronosyltransferase (UGT) isoforms involved in CA-4 glucuronidation by incubation with human liver microsomes and a panel of nine liver-expressed recombinant UGT Supersomes (1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B15, and 2B17). As we observed, the high rate of formation of CA-4 glucuronide (Vmax = 12.78 ± 0.29 nmol/min/mg protein) and the low Km (6.98 ± 0.65 μM) denoted that UGT1A9 was primarily responsible for the in vitro glucuronidation of CA-4. UGT1A6 was also a significant contributor to CA-4 glucuronidation (Vmax = 3.95 ± 0.13 nmol/min/mg protein and S50 = 44.80 ± 3.54 μM). Furthermore, we demonstrated that the kinetics of CA-4 glucuronidation with liver microsomes but also with a panel of recombinant UGTs is atypical as it fits two different models: the substrate inhibition and also the sigmoidal kinetic model. Finally, experiments conducted to inhibit the glucuronosyltransferase activity in the human liver microsomes assay showed that phenylbutazone, trifluoperazine, propofol, and 1-naphthol effectively inhibited CA-4 glucuronidation.

Development and validation of a solid-phase extraction and gas chromatography–tandem mass spectrometry method for the determination of isopropyl-9H-thioxanthen-9-one in carton packaged milk

A simple and efficient method for the determination of isopropyl-9H-thioxanten-9-one (ITX) in different fat content milk samples and baby milk samples stored in packaged cartons was developed and validated. Samples were extracted using solid-phase extraction (SPE) and analysed by gas chromatography-tandem mass spectrometry operated in selected reaction monitoring mode (SRM). Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision and trueness. LOD and LOQ values in the low microg/L were achieved, whereas linearity was established within 0.5-500 microg/L range. Good precision was obtained both in terms of intra-day repeatability and inter-day precision on two concentration levels (RSD% lower than 2%). Good percentage recoveries were obtained (92.0-102.0%) even in the presence of high amount of fat. Finally, the developed method was successfully applied to analyse a number of commercial milk samples with different fat content and baby milk samples.

Development and validation of a stability-indicating HPLC-UV method for the determination of alizapride and its degradation products

A stability-indicating high-performance liquid chromatography procedure has been developed for the determination of alizapride (AL) and its main degradation products alizapride carboxylic acid (AL-CA) and alizapride N-oxide (AL-NO2) in drug substance and product. The method was developed based on forced degradation data obtained by HPLC-MS analysis. Indeed AL underwent chemical degradation by acid/base catalyzed hydrolysis and oxidation the main degradation products being AL-CA and AL-NO2 respectively. The separation and quantisation were achieved on a 150-mm reverse phase column with a hydrophilic linkage between silica particles and hydrophobic alkyl chains. The mobile phase was constituted (flow rate 1.5mLmin(-1)) of eluant A: aqueous acetate buffer (pH 4.0; 20mM) and eluant B: CH(3)OH using a gradient elution and detection of analytes at 225nm. The method showed good linearity for the AL, AL-CA, AL-NO2 mixture in the 25-75, 1-15 and 1-15microgmL(-1) ranges respectively, being all the square of the correlation coefficients greater than 0.999. The precision, determined in terms of intra-day and inter-day precisions and expressed as RSDs were 0.8, 1.3 and 2.1% and 1.0, 1.7, 4.8% for AL, AL-CA and AL-NO2 respectively. The method demonstrated also to be accurate; indeed the average recoveries, at 100% and 0.2% of the target assay concentration, were 100.5, 98.6, and 96.8% for AL, AL-CA and AL-NO2 respectively. The robustness was also evaluated by variations of mobile phase composition and pH. Finally, the applicability of the method was evaluated in commercial dosage form analysis as well as in stability studies.

The metabolic fate of isocombretastatin A-4 in human liver microsomes: identification, synthesis and biological evaluation of metabolites.

Combretastatins, naturally occurring stilbenes, were isolated from Combretum caffrum by Pettit in 1989. Among them, combretastatin A-4 (CA-4) was shown to strongly inhibit the polymerization of tubulin by binding to the colchicine binding site and was the most cytotoxic agent. CA-4 showed toxicity at a nanomolar level against a variety of human cancer cell lines, including multiple drug-resistant cancers. CA-4 also exerts highly selective toxicity in proliferating endothelial cells and, as a consequence, demonstrates strong suppressive activity on tumor blood flow leading to cell death. Moreover, in contrast to colchicine, the antivascular effects of CA-4 are apparent well below the maximal tolerated dose, offering a wide therapeutic window. However, there are two major disadvantages with CA4 as a drug candidate: its low aqueous solubility limits its efficacy in vivo, and its low chemical stability due to the cisdouble bond isomerization to the more thermally stable but nonactive trans form during storage and administration. The first problem was solved by the synthesis of water-soluble prodrugs, including the disodium phosphate prodrug CA-4P (1) 5] and its serinamido derivative AVE-8062 (2). Currently, CA-4P, either as a single agent or in combination therapy, is undergoing several advanced clinical trials worldwide for the treatment of age-related macular degeneration (AMD) or anaplastic thyroid cancer. To overcome the second problem related to the double bond isomerization, several approaches have been undertaken to rigidify the olefin using a heterocyclic compound. In an ongoing project aimed at developing novel tubulin assembly inhibitors, we recently discovered isocombretastatin A-4 (isoCA-4), a structural isomer of the natural product that displays biological activities comparable to those of CA-4. isoCA-4 contains a 1,1-diarylethylene scaffold and is easy to synthesize on a multigram scale without the need to control the olefin geometry, and then definitively solves the problem of the double bond isomerization. Recently, we studied the metabolic fate of CA-4 in rat and human microsomal preparations, and several phase I metabolites (resulting from O-demethylation, aromatic hydroxylation and quinones formation), as well as phase II conjugates, in both cis and trans configuration, were characterized. The role of these metabolites in the pharmacodynamic activity of CA-4 is still unknown. These considerations together with the structural differences between CA-4 and isoCA-4, due to the presence of a 1,1-diarylethylene scaffold, prompted us to examine the metabolic profile of isoCA-4, identifying and synthesizing the metabolites, and then evaluating their biological properties. Herein, we report the results of this study. Possible metabolites of isoCA-4, demethylated/hydroxylated on the Aand B-rings, hydroxylated on the B-ring, or transformed into p-quinones, were prepared according to Scheme 1. Hypothetical metabolites 3–6 arising from O-demethylation or hydroxylation of the Aand B-rings were prepared by the coupling of N-tosylhydrazones with aryl iodides under palladium catalysis. Then, the crude coupling products were O-desilylated using a tetrahydrofuran (THF) solution of tetra-nbutylammonium fluoride (TBAF) to afford the desired phenols 3 and 4, as well as catechols 5 and 6, in good yields. The use of Fremy’s salt as a biomimetic oxidant with isoCA-4 in a mixture of water and dichloromethane furnished p-quinone 7 in an acceptable yield (50 %). Finally, the reduction of 7 using sodium borohydride (NaBH4) afforded the putative metabolite 8, which was found to be unstable and oxidizes to p-quinone 7 in air. This compound must be prepared carefully just before use and stored under an inert atmosphere at 40 8C. isoCA-4 was incubated in human liver microsomal (HLM) fractions in the presence of a NADPH regenerating system. isoCA-4 and its metabolites were separated by HPLC using a C18 reverse-phase column and a mixture of water and acetonitrile acidified with 1 % formic acid as the eluent. The LC/MS [a] M. A. Soussi, Dr. S. Messaoudi, Dr. O. Provot, Prof. Dr. J.-D. Brion, Dr. M. Alami Univ Paris-Sud, Facult de Pharmacie, CNRS, BioCIS-UMR 8076 Laboratoire de Chimie Th rapeutique 5 Rue J.-B. Cl ment, Ch tenay-Malabry 92296 (France) E-mail : mouad.alami@u-psud.fr [b] Dr. S. Aprile, Dr. E. Del Grosso, Dr. G. Grosa Dipartimento di Scienze Chimiche Alimentari Farmaceutiche e Farmacologiche and Drug and Food Biotechnology Center Universit degli Studi del Piemonte Orientale “A. Avogadro” Largo Donegani 2, 28100 Novara (Italy) E-mail : Giorgio.Grosa@pharm.unipmn.it [c] Dr. J. Bignon, Dr. J. Dubois Institut de Chimie des Substances Naturelles, UPR 2301, CNRS Avenue de la Terrasse, 91198 Gif-sur-Yvette (France) [] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201100193.

In vitro and in vivo phase II metabolism of combretastatin A-4: Evidence for the formation of a sulphate conjugate metabolite

Combretastatin A-4 (CA-4), is a natural compound with a potent tubulin polymerization and cell growth inhibitor properties. For these reasons CA-4 is one of the most potent anti-vascular agents that shows strong cytotoxicity against a variety of human cancer cells, including multi-drug-resistant cancer cell lines. In order to complete the knowledge of metabolic fate of CA-4, the in vitro and in vivo phase II metabolism was investigated. Both in incubation with rat and human liver S9 preparation in the presence of 39-phosphoadenosine-5´-phosphosulfate (PAPS) as a cofactor the formation of a previously no reported sulphate metabolite was demonstrated through liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) data and comparison with a synthetic reference sample. In incubation of CA-4 using rat and human liver microsomes, the formation of CA-4 glucuronide was observed and chromatographic and mass spectral properties of the metabolite were achieved and compared with those of a synthetic reference sample. Incubation of CA-4 with rat and human liver S9 preparation in the presence of uridine-5´-diphosphoglucuronic acid trisodium salt (UDPGA) and an β-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-regenerating system as cofactors resulted in the formation of glucuronides arising from phase I CA-4 metabolites. When CA-4 was administered intraperitoneally to rat at a dose of 30 mg kg−1, both glucuronide and sulphate metabolites were observed in LC-ESI-MS-MS chromatograms and their mass spectral data were identical to those obtained from synthetic standards.

Lack ofin vitro interactions using human liver micro-somes between rabeprazole and anticancer drugs

SummaryThe potential interactions between rabeprazole, a widely used proton pump inhibitor, and anticancer drugs (5-fluorouracil, docetaxel, cyclophosphamide, gemcitabine, methotrexate, doxorubicin, etoposide) or drugs commonly present in the therapy of oncological patients (fluoxetine and ondansetron), were studied using in vitro human liver microsomes. The interactions between rabeprazole and the anticancer drugs were evaluated by measuring their concentrations in test and control incubations with HPLC-DAD-UV methods. To achieve this aim, nine HPLC-DAD-UV methods were developed using different stationary and mobile phases. The methods were then validated for the following parameters: selectivity, linearity, precision, and accuracy. As expected rabeprazole did not significantly inhibit the metabolism of the evaluated drugs in human liver microsomal preparations at the selected concentrations. These results shows that rabeprazole probably could be devoid of pharmacokinetic interactions with common drugs used during chemotherapy.

New insights in oxybutynin chemical stability: Identification in transdermal patches of a new impurity arising from oxybutynin N-oxide rearrangement.

Oxybutynin hydrochloride (Oxy), the first choice drug used for the management of urinary incontinence, is available in different types of formulations. However, due to its better lipophylicity and permeability, Oxyfree base was used in the new topical formulations such as transdermal patch and gel. The presence of an unprecedented impurity (Oxy-EK) in transdermal patches led to reinvestigate the chemical stability of Oxyfree base in oxidative conditions assigning, to Oxy-EK, the structure of (3E)-4-(N,N-diethylamino)-2-oxo-3-buten-1-yl 1-cyclohexyl-1-phenylglycolate. Oxy-EK arises from the prototropic rearrangement of oxybutynin N-oxides leading to the formation of an enamino ketone function which shows a long-wavelength UV-absorption. The total synthesis of Oxy-EK was performed, allowing to propose it as the indicator of stability for oxidative degradation of Oxy free base in transdermal formulations. The presence in the structure of Oxy-EK of an α,β-unsaturated carbonyl function, a potential Michael acceptor, suggested the need of evaluating its possible mutagenic power. Accordingly, the Ames test was performed: at nontoxic concentrations, Oxy-EK did not increase the number of revertant colonies in all strains tested both in the absence and presence of the exogenous metabolic activator S9.

New insights in the metabolism of oxybutynin: evidence of N-oxidation of propargylamine moiety and rearrangement to enaminoketone

Abstract 1. Oxybutynin hydrochloride is an antimuscarinic agent prescribed to patients with an overactive bladder (OAB) and symptoms of urinary urge incontinence. Oxybutynin undergoes pre-systemic metabolism, and the N-desethyloxybutynin (Oxy-DE), is reported to have similar anticholinergic effects. 2. We revisited the oxidative metabolic fate of oxybutynin by liquid chromatography–tandem mass spectrometry analysis of incubations with rat and human liver fractions, and by measuring plasma and urine samples collected after oral administration of oxybutynin in rats. This investigation highlighted that not only N-deethylation but also N-oxidation participates in the clearance of oxybutynin after oral administration. 3. A new metabolic scheme for oxybutynin was delineated, identifying three distinct oxidative metabolic pathways: N-deethylation (Oxy-DE) followed by the oxidation of the secondary amine function to form the hydroxylamine (Oxy-HA), N-oxidation (Oxy-NO) followed by rearrangement of the tertiary propargylamine N-oxide moiety (Oxy-EK), and hydroxylation on the cyclohexyl ring. 4. The functional activity of Oxy-EK was investigated on the muscarinic receptors (M1-3) demonstrating its lack of antimuscarinic activity. 5. Despite the presence of the α,β-unsaturated function, Oxy-EK does not react with glutathione indicating that in the clearance of oxybutynin no reactive and potentially toxic metabolites were formed.

Identification of a Potent Phosphoinositide 3-Kinase Pan Inhibitor Displaying a Strategic Carboxylic Acid Group and Development of Its Prodrugs

Activation of the phosphoinositide 3‐kinase (PI3K) pathway is a key signaling event in cancer, inflammation, and other proliferative diseases. PI3K inhibitors are already approved for some specific clinical indications, but their systemic on‐target toxicity limits their larger use. In particular, whereas toxicity is tolerable in acute treatment of life‐threatening diseases, this is less acceptable in chronic conditions. In the past, the strategy to overcome this drawback was to block selected isoforms mainly expressed in leukocytes, but redundancy within the PI3K family members challenges the effectiveness of this approach. On the other hand, decreasing exposure to selected target cells represents a so‐far unexplored alternative to circumvent systemic toxicity. In this manuscript, we describe the generation of a library of triazolylquinolones and the development of the first prodrug pan‐PI3K inhibitor.