Mohammed Jaffar

Menadione-induced reactive oxygen species generation via redox cycling promotes apoptosis of murine pancreatic acinar cells

Oxidative stress may be an important determinant of the severity of acute pancreatitis. One-electron reduction of oxidants generates reactive oxygen species (ROS) via redox cycling, whereas two-electron detoxification, e.g. by NAD(P)H:quinone oxidoreductase, does not. The actions of menadione on ROS production and cell fate were compared with those of a non-cycling analogue (2,4-dimethoxy-2-methylnaphthalene (DMN)) using real-time confocal microscopy of isolated perfused murine pancreatic acinar cells. Menadione generated ROS with a concomitant decrease of NAD(P)H, consistent with redox cycling. The elevation of ROS was prevented by the antioxidant N-acetyl-l-cysteine but not by the NADPH oxidase inhibitor diphenyliodonium. DMN produced no change in reactive oxygen species per se but significantly potentiated menadione-induced effects, probably via enhancement of one-electron reduction, since DMN was found to inhibit NAD(P)H:quinone oxidoreductase detoxification. Menadione caused apoptosis of pancreatic acinar cells that was significantly potentiated by DMN, whereas DMN alone had no effect. Furthermore, bile acid (taurolithocholic acid 3-sulfate)-induced caspase activation was also greatly increased by DMN, whereas DMN had no effect per se. These results suggest that acute generation of ROS by menadione occurs via redox cycling, the net effect of which is induction of apoptotic pancreatic acinar cell death. Two-electron detoxifying enzymes such as NAD(P)H:quinone oxidoreductase, which are elevated in pancreatitis, may provide protection against excessive ROS and exert an important role in determining acinar cell fate.

Hypoxia Targeted Gene Therapy to Increase the Efficacy of Tirapazamine as an Adjuvant to Radiotherapy: Reversing Tumor Radioresistance and Effecting Cure

Solid tumors are characterized by regions of hypoxia that are inherently resistant to both radiotherapy and some chemotherapy. To target this resistant population, bioreductive drugs that are preferentially toxic to tumor cells in a hypoxic environment are being evaluated in clinical trials; the lead compound, tirapazamine (TPZ), is being used in combination with cisplatin and/or with radiotherapy. Crucially, tumor response to TPZ is also dependent on the cellular complement of reductases. In particular, NADPH:cytochrome P450 reductase (P450R) plays a major role in the metabolic activation of TPZ. In a gene-directed enzyme prodrug therapy (GDEPT) approach using adenoviral delivery, we have overexpressed human P450R specifically within hypoxic cells in tumors, with the aim of harnessing hypoxia as a trigger for both enzyme expression and drug metabolism. The adenovirus used incorporates the hypoxia-responsive element (HRE) from the lactate dehydrogenase gene in a minimal SV40 promoter context upstream of the cDNA for P450R. In a human tumor model in which TPZ alone does not potentiate radiotherapeutic outcome (HT1080 fibrosarcoma), we witnessed complete tumor regression when tumors were virally transduced before treatment.

Fatty acid ethyl ester synthase inhibition ameliorates ethanol-induced Ca2+-dependent mitochondrial dysfunction and acute pancreatitis

Objective Non-oxidative metabolism of ethanol (NOME) produces fatty acid ethyl esters (FAEEs) via carboxylester lipase (CEL) and other enzyme action implicated in mitochondrial injury and acute pancreatitis (AP). This study investigated the relative importance of oxidative and non-oxidative pathways in mitochondrial dysfunction, pancreatic damage and development of alcoholic AP, and whether deleterious effects of NOME are preventable. Design Intracellular calcium ([Ca2+]C), NAD(P)H, mitochondrial membrane potential and activation of apoptotic and necrotic cell death pathways were examined in isolated pancreatic acinar cells in response to ethanol and/or palmitoleic acid (POA) in the presence or absence of 4-methylpyrazole (4-MP) to inhibit oxidative metabolism. A novel in vivo model of alcoholic AP induced by intraperitoneal administration of ethanol and POA was developed to assess the effects of manipulating alcohol metabolism. Results Inhibition of OME with 4-MP converted predominantly transient [Ca2+]C rises induced by low ethanol/POA combination to sustained elevations, with concurrent mitochondrial depolarisation, fall of NAD(P)H and cellular necrosis in vitro. All effects were prevented by 3-benzyl-6-chloro-2-pyrone (3-BCP), a CEL inhibitor. 3-BCP also significantly inhibited rises of pancreatic FAEE in vivo and ameliorated acute pancreatic damage and inflammation induced by administration of ethanol and POA to mice. Conclusions A combination of low ethanol and fatty acid that did not exert deleterious effects per se became toxic when oxidative metabolism was inhibited. The in vitro and in vivo damage was markedly inhibited by blockade of CEL, indicating the potential for development of specific therapy for treatment of alcoholic AP via inhibition of FAEE generation.

Oxygen-sensitive enzyme-prodrug gene therapy for the eradication of radiation-resistant solid tumours.

Overwhelming clinical and experimental data demonstrate that tumour hypoxia is associated with aggressive disease and poor treatment outcome as hypoxic cells are refractive to radiotherapy and some forms of chemotherapy. However, hypoxia is rare in physiologically normal tissues representing a tumour-specific condition. To selectively target this therapeutically refractive cell population, we have combined bioreductive chemotherapy with hypoxia-directed gene therapy. We have transfected the human fibrosarcoma cell line, HT1080, with a hypoxia-regulated expression vector encoding the human flavoprotein cytochrome c P450 reductase (HRE-P450R). This conferred hypoxia-dependent sensitivity to the alkylating nitroimidazole prodrug RSU1069 in vitro, with a greater than 30-fold increase in oxic/hypoxic cytotoxicity ratio compared with controls. Xenografts of both the HRE-P450R and empty vector transfectants had comparable hypoxic fractions and were refractive to single dose radiotherapy of up to 15 Gy. However, combining a prodrug of RSU1069 with a reduced radiotherapy dose of 10 Gy represents a curative regimen (50% tumour-free survival; day 100) in the HRE-P450R xenografts. In complete contrast, 100% mortality was apparent by day 44 in the empty vector control xenografts treated in the same way. Thus, an oxygen-sensitive gene-directed enzyme prodrug therapy approach may have utility when incorporated into conventional radiotherapy and/or chemotherapy protocols for loco-regional disease in any tissue where hypoxia is a contra-indication to treatment success.

Synthesis of N -Benzyl- and N -Phenyl-2-amino-4,5- dihydrothiazoles and thioureas and evaluation as modulators of the isoforms of nitric oxide synthase

Inhibition of the isoforms of nitric oxide synthase (NOS) has important applications in therapy of several diseases, including cancer. Using 1400 W [N-(3-aminomethylbenzyl)acetamidine], thiocitrulline and N(delta)-(4,5-dihydrothiazol-2-yl)ornithine as lead compounds, series of N-benzyl- and N-phenyl-2-amino-4,5-dihydrothiazoles and thioureas were designed as inhibitors of NOS. Ring-substituted benzyl and phenyl isothiocyanates were synthesised by condensation of the corresponding amines with thiophosgene and addition of ammonia gave the corresponding thioureas in high yields. The substituted 2-amino-4,5-dihydrothiazoles were approached by two routes. Treatment of simple benzylamines with 2-methylthio-4,5-dihydrothiazole at 180 degrees C afforded the corresponding 2-benzylamino-4,5-dihydrothiazoles. For less nucleophilic amines and those carrying more thermally labile substituents, the 4,5-dihydrothiazoles were approached by acid-catalysed cyclisation of N-(2-hydroxyethyl)thioureas. This cyclisation was shown to proceed by an S(N)2-like process. Modest inhibitory activity was shown by most of the thioureas and 4,5-dihydrothiazoles, with N-(3-aminomethylphenyl)thiourea (IC(50)=13 microM vs rat neuronal NOS and IC(50)=23 microM vs rat inducible NOS) and 2-(3-aminomethylphenylamino)-4,5-dihydrothiazole (IC(50)=13 microM vs rat neuronal NOS and IC(50)=19 microM vs human inducible NOS) being the most potent. Several thioureas and 4,5-dihydrothiazoles were found to stimulate the activity of human inducible NOS in a time-dependent manner.

The relative importance of NADPH: cytochrome c (P450) reductase for determining the sensitivity of human tumour cells to the indolequinone EO9 and related analogues lacking functionality at the C-2 and C-3 positions

Analogues of EO9 (3-hydroxymethyl-5-aziridinyl-1-methyl-2[1H-indole-4-7-dione]prop-2-e n-1-ol) which lack functionality at either the C-2 or C-3 position were synthesised. The aim was to establish the importance of each group towards toxicity and to give an indication as to whether substitution at either position altered activation and toxicity after metabolism by cellular NADPH: cytochrome c (P450) reductase (P450R). MDA231 breast cancer cells were transfected with the cDNA for human P450R and stable clones were isolated. These high P450R-expressing clones were used to determine the aerobic and hypoxic toxicity of EO9 and the two analogues that lacked functionality at either C-2 or C-3. The results showed that P450R was strongly implicated in the bioactivation of EO9 and its analogues under both of these conditions. This data also showed that the C-3 functionality was primarily implicated in hypoxic toxicity.

Chemical synthesis and biological evaluation of a NAD(P)H:quinone oxidoreductase-1¿targeted tripartite quinone drug delivery system.

NAD(P)H:quinone oxidoreductase-1 (NQO1) is a potential target for therapeutic intervention but attempts to exploit NQO1 using quinone-based bioreductive prodrugs have been largely compromised by toxicity to organs that inherently express high levels of NQO1. In an attempt to circumvent this problem, this study describes the development of a tripartite quinone-based drug delivery system, the ultimate objective of which is to release a targeted therapeutic agent following the reduction of a quinone “trigger” by NQO1. Molecular modeling of drug/NQO1 interactions were conducted prior to the synthesis of N-{4-[bis-(2-chloroethyl)-amino]-phenyl}-β,β,2,4,5-pentamethyl-3,6-dioxo-1,4-cyclohexadiene-1-propanamide (prodrug 1). Prodrug 1 is a good substrate for purified NQO1 (Vmax and Km values of 11.86 ± 3.09 μmol/min/mg and 2.70 ± 1.14 μmol/L, respectively) and liquid chromatography-mass spectrometry analysis of the metabolites generated showed that lactone 3 and aniline mustard 4 were generated in a time- and NQO1-dependent manner. Chemosensitivity studies showed that prodrug 1 is selectively toxic to cells that overexpress NQO1 under aerobic conditions, and comet assay analysis confirmed the presence of elevated interstrand cross-links in NQO1-rich compared with NQO1-deficient cells. Hypoxic sensitization (hypoxic cytotoxicity ratio = 15.8) was observed in T47D cells that overexpress cytochrome P450 reductase. In conclusion, the results of this study provide mechanistic proof of principle that a tripartite benzoquinone drug delivery system is enzymatically reduced to release an active therapeutic agent. Further development of this concept to fine-tune substrate specificity for specific reductases and/or the inclusion of alternative therapeutic agents is warranted. [Mol Cancer Ther 2007;6(12):3122–30]

Synthesis and Enzymatic Evaluation of Pyridinium-Substituted Uracil Derivatives as Novel Inhibitors of Thymidine Phosphorylase

A series of water soluble N(1)- and C(6)-substituted uracil pyridinium compounds were prepared as potential inhibitors of thymidine phosphorylase (TP). The C(6)-uracil substituted derivatives were the most active. 1-[(5-Chloro-2,4-dihydroxypyrimidin-6-yl)methyl]pyridinium chloride, was identified as the best inhibitor being 5-fold more potent than the known inhibitor, 6-amino-5-bromouracil.

Prodrugs for targeting hypoxic tissues : Regiospecific elimination of aspirin from reduced indolequinones

A series of regioisomeric derivatives of a 1-methylindole-4,7-dione were synthesised, substituted with a 2-acetoxybenzoate leaving group linked through the (indol-2-yl)methyl or (indol-3-yl)methyl (or propenyl) positions. Reductive elimination of the leaving group occurred from the (indol-3-yl)methyl derivatives but not the 2-substituted regioisomers, indicating that only the C-3 position may be utilised in bioreductively-activated drug delivery, which was demonstrated with an aspirin prodrug.

3-Substituted-5-aziridinyl-1-methylindole-4,7-diones as NQO1-directed antitumour agents: mechanism of activation and cytotoxicity in vitro

Indolequinone agents are a unique class of bioreductive cytotoxins that can function as dual substrates for both one- and two-electron reductases. This endows them with the potential to be either hypoxia-selective cytotoxins or NAD(P)H:quinone oxidoreductase 1 (NQO1)-directed prodrugs, respectively. We have studied the structure-activity relationships of four novel indolequinone analogues with regard to one- and/or two-electron activation. Single-electron metabolism was achieved by exposing the human carcinoma cell line T47D to each agent under hypoxic conditions, whilst concerted two-electron metabolism was assessed by stably expressing the cDNA for human NQO1 in a cloned cell line of T47D. The C-3 and C-5 positions of the indolequinone nucleus were modified to manipulate reactivity of the reduction products and the four prodrugs were identified as NQO1 substrates of varying specificity. Two of the four prodrugs, in which both C-3 and C-5 groups remained functional, proved to be NQO1-directed cytotoxins with selectivity ratios of 60- to 80-fold in the T47D (WT) versus the NQO1 overexpressing T47D cells. They also retained selectivity as hypoxic cytotoxins with oxic/hypoxic ratios of 20- to 22-fold. Replacement of the C-3 hydroxymethyl leaving group with an aldehyde group ablated all selectivity in air and hypoxia in both cell lines. Addition of a 2-methyl group on the C-5 aziridinyl group to introduce steric hinderance reduced but did not abolish NQO1-dependent metabolism. However, it enhanced single-electron metabolism-dependent DNA cross-linking in a manner that was independent of cytotoxicity. These data demonstrate that subtle structure-activity relationship exists for different cellular reductases and under certain circumstances distinct forms of DNA damage can arise, the cytotoxic consequences of which can vary. This study identifies a candidate indolequinone analogue for further development as a dual hypoxia and NQO1-directed prodrug.