Synthesis of novel ribose-based thioimidate n-oxides

This work is based on the study of new synthetic paths to obtain thioimidate N-oxides (TINOs) from D-ribose and to study their reactivity with the purpose to obtain ketonitrones. TINOs, aren’t well known molecules, but these enantiomerically pure backbones could be valuable intermediates in the synthesis of novel ketonitrones which are key intermediates in the synthesis of iminosugars. TINOs were discovered from the study of glucoraphanin, a particular glucosinolate, that unexpectedly cyclized into a TINO after desulfatation, by a spontaneous intramolecular Michael addition. The first part of this work was to synthetize the TINO 3 from D-ribose 1. The key step was the desilylative cyclisation of a suitably functionalized thiohydroximate 2. Based on precedent work developed in the laboratory, we could obtain the thiohydroximate from D-ribose. We then focused our studies on the cyclisation step trying to find the suitable substituents that could give the TINO in good yield by desilylative cyclisation. The second part of the project is to obtain ketonitrones 4 by palladiumcatalyzed coupling reaction.

Pentitol phosphate dehydrogenases: Discovery, characterization and use in D-arabitol and xylitol production by metabolically engineered Bacillus subtilis

The ultimate goal of this study has been to construct metabolically engineered microbial strains capable of fermenting glucose into pentitols D-arabitol and, especially, xylitol. The path that was chosen to achieve this goal required discovery, isolation and sequencing of at least two pentitol phosphate dehydrogenases of different specificity, followed by cloning and expression of their genes and characterization of recombinant arabitol and xylitol phosphate dehydrogenases. An enzyme of a previously unknown specificity, D-arabitol phosphate dehydrogenase (APDH), was discovered in Enterococcus avium. The enzyme was purified to homogenity from E. avium strain ATCC 33665. SDS/PAGE revealed that the enzyme has a molecular mass of 41 ± 2 kDa, whereas a molecular mass of 160 ± 5 kDa was observed under non-denaturing conditions implying that the APDH may exist as a tetramer with identical subunits. Purified APDH was found to have narrow substrate specificity, converting only D-arabitol 1-phosphate and D-arabitol 5-phosphate into D-xylulose 5-phosphate and D-ribulose 5-phosphate, respectively, in the oxidative reaction. Both NAD+ and NADP+ were accepted as co-factors. Based on the partial protein sequences, the gene encoding APDH was cloned. Homology comparisons place APDH within the medium chain dehydrogenase family. Unlike most members of this family, APDH requires Mn2+ but no Zn2+ for enzymatic activity. The DNA sequence surrounding the gene suggests that it belongs to an operon that also contains several components of phosphotransferase system (PTS). The apparent role of the enzyme is to participate in arabitol catabolism via the arabitol phosphate route similar to the ribitol and xylitol catabolic routes described previously. Xylitol phosphate dehydrogenase (XPDH) was isolated from Lactobacillus rhamnosus strain ATCC 15820. The enzyme was partially sequenced. Amino acid sequences were used to isolate the gene encoding the enzyme. The homology comparisons of the deduced amino acid sequence of L. rhamnosus XPDH revealed several similar enzymes in genomes of various species of Gram-positive bacteria. Two enzymes of Clostridium difficile and an enzyme of Bacillus halodurans were cloned and their substrate specificities together with the substrate specificity of L. rhamnosus XPDH were compared. It was found that one of the XPDH enzymes of C. difficile and the XPDH of L. rhamnosus had the highest selectivity towards D-xylulose 5-phosphate. A known transketolase-deficient and D-ribose-producing mutant of Bacillus subtilis (ATCC 31094) was further modified by disrupting its rpi (D-ribose phosphate isomerase) gene to create D-ribulose- and D-xylulose-producing strain. Expression of APDH of E. avium and XPDH of L. rhamnosus and C. difficile in D-ribulose- and D-xylulose-producing strain of B. subtilis resulted in strains capable of converting D-glucose into D-arabitol and xylitol, respectively. The D-arabitol yield on D-glucose was 38 % (w/w). Xylitol production was accompanied by co-production of ribitol limiting xylitol yield to 23 %.

Engineering the pentose phosphate pathway of Saccharomyces cerevisiae for production of ethanol and xylitol

The baker s yeast Saccharomyces cerevisiae has a long tradition in alcohol production from D-glucose of e.g. starch. However, without genetic modifications it is unable to utilise the 5-carbon sugars D-xylose and L arabinose present in plant biomass. In this study, one key metabolic step of the catabolic D-xylose pathway in recombinant D-xylose-utilising S. cerevisiae strains was studied. This step, carried out by xylulokinase (XK), was shown to be rate-limiting, because overexpression of the xylulokinase-encoding gene XKS1 increased both the specific ethanol production rate and the yield from D xylose. In addition, less of the unwanted side product xylitol was produced. Recombinant D-xylose-utilizing S. cerevisiae strains have been constructed by expressing the genes coding for the first two enzymes of the pathway, D-xylose reductase (XR) and xylitol dehydrogenase (XDH) from the D-xylose-utilising yeast Pichia stipitis. In this study, the ability of endogenous genes of S. cerevisiae to enable D-xylose utilisation was evaluated. Overexpression of the GRE3 gene coding for an unspecific aldose reductase and the ScXYL2 gene coding for a xylitol dehydrogenase homologue enabled growth on D-xylose in aerobic conditions. However, the strain with GRE3 and ScXYL2 had a lower growth rate and accumulated more xylitol compared to the strain with the corresponding enzymes from P. stipitis. Use of the strictly NADPH-dependent Gre3p instead of the P. stipitis XR able to utilise both NADH and NADPH leads to a more severe redox imbalance. In a S. cerevisiae strain not engineered for D-xylose utilisation the presence of D-xylose increased xylitol dehydrogenase activity and the expression of the genes SOR1 or SOR2 coding for sorbitol dehydrogenase. Thus, D-xylose utilisation by S. cerevisiae with activities encoded by ScXYL2 or possibly SOR1 or SOR2, and GRE3 is feasible, but requires efficient redox balance engineering. Compared to D-xylose, D-glucose is a cheap and readily available substrate and thus an attractive alternative for xylitol manufacture. In this study, the pentose phosphate pathway (PPP) of S. cerevisiae was engineered for production of xylitol from D-glucose. Xylitol was formed from D-xylulose 5-phosphate in strains lacking transketolase activity and expressing the gene coding for XDH from P. stipitis. In addition to xylitol, ribitol, D-ribose and D-ribulose were also formed. Deletion of the xylulokinase-encoding gene increased xylitol production, whereas the expression of DOG1 coding for sugar phosphate phosphatase increased ribitol, D-ribose and D-ribulose production. Strains lacking phosphoglucose isomerase (Pgi1p) activity were shown to produce 5 carbon compounds through PPP when DOG1 was overexpressed. Expression of genes encoding glyceraldehyde 3-phosphate dehydrogenase of Bacillus subtilis, GapB, or NAD-dependent glutamate dehydrogenase Gdh2p of S. cerevisiae, altered the cellular redox balance and enhanced growth of pgi1 strains on D glucose, but co-expression with DOG1 reduced growth on higher D-glucose concentrations. Strains lacking both transketolase and phosphoglucose isomerase activities tolerated only low D-glucose concentrations, but the yield of 5-carbon sugars and sugar alcohols on D-glucose was about 50% (w/w).

Thymidine phosphorylase in cancer; Enemy or friend?

Thymidine phosphorylase (TP) is a nucleoside metabolism enzyme that plays an important role in the pyrimidine pathway.TP catalyzes the conversion of thymidine to thymine and 2-deoxy-α-D-ribose-1-phosphate (dRib-1-P). Although this reaction is reversible, the main metabolic function of TP is catabolic. TP is identical to the angiogenic factor platelet-derived endothelial-cell growth factor (PD-ECGF). TP is overexpressed in several human cancers in response to cellular stressful conditions like hypoxia, acidosis, chemotherapy and radiotherapy. TP has been shown to promote tumor angiogenesis, invasion, metastasis, evasion of the immune-response and resistance to apoptosis. Some of the biological effects of TP are dependent on its enzymatic activity, while others are mediated through cytokines like interleukin 10 (IL-10), basic fibroblast growth factor (bFGF) and tumour necrosis factor α (TNFα). Interestingly, TP also plays a role in cancer treatment through its role in the conversion of the oral fluoropyrimidine capecitabine into its active form 5-FU. TP is a predictive marker for fluoropyrimidine response. Given its various biological functions in cancer progression, TP is a promising target in cancer treatment. Further translational research is required in this area.

Total Synthesis of (+)-Cladospolide A

A stereoselective total synthesis of (+)-cladospolide A from D-ribose is described. Key features of the synthesis include olefin cross metathesis and Yamaguchi lactonization.

N-烷基氮杂糖及其衍生物的合成

多羟基哌啶类化合物通常称为氮杂糖，由于与糖结构的相似性，亚胺基环醇表现出强的糖苷酶和糖基转移酶抑制活性，可调控在生物识别及酶结构控制中起到重要作用的糖蛋白的生物合成与水解。因此这类抑制剂有望成为与糖代谢紊乱有关的疾病的治疗药物，如：抗糖尿病、抗肿瘤、抗溶酶体贮积症及抗病毒感染（包括艾滋病）等药物。正是由于氮杂糖的重要生物活性及诱人的药用开发前景，近年来，有关氮杂糖及其衍生物的合成、生物活性及应用研究备受关注。 本论文探索了一系列的作为潜在的迈克加成中间体1-C-乙酰甲基/甲氧羰基甲基-5-N-取代呋喃核糖碳苷衍生物在碱的作用下先发生β-消除反应，接着发生分子内的迈克加成反应生成1-C-乙酰甲基-N-取代氮杂吡喃糖碳苷衍生物及1-C-甲氧羰基甲基-N-取代氮杂吡喃糖碳苷衍生物的方法，该转变过程为先通过β-消除得到非环状的α/β不饱和共轭酮或酯的中间体，接着5-N-取代氨基与分子内的α/β不饱和共轭酮或酯发生分子内的1,4-亲核加成，其中，2'-酯的环加成立体选择性的得到β型1-C-乙酰甲基-N-取代氮杂吡喃糖碳苷衍生物，而2'-酮的环加成得到立体异构体1-C-乙酰甲基-N-取代氮杂吡喃糖碳苷衍生物。此外，该类N-取代氮杂吡喃糖碳苷衍生物进一步脱除保护基，得到了一系列新的N-取代氮杂吡喃糖衍生物，拓展了氮杂吡喃糖碳苷分子库。 中间体1-C-(2'-oxoalkyl)-5-N-alkylated glycoribofuranoside的合成是由核糖为原料，通过对其结构修饰，在C-5氮原子上先引入不同的取代基，在C-1上引入乙酰甲基或甲氧羰基甲基。C-5取代氨基的引入通过两种方法：(a) 5-取代链状脂肪氨基可由链状的伯胺直接与5-甲磺酰基发生SN2亲核取代得到；(b) 5-取代芳香氨基可通过芳香醛与C-5氨基缩合再由硼氢化钠还原得到。2'-酰基的引入通过烯丙基氧化得到：2'-酮羰基由醋酸汞和琼斯试剂氧化得到；2'-酯基由高锰酸钾氧化再碘甲烷的作用下得到。 The polyhydroxylated piperidines, commonly be called azasugars. Iminocyclitols and their derivatives have exhibited remarkable biological activity to inhibit glycosidase-processing enzymes, with resulting potential chemotherapeutic applications against diabetes, cancer, lysosomal storage disorders and viral infections including AIDS. Recently, because of the important biological activity and excellent foreground on pharmaceutical application, great attention has been attracted to the synthesis of the new derivatives and analogues. In this dissertation, 1-C-(2'-oxoalkyl)-5-N-substituted-glycoribofuranosides, which used as latent substrates for intramolecular hetero-Michael addition, were converted to 2-ester and 2-ketone aza-C-glycopyranosides by base treatment. The transformation was achieved through β-elimination to an acyclic α/β-conjugated ketone or ester, followed by an intramolecular hetero-Michael addition by the 5-N-alkylated amino group. The 2-ester cycloaddition was highly stereoselective in favor of an equatorial 1-C-substitution while the 2-ketone cycloaddition was produced a pair of stereoisomers of 2′-ketonyl aza-C-glycoside. Additionally, the resultant different N-alkylated aza-C-glycopyranosides could be further prepared for various azasugar library constructions by removal of protecting groups. Synthesis of the key intermediate 1-C-(2'-oxoalkyl)-5-N-alkylated glycoribo- furanoside involved the introduction of 5-substituted amino and 1-C-2′-oxoalkyl groups from D-ribose. The 5-alkylated amino was introduced through two methods: (a) the 5-aliphatic series amino synthesized by the nucleophilic substitution of 5-mesylate using neat ethylamine, propylamine, butylamine, and hexylamine, (b) the 5-aromatic series amino synthesized by various aromatic aldehydes with C-5 amino under NaBH4 reduction. The 1-C-2′-oxoalkyl groups were introduced through oxidation of the ally group: the 1-C-allyl group was oxidized with Hg(OAc)2 and Jones reagent to the 2-ketonyl C-glycoside; the 1-C-allyl group was oxidized with KMnO4 and CH3I/NaHCO3 to 1-C-methyl acetate glycoside.

Double-strand DNA cleavage induced by oxindole-Schiff base copper(II) complexes with potential antitumor activity

Some oxindole-Schiff base copper(II) complexes have already shown potential antitumor activity towards different cells, inducing apoptosis in a process modulated by the ligand, and having nuclei and mitochondria as main targets. Here, three novel copper(II) complexes with analogous ligands were isolated and characterized by spectroscopic techniques, having their reactivity compared to the so far most active complex in this class. Cytotoxicity experiments carried out toward human neuroblastoma SH-SY5Y cells confirmed its proapoptosis property. DNA cleavage studies were then performed in the presence of these complexes, in order to verify the influence of ligand structural features in its nuclease activity. All of them were able to cause double-strand DNA scissions, giving rise to nicked circular Form II and linear Form III species, in the presence of hydrogen peroxide. Additionally, DNA Form II was also detected in the absence of peroxide when the most active complex, [Cu(isaepy)(2)](2+) 1, was used. In an effort to better elucidate their interactions with DNA, solutions of the different complexes titrated with DNA had their absorption spectra monitored. An absorbance hyperchromism observed at 260 nm pointed to the intercalation of these complexes into the DNA structure. Further, investigations of 2-deoxy-D-ribose (DR) oxidation catalyzed by each of those complexes, using 2-thiobarbituric acid reactive species (TBARS) method, and detection of reactive oxygen species (ROS) formation by spin-trapping EPR, suggested that their mechanism of action in performing efficiently DNA cleavage occurs preferentially, but not only by oxidative pathways. (C) 2007 Elsevier Inc. All rights reserved.

Synthese von optisch aktivem 9,14-Methylen-Lipoxin A 4

In der vorliegenden Arbeit wurde die Synthese von optisch aktivem 9,14-Methylen-Lipoxin A4 untersucht. Lipoxin A4 und seine Derivate leiten sich von der Arachidonsäurekaskade ab wie die Prostaglandine, Prostacycline, Thromboxana und Leucotriene. Alle diese Verbindungen sind biologisch aktive Eicosanoide, die aus 20 C-Atomen bestehen. Lipoxine können im Organismus auf verschiedenen Biosynthesewegen entstehen: über die Lipoxygenase sowie über die Zell-Zell-Wechselwirkungen. Untersuchungen ergaben, dass die Lipoxine selektive biologische Wirkungen zeigen, die eine wichtige Rolle vor allem in Entzündungsprozessen und Infektabwehr zeigen. Da aber diese Moleküle rasch isomerisieren, sind diese hohen Aktivitäten mit einer sehr geringen Stabilität gekoppelt. Aufgrund dieser Instabilität konnte die biologisch aktive Konformation des Lipoxin A4 am Rezeptor nicht eindeutig bestimmt werden. Zur besseren Untersuchung der Lipoxinrezeptor-Anordnung wurden deshalb stabile Analoga synthetisiert. Die biologische Aktivität des nach Nokami et al hergestellten Lipoxin A4-Analogons weicht sehr stark von der des Lipoxins ab. Angeregt durch diese Arbeiten wurde in der Arbeitsgruppe Nubbemeyer die Idee entwickelt, das konjugierte Tetraensystem des Lipoxin A4-Moleküls durch das Cycloheptatrien nachzuahmen. Die CH2-Gruppe bildet eine Brücke, die die Isomerisierung vom aktiven cis-Isomer zu den inaktiven trans-Isomeren verhindern soll. Mit diesem Cycloheptatriengerüst als Lipoxin A4-Analogon hoffen wir das Lipoxingerüst unwesentlich zu verändern und die damit verbundene biologische Aktivität zu erhalten. Die Synthese des 9,14-Methylen-LXA4 soll möglichst konvergent erfolgen, so dass gegebenenfalls auf Bausteine zurückgegriffen werden kann, deren Aufbau bereits optimiert wurde. Eine derartige Strategie ermöglicht darüber hinaus die Herstellung einer großen Zahl von weiteren potentiell interessanten Verbindungen ohne komplettes Umstellen der Synthese. Wichtige Reaktionen im Verlauf dieser Synthese sind: bei der Synthese des C8-C20-Bausteins: Friedel-Crafts-Acylierungen, Haloform-Reaktion, Veresterung mittels Standardmethoden, enantioselektive Reduktion mit dem chiralen CBS-Katalysator und Schutzgruppenoperation. bei der Synthese des C1-C7-Bausteins: ex-chiral-pool-Synthese aus 2-Desoxy-D-ribose, Wittig-Reaktion, Hydrierung mit Pd/C, Schutzgruppenoperation, Abspaltung von Schutzgruppen und Swern-Oxidation zum Aldehyd. Die Schlüsselreaktion der ganzen Synthese ist die Eintopf-Variante der Julia-Olefinierung nach Kocienski: selektiver Aufbau des trans-Olefins durch Verknüpfung der beiden Bausteine. Nach weiteren Schutzgruppenoperationen (und Öffnung des Valerolactons) wird der Methylester des 9,14-Lipoxin A4 erhalten, dessen biologische Aktivität zweifelsfrei bewiesen wurde.

A Method for Preparing Oligodeoxynucleotides Containing an Apurinic Site

10.1002/hlca.19900730309.abs In three steps, 2-deoxy-D-ribose has been converted into a phosphoramidite building block bearing a (t-Bu)Me2Si protecting group at the OH function of the anomeric centre of the furanose ring. This building block was subsequently incorporated into DNA oligomers of various base sequences using the standard phosphoramidite protocol for automated DNA synthesis. The resulting silyl-oligomers have been purified by HPLC and selectively desilylated to the corresponding free apurinic DNA sequences. The hexamer d (A-A-A-A-X-A) (X representing the apurinic site) which was prepared in this way was characterized by 1H- and 31P-NMR spectroscopy. The other sequences as well as their fragments, which formed upon treatment with alkali base, were analyzed by polyacrylamide gel electrophoresis.

Nucleophilic substitution reactions of pyranose polytosylates

The 2,3,4-tri-toluenesulfonate ester derivatives of the methyl pyranosides of L-arabinose, D-ribose, D-lyxose, and D-xylose have been prepared, and their substitution reactions with various nucleophiles have been examined. For arabinose, xylose, and ribose, highly regioselective monosubstitutions were observed with benzoate, nitrite, and azide anions. These reactions have led to short and simple routes from D-xylose to L-arabinose derivatives, from L-arabinose to D-xylose derivatives, and from D-ribose to L-lyxose derivatives. The tritosylate derived from methyl alpha-D-lyxopyranoside was unreactive toward nucleophilic substitution reactions, giving instead a dihydropyran product arising from an initial E2 elimination reaction of the 2-tosylate.

Design and synthesis of S-ribosylhomocysteine analogues

Bacteria are known to release a large variety of small molecules known as autoinducers (AI) which effect quorum sensing (QS) initiation. The interruption of QS effects bacterial communication, growth and virulence. ^ Three novel classes of S-ribosylhomocysteine (SRH) analogues as potential inhibitors of S-ribosylhomocysteinase (LuxS enzyme) and AI-2 modulators of QS were developed. The synthesis of 2-deoxy-2-bromo-SRH analogues was attempted by coupling of the corresponding 2-bromo-2-deoxypentafuranosyl precursors with the homocysteinate anion. The displacement of the bromide from C2 rather than the expected substitution of the mesylate from C5 was observed. The synthesis of 4-C-alkyl/aryl-S-ribosylhomocysteine analogues involved the following steps: (i) conversion of the D-ribose to the ribitol-4-ulose; (ii) diastereoselective addition of various alkyl or aryl or vinyl Grignard reagents to 4-ketone intermediate; (iii) oxidation of the primary hydroxyl group at C1 followed by the intramolecular ring closure to the corresponding 4-C-alkyl/aryl-substituted ribono-1,4-lactones; (iv) displacement of the activated 5-hydroxyl group with the protected homocysteinate. Treatment of the 4-C-alkyl/aryl-substituted SRH analogues with lithium triethylborohydride effected reduction of the ribonolactone to the ribose (hemiacetal) and subsequent global deprotection with trifluoroacetic acid provided 4-C-alkyl/aryl-SRHs. ^ The 4-[thia]-SRH were prepared from the 1-deoxy-4-thioribose through the coupling of the &agr;-fluoro thioethers (thioribosyl fluorides) with homocysteinate anion. The 4-[thia]-SRH analogues showed concentration dependent effect on the growth on las (50% inhibitory effect at 200 µg/mL). The most active was 1-deoxy-4-[thia]-SRH analogue with sufur atom in the ring oxidized to sulfoxide decreasing las gene activity to approximately 35% without affecting rhl gene. Neither of the tested compounds had effect on bioluminescence nor on total growth of V. harveyi, but had however slight inhibition of the QS.^

Excitotoxicity in the lung: N-methyl-D-aspartate-induced, nitric oxide-dependent, pulmonary edema is attenuated by vasoactive intestinal peptide and by inhibitors of poly(ADP-ribose) polymerase.

Excitatory amino acid toxicity, resulting from overactivation of N-methyl-D-aspartate (NMDA) glutamate receptors, is a major mechanism of neuronal cell death in acute and chronic neurological diseases. We have investigated whether excitotoxicity may occur in peripheral organs, causing tissue injury, and report that NMDA receptor activation in perfused, ventilated rat lungs triggered acute injury, marked by increased pressures needed to ventilate and perfuse the lung, and by high-permeability edema. The injury was prevented by competitive NMDA receptor antagonists or by channel-blocker MK-801, and was reduced in the presence of Mg2+. As with NMDA toxicity to central neurons, the lung injury was nitric oxide (NO) dependent: it required L-arginine, was associated with increased production of NO, and was attenuated by either of two NO synthase inhibitors. The neuropeptide vasoactive intestinal peptide and inhibitors of poly(ADP-ribose) polymerase also prevented this injury, but without inhibiting NO synthesis, both acting by inhibiting a toxic action of NO that is critical to tissue injury. The findings indicate that: (i) NMDA receptors exist in the lung (and probably elsewhere outside the central nervous system), (ii) excessive activation of these receptors may provoke acute edematous lung injury as seen in the "adult respiratory distress syndrome," and (iii) this injury can be modulated by blockade of one of three critical steps: NMDA receptor binding, inhibition of NO synthesis, or activation of poly(ADP-ribose) polymerase.

Phase I Study Of The Poly(ADP-Ribose) Polymerase Inhibitor, AG014699, In Combination With Temozolomide in Patients with Advanced Solid Tumors

Purpose: One mechanism of tumor resistance to cytotoxic therapy is repair of damaged DNA. Poly(ADP-ribose) polymerase (PARP)-1 is a nuclear enzyme involved in base excision repair, one of the five major repair pathways. PARP inhibitors are emerging as a new class of agents that can potentiate chemotherapy and radiotherapy. The article reports safety, efficacy, pharmacokinetic, and pharmacodynamic results of the first-in-class trial of a PARP inhibitor, AG014699, combined with temozolomide in adults with advanced malignancy.

Experimental Design: Initially, patients with solid tumors received escalating doses of AG014699 with 100 mg/m2/d temozolomide × 5 every 28 days to establish the PARP inhibitory dose (PID). Subsequently, AG014699 dose was fixed at PID and temozolomide escalated to maximum tolerated dose or 200 mg/m2 in metastatic melanoma patients whose tumors were biopsied. AG014699 and temozolomide pharmacokinetics, PARP activity, DNA strand single-strand breaks, response, and toxicity were evaluated.

Results: Thirty-three patients were enrolled. PARP inhibition was seen at all doses; PID was 12 mg/m2 based on 74% to 97% inhibition of peripheral blood lymphocyte PARP activity. Recommended doses were 12 mg/m2 AG014699 and 200 mg/m2 temozolomide. Mean tumor PARP inhibition at 5 h was 92% (range, 46-97%). No toxicity attributable to AG014699 alone was observed. AG014699 showed linear pharmacokinetics with no interaction with temozolomide. All patients treated at PID showed increases in DNA single-strand breaks and encouraging evidence of activity was seen.

Conclusions: The combination of AG014699 and temozolomide is well tolerated, pharmacodynamic assessments showing proof of principle of the mode of action of this new class of agents.

Vasoactivity of AG014699, a clinically active small molecule inhibitor of poly(ADP-ribose) polymerase: a contributory factor to chemopotentiation in vivo?

Purpose: Poly(ADP-ribose) polymerase (PARP) plays an important role in DNA repair, and PARP inhibitors can enhance the activity of DNA-damaging agents in vitro and in vivo. AG014699 is a potent PARP inhibitor in phase II clinical development. However, the range of therapeutics with which AG014699 could interact via a DNA-repair based mechanism is limited. We aimed to investigate a novel, vascular-based activity of AG014699, underlying in vivo chemosensitization, which could widen its clinical application.

Experimental Design: Temozolomide response was analyzed in vitro and in vivo. Vessel dynamics were monitored using “mismatch” following the administration of perfusion markers and real-time analysis of fluorescently labeled albumin uptake in to tumors established in dorsal window chambers. Further mechanistic investigations used ex vivo assays of vascular smooth muscle relaxation, gut motility, and myosin light chain kinase (MLCK) inhibition.

Results: AG014699 failed to sensitize SW620 cells to temozolomide in vitro but induced pronounced enhancement in vivo. AG014699 (1 mg/kg) improved tumor perfusion comparably with the control agents nicotinamide (1 g/kg) and AG14361 (forerunner to AG014699; 10 mg/kg). AG014699 and AG14361 relaxed preconstricted vascular smooth muscle more potently than the standard agent, hydralazine, with no impact on gut motility. AG014699 inhibited MLCK at concentrations that relaxed isolated arteries, whereas AG14361 had no effect.

Conclusion: Increased vessel perfusion elicited by AG014699 could increase tumor drug accumulation and therapeutic response. Vasoactive concentrations of AG014699 do not cause detrimental side effects to gut motility and may increase the range of therapeutics with which AG014699 could be combined with for clinical benefit.