CTLA4-Ig and anti-CD40 ligand prevent renal allograft rejection in primates

Selective inhibition of T cell costimulation using the B7-specific fusion protein CTLA4-Ig has been shown to induce long-term allograft survival in rodents. Antibodies preventing the interaction between CD40 and its T cell-based ligand CD154 (CD40L) have been shown in rodents to act synergistically with CTLA4-Ig. It has thus been hypothesized that these agents might be capable of inducing long-term acceptance of allografted tissues in primates. To test this hypothesis in a relevant preclinical model, CTLA4-Ig and the CD40L-specific monoclonal antibody 5C8 were tested in rhesus monkeys. Both agents effectively inhibited rhesus mixed lymphocyte reactions, but the combination was 100 times more effective than either drug alone. Renal allografts were transplanted into nephectomized rhesus monkeys shown to be disparate at major histocompatibility complex class I and class II loci. Control animals rejected in 5–8 days. Brief induction doses of CTLA4-Ig or 5C8 alone significantly prolonged rejection-free survival (20–98 days). Two of four animals treated with both agents experienced extended (>150 days) rejection-free allograft survival. Two animals treated with 5C8 alone and one animal treated with both 5C8 and CTLA4-Ig experienced late, biopsy-proven rejection, but a repeat course of their induction regimen successfully restored normal graft function. Neither drug affected peripheral T cell or B cell counts. There were no clinically evident side effects or rejections during treatment. We conclude that CTLA4-Ig and 5C8 can both prevent and reverse acute allograft rejection, significantly prolonging the survival of major histocompatibility complex-mismatched renal allografts in primates without the need for chronic immunosuppression.

Apoptosis induced in Jurkat cells by several agents is preceded by intracellular acidification.

We have previously shown that in neutrophils deprived of granulocyte colony-stimulating factor, apoptosis is preceded by acidification and that the protection against apoptosis conferred on neutrophils by granulocyte colony-stimulating factor is dependent upon delay of this acidification. To test the hypothesis that acidification could be a general feature of apoptosis, we examined intracellular pH changes in another cell line. Jurkat cells, a T-lymphoblastoid line, were induced to undergo apoptosis with anti-Fas IgM, cycloheximide, or exposure to short-wavelength UV light. We found that acidification occurred in response to treatment with these agents and that acidification preceded DNA fragmentation. Jurkat cells were also found to possess an acid endonuclease that is active below pH 6.8, compatible with a possible role for this enzyme in chromatin digestion during apoptosis. Incubation of the cells with the bases imidazole or chloroquine during treatment with anti-Fas antibody or cycloheximide or after UV exposure decreased apoptosis as assessed by nuclear morphology and DNA content. The alkalinizing effect of imidazole and chloroquine was shown by the demonstration that the percentage of cells with an intracellular pH below 6.8 after treatment with anti-Fas antibody, cycloheximide, or UV was diminished in the presence of base as compared with similarly treated cells incubated in the absence of base. We conclude that acidification is an early event in programmed cell death and may be essential for genome destruction.

Conjugates of folate and anti-T-cell-receptor antibodies specifically target folate-receptor-positive tumor cells for lysis.

High-affinity folate receptors (FRs) are expressed at elevated levels on many human tumors. Bispecific antibodies that bind the FR and the T-cell receptor (TCR) mediate lysis of these tumor cells by cytotoxic T lymphocytes. In this report, conjugates that consist of folate covalently linked to anti-TCR antibodies are shown to be potent in mediating lysis of tumor cells that express either the alpha or beta isoform of the FR. Intact antibodies with an average of five folate per molecule exhibited high affinity for FR+ tumor cells but did not bind to FR- tumor cells. Lysis of FR+ cell lines could be detected at concentrations as low as 1 pM (approximately 0.1 ng/ml), which was 1/1000th the concentration required to detect binding to the FR+ cells. Various FR+ mouse tumor cell lines could be targeted with each of three different anti-TCR antibodies that were tested as conjugates. The antibodies included 1B2, a clonotypic antibody specific for the cytotoxic T cell clone 2C; KJ16, an anti-V beta 8 antibody; and 2C11, an anti-CD3 antibody. These antibodies differ in affinities by up to 100-fold, yet the cytolytic capabilities of the folate/antibody conjugates differed by no more than 10-fold. The reduced size (in comparison with bispecific antibodies) and high affinity of folate conjugates suggest that they may be useful as immunotherapeutic agents in targeting tumors that express folate receptors.

Inhibition of the integrase of human immunodeficiency virus (HIV) type 1 by anti-HIV plant proteins MAP30 and GAP31.

MAP30 (Momordica anti-HIV protein of 30 kDa) and GAP31 (Gelonium anti-HIV protein of 31 kDa) are anti-HIV plant proteins that we have identified, purified, and cloned from the medicinal plants Momordica charantia and Gelonium multiflorum. These antiviral agents are capable of inhibiting infection of HIV type 1 (HIV-1) in T lymphocytes and monocytes as well as replication of the virus in already-infected cells. They are not toxic to normal uninfected cells because they are unable to enter healthy cells. MAP30 and GAP31 also possess an N-glycosidase activity on 28S ribosomal RNA and a topological activity on plasmid and viral DNAs including HIV-1 long terminal repeats (LTRs). LTRs are essential sites for integration of viral DNA into the host genome by viral integrase. We therefore investigated the effect of MAP30 and GAP31 on HIV-1 integrase. We report that both of these antiviral agents exhibit dose-dependent inhibition of HIV-1 integrase. Inhibition was observed in all of the three specific reactions catalyzed by the integrase, namely, 3' processing (specific cleavage of the dinucleotide GT from the viral substrate), strand transfer (integration), and "disintegration" (the reversal of strand transfer). Inhibition was studied by using oligonucleotide substrates with sequences corresponding to the U3 and U5 regions of HIV LTR. In the presence of 20 ng of viral substrate, 50 ng of target substrate, and 4 microM integrase, total inhibition was achieved at equimolar concentrations of the integrase and the antiviral proteins, with EC50 values of about 1 microM. Integration of viral DNA into the host chromosome is a vital step in the replicative cycle of retroviruses, including the AIDS virus. The inhibition of HIV-1 integrase by MAP30 and GAP31 suggests that impediment of viral DNA integration may play a key role in the anti-HIV activity of these plant proteins.

Design and synthesis of constrained azacyclic pyrrolidine analogues of FTY720 as anticancer agents & metal coordination-controlled and bifunctional catalysis toward tertiary β-Ketols

Cette thèse se compose en deux parties: Première Partie: La conception et la synthèse d’analogues pyrrolidiniques, utilisés comme agents anticancéreux, dérivés du FTY720. FTY720 est actuellement commercialisé comme médicament (GilenyaTM) pour le traitement de la sclérose en plaques rémittente-récurrente. Il agit comme immunosuppresseur en raison de son effet sur les récepteurs de la sphingosine-1-phosphate. A fortes doses, FTY720 présente un effet antinéoplasique. Cependant, à de telles doses, un des effets secondaires observé est la bradycardie dû à l’activation des récepteurs S1P1 et S1P3. Ceci limite son potentiel d’utilisation lors de chimiothérapie. Nos précédentes études ont montré que des analogues pyrrolidiniques dérivés du FTY720 présentaient une activité anticancéreuse mais aucune sur les récepteurs S1P1 et S1P3. Nous avons soumis l’idée qu’une étude relation structure-activité (SARs) pourrait nous conduire à la découverte de nouveaux agents anti tumoraux. Ainsi, deux séries de composés pyrrolidiniques (O-arylmethyl substitué et C-arylmethyl substitué) ont pu être envisagés et synthétisés (Chapitre 1). Ces analogues ont montré d’excellentes activités cytotoxiques contre diverses cellules cancéreuses humaines (prostate, colon, sein, pancréas et leucémie), plus particulièrement les analogues actifs qui ne peuvent pas être phosphorylés par SphK, présentent un plus grand potentiel pour le traitement du cancer sans effet secondaire comme la bradycardie. Les études mécanistiques suggèrent que ces analogues de déclencheurs de régulation négative sur les transporteurs de nutriments induisent une crise bioénergétique en affamant les cellules cancéreuses. Afin d’approfondir nos connaissances sur les récepteurs cibles, nous avons conçu et synthétisé des sondes diazirine basées sur le marquage d’affinité aux photons (méthode PAL: Photo-Affinity Labeling) (Chapitre 2). En s’appuyant sur la méthode PAL, il est possible de récolter des informations sur les récepteurs cibles à travers l’analyse LC/MS/MS de la protéine. Ces tests sont en cours et les résultats sont prometteurs. Deuxième partie: Coordination métallique et catalyse di fonctionnelle de dérivés β-hydroxy cétones tertiaires. Les réactions de Barbier et de Grignard sont des méthodes classiques pour former des liaisons carbone-carbone, et généralement utilisées pour la préparation d’alcools secondaires et tertiaires. En vue d’améliorer la réaction de Grignard avec le 1-iodobutane dans les conditions « one-pot » de Barbier, nous avons obtenu comme produit majoritaire la β-hydroxy cétone provenant de l’auto aldolisation de la 5-hexen-2-one, plutôt que le produit attendu d’addition de l’alcool (Chapitre 3). La formation inattendue de la β-hydroxy cétone a également été observée en utilisant d’autres dérivés méthyl cétone. Étonnement dans la réaction intramoléculaire d’une tricétone, connue pour former la cétone Hajos-Parrish, le produit majoritaire est rarement la β-hydroxy cétone présentant la fonction alcool en position axiale. Intrigué par ces résultats et après l’étude systématique des conditions de réaction, nous avons développé deux nouvelles méthodes à travers la synthèse sélective et catalytique de β-hydroxy cétones spécifiques par cyclisation intramoléculaire avec des rendements élevés (Chapitre 4). La réaction peut être catalysée soit par une base adaptée et du bromure de lithium comme additif en passant par un état de transition coordonné au lithium, ou bien soit à l’aide d’un catalyseur TBD di fonctionnel, via un état de transition médiée par une coordination bidenté au TBD. Les mécanismes proposés ont été corroborés par calcul DFT. Ces réactions catalytiques ont également été appliquées à d’autres substrats comme les tricétones et les dicétones. Bien que les efforts préliminaires afin d’obtenir une enantioselectivité se sont révélés sans succès, la synthèse et la recherche de nouveaux catalyseurs chiraux sont en cours.

Design and synthesis of constrained azacyclic pyrrolidine analogues of FTY720 as anticancer agents & metal coordination-controlled and bifunctional catalysis toward tertiary β-Ketols

Cette thèse se compose en deux parties: Première Partie: La conception et la synthèse d’analogues pyrrolidiniques, utilisés comme agents anticancéreux, dérivés du FTY720. FTY720 est actuellement commercialisé comme médicament (GilenyaTM) pour le traitement de la sclérose en plaques rémittente-récurrente. Il agit comme immunosuppresseur en raison de son effet sur les récepteurs de la sphingosine-1-phosphate. A fortes doses, FTY720 présente un effet antinéoplasique. Cependant, à de telles doses, un des effets secondaires observé est la bradycardie dû à l’activation des récepteurs S1P1 et S1P3. Ceci limite son potentiel d’utilisation lors de chimiothérapie. Nos précédentes études ont montré que des analogues pyrrolidiniques dérivés du FTY720 présentaient une activité anticancéreuse mais aucune sur les récepteurs S1P1 et S1P3. Nous avons soumis l’idée qu’une étude relation structure-activité (SARs) pourrait nous conduire à la découverte de nouveaux agents anti tumoraux. Ainsi, deux séries de composés pyrrolidiniques (O-arylmethyl substitué et C-arylmethyl substitué) ont pu être envisagés et synthétisés (Chapitre 1). Ces analogues ont montré d’excellentes activités cytotoxiques contre diverses cellules cancéreuses humaines (prostate, colon, sein, pancréas et leucémie), plus particulièrement les analogues actifs qui ne peuvent pas être phosphorylés par SphK, présentent un plus grand potentiel pour le traitement du cancer sans effet secondaire comme la bradycardie. Les études mécanistiques suggèrent que ces analogues de déclencheurs de régulation négative sur les transporteurs de nutriments induisent une crise bioénergétique en affamant les cellules cancéreuses. Afin d’approfondir nos connaissances sur les récepteurs cibles, nous avons conçu et synthétisé des sondes diazirine basées sur le marquage d’affinité aux photons (méthode PAL: Photo-Affinity Labeling) (Chapitre 2). En s’appuyant sur la méthode PAL, il est possible de récolter des informations sur les récepteurs cibles à travers l’analyse LC/MS/MS de la protéine. Ces tests sont en cours et les résultats sont prometteurs. Deuxième partie: Coordination métallique et catalyse di fonctionnelle de dérivés β-hydroxy cétones tertiaires. Les réactions de Barbier et de Grignard sont des méthodes classiques pour former des liaisons carbone-carbone, et généralement utilisées pour la préparation d’alcools secondaires et tertiaires. En vue d’améliorer la réaction de Grignard avec le 1-iodobutane dans les conditions « one-pot » de Barbier, nous avons obtenu comme produit majoritaire la β-hydroxy cétone provenant de l’auto aldolisation de la 5-hexen-2-one, plutôt que le produit attendu d’addition de l’alcool (Chapitre 3). La formation inattendue de la β-hydroxy cétone a également été observée en utilisant d’autres dérivés méthyl cétone. Étonnement dans la réaction intramoléculaire d’une tricétone, connue pour former la cétone Hajos-Parrish, le produit majoritaire est rarement la β-hydroxy cétone présentant la fonction alcool en position axiale. Intrigué par ces résultats et après l’étude systématique des conditions de réaction, nous avons développé deux nouvelles méthodes à travers la synthèse sélective et catalytique de β-hydroxy cétones spécifiques par cyclisation intramoléculaire avec des rendements élevés (Chapitre 4). La réaction peut être catalysée soit par une base adaptée et du bromure de lithium comme additif en passant par un état de transition coordonné au lithium, ou bien soit à l’aide d’un catalyseur TBD di fonctionnel, via un état de transition médiée par une coordination bidenté au TBD. Les mécanismes proposés ont été corroborés par calcul DFT. Ces réactions catalytiques ont également été appliquées à d’autres substrats comme les tricétones et les dicétones. Bien que les efforts préliminaires afin d’obtenir une enantioselectivité se sont révélés sans succès, la synthèse et la recherche de nouveaux catalyseurs chiraux sont en cours.

Design and synthesis of potential antimicrobial agents

Chorismate mutase is one of the essential enzymes in the shikimate pathway and is key to the survival of the organism Mycobacterium tuberculosis. The x-ray crystal structure of this enzyme from Mycobacterium tuberculosis was manipulated to prepare an initial set of in silico protein models of the active site. Known inhibitors of the enzyme were docked into the active site using the flexible ligand / flexible active site side chains approach implemented in CAChe Worksystem (Fujitsu Ltd). The resulting complexes were refined by molecular dynamics studies in explicit water using Amber 9. This yielded a further set of protein models that were used for additional rounds of ligand docking. A binding hypothesis was established for the enzyme and this was used to screen a database of commercially available drug-like compounds. From these results new potential ligands were designed that fitted appropriately into the active site and matched the functional groups and binding motifs founds therein. Some of these compounds and close analogues were then synthesized and submitted for biological evaluation. As a separate part of this thesis, analogues of very active anti-tuberculosis pyridylcarboxamidrazone were also prepared. This was carried out by the addition and the deletion of the substitutions from the lead compound thereby preparing heteroaryl carboxamidrazone derivatives and related compounds. All these compounds were initially evaluated for biological activity against various gram positive organisms and then sent to the TAACF (USA) for screening against Mycobacterium tuberculosis. Some of the new compounds proved to be at least as potent as the original lead compound but less toxic.

The serological diagnosis of staphylococcal infective endocarditis

Objectives: Establishing the diagnosis of infective endocarditis (IE) can be difficult when blood cultures remain sterile or echocardiography is inconclusive. Staphylococcus aureus is a common aetiological microorganism in IE and is associated with severe valvular destruction and increased mortality. Early diagnosis using culture and antibiotic independent tests would be preferable to allow prompt antibiotic administration. We have developed and evaluated 2 serological assays for the rapid identification of a staphylococcal aetiology in infective endocarditis. The assays measure IgG against whole cells of S. aureus and IgG against lipid S, a novel extracellular antigen released by Gram-positive microorganisms. Methods: Serum was collected from 130 patients with IE and 94 control patients. IgG against whole cells of S. aureus and against lipid S was measured by enzyme linked immunosorbent assay (ELISA). Results: Anti-lipid S IgG titres were higher in IE caused by Gram-positive microorganisms than in controls (p < 0.0001) and higher in staphylococcal IE than in both controls and IE caused by other microorganisms (p = 0.0003). Anti-whole cell staphylococcal IgG was significantly higher in serum from patients with staphylococcal IE than in IE caused by other microorganisms and control samples (p < 0.0001). Conclusion: High anti-whole cell IgG titres are predictive of a staphylococcal aetiology in IE. Elevated serum anti-lipid S IgG titres are predictive of Gram-positive infection compared to controls, very high titres being associated with staphylococcal IE. © 2005 The British Infection Society.

Chemical and physical methods of enhancing the percutaneous absorption of antimicrobial agents

Contrary to previously held beliefs, it is now known that bacteria exist not only on the surface of the skin but they are also distributed at varying depths beneath the skin surface. Hence, in order to sterilise the skin, antimicrobial agents are required to penetrate across the skin and eliminate the bacteria residing at all depths. Chlorhexidine is an antimicrobial agent with the widest use for skin sterilisation. However, due to its poor permeation rate across the skin, sterilisation of the skin cannot be achieved and, therefore, the remaining bacteria can act as a source of infection during an operation or insertion of catheters. The underlying theme of this study is to enhance the permeation of this antimicrobial agent in the skin by employing chemical (enhancers and supersaturated systems) or physical (iontophoresis) techniques. The hydrochloride salt of chlorhexidine (CHX), a poorly soluble salt, was used throughout this study. The effect of ionisation on in vitro permeation rate across the excised human epidennis was investigated using Franz-type diffusion cells. Saturated solutions of CHX were used as donor and the variable studied was vehicle pH. Permeation rate was increased with increasing vehicle pH. The pH effect was not related to the level of ionisation of the drug. The effect of donor vehicle was also studied using saturated solutions of CHX in 10% and 20% ethanol as the donor solutions. Permeation of CHX was enhanced by increasing the concentration of ethanol which could be due to the higher concentration of CHX in the donor phase and the effect of ethanol itself on the membrane. The interplay between drug diffusion and enhancer pretreatment of the epidennis was studied. Pretreatment of the membrane with 10% Azone/PG demonstrated the highest diffusion rate followed by 10% olcic acid/PG pretreatment compared to other pretreatment regimens (ethanol, dimethyl sulfoxide (DMSO), propylene glycol (PG), sodium dodecyl sulphate (SDS) and dodecyl trimethyl ammonium bromide (DT AB). Differential Scanning Calorimetry (DSC) was also employed to study the mode of action of these enhancers. The potential of supersaturated solutions in enhancing percutaneous absorption of CHX was investigated. Various anti-nucleating polymers were screened in order to establish the most effective agent. Polyvinylpyrrolidone (PVP, K30) was found to be a better candidate than its lower molecular weight counterpart (K25) and hydroxypropyl methyleellulose (HPMC). The permeation studies showed an increase in diffusion rate by increasing the degree of saturation. Iontophoresis is a physical means of transdemal drug delivery enhancement that causes an increased penetration of molecules into or through the skin by the application of an electric field. This technique was employed in conjunction with chemical enhancers to assess the effect on CHX permeation across the human epidermis. An improved transport of CHX, which was pH dependant was observed upon application of the current. Combined use of iontophoresis and chemical enhancers further increased the CHX transport indicating a synergistic effect. Pretreatment of the membrane with 10% Azone/PG demonstrated the greatest effect.

Design and synthesis of potential antimicrobial agents

Tuberculosis is one of the most devastating diseases in the world primarily due to several decades of neglect and an emergence of multidrug-resitance strains (MDR) of M. tuberculosis together with the increased incidence of disseminated infections produced by other mycobacterium in AIDS patients. This has prompted the search for new antimycobacterial drugs. A series of pyridine-2-, pyridine-3-, pyridine-4-, pyrazine and quinoline-2-carboxamidrazone derivatives and new classes of carboxamidrazone were prepared in an automated fashion and by traditional synthesis. Over nine hundred synthesized compounds were screened for their anti mycobacterial activity against M. fortutium (NGTG 10394) as a surrogate for M. tuberculosis. The new classes of amidrazones were also screened against tuberculosis H37 Rv and antimicrobial activities against various bacteria. Fifteen tested compounds were found to provide 90-100% inhibition of mycobacterium growth of M. tuberculosis H37 Rv in the primary screen at 6.25 μg mL-1. The most active compound in the carboxamidrazone amide series had an MIG value of 0.1-2 μg mL-1 against M. fortutium. The enzyme dihydrofolate reductase (DHFR) has been a drug-design target for decades. Blocking of the enzymatic activity of DHFR is a key element in the treatment of many diseases, including cancer, bacterial and protozoal infection. The x-ray structure of DHFR from M. tuberculosis and human DHFR were found to have differences in substrate binding site. The presence of glycerol molecule in the Xray structure from M. tuberculosis DHFR provided opportunity to design new antifolates. The new antifolates described herein were designed to retain the pharmcophore of pyrimethamine (2,4- diamino-5(4-chlorophenyl)-6-ethylpyrimidine), but encompassing a range of polar groups that might interact with the M. tuberculosis DHFR glycerol binding pockets. Finally, the research described in this thesis contributes to the preparation of molecularly imprinted polymers for the recognition of 2,4-diaminopyrimidine for the binding the target. The formation of hydrogen bonding between the model functional monomer 5-(4-tert-butyl-benzylidene)-pyrimidine-2,4,6-trione and 2,4-diaminopyrimidine in the pre-polymerisation stage was verified by 1H-NMR studies. Having proven that 2,4-diaminopyrimidine interacts strongly with the model 5-(4-tert-butylbenzylidene)- pyrimidine-2,4,6-trione, 2,4-diaminopyrimidine-imprinted polymers were prepared using a novel cyclobarbital derived functional monomer, acrylic acid 4-(2,4,6-trioxo-tetrahydro-pyrimidin-5- ylidenemethyl)phenyl ester, capable of multiple hydrogen bond formation with the 2,4- diaminopyrimidine. The recognition property of the respective polymers toward the template and other test compounds was evaluated by fluorescence. The results demonstrate that the polymers showed dose dependent enhancement of fluorescence emissions. In addition, the results also indicate that synthesized MIPs have higher 2,4-diaminopyrimidine binding ability as compared with corresponding non-imprinting polymers.

Anti-obesity fixed-dose combinations

The management of hypertension, dyslipidaemia and hyperglycaemia often requires multiple medications that combine two or more agents with different modes of action to give additive efficacy. In some situations lower doses of two agents with different modes of action can achieve greater efficacy than a high dose of one agent. This is achieved by addressing different pathophysiological features of the disease, whilst at the same time producing fewer side effects than a high dose of one agent. Several examples of this have been described for combinations of blood glucose-lowering therapies in type 2 diabetes. However, the pill burden associated with multiple medications can reduce patient adherence and compromise the potential value of the treatments. To reduce the number of daily doses, single-tablet (‘fixed-dose’) combinations have been introduced to offer greater convenience. There are several ant-diabetic FDCs, mostly combining metformin with another type of glucose-lowering agent. The UK has been less enthusiastic about FDCs than many other parts of the world, and does not have most of these combinations available. One of the concerns expressed about FDCs is a reduced flexibility to select desired doses of the two agents for dose titration. However, in practise the variety of dosage strengths for most FDCs matches the dosages available as separate tablets. Another concern has been the preference to add drugs one at a time to be able to attribute any adverse effects. In most cases the FDC is used when a second drug has been added to a monotherapy that is already a component of the FDC, so it is only the same as adding one agent but without increasing the pill burden.

Identification of aspirin analogues that repress NF-κB signalling and demonstrate anti-proliferative activity towards colorectal cancer in vitro and in vivo

Substantial evidence indicates that aspirin and related non-steroidal anti-inflammatory drugs (NSAIDs) have potential as chemopreventative/therapeutic agents. However, these agents cannot be universally recommended for prevention purposes due to their potential side-effect profiles. Here, we compared the growth inhibitory and mechanistic activity of aspirin to two novel analogues, diaspirin (DiA) and fumaryl diaspirin (F-DiA). We found that the aspirin analogues inhibited cell proliferation and induced apoptosis of colorectal cancer cells at significantly lower doses than aspirin. Similar to aspirin, we found that an early response to the analogues was a reduction in levels of cyclin D1 and stimulation of the NF-κB pathway. This stimulation was associated with a significant reduction in basal levels of NF-κB transcriptional activity, in keeping with previous data for aspirin. However, in contrast to aspirin, DiA and F-DiA activity was not associated with nucleolar accumulation of RelA. For all assays, F-DiA had a more rapid and significant effect than DiA, identifying this agent as particularly active against colorectal cancer. Using a syngeneic colorectal tumour model in mice, we found that, while both agents significantly inhibited tumour growth in vivo, this effect was particularly pronounced for F-DiA. These data identify two compounds that are active against colorectal cancer in vitro and in vivo. They also identify a potential mechanism of action of these agents and shed light on the chemical structures that may be important for the antitumour effects of aspirin.

The use of computational QSAR analysis in the toxicological evaluation of a series of 2-pyridylcarboxamidrazone candidate anti-tuberculosis compounds

A series of N1-benzylidene pyridine-2-carboxamidrazone anti-tuberculosis compounds has been evaluated for their cytotoxicity using human mononuclear leucocytes (MNL) as target cells. All eight compounds were significantly more toxic than dimethyl sulphoxide control and isoniazid (INH) with the exception of a 4-methoxy-3-(2-phenylethyloxy) derivative, which was not significantly different in toxicity compared with INH. The most toxic agent was an ethoxy derivative, followed by 3-nitro, 4-methoxy, dimethylpropyl, 4-methylbenzyloxy, 3-methoxy-4-(-2-phenylethyloxy) and 4-benzyloxy in rank order. In comparison with the effect of selected carboxamidrazone agents on cells alone, the presence of either N-acetyl cysteine (NAC) or glutathione caused a significant reduction in the toxicity of INH, as well as on the 4-benzyloxy derivative, although both increased the toxicity of a 4-N,N-dimethylamino-1-naphthylidene and a 2-t-butylthio derivative. The derivatives from this and three previous studies were subjected to computational analysis in order to derive equations designed to establish quantitative structure activity relationships for these agents. Twenty-five compounds were thus resolved into two groups (1 and 2), which on analysis yielded equations with r2 values in the range 0.65-0.92. Group 1 shares a common mode of toxicity related to hydrophobicity, where cytotoxicity peaked at logP of 3.2, while Group 2 toxicity was strongly related to ionisation potential. The presence of thiols such as NAC and GSH both promoted and attenuated toxicity in selected compounds from Group 1, suggesting that secondary mechanisms of toxicity were operating. These studies will facilitate the design of future low toxicity high activity anti-tubercular carboxamidrazone agents. © 2003 Elsevier Science B.V. All rights reserved.

Rethinking the national question: Anti-statist discourses within the Kurdish national movement

Why and under what conditions have the Kurds become agents of change in the Middle East in terms of democratization? Why did the Kurds' role as democratic agents become particularly visible in the 1990s? How does the Kurdish movement's turn to democratic discourse affect the political systems of Turkey, Iran, Iraq and Syria? What are the implications of the Kurds' adoption of "democratic discourse" for the transnational aspect of the Kurdish movement? Since the early 1990s, Kurdish national movements in Turkey, Iran, Iraq and Syria have undergone important political and ideological transformations. As a result of the Kurds' growing role in shaping the debates on human rights and democratization in these four countries, the Kurdish national movement has acquired a dual character: an ethno-cultural struggle for the recognition of Kurdish identity, and a democratization movement that seeks to redefine the concepts of governance and citizenship in Turkey, Iran, Iraq and Syria. The process transformation has affected relations between the Kurdish movements and their respective central governments in significant ways. On the basis of face-to-face interviews and archival research conducted in Turkey, Iraq and parts of Europe, the present work challenges the current narrative of Kurdish nationalism, which is predominantly drawn from a statist interpretation of Kurdish nationalist goals, and argues instead that the Kurdish question is no longer a problem of statelessness but a problem of democracy in Turkey, Iran, Iraq and Syria. The main contributions of this work are three fold. First, the research unfolds the reasons behind the growing emphasis of the Kurdish movement on the concepts of democracy, human rights, and political participation, which started in the early 1990s. Second, the findings challenge the existing scholarship that explains Kurdish nationalism as a problem of statelessness and shifts the focus to the transformative potentials of the Kurdish national movement in Turkey, Iran, Iraq and Syria through a comparative lens. Third, this work explores the complex transnational coordination and negotiations between the Kurdish movements across borders and explains the regional repercussions of this process.

Avaliação da atividade anti-inflamatória do extrato aquoso, frações e compostos identificados nos frutos de Hancornia Speciosa Gomes (Apocynaceae)

Hancornia speciosa Gomes (Apocynaceae), popularly known as ‘mangabeira’, has been used in folk medicine to treat inflammatory disorders, hypertension, dermatitis, diabetes, liver diseases and stomach disorders. Regarding the Hancornia speciosa fruits, the ethnobotany indicates its use especially for treating inflammation and tuberculosis. However, no study has been done so far to prove such biological activities. The objective was evaluation anti-inflammatory activity from the fruits of Hancornia speciosa Gomes (mangabeira). Aqueous extract was prepared by decoction, subsequently submitted the liquid-liquid fractionation. The secondary metabolites were identified by high performance liquid chromatography coupled with detector diode array (HPLC-DAD) and liquid chromatography diode array detector coupled with mass spectrometry (LC-DAD-MS). The anti-inflammatory properties of the aqueous extract, dichloromethane (CH2Cl2), ethyl acetate (EtOAc) and n-butanol (n-BuOH) fractions of the fruits from H. speciosa, as well as rutin and chlorogenic acid were investigated using in vitro and in vivo models. In vivo tests comprised the xylene-induced ear edema that was measured the formation of edema, carrageenan-induced peritonitis was evaluated the total leukocytes at 4h and zymosan-induced air pouch was measured the total leukocytes and differential cell count at 6, 24 and 48 hours, whereas in vitro tests were evaluated levels of cytokines IL-1β, IL-6, IL-12 and TNF-α using ELISA obtained of carrageenan-induced peritonitis model. The results showed the presence of rutin and chlorogenic acid were detected in the aqueous extract from H. speciosa fruits by HPLC-DAD and LC-DAD-ME. Furthermore, the aqueous extracts and fractions, as well as rutin and chlorogenic acid significantly inhibited the xilol-induced ear edema and reduced cell migration in the animal models such as carrageenan-induced peritonitis and zymosan-induced air pouch. In addition, reduced levels of cytokines IL-1β, IL-6, IL-12 and TNF-α were observed. This is the first study that demonstrated the anti-inflammatory effect of aqueous extract from Hancornia speciosa fruits against different inflammatory agents in animal models, suggesting that their bioactive molecules, especially rutin and chlorogenic acid contributing, at least in part, to the anti-inflammatory effect of aqueous extract. These findings support the widespread use of Hancornia speciosa in popular medicine and demonstrate that this aqueous extract has therapeutic potential for the development of a herbal drugs with anti-inflammatory properties.