Epigenetic Therapy Using 5-Aza-2'-Deoxycytine: A Potential Magic Bullet Against Cancer
MOMPARLER RL
Université de Montréal & Centre de recherche, Hôpital Sainte-Justine, Montreal, Quebec, Canada
Aberrant DNA methylation, an epigenetic event, plays an important role in tumorigenesis by silencing genes that suppress malignancy, such as tumor suppressor genes (TSGs). Since this epigenetic change is reversible, it is a potential target for 5-aza-2’-deoxycytidine (DAC), a potent inhibitor of DNA methylation that can reactivate TSGs. There are several properties of DAC that make it a potential “magic bullet” to treat cancer: 1) Each tumor has many TSGs that are silenced by DNA methylation. 2) Since DAC is an S phase specific agent, it will only target proliferating cells. 3) The concentrations of DAC that induce a loss of clonogenicity of tumor cells are low (< 1 micromolar). 4) Due to its small molecular size (< 300 Da) and its physicochemical properties, DAC has the great potential to penetrate tumors. One of major reasons for the failure of many cytotoxic drugs to cure cancer is due to their poor penetration into tumors at therapeutic concentrations. 5) The antineoplastic action of DAC can be enhanced by other epigenetic agents, such as inhibitors of histone deacetylase. 6) Drug resistance to DAC can be prevented by the use of agents that inhibit cytidine deaminase, the enzyme that inactivates DAC or agents that are cytotoxic to tumor cells deficient in deoxycytidine kinase, the enzyme that activates the prodrug, DAC. 7) In animal models DAC was shown to be a potent antileukemic agent, more active than cytosine arabinoside (ARA-C) (Momparler et al. Leuk Res 8: 1043, 1984), the major drug for the treatment of acute myeloid leukemia. 8) Resting, non-proliferating, hematopoietic stem cells are resistant to DAC and are rapidly recruited into the cell cycle after treatment with this agent to overcome the problem of neutropenia, its major side effect. In preliminary clinical trials on patients with acute leukemia (Rivard et al. Leuk Res 5: 453, 1981) and patients with lung cancer (Momparler et al., Anticancer Drugs 8:358, 1997), DAC showed promising activity. Preclinical studies show that the potent antineoplastic action of DAC is highly dependent on its dose-schedule (Lemaire et al., BMC Cancer 8: 128, 2008). For clinical therapy of cancer the optimal dose-schedule for this interesting epigenetic agent remains to be determined. At the present moment DAC has only been approved for the treatment of the hematological malignancy, myelodysplastic syndrome (MDS). Why does an agent like DAC with so much potential in cancer therapy have only limited clinical use? The major problem is the lack of knowledge on how to translate preclinical results to clinical therapy. This is the same problem that Paul Ehrlich faced in his research on chemotherapy a century ago. (Supported by grants from Canadian Cancer Society & Cancer Research Society).
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Ketanserin And Dexfenfluramine: Angels Or Demons For 5-HT2B Receptors?
MONASSIER L1, MAROTEAUX L2
1Faculty of Medicine Inserm715, Strasbourg, France; 2Institut du Fer à Moulin Inserm839, Paris, France
Background: Ketanserin and dexfenfluramine are two old drugs that both target the 5-HT2 Gq-coupled subtypes of serotoninergic receptors. The first was developed as an 1-adrenergic/5-HT2A antagonist for the treatment of hypertension and withdrawn for the risk of cardiac arrhythmias and sudden death due to QT interval prolongation on the electrocardiogram. This effect is not due to the 5-HT2 receptor antagonism but to the blockade of HERG potassium channels. The second was employed as an appetite suppressant and is metabolized in norfenfluramine, a powerful 5-HT2B/2CR (5-HT2B/2CR) agonist. Its agonist property at 5-HT2BR explains the main adverse effects of the drug in heart and lung (i.e. pulmonary hypertension and cardiac valves fibrosis). In the presentation, we will show the main cardioprotective effects associated with 5-HT2BR blockade.
Methods: Standard cardiovascular phenotyping methods were applied to 5-HT2BR-/- mice and animals overexpressing the 5-HT2BR in cardiomyocytes. Then wild-type treated with 5-HT2BR antagonists and 5-HT2BR-/- mice were submitted to chronic infusions with a -adrenergic agonist (isoproterenol) or angiotensin II. Cardiac function and anatomy were measured by echocardiography together with the measurement of blood pressure parameters. We also measured plasma inflammatory cytokines and oxidative stress in heart and vessels. Finally, the cellular-coupling of the 5-HT2BR was analyzed in left-ventricular fibroblasts.
Results: The 5-HT2BR is crucial in adult cardiac hypertrophy due to pathological stimuli such as isoproterenol and angiotensin II. Its blockades reduces cardiac production of inflammatory cytokines and myocardial oxidative stress. Most of these actions are taking their origin in ventricular fibroblasts.
Conclusions: For cardiac remodelling, the 5-HT2BR appears as a magic bullet. Nevertheless, the “power of the past” has yet limited the clinical development for selective 5-HT2BR antagonists.
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MONTALBÁN C,1DOMINGO-DOMENECH DE, 2ESTANY C, 3CANALES MA, 4SALAR A5
Department of Clinical Hematology, Hospital del Mar;5 Department of Clinical Hematology (ICO), Hospitalet de Llobregat,2 Department of Hematology, Mutua de Terrasa;3 Barcelona and Department of Hematology, Hospital La Paz, Madrid4 and Department of Internal Medicine, Hospital de Ramón y Cajal, Madrid;1 Spain
Background: The addition of Rituximab has improved the outcome B-cell NHL and Fludarabine, alone or in combination, has been effective in MALT lymphoma. Also, our ex vivo preliminary results indicate a synergistic antitumor effect on MALT cells with combination of Fludarabine and Rituximab. This work evaluates the safety and efficacy of the combination of Fludarabine and Rituximab in the first-line treatment of extranodal MALT lymphoma.
Methods: This study enrolled 22 adult patients with extranodal MALT lymphoma, who had not received previous chemotherapy and who require systemic treatment. Patients received Rituximab 375 mg/m2 intravenously (IV) on day 1 and Fludarabine 25 mg/m2 (IV) on days 1-5 (days 1-3 when > 60 years), every 4 weeks; after the first cycle, oral Fludarabine was allowed to be given orally at 40 mg/m2 with the same schedule. An evaluation was done after three cycles and patients in complete emission (CR) received an additional cycle and those achieving only partial response (PR), a total of 6 cycles.
Results: 18 included patients have been already started on therapy: median age: 59 years (range: 32-83); 7 male, 11 female; PS 0 (94%); site of lymphoma origin: stomach (61%), skin (16%), lung (11%), parotid gland (11%); stage: I (66%), II (16%) and IV (16%). 2 pts. received 2 cycles, 9 pts. 4 cycles, 7 pts. 6 cycles. 17 pts. were evaluable for response. Overall response rate was 100% with 94% achieving CR and another one PR. One patient with initial parotid gland involvement relapsed after 6 months in two previously unaffected areas (breast and bone marrow). Median follow-up was15 months (range: 17-27). PFS rate is 93% (CI95%: 79-100%) at 12 m and OS rate 100% at 12 m. Tolerance to oral Fludarabine was excellent. Mild neutropenia was the most common toxicity, usually occurring after the third cycle.
Conclusions: These preliminary data indicate that the RF regimen, both with intravenous or oral Fludarabine, was well tolerated and is very active in the first line treatment of extranodal MALT lymphoma, even with fewer cycles than initially planned.
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Synthesis, In Silico ADME-Tox Study And Antimicrobial Evaluation Of Some Small Molecules
MOORTHY NSHN, VITTAL UB, KARTHIKEYAN C, TRIVEDI P
School of Pharmaceutical Sciences, RGPV, Bhopal, MP, India
Background: The development of antimicrobial agents from 1,2,4 triazole (TZ), 1,3 dihydrobenzimidazole-2-one (DBO) and 1,3 dihydrobenzimidazole-2-thione (DBS) derivatives were attempted to synthesized and evaluated for antifungal and bacterial activity. In silico pharmacokinetic study (PK) on the synthesized compounds were performed for its drug likeliness.
Methods: A novel Schiff bases of TZ derivatives was prepared from thiocarbohydrazide and 3,5,-dimethoxy benzoic acid followed by treating with corresponding substituted benzaldehydes. DBO and DBS derivatives were prepared by microwave method. The structures of all synthesized compounds were conformed by analytical methods. Antimicrobial activity was finding out in gram positive, gram negative and fungal strains. In silico PK study was done on Pallas software.
Results: Antimicrobial activity of the DBO compounds showed that aromatic substitutions have considerable activity against S. aureus and C. albicans.
The 2-substituted DBS derivatives posses better activity against all the strains (S. aureus, S. epidermitis, B. subtilis, E. coli, K. pneumoniae, P. vulgaries, C. albicans) in 50 mg/ml. All the compounds have significant activity against C. albicans.
In silico PK prediction shows that all the compounds obeyed Lipinski rule of 5 and have free of toxicity and metabolically stable.
Conclusion: From the study, it was concluded that Bulky substitutions in the 2nd position of DBO increase the activity than parent compound.
In DBS, 2 substituted compounds have significant activity against the microbial strains than substitution in 1st position. Except napthylamine, other compounds have negligible activity against E. coli and maximum activity with B. subtilis.
Chloro, dichloro and hydroxyl derivatives of TZ have significant activity at 64 mg/ml against C. albicans.
In silico PK study shows that the synthesized compounds except oxiranyl and napthylamine derivatives of DBO and Amino, methyl derivatives of TZ have probability of toxicity and others are free from toxicity.
From the study it is concluded that the bulky ring is necessary for the activity while the ring should not undergo any epoxide metabolite formation.
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One Bullet, Two Targets! A Nano-Particle With Two Key Medical Applications
MOREIN B1, HU K1, LARSSON R2, NYGREN P3, LÖVGREN K4
1Department of Medical Sciences, Section of Virology, Uppsala University; 2Department of Sciences, Uppsala University; 3 Department of Oncology, Uppsala University; 4Isconova AB, Uppsala, Sweden
Background: How can an efficient low-to-no toxic adjuvant nano-particle concept rest on the same concept as a cancer cell killing drug? The 40 nm ISCOM nano-particles are based on acyl-saponin (ASAP) and desacyl-saponin (DSAP) from Quillaja saponin (QS). They exert their activities by guiding cells of lymphoid origin to activation measured by down regulation of CD14 on immature dendritic cells (DCs) and expression of CD83 to differentiation measured by production of proinflammatory cytokines and expression of communication molecules e.g. CD86. That is a normal pathway leading to the programmed cell death i.e. apoptosis virtually without any side effects. More than 450 publications have demonstrated that ISCOM formulations are potent adjuvants with strong immune modulating capacity. ISCOMs are now in human phase 3 studies. To note the free i.e. non-particulate QS and the ASAP fraction are highly cell lytic due to interaction between the saponin and the cholesterol in the cell membrane. By saturating the acyl-saponin with cholesterol non-lytic particles can be formed like ISCOMs that are well tolerated by man and animal.
Methods-Concepts: The present immune adjuvant and cancer cell killing concept is developed from the ISCOM technology used as adjuvant formulations. Two 40 nm particles i.e. KGI containing ASAP and BBE containing DSAP were formulated using the same technology. A blocking technique was applied to analyse interaction of QS nano- particles with cancer cells revealing two receptor systems. Various methods were used to demonstrate lytic, necrotic and to apoptotic cell death as well as the exit of the cancer cells from the cell cycle. Various cancer cells including leukemic cells and solid tumour cells were exposed to the KGI particles and the cell survival was measured by the Alamar Blue and also other methods.
Results: The nano-particles KGI and BBE mediate their effects by communicating with the target cells by a primary attachment receptor common to ASAP and DSAP having a functional modulating capacity and a second receptor unique for ASAP most likely located on the acyl-chain. ASAP in the KGI has an additional strong modulating device for apoptosis different from the device of the common receptor. KGI particles killed cancer cells at 30 to 40 fold lower concentrations than normal cells by apoptosis after taking the cancer cells out of the cell cycle, causing activation differentiation including cytokine production. The immune modulatory and cancer cell killing effects of ASAP (KGI) and DSAP (BBE) particles differ resulting in synergistic adjuvant and cancer killing effects and also they potentiate the effect of standard anticancer drugs measured in vitro. The KGI and BBE particles have shown cancer cell killing effect on cells from about 10 different lines including cells from solid tumours.
Conclusion: QS particles can be used as vaccine adjuvant and as anti-cancer drug following the same biological mechanisms including activation, differentiation and eventually apoptosis. 1) The dual KGI - BBE system is a new low-to non-toxic concept applicable for immune adjuvants, which has already been well established, as well as for efficient induction of apoptosis of cancer cells with a high therapeutic index, which is a new discovery. 2) The dual particle system has been shown in vitro to kill a variety of cancer cells including cells from solid tumours. 3) The particles can also act synergistically with other standard anti-cancer drugs. In short, the dual ASAP and DSAP particle concept is an efficient, low-toxicity, high bioavailability vaccine adjuvant and cancer killing system based on initiating normal cell differentiation. The stand alone and synergistic effects between the components of the system, provides great promise for strong developments in two medical fields.
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A Unique SPE-LC-MS/MS Platform For Fully Automated Analysis Of Drugs In Native Whole Blood
MORELLO R, MILOJKOVIC J, BERGER I, BOOS KS
Laboratory of BioSeparation, Institute of Clinical Chemistry, Medical Center of the University of Munich, Munich, Germany
Background: Current protocols for the pretreatment of whole blood samples upfront to LC-MS/MS analysis of drugs or endogenous compounds involve manually performed multi-step operations such as protein precipitation and centrifugation or drying/elution of blood spots on filter paper. All these clean-up methods hardly can be automated or integrated into an on-line analysis procedure. In order to achieve this goal we developed a unique procedure which converts anticoagulated whole blood into a novel matrix which can be further processed on-line by different sample clean-up methods, such as Solid Phase Extraction (SPE).
Methods: For the transformation of whole blood into so called cell-disintegrated blood (CDB) we apply a heat-shock treatment. After mixing and injection of 20 µL of anticoagulated blood by an appropriate autosampler system (Symbiosis Pharma, Spark Holland) the sample is pumped at a defined flow-rate through a heated stainless-steel capillary (300 x 0.5 mm ID) set at 75°C. The resulting CDB then is flushed on-line onto a SPE-cartridge (20 x 1 mm ID, packed with Oasis HLB) and further processed. The extracted analyte fraction (immunosuppressants) finally is eluted onto a LC-column (LiChrospher 100 RP 18 EC, 125 x 2 mm ID), separated and detected by a tandem mass spectrometer (Quattro Micro, Waters) operated in ESI (+) MRM mode.
Results: The described in-line processing, i.e. the conversion of an anticoagulated whole blood sample into CDB yields a homogeneous, red-coloured biofluid composed of subcellular particles (≤ 1 µm) which do not sediment. The heat-shock quantitatively disintegrates erythrocytes, leucocytes and up to 60% of thrombocytes. A comparison of drug levels in whole blood samples of immunosuppressed patients measured by the described fully automated method and those obtained with the established manually performed precipitation (ZnSO4/MeOH) procedure revealed a very good correlation.
Conclusions: We developed a fully automated method by which an anticoagulated whole blood sample is processed in such a way that the generated cell-disintegrated blood (CDB) fluid can be further subjected on-line to conventional bioanalytical extraction, fractionation and / or separation methods (e.g. SPE, LC and CE) as well as detection modes.
Supported by EUREKA, Project E!4112
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Pharmacokinetics (PK)/Pharmacodynamics (PD) Of Infliximab In Treatment For Patients With Rheumatoid Arthritis: Characterization Of Infliximab-Resistant Cases And PK-Based Modified Therapy
MORI S
National Kumamoto Saishunsou Hospital, Kohshi, Japan
Background: Infliximab, a chimeric anti-tumor necrosis factor (TNF) monoclonal antibody, has provided a significant impact on management of rheumatoid arthritis (RA); however, a subset of patients shows poor therapeutic responses. The purpose of this study is to understand the mechanism underlying such unresponsiveness, by considering clinical PK/PD properties of infliximab and to present modified therapy for such poor responders.
Methods: Twenty-one patients with active RA were scheduled to receive an intravenous infusion of infliximab (3 mg/kg or 200 mg) at weeks 0, 2, and 6, followed by maintenance therapy every 8 weeks. We examined a relationship between clinical responses and trough serum concentrations of infliximab at week 14. We also measured time-serum concentration profiles and values of PK properties in individual patients for each infusion of infliximab and compared them with disease activities in their clinical courses.
Results: Fifteen cases achieved good or moderate responses in the European League Against Rheumatism criteria, and 3 cases resulted in nonresponders at week 14. The means of distribution volume and elimination half-life (t1/2) during the first 2 weeks were 0.05 L/kg and 9.5 days, respectively. Through 14 weeks, most good and moderate responders maintained trough serum concentrations of more than 1 g/ml. Only 3 cases showed undetectable levels of trough serum concentration at week 14. By contrast, the PK profiles of all nonresponders except one showed rapid clearance during therapy. We also found that the t1/2 during the first 2 weeks is inversely correlated to the disease activity scores at the start of therapy. For patients with a rapid clearance of infliximab, the increased use of prednisone or methotrexate was a useful way to achieve sufficient clinical responses. The addition of tacrolimus was effective to improve the clinical outcomes of nonresponders.
Conclusions: (1) Maintaining the trough serum concentrations of infliximab above therapeutic limit levels is beneficial for favorable clinical outcomes. (2) The rapid clearance appears to be the main course of unresponsiveness of infliximab. (3) PK data apparently offer guidance when an optimal treatment for infliximab-resistant RA patients is being considered.
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Cytotoxic Diterpenes From Australian Flacourtiaceae As A Good Source Of Antineoplastic Agents
MOSADDIK MA1,2, WATERMAN PG2, LEACH D2, BRUSHETT D2, BANBURY L2, FORSTER P3, BOOTH R3
1Laboratory of Natural Product Chemistry, Pharmacy Department, Rajshahi University, Rajshahi, Bangladesh; 2Center for Phytochemistry and Pharmacology, Southern Cross University, Lismore, NSW, Australia; 3Queensland Herbarium, Mount Coot-tha, Brisbane, Australia
Background: Search for new anticancer agents (e.g. taxol) from Plant is still an important field to Phytochemist and other related scientist. Flacourtiaceae is an under exploied family and no chemical and biological works have previously been reported on any Australian Flacourtiaceae. Therefore the main objective of our study was 1) To evaluate the cancer cell line based (in vitro) cytotoxic effects of diterterpens isolated from Australian Flacourtiacae 2) To provide a useful lead compound for future antineoplastic drug development 3) To gather more data to contribute to a better understanding of the Flacourtiaceae.
Methods: In this study, total threeteen novel diterpenes had been isolated from three Casearia genera of Australian Flacourtiaceae.Two rare dialdehyde clerodane diterpenes [1] and [2] and one unusual mixed acetoxy and methoxy acetal clerodane diterpenens had been isolated from root of Casearia multinervosa by column chromatography followed by semi-preparative HPLC using an RP C18 column. Similarly, leaves and stems of C.grayi and C. grewiifolia yielded seven and five clerodane diterpenes respectively, among them two were common in both species and an excepmtional optical active unsaturated keto-diacetal [3] clerodane diterpenes was obtained from Ethyl acetate extract of narrow stem of C. grayi, by preparative HPLC employing variations on a gradient elution binary system of acetonitrile and water. All the isolated diterpenes were characterized with means of various spectroscopic methods (e.g. UV, IR, NMR, LC-MS etc). All the diterpenes were tested for cytotoxicity against various cancer cell line using ATP Lite-M assay method with a reference compound chlorambucil. All the cells were obtained from American Type Culture Collection (ATCC) and were routinely cultered with essential medium. Each sample was tested in triplicate and resultes were expressed as LC50 values (µM).
Results: All the diterpenes were tested for cytotoxicity against P388 cell line and all showed cell inhibition in excess of 92% at a concertration of 0.02 mg mL-1. The most post potent dials [1 and 2] and keto-acetal [3] diterpenes, were futher examined against five cancer cell line (A375, HepG2, Hs27, P388 and PC3) and exhibited significant cytotoxicity compared to that of the reference compound chlorambucil (see the following Table).
Compound
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Cell line
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A375
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HepG2
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Hs27
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P388
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PC3
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1
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2.1
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2.0
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1.9
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2.0
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2.1
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2
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1.5
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1.6
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1.5
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2.0
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2.0
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3
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2.0
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2.0
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1.5
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1.9
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1.8
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Chlorambucil
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70.1
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27.4
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93.0
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24.5
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39.7
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