Conclusions: XRP44X inhibits various cellular properties, including cell growth, cell cycle progression and aortal sprouting, similar to other molecules that bind to the tubulin colchicine site. XRP44X has the potentially interesting property of connecting two important pathways involved in cell transformation. It is an interesting molecule for the development ofcancer treatment.
Targeted Therapy of Human Neuroblastoma Cells using Auger Electrons of Indium-111-labeled N-myc Antisense Oligonucleotide
WATANABE N
National Institute of Radiological Sciences, Chiba, Japan, International Atomic Energy Agency, Human Health Division, Vienna, Austria
Background: Auger electrons may destroy nucleic acids in the close vicinity, and thus targeted therapeutic effect of Auger electrons of indium-111 (In-111)-phosphorothioate antisense oligonucleotides on human neuroblastoma cells with the overexpression of N-MYC was studied.
Methods: Human neuroblastoma cells of SK-N-DZ (5 X 106 cells) were treated for 20 hrs with cationic reverse-phase vesicles encapsulating In-111-antisense oligonucleotides (AS) (average specific radioactivity of vesicle of 40 MBq/2 nmol of oligonucleotides/µmol of total phospholipids) with average diameter of 250 nm. The expression of N-MYC and proliferation of the treated tumor cells were investigated as well as the existence of In-111-AS hybridized with intracellular N-myc mRNA. Tumorigenecity o the treated tumor cells was examined in nude mice. As a control, non-radiolabeled AS or In-111-sense oligonucleotides (S) were employed.
Results: The In-111-AS hybridized with intracellular N-myc mRNA were detected from the treated tumor cells at 12 and 24 hrs after initiation of the treatment. Reduction in the expression of N-MYC (average 27% to control) and inhibited cell proliferation (average 59% to control) were shown in cells at 48 hrs after treatment of In-111-AS.The N-MYC-suppressed cells however continued to produce tumor although a significant decrease in average weight of tumors was demonstrated in intraperitoneal cavity of nude mice. Neither of non-radiolabeled AS nor In-111-S caused any effect in tumor cells.
Conclusions: Auger electrons of In-111 in the close vicinity of their target N-myc mRNA may cause reduction of cell proliferation rate of human neuroblastoma cells with the overexpression of N-MYC, and In-111-antisense oligonucelotides could potentially be a tool for internal molecular radiotherapy at a level of mRNA of a tumor cell.
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The Tissue Specific Choice of Anti-caner Drugs on the Basis of Polyamine Level
WATANABE S1, NAGASE S2, SATO S2, OHKUMA S1
1Kawasaki Medical School, 577 Matsushima, Kurashiki City, Okayama Japan
2Kawasaki College of Allied Health Professions, 316 Matsushima, Kurashiki City, Okayama Japan
Background: Polyamines (putrescine:put, spermidine:spd and spermine:spm) are essential to neoplastic cell growth. Therefore, analysis of the effects of the anti-cancer drugs on the polyamine concentration of individual tissues of intact rats provides tissue specific treatment of the drug on the basis of polyamine level and could improve the management of cancer patient by allowing development of new strategies of the drug use that targets the tumor-bearing tissues.
Methods: The experimental groups of rats (7) received intraperitoneal injections of the drugs and volume of a solution successive daily for 5 days. All rats were euthanized with enflurane on the sixth day, and the tissues were immediately removed, weighed and kept in 2.0 ml of an aqueous 10% trichloroacetic acid solution (TCA) containing 0.1 mmol/L 1,4-diaminohexane as an internal standard in an ice bath. Each tissue was homogenized and then centrifuged at 2500 rpm for 15 min. The supernatants were washed twice with 5 ml diethyl ether to eliminate the TCA in the water layer. Determination of polyamine concentration in the water layer was carried out using HPLC and the amount of each polyamine was calculated from the peak area relative to the internal standard 1,6-diaminohexane
Results: All polyamines in 5-FU- and ara-C-treated small intestine and 5-FU-treated seminal vesicles, and spd and spm in 5-FU-treated prostate and testis, ara-C-treated stomach, adriamycin-treated seminal vesicles and large intestine and cisplatin-treated seminal vesicles of intact rats increased. On the other hand, all polyamines in the adriamycin-treated heart, methotrexate-treated thymus and spleen, cyclophosphamide-treated prostate, seminal vesicles, thymus, spleen, kidney, heart and small intestine of rats, and etoposide-treated thymus and put and spd in adriamycin-treated prostate, testis and thymus and in etoposide-treated skeletal muscle and lung decreased. At the regional brain, all polyamines in etoposide-treated cortex decreased, and spd and spm in etoposide-, ranimustine- and nimustine-treated hippocampus, cortex and corpus striatum of rats decreased, respectively.
Conclusions: (1) Anti-cancer drugs, adriamycin, methotrexate, cyclophosphamide and etoposide is suitable to the treatment of the cancers bearing the skeletal muscle, thymus and spleen, small intestine, prostate, seminal vesicles, thymus, spleen, kidney and heart, and thymus, respectively. (2) 5-FU and Ara-C, respectively, should not use the treatment of the cancer bearing the seminal vesicles and small intestine, and the small intestine because of increase of polyamines that stimulate the growth of the cancer cells. (3) Etoposide is suitable to the treatment of the cancers bearing the hippocampus and cortex, and ranimustine and nimustine to the cortex and corpus striatum, respectively.
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When More is Not Necessarily Better: Interdisciplinary Inquiry into the Implications of U-Shaped Dose Responses for Personalizing Anticancer Interventions
WATERS DJ
Purdue University Center on Aging and the Life Course, and the Gerald P. Murphy Cancer Foundation, West Lafayette, IN, USA
Background: The perception that is pervasive among the public is that, when it comes to taking cancer-fighting dietary supplements, more is better. Whether or not this concept is valid is especially relevant to health-conscious men and women, who are ironically at highest risk for the ill-effects of oversupplementation because they are already consuming high quality diets rich in vitamins and minerals. In 2001, the National Cancer Institute launched SELECT to evaluate whether daily supplementation with selenium (Se) or vitamin E prevents prostate cancer. But very little was known about what dose of Se might offer the most potent cancer-protective effects. We hypothesized that Se regulates the accumulation of genotoxic damage within the prostate and that the relationship is non-linear, i.e. more Se is not better.
Methods: We conducted a randomized feeding trial in which 69 elderly beagles (equivalent to 65 year-old men) received adequate or supranutritional Se intake for 7 months. We used the aging dog prostate to mimic the aging human prostate, enabling us to study the effects of Se on prostatic cells in an appropriate context.
Results: Se supplementation significantly decreased the accumulation of DNA damage in the prostate (alkaline Comet assay). When we examined the relationship between toenail Se level and prostatic DNA damage, we discovered an intriguing U-shaped dose response curve: more was not better. Further, we showed that the Se level that minimizes DNA damage in the aging dog prostate remarkably parallels the Se level that minimizes prostate cancer risk in 2 large human studies.
Conclusions: Now, more than ever, we need a new approach to cancer prevention — personalized cancer prevention (Waters et al, Nutrition and Cancer 2008; 60:1-6). Defining the U-shaped relationship between DNA damage and cancer-modulating nutrients addresses one of the major obstacles to developing personalized cancer-reducing interventions. It follows from this understanding that not all individuals will necessarily benefit from increasing their nutrient intake. Baseline nutrient status should be required for all individuals in prevention trials to avoid oversupplementation.
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Early Results of Imatinib as the Active Agent in Control of Aggressive Fibromatosis. An Attempt of Metaanalysis
WCISLO G, BODNAR L, SZARLEJ-WCISLO K, SZCZYLIK C
Department of Oncology, Military Institute of Medicine, Warsaw, Poland
Email: gabrielwcislo@yahoo.pl
Background: Aggressive fibromatosis (AF) is a neoplasm arising from musculoaponeurotic structures with fibroproliferative characteristics. Most patients suffer from the abdominal tumors and other locations. Molecular pathobiology appears to be of clinical value especially when there is a possibility to use targeted therapy with imatinib. This drug can block several molecules to imply clinical benefits in patients whose advanced AF tumors are unresectable and progressed after completion of radiation, and systemic therapy.
Methods: We have described our clinical experience and have searched databases (PubMed, and Medline) to find publications focused on the imatinib therapy for advanced AF. Early results have shown the clinical activity of imatinib as the salvage therapy in patients with advanced AF.
Results: We found only a few publications focused on the role of imatinib as the salvage therapy in advanced AF. The total number of 175 patients with advanced AF were given imatinib at the dose of 400 mg orally daily for more than 2 months. Median treatment time was 9 months. Median time to progression of AF on imatinib was 6.8 months. 13 patients were given 800 mg orally daily when progression of AF on 400 mg of imatinib was recorded. Among of them in 7 patients SD (stabilization disease) was noted. There were following responses to imatinib assessed in 175 patients: 2 CR-1.1% (complete response), 26 PR-14.8% (partial response), 99 SD-56.5%. 127 patients (72.5%) experienced clinical benefits interpreted as disease control. 47 patients had toxicity profile of G1-3 (grade) (no G-4): asthenia-44.6%, nausea and vomiting-40.4%, diarrhoea-25.5%, oedema-25.5%, abdominal pain-12.7%, rash-12.7%. There were also attempts to characterize AF at the molecular level by expression of following KIT-26 positive/44 patients, PDGF receptor alfa-24 positive/44 patients, PDGF receptor beta-31/44 patients, mutations within PDGF receptor alfa exon 12-1case/22 patients, within PDGF receptor alfa exon 18-3 cases/22 patients. Additionally, WNT was mutated in 84% (16 cases/19 patients) but no correlation between mutations and responses to imatinib was noted.
Conclusion: Imatinib is an active drug that is able to be used to control advanced AF remaining unresectable and after completion other therapeutic modalities as radiation, and systemic treatment with anti-inflammatory drugs, endocrine therapy, and chemotherapy.
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Antiproliferative Properties of Quinidine
WEIGER TM, HERMANN A
University of Salzburg, Salzburg, Austria
Background: Quinidine reduces sodium channel activity and is a general potassium channel blocker. While its antiarrhythmic class I properties in curing heart diseases are well known, less information is available about its antiproliferative properties.
Results: It is established that K+ channel blockers inhibit cell proliferation by leading to membrane depolarization and an arrest in early G1 phase. Recent work demonstrates that quinidine prevents the IGF-1 stimulated cell growth in MCF-7 cells. It interferes with the IGF-1 (Insulin-like growth factor) stimulated activity and expression of hEAG (human ether à go-go) potassium channels through an Akt-phosphorylation dependent pathway. In C6 glioma cells quinidine was found to reduce polyamine levels which are essential for cell proliferation by a block of ODC (ornithine decarboxylase) activity. Further downstream quinidine reduces protein levels of the G1- S transcription factor, E2F1. In addition to its antiproliferative action in breast cancer cell lines or glioma cells lines quinidine was also found to be of potential therapeutic interest for a subset of mesothelioma tumors. While normal Schwann cells remained unaffected in proliferation by quinidine, neurofibromatosis type 2 Schwann cells were down regulated. In addition quinidine was found to be a proliferation modulating agent for human liver cells. Furthermore, quinidine is used as one of the cinchona alkaloids against Plasmodium falciparum growth.
Conclusions: Quinidine has antiproliferative qualities which may be beneficial for a selected number of diseases which are characterized by over stimulated cell proliferation.
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Operation as a motivation for smoking cessation
WEISS-GERLACH E, TURINI S, LINNEN H, SPIES CD
Charité – Universitaetsmedizin, Department of Anesthesiology and Intensive Care Medicine, Berlin, Germany
Background: From the anesthesiologist’s view, the hospital stay should be used to aid the patient in smoking cessation, as smoking may lead to prolonged length of stay and numerous cardiopulmonary complications. In addition significantly more general and wound infections that lead to an increased length of stay. In addition the time of the preanesthetic evaluation is perfect as a teachable moment, and should be used for health promoting measures (i.e.: smoking cessation).
Methods: We conducted a survey of 2516 adults in the pre-anaesthesia clinic before surgery. Patients answered questions on a laptop regarding their consumption of alcohol, nicotine and illicit drugs, social and educational background. A total of 1169 patients were asked for the BA.
Results: The percentage of smoker is almost identical in both settings (34.8 % in the PC and 34.5% in BA). The degree of nicotine dependence differs between both settings. The BA revealed 37.2 % of patients with a FTND > 4, while the PC showed 47 %. The share of smokers with an action stage according to the transtheoretical model is much higher in the PC despite the lower nicotine dependence with an average of 27.6 % compared to 4.2 % in the control group.
Conclusions: As in hospital patients have a higher motivation for smoking cessation, the preanesthetic clinic is the ideal place for explicit medical advice and the offer to support in smoking cessation.
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Intracellular and organelle-specific drug delivery: „The new frontier for Magic Bullets”
WEISSIG V
Midwestern University College of Pharmacy Glendale, Glendale, AZ 85308, USA
vweiss@midwestern.edu
Sub-cellular, i.e. organelle-specific drug delivery is emerging as the new frontier in drug delivery [1]. It has become more and more evident that the efficiency and efficacy of drug action is dependent largely on how well an unaided drug molecule is able to reach its intracellular target. Subsequently, specific delivery of a drug to its site of action inside cells will dramatically improve its action [2]
Mitochondria play a key role in apoptosis and several clinically used as well as experimental drugs are know to trigger apoptosis by directly interacting with target site at or inside mitochondria. Therefore we hypothesized that that the mitochondria-targeted delivery of such drugs will dramatically increase their pro-apoptotic activity.
We have been developing during the last years a variety of mitochondria-specific pharmaceutical nanocarriers for the purpose of delivering therapeutic DNA or low-molecular weight compounds to mitochondria inside living mammalian cells [3, 4].
Here we summarize our efforts and introduce new data demonstrating the applicability of our mitochondria-targeted nanocarriers for increasing the apoptotic activity of two drugs, paclitaxel [5] and ceramide [6].
Paclitaxel is a molecule that has recently been shown to have pro-apoptotic biological targets on the mitochondria but has a QSAR-predicted cytosolic accumulation and no affinity for mitochondria. Using a mitochondria-specific nanocarrier system (DQAsomes) prepared from the amphiphilic quinolinium derivative dequalinium chloride to deliver paclitaxel to mitochondria in cells we report that it is possible to improve the pro-apoptotic action of paclitaxel. Ceramide is a sphingolipid signaling molecule that has been shown to mediate a diverse range of biological responses to extracellular stimuli. It is well known that mitochondria are the link between the increased levels of ceramide generated by chemotherapeutic drugs and the induction of apoptosis. We report that the specific delivery of ceramide using mitochondriotropic liposomes to the mitochondria elicited a robust apoptotic response while non-targeted delivery failed to do so. In summary, our studies cclearly demonstrated that organelle-specific drug-loaded nanocarriers can significantly enhance therapeutic drug effects.
[1] C. S. Lim, Organelle-specific targeting in drug delivery and design, Adv Drug Deliv Rev 59 (2007) 697.
[2] V. P. Torchilin, Recent approaches to intracellular delivery of drugs and DNA and organelle targeting, Annu Rev Biomed Eng 8 (2006) 343-375.
[3] V. Weissig, S. V. Boddapati, S. M. Cheng, and G. G. D'Souza, Liposomes and liposome-like vesicles for drug and DNA delivery to mitochondria, J Liposome Res 16 (2006) 249-264.
[4] V. Weissig, Boddapati, S.V., Jabre, L., D'Souza, G.G.M., Mitochondria-specific nanotechnology, Nanomedicine 2 (2007) 275-285.
[5] S. M. Cheng, D'Souza, G.G.M., Boddapati, S.V., Horobin, R.W., Weissig, V., Nanocarrier-assisted sub-cellular targeting to the site of mitochondria improves the pro-apoptotic activity of paclitaxel, J Drug Target, in press (2008).
[6] S. V. Boddapati, D'Souza, G.G.M., Erdogan, S., Torchilin, V.P., Weissig, V., Organelle-targeted nanocarriers: Specific delivery of liposomal ceramide to mitochondria enhances its cytotoxicity in vitro and in vivo, submitted (2008).
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Beyond cholesterol lowering: Immunomodulatory effects of lovastatin
WEITZ-SCHMIDT G, DAWSON J, HOMMEL U, KALLEN J, MEINGASSNER J, WELZENBACH K
Novartis Institutes for BioMedical Research, Basel, Switzerland
Background: For decades 3-hydroxy-3-methylglutaryl coenzym A (HMG-CoA) reductase inhibitors (statins) such as lovastatin have been used to lower plasma cholesterol levels and in consequence the risk of cardiovascular disease. More recently, there has been an increasing interest in the immunomodulatory effects of statins. These latter effects have been largely attributed to the modulation of the HMG-CoA reductase pathway. Our group discovered an anti-inflammatory mechanism of lovastatin action entirely unrelated to HMG-CoA reductase inhibition.
Methods and Results: We demonstrated that lovastatin selectively blocked the binding of the integrin lymphocyte function-associated antigen-1 (LFA-1) to its ligand intercellular adhesion molecule-1 (ICAM-1). Unexpectedly, lovastatin was found to bind to a hitherto unknown site within LFA-1 distant from the ligand binding site, suggesting an allosteric mode of inhibition (Fig.1). LFA-1 is one of the key adhesion molecules involved in leukocyte migration during an immune response. In agreement with this function lovastatin inhibited leukocyte migration in a murine model of peritonitis and exhibited beneficial effects in murine allergic contact dermatitis. Subsequent optimisation of lovastatin for LFA-1 binding resulted in novel allosteric LFA-1 inhibitors which are under preclinical evaluation for the treatment of autoimmune diseases and transplant rejection.
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Fig.1: Crystal structure of the complex between the LFA-1 binding domain (I-domain) and lovastatin. The crystal structure shows that the allosteric ‘lovastatin site’ (L-site) of LFA-1 is located distant from the metal-ion dependent adhesion site (MIDAS) implicated in ligand binding.
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Conclusions: Our demonstrate that lovastatin inhibits LFA-1 function by binding to a novel allosteric integrin site. Further investigations are needed to assess whether and to which extent the lovastatin effect on LFA-1 contributes to the overall benefit of the drug in patients.
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Residue Networks and Drug Resistance in Hepatitis C Virus Antiviral Therapy
WELSCH C1,2, DOMINGUES FS2, ALBRECHT M2, LENGAUER T2, SARRAZIN C1, ZEUZEM S1
1J. W. Goethe University Hospital, Frankfurt/Main, Germany; 2Max Planck Institute for Informatics, Saarbrücken, Germany
Background: Telaprevir (VX-950) and Boceprevir (SCH 503034) are hepatitis C virus (HCV) NS3-4A protease inhibitors with strong antiviral activity in recent phase 1 clinical trials and currently in phase 2 development. We revealed amino acid mutations conferring varying degrees of drug resistance by direct sequencing and clonal analysis for both inhibitors. We apply in-silico approaches and the HCV replicon system to study the molecular mechanisms of drug resistance.
Methods: We use available experimental structures of NS3-4A, together with protein-ligand docking, rotamer analysis and molecular dynamics simulations. Furthermore, we introduce a novel approach using a 2D network formed by the non-covalent interactions between amino acids of the 3D protease structure to interpret the effect of important mutations on replication efficacy and resistance development. An HCV replicon assay was used for IC50 determination of mutant NS3-4A proteases.
Results: We investigate the conformational variability of the protease in alternative ligand-binding models and predict the binding mode of VX-950. We use the 2D network of non-covalent interactions to discover mechanisms of drug resistance in VX-950 and SCH 503034. Mutations at positions 36 and 54 are located spatially close to a hydrophobic cavity in the ligand-binding pocket. We show that the cyclopropyl group in VX-950 is oriented towards this hydrophobic cavity. We also describe the potential impact of mutations at V36 and T54 on the side-chain and backbone conformations and provide possible explanations for their effects on antiviral efficacy and viral fitness.
Conclusions: 1) T54 mutants are expected to interfere with the catalytic triad of the protease and thereby are assumed to affect the viral replication efficacy to a larger degree than V36 mutants. Molecular dynamics simulations of T54A/S mutants and rotamer analysis of V36A/G/L/M side-chains support our interpretations. 2) Mutations at V36 and/or T54 result in impaired interaction with the VX-950 cyclopropyl group, which explain the development of viral breakthrough variants in VX-950 but not in SCH 503034.
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On the Need for Fractal Modeling of Renal Clearance, G (ml/min), of Radiolabled Pentatate
WESOLOWSKI CA, PUETTER RC, BABYN PS
MUN, St. John’s, Canada; UCSD, La Jollia, USA; The Hospital for Sick Children, Toronto, Canada
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