2 Free University of Berlin, Germany
Background: Nonviral gene therapy could offer the opportunity to cure various inherited and acquired pulmonary and hematopoietic diseases. The aim of gene therapy is to: (1) design effective nonviral gene vectors, (2) optimize efficiency and specificity of transfer of genetic material into target cells, (3) minimize adverse toxicity and immune responses, (4) maximize the therapeutic potential.
Methods: Nonviral vectors, both episomal-replicating (S/MAR) and integration (C31 integrase) based, were designed. Well-defined gene transfer agents possessing biodegradability, targeting functionalities and reduced toxicity were synthesized and applied via different routes into mice. Aerosol-based targeting of lung regions by combining gene vectors with magnetic gradient fields was investigated in vivo.
Results: Stable gene expression was achieved using different nonviral vectors together with plasmid DNA containing Ubiquitin C or Ubiquitin B promoters and S/MAR elementsin hematopoietic cells in vitro. Stable expression in the lungs of mice was obtained with the co-delivery of C31 integrase expression plasmid. When delivered as a fusion protein, recombinant C31 integrase-TAT, mediated site-specific recombination in mammalian cells in vitro. Using lactoferrin, insulin and clenbuterol as ligands coupled to PEI, selectivity towards specific cell types was achieved. Intranuclear trafficking of the plasmid into the nuclear matrix, resulted in higher and stable long-term expression in lung cells in vitro. High toxicity and non-biodegradability of PEI limit its in vivo application. Therefore, well-defined polymethacrylate based copolymers were characterized as gene transfer agents with low cytotoxicity, high colloidal stability and comparable transfection efficiency in vitro. Efficient methods for targeting of gene vectors to localized regions of the lung have been successfully established in mice by application of an external magnetic gradient field during inhalation of aerosols containing superparamagnetic iron oxide nanoparticles.
Conclusions: The standard requirements for clinical use of nonviral vectors have not been met yet in terms of efficiency and specificity. Future research will focus on improving the efficiency, specificity and safety of the gene delivery systems.
Pyrrole Hydrazones as a reliable Starting Platform in anti-tuberculosis Drugs Development BIJEV A, PRODANOVA P, VLADIMIROVA S, GEORGIEVA M University of Chemical Technology and Metallurgy, Sofia, Bulgaria
Background: The return of tuberculosis was declared by WHO as a global emergency. In the search for a synergic combination between the recognized anti-tubercular activity of both pyrrole derivatives and hydrazones, the current research offers sixty pyrrole-containing hydrazones synthesized and evaluated as potential tuberculostatics and 28 perspective new products designed thereof.
Methods: Both diversity and intermediate screening results directed the design of 88 hydrazones, prepared by condensation of Isoniazid or nine 1H-1-pyrrolylcarbohydrazides with 24 carbonyl compounds:
The products were evaluated at two levels High-throughput Screening (HTS): Level 1-screening against M. tuberculosis H37Rv (ATCC 27294) at 6.25 μg/mL in 12В medium using the Microplate Alamar Blue Assay and Level 2-determination of Minimum Inhibitory Concentration (MIC) of compounds with inhibition ≥ 90 %.
Results: 60 hydrazones exhibited inhibitory activity in Level 1 in the range of 0-100% and 12 hits were identified with 92-100% activity. Level 2 HTS pointed out 8 products with MICs=0.10-0.78, IC50=0.200-0.966, IC90=3.236-22.581 μg/mL and SIs=12.82-100 as candidates for further developments.
Some structure-activity correlations and a simplified second order QSAR-model were derived. Both R1=CH3 and electrono-withdrawing substituents in the aromatic ring decreased the activity. “Drug likeness” was assessed.
The architecture of some active hits was used as a template in the synthesis of new hydrazones of carbohydrazides 1, 2 and 4 and 28 perspective new analogs were prepared.
Conclusion: With inhibitory activities of 92-100% 12 compounds may serve as reliable prototypes in further ligand based design and optimization of potent anti-tubercular agents favoring compounds with moderate molecular sizes, polarity and hydrophobic parameters.
Authors’ disclosure statement
The current research was performed in collaboration with the Tuberculosis Antimicrobial Acquisition & Coordinating Facility (TAACF), Southern Research Institute, Frederick, USA, which organization performed the screening of compounds synthesized in our laboratory (acknowledged in the text). The results regarding the synthesis and evaluation of the disclosed 60 hydrazones were published recently in the following international journals:
Bijev, А, Synthesis and preliminary screening of carbohydrazides and hydrazones of pyrrole as potential tuberculostatics, Arzneimittelforschung. 2006; 56(2): 96-103.
Bijev A. New hydrazones as pyrrole derivatives with higher inhibitory activity against Mycobacterium tuberculosis. Lett Drug Des Discov. 2006;3(7):506-512.
Bijev A. Synthesis, in vitro evaluations and structure-activity assessment of pyrrole hydrazones. Lett Drug Des Discov. 2008;5(1):15-24.
Bijev А. Synthesis and in vitro Evaluation of New Hydrazones as Pyrrole Derivatives with Anti-tubercular Activity. Arzneim-Forsch/Drug Res. (2008, in press)
The essence of some latest unpublished results comprising structures and synthesis of 28 new perspective analogs whose design was based on the primary results completes the study.
Adoptive immunotherapy with Streptamer-selected HCMV specific T-cells after allogeneic stem cell transplantation GRIGOLEIT GU1, BUSCH DH2, ODENDAHL M2, ANDERL F2, GERMEROTH L3, EINSELE H1, TONN T4 Human cytomegalovirus (CMV) infection continues to be one of the most important and life threatening complications after allogeneic stem cell transplantation (SCT).
Selective restoration of anti-CMV cellular immunity by CMV-specific adoptive T-cell transfer is an attractive alternative therapeutic approach, since it is an effective, non-toxic treatment. The finding that recovery of CD8+ CMV-specific cytotoxic T-cell (CTL) responses conferred protection against the development of CMV disease following allogeneic SCT stimulated attempts to restore antiviral immunity in humans. Different groups could demonstrate that chemotherapy-resistant CMV infections can be treated successfully with the adoptive transfer of ex vivo expanded CMV-specific T-cells. In principle, two different strategies to obtain sufficient amounts of T cells with defined specificity have been developed: in vitro expansion of T cell lines or clones, or the direct ex vivo purification of epitope-specific T-cells by multimeric HLA complexes. In vitro expansion is time consuming, and leads to a loss of antiviral activity and limited persistence of transferred immunity. Binding of multimeric complexes causes functional alterations of T-cells, and in addition, the cell preparation still contains the multimeric complexes used for isolation. Recently, the Streptamer technology was developed to overcome these problems. Streptamers are reversible multimers, which are unlikely to interfere with T- cell function, since Streptamer reagents can be rapidly dissociated from the T-cell receptor. Furthermore, T-cells selected by the Streptamer technology do not contain any reagents used for isolation. In this first clinical trial, we applied Streptamers to isolate functional CMV-specific CD8+ T-cells from stem cell donors. After complete dissociation of Streptamer reagents, the isolated T-cells are transferred directly - without any in vitro expansion - to the respective CMV-infected patients. First results of the ongoing phase I/II clinical trial show that sufficient numbers of specific T-cells could be selected from all CMV sero-positive stem cell donors. In addition, we initiated extensive clonal analyses to demonstrate that the transferred T-cells participate in cellular immune responses against CMV after transfer. In conclusion, transfer of specific T-cells selected by Streptamers is a safe and feasible method to restore CMV specific cellular immunity after allogeneic SCT.
Photodynamic therapy as a new method for the treatment of
1Parasitology Dept., School of medical science, Tarbiat Modarres University, Tehran, Iran
2Department of Dermatology , Tehran Medical University, Medical School, Razi hospital , Laser clinic Tehran, Iran
Background: Leishmaniasis is an important disease caused by Leishmania spp. Cutaneous leishmaniasis occurs within a few weeks after with a small papule on the exposed site and finally ulcerates. The drugs of choice, pentavalent antimony or meglumine antimonite are characteristically moderately toxic and there are risks of recurrence and unsatisfactory side effects. Leishmania was found deficient in at least five enzymes in the heme biosynthesis pathway. The first enzymes was delta-aminolevulinic acid (ALA). During the irradiation with red light the porphyrin-enriched tissues led to the cell death.
Methods: In this study, we used photodynamic therapy (PDT) for the treatment of cutaneous leishmaniasis caused by leishmania major..
In this study, the Leishmania lesions of five patients was applied locally with ALA 10%, then after 4 hours per treatment session was delivered, using red light (570– 670 nm), 100 J/cm2 at a light intensity of 150 mW/cm2 (approximately 21 min). Treatments were repeated weekly for 4 times.
Results: In direct staining smears were showed no amastigotes after one or two sessions. The follow up continued for four months. The results showed that Photodynamic therapy to remodeling flattening and filling the sores.
Conclusion: Treatment of cutaneous leishmaniasis is directed toward the eradication of amastigotes and the reduction of the size of the lesions to promote healing and achieve maximum efficacy with minimal scarring and toxicity. Photodynamic therapy in contrast to all systemic treatment modalities has no risk of toxicity but only mild local inflammatory reaction with an excellent cosmetic outcome. So PDT might offer a new promising treatment modality for the disseminated lesions of cutaneous leishmaniasis.
Designing Material of Particular Equilibrium and Transport Properties by Ab Initio Molecular Dynamics GHATEE, MH 1 and ZARE, M, JAHROMI, F, MOOSAVI, F1 1Shiraz University, Shiraz, Iran
Background: Prediction and study of equilibrium and transport properties of ionic liquids as green solvent are currently of high interest. These liquids are used in organic synthesis without harmful environmental effect of ordinary volatile solvent. Application of atomistic simulation methods to design 1-alkyl-3-methyl imidazolium based ionic liquids of suitable equilibrium and transport properties as well as understanding the aggregation mechanism within the ionic liquid assembly forms the aims of the present study.
Methods: ab initio Car-Parrinello molecular dynamic (CPMD) simulation was used to simulate the transport properties including viscosity and diffusion constant of 1-alkyl-3-methyl imidazolium based ionic liquids. Simple anion like chloride (Cl-) and iodide (I-) and complex ones like and was studied. All the simulations were made at 300K. In the same way the aggregation of the ionic liquids with long chain alkyl group was studied. A thorough understanding of the dynamic and the structure were followed by studying the static properties by using Gaussian program.
Results: Structural and the dynamic properties were studied by the results of simulation based on statistical mechanics of the liquid state. The studies show a slow dynamic, higher viscosity, smaller coordination number for chloride compounds than the iodide one. The I- compound form larger aggregates than the Cl- one. Contrary to the ionic liquids with Cl- and I- anions, the and compounds show hydrophobic properties, and therefore they show higher dynamics, smaller viscosity, and those properties characteristics of tightly bonded ion pair salts.
Conclusions: The slow dynamics and high viscosity of the imidazolium based ionic liquids containing simple anions are due to the strong electrostatic cation-anion interaction. The more complex anions make a more complex interaction leading to the hydrophobic property. The details knowledge of these properties enables to design system of interest with desire equilibrium and transport characters.
Buccal Delivery of Carbamazepine (CBZ): a New Scenario in Management of Trigeminal Neuralgia (TN) GIANNOLA LI, DE CARO V, GIANDALIA G, SIRAGUSA MG
University of Palermo, Italy
Background: CBZ is one of the most effective drugs in treatment of TN; however, on chronic dosing, the drug presents a decrease in its half-life due to metabolism autoinduction. Following peroral administration, bioavailability of CBZ is limited by poor water solubility. Moreover, in medications, the various coexisting polymorphs of CBZ generate great fluctuations in drug solubility and absorption. The entrapment of CBZ into polymeric microspheres (MS) might alter the crystal habit of CBZ and provide regular dissolution and improved bioavailability.
The oral cavity is an attractive site for drug delivery due to ease of administration, and avoidance of first-pass metabolism and possible drug degradation in gastro-intestinal tract. Buccal tablets prepared with CBZ loaded MS were developed to obtain slow and regular drug release.
Methods: The aptitude of CBZ to penetrate porcine buccal mucosa and reconstituted human oral epithelium (HOE) was evaluated using Franz diffusion cells and Transwell diffusion cells system respectively (donor phase drug solutions in artificial saliva, acceptor phase artificial plasma). CBZ loaded MS were prepared by the emulsion solvent evaporation method using Eudragit® L-100 as polymer. CBZ-polymer interactions were measured by differential scanning calorimetric analysis. Tablets for transbuccal CBZ administration were prepared by direct compression of drug loaded MS. CBZ release from tablets was performed in vitro using aflow through system in conditions simulating the oral cavity environment.
Results: CBZ well penetrates the buccal membrane: drug fluxes and permeability coefficients were calculated as 7·10-2 mg/cm2h and 0.23 cm/h for HOE and 1.81·10-2 mg/cm2h and 4.57·10-2 cm/h for porcine mucosa respectively, thus suggesting that buccal mucosa does not appear a limiting step to the drug absorption. The thermogram of CBZ loaded MS confirmed the complete drug amorphization that implies regular drug solubilization. The release of CBZ from tablets showed a reproducible Higuchian pattern.
Conclusions: Buccal mucosa does not block diffusion of CBZ and could represent an alternative way of the drug administration. CBZ can be successfully transformed into loaded Eudragit® L-100 MS that, in turn, after direct compression, can form tablets useful for slow pre-programmed drug delivery on buccal mucosa.
Treatment of Intravenous Drug Users with Chronic Hepatitis C: Treatment Response, Compliance and Side Effects GIGI Ε1, SINAKOS Ε1, LALLA ΤH1, VRETTOU E2, ORPHANOU E1, RAPTOPOULOU M1 1 2ndDpt of Medicine, Thessaloniki University Medical School, Greece,
2 Dpt of Pathology, Thessaloniki University Medical School, Greece.
Background:Although IVDUs comprise the majority of patients with chronic hepatitis C, most of them are excluded from treatment because of concerns about adherence to treatment and side effects.
Methods:In this study we retrospectively evaluated safety, compliance to treatment and efficacy of treatment in IVDUs with HCV infection in 163 former IVDUswith chronic hepatitis C, who were not in methadone substitution and were attending our clinics during 1997-2004. All subjects were HCVRNA (+), had ALT levels >X1,5 UNL and were treated for their HCV infection. Treatment consisted of three different regimens: IFN-α monotherapy (39,8%), IFN-α/ribavirin combination therapy (30,1%) and pegylated IFN-α/ribavirin combination therapy. 87/163 patients (53,3%) discontinued treatment early due to drug abuse relapse (62%), side effects (32,1%, 10% psychiatric) and 5,7% for other reasons. 80% of those who discontinued treatment had pre-treatment drug abstinence ≤ 9 months. 70/76 patients who completed therapy had an end-of-treatment virologic response (ETR, 92%). 54/76 patients showed sustained virologic response (SVR, 71,05%).
Results: ETR and SVR were significantly higher in both combination therapies compared to IFN-α monotherapy. The most prevalent HCV genotype was 3 (65%) and mild histological lesions were detected in the majority of subjects. In conclusion our findings show that treatment for chronic hepatitis C was reasonably safe and sufficiently effective in our group of non methadone-substituted IVDUs, despite the fact that more than half of them discontinued treatment early and many relapsed to drug abuse. We suggest that the optimal duration of pretreatment abstinence from drug abuse should be > 9 months.
Evidence for Anti-Cancer activity for the Antidepressant Sertraline, In-Vitro and In-Vivo Effect in Nude Mice Xenografted with HT29 cells. GIL-AD I1, ZOLOKOV A1, LOMNITSKI L2, TALER M, BAR M1, WEIZMAN A1,.
1Lab Biological Psychiatry, Tel-Aviv University, Felsenstein Institute, Campus Rabin, Petah-Tiqva, Israel , and 2 Perrigo Israel Pharmaceuticals Ltd. Bnei Brak, Israel
Background: Recent reports provide evidence for a pro-apoptotic activity of some antidepressants, mainly the serotonin reuptake inhibitors (SSRIs). Oncogenes like Bcl2, and proteins of the mitogen activated protein kinases (MAPK) pathway, participate in the pathogenesis of the disease. Objectives: 1. Evaluation of the effect of SSRIs on apoptosis markers 2. Determination of the molecular mechanism of the drugs. 3. Evaluation of the in vivo effect of the SSRIs in a nude mouse model xenografted s.c with human colorectal carcinoma cells HT29.
Methods: Human colorectal carcinoma HT29, and LS1034, a multi drug resistant (MDR) cell lines (ATCC) were studied. Cell viability (neutral red) and cell proliferation (3H-thymidine incorporation) were determined. Apoptosis was studied using flow cytometry of propidium iodide stained cells and caspase 3 determination by an enzymatic fluorimetric assay. Protein expression was determined by western blot analysis. Tumor growth was determined in CD1 nude mice xenografted s.c and treated ip with the drugs.
Results: The SSRIs paroxetine and sertraline induced a dose-dependent inhibition of cell viability and proliferation in both cell-lines (IC50 8-15mcM). When compared to some cytotoxic agents e.g doxorubicin, vincristine and 5FU, the SSRI’s activity demonstrated a similar (HT29) or stronger effect (LS1034). Both agents stimulated DNA fragmentation and increased caspase-3 activation, suggesting a proapoptotic mechanism. Western blot analysis revealed an increase 24hr later in c-Jun and p-ERK and decreased Bcl2 expression. For in vivo experiments, we used CD1 nude mice xenografted subcutaneously with HT29 cells. Sertraline (3 times/week 15mg /kg s.c or i.p), but not paroxetine, induced a significant inhibition of tumor growth the animals.
Conclusions: Collectively, our results suggest that the widely used and safe antidepressant sertraline possesses potential anti-tumor activity, which circumvents the MDR mechanism and thus could be valuable in the arsenal of colon carcinoma therapy. Since SSRI therapy is frequently indicated in cancer patients as an antidepressant, this possibility seems attractive.
Serotonin Transporter: Mechanisms of Inhibition by Enteropathogenic E. coli (EPEC) GILL R, ESMAILI A, NAZIR S, BORTHAKUR A, TURNER J, ALREFAI W, HECHT G, DUDEJA P.
Background: Serotonin transporter (SERT) plays a critical role in regulating serotonin availability by its reuptake through a Na+ and Cl- coupled mechanism. Elevated levels of serotonin are associated with several diarrheal conditions including inflammatory bowel diseases and enteric infections. However, whether alteration in SERT activity contributes to the pathophysiology of diarrhea induced by food-borne pathogens such as enteropathogenic E coli (EPEC) is not known. EPEC is non-toxigenic but houses a pathogenicity island encoding a type III secretory system (T3SS) that translocates bacterial proteins directly into host cells. We hypothesized that EPEC decreases SERT activity to contribute to the associated rapid diarrhea. Therefore, present studies were aimed at examining the effects of EPEC infection on SERT activity and expression and delineating the underlying mechanisms.
Methods: Caco-2 cells were used as an in vitro model of human intestinal epithelia and were infected with EPEC strain E2348/69 or commensal E. coli. SERT activity was measured as fluoxetine-sensitive 3[H]-5-HT uptake. Infection of Caco-2 cells with EPEC for 30-90 min decreased luminal SERT activity (~50-60% inhibition at 30 min; P<0.005); however, infection with commensal E. coli had no impact. Kinetic analysis revealed that EPEC infection inhibited SERT activity via a decrease in Vmax (~ 3 fold). In parallel, EPEC infection caused internalization of SERT from the plasma membrane to endocytic vesicles as assessed by live cell imaging of SERT-GFP construct in transfected Caco-2 cells. Mutation of escN, which encodes the ATPase for T3SS, ablated the effect of EPEC on luminal SERT activity indicating that effects of EPEC were T3SS dependent. Inhibitory effect of EPEC on SERT activity was abolished in the presence of tyrosine phosphatase inhibitors (phenyl arsine oxide and dephostatin). EPEC infection in vivo significantly reduced mucosal 5-HT content in the mouse small intestine.
Conclusion: Infection of intestinal epithelial cells with EPEC decreases SERT via a T3SS dependent mechanism and involvement of tyrosine phosphatases. These data further highlight the interactions of a common enteric pathogen with the expression and function of SERT and provide mechanistic insights into development of a potential new pharmacotherapy to modulate the serotoninergic system in treatment of diarrheal diseases.
A new mode of cell death for tumor cells after ascorbate : menadione treatment in vitro and in vivo. GILLOTEAUX J 1, JAMISON JM 2, TAPER HS 3, SUMMERS JL 2 1 St George’s International School of Medicine, Newcastle-upon-Tyne, NE1 8ST, U.K.,
2 Department of Uro-Oncology, Summa Research Foundation, Akron OH., U.S.A.
3 Unité de Pharmacologie, Métabolisme, Nutrition & Toxicologie, Université Catholique de Louvain, B-1200 Brussels, Belgium
Background: Dietary supplements can be used as adjuvants to treat several kinds of cancers. Nucleases are repressed in tumors. Reactivating nucleases in tumor cells would assist in killing those cells. Tumor-bearing rodents fed by a mixture of ascorbate (VC) + menadione (VK3) before irradiation or chemotherapy brings a higher rate of survival and significant decrease of tumor size.
Methods: Series of 106 human carcinoma cells (DU145, T24, RT4, MDAH, etc) grown on 12 mm diam. titer dishes with 24 h in M5A milieu, washed with PBS saline, overlaid for 1, 2, 4, or 6h with 2 mL M5A with VC, VK3 or VC+VK3 + 2 mL PBS ( cytotoxic doses CD99) washed by PBS, then prepared for electron microscopy. Male 4 mths old, NCr-nu/nu mice injected with 106 DU145 cells in 100 μL M5A medium were injected sc. After 4 wks, given 100 μL PBS or 100 mL VC+VK3 and killed 1, 2, 4, 8 and 24h after injection, and 1-4 mm tumors on diaphragm were prepared for TEM.
Results: Carcinoma cell treated by Sham-PBS, VC, VK3, VC+VK3 combinations for 1, 2 and 4h show the cytotoxic damages by electron microscopy, biochemistry and flow cytometry were treatment-dependent as VC+VK3 > VC > VK3 > Sham. Oxidative stress induces alterations of cytoskeleton, mitochondria, lysosomes lead to cytoplasm self-excisions without organelles. Cell size reduction and other nuclear damages, DNAses I + II reactivation, with DNA gel electrophoreses smears patterns are data consistent with injuries of autoschizic cell death, not apoptosis. In vivo DU145 tumors treated reactivated DNAses and morphology reveals tumor demise by autoschizis causing tumor shrinkage and significant mice survival.
Conclusions: VC: VK3 (or VC) exerts antitumor activities through a wide array of mechanisms: oxidative stress, nucleases’ reactivation, cell cycle blocks and induction of autoschizic cell death (Gilloteaux et al., 1995-2006). Metalloproteinases inhibition and potentiation of the immune system make VC+VK3 combination, or Apatone ® , a magic bullet to be used safely in patients against many tumor cells.
Erythropoietic Stimulating Proteins - What is the Optimal, Safe Hemoglobin Target? GILMARTIN C Univ. of Illinois, Chicago, USA
Background: Erythropoietic stimulating proteins (ESPs) have been used in the treatment of anemia of chronic kidney disease (CKD) and chemotherapy-induced anemia (CIA) since 1989 and the early the 1990s, respectively to a target hemoglobin (HgB) between 11 to 12 g/dL. Recent clinical trials have examined HgB targets > 12 g/dL that have demonstrated increased mortality and thrombotic events in both anemia of CKD and CIA. ESPs have also been utilized for the off-label indication in anemia of cancer.
Methods: A review of the clinical trials prompting the addition of the boxed warning to the ESPs product labeling in the United States was performed. An evaluation of the trials including target Hgb, primary endpoints and outcomes in both CKD, CIA and anemia of cancer was assimilated.
Results: The following table summarizes the trials in CKD anemia.
The table below summarizes the trials for CIA and anemia of cancer
[Regrettably not enough space on this page.]
Conclusions: Targeting Hgb levels ≥ 12 g/dL CKD and CIA utilizing ESPs has not shown benefit and may increase the risk of death and thrombotic events. CIA trials demonstrated a decreased progression free and overall survival and decreased locoregional tumor control with ESP use. One trial failed to appreciate a reduction in RBC transfusions with ESP therapy. ESPs for CIA are indicated only concurrently with mylosuppresive therapy when cure is not the goal. Current guidelines recommend initiation when the Hgb < 10g/dL. Guidelines in CKD recommend initiation when < 11 g/dL, and caution when approaching 13 g/dL.
High-dose cyclophosphamide is active in immune-mediated illnesses. GLADSTONE D Stony Brook University, New York, USA
Background: High-dose cyclophosphamide is active in immune-mediated illnesses.
Objective: To describe the effects of high-dose cyclophosphamide on severe refractory multiple sclerosis.
Design, Setting, and Patients: Patients with multiple sclerosis with an Expanded Disability Status Scale (EDSS) score of 3.5 or higher after 2 or more Food and Drug Administration-approved disease-modifying therapy regimens were evaluated.
Intervention: Patients received 200 mg/kg of cyclophosphamide over 4 days.
Main Outcome Measures: Patients had brain magnetic resonance imaging ad neuro-ophthalmologic evaluations every 6 months and quarterly EDSS and quality-of-life evaluations for 2 years.
Results: Twelve patients were evaluated for clinical response (median follow-up,15.0 months; follow-up range, -24 months). During follow-up, no patients increased their baseline EDSS scores by more than 1.0. Five patients decreased their EDSS scores by 1.0 or more (EDSS score decrease range, 1.0-5.0). Two of 11 patients had a single enhancing lesion at baseline; these lesions resolved after high-dose cyclophosphamide treatment. At 12 months, 1 patient showed 1 new enhancing lesion without a corresponding high-intensity T2-weighted or fluid-attenuated inversion recovery signal. Patients reported improvement in all of the quality-of-life parameters measured. Neurologic improvement involved changes in gait, bladder control, and visual function. Treatment response was seen regardless of the baseline presence or absence of contrast lesion activity. Patient quality-of-life improvement occurred independently of EDSS score changes. In this small group of patients with treatment-refractory multiple sclerosis, high-dose cyclophosphamide was associated with minimal morbidity and improved clinical outcomes.
Conclusions: High-dose cyclophosphamide treatment in patients with severe refractory multiple sclerosis can result in disease stabilization, improved functionality, and improved quality of life. Further studies are necessary to determine the most appropriate patients for this treatment.
Vancomycin Resistant Enterococcus faecium Under Constant Linezolid Exposure GLOEDE J, SCHEERANS, C., KLOFT C
Martin-Luther-Universitaet Halle-Wittenberg, Halle, Germany
Background: The occurence of vancomycin resistant Enterococcus (VRE) has been increasing from 2.7% to 13.5% in Germany (2002-2004). This is due to too high, too low and inadequate dosing of antibiotics.[1] For VRE colonised patients linezolid offers an alternative treatment. The prediction of the antibacterial effect by a pharmacokinetic/pharmacodynamic (PK/PD) analysis could guide the antibiotic therapy, shorten the duration of disease and reduce the extent of bacterial resistance.
Methods: The killing behaviour of VRE (ATCC 700221) over time under various linezolid concentrations was investigated in a static in vitro model. An inoculum of 106 cfu/mL VRE in Mueller-Hinton (MH) broth was spiked with linezolid concentrations of 0.5, 1, 2, 4, 8, 16, 32 µg/mL, incubated at 37 °C and continuously shaken (62 min-1). The time-killing process was monitored via viable cell counting over 24 h. Bacteria samples were taken at 0, 1, 2, 4, 6, 8, 10, 12, 16, 20 and 24 h and plated on MH agar plates with 5% sheep blood. After 24 h of incubation all plates were counted by a digital automatic colony counter (ColonyQuant, Schuett Biotec, Goettingen, Germany). Geometric means and confidence intervals of the respective bacterial concentrations were numerically calculated via bootstrapping in Excel (Mircosoft).
Results: The time-kill curves of VRE under various linezolid concentrations were investigated. Linezolid concentrations of 4 and 8 µg/mL were identified as bacteriostatic and 17 and 36 µg/mL as bactericidal. For concentrations lower than 4 µg/mL bacterial regrowth after 1h was visible. Moreover, 17 and 36 µg/mL achieved very simlar rates in bacterial killing.
Conclusions: Longer-term in vitro time-kill curves for VRE under various linezolid concentrations describe the bacterial growth. LZD displayes time- and concentration-dependent effects of killing with respect to VRE.
1. Kresken, M., et al., PEG-Resistenzstudie 2004. 2006, Paul-Ehrlich-Gesellschaft für Chemotherapie e.V.: Rheinbach. p. 1-90.
Antibody responses in cancer vaccines and immunotherapies: from cancer/testis antigens to new targets discovered by protein arrays GNJATIC S1, ODUNSI K2, ALTORKI NK3, RITTER G1, BUECHLER MW4, JAEGER D4, OLD LJ1 1Ludwig Institute for Cancer Research, New York NY, USA; 2Roswell Park Cancer Institute, Buffalo NY, USA; 3Weill Medical College of Cornell University, New York NY, USA; 4 Nationales Centrum für Tumorerkrankungen, Heidelberg, Germany
Background: Analysis of antibody responses to self-antigens has driven the development of the field of tumor immunology. While cancer/testis antigens have been put to the test in clinical trials, more targets are needed. Protein microarray technologies offer an unprecedented platform to assay the serological response of cancer patients to tumor antigens in a comprehensive fashion.
Methods: Sera from patients with non-small cell lung cancer (NSCLC), epithelial ovarian cancer (EOC), and pancreatic cancer, as well as healthy donor sera from the New York Blood Bank, were collected and tested by Enzyme-Linked ImmunoSorbent Assay (ELISA) against known recombinant tumor antigens, as well as by antibody profiling using commercially available protein microarrays containing >8200 antigens.
Results: We first validated our approach by using sera with known immunoreactivity in ELISA to antigens present on microarrays. We found 197 antigens reacting frequently and strongly with EOC patient sera compared to healthy donor sera. The same study with pancreatic cancer patient sera returned 28 antigens with preferred immunogenicity in cancer, 21% of which overlapped with antigens immunogenic in EOC.
Conclusions: With a stringent strategy for data analysis and normalization designed to determine antigen-specific serum antibody responses using protein arrays, we describe new antigens immunogenic in cancer patients and propose that this approach is suitable for defining potential antigenic targets for cancer vaccine development, serum antibody signatures with clinical value, characterization of predictive serum markers for experimental therapeutics, and eventually for the serological definition of the cancer proteome (seromics).
Intravascular fluid replacement in the critically ill: Is it the substance or the timing that makes the difference? GOEPFERT MS, KUBITZ JC, GOETZ AE, REUTER DA
Department of Anesthesiology, Hamburg-Eppendorf University Hospital, Universiy of Hamburg, Germany
Background: The ideal infusion solution used for intravascular fluid replacement in critically ill patients is still not determined in order to improve patients´outcome. Further, inconsistent opinions exist whether restrictive or liberal fluid management is beneficial in critical ill patients.
In this regard, also the “timing aspect”, and how to guide fluid therapy in critically ill patients has not been elucidated yet satisfactorily. We therefore performed a prospective study in cardiac surgery patients where the timing and the amount of fluid replacement was guided by an early goal directed hemodynamic algorithm using volumetric parameters of cardiac preload for optimization of timing and quantity of intravascular fluid replacement.
Methods: 40 patients undergoing cardiac bypass surgery were included prospectively (study group, SG) and compared with a control group (CG). In the SG, from induction of anaesthesia until 48 hours after surgery, hemodynamic management was continuously guided by an algorithm based on the measurement of global enddiastolic volume index (GEDVI) and cardiac index (CI). Hemodynamic goals were a GEDVI > 640 ml m-2, CI > 2.5 l m-2, and mean arterial pressure (MAP) > 70 mmHg. The CG was treated at the discretion of the attending physician based on central venous pressure (CVP), MAP and subjective clinical evaluation.
Results: Total duration of catecholamine and vasopressor dependency was significantly shorter in the SG (187 ± 70 min vs. 1458 ± 197 min, p < 0.001). Less vasopressors (0.73 ± 0.32 mg vs. 6.67 ± 1.21 mg, p < 0.001) as well as catecholamines (0.01 mg ± 0.01 mg vs. 0.83 ± 0.27 mg, p < 0.001) were administered in the SG. In cristalloid infusions no differences were detected at any time. In the SG significantly more colloids (HES 130/0.4, gelatin solution 3.5%) were used during surgery and ICU-therapy (1515 ± 60 ml of colloids vs. 1327 ± 50 ml during surgery and 5403 ± 222 ml vs. 4187 ± 167 ml during ICU therapy). Regarding allover fluid bilance (6509 ± 240 ml SG vs. 6403 ± 184 ml CG; p < 0.05) there was no clinical relevant difference. SG patients reached ICU-discharge criteria significantly earlier (25 ± 13 vs. 33 ± 17 h).
Conclusions: Not only the substance used for fluid replacement but continuous preload optimization and the optimal timing for fluid application are necessary to improve critical ill patients outcome.
Changing the dosing frequency of ceftazidime transforms this “Daisy cutter” into an antibiotic with limited collateral damage while retaining its efficacy GOESSENS WHF1, MOUTON JW2, TEN KATE MT1, OTT A1, BAKKER-WOUDENBERG IAJM1 1Erasmus University Medical Center Rotterdam, Rotterdam; 2Canisius Wilhelmina Ziekenhuis, Nijmegen, The Netherlands
Background: During treatment of infection the host’s normal flora is unintentionally exposed to antibiotics, which may lead to secondary colonization by potentially pathogenic, often multiple antibiotic-resistant, organisms. The aims of the present investigation is to study in rats the effect of ceftazidime dosing increments and frequency of dosing on the selection of ceftazidime-resistant Enterobacter cloacae in the intestine during treatment of a pulmonary infection caused by Klebsiella pneumoniae.
Methods: Rats with pulmonary infection (n=10 per group) received therapy with doses of 3.1 to 400 mg/kg/day of ceftazidime at a frequency of every 6,12 or 24 h during 18 days, starting 24 h after bacterial inoculation of the lung. Emergence of resistance in intestinal E. cloacae was monitored by culturing fresh stool specimens at days 0, 8, 15, 22, 29, 36 and 43 on agar plates with (6.4 mg/L) and without ceftazidime. Pharmacodynamic indices and time within the mutation selection window (MSW) were assessed in infected rats for each regimen. Ceftazidime-resistant E. cloacae mutants were characterized by determination of the ß-lactamase activity under cefoxitin-induced and non-induced conditions.
Results: A reduction of intestinal ceftazidime-susceptible E. cloacae was observed and showed a significant correlation with the fAUC/MIC at days 8, 15 and 22 and with the fCmax on days 8, 15, 22, 29 and 36, respectively. More rats treated with 12-25 and 50-100 mg/kg/day every 6 h were found colonised with ceftazidime-resistant E. cloacae mutants than animals treated every 12 h or every 24 h. The proportion of rats colonised with ceftazidime-resistant E. cloacae mutants at days 15, 36 and 43 correlated with the time during which ceftazidime-plasma concentrations were within the boundaries of the MSW. Ceftazidime-resistant E. cloacae mutants (MIC > 128 mg/L) were characterized as stable derepressed mutants. Furthermore, the therapeutic efficacy on the K. pneumonia pulmonary infection by long ceftazidime treatment appeared to correlate with the fAUC/MIC ratio (AUC is the area under the concentration time curve).
Conclusions: Colonization with stable derepressed ceftazidime-resistant E. cloacae mutants particularly occurred when rats were exposed to moderate doses of ceftazidime (12-25 or 50-100 mg/kg/day) administered every 6 h. Emergence of resistance was correlated to time within the MSW.
Frovatriptan - the triptan with the least drug/drug interactions GOLDSTEIN J Director, San Francisco Clinical Research Center, San Francisco Headache Clinic, and San Francisco Alzheimer's and Dementia Clinic
Frovatriptan was the sixth triptan to be approved since the approval of Sumatriptan (Imitrex/Imigran) in 1993. Concern over cardio-toxicity and drug/drug interactions has prompted development of other triptans. Also the short duration of action of most triptans (2-4 hours) has encouraged the development of other triptans. In addition to the cerebro-selective nature of Frovatriptan multiple clinical trials have confirmed tolerability and efficacy with statistically significant results of efficacy. Frovatriptan has no inhibiting or inducing effects on cytochrome P450 enzymes and therefore has the lowest potential for drug/drug interactions of all the triptans. Clinical trials confirm that Frovatriptan can be administered safely in patients with hepatic and renal impairment and there is no specific age limit to administration of the drug. Low recurrence rate has also been confirmed in clinical trials and Frovatriptan has been studied in menstrual migraine headache with demonstrable efficacy in that condition. Thus, Frovatriptan is the triptan best suited for long duration headaches, headaches prone to recurrence, and those patients with significant triptan side effects.
Molecular Imaging of Multidrug Resistance by ABC Transporters in Osteosarcoma GOMES CMF1, WELLING MM2, QUE I2, HENRIQUEZ N2, PLUIJM G2, ROMEO S2, ABRUNHOSA AJ1, BOTELHO MF1, HOGENDOORN PCW2, PAUWELS EKJ2, CLETON-JANSEN AM2 1IBILI-Faculty of Medicine, Coimbra, Portugal
2Leiden University Medical Center, Leiden, The Netherlands
Background: Multidrug resistance (MDR) is a significant obstacle to successful chemotherapy and a major prognostic factor in osteosarcoma (OS) patients. We have previously demonstrated that the sensitivity of OS cell lines to doxorubicin and cisplatin depends on MDR-related ABC-transporters and can be predicted based on functional assays using 99mTc-Sestamibi (MIBI). These observations prompted us to develop an orthotopic model of OS to evaluate the role of MIBI imaging in the assessment of MDR and pharmacological inhibition.
Methods: Sensitive (143B) and resistant (MNNG) OS cell lines expressing different levels of P-glycoprotein carrying a luciferase reporter gene were inoculated into the tibia of nude mice. Local tumour growth was monitored weekly by whole-body bioluminescent reporter imaging and by radiography. After primary tumour growth, the animals were imaged with MIBI during 60 min. A group of animals were pre-treated with a Pgp inhibitor (PSC833). Images were analyzed for calculation of MIBI washout half-life (t1/2), % of washout rate (%WR) and uptake ratio.
Results: A faster washout rate of [99mTc]MIBI was observed in resistant tumours has demonstrated by the shorter t1/2 and higher %WR in MNNG-resistant tumors (t1/2=87.3±15.7min; %WR=37.5±4.0%) compared with those in 143B-sensitive tumors (t1/2=161.0±47.4min; %WR=24.6±7.5%). Administration of PSC833 increased significantly the retention of MIBI in MNNG tumors (t1/2=173.0±24.5min, %WR=23.8±4.3%, p<0.05) and had no significant effects on 143B-sensitive tumours.
Conclusions: 1) The orthotopic injection of cancer cells provides an animal model closely resembling the clinical situation of OS that can be used for functional imaging of MDR. 2) The kinetic analysis of MIBI washout provides information on the functional activity of MDR-related to Ppg expression and its pharmacological inhibition. 3) Functional imaging with MIBI might be a valuable clinical tool to predict chemotherapy response in OS.
Antioxidant Supplementation Impacts upon Electronegative Low Density Lipoprotein Plasma Levels and Cardiovascular Risk GOMES LF1, SIMON KA2, MELLO-ALMADA FILHO C2, CENDOROGLO MS2, QUIRINO LM2, RAMOS LR2, JUNQUEIRA VBC2 1Universidade de São Paulo; 2Universidade Federal de São Paulo, São Paulo, Brazil.
Oxidative stress has been considered determinant of aging and other pathological processes. Indeed a number of authors have shown a decrease in non-enzymatic antioxidants during aging. Moreover, human chronic diseases, as cardiovascular and neurodegenrative diseases are also proposed to have an oxidative stress component. Despite various attempts to prove the above statement, researchers have been poorly successful. The failure to demonstrate the role of antioxidant strategies against the mentioned diseases has many causes, mainly the lack of the antioxidant network knowledge to design an appropriate strategy. The present paper deals with decreasing levels of minimally oxidized LDL in urban hypercholesterolemic elderly patients. Antioxidant supplementation to hypercholesterolemic patients, had the following composition: Tablet 1:600 mg Vitamin C, 200 mg Vitamin E, 0.6 mg β-carotene; 40 mg Zinc; 1.0 mg Copper; and 100 g Selenium; Tablet 2: 10 mg Coenzyme Q10. Participants were divided into five groups (20 per group) and received a combination of the two tablets or placebo for 180 days. It was demonstrated that plasma levels of antioxidant vitamins E, C and -carotene were increased among the subjects taking two tablets 1. Q10 coenzyme was increased only among those taking two tablets 1 and one tablet 2. Minimally oxidized LDL levels decrease was dependent on the intake of two tablets 1. Lower intakes were ineffective. In conclusion, an adequate antioxidant strategy decreases oxidative stress. Whether it may offer a potential protection against vascular diseases, it remains to be studied in large controlled trials. Financial support: Fapesp, CNPq, CAPES, and Marjan Indústria Farmacêutica.
NO-Donors As Modulators of Antitumor Drugs Resistance. GONCHAROVA SA, RAJEWSKAYA TA, KONOVALOVA NP
Institute of Problems of Chemical Physics, RAS, Russia
One of the major causes of anticancer chemotherapy failure is connected with development of resistance of tumors to those drugs to which they were high sensitive at first. According to famous P.Ehrlich, “tumor resistance follows chemotherapy like a shadow, and brings to naught all its achievements and successes”. And it is indeed so. The resistance is developing to all known drugs, including the latest targeted ones. Based on modern knowledge of drug resistance (DR) mechanisms, many compounds were proposed to overcome it. But, almost all of them did not found the use because of high toxicity. And the quest for DR modulators is continued.
Last time an important role of nitric oxide (NO) in different biological processes, including tumor growth, was elucidated. NO participates in the effect of antitumor drugs. Some works link NO and DR. These data stimulated the use of exogenous NO-donors in chemotherapeutic studies, including those concerning DR.
We investigated the possibility of using two NO-donating compounds: 1)AK-2123 {N-(2'-me-thoxyethyl)-2-[3"-nitro-1"-triazolyl]acetamide}, has 1 NO-group; 2) NMO [3,3-bis(nitroxymethyl)ok-setan}, has 2 NO-groups.
We showed that AK-2123 significantly enhanced the sensitivity of multidrug-resistant (MDR) strains of P388 mouse leukemia (developed and characterized on phenotype and genotype by us) to mitomycin C (MMC). The modulating effect was dependent on the initial sensitivity of resistant tumors to MMC, which was correlated with existence or absence in MDR-amplicon of sorcin gene coamplification. Moreover, AK-2123, used in extra low doses (10-6–10-10 mg/kg) also increased the sensitivity MDR-tumors to MMC.
NMO essentially slows down of resistance forming of parent P388 tumor to cyclophosphamide (CPA), from 8th to 14th transplant generation.
On different experimental models we discovered that both compounds increased the antitumor and antimetastatic effect of various traditional anticancer drugs (CPA, doxorubicin, cisplatin), used in low uneffective doses.
Thus, the combined employ of NO-donors and cytostatics may slow the development of DR to the latter and help in cure of MDR-tumors. Beside, NO-donors may be used as adjuvant agent in the chemotherapy of tumors.
In our opinion, our data show that NO-donating compounds could be excellent supplement to P. Ehrlich’s MAGIC BULLET, when it is found.
Development of Prognostic and Predictive Variables in Breast Cancer: Personalized Approach Using Gene Expression Profiling Microarray GONG Y1, YAN K1, LIN F1, ANDERSON K1, SOTIRIOU C2, ANDRE F3, HOLMES FA4, VALERO V1, BOOSER D1, PIPPEN JE5, VUKELJA S6, GOMEZ H7, MEJIA J1, BARAJAS LJ8, HESS KR1, SNEIGE N1, HORTOBAGYI GN1, PUSZTAI L1, SYMMANS WF1 1The University of Texas M. D. Anderson Cancer Center, Houston TX, USA; 2Institut Jules Bordet, Brussels, Belgium; 3Institut Gustave Roussy, Villejuif, France; 4US Oncology-Texas Oncology, Houston, TX, USA; 5US Oncology-Texas Oncology, Sammons Cancer, Dallas, TX, USA; 6US Oncology-Texas Oncology, Tyler Cancer Center, Tyler, TX; 7Instituto Nacional de Enfermedades Neoplásicas, Lima, Peru; 8Instituto Mexicano del Seguro Social, Guadalajara, Jalisco, Mexico
Background: Despite a considerable decline in the mortality from breast cancer following systemic therapy, the biology of breast cancer remains poorly understood. Unfortunately, the routinely-used clinicopathologic variables fail to fully capture the biologic heterogeneity. As a result, many patients are overtreated whereas others may not receive the necessary therapy. Gene expression microarrays may provide more sophisticate information than conventional biomarkers in predicting disease outcome and response to a specific systemic therapy on an individual basis.
Methods: 1).To identify the molecular signature that predict pathologic complete response (pCR) to sequential treatment of paclitaxel, 5-fluorouracil, doxorubicin and cyclophosphamide (T/FAC) neoadjuvant chemotherapy, our group profiled 82 breast carcinomas and searched for the gene signature. Predicting accuracy of the signature was then validated on an independent set of 51 tumors. 2). To evaluate whether ER and HER2 status can be reliably measured from the comprehensive microarray data, we used gene expression data of 495 breast carcinomas to assess the correlation between ER and HER-2 mRNA levels and clinical status of these genes (as determined by immunohistochemical and/or fluorescence in situ hybridization). Data from 195 fine-needle aspiration (FNA) samples was used to define mRNA cutoff values and the accuracy of these cutoffs was assessed in two independent data sets: 123 FNA samples and 177 tissue specimens (ie, resected or core-needle biopsied tissues).
Results: 1). A 30-gene predictor of pCR was developed which could predict pCR with an overall accuracy of 76%. 2). ER and HER-2 mRNA levels correlated closely with routine receptor status measurements in all three data sets. Spearman’s correlation coefficients ranged from 0.62 to 0.77. The defined ER mRNA cutoff identified ER-positive status with the overall accuracy of 88-96%; and the defined HER-2 mRNA cutoff identified HER-2-positive status with the overall accuracy of 89-93%.
Conclusions: 1). Although the gene signature may need to be refined and validated by large studies, this approach potentially allows us to tailor therapy based on expression microarray data. 2). ER and HER2 gene expression can be reliably measured from the comprehensive microarray data. Integration of ER and HER2 mRNA expression with multigene signatures from the same microarray data may refine and improve their predictive power. These findings may represent an important step towards personalized treatment.
Medicinal effects of maca: Experimental data on reproduction, memory and learning GONZALES GF1, VILLEGAS L1, GONZALES C1, GONZALES-CASTAÑEDA C1, GASCO M1, YUCRA S1, RUBIO J1.
1Faculty of Sciences and Philosophy, Universidad Peruana Cayetano Heredia, Lima, Peru.
Background: Peru has important tradition in the use of medicinal plants. Lepidium meyenii (maca), is a cruciferous cultivated plant growing over 4,000 m altitude in the central Andes of Peru traditionally used by its nutritional and supposed medicinal properties. The hypocotyl, the edible part of the plant is observed in different varieties (color). In the last years the interest for maca has been raised in many parts of the world. We have developed experimental and clinical studies to demonstrate the biological effects of this plant.
Methods: Clinical study was performed in 60 healthy adult men. Experimental studies were performed in rats (300 g BWt) and mice (30 g BW). Maca was boiled as traditionally used before administered. Red (RM) and Black (BM) variety were used in experimental studies. Maca was administered as boiled aqueous or hydroalcoholic extracts. Some experiments included the mixture of maca and another plant extract (UPCH). Toxicological studies were also performed. Data were assessed using parametric and non parametric statistics.
Results: In men gelatinized maca has favorable effects on energy, mood, decreasing anxiety, blood pressure, sexual desire, sperm production, sperm motility and semen volume without affecting sexual hormone levels. Experimental studies showed that BM increase sperm count and memory and learning in rats. Red maca reversed testosterone-induced prostatic hyperplasia and osteoporosis induced after ovariectomy in rodents. RM had more polyphenols and antioxidant activity than BM. The mixture BM+UPCH increased sperm count and reduced glycemia and serum Cholesterol in normal rats. Maca acute median lethal dose (LD50) for the oral route was >9 g/kg bw. Hydroalcoholic maca extract was not mutagenic in vivo and in vitro assays. Maca extract administered for 8 months to mice was not hepatotoxic rather than looks as hepatoprotective.
Conclusion: Gelatinized maca and extracts of black maca and red maca had acceptable safety profile and they are potential candidates as therapeutic agents.
7 days
Control
83.46±5.87
BM (160 mg/Kg)
106.63±3.35*
UPCH 0.01 g/Kg + BM
107.75±6.07*
UPCH 0.1 g/Kg+BM
108.43±4.72*
Table 1. Epididymal sperm count (x106) after treatment with black maca (BM) alone or with UPCH (Plant extract). Data are mean ±SEM. Number of rats: 10 pergroup.*P<0.01
and 
was studied. All the simulations were made at 300K. In the same way the aggregation of the ionic liquids with long chain alkyl group was studied. A thorough understanding of the dynamic and the structure were followed by studying the static properties by using Gaussian program.
