Ehrlich II –2nd World Conference on Magic Bullets

Using Chimeras With C-Terminal Tails Of Noss For Probing Of The Electron Traffic In CYPOR JÁCHYMOVÁ M^1, 2, MARTÁSEK P^3, SHEA TM^2, ROMAN LJ^2, MASTERS BS^2 ^1Institute of Clinical Biochemistry and Laboratory Diagnostic, Charles University 1^st Medical Faculty, Prague, Czech Republic ^2Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas ^3Department of Pediatrics, Charles University 1^st Medical Faculty, Prague, Czech Republic The three NOS isoforms contain unique sequences that modulate electron transfer: the calmodulin-binding sequence, the C-terminal extension, and the autoregulatory loop in the FMN-binding module of the reductase domain. In the present studies, we have transfered the control conferred by the C-termini of NOS to NADPH-cytochrome P450 oxidoreductase (CYPOR), which does not contain any of these regulatory elements. The effect of the addition of the C-terminal sequences, specific for each isoform of NOS (21-mer, 33-mer, and 42-mer for iNOS, nNOS, and eNOS, respectively), on the catalytic activity and properties of CYPOR was determined. The aim was to ascertain the possible evolutionary origin of NOS and to address the effect of new peptide recruitment on the development of new functions for CYPOR. Compared to the soluble CYPOR construct to which each of the C-termini was attached by genetic engineering, CYPOR-iNOS (+ iNOS 21-mer) was ~20% inhibited, CYPOR-nNOS (+ nNOS 33-mer) was ~26% inhibited and CYPOR-eNOS (+42-mer) was ~42% inhibited. While similar reduction in 2,6-dichlorophenolindophenol activities was obtained, ferricyanide reduction was affected much less to negligibly. In addition examination of the kinetic constants showed no significant changes in Km for NADPH (1.88 ± 0.49 to 2.55 ± 0.48mM) at 100mM cytochrome c or for cytochrome c (19.22 ± 2.13 to 27.77 ± 2.43mM) at 50mM NADPH for all of the constructs. However, reduction of molecular O2 was increased by the addition of C-terminal sequences, suggesting a shift in the rate-limiting step caused by interference of electron flow between FAD and FMN by the extension of the C-termini over the FAD-FMN interface in the NOS isoform structures. This conclusion has been supported by the published structures of CYPOR and of the nNOS reductase. The modulation of CYPOR by the addition of the NOS C-termini is also supported by flavin reoxidation and fluorescence-quenching studies and antibody recognition of the C-terminal extension. These experiments support the origin of the NOS enzymes from modules consisting of a heme domain and CYPOR or ferredoxin-NADP(+) reductase- and flavodoxin-like subdomains that constitute CYPOR, followed by further recruitment of smaller modulating elements into the flavin-binding domains.	Key Aspects for the Compilation and Enhancement of a Comprehensive Chemogenomics and Drug Discovery Compound Screening Collection JACOBY E Novartis Institutes for BioMedical Research, CH-4002 Basel, Switzerland The NIBR (Novartis Institutes for BioMedical Research) compound collection enrichment and enhancement project integrates corporate internal combinatorial compound synthesis and external compound acquisition activities in order to build up a comprehensive screening collection for a modern drug discovery organization. The main purpose of the screening collection is to supply the Novartis drug discovery pipeline with hit-to-lead compounds for today?s and the future's portfolio of drug discovery programs, and to provide tool compounds for the chemogenomics investigation of novel biological pathways and circuits. As such, it integrates designed focused and diversity-based compound sets from the synthetic and natural paradigms able to cope with druggable and currently deemed undruggable targets and molecular interaction modes. Herein, we will summarize together with new trends published in the literature, scientific challenges faced and key approaches taken at NIBR to match the chemical and biological spaces.
The Outer Membrane Vesicles Of An Antarctic Bacterium Pseudomonas Syringae Lz4W JAGANNADHAM MV, DHOPLE VM and SUNDARAM CS Centre for Cellular and Molecular Biology, Uppal road, Tarnaka, Hyderabad-500 007, India. Background: Outer membrane vesicles are constantly discharged from Gram –ve bacteria during cell growth. The outer membrane vesicles perform several biological functions, such as DNA transfer, and protein delivery to eukaryotic or prokaryotic cells. Characterization of these vesicles may facilitate the design of protein, DNA or other drug carriers. Studies on cold adapted bacteria might further facilitate the detection of biotechnologically important molecules such as enzymes. The main objective of these studies is to characterize the naturally releasing vesicle from bacteria, that may help to design new vesicle with required protein /drug molecules and use them as transporters. Membrane vesicles of the Antarctic bacterium Pseudomonas syringae Lz4W were prepared and characterized. Methods: The bacterium was grown at 22^0C harvested the cells by centrifugation, the supernatant was filtered through a 45 μm filter, and the filtrate was ultra centrifuged, to obtain the vesicles as a pellet. The proteins of these vesicles were identified with the help of mass spectrometry and using NCBI data base of the Pseudomonas sp. Results: Transmission electron microscopy revealed that the size of the proteo-lipids ranged from 90 to 160nm.The vesicles contained ~10 kb DNA fragment. Vesicle proteins were fractionated on a 10% SDS-gel and the trypsin digests of the bands were analyzed by liquid chromatography-matrix assisted laser desorption/ionization. The vesicles contained about 100 proteins. The subcellular proteins present were from cytoplasm, inner membrane and outer membrane. Conclusions: 1) The outer membrane vesicles of the Pseudomonas syringae Lz4W were prepared and characterized. 2) The proteins identified from these vesicles were from different locations of the bacterium and have diverse functions such as transport, metabolism, antimicrobial, anti parasitic and several other functions.	Apatone^®, A Combination Of Vitamins, With In Vitro, In Vivo And Clinical Effectiveness Against Prostate Cancer JAMISON JM^1, GILLOTEAUX J^2, TAPER HS^3, VERRAX J^3, NEAL D^1, MCGUIRE K^1, TAREEN B^1, GERSON L^1, BUC-CALDERON P^3, DIOKNO A^4, SUMMERS JL^1 ^1 Summa Health System, Akron, OH, USA ^2 St. George’s International School of Medicine, Newcastle-upon-Tyne, UK ^3 Université Catholique de Louvain, Brussels, Belgium ^4 William Beaumont Hospital, Royal Oak, MI, USA Background: When vitamin C (VC) and vitamin K3 (VK3) were combined in a 100:1 ratio (Apatone®), the cytotoxicity against DU145 and PC-3 prostate cancer cells was potentiated 4- to 20-fold. Oral Apatone also significantly reduced the growth rate of the solid tumors in nude mice (p < 0.05) without inducing significant bone marrow toxicity, changes in organ weight or pathological changes in these organs. Electron micrographs revealed tumor cell death by autoschizis (self-excision of organelle-free cytoplasmic pieces which left an intact, pyknotic, nucleus surrounded by a narrow rim of cytoplasm which contained most organelles). Apatone treatment induced a G1/S and G2/M block, diminished DNA synthesis, increased hydrogen peroxide production, decreased cellular thiol levels and increased intracellular Ca^2+levels. Electrophoretic analysis of DNA revealed a spread pattern of degradation due to the sequential activation of DNase I and DNase II and was independent of cytochrome C release and caspase-3 activation. The current study was designed to evaluate the safety and efficacy of oral Apatone administration in the treatment of prostate cancer in patients who failed standard therapy. Materials and Methods: Seventeen patients with 2 successive rises in PSA after failure of standard local therapy were treated with (5,000 mg of VC and 50 mg of VK3 each day) for a period of 12 weeks. Prostate Specific Antigen (PSA) levels, PSA velocity (PSAV) and PSA doubling times (PSADT) were calculated before and during treatment at 6 week intervals. Following the initial 12 week trial, 15 of 17 patients opted to continue treatment for an additional period ranging from 6 to 24 months. PSA values were followed for these patients. Results: At the conclusion of the 12 week treatment period, PSAV decreased and PSADT increased in 13 of 17 patients (p ≤ 0.05). There were no dose-limiting adverse effects. Of the 15 patients who continued on Apatone after 12 weeks, only 1 death occurred after 14 months of treatment. Conclusion: Apatone showed promise in delaying biochemical progression in this group of end stage prostate cancer patients.
Alpha1-Antitrypsin Augmentation Therapy: New Insights Into The Molecular Basis Of Efficacy JANCIAUSKIENE S, NITA I Wallenberg Laboratory Entrance 46, 2nd floor, Lund University, University Hospital Malmö, 20502 Malmö, Sweden. Background: The augmentation therapy with human purified plasma α1-antitrypsin (AAT) is approved for emphysema patients with severe inherited AAT deficiency. The major concept behind augmentation therapy is that a rise in the levels of blood and tissue AAT will restore protease/anti-protease balance and will protect lungs from the destruction by neutrophil elastase. However, AAT also appears to function as an endogenous inhibitor of inflammatory cytokines, apoptosis and anthrax toxin induced cytolytic activity. In an in vivo model of murine islet cell allograft rejection, AAT blocked rejection when used as a single agent. Our aim is to characterise the pleitropic functions of AAT. Methods: Human blood monocytes were isolated from blood donors (n=65).Cells were stimulated with lipopolysaccharide(LPS, 10 ng/ml, J5 Rc mutant), 10–50µM rolipram or forskalin, or 0.5 mg/ml AAT, separately and in combination for 30 min and 1 and 18h at 37°C, 5% CO2.Cells and cell culture supernatants were analyzedto determine tumor necrosis factor-α (TNFα) and IL-10 expression and release by using enzyme-linkedimmunosorbent assays and Quantitative Real Time Reverse Transcription. Total cAMP levels and protein kinase A (PKA) activity weredetermined using commercial kits. Results:  1) AAT inhibited LPS-stimulated TNFα release and expression, and enhanced IL-10 release (see Figure ); 2) In monocytes pre-treated with LPS for 1 h followed by addition of AAT(0.1–4 mg/ml) for 2 min, cAMP levels increased 82–190%, p<0.001; 3) Pre-treatmentof the monocytes with rolipram alone resulted in an 87% (p <0.01) increase in cAMP level and caused anaugmentation in cAMP levels in response to AAT (52% increase,p < 0.01, n = 3 experiments) compared with rolipram alone; 4) BothAAT (0.5 mg/ml) and forskolin (as positive control) increasedPKA activity 213 and 256% (p < 0.001), respectively, comparedwith control. Conclusions: 1) Theanti-inflammatory activities of AAT in vitro, namely inhibition of endotoxin-stimulatedTNFα and enhancement of interleukin-10 inhuman monocytes, are mediated by an elevation of cAMP and activationof cAMP-dependent PKA; 2) Elucidation of the pleitropic functions of AAT may help to improve efficacy of augmentation therapy and to design novel therapeutic drug candidates to exploit the therapeutic potential of AAT-like molecules.	Is Levodopa The Magic Bullet For Parkinson's Disease? JANKOVIC J Parkinson's Disease Center and Movement Disorders Clinic, Department of Neurology, Baylor College of Medicine, Houston, Texas (www.jankovic.org) Background: Much progress has been made since James Parkinson first described the disease in 1817, but Parkinson’s disease (PD) continues to be one of the most common causes of disability, particularly among the elderly. Our understanding of the etiopathogenesis of PD has improved markedly with the discovery of genetic causes and with growing knowledge of mechanisms underlying neurodegeneration in the substantia nigra and resulting depletion of dopamine and other neurotransmitters (Pan et al. Brain 2008;131:1969-78). Dramatic improvement in the cardinal signs of PD, tremor, rigidity, akinesia, and postural instability (TRAP) and other motor symptoms, in response to levodopa has been recognized since 1961, when Birkmayer and Hornykiewicz (Wien Klin Wochenschr 1961;73:787-8) first treated parkinsonian patients with the dopamine precursor. Methods: Evidence-based literature on the efficacy and safety of levodopa was critically reviewed and analyzed in the context of long-term experience. Videos of patients treated with levodopa will be presented to illustrate its efficacy and motor complications. Results: The therapeutic options for patients with PD have been expanding with the introduction of dopamine agonists, MAO-B inhibitors, and other medical and surgical strategies, but levodopa continues to be the “gold standard” and is considered to be the most effective drug in the symptomatic treatment of PD. Psychiatric and motor complications, including fluctuations and dyskinesias, can be managed effectively until the advanced stages of the disease. There is no evidence of levodopa-related neurotoxicity from in vivo studies or long-term clinical experience. Conclusions: Levodopa is not only the most effective drug in the treatment of PD, but its introduction revolutionized treatment of neurodegenerative disorders and transformed Neurology from a primarily a diagnostic specialty to a therapeutic discipline and as such it may be considered “the magic bullet” in Neurology. Despite its extraordinary impact on the quality of life of patients with PD, there are, however, many limitations to levodopa, including the various acute and chronic complications and its lack of efficacy in certain “axial” motor signs, particularly freezing of gait and postural instability, and in most non-motor symptoms associated with PD, such as behavioral, cognitive, sensory, autonomic and sleep disorders (Jankovic J. J Neurol Neurosurg Psychiatry 2008;79:368-76). Because of the broad diversity of symptoms associated with PD and the growing recognition that non-dopaminergic neurotransmitters are also involved in PD, future therapies will likely involve not just a single bullet but a shot-gun approach targeting many systems.
Clusters of Free Radicals from Dihydroartemisinin Cure Several Parasitic and Viral Diseases. They Attack Cancers by Activating a Series of Apoptotic Pathways and by Causing Strong Angiogenesis Inhibition JANSEN FH^1, SOOMRO SA^1, EFFERTH T^2; RULISA S^3 ^1Department of Research and Development, Dafra Pharma R&D, Turnhout, Belgium, ^2Institut für Krebsforschung Heidelberg, Germany and ^3Department of Gynaecology, Kigali, Rwanda. Dihydroartemisinin (DHA); a reduced form of artemisinin; is a sesquiterpen which posses peroxide in the form of seven-membered ring and a lactol which makes it a highly energetic and unstable molecule but at the same time this instability makes it useful for number of diseases including actions against parasites such as malaria and schistosomiasis, also it is effective agains bacteria, fungi and some selected virus. DHA is unstable so that it is hardly suitable for use pharmaceutically as its own, certain derivatives are made to increase the stability and ease in formulation. Artesunate, an ester of DHA and artemether, a methyl ether of DHA are common in use and further research is going on to explore other derivatives. The mechanism of artemisinin is complex. It is reported that peroxide moiety is responsible for its reactivity. Upon contacting with iron of the cell it generates free radicals which are believed to react with the biomolecules of parasites. Based upon this mechanism the synthetic peroxides have also been investigated but none of them has been in market upto now. Variety of other possible mechanisms are also proposed that included targteting the SERCA or its effect on immune system. Artemisinin derivatives can also selectively kill cancer cells and retard the growth of tumour. These derivatives induce the apoptesis via generation of reactive oxygen species (ROS). This mechanism is different from common anticancer drugs such as Doxorubicin and therefore Artemisinins could be used in paralled to doxorubicin or other DNA intercalators or Doxorubicin-resistant cells. The induction of apoptosis by artemisinins was shown for human KS-IMM Kaposi sarcoma cells, whereas normal endothelial cells don’t undergo apoptosis in the presence of artemisinins. Artemisinin derivatives are also reported to inhibit the antiogenesis which is required by tumor cells to get the oxygen and nutritions. Artemisinin derivatives significantly inhibit angiogenesis in a dose dese-dependent manner. Recently clinically relavent anti-tumour effects were clearly demonstrated in man.	Sulfasalazine Revisited: A Multi-Targeted Magic Bullet JANSEN G, VAN DER HEIJDEN JW, DIJKMANS BAC Dept. of Rheumatology, VU University Medical Ctr, Amsterdam, The Netherlands Background: Sulfasalazine (SSZ) is commonly prescribed for the treatment of patients with chronic inflammatory diseases, such as those with rheumatoid arthritis (RA). Here we report on the novel mechanisms of action of SSZ, SSZ-drug interactions with the folate antagonist methotrexate (MTX), and mechanisms of acquired resistance to SSZ. Methods: In vitro cell line model systems of immune effector cells (T-cells, macrophage cells) implicated in the pathophysiology of RA were used to evaluate anti-inflammatory properties of SSZ after short-term drug administration or after chronic exposure to stepwise increasing concentrations of SSZ, which provoked acquired resistance to SSZ. Results: SSZ proved to be a potent inhibitor (IC50: 0.55 mM) of the production of pro-inflammatory cytokine tumor necrosis factor  via inhibition of the nuclear transcription factor NFB. This capacity was 3-fold reduced for cells with acquired resistance to SSZ. The mechanistic basis underlying SSZ resistance involved the overexpression of a cell membrane-associated drug efflux transporter, ABCG2, which reduces intracellular SSZ concentrations. Further studies revealed that SSZ was a potent, non-competitive inhibitor (IC50: 0.3 mM) of the Reduced Folate Carrier (RFC), the dominant transporter for the cellular uptake of MTX and natural folates. Concurrently, this SSZ-RFC interaction provokes an intracellular folate depletion that could further enhance the therapeutic effect of MTX in SSZ+MTX drug combinations, but only when SSZ administration precedes and is spaced in time from MTX administration. Finally, chronic exposure of cells to stepwise increasing concentrations of SSZ markedly increased the cellular sensitivity for the glucocorticoid (GC) drugs prednisolone and dexamethasone. In fact, after SSZ exposure, primary GC-sensitive T cells displayed 10-20 fold greater sensitivity to GCs, while primary GC-resistant macrophage cells resumed full GC-sensitivity due to a greatly enhanced upregulation and stabilization of the GC-receptor-, facilitating enhanced GC-induced apoptosis. Conclusions: SSZ not only elicits potential anti-inflammatory activity as a direct inhibitor of the NFB pro-inflammatory signalling pathway, by targeting other cellular processes (a.o. folate and glucocorticoid metabolism), SSZ allows a rational utilization in drug combinations.
Comparison of the Pharmacodynamics of Imipenem in Patients with Ventilator-Associated Pneumonia following Administration by 2 h or 0.5 h Infusion JARURATANASIRIKUL S, SUDSAI T Prince of Songkla Univ., Songkla, Thailand. Background: The time that concentrations in plasma are above the MIC (t>MIC) is the pharmacokinetic/pharmacodynamic parameter correlating with the therapeutic efficacy of β-lactam antibiotics. The aim of this study was to compare the t>MICs of imipenem between administration by a 2 h infusion with a 0.5 h infusion. Methods: The study was a randomized three-way crossover in nine patients with ventilator-associated pneumonia. Each subject received imipenem in three regimens consecutively: (i) 0.5 h infusion of 0.5 g every 6 h for 24 h; (ii) 2 h infusion of 0.5 g every 6 h for 24 h; and (iii) 2 h infusion of 1 g every 6 h for 24 h. Results: Following the 0.5 h infusion of 0.5 g of imipenem, the percentages of the t>4MICs of 4, 2, and 1 mg/L were 20.32%  9.32%, 44.11%  16.40%, and 64.67%  20.56% of a 6 h interval, respectively. For the 2 h infusion of 0.5 g of imipenem, the percentages of the t>4MICs of 4, 2, and 1 mg/L were 17.71%  19.27%, 53.75%  19.30% and 76.54%  17.36% of a 6 h interval, respectively. For the 2 h infusion of 1 g of imipenem, the percentages of the t>4MICs of 4, 2, and 1 mg/L were 60.26%  23.96%, 77.78%  20.11% and 93.35%  8.26% of a 6 h interval, respectively. Conclusions: 1) The 2 h infusions of imipenem resulted in greater t>MICs than the 0.5 h infusion. 2) For infections caused by pathogens with high MIC, a 2 h infusion of 1 g of imipenem every 6 h can provide plasma concentrations above the MIC of 4 mg/L for 60% of a 6 h interval.	Decontamination of Cardiovascular Allografts in European Homograft Bank (EHB). Comparison of different Antibiotic Cocktails in low Concentration low Temperature Conditions JASHARI R European Homograft Bank, International Association, Brussels, Belgium Background: Heart valve and vascular allografts have been processed in the EHB since 1989 and 1991, respectively. Low temperature (+4°C), low concentration cocktail of four different antibiotics (cefoxitin, lincocin, vancocin and polymixin B) during respective 24 and 48 hours for heart-beating and non heart-beating donors was classical decontamination protocol. We have compared the efficiency of different incubation periods as well as antibiotic cocktails (4 versus 3 antibiotics) in allograft decontamination. Methods: Donors were of three categories: living, heart-beating multiorgan donors (MOD) and recipients of heart transplantation (RHT) and non heart-beating (NHBD) or cadavers with age up to 65 years. Tissue preparation is performed in grade A laminar flow. First part of the study included 948 allografts from 541 donors, assessing decontamination efficiency depending on duration of incubation (below 30 versus above 30h). Second part inluded 80 donors during first step and 366 other donors during second step for allograft incubation in classical (4 antibiotics) versus modified cocktail (3 antibiotics). Bacteriological examination is carried out during dissection (a-sample), following the period of incubation (b-sample) and just before cryopreservation (c-sample). Examination for aerobe and anaerobe bacteria is carried out during 14 days at 37°C in two enriched culture media, according to the European Pharmacopoeia. For fungi and yeasts the medium was enriched with trypto-caseine and soja at 20-25°C during 14 days. Different data groups are statistically compared using Fisher’s exact test considering p value of <0.05 as significant. Results: Initial contamination rate during first part of the study was 36.4% (NHBD-78.1, MOD-36%, RHT-21.6%) with significantly higher incidence for NHBD than HBD group (p<0.001). Final sterility rate was 94.0% (MOD-95.4%, RHT-96.8%, NHBD-86.3%, p>0.5). Difference between contamination rate in the beginning and end stage of allograft processing was significant (p<0.001). Difference between duration of incubation and decontamination rate was not significant (p<0.1). During second part of the study, among 80 donor tissues of first step, 23.75% were initially contaminated (RHT-0.0%, MOD-21.66%, NHBD-33.3%). Tissues incubated in classical cocktail of antibiotics were sterile in 93.75% and those in modified cocktail 100% (p=0.058). During second step, initial contamination rate of group one was 25.54% and group two 30.77%. Final decontamination rate was 90.22% and 90.11% for group one and two, respectively (p=0.8). Conclusions: Initial tissue contamination was significantly higher among NHBD than HBD. Effectiveness of decontamination rate in the end of processing was significantly higher in all donor groups. However, highest final contamination rate was among NHBD group. Duration of incubation did not influence final sterility rate of allografts. Modified antibiotic cocktail was as efficient as classical one in allograft decontamination.
A New Physiological Role for Dopamine and its Transporter in the Pituitary: Induction of Prolactin Cells Apoptosis at Weaning. JAUBERT A^1, DRUTEL G^2, LESTE-LASSERRE T^2, ICHAS F^3, and BRESSON-BEPOLDIN L^3 ^1Division of Molecular Neuroendocrinology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, UK; ^2Physiopathologie de la plasticite neuronale, INSERM U862, Institut Europeen de Chimie et de Biologie, Universite Bordeaux 2, Pessac, France; ^3VINCO, INSERM U916, Institut Bergonié and Université Victor Segalen Bordeaux 2, Bordeaux, France. Background: In the rat pituitary gland, cessation of lactation causes a massive loss of prolactin (PRL) cells, eliminating the surplus of cells coming from previous proliferation occurring during pregnancy and lactation. The factors and mechanisms involved in this phenomenon were unknown, but our study provides the first evidences that dopamine (DA) plays a key role in this process. Results: We tested the pro-apoptotic effect of DA on pituitary primary cells from virgin, lactating, and post-lactating rats. By measuring several apoptotic markers as mitochondrial membrane potential loss, caspase-3 activation, and nuclear fragmentation, we show that DA induces apoptosis specifically in PRL cells from post-lactating rats. Surprisingly, although the DA receptor (D2R) expressed in PRL cells has been linked to inhibition of cell proliferation, and D2R agonists are used in therapy against pituitary tumors, the D2R is not involved in DA induced-apoptosis in our context. We then determined that this effect was mediated by the DA transporter (DAT), as revealed by a pharmacological study corroborated by detection of the DAT expression exclusively in PRL cells from post-lactating rats. In the same time, we also observed the expression of tyrosine hydroxylase (TH, the major enzyme of the DA synthesis) in post-lactating PRL cells which was accompanied by an increase in DA content in the AP gland of post-lactating as compared to virgin rats. Finally, we observed that cells expressing TH co-expressed DAT and cleaved caspase-3. Finally, we studied in a PRL cell line model (GH3 cells) the DA-induced apoptotic pathway and showed that, as described in the neuronal model, transported DA induces oxidative stress, leading to stimulation of pro-apoptotic proteins involved in the mitochondrial pathway (Bax, Cytochrome c) and finally to caspase activation. Conclusions: These findings show that DA may play an important role in lactotroph regression during the post-lactation period by inducing apoptosis. The fact that this process requires DAT and TH expression by lactotrophs themselves suggests that it may be “autocrine” in nature. This mechanism, already described in neuronal model of parkinson desease, could be the first physiological example of a regulatory expression of DA and DAT to specifically induce apoptosis. A better understanding of the mechanisms inducing these regulations could help us to explain pituitary tumors formation (particularly prolactinoma), no convincing explanation having never been caracterised so far.	Combined Targeting of IL6 and VEGF Potently Inhibits Glioma Growth and Invasiveness SAIDI A^1,2, HAGEDORN M^1,2, ALLAIN N^1,2, BIKFALVI A^1,2, JAVERZAT S^1,2 ^1 INSERM U920, ELAT (European Laboratory for Angiogenesis and Translational Research), Talence, F-33405, France; Univ Milan, Milan I-20122, Italy; ^2 Univ Bordeaux 1, Talence, F-33405, France Background: Interleukin-6 (IL6) and Vascular Endothelial Growth Factor (VEGF) are abundantly produced by glioma cells and contribute to malignancy by promoting angiogenesis-dependent proliferation and resistance to apoptosis. We compared the effect of RNA interference against IL6 and VEGF on the growth of experimental glioma. Methods: U87 human glioma cells with IL6, VEGF or IL6/VEGF knockdown were analyzed in vitro and implanted on the chick chorio-allantoic membrane (CAM). Tumor growth was monitored by biomicroscopy and immunohistology. Molecular response of tumor cells to single or combined treatment was studied by transcriptomic profiling. Results: In vitro, IL6 knockdown had no effect on proliferation but substantially enhanced invasion. In vivo, IL6 knockdown reduced growth and vascularization of the tumors but revealed tissue-invasion properties of the resistant tumor cells. By contrast, IL6/VEGF knockdown not only had a greater inhibition effect on the tumor mass and on angiogenesis but also prevented resistant cells to invade the host tissue. Interestingly, cell cycle promoting genes as well as modulators of blood vessel morphogenesis were specifically downregulated in the double knockdown, illustrating a synergistic effect of the combined treatment. Invasive behavior of tumor cells under IL6 inhibition was explicited at the molecular level with the induction of known marker genes (e.g. CYR61, IL6ST). Conclusions: Our results show that treatment of glioma with a combination of IL6 and VEGF inhibitors brings synergistic antitumoral benefit and reduces the risk of activating major pathways of cell survival, proliferation and invasiveness in remaining tumor cells that may be promoted by using IL6 inhibitors alone.
Impact of Cisplatin potentiation by Cytarabine in the 5-FU-CDDP regimen for dismal- prognosis head and neck cancer (HNC) patients; a meta-analysis of 3 local trials involving 492 patients. JELIC S, KREACIC M, URSLOVIC T, VUCICEVIC S, GAVRILOVIC D, JOVANOVIC N, BABOVIC N, PETROVIC Z, MIKIC A Institute for Oncology and Radiology of Serbia, Belgrade Further to our randomized study demonstrating response and survival benefit for Cytarabine (CAR) 1000 mg/m2 potentiating Cisplatin (CDDP) in the standard 5-FU-CDDP regimen (Eur J Cancer 2002) in dismal-prognosis HNC patients (unresectable T4 N2c-3 or relapsing or metastatic) two further studies were done. One compared potentiation with CAR 500 mg/m2 versus 1000 mg/m2; the other compared the CAR 500 mg/m2 with 5-FU administered as bolus versus continuous infusion (RR and OS were identical in both studies). The present report is a meta-analysis of the 3 trials with response and survival as main issues. The three studies included a total of 482 patients. Cohort 1 received the standard 5-FU-CDDP regimen (83 pts), Cohort 2 CAR-1000-5-FU-CDDP (153 pts) and Cohort 3 CAR-500-5-FU-CDDP (246 patients). All there regimens were applied both in palliative and neoadjutvant setting, the neoadjuvant preceding radiotherapy with 70 Gy. RR and PD rates were assessed on evaluable patient basis and survival on intend-to treat basis. Statistical analysis included the chi-square test, the log-rank test, determination of the death hazard radio and Cox regression analysis. Significance was assessed by the t-test with Bonferroni correction. The RRs were significantly higher in CAR-potentiated Cohorts (Cohort 1 44%, Cohort 2 62%, Cohort 3 66%, p=0,0031) and PD rates in the standard 5-FU-CDDP Cohort (Cohort 1 43%, Cohort 2 21%, Cohort 3 15%, p<0.001. The median survival in Cohort 1 was 7 months and, in Cohorts 2 and 3, 11 months. The one and two years survivals were for the Cohort 1 26% and 6%, for Cohort 2 42% and 14% and for the Cohort 3 44% and 24%. The difference in survival with the log rank test was highly in the favor of both CAR-potentiated Cohorts (p<0.0001) with the power of over 90% for p=0.01. Cox regression analysis showed that both performance status, primary tumor localization and treatment schedule were significant predictors of survival. The highest impact on survival had the administration of the CAR-potentiated regimens with a death hazard ratios of 0.58 an 0.53 (CI respectively 0.44-0.77 and 0.40-0.70) as compared to standard 5-FU-CDDP regimen. Potentiation of CDDP by CAR improves both RR and survival in dismal-prognosis HNC patients. The choice of the neoadjuvant regimen prior irradiation is crucial in judging its benefit impact in otherwise dismal-prognosis HNC patients.	Therapeutics: A New Control Strategy Using The Doses As Well As The Serum Concentrations To Optimize Learning About The Patient While Treating Him/Her At The Same Time BAYARD D^1,2, SCHUMITZKY A^1,3, JELLIFFE R^1 ^1Laboratory of Applied Pharmacokinetics, USC Keck School of Medicine, Los Angeles, CA, USA. ^2Guidance and Control Section, Jet Propulsion Laboratory, Pasadena CA, USA. ^3Department of Mathematics, USC, Los Angeles CA USA. Our laboratory has developed a new dual controller that combines, for the first time, particle filtering for nonlinear estimation with the Iteration-in-Policy Space (IPS) algorithm for approximating the Stochastic Dynamic Programming (SDP) equations of Bellman. Dual controllers optimally proportion their effort between controlling the patient, and actively probing the patient to extract useful information. This probing action is known to systematically improve controller performance compared to controllers that do not probe. Translated into clinical practice, dual controllers provide better drug regimen design and less variability in patient response. We tested the method first by applying it to a problem of controlling a model of a swinging pendulum. That model provides a non-trivial yet physically interpretable example. The model contains process noise (a child’s swing blows in the wind) and is controlled by pulling and pushing on it at allowable times (analogous to the doses). The controller goal is defined by having the pendulum achieve a prescribed excursion at a specified time. The problem is made difficult by taking both the length and mass of the pendulum to be unknown parameters, the sign of the control influence parameter (push or pull) to be unknown, and the pendulum position measurements to be in error (i.e., measurement noise). These complexities make the problem challenging, yet similar to the clinical problem of actively controlling a 1 or 2 compartmental aminoglycoside model. Results of a Monte Carlo study of the pendulum are shown in Table 1. The Heuristic Certainty Equivalence (HCE) controller, using Maximum Aposteriori Probability (MAP) Bayesian adaptive control, incurs an expected cost of 18.8 compared to 16.5 for the active 1-IPS(HCE) algorithm. This represents an improvement of 2.3 expected cost units. On the same example, the OLF policy using multiple model (MM) Bayesian control incurs a cost of 15.9 compared to the 14.9 for the 1 IPS(OLF) algorithm. CONTROLLER PERFORMANCE COMPARISON Control Law Expected Cost CPU Time (s) HCE 18.794 .05 1-IPS(HCE) 16.536 20 OLF 15.874 .05 1-IPS(OLF) 14.873 20