Ehrlich II –2nd World Conference on Magic Bullets

The Therapeutic “Cure” of Xenograft Tumors by the Third Generation Epothilone: Iso-oxazole-Fludelone CHOU TC Memorial Sloan-Kettering Cancer Center, New York, NY, USA 17-Iso-oxazole-26-F3-9,10-dehydro-12,13-desoxy-epothilone B (Iso-Flu or KOS-1803) is designed by diverted total synthesis based on pharmacological property. In vitro, it has an IC50 of 0.27nM against CCRF-CEM cells, which is 4-x more potent than paclitaxel (taxol). It is 638-x and 637-x more potent than taxol against drug-resistant CCRF-CEM/taxol and CCRF-CEM/vinblastine cells, respectively. It showed microtubule-stabilization activity similar to taxol. When compared with dEpoB and dehydelone, Iso-Flu is metabolically stable in mouse plasma when delivered as either a 6 hr-i.v. infusion or oral administration; has improved water solubility; has increased bioavailability and tissue penetration for prolonged tumor tissue drug retention; and demonstrates favorable pharmacokinetic and pharmacodynamic properties. The critical-optimal conditions for therapy and low toxicity are 6 hr-i.v. infusion at 25-30mg/kg every 14 days, 3x doses. In human xenograft tumors: for CCRF-CEM/taxol at optimal conditions, Iso-Flu led to 4/4 complete remission (CR) without any relapses for over 3 months, whereas taxol had no therapeutic effect, and cyclophosphamide (CTX) suppressed tumor without a CR. For CCRF-CEM xenograft, Iso-Flu, taxol and CTX all achieved CR, but 2/4 of CTX treated relapsed in one month. For the pancreatic Bx-CP-3 xenograft, Iso-Flu (25mg/kg, Q12Dx3, i.v. infusion) led to suppression and shrinkage but no CR, whereas taxol (20mg/kg, Q2Dx8, i.v.) and gemcitabine (40mg/kg, Q2Dx10, i.v.) suppressed tumor growth only 90% and 60%, respectively. For hepatoma Hep, Iso-Flu, (30mg/kg, Q16x2, i.v. infusion) led to growth suppression and shrinkage but no CR, whereas taxol (25mg/kg, Q2Dx4, i.v.) and CTX (50mg/kg, Q2Dx3, i.v.) and 5-fluorouracil (40mg/kg, QDx5, i.v.), led to only 15%, 55%, and 30% growth suppression, respectively. The superior data are in addition to early results for Iso-Flu against mammary MX-1 (i.v. infusion or oral), neuroblastoma SK-NAS, lung A549 and A549/taxol, ovarian SK-OV-3, mammary MCF-7/Adr xenografts over other chemotherapeutic agents [Chou, et al. Proc. AACR 47: 115, 2006; 48: 342, 2007, and Chou, et al. PNAS (in press)]. Thus, so far, we have not been able to find any other cancer therapeutic agent that is even approximate to Iso-Flu in overall therapeutic efficacy in xenograft experimental systems. Authors’ disclosure statement: The intellectual property rights for epothilones at Memorial Sloan-Kettering Cancer Center have been licensed to Kosan/Bristol-Myers Squibb Pharmaceuticals.

Deferoxamine and Defersirox as magic bullets against iron overload CHOUDHRY VP (M.D. FIAP, FIMSA, FISHTM, FIACM) Director, SUNFLAG PAHUJA CENTRE FOR BLOOD DISORDERS, Sunflag Hospital, Faridabad, Haryana-121002, India. Patients with anemias such as thalassemia, sickle cell disease, refractory anemias and myelodysplastic syndromes, aplastic anemia, requiring multiple blood transfusions for their survival develop hemosiderosis and/or hemochromatosis. The excess iron gets deposited in the liver, heart and endocrine organs leading to multiple organ failure. It is a major cause of morbidity and mortality in these patients. Iron overload is manifest as elevated liver iron concentration (LIC) and elevated serum ferritin levels. An increased risk of iron-induced cardiac disease has been observed when LIC values exceed 15 mg of iron per gram of dry weight (15 mg/Fe/g/dw), and serum ferritin values above 2500µg/L. Liver biopsy has been the “gold standard” for iron balance studies, but the technique is invasive procedure, expensive, and subject to variability within and between research subjects. Liver iron concentration (LIC) has been measured non-invasively by biomagnetic susceptometry using a low critical temperature (low-TC) superconducting quantum interference device (SQUID) biomagnetometer, which allows the measurement of the paramagnetic susceptibility of the iron stored in the liver as hemosiderin and ferritin. The results of biomagnetic susceptometry measurements of hepatic non-heme iron have been reported to be strongly correlated with those obtained by conventional analysis of liver biopsy. Another technique used is T2* imaging of heart. Low T2* values {<8 ms compared with (>20ms)} are related to risk of heart failure and death in iron-overloaded thalassemic patients. Bullets as Iron Chelators: Deferoxamine: It was the first chelator developed and has been used extensively in multiple disorders needing iron chelation. It is a hexadentate chelator, it binds iron tightly, and the iron-DFO complex is excreted in both urine and stool. More than 40 years of clinical experience with deferoxamine (Desferal, DFO) in iron-overloaded patients it reduces iron-related complications and thereby improves quality of life and overall survival, when administered in adequate doses over long periods. The standard regimen to remove excess iron is by subcutaneous (sc) infusions of DFO over 8-12 hours, on 5 to 7 days each week because of short plasma half-life. The DFO-iron chelate is charged and does not readily enter and leave cells. Parenteral administration with use of an infusion pump has been the major limitations. The studies revealed that the compliance decreased significantly with the increasing age. Deferasirox (Exjade®, ICL670) is an N-substituted bis-hydroxyphenyl-triazole was developed under a rational drug development program from over700 compounds. It represents a new class of tridentate iron chelators with a high specificity for iron with a plasma half-life of 8 to 16 hours. It is practical to administer the drug once a day orally and to maintain effective plasma levels. It is able to scavenge non-transferrin-bound “liable plasma iron, responsible for tissue damage”. Two molecules of the drug form a stable iron complex which is excreted in the feces. Iron is chelated, both from the reticuloendothelial cells (RE cells) as well as various parenchymal tissues. The chelated iron is cleared by the liver and excreted through the bile. ICL670 produced a linear dose-dependent rise in net iron excretion, with wide variation seen at 40 mg/Kg/day while variation was much less in the dose of 20 mg / Kg. It is highly selective for iron and does not induce the excretion of zinc or copper. Clinical Trials: In patients receiving 20 mg/Kg deferasirox the iron burden was essentially unchanged if the baseline LIC values were between 7 and ≤14mg Fe /g dw. While the patients receiving 30 mg/Kg the iron burden was reduced. In frequently transfused patients, defined as individuals receiving 2 to 4 units per month (or 7-14 ml/Kg/month) of packed RBCs, oral deferasirox once daily in dose of 20 mg/Kg led to maintenance of LIC, neutral iron balance, and stable serum ferritin levels. In another large phase 2 study, patients were randomized to receive once-daily deferasirox (10 or 20 mg / Kg; n=24 in both groups) or DFO (40 mg / Kg, 5 days / week; n=23) for 48 weeks. Decrease in liver iron concentration (LIC) were comparable in the deferasirox 20 mg/Kg/day and DFO groups; baseline values of 8.5 and 7.9 mg Fe / g dw fell to 6.6 and 5.9 mg Fe / g dw, respectively, by week 48. Hishet and his colleagues observed daily single dose of 20 mg/ Kg was well tolerated and was effective as an iron chelator. Most common adverse events with an apparent relationship to deferasirox were transient, gastrointestinal events were observed in 15.2% of patients that included abdominal pain, nausea and vomiting, diarrhea, and constipation. Skin rash was seen in 10.8% of patients. Deafness, neurosensory deafness, were observed infrequently. Mild transient dose dependent increase in serum creatinine was seen in nearly 1/3 of cases. There were no episodes of neutropenia, agranulocytosis or thrombocytopenia in any of the treatment groups. No adverse effects on growth or development in pediatric patients were observed. It should also facilitate patient compliance, a critical factor in effective patient management to maintain low iron burdens in patients requiring frequent blood transfusions. This drug meets all ideal properties of effective single oral iron chelators with a practical minimal side effects. Thus it serves as a sensational magic bullet of the century against hemosiderosis and hemochromatosis.

Nanomedicines - Self Nanoemulsifying Drug Delivery System (SNEDDS) and Nanosuspension for Oral and Parenteral Formulations in Cancer Therapy with Significant Impacts on Pharmacokinetics and Biodistributions CHOW DSL^1, GUPTA P^1,2, QI Y^1,3, LIANG D^4, WISNIECKI P^5, SHAH JC^5 ^1Univ. Houston, Houston, TX, USA; ^2Schering-Plough, Kenilworth, NJ, USA; ^3BioMarin, Novato, CA, USA; ^4Texas Southern Univ., Houston, TX, USA; ^5Pfizer Global Research and Development, Groton, CT, USA Background: Mebendazole (Mbz), a highly lipophilic new anti-neoplastic agent (aq. Solubility: 0.7 μg/ml), is challenging for pharmaceutical formulation development. Aims: 1) To develop SNEDDS and Nanosuspension (NS) for oral and parenteral administrations in cancer therapy. 2) To characterize their pharmacokinetics (PK) and biodistributions (BD). 3) To establish the impacts of droplet/particle size on PK and BD of Mbz. Methods: For SNEDDS, systematic approaches in excipient selections and SNEDDS-based nanoemulsion (NE) region identifications, using ternary phase diagram enabled the effective formulation optimizations. NS of sizes of 128-167, 250-253, 739-891 and 1552-1781 nm, were prepared using milling technique. The PK and BD of the nanoformulations were comparatively characterized in Sprague Dawley rat and Swiss athymic nu-mouse models. Results: The SNEDDS-based NE of 35 and 143 nm significantly improved the oral bioavailability of Mbz, 228 and 120 times of that of unformulated suspension, respectively. With parenteral administrations of the NE, Mbz yielded prolonged half-life, 644 min vs 173 min, and sustained exposures in organs, especially in the lung, AUC of 12.4 vs 1.8 (hr*ug/g)/(mg/kg), from the cosolvent reference (CS). Droplet sizes (35 and 478 nm) impacted PK and BD, with an even higher concentration and a longer retention half-life with 35 nm than 478 nm NE. The Mbz from NS exhibited prolonged half-lives, 13-30 hr vs 3 hr of CS. The Vss were significantly larger, 1.19-1.69 L vs 0.06 L of CS, and peripheral V2 were substantially larger, 24-49 L vs 0.4 L of CS. The size impacts were also demonstrated. Conclusions: 1) SNEDDS of Mbz with droplet sizes of 35 – 478 nm were formulated for oral and parenteral administrations. 2) Nanosuspensions of Mbz with 4 particle sizes ranging from 128 to 1781 nm were developed. 3) Greater tissue distributions and slower in vivo dissolution of NS were key parameters responsible for the size-dependent PK distinction of Mbz. 4) Both nanoformulations offer potential merits of sustained and targeted cancer therapies, in lung and liver.

Akt Inhibitors: A New Strategy Targeting Long-Living HIV Macrophage Reservoirs CHUGH P, RIVERA-PABON O, KIM B University of Rochester, Rochester, NY, USA Background: The infection of CD4+ T cells with Human Immunodeficiency Virus (HIV-1) is well-studied and typically leads to virally-induced cytolysis. In contrast, infected macrophages are able to uniquely evade the apoptotic effects of HIV-1 infection. These cells become infected early on and persist throughout viral pathogenesis. In addition, brain macrophages, or microglia, are thought to significantly contribute to the development of HIV-1 associated neurological problems. Since these cells seem to play a key role in HIV-1 pathogenesis, the study of the interactions between macrophages and HIV-1 is vital to understanding their role as persistent viral reservoirs. Here, the pro-survival effect of HIV-1 is presented as a mechanism for establishing these cells as viral reservoirs. Methods: Primary human macrophages were infected with HIV-1 M-tropic YU-2 virus or a pseudotyped vector system HIV-GFP and used to perform experiments pertaining to cell survival and the role of PI3K signaling pathway. Miltefosine along with related PI3K/Akt inhibitors were used to antagonize the effects of HIV. Viral production and cell death were monitored. Results: We previously found that HIV-1 infection and the expression of HIV-1 Tat protein were able to induce a pro-survival effect in primary human macrophages. Subsequent studies showed that these pro-survival effects could be attributed to the activation of the Akt survival pathway. A series of mechanistic studies further revealed that the modulation of the Akt pathway was key in establishing the extended survival phenotype in these cells following infection. Specifically, we observed a reduction in PTEN protein levels and a significant increase in Akt activity. Based on these data, the role of Akt inhibitors as an anti-HIV therapy was tested. Interestingly, Akt inhibitors and specifically Miltefosine were found to block the cytoprotective effect of HIV-1 infection and Tat expression in primary human macrophages. Furthermore, a drastic decrease in viral production was observed in HIV-1 infected macrophages following treatment with Miltefosine and related PI3K/Akt inhibitors. Conclusions: Collectively, these data suggest that Akt inhibitors like Miltefosine may provide a means of targeting long-lived viral reservoirs and may offer further insight into novel therapeutic targets for anti-HIV therapy without concerns of viral resistance.

The urokinase system is a natural inducer of cancer cell drug resistance CHUN MH, HOFFMANN MK ApoTech, Seocho gu, Banpo dong, Gu-ganpo 106-505, Seoul Korea Background: Cell-bound urokinase converts serum plasminogen to plasmin. High membrane urokinase expression is associated with enhanced cancer cell metastasis formation and poor disease prognosis. Experiments were conducted to explore the relationship between urokinase activity and cancer cell drug sensitivity. Methods: MCF7 Human breast cancer cells were cultured for 2 days in the presence or absence of plasmin, and subsequently exposed for additional two days to doxorubicin, paclitaxel, or tumor necrosis factor. Cell survival was determined. Results: After culturing in the presence of plasminogen-containing human serum, 50% MCF7 cells were killed with 15 ng/ml doxorubicin. When cells were cultured in the presence of plasminogen-depleted human serum and thus blocked urokinase activity, as little as 5 ng/ml had an equal cytotoxic effect. Addition of exogenous plasmin to plasminogen-depleted culture restored the drug resistance of cancer cells. Blocking cancer cell’s urokinase activity by anti-uPA antibody enhanced the susceptibility to doxorubicin toxicity. The life preserving effect of plasmin is demonstrated to be mediated by the insulin-like growth factor (IGF). IGF is known to induce drug resistance in cancer cells. Blockage the IGF pathway abrogated the ability of plasmin to render cancer cells drug resistant. Other apoptosis-inducing cancer drugs were tested in the presence or in the absence of plasmin. Plasmin-dependent drug resistance was observed in each case. Conclusion: 1. Cancer cells utilize the urokinase system to acquire drug resistance. 2. Drug sensitivity is restored when the urokinase system is inhibited 3. Insulin-like growth factor (IGF) is identified as a mediator of this plasmin effect.

Magnetic Alginate Nanospheres: a Novel Vector for Targeted Drug Delivery CIOFANI G^1, RAFFA V^1, MENCIASSI A^1,2, MICERA S^1, CUSCHIERI A^1 ^1Scuola Superiore Sant’Anna, Pisa, Italy; ^2IIT Network, Genova; Italy. Background: The explosive growth of nanotechnology in the last years has produces dramatic innovations in pharmacology. Precise targeting of drugs to diseased cells or locations within organs is considered the “magic bullet” in medical therapy, but it is not yet achieved by current drug delivery methods. Magnetic materials have been proposed for biomedical purposes to a large extent for several years. In this paper, the development, characterization, and in vitro test of alginate nanoparticles, embedding magnetite, which responds to externally applied magnetic fields, are presented. Methods: Magnetite powder was obtained reacting iron(II) and iron(III) ions in an aqueous ammonia solution. Magnetite is then isolated from the reaction suspension and completely dissolved in an alginate solution. Alginate magnetic particles were realized by a homogenization process and reticulation with calcium ions. Such microparticles were characterized in terms of external morphology, size distribution, zeta potential, magnetic properties and drug release behaviour. In vitro testing was performed with NIH/3T3 and PC12 cells. Results: Concerning the magnetic properties, magnetization curves show the typical trend of superparamagnetic materials. Important parameters, such as magnetic permeability (μr = 12.3) and magnetic momentum (μ = 2.25∙10^-25 A∙m^2), were derived by employing Langevin theory. A drug release of about 5-6 days was assessed usig albumin as protein model. Nerve growth factor (NGF) loaded nanoparticles were tested on PC12 cells. Cells showed neuronal phenotype and developed neuritis with length strictly dependent on the distance from the nanospheres, fixed in a region of the Petri dish thanks to an external magnetic field. Finally, dynamic colture of NIH/3T3 cells incubated with fluorescein loaded magnetic nanoparticles definitely demonstrated the possibility to guide these nanovectors through a circuit simulation blood circulation. Conclusions: 1) Alginate nanoparticles with a core of magnetite and filled with specific drugs were realized and fully characterized. 2) Magnetic nanospheres loaded with neurotrophic factors were able to trigger cell differentiation strictly depending on the distance of the release source. 3) The proposed system was successfully tested on dynamic cultures simulating blood circulation.

Treatment and relocation of wheat flour sensitized workers by an allergenic vaccine CIRLA AM, LORENZINI RA, CIRLA PE Italian Center Environmental Occupational Health (CIMAL), Milan-Cremona, Italy Background: Proteins binding IgE are natural components of wheat flour (WF) and the inhalation of particles during working is a major cause of WF occupational allergy (WOA) for bakers, pastry-makers or pizza-makers with workplace-related respiratory symptoms. They develop rhinitis and subsequently asthma with unfavourable consequences on quality of life, often being forced to give up their jobs and to claime compensation. Although drugs treatment could be useful for limited periods, attempts to realize a desensitization by continous administration of incremental doses of an allergen-specific vaccine (AV) were carried out in the clinical practice of recent years. An AV containing the most relevant fraction for WOA (water/salt soluble albumins and globulins) and standards related to this subcutaneous immunotherapy (SIT) are the remarks of the author’s experience, whose evidence is proposed. Methods: 43 subjects were AV-treated (39 M, 4 F, mean age 39 yr, range 23-67); bakers 27, pastry makers 13, pizza makers 4. All had a diagnosis of WOA with rhinitis and 60% suffered also of persistent mild asthma (prick test and specific-IgE positive, occupational challenge confirmed). WF-AV (Lofarma Laboratories, Milan) was characterized by SDS-PAGE and immunoblot; content of 14-17, 36 and 50 Kd proteins as major allergens was documented. The product was a retard preparation in aluminium hydroxide; an in-house reference standard (RAST Units, RU) and quality control were developed. SIT was always performed by the same single-patient management plan, without stopping the work, basing on a slow induction schedule of 4 months and a montly maintainance with 8000 RU for 4-7 yrs. Subjects were retrospectively interviewed after SIT by questionnaire and data were registered about symptoms, SIT-compliance, job conditions, global efficacy. Results: SIT was tolerated without adverse effects; only 7 cases reported local reactions during starting months of SIT. Above all 34 subjects keepped up regularly his own job, 16 of them being at work after WF-SIT was discontinued from several yrs. Nobody claimed persistent asthma, while many sneezed again sometimes during work in the last year, but feeling rarely poor nasal occlusion or runny nose (see table). Only 9 subjects changed the activity at risk, because of better engagements elapsed or being retired for ageing; nobody for worsening of symptoms. The immunological paramethers (reduction of cell reactivity and of wheat-specific IgE) are reported elsewere. Conclusions: 1) WOA may be treated by SIT with an AV. 2) A WF-AV was proved to be effective by a standardized protocol, duration at least 4 years. 3) Giving up a qualified job by bakers and similar exposed ones may be prevented, with economic and social advantages. 4) Compliance of SIT reduces the sensitization, improves symptoms and promote the work relocation in most of cases.

Management of Deep Space Neck Infection. Five Year Experience. ČIZMAREVIČ B, DEBEVC D, LEVART P Univ. Clinical Center Maribor, Department for otorhinolaryngology and maxillofacial surgery, Maribor, Slovenia. Background: Deep neck space infections present as deep neck abscess or necrotizing fasciitis.They have severe potential for complication which can be life threatening. Aims: To find out predictive factors for more fulminant progression of infection. Methods: We retrospectively analyzed medical data for patients surgically treated for deep neck space infection between 2002 and 2006. Our group of patients consists of 79 male and 53 female. We divided them in two groups: deep neck abscess and necrotizing fasciitis. Results: 115 patients had deep neck abscess and 17 necrotizing fasciitis. Infection started with nondental origin (angina, epiglotitis, infected haemathoma) in 8 patients in first group and in 6 patients in second group. Among comorbidities Diabetes mellitus was the most common, others were prolonged treatment with corticosteroids, cirrhosis and chemotherapy. Conclusions: Dental infections are the most common cause of deep neck abscesses and necrotizing fasciitis. Nondental origin is more likely to cause fulminant infection and necrotizing fasciitis. Diabetes mellitus was the most common comorbidity.

Zolpidem after brain Damage CLAUSS RP^1, NEL HW^2 ^1Royal Surrey County Hospital, Guildford, Surrey, UK ^2Family Practice, Springs, South Africa 11 January 2009 marks the tenth anniversary of the awakening of Louis from his state of impaired consciousness 30 minutes after swallowing 10mg Zolpidem. In the past decade, Zolpidem has been effective in a multitude of patients with brain damage ranging from birth injury to traumatic brain injury to stroke and others. SPECT and PET studies in these patients show reactivation of brain metabolism in quiescent brain regions after injury, designated functional neurodormancy. MEG studies show that these suppressed neurodormant brain areas have a characteristic slow wave magnetic rhythm that normalises after Zolpidem, but not after placebo or other sleeping drugs such as Zopiclone. Neurodormancy arises as a physiological thread that is present in a multitude of unrelated brain pathologies, possibly due to a basic physiological protection mechanism that is initiated after brain damage. It forms a target for Zolpidem which re- activates neurodormant tissue and normalises clinical features that occur because of the suppressed neurodormant brain.

New Applications for Micro- and Nanoscaled Drug Delivery Systems COESTER C Ludwig Maximilians University, Munich, Germany Our group comprises four members located in the Pharmaceutical Technology of the Department Pharmacy, LMU-Munich, headed by Mr. Gerhard Winter. We are concerned with the pharmaceutical development of colloidal carriers for the targeted gene and tumor therapy by delivery of siRNA, RNA, plasmid DNA, and classical chemotherapeutics. The idea behind is to use simple and straight forward formulation approaches, which are easy to implement on a larger scale e.g. for the industrial production. Our colloidal carriers are produced solely out of biocompatible, biodegradable and low toxic materials like phospholipids, porcine gelatin and chemically modified derivatives of them. Besides our focus for advanced pharmaceutical quality, we chase for a high therapeutical efficacy in numerous clinical studies. Our research is based on proper inventions and is one of the worldwide leading groups in the field of gelatin nanoparticles. The group’s focus here is on high-end polymer and particle analysis with asymmetrical flow field flow fractionation (AF 4), novel in-vitro simulation models and the use of fluorescent or radioactive labeled nanoparticles for whole body in-vivo imaging. For several years the group holds a strong knowledge in the field of immunity based tumor therapy with CpG oligonucleotides delivered by nanoparticles made of gelatin and other natural polymers. A new matter of our group is the knockdown of disease - related genes by siRNA. This is anticipated to gain momentum with the aid of our newly established cell culture facility. A further challenge we have recently taken is the development of acoustically active gas-filled microparticles, also called microbubbles. Microbubbles are known for over 15 years as contrast agents for the ultrasound diagnostic imaging. Recently, they have also proven promising carriers for drug and genes. After being injected, microbubbles can be visualized on the target site by using diagnostic ultrasound. Following, by increasing ultrasound intensity they can be destroyed and release their therapeutic load. Our research on microbubble carriers diverges in the fields of targeted therapy of solid tumors and targeted gene therapy.

The ADAM9 Disintegrin Domain (ADAM9D) Inhibits Platelet and Tumor Cell Adhesion to Subendothelial Matrix under Dynamic Flow Conditions COMINETTI MR^1, MARTIN ACBM^1, RIBEIRO JU^1, FAUVEL-LAFÈVE F^2, CRÉPIN M^2, SELISTRE-DE-ARAUJO, HS^1 ^1Federal University of São Carlos, SP, Brazil; ^2INSERM Unit 553, Paris, France. Background: Members of the ADAM (A Disintegrin And Metallopeptidase) protein family are composed by a series of conserved protein domains including a disintegrin domain, which interacts with cell surface integrins. Adhesion to and extravasation through the endothelial lining of blood vessels is critical for tumor cells to establish metastasis. The objective of this work was to verify the effects of the disintegrin domain of ADAM9 (ADAM9D) on the adhesion of tumor cells (MDA-MB-231) and platelets to collagen type I, under flow to simulate shear conditions found in the circulation. Methods: The recombinant ADAM9D was produced by cloning into a pGEX-4T-1 vector which was used transform E. coli AD494(DE3) cells. The synthesis of GST/ADAM9D was induced by IPTG (0.1mM, 4h). After purification on a Glutathione-Sepharose 4B resin, the ADAM9D was released from GST by cleavage with thrombin and further purified in a Benzamidine-Sepharose 4B column. MDA-MB-231 breast tumor cells labeled with cell tracker red were previously incubated with ADAM9D (5µM) or PBS (control) and then mixed with whole blood prepared labeled with calcein green. The mixture was perfused at a shear rate of 1500sec^-1 in a flow chamber on a collagen type I-coated coverslip. Adhered platelets and cells in each field were differentially counted using the software Image J. The results were statistically compared with a two-way analysis of variance (ANOVA), followed by Bonferroni’s significant difference post hoc analysis. Results: Recombinant ADAM9D was able to inhibit about 50% of breast tumor cells and platelet adhesion to collagen type I, under flow conditions. Conclusions: ADAM9D can be used as a tool for investigating the role of ADAMs in metastasis and cancer progression and for the design of selective inhibitors against the adhesion and extravasation of cancer cells. Financial support: FAPESP, CNPq, INSERM.

In situ Photopolymerized Coatings for pH-Specific Drug Delivery from Pellets CONCHEIRO A, MAYO-PEDROSA M Univ. Santiago de Compostela, Santiago de Compostela, Spain. Background: Coating of drug-containing pellets enables the delivery to be sustained or site-specific. As an alternative to the traditional coating procedure consisting of deposition of successive layers of preformed polymers, tailored coatings can be obtained by photopolymerization/ cross-linking of acrylic monomers on the pellet surface. Particularly, we have applied this new approach for preparing hydrogel coatings able to provide theophylline pellets with pH-dependent release rate. Methods: Pellets of theophylline (20%), powdered cellulose (60%) and poly(vinylpyrrolidone) (20%) were obtained by extrusion-spheronization and then placed on a tray adapted to a vortex stirrer, pulverized with solutions of cross-linker and acrylic acid (AA) or AA:lauryl acrylate (LA) 88:12 molar ratio in ethanol:water medium and immediately irradiated with 366 nm UV-light. The coating process involved several sessions of pulverization and irradiation. Theophylline release was tested in 900 ml of 0.1N HCl or pH 7.4 phosphate buffer at 37ºC. Results: The new photopolymerization process was particularly adequate for the coating of pellets since enabled: i) an easy and homogenous spraying of the monomeric solution on the pellets, ii) a quasi-instantaneous polymerization, and iii) a deep-thorough cure resulting in the formation of a well-structured network, of a high consistency but still flexible. No significant increase in pellet size (ca. 1 mm) was observed after coating. Non-coated pellets rapidly disintegrated and released all drug in few minutes. When only AA was used for the coating, the release process was sustained for 4 hours in 0.1 M HCl and finished in 2 hours in pH 7.4 medium. By contrast, coating with AA:LA mixtures and a cross-linker notably hindered the release at acid pH (only 50% released at 4 hours), without significantly compromising the fast delivery at neutral pH. Conclusions: Photopolymerization/cross-linking of AA:LA on pellet surface enables site specific delivery as a function of pH and opens a wide range of possibilities for preparing tailored coatings adapted to specific triggering pH values. The coating conditions used in the present work could be easily adapted to industrial scale. Authors disclosure statement: Some information described in this abstract is the subject of patent applications filed by the University of Santiago de Compostela (ES 200600757).

Role of Drug Metabolism in the Development of Eplerenone (EP): A Lesson Learned from Spironolactone (SP). COOK CS^1, BERRY LM^2ZHANG L^3 ^1Baxter Healthcare, Round Lake IL, USA, ^2Amgen Inc Cambridge MA, USA, ^3Pfizer Inc, Groton CT, USA Background: EP is a highly selective aldosterone blocker approved in the US for the treatment of hypertension and heart failure. During development of the drug, extensive metabolism studies were necessary since another competitive aldosterone antagonist, potassium canrenoate (PC), that is structurally related to EP, was shown to be metabolized to potent mutagenic metabolites which appear to be associated with myelogenous leukemia. PC is a potassium salt of canrenoic acid formed from SP. However, SP did not produce these mutagenic metabolites because a thiol metabolite of SP inhibited formation of mutagenic metabolites from canrenoate. Methods: In vitro metabolism studies of EP were carried out using human liver microsomes and cDNA-expressed CYP450 isozymes. Major metabolites were isolated and identified using MS and NMR. Inhibition of metabolism in human liver microsomes was examined in the presence of various chemical inhibitors at a concentration of 10 M and with human CYP450 antibodies. Urinary and fecal metabolites were identified following oral administrating of [^14C]EP to healthy subjects at dose of 100 mg/person. Results: Metabolic pathways of EP were 6- and/or 21-hydroxylation by CYP3A4/5 and 3-keto reduction. The major metabolite identified was 6-OH EP. There was no evidence for any alteration of the 9,11-epoxide ring or carboxy methyl ester. In contrast to canrenoate metabolism, no 6,7-epoxy metabolite, a precursor to mutagenic metabolites, was found with EP. Conclusions: 1) Major metabolic pathway of EP was hydroxylation by CYP3A4/5. 2) Unlike PC, there was no 6,7-epoxide metabolite formed with EP. 3) Demonstration of stability of 9,11-epoxide ring and absence of 6,7-epoxy metabolite was essential in the development of EP.

Is there a magic bullet for prostate cancer? COREY E^1, KORECKIJ T^1, MORRISSEY C^1, VESSELLA RL^1,2 ^1University of Washington, Seattle, WA, USA ^2Veteran Administration, Seattle, WA, USA Background: Patients with advanced prostate cancer (CaP) frequently suffer from significant morbidity secondary to bony metastases. Therapeutic modalities that would eliminate or slow the growth of metastatic CaP and improve quality of life are of great interest. Although current docetaxel-based regimens offer superior survival advantage over previous standard therapies for men with castration-resistant metastatic CaP, this improvement resulted in an overall survival of only 18-19 months. Novel therapies with greater efficacy, either alone or in combination with docetaxel, are needed to continue to move the field forward to benefit afflicted men. The objectives of our studies were to characterize CaP metastases and test new treatment modalities. Methods: Metastases of prostate cancer were obtained from patients who had died from advanced CaP and had consented to be part of the rapid autopsy program. There are two prevalent ways of selecting agents for combination therapy: utilize compounds that act by clearly distinct mechanisms (e.g. affecting either the tumor or microenvironment) or select compounds that affect different points in a specific signaling pathway critical to cancer progression. In preclinical testing, a model of experimental bone metastases was used whereby CaP tumor cells were injected directly in to tibiae of SCID mice. Animals were treated with various agents including docetaxel, zoledronic acid, RAD001, and the effects on tumor growth and bone remodeling were monitored. Results: Our results clearly show that metastases of prostate cancer are phenotypically heterogeneous not only between different patients but also within a single patient. Our preclinical studies showed that while multiple agents are able to slow the growth of CaP in the bone, no single agent resulted in eradication of the tumor. Our further efforts have demonstrated that combinations of various agents inhibit the growth on CaP in the bone more than any single agent alone. Conclusions: 1) CaP is an extremely heterogeneous disease; 2) Significant heterogeneity of CaP tumors makes treatment with a single agent unlikely to result in prolonged clinical responses; 3) Efficacy of drugs is affected by the site of metastases; 4) None of the tested agents was capable of eradicating the disease; and 5) Agents used in combination had more pronounced inhibitory effects.

Corticotrophin releasing factor as drug target for modifying dopaminergic system neuroplasticity in cocaine addiction COROMINAS M^1, RONCERO C^1, CASTELLS X^2, CASAS M^1. ^1Department of Psychiatry, Vall d'Hebron University Hospital, Barcelona, Spain; ^2Department of Clinical Pharmacology, Vall d'Hebron University Hospital, Barcelona, Spain. Background: Cumulative evidence has demonstrated that stress plays a crucial role in cocaine addiction by enhancing the rewarding properties of the drug and inducing relapse during withdrawal. Corticotropin Releasing Factor (CRF) is one of the major effectors of stress in different structures of the reward circuitry, such the ventral tegmental area (VTA) and the amygdala. Aims: This paper explores the pattern of interaction between CRF, dopamine (DA) and glutamate (GLU) in different structures of the mesocorticolimbic circuitry during cocaine consumption. Understanding the neurochemical and cellular mechanisms involved in these processes would be useful in the development of new pharmacological strategies for the treatment of cocaine addiction. Methods: A systematic literature review was conducted of animal and human research on the effects of stress through the CRF system. The interaction between CRF, dopamine and glutamate in VTA and the amygdala during repeated cocaine consumption and withdrawal were the focus of this review. Results: In the VTA, stress-induced increase of CRF potentiates the activity of GLU transmission, which in turn increases the excitability of the DA system in cocaine-experienced animals. Blockade of CRF-R2 prevents stress-induced increase of synaptic plasticity in the DA system and relapse during withdrawal. In the amygdala, during early and late cocaine withdrawal, there is a progressive increase of CRF release that coincides temporally with behavioral anxiety during cocaine withdrawal and contributes to relapse. This increase in CRF activity potentiates GLU transmission, long-term potentiation, and synaptic plasticity. Selective antagonists of CRF-R1 are useful for preventing anxiety during withdrawal and reinstatement of cocaine seeking. Conclusions: 1) Both CRF-R1 and CRF-R2 inhibitors may be useful for preventing withdrawal symptoms and relapse in cocaine addicts. 2) Bearing in mind the interactions between CRF, glutamate and DA in the different structures of the mesocortilimbic system, the use of combined pharmacological strategies involving all these neurotransmitters should be considered in the treatment of cocaine addiction.

BDNF and Its Intracellular Signaling Pathways as Drug Targets in Addiction COROMINAS M^1, RONCERO C^1, RIBASES M^1, CASTELLS X^2, CASAS M^1. ^1Department of Psychiatry, Vall d'Hebron University Hospital, Barcelona, Spain; ^2Department of Clinical Pharmacology, Vall d'Hebron University Hospital, Barcelona, Spain. Background: Craving and relapse, the most challenging aspects in the treatment of cocaine addiction, still lack an effective pharmacological treatment. Aims: This review examines the evidence supporting the involvement of brain-derived neurotrophic factor (BDNF) and its intracellular signaling pathways, mitogen-activated protein kinase (MAPK/ERK) and phosphatidylinositol 3-kinase (PI3-K), in the cerebral neuroplastic mechanisms in different structures of the mesocorticolimbic dopamine system underlying psychostimulant abuse and dependence, and also explores the possibility of using pharmacological inhibitors of these intracellular pathways as a new strategy to prevent cocaine craving and relapse during withdrawal. Methods: A systematic literature review was conducted of animal and human research on BDNF and its intracellular pathways in acute and repeated cocaine consumption and withdrawal. Results: Animal research demonstrates the involvement of BDNF, ERK and PI3-K in cocaine addiction. Repeated cocaine exposure and withdrawal induce an increase of BDNF and ERK in different cerebral areas, including the ventral tegmental area, nucleus accumbens (NAc) core and shell, amygdala and hippocampus. After repeated cocaine administration there is also a PI3-K up-regulation and a down-regulation in the NAc and prefrontal cortex, respectively. These molecular changes are associated with experimental measures of cocaine addiction, craving and relapse during withdrawal. Pharmacological blockade of the ERK a) prevents the acquisition of behavioral sensitization (increase of some behavioral responses), b) abolishes the association of environmental cues to cocaine, and c) decreases cocaine seeking during withdrawal. Pharmacological PI3-K inhibition after repeated cocaine prevents the expression of behavioral sensitization. Conclusions: 1) BDNF modulates synaptic plasticity in cocaine addiction, especially during withdrawal, contributing to the transition from sporadic cocaine consumption to addiction and relapse. 2) ERK and PI3-K cascade inhibitors are potential therapeutic strategies to be further investigated in the context of addiction.