Ehrlich II –2nd World Conference on Magic Bullets

Signal Transduction Therapy Of Cancer
LEVITZKI A
Unit of Cellular Signaling, Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel
Background: Signal transduction therapy was introduced into cancer therapeutics in the early 1990s after the first tyrphostins and anti-Her2 antibodies were developed. Currently there are a few antibodies like Herceptin, Avastin and Erbitux in the clinic as well as eight tyrosine phosphorylation inhibitors.

Methods: The design and synthesis of novel protein kinase inhibitors that are ATP non-competitive inhibitors will be presented. We shall demonstrate the successful use of a combination of an EGFR kinase inhibitor with our novel PKB/Akt inhibitor and a DNA damaging agent to strongly inhibit glioblastoma, which overexpreses EGFR and lacks PTEN. We shall also present the design, synthesis and performance of a chemical vector homing for the EGF receptor. The vector is loaded with PolyIC and due to the ability of the EGFR to internalize the vector; PolyIC is inserted into the cell, eliciting a strong anti-tumor response. Here we shall specifically discuss an EGFR homing vector carrying PolyIC.

Results: In view of the success of targeted cancer therapy since the early studies on tyrphostins (1988) we have enhanced our efforts to improve the performance of targeted therapies by following new paradigms. In this talk I will present three ongoing studies in our laboratory (1) the development of novel substrate competitive PKB/Akt inhibitors and how they perform in the treatment of prostate cancer and brain cancer in nude mice. (2) The development of novel allosteric IGF1R inhibitors that lead to the degradation of the IRS proteins and their highly potent in vivo efficacy against disseminated ovary cancer, breast cancer, and prostate cancer in nude mice and (3) the eradication of EGFR over-expressing disseminated tumors by EGFR targeted long chain dsRNA (PolyIC). This approach is highly effective due to the targeted “bystander” effect induced by the PolyIC that is inserted selectively into the EGFR over-expressing tumor cells. This is by far the most effective signal transduction therapy regimen in the treatment of EGFR over-expressing tumors, in experimental animals.

Conclusions: (1) Non-ATP competitive protein kinase inhibitors are highly effective agents and may be used with success to treat various cancers. (2) A chemical vector homing to a receptor, which is over-expressed in tumors and which is capable of internalization can induce complete eradication of the tumor if loaded with PolyIC. This approach can be in principle be utilized with many receptors as a target, where the example given here is an EGFR homing vector.

Molecular Targeting of the bcl-2 Oncogene for Staging and Therapy of B-Cell Lymphoma LEWIS MR^1, JIA F^2, BALKIN ER^2, DAIBES FIGUEROA S^1, BALAJI BS^2, GALLAZZI F^3, STATHAM KA^2, HOFFMAN TJ^1 ^1Research, Harry S Truman Memorial Veterans’ Hospital, Columbia, MO, USA; ^2Veterinary Medicine and Surgery, ^3Molecular Biology, University of Missouri, Columbia, MO, USA. Background: The B-cell lymphoma/leukemia-2 (bcl-2) oncogene is a dominant inhibitor of apoptosis, correlating with resistance to radiation and chemotherapy, high relapse rate, and poor survival in non-Hodgkin’s B-cell lymphoma (NHL). NHL also expresses type 2 somatostatin receptors (SSTR2) in 87% of cases, making it attractive for delivery of intracellular tumor-targeting agents. Methods: A bcl-2 antisense peptide nucleic acid (PNA) conjugated to a SSTR2-targeting peptide, anti-bcl-2-Tyr^3-octreotate, was evaluated for ^111In gamma scintigraphy and single photon emission computed tomography (SPECT), ^64Cu positron emission tomography (PET), and ^177Lu targeted radiotherapy (TRT) in NHL cells in culture, mouse models of human NHL, and dogs with spontaneously occurring NHL. SCID mice bearing bcl-2 mRNA-positive Mec-1 (n = 3) or bcl-2 mRNA-negative Ramos (n = 3) xenografts were used for ^111In microSPECT or ^64Cu microPET imaging. The ^111In conjugate was also used for gamma scintigraphy of canine NHL patients (n = 15). The ^177Lu conjugate was evaluated in vitro for TRT in Mec-1 cells (n = 3). Results: Incubation of Mec-1 cells with anti-bcl-2-Tyr^3-octreotate showed a 51% decrease in bcl-2 protein synthesis, suggesting that the target mRNA function had been perturbed by a specific antisense effect. Both ^111In microSPECT and ^64Cu microPET could detect Mec-1 tumors, but not Ramos tumors, in SCID mice (p < 0.05). Gamma scintigraphy demonstrated the utility of the ^111In conjugate for molecular staging of bcl-2 in canine NHL. In vitro Mec-1 cell studies showed that the ^177Lu conjugate had at least an additive effect on cell viability, compared to controls for targeted radioactivity and bcl-2 antisense activity. Conclusions: 1) Imaging studies demonstrated that ^111In- and ^64Cu-anti-bcl-2-Tyr^3-octreotate were specific for bcl-2 mRNA-positive NHL xenografts. 2) Imaging of canine NHL established bcl-2 expression as a clinical and molecular model relevant to human disease. 3) TRT studies of the ^177Lu conjugate in Mec-1 cells demonstrated down-regulation of bcl-2 with radiation insult, creating a NHL therapy agent acting through two targeted anti-tumor mechanisms.	Development of Novel -Conopeptide Inhibitors of the Norepinephrine Transporter for the Treatment of Severe Pain LEWIS RJ^1, BRUST A^1, PALANT E^1, COLLESS B^1, SCHMIDT P^1,DRINKWATER R^1, GIBBONS S^1, MARTIN W^2 ^1Xenome Ltd, Brisbane, Queensland, Australia; ^2Xenome Inc, San Diego, CA, USA Background: Venoms are a valuable source of novel bioactive peptides. The venom of ~500 species of cone snails contain 500,000+ small bioactive peptides (venom peptides) that target specific ion channels, transporters and receptors. A number of classes of conotoxins have emerged with therapeutic potential in the treatment of neuropathic and inflammatory pain states, including the -conotoxins that inhibit the norepinephrine transporter. The aim of the present study was to evaluate a series of -conotoxins for their ability to inhibit the norepinephrine transporter (NET) and reverse pain behaviors in a rat model of neuropathic pain. Methods: -Conopeptide analogues were assembled by solid phase peptide synthesis and were assessed for their ability to inhibit [^3H]-NE uptake through human NET. Selected analogues were further evaluated for target specificity and ability to reverse allodynia in a rat model of neuropathic pain when delivered intrathecally. Results: Over 300 -conotoxin analogues were synthesized. Their potency to inhibit NE uptake ranged from >80-fold enhanced to >1000-fold reduced compared with the parent conopeptide, helping define the -conotoxin pharmacophore. Sixteen analogues were selected across the series of active leads and tested in a neuropathic pain model. Several potent analogues produced less than expected efficacy and one related series produced side effects of unknown origin. Based on superior animal efficacy, side effect profile and chemical stability, Xen2174 was progressed into the clinic. An open-label clinical study of pain in oncology patients revealed that (i) a single intrathecal bolus dose of Xen2174 was safe and well-tolerated up to a dose of 30 mg, and (ii) some patients experienced multiple-day pain relief. Conclusions: These studies indicate an important role of the norepinephrine transporter in controlling the effects of norepinephrine released from spinal descending inhibitory pathways to reduce pain states in rats and possibly in humans.
Antibody-Therapeutic Targeting Of Tolc For Growth-Combating Of Antibiotic-Resistant Escherichia Coli HUI LI^1, LIN XIANG-MIN^1,SAN-YING WANG^2, XUAN-XIAN PENG^1, ^1Center for Proteomics, State key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, University City, Guangzhou, China; ^2School of Life Sciences, Xiamen University, Xiamen, China. Background: Bacterial resistance to an antibiotic may result from survival in a suddenly strong antibiotic or in sub-minimum inhibitory concentration of the drug. Their shared proteins responsible for the resistance should be potential targets for designing of new drugs to inhibit the growth of the antibiotic-resistant bacteria. Aims: 1) To identify sharedly altered outer membrane proteins (OM proteins) that are responsible for chloramphenical (CAP)-resistant Escherichia coli and for survival in medium with suddenly strong CAP treatment. 2) To develop a novel method of specific antibody combating bacterial growth based on these shared OM proteins. Methods: Comparative sub-proteomic methodologies were used to identify differentially expressed OM proteomes from E. coli respond to suddenly increased CAP treatment and from CAP-resistant E. coli selected from survivals after ten passages of subculture with the sub-inhibitory concentration of CAP. After the shared OM proteome responsible for CAP between the two ways of the exposures to the antibiotic treatment were determined, their capacity in antibiotic resistance was further investigated by their mutants. A specific antibody combating growth assay was developed to inhibit the activity of CAP-resistant OM proteins. The inoculums of CAP-R, CAP-R-O, △tolC, △ompC, △ompT and △ompW were separately cultured in 5 mL fresh LB medium at 37 ^0C overnight, and then the cultures were diluted 1:100 into 5 ml fresh LB medium to obtain the desired cell density (OD600nm=0.5, 10^8CFU/ml). Pellet from 200 µL of each of six cultures was obtained by centrifugation at 9000×g for 4 min and was separately incubated with 50µl of rabbit pre-immune serum and immune serum against TolC, OmpC, OmpT or OmpW at 37 ^0C for 1h. After centrifugation at 9000×g for 4 min, the bacteria were suspended in 200 µL fresh LB medium and then were diluted 1:1000 into 5 mL LB medium without or with 1/8 MIC CAP. These cultures were incubated at 37 ^0C for 9 h and were measured at OD600nm for survival capacity. Results: Six differential OM proteins and an unknown location protein were determined to be sharedly CAP-resistant-related proteins with the use of 2-DE/MS, Western blotting and gene mutant methods, in which TolC, OmpT, OmpC and OmpW were critical altered proteins and potential targets for the designation of the new drugs. Furthermore, only anti-TolC showed a very significant inhibition on bacterial growth in medium with CAP when antisera to TolC, OmpC, OmpT and OmpW were separately applied. The growth of CAP-resistant E. coli and its original strain was completely inhibited when they bound with anti-TolC and survived in 1/8 MIC of CAP, which was equal to behavior of △tolC when it did in the same concentration of the antibiotic. Conclusions: 1) Bacterial growth can be combated using the antibody specific to TolC, suggesting a novel insight into therapy to infection by antibiotic-resistance bacteria. 2) Combination therapy involving antibiotics that enhance the expression of an antibody target could be far more effective than either drug alone, which gives a novel insight into therapy to infection by antibiotic-resistant bacteria. This work was sponsored by grants from NSFC project 30530610, Guangzhou Key Project 2006Z3-E0251 and Guangdong NSF key project (7117645)..	Funding New Technologies For The Development Of Seasonal And Pandemic Influenza Vaccines LI S Biomedical Advanced Research and Development Authority, US Department of Health and Human Services Influenza viruses, both seasonal and pandemic, pose an ongoing threat to global health. Vaccines remain the best means of controlling influenza infection and spread, but the current worldwide capacity for seasonal influenza vaccine production falls far short of the capacity needed to provide sufficient vaccine for mass immunization in the event of a pandemic outbreak. To increase the supply of seasonal vaccine and the surge capacity for producing pandemic vaccine, the U.S. Government has established programs to facilitate the development of new technologies for enhancing the domestic and international influenza vaccine manufacturing infrastructure. Three major initiatives have been undertaken to support advanced development of: i) cell culture-based influenza vaccines; ii) novel dose-sparing adjuvant technologies for pandemic influenza vaccines; and iii) recombinant influenza vaccines. Funding has been provided under these programs for process development, clinical studies, establishment of manufacturing facilities, and other activities leading to licensure of the vaccines by U.S. Food and Drug Administration. The U.S. Government has awarded six contracts for the development of cell-based influenza vaccines. The cell-based technologies will complement the current licensed egg-based manufacturing technology to meet the USG’s stated goal of producing 450 million doses of vaccines within six months. Three contracts have been awarded for the development of novel adjuvants that would allow antigen-sparing in H5N1 vaccines. As the non-adjuvanted H5N1 vaccine is poorly immunogenic, and thus requires high doses of antigen to be effective in humans, development of these adjuvants may be critical for meeting potential pandemic needs. The successful conclusion of these programs will fundamentally alter the U.S. and global influenza vaccine manufacturing base and provide increased vaccine production capacity for pandemic preparedness.
Pharmacogenomic Targeting Of Ehrlich’s Magic Bullet LI-WAN-PO A National Genetics Education and Development Centre, Morris House, Birmingham Women’s Hospital, Edgbaston, Birmingham, UK, Centre for Evidence-Based Pharmacotherapy, Nottingham, UK. Background: Paul Ehrlich focused on targeting microbial invaders and renegade (cancer) cells. Despite his valiant efforts, both the enemies within, and the, invaders have proved resilient. However, considerable progress has been made since and we demonstrate the wide applicability of his vision and approach. Methods: We surveyed drugs in current and investigational use, and highlight the dramatic progress made through application of Ehrlichian principles of drug discovery and optimization. Results: Ehrlich, the visionary, was visual in his approach. His genius was to recognize that selective staining of microbes and cancer cells highlighted a route to drug selectivity. We still use the same approach but probe at a deeper genomic level, and on a vastly wider scale using gene expression microarrays. Ehrlich used both small molecules and macromolecules in his armamentarium. We maintain this mix to include an array of selective small molecules for targeting specific enzymes (e.g. imatinib) and disease pathways, and highly selective macromolecular drugs (e.g. monoclonal antibodies) for virtually every degenerative disease. Moreover, we can now identify the enemy with much greater precision (e.g. fine molecular classification of clinically similar diseases, and identification of resistance genes and epitopes to develop effective vaccines against enemies, both seen and unseen). Use of combinations of magic bullets causes less collateral damage (e.g. infections, rheumatoid arthritis), and lowers the risk of resistance. Unravelling more of the mysteries of our DNA self, helps us develop new magic bullets (e.g. siRNA), and new shuttles for our wounded troops (e.g. gene vectors) and sometimes to act as decoys against renegades (e.g. suicide gene therapy). Learning how to navigate in and out of tunnels (transmembrane transporter pharmacogenetics) ensures that our magic bullets are aimed more precisely. We illustrate these visually in our presentation. Conclusions: Ehrlich’s magic bullet has undergone considerable development. With our ageing populations, Ehrlich’s disciples have to contend with an increasing number of enemies from within as well as the persistently challenging invaders. It is of course ordained that the enemy will always win in the end but new Ehrlichian weaponry is allowing us to prolong life and improve its quality.	Enhancement Of Radiation Or Chemotherapeutic Effects Of Para-Aminobenoic Acid (PABA) And A Novel Analog On Melanoma: Preclinical Studies And Phase I Studies LIEBES L^1, PAVLICK^1 A, MUGGIA F^1, HARDIN E^1, MENDOZA S^1, ROTH J^2, BROOKS P^2 ^1NYU Langone School of Medicine, New York, NY, ^2Maine Medical Center Research Inst., Scarborough, USA. Background: B16F10 melanoma cells cultured in media containing PABA resulted in a depigmentation and inhibition of tyrosinase activity relative to cells grown in PABA deficient media. Following PABA’s potentiation of radiation or paclitaxel cytotoxicity, mechanistic studies showed inhibition of cell cycle arrest by up-regulation of CDC25A and down-regulation of p21^CIP! and BRCA2. A Phase I study of combination PABA, Paclitaxel (P) and carboplatin(C) in metastatic melanoma patients who had failed first-line therapy was initiated. Structure-activity studies utilizing tyrosinase activity identified C45, as a more potent PABA analog. Methods: Cell culture and murine models assessed the effects of PABA and C45 on melanoma tumor growth and gene expression. Cell cycle modulation and potentiation of cytotoxicity was greatest for P. We then enrolled patients (pts) with metastatic melanoma who had failed at least one prior chemotherapy regimen to receive PABA in combination with C and dose-escalated P. Measurement of plasma pharmacokinetics (PK) of PABA prior to, and 24 hours (h) after carboplatin/paclitaxel were obtained as well as PK of P after PABA administration. Results: C45 showed modulation of cellular proliferation in Lewis Lung and B16F10 melanoma. It potentiated the in-vivo activity of P, Temozolomide and Alimta and external beam radiation in M21 and B16F10 models. In the phase I, 19 pts were enrolled with 18 available for assessment. One grade IV neutropenia was seen and no dose-limiting toxicities even at doses of C AUC 5, and P 175 mg/m2 that are in common use. PABA PK for dose levels I-V showed a mean Cmax of 14 µg/ml + 7.9 g/ml at 0.5 h, and a mean elimination half-life of 2 h. In the presence of P, the mean half-life of PABA was 4.6 h. Four partial responses were seen and 2 had stable disease, with duration of response ranging from 12-32 weeks. Conclusions: PABA is safely administered in combination with full dose P and C in pts with refractory metastatic melanoma. Our enhanced therapeutic activity with low toxicity, justifies a phase II efficacy trial. Studies are continuing with the evaluation of the dose response and pharmacokinetics of the PABA analog, C45. Authors’ disclosure statement (not counting towards the character count): Supported in part by grants from NIH CA 91645, 2P30 CA16087-28, The Chemotherapy Foundation, and from Cancer Innovations, Inc.
Advantages of Multiple Drug Interactions: Combinatorial Treatments Using Neural Networks LIEBOVITCH LS, PANDYA A Florida Atlantic University, Boca Raton, FL, USA. Background: There are few Magic Bullets since drugs designed for one specific effect also interact with many other molecules in a cell. Instead of a disadvantage, we can turn this network of interactions into an advantage. If we can infer enough information about this network, from a limited set of experiments, we can predict which combinations of drugs will interact with each other, in just the right way, to have the most targeted effect with the fewest side effects. We call this approach Combinatorial Multi-Component Thearpy (CMCT). Methods: To illustrate this approach we constructed model test networks of linear or non-linear drug interactions. We then trained an artificial neural network (ANN) on a limited data set of drug inputs presented one-at-time and pairs-at-a-time and their output effect predicted by these models. We optimized the performance of the ANN by using only one output, softening the transfer function between the input and hidden layer, using four times as many units in the hidden layer as the input, and taking the logarithm of the output values. Results: For the model test networks, the ANN, trained on only very limited data, accurately computed the output effect for different combinations of input drugs. For example, 99% of the outputs had errors of less than 10% for all 32,768 combinations of 15 inputs that are either {0,1} in a highly nonlinear model of drug interactions. Conclusions: These test results suggest that this CMCT approach may be of value in determining how combinations of drugs can be effectively used to achieve specific therapeutic results.	Ecstasy – The Pharmacology of Happiness LIECHTI ME Clinical Pharmacology, University Hospital, Basel, Switzerland. Millions of young people consume „Ecstasy“ (MDMA), indicating that MDMA has strong rewarding effects. MDMA induces a state of well-being, elevate mood, moderate psychomotor stimulation, few perceptual changes and little anxiety. MDMA acts at the presynaptic monoamine transporter and releases serotonin, dopamine, and norepinephrine by reversing the transport of these monoamines. While the neurochemical effects of MDMA have been well described in preclinical models, it is less clear how the neurochemistry translates into to the psychotropic effects in humans. The rewarding effects of drugs of abuse are commonly attributed to the release of dopamine in the mesolimbic system. Indeed, pharmacological blockade of dopaminergic D2 receptors with haloperidol attenuated the positive mood effects of MDMA and produced increased adverse effects including anxiety. Serotonin uptake inhibitors (SSRIs) decrease MDMA-induced serotonin release by blocking the interaction of MDMA with the serotonin uptake site. In humans, administration of an SSRI prior to MDMA markedly decreased all subjective and physiological effects of MDMA including its positive effects, negative/adverse effects, the slight perceptual changes and the increase in blood pressure and heart rate. Further, blockade of the postsynaptic serotonergic 5-HT2 receptors selectively attenuated MDMA-induced perceptual chances. Together these results indicate that the MDMA-induced effects are overall due to release of endogenous serotonin with contributing effects of dopamine release to positive mood. MDMA-induced hallucinogen-like perceptual changes can be linked to 5-HT2 receptor stimulation. Ecstasy use is associated with serious adverse effects including hyperthermia, liver failure, hyponatremic brain edema and cardiovascular complications. Furthermore, there are concerns that heavy chronic Ecstasy use may lead to lasting cognitive impairment due to serotonergic neurotoxicity. So far, the magic Ecstasy pills induce short-term happiness but severe adverse effects and potentially persisting cognitive effects are a high prize to pay. Recent rodent studies indicate that the Alzheimer treatment memantine prevented MDMA-induced neurotoxicity and cognitive deficits. The question is open whether the magic bullet – the safe pill for happiness – will contain a psychostimulant and a neuroprotective treatment for Alzheimer’s disease.
Engineering Human Bak Proteoliposomes: a New Approach for the Treatment of Glioblastoma LIGUORI L, LENORMAND J-L HumProTher laboratory, TheRex-GREPI, TIMC-IMAG Laboratory, University Joseph Fourier, UFR de Medecine, Domaine de la Merci, 38706 La Tronche, France. Background. Specific delivery by nanoparticles of functional therapeutic proteins into targeted living cells is one of the most promising strategies for cancer treatment. The use of liposomes in the delivery of therapeutic membrane proteins directly into cells have not been tested because methods for producing membrane proteins and functional proteoliposomes are still difficult to carry out. Methods. One-step expression of human Bak and mutant Bak (BakBH3) proteoliposomes was tested using an optimized E. coli cell-free synthesis system in the presence of PEGylated liposomes. Subcutaneous inoculations of brain tumor (GL 26) on C/57/BL mice were performed into the rear left flanks and on day 20 post cell injection the tumor reaches 1mm^3, the tumor-bearing mice were injected with 75 µg of recombinant Bak or BakBH3 proteoliposomes. The mice were monitored for tumor regression and survival rate. Results. We demonstrate that the yields of pure Bak proteoliposomes are up to 2 mg/ml of reaction. Pre-clinical experiments indicate a significant regression in the tumor mass when mice were treated with lipo-Bak from day 15 post-injections and total absence of tumor in 60% of the mice treated after 30 days. The same treatment with lipoBakBH3 shows a plateau in the tumor growth followed by an exponential proliferation of the tumor. Survival analysis indicates that 60% of mice treated with lipoBak were still alive after 80 days after injection. Conclusions Here we report the therapeutic effect of long-circulating Bak proteoliposomes produced with an innovative cell-free expression system. We can conclude that: 1) Cell free expression system is a powerful tool to produce proteoliposomes with inserted therapeutic proteins (hBak). 2) In vitro experiments on different cancer cell lines demonstrate that lipo-Bak is able to be internalized and to exert an apoptotic effect. 3) Results on mice subcutaneous glioblastoma model show a survival of 60% of the treated mice with a total regression of the tumor. Considering this proof of concept, we are convinced that engineering human Bak proteoliposomes can be an effective new approach for the treatment of glioblastoma.	Peripherally Administered TrkB Agonists Cause Appetite Enhancement and Weight Gain in Non-Human Primates LIN JC^1*, TSAO D^1, BARRAS P^3, BASTARRACHEA R^2, BOYD B^4, CHOU J^1, ROSETE R^1, LONG H^1, FORGIE A^1, ABDICHE Y^1, DILLEY J^1, STRATTON J^1, GARCIA C^1, SLOANE D^1, COMUZZIE A^2, ROSENTHAL A^1 1 Rinat, Pfizer Inc., S. San Francisco, USA, 2 SW Foundation for Biomedical Research, San Antonio, USA, 3 Alpha Genesis, Inc., Yemassee, USA, 4 Northern Biomedical Research, Muskegon, USA Background: Brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4), ligands of receptor tyrosine kinase trkB, are anorexigenic when administered peripherally or centrally in mice. We hypothesized that like rodents, peripheral or central administration of TrkB agonists could reduce body weight and food intake in non-human primate species. If true, TrkB agonism may be a therapeutic approach for human obesity. Methods: 8 rhesus monkeys received a dose escalation of BDNF (n=4) or NT4 (n=4) by intracerebroventricular (ICV) delivery. 24 cynomolgus macaques were administered with NT4 either daily intravenously (IV), or daily subcutaneously (SC), or with a TrkB agonist antibody twice weekly IV. 6 obese baboons were given NT4 (n=3) or vehicle (n=3) daily IV. These baboons were later administered NT4 (n=3) or vehicle (n=3) IV twice weekly. Results: ICV administration of NT4 or BDNF into rhesus monkeys resulted in a dose-dependent suppression of food intake. Daily SC dosing of 2mg/kg of NT4, or IV dosing of 2mg/kg in cynos monkeys resulted in a 2 to 3-fold increase in daily food intake, and a 1.6 to 2.3-fold increase in cumulative food intake respectively. Daily SC or IV injections resulted in a 16% (SC for 21 days, p<0.001)) or 33% (IV for 30days, p<0.001) increase of body weight respectively. Twice a week IV dosing (21 days) of the TrkB agonist antibody (5mg/kg) in cynos monkeys resulted in a 40% increase in cumulative food intake (p<0.001) and a 10% increase in body weight (p<0.01). Obese baboons given daily IV injections of NT4 (2mg/kg) increased their daily food intake by 2 to 3-fold, and their cumulative food intake (25 days) by 2.5 fold. Body weight increased by 16% (p<0.001). Conclusions: 1) We observed a novel orexigenic response to peripheral administration of TrkB agonists, that is contrary to the anorexigenic response of peripheral or central TrkB agonism in mice. 2) Peripheral administration of NT4 was well-tolerated, suggesting that TrkB agonism could be a feasible therapeutic for anorexia or cachexia.
Hyaluronan-Mediated Transformation And Relapse Of Prostate Cancer LIN S-L Department of Cell and Neurobiology, Keck School of Medicine, University of Southern California, Los Angeles, California, U.S.A. INTRODUCTION: Interaction between extracellular matrices (ECM) and cancer cell receptors frequently alters signal transduction pathways, which lead to malignant transformation and metastasis. Hyaluronan (HA), an ECM tumor promoter and enhancer, is synthesized by stromal fibroblasts in response to paracrine factors produced by tumor cells. This type of tumor-stromal interaction plays a crucial role in the stimulation and promotion of cancer cell tumorigenecity. However, the molecular mechanism remains unclear. MATERIALS &METHODS: Human microarray analysis and immunodetection in human CaP tissue arrays were used to screen differentially expressed oncogene markers from patients with various stages of prostate cancer (CaP). In vitro tumorigenecity assays were performed to evaluate the loss-of-oncogene function, including cell-cycle flow cytometry (cell proliferation), cell invasion chamber (migration and invasion), and adhesion to a human bone marrow endothelial cell (hBMEC) monolayer (metastasis). RESULTS: We observed that HA-mediated CD168, a receptor for HA-mediated motility (RHAMM), and its downstream signal molecules, including ROCK1, Gab-1, PI3K•p110 and eIF4E, promote the malignant progression of hormone-refractory CaP (Carcinogenesis 28: 310-320, 2007). In normal prostate, androgen receptor (AR) serves as a tumor suppressor against the HA-stimulated CD168 signaling by binding and inactivating CD168 (Carcinogenesis 29: 282-290, 2008). AR is also found to regulate the transcription of CD168 mRNA in the presence of androgen. The results of in-vitro tumorigenecity assays further showed that CaP cells with deficiency or mutation of AR significantly increase the malignancy of cancer tumorigenecity in terms of cell proliferation, cell invasion and metastasis into the hBMEC monolayer. The expression of mir-146 against the key kinase ROCK1 of the HA–CD168 signaling pathway can reverse the HA-stimulated malignancy (RNA 14: 417-424, 2008). CONCLUSION: HA activates the signal transduction cascade of CD168–ROCK1– PI3K–eIF4E in CaP, which can be prevented by ROCK depletion using mir-146. Therefore, our study suggests that the combination of current cancer therapy with an anti-ROCK agent may lead to beneficial results in preventing HA-mediated cancer transformation and relapse.	Verotoxin (Shiga toxin) binding to its receptor glycolipid, globotriaosyl ceramide, provides a new antineoplastic tool and physiologically-based approaches to tumour cell drug resistance LINGWOOD C Research Institute, Hospital for Sick Children, Toronto, Canada. Background Verotoxin 1 (Shiga toxin) kills cells expressing its receptor glycosphingolipid(GSL), globotriaosyl ceramide (Gb3 ) expressed within detergent resistant plasma membrane (DRM) domains. Gb3 is upregulated in many human cancers including breast, ovarian, colon, glioblastoma, meningioma, renal, testicular. Although Gb3 is expressed on a few normal tissues these are not targeted in a primate model, suggesting that Gb3 may not be in DRM in such cases. Intratumoral VT1 injection reduces growth rate and eliminates human Gb3 positive tumors xenografts(astrocytoma, meningioma, colon and renal carcinomas) grown in mice. Gb3 is also expressed in tumor neovasculature, indicating that VT1 has antineoplastic and antiangiogenic activity. Primate studies established a safe VT1 dosage for clinical trials. Methods MDR1 transfected MDCK cells were used to assess MDR1 inhibition and link between MDR1 processing and GSL biosynthesis and polarized C2BBe1 gastrointestinal epithelial cells use to measure MDR1 mediated drug fluxes. Results Gb3 is particularly elevated in MDR1 expressing drug resistant tumour variants which led us to determine that MDR1 is a Golgi GSL flippase, involved in neutral GSL biosynthesis ^1. Inhibition of GSL biosynthesis prevents cell surface MDR1 expression in cell lines from drug resistant tumors, though intracellular MDR1 accumulates. Cell surface MDR1 showed significant colocalization with Gb3 as monitored by VT1 binding. MDR1 is found within DRMs and treatment of drug resistant cells with VT1, or its receptor binding B subunit, internalized Gb3 (in DRMs) to prevent MDR1–mediated rhodamine efflux. Thus the glycolipid environment of MDR1 is important to function and cell surface trafficking. AdamantylGb3 is a soluble Gb3 mimic we designed which competes for VT1-Gb3 binding. AdamantylGb3 was found to bind deep between the 6^th and 7^th membrane-spanning -helixes of MDR1, and proved the first physiologically-based inhibitor of MDR1 ^2. MDR1-mediated rhodamine efflux was prevented and adamantylGb3 reversed cell resistance to vinblastin. Gastrointestinal MDR1 also reduces oral drug bioavailability. MDR1-mediated digoxin and vinblastin efflux in human intestinal epithelial cells was prevented by adamantylGb3. Conclusion Gb3 provides an antineoplastic target and new insight into tumour drug resistance and drug oral bioavailability. 1. De Rosa MF, Sillence D, Ackerley C, Lingwood C. Role of Multiple Drug Resistance Protein 1 in neutral but not acidic glycosphingolipid biosynthesis. J. Biol. Chem. 2004; 279: 7867-7876. 2. De Rosa MF, Ackerley C, Wang B, Ito S, Clarke D, Lingwood C. Inhibition of multidrug resistance 1 (MDR1) by adamantylGb3, a globotriaosylceramide analog. J Biol Chem 2008; 283: 4501-4511. 
Dodging the bullets: an update on the multi-drug efflux pump P-glycoprotein ZOLNERCIKS JK, LINTON KJ MRC Clinical Sciences Centre, Imperial College, Hammersmith Hospital, Du Cane Road, London UK. Background: Human P-glycoprotein (Pgp, ABCB1) is a clinically-relevant drug exporter of the ATP Binding Cassette transporter family. The protein has two transmembrane domains (TMD) that contain the drug binding sites, and two nucleotide binding domains (NBD) that bind and hydrolyse ATP to drive the drug transport cycle. Structural studies on bacterial homologues indicate that the two NBDs interface to form two composite sites of highly conserved motifs to co-ordinate ATP. Each site comprises a Walker A and B motif, stacking aromatic and H-loop of the core subdomain of one NBD, and the ABC Signature and D-loop of the -helical subdomain of the second. The highly conserved glutamine of the Q-loop also contacts the -phosphate of ATP via a water molecule. The Q-loop links the core and -helical subdomains of the NBD, and also interdigitates with the TMDs. Methods: We systematically examined the importance of these motifs in the function of Pgp by mutating the most highly conserved residue in each motif in either or both NBDs. The proteins were expressed in HEK293T cells and measured surface expression and drug efflux in real time by flow cytometry. Drug-stimulated ATPase activity of purified Pgp was measured by colorimetric assay. Results: Single mutations introduced into the Walker A or B motifs render the protein virtually inactive. However, for the stacking aromatic, the ABC signature and the D-, H- and Q-loop motifs, mutation of one NBD has no, or minimal effect, on drug transport, but mutation of the motif in both NBDs has a strong, synergistic, negative effect. The high level of activity in the single Q-loop mutants was particularly surprising as data from mouse Pgp published previously suggests that these should be severely debilitated. However, on purification, the single Q-loop mutants of human Pgp exhibit <10% of the drug-stimulated ATPase activity of wild type Pgp and the double mutant was inactive. Conclusions: The emerging picture is that canonical Walker A and B motifs are essential for drug transport, however, there is redundancy in the mechanism for the stacking aromatic, the ABC signature and the D-, H- and Q-loop motifs. In the absence of either Q-loop glutamine, Pgp appears sensitive to detergent. Together with structural data, this suggests that the Q-loop is the fulcrum of the molecular mechanism and an important conduit for energy transduction between domains. Authors’ disclosure statement: The abstract describes unpublished data	Fluorescent Biosensors to Detect Magic Bullets against Multi-Drug Resistant Bacteria MECKLENBRÄUKER I, SCHLEBERGER C, HAHN C, WILLER Y,. LE NQ, KÖNIG C, WILKEN L, BECHTHOLD A, BUMANN D and LIPPUNER C Albert-Ludwigs Universität Freiburg, Freiburg, Germany Background: Rising antimicrobial resistance of major pathogens limits therapeutic options. Identifying novel antimicrobial targets through genomic-derived, target-based approaches have widely failed in the past decades. Whole-cell bacterial biosensors with promoter-inducible fluorescent reporters provide an attractive method to discover novel drugs. However, there is a bottleneck in finding suitable pathway-specific biosensors because of insufficient specificity and poor signal to noise ratio. Methods: E. coli promoter trap libraries were constructed containing random bacterial DNA in front of the reporter gene green fluorescent protein. Several fluorescent biosensors that report specific disturbance in cell wall and protein biosynthesis pathways were found. An optimal assay protocol for screening in a 96-well microtiter plate format was developed. Results: These biosensors react only to inhibitors that reach significant concentrations in live bacteria and not to unspecific stresses. We optimized these biosensors for HTS screening in a 96-well plate format. In comparison to luciferase or beta-galactosidase based biosensors, we can combine in a single well up to six fluorescent biosensors expressing different variants of GFP and simultaneously detect distinctive responses of each individual biosensor in the composite using automated multi-color FACS analysis. Our biosensors are more sensitive than growth assays and highly specific for well-defined targets. They are compatible for high-throughput-screening with a LSR2 flow cytometer with a high throughput sampler. The system measures 13 parameters per well. Automated high-content analysis records then individual information about bacterial growth, cell wall permeability and fluorescent protein expression in up to six different biosensors incubated with compounds. Conclusions: A new sophisticated screening system was developed. The system can detect specific groups of antimicrobial compounds. Incubation times are short and only low compound concentrations are needed. This opens up new perspectives for antimicrobial screening.
Alpha-1-antitrypsin and IgA in Serial Meconium and Faeces for Date Newborn`s Faeces Formed during Intrauterine and Extrauterine Maturation. LISOWSKA-MYJAK B, PACHECKA J Medical University, Warsaw , Poland Background: Meconium is a series of layers formed in the foetal intestine from the 12^th week of gestation. High content of meconial alpha-1-antitrypsin (AAT) , decreasing within the first days of extrauterine life appears to reflect the meconium clearance of the gut. At birth IgA is not present in the meconium and breast-fed infants receive this antibody postnatally with human milk. Methods: 24 healthy breast-fed newborns were studied prospectively during the first 4 days of postnatal life. AAT and IgA concentrations in meconial and faecal samples and IgA concentration in mother`s milk taken on the third day after delivery were determined by radial immunodiffusion. Results: The medians (range) of AAT concentrations in milligrams per gram of dry meconium or faeces were: 68,8 (29,2-138,4) (day 1), 56,9 (30,8-112,8) (day 2), 26,2 (6,8-80,7) (day 3), and 6,6 (1,4-27,1) (day 4). The median (range) of IgA concentration in mothers’ milk was 715 mg/dl (420-890). IgA was absent in meconium portions from the first day of life while on the successive days the medians (range) of IgA concentration in mg/g dry mass of meconium and faeces were as follows: 0 (0-2,90) (day 2), 2,50 (1,10-9,60) (day 3), 7,05 (4,10-30,60) (day 4). On the day 4 of extrauterine life a negative correlation was found between AAT and IgA concentrations in faeces of the newborns (r= - 0,46 ). Conclusions: 1) Analysis of the systematic decrease in AAT and increase of IgA concentration in serial portions of meconium and faeces over the first days of extrauterine life of breast –fed newborns can be a new marker for identifying meconium formed in utero. 2) Taking into account, that the use of illicit and legal drugs during pregnancy is common, meconium formed in utero can be a new clinical material for identifying exposure of infants to a number of illicit and legal drugs during 12-40 weeks of gestation.	Antibiotic “Magic Bullets”: The Promise and the Reality of the Past Thirty Years LIU HH Bryn Mawr Medical Specialists & Thomas Jefferson University, Philadelphia, PA, USA Background: Antibiotics, vaccines, and biologic mediators have been true “magic bullets” against infections over the past 30 years. However, benefit has been tempered by toxicity, side effects, cost, and rising antimicrobial resistance. Even their successes have led to physician inappropriate use and public complacency about infectious diseases. Methods: Local surveys of infections and resistant microbe prevalence was compared with healthcare professionals’ attitudes on antibiotic and vaccine use. This was correlated with national and global data searching for broad trends. Literature was reviewed for approaches to countering resistance development and minimizing adverse events while promoting appropriate antibiotic use. Results: In Philadelphia, pneumococcal penicillin resistance has risen from <10% to 29% over three decades; macrolide resistance is now >35%. Extended spectrum cephaloporins must be used carefully against enteric gram-negative bacilli due to extended spectrum beta-lactamases. Fluoroquinolones are much less effective against Pseudomonas a. than 30 years ago (0% resistance vs. 38% now). Vaccine uptake in children is only 80% currently, and some parents/patients decline inoculations as “not needed” (measles, mumps) or “not effective or dangerous” (influenza). Clostridum difficile colitis cases have risen dramatically (to 546 cases / 100,000 population) with a 2-1/2 fold rise in mortality just over 1993-2003. There is rising public awareness of antibiotic “collateral damage” but relatively little impact on demand for antibiotics. Conclusions: 1) The great value of antibiotic “magic bullets” and vaccines has been countered by adverse effects and rising antibiotic resistance; 2) Future hope rests on: education (physician & public, elective & mandatory), disease manage-ment initiatives (practice guidelines), point-of-service behavior modification (physician computer order entry), and incorporating appropriate antibiotic use initiatives into clinical trials and marketing.
Trans-lymphatic Chemotherapy LIU J Princess Margaret Hospital, Toronto, ON, Canada Background: Lymph node metastasis is a significant prognostic factor for most cancers. Failure to control lymphatic metastasis may result in local recurrence and systemic metastasis. A trans-lymphatic chemotherapy technology was developed to control lymphatic metastasis. Methods: The technology involves an implantable gelatin sponge impregnated with biodegradable polymer microparticulate anticancer agent and is referred as microparticulate lymphatic targeting system (MLTS). When the system is formulated for paclitaxel (PTX) or doxorubicin (Dox), it is designated as MLTS-PTX or MLTS-Dox respectively. The systems were characterized in vitro. The pharmacokinetics (PK) of MLTS-PTX was studied in rats with comparison to injectable PTX given iv or intrapleurally (ipl). The therapeutic efficacy was examined in an orthotopic lung cancer model. MLTS-PTX was placed into the pleural cavity when the tumor lung was resected 14 days after orthotopic tumor implantation. Tumor recurrences were assessed 32 days following the procedure. The therapeutic efficacy of MLTS-Dox was examined in SCID mice bearing DLD1 orthotopic colon cancer with 100% incidence of lymphatic metastasis. Seven days after tumor implantation on the cecal wall, animals were treated with either ip implantation of MLTS-Dox, placebo sponge or no treatment. Lymphatic metastasis was examined in 40 days. Results: Both systems exhibits controlled drug release properties in vitro. The microspheres were selectively taken up by the lymphatics and delivered to the regional lymph nodes as the sponge disintegrated. PK studies revealed a significantly higher AUC in mediastinal lymph nodes with ipl placement of MLTS-PTX as compared to iv or ipl administration of PTX. This represents approximately a 400-fold increase in lymphatic drug exposure as compared to iv dosing. Peak plasma concentration was significantly reduced. There was an 80% reduction in lymph node metastasis with MLTS-PTX treatment. The microparticulate PTX was microscopically evident in the targeted lymph nodes. Similarly, MLTS-Dox significantly decreased the incidence of lymph node metastasis in the treatment arm (20%) as compared to the controls (100%). Microparticulate Dox were seen in the targeted lymph nodes. Conclusions: Trans-lymphatic targeted chemotherapy reduces lymph node metastasis in both lung cancer and colon cancer models. This effect may be attributed to the improved lymphatic distribution of the therapeutic agents.	An enzymatic approach for developing heparan sulfate-based drugs LIU Jian Division of Medicinal Chemistry and Natural Products, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina, USA Background: Heparan sulfate (HS) represents a substantial portion of glycans that perform essential physiological functions. Heparin, a special form of HS, is a commonly used anticoagulant drug. The wide range of biological functions of HS attract considerable interest to exploit heparin or heparin-like molecules for the development of anticancer, antiviral drugs and a better anticoagulant drug. Heparin is currently isolated from pig intestine. A contaminant in the heparin could lead to severe allergic reactions and deaths as it happend recently in US and Germany. Thus, a cost effective method for preparing synthetic heparin is highly desirable. HS is a sulfated polysaccharide, and the unique sulfation patterns dictate the biological activity. Chemical synthesis of HS oligosaccharides that are larger than hexasaccharides is extremely difficult. Methods: We have developed an enzyme-based approach to synthesize heparin and heparan sulfate. There are total of 13 specialized sulfotransferases and one epimerase involved in the biosynthesis of HS. We have expressed most of these enzymes in E. coli, permitting the access of a large amount of proteins. A low cost sulfo donor system was also successfully coupled with the synthesis, which reduced the cost of the synthesis by more than 1000-fold. Results: Our method has demonstrated the feasibilities of the synthesis of the heparan sulfate with different biological functions in mutli-milligram scales. This method was employed to identify novel structures of anticoagulant HS, known as Recomparin. In addition, using structurally based mutagenesis approach, we are able to alter the susbtrate specificities of sulfotransferases. The engineered sulfotransferases allowed to synthesize those polysaccharides that can not be achieved by wild type proteins. Our results have demonstrated the potential of the enzymatic approach to prepare HS-based therapeutic agents.
Intrinsic antibiotic resistance mechanism of Mycobacteria LIU Jun Department of Molecular Genetics, University of Toronto Mycobacteria including Mycobacterium tuberculosis are naturally resistant to most common antibiotics and chemotherapeutic agents. The underlying molecular mechanisms are not fully understood. The mycobacterial cell wall, with its unique chemical composition and physical structure, plays a major role in the natural resistance. However, our studies of hypersusceptible mutants suggest that other mechanisms also exist. I will discuss the role of these factors in the natural resistance of mycobacteria. Understanding the molecular mechanisms of natural resistance may provide insights into the development of new generation of antimycobacterial agents or novel combinations of existing drugs.	Monitor of Chemosensitivity by Bcl-2 Transcript Kinetics in Acute Myeloid Leukemias LIU KS^1, KREUZER KA^2, LASS U^2, SCHMIDT CA^2 ^1 Univ. Jinan, Guangzhou, China; ^2 Univ. Humboldt, Berlin, Germany. Background: Enforced bcl-2 gene expression suppresses apoptosis and confers resistance to anticancer drugs. Aims: 1) to develop the real time quantitative PCR that can monitor the expression of the bcl-2 transcript in the therapeutic course of AML patients; 2) to analyze the association between the bcl-2 gene expresssion and clinical chemosensitivity in acute myeloid leukemia. Methods: A total of 53 patients with acute myeloid leukemia were evaluated in this study. Mononuclear cell fractions were isolated from the peripheral blood of 5 patients and classified as T-cells, B-cells, granulocytes. A) Standard curve construction: The standard curve for bcl-2 transcripts was constructed and the house-keeping gene β-actin was prepared as an endogenous reference. B) Real time quantitative TaqMan PCR was performed in the ABI Prism^TM 7700 Sequence Detector System. C) Statistical analyses were performed using MS Excel 7.0 computer software. Results: A quantitative RT-PCR assay of the bcl-2 gene, using TaqMan^TMfluorogenic detection system was developed, which detected accurately the changes of the bcl-2 in the course of the chemotherapy for AML patients. The bcl-2/β-actin ratio from the patients with AML was various, but not related to FAB subtypes. This transcript ratio was not affected by mononucleated cell types. The rapid decrease of the bcl-2/β-actin ratio in samples by the real time quantitative PCR substantiated the early response, remission induction and the susceptibility to the chemotherapy protocols ( Figure A & B ). In the opposite, the gradual elevation of the bcl-2/β-actin ratio demonstrated the loss of effect in update-therapy protocol and the drug-resistance in AML patients ( Figure C & D ). Conclusions: By applying real time PCR to clinical samples, although the bcl-2/β-actin ratio was not related to FAB subtypes, the changing data following remission induction therapy clearly reflected drug-sensitivity. These results suggest that RT-PCR assay monitored the efficacy of the chemotherapy by quantifying the bcl-2 gene transcript in AML.
Analgesic Activity of Dragon’s Blood Caused by Interaction of Its Components—Cochinchinenin A, Cochinchinenin B, and Loureirin B CHEN S, GUO M, LIU XM South-Central University for Nationalities, Wuhan, China. Background: Dragon’s blood is a renowned traditional medicine with analgesic activity. Elucidation of mechanism and material basis for its analgesic effect is important to research and development of little side-effect analgesic medicine. Aims: 1) To clarify analgesic mechanism of dragon’s blood. 2) To identify its corresponding material basis. 3) To evaluate interaction between components of dragon’s blood in producing analgesic effect. Methods: Cochinchinenin A, cochinchinenin B and loureirin B were components extracted from dragon’s blood. According to their percentage contents in dragon’s blood, two or three component combinations were prepared. Using patch clamp technique and microelectrode extracellular recordings, effects of dragon’s blood and its component combinations on voltage-gated sodium currents in dorsal root ganglion neurons and on noxious stimulation evoked discharges of wide dynamic range (WDR) neurons in spinal dorsal horn of SD rats were observed (10 neurons per drug). With pharmacodynamic parameter values of dragon’s blood as reference, those of each combination was compared with the reference to identify which combination can take the place of dragon’s blood. Based on concept of dose equivalence, zero interaction response surfaces used to assess interaction between three drugs with dissimilar Hill coefficients was proposed. Applying it， interaction of various combinations on sodium currents and discharges of WDR neurons were evaluated. Results: Both dragon’s blood and its component combinations not only modulated tetrodotoxin-sensitive and tetrodotoxin-resistant sodium currents but also inhibited discharge frequencies of WDR neurons. Only combined effects of cochinchinenin A (0.38mmol/L), cochinchinenin B (0.19mmol/L) and loureirin B (0.08mmol/L) were similar to effects of dragon’s blood (0.05%). Inhibition rates of combination and dragon’s blood on discharge frequencies were (29.79±2.51)% and (30.56±2.09)%, respectively. The combined effects were defined as synergistic. Conclusions: 1) Dragon’s blood interfere not only with transmission of pain in primary sensory neurons but also with processing of pain in spinal dorsal horn. 2) Analgesic activity of dragon’s blood was caused by synergistic interaction of three components—cochinchinenin A, cochinchinenin B, and loureirin B. Authors’ disclosure statement: The experimental results that the effects of dragon’s blood and its component combinations on noxious stimulation evoked discharges of wide dynamic range (WDR) neurons in spinal dorsal horn of SD rats have not been reported. Due to space limitation, only the numerical results obtained in this part have been included. In addition, our study further finds that the inhibition of dragon’s blood on capsaicin (CAP)-activated currents and CAP-evoked depolarization was greater than that of cochinchinenin B. It is inferred that the above inhibition may correlate with the analgesic effect of dragon’s blood and the combination of three components may have antagonistic effect on modulation capsaicin receptor similar to dragon’s blood.	Matrix metalloproteinases at BBB and beyond in Multiple Sclerosis and HIV-dementia. New perspectives for therapeutical interventions LIUZZI GM Department of Biochemistry and Molecular Biology “E. Quagliariello”, University of Bari, Italy Matrix metalloproteinases (MMPs) are extracellular zinc-dependent neutral proteinases that play an important role in physiological processes that involve tissue remodelling. Recent evidence suggests that dysregulation and imbalance between MMPs and endogenous tissue inhibitors of MMPs (TIMPs) might contribute to different pathological conditions. Among MMPs, the subfamily of gelatinases seems to be involved in mechanisms of T cell migration into the CNS, blood-brain barrier (BBB) disruption and demyelination in the course of MS and HIV-associated neurological diseases such as AIDS Dementia Complex (ADC). Given the importance of MMPs as key mediators in the pathogenetic mechanisms of MS and ADC, targeting MMP enzyme activity may constitute a novel therapeutic strategy in the treatmentof these diseases. We studied the role of MMPs as key mediators in the pathogenesis of MS and ADC, with particular attention to the effect of therapy on MMP secretion and expression. By using an in vitro model we investigated whether IFN-as well as the antiretroviral drugs zidovudine (AZT) an indinavir (IDV), drugs used for the treatment of MS and HIV-infected patients, respectively are able to modulate the activity and the expression of MMPs in glial cell cultures. As assessed by gelatine-zymography and RT-PCR, we observed a dose-dependent inhibition of MMP-9 activity and expression in both LPS-activated astrocytes and microglia. MMP-2 inhibition by IFN- and antiretroviral drugs was observed only in astrocytes but not in microglia.