Ehrlich II –2nd World Conference on Magic Bullets

Spider silk proteins – a new generation of bioimplantable materials. ENGSTRÖM W^1, JOHANSSON J^1, VADGAMA P^2, HATTON P^3, WAGEMAKER G^4, SKAER N^5, VOLLRATH F^6, LARHED A^7, HABIBOVIC P^8, VON AULOCK S^9. SPIDERMAN CONSORTIUM. 1) Swedish University of Agricultural Sciences, 2) Queen Mary University of London, UK, 3) Sheffield University, UK, 4) Erasmus University, Rotterdam, the Netherlands, 5) Oxford Biomaterials, Newbury, UK, 6) University of Oxford, UK, 7) Q-Med Uppsala, Sweden, 8) University of Twente, The Netherlands, 9) University of Konstanz, Germany Background; Spider silk consists of fibrous proteins containing highly repetitive sequences of amino acids stored in liquid form that configure into fibres when sheared or spun at secretion. This multipartner project aimed at recombinant production of this elastic tough and tensile polypeptide fibre that can be used for development of implantable surgical devices. Methods; This study has used a vast range of recombinant technology structural chemical and immunological methodology in combination with a range of in vitro and in vivo approaches. Results: We have produced strong tensile fibres originating from a partial cDNA sequence from the Euprosthenops Spidroin I gene with biomechanical properties that allow the fibres to be used for the development of implantable surgical material. In parallel progress has been made on the development of alternative material originating from silkworm silks. This class of materials can be used as a scaffold for in vitro synthesis of certain artificially produced biological tissues. Moreover, it has been shown that the different silks form scaffolds that support differentiation and growth of mesenchymal stem cells. .Furthermore biocompatibility parameters of bona fide , reconstituted and recombinant silk will be presented. A novel in vitro system for assaying immunogenicity as measured by release of selected interleukins from cultured macrophages has been developed. The characterisation of spider silks ability to release pyrogens and interleukins from target cells has also been deployed. It has been demonstrated that biosynthetic fibers from Euprosthenops and refined silkworm silk fibers are can be virtually devoid of LPS contamination and show a promising performance in vivo. Conclusion: Novel fibrous protein biomaterials have been developed that possess the required biomechanical properties to proceed into the development of implant prototypes.

The Cancer Cell Life-Cycle: Providing Mechanism for Genotoxic Resistance ERENPREISA J^1 and CRAGG MS^2 ^1Latvian Biomedicine Research and Studies Centre, Riga, Latvia; ^2Tenovus Research Laboratory, Southampton University, UK Background. Recently, the genetic mutation theory of cancer has been complemented with the concept of "cancer stem cells". This concept recognises that only a small fraction of tumour cells appears to possess the properties of self-renewal and unlimited proliferation necessary to explain our observations of cancer treatment, resistance and re-growth. Two groups of ontogenetic genes appear to be up-regulated in parallel to the degree of malignancy and poor patient prognosis: cancer testes-associated genes and embryonal-germ-line-specific genes, the both may have relevance to the poorly known biology of cancer stem cells. Their importance in resistance to genotoxic stress remains to be fully elucidated. Over eight years ago, we observed the role of transient polyploidy in the genotoxic resistance of p53-deficient lymphoma cells (Illidge et al., 2000; Erenpreisa and al., 2000). Our next task was to see if the above mentioned ontogenetic genes are associated with transient polyploidy. Methods. Irradiated human lymphoid cell-lines. RT-PCR, Western blotting, and imunofluorescence. Results. We found previously (Kalejs et al., 2006) and extended observations in the present studies that transient polyploids induced by mitotic catastrophe in irradiated p53-mutant tumour cells express several hall-marks of meiosis and undergo reduction divisions producing mitosis-capable para-diploid cells. Conclusions. The data support our hypothesis (Erenpreisa and Cragg, 2001, 2007) that the ploidy cycle acts as a bridge from one cancer cell generation (akin to a life-cycle) to another. The slippage from mitotic cycle for the ploidy cycle is started by mitotic catastrophe. Furthermore, we suggest that this life cycle occurs constitutively in tumour cells (affording them immortality through continual rejuvenation of the cancer stem cells) and at an accelerated rate following genotoxic treatment providing them with the ability to survive genotoxic treatments. Therefore, we feel that the next strategy for Ehrlichs’ "magic bullets" should target this process.

Biopharmaceutical and Pharmacokinetic Searches of Drugs in Perinatology (1968-2008) ERIASHVILI V, KINTRAIA P, GOTSIRIDZE E, KINTRAIA N, ERIASHVILI T, DUGASHVILI N, PAPASHVILI G, KVIZHINADZE N Scientific Center of Biopharmacy, Tbilisi, Georgia. Background: As it’s known the biopharmaceutical factors influence on the pharmacokinetic parameters of medical drugs and therapeutical effectiveness. Since 1968 the aim of scientific-industrial center of biopharmacy of Georgian Ministry of Labor, Health and Social affairs is to work out the Biopharmaceutical and Pharmacokinetic searches of drugs, used in perinatal medicine and obstetric - gynecology to create the effective therapeutical schemes and rational drug forms. Methods: Our studies worked out by the following directions: To work out the sensitive methods of determination of medical drugs in biological fluids and experimental animals’ organs; To implement the permanent method of vaginal and rectal suppositories; To select the release the pain, mastimulate and anti trichomikocidi drugs to create the property material; prepare rational drug forms, investigation their pharmacokinetic and biopharmaceutical data. For the determination of pharmacokinetic parameters and study the biopharmaceutical factors of drugs we used the following compounds: Aminaloni (^15N), Oxybutirat Natrium (^18O), Ethmozini (^34S) for mass spectral analyze and kavintoni, furazolidoni, ASA and their metabolites, Etazoli and its’ modifications, aloe and placenta extracts, “superini”, biogenic stimulator – “Amnitini”. “Trichomikocidi” and “Alferoli” were studied by chromatofotocolorimetric and spectrofotometric methods modified by us. Results: Preparations “Supetini” and biostimulator “Placenta extract” is registered by Ministry of Health in Georgia. The rest medical drugs are fulfilled extemporal, confirmed on the base of methodical recommendations and they have use in gynecology, to treat the urological, proctologic, oncological etc diseases. Conclusions: According to the fulfilled biopharmaceutical, pharmacokinetic and pharmacodinamic searches created about 40 medical drugs mostly in suppositories forms by original permanent methods. Among them “Trichomikocidi” passed clinical approbation and is producing in the Ministry of Health for registration, anti anemia suppositories “Alferol” is in the clinical approbation process, the technical documentations for amnitini suppositories are preparing to get permission for clinical approbation.

Novel Phyto-antiviral Leads from Aglaia Species ESIMONE CO^1,3, ECK G^2, DUONG TN^4, ÜBERLA K^3, PROKSCH P^2 ^1University of Nigeria, Nsukka, Nigeria, ^2Heinrich-Heine University, Düsseldorf, Germany, ^3Ruhr University, Bochum, Germany, ^4Vietnamese Academy of Science and Technology, Hanoi, Vietnam. Background: Medicinal plants have consistently served as suitable lead sources for potent antiviral, antimicrobial and other pharmacological agents. The genus aglaia contains more than 100 species, many of which have been shown to possess diverse pharmacological activity. In our current search for novel plant-derived antiviral compounds effective against HIV and other human pathogenic viruses, we focused on 18 triterpenoidal compounds from 5 Aglaia species. Methods: A total of 18 pure triterpenoidal compounds isolated from 5 Aglaia species (A. ignea, A. duppereana, A. cucculata, A. euphoroides and A. tsangii) were screened against the human immunodeficiency virus type 1 (HIV-1), human adenovirus type 5 (hAd5) herpes simplex virus type 1 (HSV-1) and the respiratory syncytial virus (RSV) using cell lines permissive for these viruses. Toxicity of the compounds to the cell lines was assessed in parallel using either the MTT method or a standard ATP-based assay. Mechanistic evaluation of the potent compounds was carried out using time-of-addition studies. Results: While only one compound (dammarenolic acid) displayed very potent and selective activity against HIV-1 and HSV-1, three compounds (dammarenolic acid, aglaiol and niloticin) displayed selective anti-RSV activity. Time of addition studies showed that dammarenolic acid (DA) and aglaiol (AG) targeted both entry and post-entry steps in the viral replication cycle of test virus (against HIV-1 and RSV for DA and RSV for AG). Conclusions: Dammarenolic acid, aglaiol and niloticin represent novel plant-derived compounds from Aglaia that could be further exploited as suitable leads for the development of potent anti-HIV-1, anti-HSV and anti-anti-RSV agents.

Recombinant Viral Vectors as Suitable Surrogates for Antiviral Screening Studies ESIMONE CO^1,3, ECK G^2, DUONG TN^4, ÜBERLA K^3, PROKSCH P^2 ^1University of Nigeria, Nsukka, Nigeria, ^2Heinrich-Heine University, Düsseldorf, Germany, ^3Ruhr University, Bochum, Germany, ^4Vietnamese Academy of Science and Technology, Hanoi, Vietnam. Background: For high throughput antiviral screening studies, safe, rapid, reproducible and cheap screening techniques are needed. Reporter gene-based screening assays are increasingly being recognized as suitable probes for such assays. Here we describe the use of recombinant retroviral and adenoviral vectors expressing either the luciferase (Luc) or green fluorescent protein (GFP) reporter genes as suitable surrogates for the efficient high throughput screening of antiviral compounds from plant or synthetic sources. Methods: About fifty plant-based and two synthetic compounds were screened for anti-HIV activity using recombinant single-cycle infectious lentiviral vectors expressing either the Luc or GFP gene. To increase tropism the vectors were pseudotyped with the vesicular stomatitis virus Glycoprotein envelop. Infectious lentiviral vector particles generated by transient co-transfection of the vector plasmid with packaging plasmids were incubated with various concentrations of the antiviral compounds and then used to transduce HeLa cells in 96-well plates. Cytotoxicity was assessed in parallel using similar cell lines stably expressing Luc. After two days, the reporter gene read-out of treated cells were analysed and the effect of compounds relative to untreated control was expressed as a percentage. Replication-competent and defective adenoviral type 5-based (Ad5) vectors expressing the Luc or GFP genes were similarly screened with antiviral compounds. Results: Compounds from Aglaia species, Ramalina farinacea, Jatropha tanjorensis and Nymphae lotus displayed potent anti-HIV activity, with IC50 ranging between 2.7 and 18.2 µg/ml. Only selected compounds from Aglaia and R. farinacea did show potent anti-Ad5 activity. The anti-HIV effect against the viral vectors was confirmed with 10 times potency against the wild-type HIV-1. The anti-HIV effect and antiretroviral spectrum of activity of nevirapine was authenticated using both wild-type and HIV-1 vector. Conclusions: 1) The recombinant viral vectors are safe and reproducibly mimick the wild-type viruses. 2) Several plant-based and two standard synthetic compounds were appropriately screened using the vector-based technique.

Magic Bullets and the Nosocomial Nemesis – the KwaZulu-Natal Experience ESSACK SY School of Pharmacy and Pharmacology, University of KwaZulu-Natal, Durban, South Africa Background: The impact of bacterial species, antibiotic drug and hospital type on the empiric therapy of nosocomial infections was evaluated within the public health care system in Kwazulu-Natal. Methods: A multi-centre surveillance studies instituted in 3 hospitals at 3 progressive levels of health care (district, regional, and tertiary) collected consecutive, non-repetitive isolates commonly implicated in nosocomial infections, viz., Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. Isolates were subjected to susceptibility testing against antibiotics recommended as empiric therapy for nosocomial infections in treatment guidelines formulated by the National department of Health using the Kirby Bauer disc diffusion method advocated by the CLSI. Percentage susceptibility across (1) bacterial species, (2) antibiotics and (3) hospital levels was statistically analysed. Results: Susceptibility to antibiotics recommended in the treatment guidelines and hence potentially successful empiric therapy ranged from 5% to 95% with multi-resistance evident in all isolates. Statistically significant differences in overall susceptibility were observed (1) across bacterial species, (2) within 2 of the 3 bacterial species for different antibiotics and (3) across hospital levels for 2 antibiotics with p values <0.001 for (1), ranging from 0.003 to <0.001 for (2) and ranging from 0.001 to <0.001 for (3). Conclusions: This study showed that the success of empiric therapy would vary depending upon the bacterial species, the antibiotic used and the hospital, thus making a strong case for institution-specific guidelines based on evidence from well-executed surveillance.

Targeting Cellular Mechanisms with Natural Antioxidants from Red Palm Oil in order to enhance Cardioprotection ESTERHUYSE AJ^1, BESTER DJ^1, DU TOIT EF^2, ENGELBRECHT AM^2, TRUTER EJ^1, VAN ROOYEN J^1 ^1Univ. Cape Peninsula, Bellville, SA; ^2Univ. Stellenbosch, Stellenbosch, SA. Background: Activation of the NO-cGMP pathway is associated with myocardial protection against ischaemia/reperfusion injury. However, high-cholesterol diets alter function of this pathway and these alterations have been implicated in both ischaemic/reperfusion injury and the development of ischaemic heart disease. Little is known about the effects of supplements such as Red Palm Oil (RPO) on the myocardial NO-cGMP-signalling pathway. Aims: 1) to determine whether dietary RPO-supplemention protects against ischaemia/reperfusion injury in rats fed a standard rat chow (control) and cholesterol-enriched diets and 2) if so, to investigate possible mechanisms for this protection. Methods: Male Long-Evans rats were fed a standard rat chow or a standard rat chow plus cholesterol and/or RPO-supplementation for 6 weeks. Myocardial mechanical function, NO-cGMP signalling pathway intermediates and MAPK phosphorylation were determined before, during and after ischaemia. Results: Cholesterol supplementation caused a poor aortic output (AO) recovery compared with the control group (35·5 % (SEM 6·2) v. 55·4 % (SEM 2·5)), but when RPO was added, the percentage AO increased significantly when compared with the cholesterol group (63·2 % (SEM 3·1); p<0·05). RPO-supplementation also increased myocardial ischaemic cGMP concentrations. Simulated ischaemia increased intracellular cardiomyocyte nitric oxide levels in the RPO-supplemented group, but not in the control non-supplemented group. Furthermore, we demonstrated for the first time that RPO-supplementation protects the isolated perfused working rat heart during reperfusion from ischaemia/reperfusion-induced injury through a MAPK-dependent pathway. Conclusions: 1) From our work it is evident that most of the RPO-induced changes occurred during the ischaemic period with the NO-cGMP pathway being a major role player. 2) Our results also suggest that hearts of cholesterol-fed animals were protected through a different mechanism (may possibly include the antioxidant capacity of RPO). 3) The proposed mechanisms include RPO protection in ischaemia via the NO-cGMP pathway and MAPK and PKB/Akt signalling pathways during reperfusion. It is therefore postulated that RPO might offer an alternative, dietary route to protect the heart against ischaemia-reperfusion induced injury.

The Use of Permeability and Reference Compound Data for Predictions and Understanding of Human Pharmacokinetics. FAGERHOLM U AstraZeneca R&D, Södertälje, Sweden. Background: Good predictions of human pharmacokinetics (PK) and drug-drug interactions (DDIs) require that permeability (Pe) is considered, the interplay between metabolism and Pe is understood, relationships between Pe vs fraction absorbed (fa) or reabsorbed (freabs) in various important organs have been established, and that reference compound data are available and used. Methods: Passive in vitro Pe and in vivo PK-data were collected from the literature. These were used for establishment of Pe vs fa or freabs-relationships in the human intestines, liver, kidneys and brain, and finding suitable reference compounds for drug absorption (intestines, brain and renal), metabolism (liver and gut-wall) and excretion (renal, intestinal and biliary). Results: Pe vs fa and freabs-relationships were established, and based on these, a Pe-based classification system (PCS) was developed. By combining the PCS and in vivo and in vitro metabolism and excretion data for reference compounds with known in vivo PK-properties, prediction of fa and freabs in various organs, hepatic clearance (CLH), gut-wall extraction ratio (EGW), excretion CL, major elimination routes, DDIs, and drug and metabolite organ/cell retention are enabled. Reference probes include atenolol (for intestinal and hepatic fa, renal freabs, EGW, gut-wall metabolism induction and inhibition potential), metoprolol (intestinal and hepatic fa, CLH, renal freabs, biopharmaceutical Pe-classification), propranolol (CLH and oral bioavailability (F)), verapamil (EGW, CLH, and brain uptake and efflux DDI), midazolam (EGW), digoxin (intestinal, renal and bile excretion CL and DDIs, hepatic uptake, and F), enalaprilat (intestinal and hepatic absorption), rosuvastatin (intestinal excretion CL) and cimetidine (renal excretion CL and PCS Pe-classification). The PCS includes 15 additional substances for Pe-categorization. Conclusions: Pe vs fa and freabs-relationships (in the PCS) and reference compound data are believed to be very useful for improved predictions and understanding of PK and DDI-potentials in man.

Primary CMF Chemotherapy in Operable Breast Cancer. FALO C, MORENO A, BENITO E, LLOVERAS B, VARELA M, Mª SERRA J, PRIETO L, AZPEITIA D, ESCOBEDO A. UNITAT FUNCIONAL DE MAMA. Institut Català d'Oncologia. Hospital Duran i Reynals, Hospital Universitari de Bellvitge (Barcelona). Spain. Background: Primary chemotherapy (PC) is becoming an accepted practice for large tumors in order to avoid mastectomies and as a surrogate of outcome. Methods: a series of 305 patients with tumors > 3cm T2-3/N0-1M0 were treated according to a multimodal approach that consisted on three courses of primary CMF followed by appropriate local treatment and three further courses of CMF or four courses of doxorubicin. Response was assessed by mammography. Results: the overall response rate was 48% (3% pathological complete response). Conservative surgery was achieved in 79.64% of the patients with a low rate of local recurrences ( 5%). Toxicity was minimal. With a median follow-up of 104 months, the 8y-DFS was 57.63% and 8y-OS was 67.65%. DFS and OS for cases with clinical response were significantly longer: 70% (p= 0.0048) and 90% (p=0.0042) respectively. Conclusion: PC with CMF is feasible. A high rate of breast conservative surgery has been achieved. Our results stress the value of PC to increase conservative surgery and as a predictor of outcome.

Synergistic or Antagonistic Interaction between Taxanes and G1/S Arresting Agents in Combination Therapy FAN W^1,2 , SUI M^2 , ZENG S^1 and ZHENG SS^1 ^1Zhejiang Univ., Hangzhou, China; ^2Med. Univ. of SC, Charleston, USA Taxanes (paclitaxel and docetaxel), a novel class of naturally occurring antimicrotubule agents, may represent the most active chemotherapeutic agents developed in the last decade for the treatment of advanced breast cancer and many other types of solid tumors. The promising clinical profiles of taxanes have also promoted considerable interest in combining them with other therapeutic agents. However, accumulating clinical data show that such taxane-based chemotherapy or modality therapy may not always increase the therapeutic efficacy. Through development of appropriate in vivo and in vitro model systems, we have evaluation of many clinically used protocols of taxane-based combination therapy. Our results revealed that some of the taxane-containing combination therapy may result in antagonistic interactions so that the actual therapeutic activity produced by two agents is less than their expected synergistic or additive effects. Specifically, our studies show that the combination of paclitaxel or docetaxel with G1-S arresting agents such as 5-fluorouracil, doxorubicin, cisplatin, gemcitabine and gamma-radiation may produce schedule-dependent antagonistic interactions. Further, we have investigated the potential mechanism by which G1-S arresting agents interfere with therapeutic efficacy of taxanes. Data obtained from a variety of assays demonstrated that G1-S arresting agents interfere with the cytotoxic effects of taxanes on both mitotic arrest and apoptotic cell death unless taxanes are administered before G1-S arresting agents. In addition, biochemical examinations revealed that paclitaxel and docetaxel could regulate several apoptosis- and mitotic arrest-related proteins such as phosphorylation of bcl-2, c-raf-1 and activation of NF-kB pathway, but these changes were inhibited when tumor cells were pretreated or simultaneously treated with G1-S arresting agents. In conclusion, our results indicate that the interaction between taxanes and G1-S arresting agents is highly schedule dependent. Exposure of tumor cells to G1-S arresting agents before taxanes could result in pronounced antagonism. The optimal schedule for this combination might be sequential exposure to taxanes followed by G1-S arresting agents. These findings suggest that careful considerations may be necessary when combining antineoplastic agents that exert their cytotoxic action at different phases of the cell cycle.

Oxystress-induced antitumor therapeutics via targeted-inhibiting heme oxygenase-1 (HSP32) in tumor FANG J^1, SEKI T^2, BHARATE GY^1, QING HB^1, NAKAMURA H^1, MAEDA H^1 ^1Sojo University, Kumamoto, Japan; ^2Venture Business Laboratory, Kumamoto University, Kumamoto, Japan. Background: Heme oxygenase-1 (HO-1), which is recently recognized as a heat shock protein (HSP32), play important roles in tumor growth. We have reported that HO-1 inhibitor, i.e., zinc protoporphyrin (ZnPP) exhibited potent anticancer activity, however its poor water solubility hamper its application. To overcome this drawbacks, we prepared water soluble micelles of ZnPP by use of poly(ethylene glycol) (PEG) and styrene-maleic acid copolymer (SMA) (PZP and SZP respecitively) and investigated their physiochemical properties and in vitro, in vivo antitumor effect. Methods: The molecular size and particle size of the micelles were measured by chromatography and dynamic light scattering. A rat splenic microsomal fraction was prepared for measurement of HO activity, by which the Ki was determined by using Line-weaver-Burk plots. In vitro cytotoxicity assay was carried out by MTT method; in vivo experiments were carried out by use of several tumor models. Results: PZP and SZP showed high water-solubility (> 200mg/ml). The molecular size of the micelle is about 144kDa, and the particle size is around 60~350nm. PZP and SZP inhibited splenic HO activity in a competitive manner, with the Ki of 0.11µM and 0.15µM, respectively, which is comparable to that of native ZnPP. MTT assay showed dose-dependent cytotoxicity in various cancer cells tested (average IC50 of 9µM), whereas normal cells showed realtive tolerance to this treatment. In vivo antitumor experiments clearly demonstrated that PZP and SZP had remarkable antitumor activities, even for the highly malignant tumor-rabbit VX-2 liver carcinoma. In addition, no apparent side effects were observed in this treatment. More important, a synergistic effect of light induced photosensitizing capabilities and HO-1 inhibitory potentials of these micelles was observed both in vitro and in vivo under localized , mild illumination conditions using a xenon light source. Conclusions: 1) Tumor-targeted inhibition of HO activity, could be achieved by using the micellar HO inhibitor based on EPR effect. Consequently, effective antitumor activity can be accomplished without any apparent toxicity in normal tissues or organs 2) PZP and SZP can also be applied for photodynamic therapy, which will further increase their antitumor activities.