Ehrlich II –2nd World Conference on Magic Bullets

A new focus on atherosclerosis treatment: transialidase from Trypanosoma cruzi as an anti-proliferative drug. HIGUCHI ML, FAGUNDES R, IKEGAMI R, REIS MM, PALOMINO S, DAMY S, SAMBIASE N, STOLF N. Heart Institute of School of Medicine of São Paulo University , Sao Paulo, Brazil Background – Chagas disease is caused by Trypanosoma cruzi that produces transialidases (TS) and Chagasic patients usually do not have atherosclerosis. Atherosclerosis is an inflammatory multifactorial disease, presenting increased levels of sialic acids (SAs), transferrin and iron in the plaques possibly related with free radical release and inflammation. TS produced by TC may remove SAs from the plaques. Aim: To report a new way for treating atherosclerosis focusing on multitargets simultaneously: removal SAs with a recombinant TC TS, a metal chelant (PDTC) and antioxidative nanoparticles derived from three plant extracts in an animal model. Methods - We compared six groups (n=5) of rabbits. GI – normal diet; GII – 1% cholesterol diet for 12 weeks; GIII – 1% cholesterol diet for 12 weeks and, in the last 4 weeks, injection of transialidase (TS) plus PDTC. Groups IV, V and VI received the same scheme of GIII plus aged extracts: Allium sativum (AL); AL+ Ginko biloba (GB) and AL+GB + Zingiber officinale (ZO), respectively. The thoracic aorta atheromas stained with Sudan IV were macroscopically detected and plaque and fat areas in cross sections were microscopically detected using an image analysis system. The LDL in the serum was also measured. Results – The combined therapy using TS+PDTC and 3 plant extracts was the most effective scheme in reducing atheromas and LDL serum levels to normal values. (Figure and Table below). Conclusion – The successful treatment of experimental animal atherosclerosis using T.cruzi transialidase, PDTC and plant extract nanoparticles combined formulation brings a new promissory way for the treatment of human atherosclerosis.

Using Immune Complexes to Enhance Antigenicity and Immunogenicity of a Neutralizing Epitope on HIV-1 Envelope gp120 HIOE CE^1, VISCIANO ML^1, GORNY MK^1, TUEN M^1 ^1New York University School of Medicine and VA New York Harbor Healthcare System, New York, USA. Background: HIV envelope gp120 is a crucial antigenic target for Ab responses against the virus; but most, if not all, of the critical gp120 epitopes are not readily accessible for Ab recognition or are poorly immunogenic. Since antibodies are known for their capacity to enhance immune responses to specific antigens, we investigated the use of immune complexes as immunogens to improve the overall Ab titers against gp120 as well as to target the Ab response toward specific neutralizing epitopes. Methods: Immune complexes were made of gp120 from different HIV-1 subtype B strains and human monoclonal Abs (mAbs) to the CD4-binding site (CD4bs) of gp120. The anti-CD4bs mAbs were selected because a) these mAbs can induce significant structural changes in gp120 that potentially expose neutralizing epitopes, and b) the gp120/anti-CD4bs complexes are relatively resistant to degradative enzymes and may serve as a durable antigen source for stimulating B cells and Ab production. The antigenicity of the gp120/anti-CD4bs complexes was examined by ELISA using biotinylated mAbs to different gp120 regions, while the immunogenicity of the complexes was evaluated by immunizing BALB/c mice intraperitoneally with the complexes. Results: HIV-1 gp120 complexed with anti-CD4bs mAbs displayed higher reactivities with mAbs specific for the neutralizing epitopes in the V3 loop. The enhanced reactivities were observed with gp120 from different HIV-1 strains. Immunization of mice with the complexes also elicited >1 log higher titers of gp120-specific serum IgG and IgA than immunization with uncomplexed gp120 or other gp120/mAb complexes. Notably, the enhanced Ab production was directed against V3, and the use of gp120JRFL bearing V3 of the HIV-1 subtype B consensus sequence increased the cross-reactivity of Abs generated. Importantly, potent neutralizing activity was observed in sera from mice immunized with the gp120/anti-CD4bs complexes, although assessment of the breath of neutralizing activities against a panel of subtype B HIV-1 isolates is still on-going. Conclusions: Overall, these results indicate that the use of immune complexes is a promising approach to improving the immunogenicity of HIV-1 envelope and to directing the Ab responses toward virus-neutralizing epitopes on this antigen.

Consequences to morphology of primary neurons as an effect of Nitric Oxide on plasma membrane fluidity HIPPE S, HEUMANN R Department of Molecular Neurobiochemistry, NC 7 / 172, Ruhr-University of Bochum, Universitätsstraße 150, D-44801 Bochum. Nitric Oxide is described as a signal molecule of special importance in the nervous system mainly acting via cyclic guanosine monophosphate which is generated by the Nitric Oxide sensitive guanylate cyclase. However, our group [Grote Westrick, Hippe et al. (in preparation)] could show that Nitric Oxide has a direct effect on the plasma membrane of living cells by demonstrating the same effect on artificial generated liposomes. In consequence of Nitric Oxide donor application membrane fluidity is increased. Initial experiments have shown that the same effect occurs on neuronal cells. By the use of NOC-18 (Nitric Oxide donor) and L-NAME (Nitric Oxide synthase inhibitor) we show expected morphological changes on primary hippocampal neurons with scanning electron microscopy and immunocytochemical staining in addition to enhanced membrane fluidity measured by fluorescence recovery after photobleaching (FRAP). Further we plan to establish solid state ^31P-NMR anisotropy measurements of artificial generated liposomes followed by electron microscopy analysis to detect changes in the membrane of those vesicles. In combination with mass spectrometry measurements we intend to demonstrate the molecular basis of the characterized direct NO effect on neuronal plasma membranes.

Changes in Plasma Protein Binding of an Extensively Bound and Highly Extracted Drug, Propofol, Have Clinical Relevance HIRAOKA H ^1, 2, HIRAOKA T^1  ^1Department of Anesthesiology, Medical & Art Office Takasaki, Takasaki, Japan, ^2Department of Clinical Pharmacology, Gunma University Graduate School of Medicine, Maebashi, Japan. Background: Changes in plasma protein binding have little clinical relevance for most drugs. However, this situation is likely to be clinically important for a limited number of highly cleared drugs that are extensively protein bound, administered intravenously and have a narrow therapeutic index. Propofol has been widely used in the clinical field of anesthesia and intensive care medicine and is one such drug. Thus, changes in plasma protein binding of propofol would be expected to alter disposition of propofol and its potency. Therefore, we have investigated propofol’s pharmacokinetics during cardiopulmonary bypass (CPB) to determine whether the predicted theoretical changes actually occur. Methods: After induction of anesthesia propofol was infused continuously during surgery. Propofol's concentration was measured by HPLC in blood samples collected from the radial artery during surgery at predetermined intervals. The drug's unbound fraction in arterial plasma was estimated via equilibrium dialysis. Bispectral index (BIS) and burst suppression ratio (BSR) were measured continuously to quantify the potency of propofol. Results: The total concentration of propofol in blood was unchanged during surgery. By contrast, the fraction of unbound propofol in blood increased by 2-fold during cardiopulmonary bypass. BIS was significantly decreased and BSR was significantly increased during CPB. Conclusions: The potency of propofol significantly increased during CPB without any alteration in the total drug concentration. The enhanced efficacy would be caused by a reduction in plasma binding of the drug. Furthermore, total drug levels of propofol are influenced by cardiac output. We also report the cases of accidental hemorrhagic shock in patients undergoing liver transplantation, where 2-fold increases in both total propofol concentrations and the unbound fraction result in 4-fold increases in the unbound concentrations. Changes in plasma protein binding of propofol would be clinically important.

Rational Design of Specific Inhibitors of -Glutamyl Transpeptidase (GGT) and -Glutamylcysteine Synthetase (GCS) for Modulating Cellular Glutathione and Redox Status HIRATAKE J^1, NAKAJIMA M^1, HAN L^1, KAWAMURA N^1, HIBI T^2, NAKAJIMA Y^2 ^1Institute for Chemical Research, Kyoto University, Uji, Japan; ^2Fukui Prefectural University, Fukui, Japan. Background: Glutathione (-Glu-Cys-Gly, GSH) is a ubiquitous tripeptide that serves as a major cellular antioxidant or detoxifying agent. Therefore cellular GSH level is deeply associated with drug resistance of cancer cells and pathogens. GSH biosynthesis is dependent on the activities of -glutamylcysteine synthetase (GCS), a second biosynthetic enzyme, and of -glutamyl transpeptidase (GGT) that hydrolyzes the extracellular GSH to supply cells with its constituent amino acids. Aims: 1) To develop rationally specific inhibitors of GGT and GCS. 2) To understand the interaction of the inhibitors with the enzymes at the molecular level. Methods: A series of electrophilic -phosphonic diester analogs of GSH were synthesized as mechanism-based inhibitors of GGT. Each inhibitor was evaluated by the second-order rate constant for enzyme inactivation. The structure-activity relationships were used for the active-site mapping. For GCS inhibitors, sulfoximine-based transition-state analogs were synthesized according to the X-ray crystal structure of E. coli GCS. The recognition of Cys by an Arg residue of E. coli GCS was utilized for the inhibitor design, and a cyano group was introduced as a SH mimic. Results: The -phosphonic diesters served as potent irreversible inhibitors of both E. coli and human GGTs by attaching covalently with the catalytic Thr. The potency was highly dependent on the structure mimicking the Cys-Gly moiety, but human GGT was far more selective, suggesting that human GGT served as a “glutathionase” in vivo. The -phosphonic diesters did not inhibit glutamine amidotransferases. The most potent GGT inhibitor exhibited ca. 6000 times as potent as acivicin, a hitherto used non-selective GGT inhibitor. The introduction of CN group significantly increased the potency of inhibitor for both E. coli and a pathogenic Streptococci GCS. The best inhibitor was ca. 2500 times more potent than BSO, a commonly used GCS inhibitor. Conclusion: The reaction mechanisms and the structure of GSH biosynthetic and degrading enzymes were highly useful for rational design of selective and potent inhibitors. This is the first step toward controlling the cellular GSH levels that is highly promising for combating drug resistance.

Effectiveness and complications of treatment for childhood liver tumors – from the experience of JPLT (Japanese Study Group for Pediatric Liver Tumor) study HIYAMA E^1^,2, YAMAOKA H^1^,2, NISHIMURA S^1^,2, HISHIKI T^2, HIYAMA K^1^,2, SASAKI F^2, HORIE H^2, OHNUMA N^2 ^1HiroshimaUniv., Hiroshima, Japan; ^2Japanese Study Group for Pediatric Liver tumor, Japan Background: Hepatoblastoma (HB), which is derived from hepatic precursor cells, is a primary liver cancer that occur predominantly in children. The prognosis of children with HB has been improved significantly during the past two decades by the induction of neoadjuvant and adjuvant chemotherapy. JPLT group launched in 1991 is running cooperative treatment studies based on the cisplatin/pirarubicin regimen (CITA) as the first line on HB. Aims: 1) To evaluate the efficacy of JPLT protocol. 2) To evaluate the late complications of the patients by JPLT regimen Methods: Until 2007, 235 HB cases have been registered in JPLT-2. According to PRETEXT classification, standard risk (SR) tumors (PRETEXT 1-3) without metastasis were 146 and high risk (HR) patients (PRETEXT-4 or metastatic cases) were 89. In JPLT-2 protocol, high dose chemotherapy with stem cell transplantation (SCT) was carried out for metastatic tumors and living-donor liver transplantion (LD-LT) for some PRETEXT-4 tumors. Late complications were evaluated in 126 cases that received complete regimen and survived more than 2 years. Results: The 3-year overall survival (OS) and event-free survival were 80% (95% in SR and 54% in HR) and 67%, respectively. The response rates of CITA regimen were 82% and 52% and the complete resectability was 87% and 41% in SR and HR cases, respectively. In 23 cases who underwent high dose regimen with SCT, only 13 cases (57%) were cured. In 31 cases who underwent LD-LT, 3-year OS was 76%. Late complications are shown in Table. Long term toxicity Grade 1-2 Grade 3-4 Grade 5 total Growth & development 3 1 - 4 Cardiac 11 2 13 Auditory 17 5 22 Secondary malignancies - 2 2 4 Conclusions: 1) SR cases had a fair to excellent outcome but, late complications are now problematic. 2) The outcome of HR cases remained poor. Reduction of chemotherapy for SR cases and more promising strategies for HR cases including LT and new targeting drugs should be developed in international collaboration because the case numbers were limited.

Genes commonly upregulated in immortal cancer cells but not in immortalized non-cancerous cells are novel molecular targets for universal anti-cancer strategy Hiyama K^1, Tanimoto K^1, Hiyama E^1, Nishiyama M^1^,2 ^1HiroshimaUniv., Hiroshima, Japan; ^2 Saitama Medical Univ. Saitama, Japan. Background: Molecular targetting anticancer drugs are quite promising in some specific cancers but usually limited to selected patients. It is widely accepted that activation of telomerase induces indefinite proliferation capacity in almost all kind of cancer cells resulting in poor progonosis of patients, but never in normal human cells in vivo, resulting in senescence. Methods: To elucidate the genes responsible for telomerase-mediated cellular immortalization in cancer cells, we established telomerase overexpressing in vitro immortalized normal and transformed cells lines by gene transfection of TERT and/or SV40 EA. By comparing the expression profiles of them and various cancer-derived cell lines with those of normal cells using microarrays, we explored the genes that are differentially expressed in immortal cancer cells but not in immortalized non-cancerous cells. The characteristics of the candidate genes were examined in vitro and in clinical cancer tissues of various organs. Results: While 3 genes were detected as commonly down-regulated, 34 genes were commonly up-regulated in the cancer cell lines of any origin, except for breast cancer (“*” in Table). Large part of these genes were also upregulated in various cancer tissues but not in in vitro immortalized normal cells. Some of them also correlated with advanced stage and/or poor progonosis of the patients. Knockdown by siRNA of these genes demonstrated inhibititory effects on cellular growth in cancer cell lines. Table. Number of the genes differentially expressed in immortal cancer cells. Cancer cell line Lung Esophagus Colon etc Breast Ovarian Pancreas Common Upregulated 570 196 958 196 935 2553 34* Downregulated 675 396 747 447 784 1504 3 Conclusions: We have found the candidate genes that may be involeved in cellular immortalization commonly and specifically in cancer cells, but not in normal cells, cooperatively with telomerase. The genes we found are expected to become the novel molecular targets of universally effective anti-cancer therapy.

System approach to Magic Bullets: tissue and cell targeted delivery of HIV and cancer drugs HO Rodney J.Y. University of Washington, Seattle, Washington 98195-7610, USA The estimated number of drug targets between 1940-1990 was about 500. In post-genomics era, over 1700 human genes express function proteins suitable for serving as drug targets. Despite the drug-target growth, approval of novel therapeutics for human diseases has not increased substantially. In addition to well-documented preclinical challenges (in drug absorption, distribution, metabolism and elimination) in the development of new drug candidates, increasing frequency in drug failures during the late-stage clinical trials are often attributed to toxicity or lack-of-efficacy. Consequently, introduction of new drugs into market is low despite increased expenditure by pharmaceutical industry and government sponsors. To improve success rate, distribution profile of drug targets in “normal” and “cancerous or virus-infected” cell are being elucidated and mapped with the help of various in vivo-imaging techniques as a part of increased collaborations among basic, preclinical and clinical scientists, as well as integration of translation research early in drug discovery and development. Even with improved understanding in “biodistribution of drug targets,” oral or systemically administered drug molecules must cross a number of physiologic—tissue, cell and enzyme (such as cytochrome P450’s metabolism in the gut and liver) barriers before reaching the virus or cancerous cells found in target tissues and cells. A significant fraction of drugs are either eliminated in the gut and liver without ever reaching systemic blood circulation or being metabolized and inactivated. Some of the metabolites also induce untoward responses in liver kidney and other tissues. As a result, current oral anti-HIV drug combination therapies could reduce virus load to undetectable levels in the blood, but could not clear the virus in the tissues such as those in lymphoid tissues and cells. Building on the physiologic and biologic understanding at systemic levels, and applying the advances in drug delivery technologies, we have made progress in improving drug localization at three progressive levels; (1) lymphoid tissues, (2) HIV host cells, (3) HIV drug targets—protease and reverse transcriptase found in HIV infected cells. This presentation will highlight our results in developing a systematic, practical, and novel approach to accomplish these goals using a HIV infected macaques model. Authors’ disclosure statement: Supported by NIH grants AI077390; AI52663; GM62883; NS39178, and Milo Gibaldi Endowment Fund.

Pharmacokinetic/pharmacodynamic considerations for inhaled glucocorticoids HOCHHAUS G Univ. of Florida, College of Pharmacy, Gainesville, FL Background: For the last 30 years, pulmonary drug delivery has been successfully employed for topical therapy of pulmonary diseases, with the goal of achieving pronounced pulmonary effect while reducing systemic side effects. The degree of pulmonary targeting is determined by a number of pharmacokinetic (PK) and pharmacodynamic (PD) factors. This presentation will discuss these relationships, and will review the pharmacokinetic and pharmacodynamic properties an inhaled glucocorticoid should provide. Methods: This presentation is based on previously published in-vitro experiments, PK/PD simulations, animal and clinical studies of glucocorticoid action including the effects of biopharmaceutical parameters on pulmonary selectivity. Results: Pharmacokinetic/dynamic simulations suggested that pharmacodynamic properties of an inhaled glucocorticoid beneficial for pulmonary targeting are low oral bioavailability, pronounced systemic clearance and distinct pulmonary residence time, while factors such as protein binding and degree of receptor affinity can be adjusted for by dose. PK/PD tools were also suitable to address the question when once-daily glucocorticoids should be administered. A pulmonary targeting model in rats was able to demonstrate the relevance of the results obtained in PK/PD based simulations and revealed the importance of biopharmaceutical optimization for the degree of pulmonary selectivity. Conclusions: The presented work suggests that the use of PK/PD tools within the rationale drug design is also beneficial when drugs for topical use, such as inhalation, are to be designed. Authors’ disclosure statement: Studies were supported in part by GSK and Astra-Zeneca.

Pharmacokinetic-Pharmacodynamic Modeling of Calcium Channel Blockers in Animal Models of Hypertension. HÖCHT C, BERTERA F, MAYER M, OPEZZO JAW, TAIRA CA. Department of Pharmacology, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina. Background: Pharmacokinetic-pharmacodynamic (PK-PD) properties of calcium channel blockers have been scarcely studied in animal models of hypertension. Aims: 1) To compare different pharmacodynamic models for PK-PD modeling of diltiazem (DTZ). 2) To evaluate pharmacokinetic properties of DTZ in two different models of hypertension, such as aortic coarctated rats (ACo) and spontaneously hypertensive rats (SHR) 2) To establish sensitivity to calcium channel blockade in SHR and ACo rats. Methods: A “shunt” microdialysis probe was inserted in carotid artery of anaesthetized SHR, ACo and normotensive control rats for determination of DTZ plasma levels and their effects on blood pressure and heart rate after drug administration (1, 3 or 6 mg.kg^-1, iv). Correlation between DTZ plasma levels and their cardiovascular effects was established by fitting the data to a conventional and modified Emax model. Results: Volume of distribution and clearance of DTZ was greater in both SHR and ACo with regards to normotensive animals. A good correlation between plasma levels of DTZ and their hypotensive and chronotropic effects was found in all experimental groups using both PK–PD models. Application of the modified Emax model for PK–PD modeling of DTZ allowed a more accurate and precise estimation of PK–PD parameters than the Emax equation do. Initial sensitivity (S0), estimated by the modified Emax model, to DTZ chronotropic effect was greater in SHR with regards to control animals after administration of 1 mg kg^−1. Conversely, no differences were observed in S0 to DTZ chronotropic effect comparing ACo and normotensive animals. Both SHR and ACo rats showed increased S0 to DTZ hypotensive effect with regards to normotensive animals. Conclusions: 1) Modified Emax model allowed both a precise and accurate estimation of PK-PD parameters, suggesting that this pharmacodynamic model is the most suitable for PK-PD modeling of DTZ. 2) ACo and SHR induced profound changes in DTZ pharmacokinetic behaviour, affecting both drug distribution and clearance. 3) Experimental hypertensive rats showed an increased sensitivity to DTZ blood pressure lowering effect.

T-cells as Magic Bullets: Recombinant Vaccine Strategies for Cancer Immunotherapy HODGE JW Director, Recombinant Vaccine Group, Laboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, Bethesda MD. Cancer vaccines can induce T-cell responses that can preferentially target tumor cells while sparing normal cells. Preclinical and clinical investigations currently underway are employing novel strategies for combining vaccines with conventional and experimental anticancer therapies. To date, the FDA has not approved a therapeutic cancer vaccine. However, the results of recent investigations suggest an increasing role for vaccines in new models of combination therapy for many types of cancer. In this talk I will discuss therapeutic cancer strategies that employ vaccines in combination with local radiation, chemotherapy, hormone therapy, and anti-CTLA-4 mAb. Preclinical studies have shown that certain anticancer agents have immune modulatory effects that result in up-regulation of surface expression of MHC molecules, tumor-associated antigens, or Fas on malignant cells, rendering them more susceptible to immune destruction. Preliminary results of clinical studies using combination strategies have demonstrated a postvaccination antigen cascade, prolonged time to disease progression, and improved overall survival. Several larger randomized trials are ongoing, and more are required to support these findings.

The Utility of the Cholestanol:Cholesterol Ratio in Predicting LDL-Cholesterol response to Atorvastatin 80 mg: A paradigm for individualized lipid-lowering therapy HOENIG MR, WALKER PJ. Department of Vascular Surgery, Royal Brisbane & Women’s Hospital Australia. Background: Cholestanol is an endogenously produced marker of cholesterol absorption that has been validated with sterol balance methodology. A retrospective analysis by the 4S investigators suggested that patients in the highest quartile of the cholestanol:cholesterol ratio (CCR), indicating a cholesterol ‘absorber’ phenotype did not benefit from statin therapy in the 4S trial. Patients with high CCR have high cholesterol absorption and low synthesis; hence statins are expected to be relatively ineffective in such patients. Since recent meta-analyses have shown that benefit from statin therapy is directly related to LDL-cholesterol (LDL-C) response, we sought to prospectively assess whether the CCR can predict LDL-C response to Atorvastatin and identify statin hypo-responders, defined as an LDL-C response <40%. Aims: To assess the predictive value of CCR in predicting LDL-C response to Atorvastatin 80mg. Hypotheses: CCR correlates with the LDL-C response to Atorvastatin. Patients who have a higher CCR will have a suboptimal LDL-C response to statin and the CCR can be used to identify statin hyporesponders (LDL-C response <40%). Methods: ~60 patients with coronary artery disease or coronary risk-equivalents were recruited into the study. Baseline measures included CCR, baseline lipid panel, insulin, CRP, assessment of metabolic syndrome criteria, blood sampling for genetic analysis and an MRI substudy for patients without contraindications. Patients were given a 6 week course of Atorvastatin 80mg before follow up to determine the lipid and CRP response to the course of therapy. Major Results: The 6 week course of Atorvastatin reduced LDL-C by a mean of 55.7% (P<0.01 for post vs pre-treatment LDL-C values); however, there was marked heterogeneity in the LDL-C response (range 12-80%). Of the baseline variables assessed, only the CCR correlated with the percent LDL-C reduction (p<0.01, R2=0.21). When the population was divided into quartiles of CCR, statin hypo-responders (LDL-C response <40%) clustered in the patients with the highest CCR (quartile 4 vs quartile 1 p<0.03). Patients in the highest quartile (quartile 4) of CCR showed less features of the obesity and the metabolic syndrome than those in the lowest quartile (quartile 1) with significant differences being found in number of metabolic syndrome criteria, HDL-C, VLDL-C, TG, BMI (all p<0.01). ROC analysis suggested that the CCR can identify statin hypo-responders (LDL-C response <40%) with a 100% sensitivity and 62% specificity (Area under curve = 0.85, p<0.01). Significance: This is a highly significant finding and highly applicable to personalized medicine in the area of lipid-lowering therapy. Of note, pharmacogenomic approaches to variability in statin response typically yield R2s of 0.01-0.02. We propose that the CCR can be used to predict LDL-C response to statin therapy and identify statin hypo-responders who might be expeditiously treated with anti-resorptive co-therapy such as ezetimibe. These findings are particularly applicable to high risk cardiovascular patients where aggressive lipid-lowering therapy is warranted.

A Genomic Parasite in the Evolution of Metazoan Development Evolvability and Evolutionary Constraints as Fingerprints of Selection HOENIGSBERG H Instituto de Genética Evolutiva & Biología Molecular and Instituto de Genética Ecológica Y Biodiversidad del Trópico Americano. Bogotá D.C., Colombia It is a challenge to understand how development emerged as a mechanism to dismantle and dismiss the intromission of foreign parasites in order to consolidate a higher-level multicellular unit of selection where more heritable variations in fitness, required for complex organization, can be procured.levels in the biological hierarchy-genes,networks of genes, chromosomes, cells, organisms etc.,possess heritable variations in fitness to varying degrees,and as such they function as units of selection in the evolutionary process.to pass from each of these levels to the next constitutes a major transition in evolutionary history.when analyzing the splendid road epitomized by these transitions in the units of selection, it is only possible to conceive of three processes:1)the molecular recognition of the convenience of exchanging the higher energy cost of cooperating cells with more fitness than single cells selection. After that first recognition the emergence of cooperation among cells is possible.2)the establishment of the mechanisms to regulate conflict, and 3)the regulation of cell differentiation and compartmentalization.

Non-Selective and Selective Adenosine Receptor Agonists in the Treatment of Radiation- and Chemotherapy-Induced Myelosuppression HOFER M, POSPÍŠIL M Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic Background: Adenosine acts as a regulator of many cellular functions including those of cell proliferation and differentiation. Regulatory role of extracellular adenosine is based on the activation of specific cell surface receptors. Adenosine receptors can be classified as A1, A2A, A2B, and A3. We have investigated the role of adenosine receptor agonists in regulation of processes proceeding in hematopoiesis suppressed by ionizing radiation or cytotoxic drugs. Methods: The studies have been performed on mice. The animals were exposed to ionizing radiation or to cytotoxic drugs. Non-selective or selective adenosine receptor agonists were administered to the mice in protective or treatment regimens. Hematopoietic status of the animals was assessed by a complex evaluation comprising parameters of hematopoietic progenitor and precursor cells, as well as of peripheral blood cells. Results: Non-selective activation of adenosine receptors has been achieved by concommitant administration of adenosine monophosphate (AMP), an adenosine prodrug, and dipyridamole (DP) which prevents cellular uptake of adenosine and potentiates, thus, its receptor-mediated action. This drug combination has been found to support hematopoiesis both in normal and radiation- or chemotherapy-exposed mice. DP+AMP combination has been also observed to potentiate hematopoiesis-stimulating effects of granulocyte colony-stimulating factor. Selective activation of adenosine A1 receptors by N^6-cyclopentyladenosine inhibited whereas that of A3 receptors by N^6-(3-iodobenzyl)adenosine-5’-N-methyluronamide (IB-MECA) stimulated proliferation of hematopoietic progenitor cells. IB-MECA has been found to support hematopoiesis in γ-irradiated mice. Conclusions: Elevation of extracellular adenosine leading to non-selective activation of adenosine receptors stimulates hematopoiesis. This effect can be utilized in the treatment of myelosuppression of various origin, as shown in animal experiments. Utilization of synthetic adenosine receptor agonists, selective for individual adenosine receptor subtypes, has revealed that adenosine A3 receptor activation is responsible for the earlier described action of elevated extracellular adenosine. These findings may be of significance also in clinical practice. Authors’ disclosure statement: This work was supported by the Academy of Sciences of the Czech Republic (grant AV0250040507) and by the Grant Agency of the Czech Republic (grants 305/06/0015 and 305/08/0158).

Heterocyclic hydrazones induce radical formation and dissipation of mitochondrial membrane potential Hofmann J^1, JennyM^1, Hermann^1, Easmon J^2, Puerstinger G^2, HeinischG1, Shtil AA^3, Condorelli DF^4, SciréS^4, Musumarra G^4 ^1Innsbruck Medical University, Innsbruck, Austria; ^2University of Innsbruck, Innsbruck, Austria; ^3Blokhin Cancer Center, Moscow, Russia; ^4Università di Catania, Catania, Italy. Background: The novel compound N-benzoxazol-2-yl-N’-1-(isoquinolin-3-yl-ethylidene)-hydrazine (EPH136) has been shown to exhibit antitumor activity in vitro and in vivo. A COMPARE analysis showed that the patterns of cellular effects of EPH136 are not related to any of 175 standard antitumor agents with a known mechanism of action. Methods: In order to help identify the mechanism of action we employed the following methods: (i) a bioinformatics approach, called partial least squares modelling in latent variables, (ii) a DNA microarray for detection of the expression of ~5000 known genes following treatment of HT-29 colon carcinoma cells with a two-fold IC50 concentration of EPH136, (iii) detection of radicals by luminol, (iv) determination of the mitochondrial membrane potential by JC-1 and by TMRM, (v) treatment of cells with EPH136 and the radical scavenger N-acetylcysteine and (vi) treatment of cells with elevated levels of glutathione. Results: The 60 genes found by the bioinformatic approach to be most important for the antiproliferative effect of EPH136 are involved in nucleoside, nucleotide, nucleic acid binding and metabolism, developmental processes, protein modification and metabolism. The genes that were up-regulated more than two-fold (in DNA microarrays) compared to untreated controls belong to the same classes as found by the bioinformatic approach. Many of these proteins are regulated by oxidation/reduction and so we concluded that formation of radicals may be involved in the mechanism of action. We found that EPH136 leads to generation of radicals, swelling of mitochondria and dissipation of the mitochondrial membrane potential. The antiproliferative activity of EPH136 was prevented by the radical scavenger N-acetylcysteine. Cells with elevated glutathione exhibited resistance to EPH136. Conclusions: The mechanism of action of the novel experimental anticancer drug EPH136 is generation of radicals and dissipation of the mitochondrial membrane potential.

Alpha1-Adrenoceptor-Mediated Pronociception Attenuates Induced Analgesia in Rat HOLDEN JE Univ. Illinois at Chicago, Chicago, IL, USA Background: Unrelieved pain is a worldwide problem. Understanding mechanisms of endogenous pain circuits may identify areas that interfere with clinical pain relief. The A7 catecholamine cell group provides the majority of noradrenergic input to the spinal cord dorsal horn in some rat strains. Stimulation of the A7 cell group produces antinociception blocked by alpha2-adrenoceptor antagonists. This finding forms the basis in part for clinical use of alpha-adrenergic agonists and reuptake inhibitors as analgesics. Yet alpha-adrenergic agents are not always effective and have major side effects. We report several studies in which we stimulated the A7 cell group directly with morphine sulfate, or indirectly by stimulating the hypothalamus, and obtained pronociception mediated by alpha1-adrenoceptors that attenuates alpha2-mediated antinociception. Methods: In the first study, morphine sulfate (5 or 10 ug) was microinjected in the A7 cell area in lighty anesthetized female sprague-dawley rats, followed by intrathecal (IT) injection of either the alpha1 antagonist WB4101, the alpha2 antagonist yohimbine (97 nmol), or saline for control. Response latencies were measured using the tail flick and foot withdrawal tests. In subsequent studies the lateral (LH) or the posterior (PH) hypothalamus was stimulated using carbachol and IT alpha-antagonists given as described above. Results: The 5 ug dose of morphine decreased foot and tail withdrawal latencies, a hyperalgesic response. IT injection of yohimbine did not alter this hyperalgesia, but WB4101 reversed the hyperalgesia and produced antinociception. Microinjection of 10 ug morphine in the A7 area did not alter nociceptive responses. However, IT injection of yohimbine produced significant pronociception, while WB401 produced significant antinociception. The LH and PH project to the A7 cell group and do not contain spinally-projecting noradrenergic neurons. Stimulation of either the LH or PH produced antinociception blocked by IT yohimbine but increased by IT WB4101. Conclusions: Stimulating the A7 cell group produces bidirectional control of nociception in which alpha1-adrenoceptors increase nociception while alpha2-adrenoceptors inhibit nociception. These findings indicate that therapies involving the descending alpha-adrenergic system may be less effective if alpha1-adrenoceptors are activated along with alpha2-adrenoceptors.