Ehrlich II –2nd World Conference on Magic Bullets

Formulation Challenges in Biologics FRIESS W Ludwig-Maximilian-University; Department of Pharmacy; Pharmaceutical Technology and Biopharmaceutics Background: Biotechnology derived products have reached a share of more than 30% of the newly approved drugs. The formulation of these compounds presents specific challenges. This is due to the parenteral application and specific instability. Additional challenges are presented by antibodies which often require high dosing. Methods: Various routes of formulation of highly concentrated antibody formulations have been tested. The pros and cons of different manufacturing methods are discussed. As a second example the formulation of hydrophobic cytokine is presented which demonstrates solutions to problems with adsorption and protein-protein interactions. Results: Ultrafiltration / Diafiltration processes like tangential flow filtration are suitable but the flow rate is reduced with increased protein concentration and viscosity. At the same time substantial shear stress can affect the protein structure. As an alternative drying via lyophilisation or spray drying followed by reconstitution with less liquid is a favoured approaches. But this process leads to enrichment of excipients next to the protein which has to be considered. Furthermore reconstitution times can be substantially increased. Precipitation and crystallisation of antibodies followed by application of a suspension or a solution after redissolution may be limited by protein stability and residual precipitation agent. Overall physical stability of antibodies may be scrutinized due to the high concentrations leading to aggregation and precipitation. Syringeability has to be assured despite the increased viscosity with protein concentrations and can be controlled e.g. by pH and ionic strength. For the formulation of hydrophobic proteins, adsorption phenomena can be resolved by adjusting pH and ionic strength in combination with surfactants and the container quality. HSA as an excipient substantially changes its properties depending on the formulation and may not be adequate for stabilization of protein drugs. Conclusions: (1) high concentration liquid formulations can be realized by drying-, TFF- and precipitation - processes; (2) go for HSA-free formulations (3) formulation development of biosimilars requires an even better understanding (4) develop appropriate analytic tools e.g. FTIR, fluorimetry.

Psoriasis [Ps]: A Possinle Candidate for Vaccination FRY L    BAKER BS Imperial College, London W2 1PG   Background  Ps is a common skin disease affecting 2-3% of the European and American populations. It is a mutifactorial disease with both genetic and  enviromental factors. Histlogically Ps is characterised by hyperproliferation of the keratinocytes. It was shown some 20 years ago that this hyperproliferation was T cell mediated  However what has eluded detection is the antigen responsible for initiating this immune  response. Methods. Clinical evidence has shown that psoriasis can be triggered by beta hemolytic streptococci [BHS]..Our group has studied T cell responses to BHS in the blood and skin. Results. T cells in the blood and skin responded to BHS extracts. Next we showed that this reponse was triggered by cell wall extracts. Further studies have now shown that the cell wall antgen was peptidoglycan [PG] and this is an adaptive immune response Conclusions. PG is also known to trigger the innate immune response and this system is known to be involved in the pathogenesis of Ps. The antigen in psoriasis may therefore be the peptide brides in streptococcal PG. The identification of this peptide opens the way to possible vaccines for Ps.

Estrogen Receptor Beta may be a Novel Target for the Beneficial Effects of Estrogens in Females, and Androgens in Males, for Anxiety, Depression, and Cognitive Function FRYE CA^1-4, WALF AA^1 ^1Psychology, ^2Biology, ^3Center for Neuroscience, ^4Center for Life Science Research, Univ. at Albany-SUNY, Albany, NY, USA Background: Hormone-replacement therapies may have some beneficial effects for cognitive or affective processes; however, their effects and mechanisms in this regard are not well-characterized. Studies in our laboratory have focused on the effects and mechanisms of estrogens, such as 17β-estradiol (E2), and androgens, such as the 5-reduced metabolite of testosterone, 3α-androstanediol (3-diol), for their anti-anxiety, anti-depressant-like, and cognitive-enhancing effects in female and male rodents. Moreover, estrogens have well-known trophic effects, which can increase risk of some cancers. As such, it is important to discern the mechanisms of steroids for their beneficial versus unwanted proliferative effects. Our laboratory has been investigating the  isoform of the estrogen receptor (ER) as a putative target for these effects. Methods: We have investigated effects on anxiety/depression and cognitive behavior utilizing the following three approaches. First, the effects of systemic or intra-brain administration of selective ER modulators (SERMs) or selective androgen receptor modulators (SARMs), which vary in their affinity for ERα or ERβ, to female and male rodents were assessed. Second, intra-brain administration of ER and/or ER antisense oligonucleotides (AS-ODNS) to rats administered SERMs or SARMs was utilized. Third, the effects of SERMs or SARMs administration to mice with targeted deletions of ER (βERKO) were assessed. Furthermore, we have determined the whether some of these treatments alter tumorigenesis following exposure to a chemical carcinogen. Results: Systemic or intra-brain administration of SERMs or SARMs reduces anxiety and depression-like behavior of rats and mice. ERβ, but not ERα, AS-ODNs attenuate the beneficial effects of E2 and 3α-diol. Effects of SERMs or SARMs to reduce anxiety-like behavior and enhance cognition are not observed in βERKO mice. We have preliminary evidence about the tumorigenic role of SERMs, and have shown that E2 increases tumor burden of young ovariectomized rats. Conclusions: These data support the notion that the beneficial effects of estrogens in females or androgens in males for psychological (improve affect/mood, cognition) may be via actions at ERβ.

Role of antiproliferative enzyme indoleamine (2,3)-dioxygenase in the impaired immune function in infectious diseases BRANDACHER G,^1 SCHROECKSNADEL K,^2 SUCHER R,^1 MARGREITER R,^1 FUCHS D^2 ^1Center of Operative Medicine, Department of Visceral, Transplant, and Thoracic Surgery, and ^2Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Austria In several pathologic conditions like infections, autoimmune syndromes, cardiovascular and neurodegenerative disorders as well as malignant disease, activation and inflammation are strongly involved. Pro-inflammatory cytokines like interferon- (IFN-) play a dominant role in the clearance of infections with viruses or intracellular bacteria and parasites but also in the development of inflammation. In various cells, the expression of tryptophan-degrading enzyme indoleamine (2,3)-dioxygenase (IDO) is induced by IFN- as a part of its antimicrobial armature. Activation of IDO restricts protein biosynthesis by deprivation of essential amino acid tryptophan and thereby growth of pathogens is halted. As a side effect, also development and proliferation of normal host cells like activated T-lymphocytes is diminished. Accordingly, IDO appears to represent a critical step within host-response directing whether immune activation is successful in controlling an intracellular infection or whether T-cell responsiveness is hampered, and consequently a persistent infection is developing. Increased degradation of tryptophan has been described, e.g., in patients with HIV infection, in Streptococcus pyogenes infection as well as in Lyme neuroborreliosis. Tryptophan deprivation as a result of the microbicidal activity of IFN- appears to be involved also in the pathogenesis of anemia when erythroid progenitor cells suffer from insufficient tryptophan supply. Also weight loss and cachexia are closely linked to inflammatory response when protein biosynthesis of the organism is restricted by diminished tryptophan availability. In the absence of any ability to synthesize tryptophan, upon shortage of tryptophan cells begin to degrade protein to sequester tryptophan for production of highly needed proteins. Finally, tryptophan shortage affects biosynthesis of neurotransmitter 5-hydroxytryptamine (serotonin) and lead to the production of potentially neurotoxic tryptophan catabolites such as quinolinic acid. Both these biochemical cascades seem to be involved in the development of neuropsychiatric symptoms like cognitive impairment and depression especially in patients suffering from severe and chronic infections. Thus, accelerated tryptophan degradation by IFN--induced IDO can give raise to an immune activation syndrome in patients suffering from infections, which is characterized by subnormal tryptophan levels and is associated with adverse outcome.

Application of pharmacokinetics of individualize antineoplastic therapy FUHR U Department of Pharmacology, Clinical Pharmacology Unit, University Hospital of Cologne, Germany Background: Antineoplastic drugs usually are administered at the mean maximum tolerated dose in the patient population. However, these doses may result in considerable interindividual exposure differences because of individual pharmacokinetic properties. These differences explain a part of individual differences in toxicity and efficacy. Such variation occurs in systemic as well as in local pharmacokinetics. Systemic pharmacokinetics in an individual may be assessed by therapeutic drug monitoring (TDM) but with the disadvantage that the information is available only after administration of the first dose. This however is useful only if there is a relationship between concentrations at selected time points and drug effects. For some drugs, key components of variability in systemic pharmacokinetics are known, such as activity of a metabolizing enzyme which may be related to an underlying genetic polymorphism, sex, or renal function. This enables a prospective dose adjustment without measuring individual concentrations. Important examples include the adjustment of doses to BSA, to renal function (carboplatin), and to UGT1A1 (irinotecan) or thiopurine S-methyltransferase genotype (thioguanin). Indeed, a more extensive assessment of enzyme activity by phenotyping and/or genotyping has the potential to further decrease the unexplained fraction of interindividual variability. In malignant disease, there are also pronounced differences in local pharmacokinetics, such as distribution of a drug into the tumor interstitium, which cannot be explained exclusively by tumor size and vascularization, as well as uptake and metabolism by the tumor cell. Local pharmacokinetics is difficult to assess. Microdialysis may be used to measure interstitial unbound drug concentrations but this tool is limited to experimental studies and cannot be applied to each patient. Biopsies are required to characterize intracellular pharmacokinetics. Thus, while occasionally CSF concentrations are measured to assess CNS exposure, currently local pharmacokinetics cannot be taken into account for treatment individualization.

Pharmacokinetics of Antimycotic Drugs during Continuous Renal Replacement Therapy FUHRMANN V^1, SCHENK P^1, THALHAMMER F^2 ^1 Medical University Vienna, Internal Medicine 3, Vienna, Austria ^2 Medical University Vienna, Internal Medicine 1, Vienna, Austria Background: Critically ill patients frequently suffer from acute renal failure requiring continuous renal replacement therapy. The risk of fungal infections is significantly increased in these patients at the intensive care unit. Knowledge of the influence of renal replacement therapy on the elimination of antimycotic drugs is essential to provide adequate dosing. Elimination of any given drug by renal replacement therapy is influenced by membrane-specific factors, due to physico-chemical properties of the drug, and characteristics of the renal replacement therapy. We review available data concerning pharmacokinetics and dosing recommendations of antimycotic drugs during continuous renal replacement therapy. Methods: Review of studies investigating the pharmacokinetics of antimycotic drugs during continuous renal replacement therapy. Results: No dosage adaptation seems to be necessary in most antifungal drugs during continuous renal replacement therapy. This may be explained by their high protein binding, high molecular weight and mainly non-renal elimination. In contrast, fluconazole is mainly eliminated via the kidneys. However, as continuous renal replacement therapy results in fluconazole clearance similar to that of individuals with normal renal function, no dosage reduction is recommended. Voriconazole´s pharmacokinetics is barely affected during continuous renal replacement therapy. However, attention should be directed to its intravenous vehicle sulphobuthylether beta-cyclodextrin which may accumulate during renal replacement therapy. Switching to the oral route seems to be rationale as soon as sufficient gastrointestinal absorption is ensured in patients requiring treatment with voriconazole. Conclusions: Continuos renal replacement therapy does not influence the dosing regimen of most antimycotic drugs. As fluconazole is effectively removed via continuous renal replacement therapy, no dosage reduction is required despite its mainly renal elimination. Voriconazole should be administrated via the oral route when sufficient gastrointestinal absorption is ensured to avoid accumulation of the intravenous vehicle sulphobuthylether beta-cyclodextrin.

The VesiVax^® System: Vaccinology’s Magic Bullet? FUJII, G^1, ERNST WA^1, ADLER-MOORE, J^2 ^1Molecular Express, Inc., Rancho Dominguez, USA; ^2California State Polytechnic University Pomona, Pomona, USA. Recently, there has been significant emphasis placed on the development of vaccines that address the effectiveness, safety and manufacturing issues associated with the classical method of pathogen based vaccines. Towards this end, the VesiVax^ vaccine system was designed, which employs a flexible and easily modified gene cassette designed to rapidly engineer and produce recombinant antigen proteins that are compatible with bilayer membranes. The recombinant antigen proteins consist of a target epitope or antigen fused to an aqueous soluble hydrophobic domain that makes purification simple yet allows for stable insertion of the immunogen within the lipid membrane. Immunogenic liposomes consisting of a well-defined set of lipid constituents incorporating the recombinant antigen protein can then be produced using industry standard manufacturing processes. Vaccines based on the VesiVax^system have been constructed against several pathogens including the influenza virus and herpes simplex type 2 virus, the causative agent of genital herpes. These vaccines have been tested in animal models and have demonstrated significant protective efficacy from microbial challenge and have elicited strong immune responses. Assays of the immunological parameters suggest that both T and B cell responses can be elicited by VesiVax^ vaccines. Taken together, the inherent flexibility of the VesiVax^ platform is expected to facilitate the rapid development of new vaccines which are effective at stimulating protective immune responses..

Comparison of susceptibility against MRSA isolates to the brand vancomycin and manufactured generic drugs FUJIMURA S^1, FUSE K^2, WATANABE A^1. ^1 Research Division for Development of Anti-Infective Agents, Institute Development Aging and Cancer, Tohoku University, Sendai, Japan. ^2 Department of Pharmacy, Tohoku KoseiNenkin Hospital, Sendai, Japan Background: The brand vancomycin for injection(VCM 1) was released for the treatment of the methicillin-resistant Staphylococcus aureus (MRSA) infection. At present, 11 generic vancomycin products are available in Japan. Of them, five have been available for several years already. The bulk of brand vancomycin is imported from the United States, and the products for injection are manufactured in Japan. However, most of the generics of vancomycin used in Japan are imported as finished product or by bulk from Taipei, France, Hungary, and Slovenia. Aim & Method: The objective of this study was to test VCM 1 for its activity against 80 clinical isolates of MRSA and to compare the results with those obtained with generic products. We investigated the susceptibility of 80 MRSA strains to brand vancomycin (VCM 1) and 5 generic products (VCM 2-6). The antibacterial activity of VCM 1 and VCM 2-6 was determined using the CLSI broth microdilution method. Furthermore, we compared the potency equivalent per vial of VCM 1 and these 5 generic products by a bioassay. Results: The MIC50 of VCM 6 was 1 mg/L, while that of VCM 1-5 was 0.5 mg/L. The MIC of generic VCM 6 was slightly behind in comparison with other vancomycin products. In this study, the potency equivalent of VCM 1 and generic VCM 6 was 495 mg/vial and 455 mg/vial, respectively. The potency equivalent of the generic products was slightly lower than that of VCM 1. Conclusions: Although the potency equivalent of the VCM used in this study was within the range accepted by the United States and Japanese pharmacopeia, the results showed that the susceptibility of one generic product was not similar to VCM 1. Because vancomycin shows side effects such as nephrotoxicity, therapeutic drug monitoring using a pharmacokinetic parameter is performed for chemotherapy. The population parameter necessary for this analysis consists of data obtained using VCM 1. Therefore, it is possible that some generic products whose potency equivalent of vancomycin is lower than that of the brand drug will have a weak effect on the infectious agent.

Endovascular Stenting for Malignant Superior Vena Cava Syndrome is Essential Therapy but not Approved in Japan FUKUDA M, MOTOSHIMA K, HISATOMI K, FUTSUKI Y, HARADA T, OGAWARA D, IMAMURA S, IKEDA K, YAMAMURA M, YAMAKAWA M, MINAMI K, NAKAMURA Y, KOHNO S Malignant superior vena cava (SVC) syndrome is difficult problems and associated with poor outcome in patients with lung cancer. Radiotherapy and/or chemotherapy could once improve the condition. Subsequently, the tumor becomes refractory and SVC syndrome appear again, constructive therapy including stent placement is abandoned since the case is viewed as terminal and treatment becomes palliative. Moreover, vascular stenting is not approved in Japan. A 66-year-old women with advanced primary lung cancer (adenocarcinoma cT1N3M1 stage IV BRA OSS) treated with 4 cycles of irinotecan/carboplatin combination chemotherapy, two times of Gamma Knife radiosurgery for brain metastasis, and radiotherapy for bone metastasis with cancer pain. Ten months later, the patient treated with oral fluoropyrimidine anticancer drug S-1 for second line, and drainage against malignant pleural effusion. For third line therapy of gefitinib, the patient maintained stable condition for a while. One year and 7 months after the onset, the patient developed severe swelling of face and both arms as SVC syndrome. We recognized that is the timing to place a self-expandable metal stent in the SVC. However, in the treatment group discussion, we attached importance to that endovascular stenting is not approved in Japan, and decided to not use the stent. The patient underwent radiation therapy (48 Gy in 20 fractions) with irinotecan (40 mg/m2/week) chemotherapy. The symptoms of SVC syndrome were resolved once, and took a turn for the worse within the chemoradiotherapy. Finally, the patient died one year and 10 months after the disease onset with miserable watched severe swelling of the upper half of the body especially face. A post mortem examination showed complete response and almost no remaining tumor, but thrombus obstruct the SVC. Recent development of stent placement therapy for the treatment of malignant constriction has improved the quality of life, and possibly survival. In cases like our patient, chemoradiotherapy reach the limit for SVC syndrome, and Stenting is essential. The approval of endovascular stenting for SVC syndrome is warranted in the worldwide.

Chelating Agents for Treatment of Uranium-induced Toxicity in Radiation Emergency Medicine FUKUDA S^1, IKEDA M^1, YAN Y^2, XIE Y^2 ^1National Institute of Radiological Sciences, Chiba, Japan, ^2Shanghai Institute of Materia Medica, Shanghai, China. Backgroud: Radiation workers treating actinides in the nuclear fuel cycle are exposed to accidental internal contamination of uranium. One of route of uranium intake is via wounds. Chelation therapy is an optimal method for reducing uranium-induced toxicity. So far, we examined on the effects of catechol-3,6-bis(methyle-imino-diacetic acid) (CBMIDA) by local treatment in simulated wounds, in which the depths contaminated with uranium in wounds are different. Methods: Male Wistar rats, 8 weeks old, were divided into three groups (n=42/group) of intracutaneous(IC), subcutaneous(SC), and intramuscular (IM) injection. After the injection of DU (4 mg/kg, pH 1) by the different routes, rats of each group were infused with 480 mg/kg (adjusted to pH 6.8 by bicarbonate, molar rate 78 times of uranium) of CBMIDA into the DU-injected site at 10, 30, 60, 120 min and 24 hours (7 rats at each point). Rats were killed 24 hours after CBMIDA treatment. Data obtained were compared with that in the corresponding no-treated group, respectively. Results: When CBMIDA was administered within 10 - 60 or 120 min after DU-injection, the uranium concentration of the DU-injected site decreased significantly (P<0.05) to 3-29 % (IC), 4-11% (SC), and 25-32%(IM) of that in the no-treated group. Amounts of excreted uranium increased to 4-5 times. Uranium concentrations in the kidney, as the target organ of uranium, decreased to 35-74% (IC), 22-42% (SC), and 12-39% (IM). Regarding to the kidney, as the target organ, the improvement of dysfunction by serum and urinary examinations and tissue damages by histological observation, were confirmed. Also, CBMIDA improved not only the damage by chemical action of uranium but also the burn by acid solution in the DU injected site. Conclusions: The results indicated that CBMIDA is the useful chelating agent, (1) to increase uranium and (2) prevent the tissue damages and dysfunctions of organ, in the treatment for the wounds contaminated accidentally with uranium, if CBMIDA applies as early as possible after the intake.

Trabectedin: a new anticancer bullet from the sea GAGO F Departamento de Farmacología, Universidad de Alcalá, E-28871 Alcalá de Henares, Spain. Background: Trabectedin (formerly ecteinascidin ET-743) is a potent antitumor tetrahydroisoquinoline alkaloid in clinical use originally derived from a marine tunicate and now obtained by synthetic modification of microbially produced cyanosafracin B. Methods: A variety of physico-chemical techniques, including the use of fluorescently labelled oligonucleotides, have been employed to test the ability of trabectedin to bind covalently to DNA. Insights about the activation and binding mechanisms have been gained both experimentally (i.e. nuclear magnetic resonance spectroscopy) and computationally (i.e. molecular dynamics simulations). The panel of 60 human tumor cell lines of the National Cancer Institute (NCI) Anticancer Drug Screen was used to reveal a rather unique activity profile that encouraged further development as an anticancer agent. Additional work has been done on mammalian and yeast cells both proficient and deficient in several DNA repair mechanisms. Results: A definite role for hydrogen bonding has been demonstrated for sequence recognition and binding orientation of trabectedin in the DNA minor groove. TGG, CGG, AGC, GGC, and AGA triplets have been identified as the preferred DNA sites for stable adduct formation with the central guanine. As a consequence of trabectedin bonding, the double helical structure is only minimally perturbed except for widening of the minor groove and a net smooth bending towards the major groove due to the introduction of positive roll. The close contacts that are established between trabectedin and both DNA strands give rise to a significant increment in the stability of the resulting drug-DNA complexes, which is thought to result in stalled replication and transcription forks that can lead to double-strand breaks. Cell sensitivity to trabectedin is dependent on the cellular status of proteins involved both in transcription-coupled nucleotide excision repair (TC-NER) and in homologous recombination (HR). Trabectedin is currently approved for the treatment of soft tissue sarcoma and is an orphan medicinal product for relapsed ovarian carcinoma. Toxicity to trabectedin is dose-related, mostly limited to bone marrow and liver, and follows a transient-reversible pattern. Conclusions: 1) Trabectedin is a novel chemical entity endowed with a new mode of action, (2) Patients harboring tumour cells with proficient TC-NER and deficient HR systems would be expected to respond best to this drug. Authors’ disclosure statement: The author’s investigations into the mechanism of action of trabectedin have been financially supported in part by the Spanish biopharmaceutical company PharmaMar.

Perspective Chemotherapeutic Combination to Combat Flu GALABOV AS, SIMEONOVA L, GEGOVA G The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria Background: Previous studies of ours demonstrated a marked synergistic combination effect of rimantadine and oseltamivir in 100:1 compounds doses ratios in experimental infection with influenza A (H3N2) in mice when the treatment course onset was on the day of virus inoculation. Considering these data we studied combination effect of both compounds in 50:1 and 25:1 ratios in order to determine the dose ratios scope preserving a high efficacy. The antiviral effect of the treatment course with the combination started 24-hours after virus inoculation was tested. Methods: White male mice 16-18 g were inoculated intranasally with 0.05 ml/mouse of influenza A/Aichi/2/68 (H3N2) virus. Rimantadine hydrochloride and oseltamivir phosphate were administered per os in five-day-treatment course beginning 4-hours before or 24 hours post-virus inoculation with 20 – 30 MLD50. Protection index (PI) and mean survival time (MST) were determined through 14 days post infection. Infectious virus titers were determined in Madine-Darby canine kidney cells. Lung consolidation score and lung index were evaluated. Results: Combinations of selected doses of 5, 10 and 20 mg/kg/day rimantadine and 0.1, 0.2, 0.4 and 0.8 mg/kg/day oseltamivir were combined in doses ratio 50:1. PI up to 82.7% and 91.3% and MST up to 13.2 and 13.6 days for certain combinations were evaluated, while the individual effects of the same doses were from 13.3% to 30.6% PI and 7.9 to 9.8 days MST, respectively. Determination of lung virus titers and lung parameters in combination-treated groups also proved the synergistic effect of both therapeutics. Conclusions: Oseltamivir and rimantadine at daily doses up to 50 times lower than optimal effective one for oseltamivir and 8-16 times lower - for rimantadine in 1:50 ratio demonstrated synergistic effect when administered in combination in experimental infection with influenza virus A (H3N2) in mice.

Anti-leishmanial effect of Hydroxyurea GALINDO-SEVILLA N^1, MARTINEZ-ROJANO H^2,3, MANCILLA-RAMIREZ J^1,2, QUIÑONEZ-DIAZ L^2,4 ^1Instituto Nacional de Perinatologia, Mexico City, Mexico;^2Escuela Superior de Medicina, Instituto Politecnico Nacional, Mexico City;^3Hospital de Gineco-Pediatria 3A, IMSS, Mexico City;^4UJAT, Villahermosa, Tabasco. Background: Protozoa are eukaryotic parasites sharing metabolic pathways with human cells, including neoplastic cells. Hydroxyurea, a drug affecting ribonucleotide reductase enzyme, has been used to treat malignant and non-malignant diseases. Aims: 1) To determine if hydroxyurea inhibit Leishmania growth in vitro. 2) To develop a more accurate method that resembles the intracellular infection in the host. 3) To determine the ED50 of hydroxyurea on Leishmania mexicana. 4) To investigate the effect of hydroxyurea on the cell cycle of Leishmania. Methods: Growth curve of M379 and Tab3 Leishmania mexicana strains were followed in the presence of 0.01, 0.1, 1, 10 and 100 g/mL of hydroxyurea. To test the effect on intracellular parasites, adherent macrophages were infected with amastigotes-like forms, exposed to hydroxyurea by 3, 6, 9 or 12 days, when they were intracellular. Then, hydroxyurea was removed and parasites transformed to promastigotes by temperature shift from 32 to 26^oC. Parasite density was monitored during 8 days. The ED50 was calculated by polynomial regression analysis. The cell cycle was studied in an EPICS-ALTRA flow cytometer. Results: Hydroxyurea eliminated Leishmania at 10 and 100 g/mL. The ED50 for intracellular parasites was 0.015 g/mL, and for promastigotes was 0.05 g/mL. Hydroxyurea at 10 and 100 g/mL arrested Leishmania cell cycle at G2/M. Conclusions: Hydroxyurea is highly effective killing promastigotes and intracellular amastigotes in vitro. At the concentrations used, HU induces parasite death and cell cycle arrest of Leishmania in G2/M.

Multiple Mechanisms of Action and Pharmacological Activities of Valproate GALLAGHER HC School of Medicine and Medical Science, Conway Institute, University College Dublin, IRELAND. Background: Although it is over 40 years since the anticonvulsant properties of valproate were discovered serendipitously, this drug remains one of the most widely prescribed anti-epileptics. Interestingly, several other indications have since emerged for valproate including mood stabilisation, prevention of migraine, treatment of mania and, most recently, chemotherapy of acute myeloid leukemias. In addition to these therapeutic effects, valproate is a known teratogen inducing neural tube defects in exposed offspring – an effect that is largely reflects its inhibition of cell proliferation. While the precise mechanisms of action that underlie these distinct pharmacological activities are unconfirmed, several different signalling pathways are influenced by valproate and related drugs. Methods: The influence of valproate on cell proliferation, differentiation and cell cycle signaling was investigated in glioma (C6) and neuroblastoma (N2A and SHSY5Y) cell lines. Cell cycle synchrony was achieved by mitotic selection and western blotting techniques were employed to investigate the expression of cyclins and related proteins. The effect of valproate on cAMP signaling was investigated in forskolin-treated cells. These signaling pathways, along with other proposed mechanisms of action that may mediate the antiproliferative activity of valproate are reviewed and discussed. Results: Valproate arrested cell cycle progression, induced ectopic expression of cyclin D3 and inhibited the accumulation of cyclic AMP via increased phosphodiesterase activity. Both these effects on cyclin and cAMP signaling involved perturbance in normal temporal G1 phase signaling mechanisms leading to cell cycle exit. Other mechanisms of action of valproate that may influence growth arrest include inhibition of histone deacetylase activity and activation of the tumour suppressor gene PTEN via peroxisome proliferator-activated receptors. The anticonvulsant activity of valproate may primarily reflect potentiation of GABAergic neurotransmission via multiple pathways. Conclusions: Valproate is a clinically useful drug with a wide spectrum of pharmacological activity. Improved understanding of the mechanisms underlying its therapeutic and toxic effects will aid the development of new generation analogues with enhanced efficacy and reduced side-effects.

Photophysics, Photochemistry and induced-Photoallergy of Tricyclic Antidepressants GARCÍA C^1, OYOLA R^1, PIÑERO LE^1, ARCE R^2 1) 100 Carr 908; University of Puerto Rico at Humacao; Department of Chemistry; Humacao, Puerto Rico 00791-4300 2) University of Puerto Rico at Rio Piedras; Faculty of Natural Sciences - Department of Chemistry; PO Box 23346; San Juan, Puerto Rico 00931-3346 Background: The major used tricyclic antidepressants (TCA) are the promazines, dibenzazepines, and dibenzodiazepines. Most of the derivatives of these drugs produce serious side effects, including allergy and photosensitization. Small changes in their structure, change their mode of action, potency and the spectrum and severity of the side effects. The molecular mechanisms for their photosensitizing ability are still unknown, even through these drugs are actually used in the world to treat thousands and thousands of psychiatric patients annually. The goals of this project are: 1) To measure the properties of their short-lived intermediates. 2) To find a molecular/photophysical descriptor for their phototoxic side effect. Methods: The photophysical properties were measured in several solvents. In this work, we present absorption, steady-state, and time-resolved emission, laser flash photolysis, and quantum theoretical results for the ground state, the first excited singlet and triplet states, and the cation radical of several TCA series. Results: The photophysical properties of the promazine family depend more on the solvent and the 2-substituents than on the dialkylaminopropyl chain. The largest effect was found for the triplet state of the 2-halogenated derivatives in phosphate buffer (PBS). The triplet state of these TCAs (^3TCA*) is efficiently quenched by a proton-transfer mechanism, and the rate of this quenching correlates very well with their phototoxicity. In the case of the imipramines, the ground-state properties are solvent-independent, while the emission maxima are red-shifted with increasing solvent polarity/polarizability. The fluorescence quantum yield is relatively low in all solvents. Conclusions: 1) The effectiveness of the ^3TCA* quenching in PBS correlates very well with their phototoxicity index (i.e., the more effective the quenching, the smaller the triplet lifetime and the more phototoxic the drug). 2) Besides the ^3TCA*, the involvement of some membrane components is required to explain the large differences in phototoxicity of similar TCAs.

Anti-mycobacterial compounds: effects on the microbe and its host cell GARCIA RC^1, OLIVEIRA RAS^1, AZEVEDO-XIMENES E^2, BANFI E^3, LUZZATI R^4 Leukocyte Biology Group, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy^1; Department of Antibiotics, Universidade Federal de Pernambuco, Recife, Brazil^2; Department of Biomedical Sciences, University of Trieste, Italy^3 and Department of Infectious Diseases, University Hospital, Trieste, Italy^4 We study potentially anti-mycobacterial compounds in a cell culture system by looking at their effect on the intracellular survival of mycobacteria and innate immune response-related functions of the host cells. We have established that lapachol, a naphthoquinone from Tabebuia sp., exhibits bacteriostatic activity toward Mycobacterium avium growing free as well as intracellularly in human THP-1 macrophage-like cells. Lapachol prevented free M. avium growth at a minimal inhibitory concentration of 32 mg/L (0.13 mM), which was also able to arrest intracellular bacterial growth while not being apoptotic toward the host cells. Regarding host cell function, we determined the effect of lapachol on the activation of THP-1 macrophages by IFN- and toll-like receptor 2 (TLR2) agonism. The induced expression of the NADPH oxidase catalytic component gp91-phox was decreased, while that of p47-phox and its translocation to the cell membrane were not affected. Some beneficial effects of lapachol on the host cells were observed: increases in IFN- receptor, ICAM and MHCII levels and a decrease in IL-10 secretion. IL-1 production was not affected. The TLR2-mediated increase in TNF- secretion and in the levels of manganese superoxide dismutase (MnSOD), which protects the host cell from self-damage and apoptosis, was not impaired by the naphthoquinone. Instead, M. avium-induced TNF- secretion was decreased, probably as a reflection of the bacteriostatic effect of lapachol. We did not detect endoplasmic reticulum (ER) stress, as indicated by unchanged levels of grp78. Altogether, lapachol appears to be a satisfactory anti-mycobacterial agent. The present report describes an in-vitro approach for the evaluation of new compounds with activity against intracellular microbes through the determination of relevant functional parameters of macrophages.

Antihormonals and liposomal carriers as novel strategies for chemo-radiotherapy based on cisplatin in cervical cancer. GARCIA-LÓPEZ P^1, JURADO R^1, GUERRERO-VARGAS N^1, MEDINA LA^2.   ^1Subdirección de Investigación Básica, Instituto Nacional de Cancerología; ^2Instituto de Física, Universidad Nacional Autónoma de México. México, D.F., México Background: Cisplatin (CP) is a widely used antineoplastic drug that has potent cytotoxic effects upon a variety of tumor types including cervical carcinoma. However, its administration is associated with nephrotoxic and neurotoxic events. On the other hand, steroid hormones are related to the development of drug resistance in cervical cancer. Upon this situation, in this work we have investigated the ability of a pure anti-estrogen ICI 182,780 (Fulvestran) and an anti-progestin Mifepristone (MF) to modulate the cytotoxic effect of CP and gamma irradiation in cervical cancer cell lines (HeLa and CaSki) and in a model of cervix cancer in athymic mice Methods: The effect of CP alone and CP combined with either ICI, MF and/or gamma irradiation (RT) on cellular death was studied using an assay based on tetrazolium dye (XTT) and a clonogenic assay. Before and after treatment with antihormonals, expression of the estrogen, progesterone receptor (ER, PR) and the proangiogenic factor -vascular endothelial growth factor (VEGF)- genes were assessed by a reverse transcriptase polymerase chain reaction (RT-PCR). Cell-cycle modifications after combined treatments were studied by flow-cytometry. RT dose was evaluated with dosimetric procedures based on Gafchromic film. Results: The analysis showed that ICI or MF alone produced no changes in cell growth; however, the combination of these antihormonals with CP and RT produced synergistic anti-proliferative effect in cervical cancer cells and significant delayed of the tumor growth without apparent toxic effect for the animals (p<0.05, n=6)). The effect of ICI and MF on the cytoxicity of CP and RT could be mediated, at least partially, by inhibition of ER, PR and VEGF gene expression, and by arresting the cell cycle at G2/M phase. Conclusions: The results suggest that the combination of antihormonal drugs can improve the efficacy of CP and RT in cancer cells and tumor xenografts of cervical carcinoma. Based on these results we have planned the use of liposomes as drug carries of the CP and MF, which potentially could be used in chemoradiotherapy treatments decreasing the secondary effects of these drugs.

Combinatorial Ribosome-Inactivating Protein Libraries: Part of a New Arsenal in Combating Cancer GARIEPY J, PERAMPALAM S, WEI X, KIARASH R, GREEN E, ZHANG QH, CHEUNG M, ABDUL-WAHID A, REVERS L University of Toronto, Ontario, Canada Background: Ribosome-inactivating proteins (RIPs) such as ricin and Shiga-Like Toxin 1 (SLT-1) are highly effective at killing eukaryotic cells. Importantly, their cytotoxic A chain lacks receptor specificity. We propose that one can evolve a RIP A subunit template alone to specifically target and kill cancer cells by inserting a random peptide element within its structure and screening the resulting combinatorial library of A chain variants for RIP A chains displaying such selective killing properties. Aims: 1) To design a combinatorial SLT-1 A chain library expressing a repertoire of cytotoxic A subunit variants, each harboring a unique peptide ligand. 2) To purify recombinant SLT-1 A chain variants and identify single chain toxins able to kill human melanoma cell lines. Methods: A combinatorial protein library was constructed by inserting a random heptapeptide element between residues 245 and 246 of the SLT-1 A chain. Single bacterial colonies were individually grown as 1mL cultures in 96-well blocks A His8 affinity purification tag located at the N-terminus of all A chain variants was used to purify A subunit mutants from bacterial lysates using magnetic nickel NTA affinity beads. Aliquots of purified A chains were then dispensed into wells containing target cancer cells insensitive to wt SLT-1. Levels of cell survival were subsequently assessed using sulforhodamine B. Results: 9,400 RIP A chain variants were individually purified and screened for their ability to kill human cancer cell lines insensitive to wt SLT-1 namely 518A2, PC-3 and CAMA-1 cells. 112 A chain variants were initially found to exhibit cytotoxicity towards one or more of these three cancer cell lines. The candidates were subsequently re-expressed and re-tested for cytotoxicity against a panel of normal and cancer cell lines. Only one variant was selectively toxic towards 518-A2 melanoma cells (CD50, 300 nM) as well as towards 7 of 8 human melanoma cell-lines subsequently tested. This A chain was also effective in vivo in terms of causing tumor regression and animal survival when injected i.v.into SCID mice bearing a 518A2 xenograft. Conclusions: The screening of combinatorial libraries of a ribosome-inactivating protein template represents a new strategy for identifying targeted protein-based anticancer agents that are distinct from antibodies.

A Climate for Change? A Statistical Analysis of General Practitioners’ Relationship with Chiropractic Care. GATES SEE ^1, COCHRAN KD ^1, RITCHIE MM ^2,3, GIBBS TJ ^4, GILMOUR A ^5, WONG V ^6. ^1Cambridge University, Cambridge, England, ^2Napier University, Edinburgh, Scotland,^3Arbroath High School, Angus, Scotland, ^4Chinese University of Hong Kong, Hong Kong, China, ^5Manchester University, Manchester, England, ^6Aberdeen University, Aberdeen, Scotland Introduction: Chiropractic is the complementary medicine dealing with correcting misaligned joints through mechanical manipulation. This study aims to identify the knowledge, attitudes and referral patterns of general practitioners (GPs) in Fife, Scotland regarding chiropractic care. Materials and Methods: A postal questionnaire was sent to 100 GPs in Fife. Reply-paid envelopes were included, and a reminder questionnaire sent out one week later. GPs from ten randomly selected practices were asked to participate in a follow-up interview. Results: Despite evidence demonstrating the usefulness of chiropractic, over 80% of GPs rated their knowledge of chiropractic as less than or equal to five out of ten (with 10 representing ‘very knowledgeable’). The average score was three. GPs’ attitudes towards chiropractic were fairly positive, though these attitudes were not reflected in GP perception of the helpfulness of chiropractic, or in their referral practices. In general, 3% of GPs found chiropractic very helpful, 14% rated it as helpful, 12% as neutral and 72% as unhelpful. GPs referred most readily for lower back pain, neck pain and sciatica. On average, 3% of GPs refer patients to chiropractors at the first consultation, 11% after failure of traditional treatments, 10% only at the patient’s request, and 76% would never refer. Thirty-six percent of GPs would never refer patients to a chiropractor for any condition. Conclusion: The results indicated an under-utilization of chiropractic treatment. GP attitudes towards chiropractic were positive; however, attitudes did not show a strong correlation with referral practices or with perceptions of helpfulness. GP perceptions of the conditions treated by chiropractic medicine do not match the evidence in this field. In general, GPs under-valued the helpfulness of chiropractic. However, for some conditions GPs perceived chiropractic to be more helpful than evidence-based medicine research supported. How helpful GPs perceived chiropractic to be was strongly correlated with the stage at which they were willing to refer patients to chiropractors. GPs seem very unwilling to refer patients to chiropractors; most will never refer or only refer at the patient’s request. GP gender, years since qualification, number of patients per GP and number of GPs per practice did not significantly affect knowledge scores, overall attitudes towards chiropractic or mean number of monthly referrals to chiropractors.

Oxfendazole: A novel strategy to control Cystic Echinococcosis by targeting the intermediate host (sheep) GAVIDIA CM^1, GONZALEZ AE^1, BARRON E^1, LLAMOSAS M^1, VERASTEGUI MR^2, GILMAN RH^3 ^1Universidad Nacional Mayor de San Marcos, Facultad de Medicina Veterinaria, Lima, Peru; ^2Universidad Peruana Cayetano Heredia, Lima, Peru; ^3Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, USA. Background: Cystic Echinococosis (CE) is a zoonotic disease caused by larval stage of the Echinococcus granulosus tapeworm. It is a major economic and public health problem worldwide. We determined the effects of high dose Oxfendazole, combination Oxfendazole/Praziquantel, and combination Albendazole/Praziquantel against CE in sheep. Methods: A randomized placebo-controlled trial was carried out on 118 randomly selected ewes. Ewes were assigned to one of the following groups: 1) control; 2) OXF 60mg/Kg of body weight (BW) weekly for four weeks; 3) ALB 30mg/Kg BW + PZQ 40mg/Kg BW weekly for 6 weeks, and 4) OXF 30mg/Kg BW+ PZQ 40mg/Kg BW biweekly for 3 times (6 weeks). Percent protoscolex viability was performed using a 0.1% aqueous eosin vital stain for each cyst. “Cured” sheep were those that had no viable protoscolices; “improved” were those that had 1% to 60% protoscolex viability; and “unchanged” were those with more than 60% protoscolex viability. We evaluated 92 of the 118 sheep at the slaughterhouse. Results: The CE prevalence was 95.7% (88/92) with a total number of cysts of 1094 (mean=12.4 cysts/animal). On average, the two-drug-combination groups had 6mm smaller pulmonary cysts than control (p<0.05) and 4.2mm smaller hepatic cysts than control (p<0.05). ALB/PZQ had the lowest PSC viability for lung cysts (12.7%), while OXF/PZQ had the greatest effect on liver cysts (13.5%). The percentage of either “cured” or “improved” sheep was 90%, 93.8% and 88.9% for OXF, ALB/PZQ and OXF/PZQ group as compared to 50% cured or improved for controls. Conclusions: We demonstrate that Oxfendazole at 60mg, combination Oxfendazole/Praziquantel and combination Albendazole/Praziquantel are successful schemas that can be added to control measures in animals and could be used for the treatment of human CE. Further investigations on different schedules of monotherapy or combined chemotherapy are needed, as well as studies to evaluate the safety of Oxfendazole in humans.

Recombinant virus-like particles and their application for vaccine development and diagnostics GEDVILAITE A, SLIBINSKAS R, ZVIRBLIENE A, SASNAUSKAS K Institute of Biotechnology, Vilnius, Lithuania. Background: Supermolecular structures, named VLPs (virus-like particles), built symmetrically from hundreds of proteins of one or more types, represent molecules useful for the development of diagnostic, prophylactic and therapeutic tools for human and non human diseases. The aim of this study was to adapt the yeast expression system for polyomavirus and paramyxovirus nucleocapsid protein synthesis and VLPs production and to use those VLPs for diagnostics, monoclonal antibody generation, immunological investigations or virus basic research applications, as structural and assembly studies, receptor identification and entry studies. Methods: A galactoseinducible S. cerevisiae yeast expression system was used. Formation of empty VLPs was confirmed by cesium chloride ultracentrifugation, agarose gel electrophoresis and electron microscopy. Recombinant VLPs were used for enzyme immunoassay studies, mice immunizations and monoclonal antibody generation using hybridoma technology. Results: The high efficiency of the S. cerevisiae-based expression system was confirmed by the production of VLPs based on the VP1 of different human (JCPyV, BKPyV), primate (SV-40), mouse, hamster and avian polyomaviruses. The expression level of most polyomavirus VP1 proteins and mumps virus NP protein was high, and yielded 1-3 mg of purified protein per 1 g of wet biomass. Measles virus N protein yield was 6 mg/g of wet biomass and this showed the effectiveness of yeast as a host for generation of biomedical preparations. Measles and mumps virus nucleocapsid proteins were applied for commercial tests optimized for detection of these virus infections using oral fluids. Polyomavirus VP1-VLPs were used for diagnostics, structural and assembly studies and as molecular carriers of selected epitopes for production of chimeric VLP and the monoclonal antibody of desired specificity. Conclusions: 1) We developed a universal expression system in yeast, for polyomavirus and paramyxovirus nucleocapsid protein synthesis and VLPs production. 2) The yeast generated recombinant viral VLPs retain biological activity of native virus proteins and were successfully used for detection and generation of specific antibodies.3). Polyomavirus VP1-VLPs were used for diagnostics, virus structure and entry studies and chimeric virus like particles production.

Development of Serpin drugs for the treatment of HIV/HCV co-transfections LETVIN NL and GEIBEN-LYNN R Division of Viral Pathogenesis, BIDMC and Harvard Medical School, Boston, MA 02115, USA. Background: Novel antivirals against HIV and HCV targeting host-cell proteins are needed to prevent the generation of multi-resistant viruses. The Serine protein inhibitors (Serpins) such as Secretory Leucocyte Protease Inhibitor (SLPI), anti-trypsin and Antithrombin III (ATIII), all display potent antiviral activity against HIV in vitro. Their in vivo potential can be demonstrated by: a) the near-absense of HIV oral transmission most likely due to the anti-viral activity of SLPI, the predominant HIV-inhibitor in saliva; b) the correlation between disease progression and certain anti-trypsin mutations; c) the observation that CD8^+ T cells of HIV long-term non-progressors produce a modified form of ATIII with high anti-viral activity. STUDY AIM: 1) Demonstrate anti-viral inhibition of ATIII for HIV and HCV; 2) Elucidate novel host-cell targets. ATIII is the first recombinant protein produced in goats and approved for human use. Due to its improved availablity, 60 h half-life and low toxicity ATIII has strong potential as a novel protein-based anti-viral against HIV and HCV. Methods: HIV inhibition was measured in cell lines and human Peripheral Blood Mononuclear Cells (PBMC). HCV inhibition was measured using a replicon system with a full-length HCV genome. Activation or inhibition of pathways and host-cell targets was measured by microarray with 84 key genes testing for 18 different pathways. Results: ATIII blocked HIV viral replication in nM and HCV in M concentrations in a dose dependent manner. Using 2.4, 12 and 24 U/ml ATIII we saw 8 genes in HIV infected PBMC upregulated (Prostaglandin-endoperoxide (PTGS2), IL-8, IL-1, CCL20, BCL2A1, MMP7, Fas and HK2). At the highest dose PTGS2 was 300-fold upregulated. IL-8 and IL-1 were both 60-fold upregulated. In the HCV replicon system seven genes were more than 10-fold downregulated. Cancer genes Jun and Myc where up to 1000-fold and 80-fold downregulated, respectively, transcription factor C/EBP was downregulated more than 600-fold. Conclusions: ATIII blocks viral replication through a mechanism of action that targets multiple host-cell proteins which might decrease the ability of the viruses to develop resistance to this modality.