Ehrlich II –2nd World Conference on Magic Bullets

Cytokine-associated neutrophil extracellular traps and antinuclear antibodies in Plasmodium falciparum infected children under the age of six BAKER V^1, IMADE G^2, MOLTA N^3, PAM S^2, OBADOFIN M^2, SAGAY S^2, EGAH D^2, IYA D^2, AFOLABI B^4, BAKER M^5, FORD K^6, FORD R^6, ROUX K^7, KELLER T^7 ^1Chipola College,FL, ^2JUTH Medical School, Nigeria; ^3Jos University, Nigeria; ^4Nigerian Institute of Medical Research, Nigeria; ^5Jackson Hospital, FL, ^6World Health Mission, PA,^7Florida State University, FL Background: In Plasmodium falciparum-infected children, relationships between blood cell histopathology, blood plasma components, development of immunocompetence and disease severity remain poorly understood. Blood from Nigerian children with uncomplicated malaria was analyzed to gain insight into these relationships. This investigation presents evidence for circulating neutrophil extracellular traps (NETs) and antinuclear IgG antibodies (ANA). The presence of NETs and ANA to double-stranded DNA along with the cytokine profiles found suggests autoimmune mechanisms that could produce pathogenesis in children, but immunoprotection in adults. Methods: Peripheral blood smear slides and blood samples obtained from 21 Nigerian children under six years of age, presenting with uncomplicated malaria before and seven days after initiation of sulfadoxine-pyrimethamine (SP) treatment were analyzed. The slides were stained with Giemsa and with DAPI. Levels of the pro-inflammatory cytokines IFN-γ, IL-2, TNF-α, CRP, and IL-6, select anti-inflammatory cytokines TGF-β and IL-10, and ANA were determined by immunoassay. Results: The children exhibited circulating NETs with adherent parasites and erythrocytes, elevated ANA levels, a Th2 dominated cytokine profile, and left-shifted leukocyte differential counts. Nonspecific ANA levels were significant in 86% of the children pre-treatment and in 100% of the children seven days after SP treatment, but in only 33% of age-matched control samples collected during the season of low parasite transmission. Levels of ANA specific for dsDNA were significant in 81% of the children both pre-treatment and post-treatment. Conclusions: The results of this investigation suggest that NET formation and ANA to dsDNA may induce pathology in falciparum-infected children, but activate a protective mechanism against falciparum malaria in adults. The significance of in vivo circulating chromatin in NETs and dsDNA ANA as a causative factor in the hyporesponsiveness of CpG oligonucleotide-based malaria vaccines is discussed.

A Novel Anti-Angiogenic Glycotherapeutic for Breast Cancer BANERJEE DK Department of Biochemistry, School of Medicine, University of Puerto Rico, Medical Sciences Campus, San Juan, PR. USA Complex etiology of breast cancer needs personalized treatment. Angiogenesis is critical for the breast tumor growth. Our objective has been to evaluate the requirement of lipid-linked oligosaccharide (LLO), a pre-requisite for asparagine-linked glycoproteins in angiogenesis and ask if anti-angiogenic glycotherapeutics could eliminate the breast tumor growth. Our angiogenesis model is a non-transformed capillary endothelial cell line with preserved physiology and anatomy responding adequately to cellular microenvironment. cAMP-signaling enhances LLO biosynthesis and results in accelerated cell cycle progression, increased capillary lumen formation and HSP-70 and HSP-90 expression. Mannosylphospho dolichol synthase (DPMS), a key regulator in the LLO biosynthesis is also increased. Cloning and sequencing identifies a PKA motif in DPMS gene and supports PKA-dependent phosphorylation. On the other hand, tunicamycin (TM, an antibiotic and an LLO inhibitor) inhibits the cell surface N-glycan expression, down regulates the expression of Bcl-2, D-type cyclins, cdks and the transcription factor E2F causing a G1 arrest and halts angiogenesis. Increased caspase-3, -9 and -12 expression, DNA laddering and Annexin V binding all support apoptosis. High expression of GRP-78/Bip indicat “ER stress” and the induction of unfolded protein response. DPMS activity as well as its protein expression is lost, and VEGF165 looses the cellular protection in TM treated cells. TM inhibits angiogenesis in Matrigel^™ implants and also reduces the humanized breast tumor growth in athymic nude mice by 50-65% in 1-3 weeks when administered orally or intravenously. Therefore, TM has a potential to succeed in the clinic as an excellent breast cancer therapeutic. Supported in parts by grants from NIH/U54-CA096297 and Susan G. Komen Breast Cancer Foundation BCTR58206.

Search for diagnostic and prognostic markers for Glioblastoma multiforme(GBM). BANERJEE HN Department of Biological & Pharmaceutical Sciences. Division of Mathematics, Sciences, Technology and Pharmaceutical Sciences. Room# 425, Jenkins Science Building. Elizabeth City State University, University of North Carolina. Background: Glioblastoma Multiforme are brain tumors which are a heterogeneous group of central nervous system neoplasms that arise within or adjacent to the brain. Some are curable by surgical resection, but many cannot be eradicated by current treatments, and, when they are, disabling neurological injury often ensues. At present these brain tumors are detected by imaging only after the onset of neurological symptoms and no early detection strategies are in used. Imaging, the obvious screening strategy for glioblastomas is extraordinarily costly, especially given the relative rarity of these tumors in comparison with breast or prostate cancer. Genetic testing is a better option and is desirable as a screening tool because it is generally based on a simple blood test. The current histopathological approach to the diagnosis and classification of glioblastomas is not satisfactory in many respects.Therefore,a gene and protein based diagnostic and classification system is best for prognosis and therapeutic approaches. Methods: The following techniques were used to analyse glioblastoma specimens and cell lines and search for biomarkers a)Microarray analysis b)SELDI studies c) Laser Optical tweezers d)DIGGE and e)Real Time PCR f)ELISA. Results: Several novel proteins and genes were identified that differentially express in Glioblastoma specimens in comparison to normal control brain samples. Conclusions: There are several genes that are either down regulated,loss of function or upregulated that we observed in our research.We are working farther on the importance of these genes and their proteins in their role as a significant biomarker by analyzing more patient population.

Synthesis and Biological Evaluation of Anticancer β-Lactams BANIK BK^1,BANIK I^2 AND BECKER FF^2 ^1Department of Chemistry, The University of Texas-Pan American, Edinburg, USA; ^2University of Texas M. D. Anderson Cancer Center; Houston, USA. Background: Systematic analyses of polyaromatic derivatives through an examination of their anticancer effects in vitro and in vivo has been demonstrated. During the course of our studies, it has been anticipated that conformationally restricted analogues might increase the potency. We have envisioned that stereodefined β-lactams will serve as improved conformationally constrained analogues of our open-chain diamides that have already been shown to possess anticancer activity. Since many β-lactams (penicillin-types of compounds) have been widely accepted as safe antibacterial agents for more than 50 years, our hypothesis is that novel drugs can be synthesized and selected within this class that will have both an enhanced anticancer activity and low toxicity to normal tissues. Aims: 1) To synthesize -lactams derived from polyaromatic imines. 2) To study their effects in vitro and in vivo. 3) To study the mechanism of action of the lead -lactams Methods: Synthesis of -lactams was achieved following cycloaddition reaction of imines with acid chloride in the presence of triethylamine. In vitro cytotoxicity and in vivo study were performed using NCI protocol. Ames assay, topoisomerase inhibition and cell cycles were also investigated. Results: A number of racemic and optically active novel β-lactams were synthesized. Synthesis of these agents using microwave irradiation was performed. An unprecedented stereochemical results was identified. Some of these compounds demonstrated promising antitumor activity in vitro and in animal tumor model systems. In some instances the anticancer activity exceeded that of cisplatin in vitro. These agents demonstrated a blockade of the G2/M checkpoint in cancer cell lines, but they had no effects on topoisomerases. The active compounds were non-mutagenic. Conclusions: 1) Synthesis of several novel anticancer β-lactams achieved. 2) One of them demonstrated antitumor activity in animal model. 3) Although the mechanism of action of the lead compounds was not established, our research on cell cycle analysis offered intriguing possibilities.

Synthesis of a Series of 8-(Substituted-Phenyl)xanthines and a Study on the Effects of Substitution Pattern of Phenyl Substituents on Affinity for Adenosine A1 and A2A Receptors- The Magic of Theophylline BANSAL R^1, KUMAR G^1, GANDHI D^1, YOUNG CL^2, HARVEY AL^2 ^1Panjab University, Chandigarh, India; ^2 University of Strathclyde, Glasgow, United Kingdom Background: Adenosine receptors are promising therapeutic targets in a wide range of conditions, including cerebral and cardiac ischaemic diseases, sleep disorders, immune and inflammatory disorders and cancer. 8-Phenyltheophylline is the parent member of a variety of potent adenosine receptor antagonists. Herein we report the synthesis of a new series of 8-(substituted-phenyl)xanthines and the effects of substitution pattern of phenyl substituents on adenosine receptors binding affinity. Methods: The three series of compounds A, B and C were prepared by treating 5,6-diamino-1,3-dimethyluracil with alkylaminoalkyl substituted derivatives of vanillin, isovanillin and 3-hydroxybenzaldehyde and subsequent cyclization in thionyl chloride. Results: The compounds were evaluated for their affinity for A1 and A2A receptors using [^3H]DPCPX and [^3H]ZM-241385 as radioligands. Table summarizes the observed affinities of various newly synthesized 8-phenylxanthine derivatives in radioligand binding assays at human A1 and A2A receptors. The presence of a methoxy substituent at 3 or 4- position of phenyl ring in A and B along with an ortho polar side chain increases selectivity for A2 over A1 receptors. However absence of a methoxy group as in C results in almost equal selectivity for both subtypes. Table: Adenosine A1 and A2A binding affinities of compounds Comp. No. A B C DPCPX ZM 241385 A1 Ki (µM) >100 >100 2.1 0.095 0.54 A2A Ki (µM) 0.7 2.7 0.8 0.13^1 0.064 Conclusions: It can be concluded that suitable selection and positioning of aryl substituents may lead to development of potent and selective xanthine based adenosine receptor antagonists.

The Modified Vaccination Technique developed by Barabas provides the "magic bullet" for the prevention and cure of chronic ailments such as autoimmune disorders and cancer specifically, without side effects BARABAS AZ^1, COLE CD ^2, LAFRENIERE R^1 1 Department of Surgery, University of Calgary, Health Sciences Centre, Calgary, Alberta, Canada 2 Department of Neurosurgery, University of Utah, Salt Lake City, Utah, USA Background: Barabas has developed and implemented a new vaccination method called modified vaccination technique (MVT) - in experimental animals - that can be employed both prophylactically and therapeutically with equal effectiveness to combat chronic disorders such as autoimmune diseases, cancer and exogenous antigen induced mishaps. This breakthrough discovery of antigen presentation by the MVT allows the induction of a predetermined beneficial immune response outcome. Presently available vaccination programs are preventative (through active immunization) or therapeutic (through passive immunization) and neither technique can evoke specific downregulatory (in autoimmune disorders) or upregulatory (in cancer and chronic infections) immune responses to correct chronic disorders. Methods: When vaccinating, using the MVT, appropriately assembled immune complexes - made up of the specific target antigen and specific homologous polyclonal or monoclonal antibodies against the target antigen - have to be prepared (the "magic bullets") for the prevention and treatment of presently drug only treatable conditions such as autoimmune diseases and cancer. The goal is to inject exactly the right immune complex (the "magic bullet") into recipients to evoke the production of the same class of immunoglobulin (i.e., antibody) with the same specificity against the target antigen that resides in the inoculum. Results: Using the MVT antibody information transfer is achieved resulting in predetermined immune responses. Through the utilization of the MVT the potential of downregulating or upregulating immune events - in humans with certain autoimmune disorders and cancer - to achieve self cure and regained tolerance to self is in sight. Conclusion: We believe absolutely in the immune system's natural ability to correct mistakes. It requires only in certain instances tailor made "instructions" for eliciting and maintaining redirected beneficial immune-response outcomes.

Bacteriocin substance producting by Lactococcus lactis against Listeria monocytogenes isolated in ready-to-use fish fillets BARILE M.^1, PALOMBA S.^2, CERES C.^1, MURRU N.^1, PEPE O^2 ^1Department of Zootechical Sciences and Food Inspection, Faculty of Veterinary Medicine, Napoli, Italy; ^2Department of Food Sciences, Faculty of Agricultural, Portici, Italy Background: Bacteriocins produced by lactic acid bacteria are important in preventing the growth of pathogenic bacteria especially in ready to use fish that could be potentially contaminated by pathogens. Methods: 26 Carnobacterium piscicola, 5 Carnobacterium divergens and 24 Lactobacillus spp. strains were screened against 22 strains of Listeria monocytogenes, 5 strains of Yersinia enterocolitica, 1 strain of Staphylococcus aureus, 1 strain of Pseudomonas spp, 1 strain of Salmonella spp. All producer and indicator strains were isolated from farmed gilthead fish fillets (Sparus auratus) held in the culture collection of Department of Zootechical Sciences. The microorganisms were previously identified by biochemical method and submitted to antagonistic activity experiments in solid and liquid media by using the spot on lawn and the agar well diffusion assay, respectively. LAB strains producers of bacteriocin-like substance in solid media, were then tested for bacteriocin producing in broth and sensitivity to heat (60-80°C/1h; 100°C/30 min.; e 121°C/10 min), and to proteolitic enzymes. Bacterial growth curves and bacteriocin production were studied for LAB strain producer. Results: On 25,4% of LAB strains showing antimicrobial activity in solid media, only 1 strain, belonging to Lactococcus lactis Sa 31, was able to produce bacteriocin in liquid medium. Lact. lactis Sa31 showed a narrow spectrum of it activity towards Listeria monocytogenes (clear inhibition zones 5-8 mm). No inhibition zones were observed over Staphylococcus aureus, Salmonella spp., Pseudomonas e Yersinia enterocolitica. The increased bacteriocin production of Lact. lactis Sa 31 was observed after 11 h of incubation at 30°C in Elliker agar with maximum titre of bacteriocin of 2,560 Arbitrary Units ml^-1. The bacteriocin was sensitive to trypsin, pronase E and papain and was inactivated by temperatures ≥ 100°C. Conclusions: The bacteriocin produced by Lactococcus lactis showed a relevant antimicrobial activity against different strains of Listeria monocytogenes and could be employed as biopreservative in ready-to-use fish fillets.

Allosteric modulation of hormone receptors in cancer treatment BARKER S, PUDDEFOOT JR, VINSON GP. School of Biological and Chemical Sciences, Queen Mary, University of London, London, United Kingdom. Background: Trilostane was originally developed and used as an inhibitor of 3-beta hydroxysteroid dehydrogenase in treating Cushing’s syndrome. It was later found to confer clinical benefit in breast cancer patients. Our studies began with the observation that trilostane (at micromolar concentrations) could effect the function of the estrogen receptor (ER) but without displacing the native ligand, estradiol, from its ligand binding site. We have proposed that trilostane can act as an allosteric modulator of the ER and our research has focused on trying to find an explanation for its efficacy in the treatment of postmenopausal breast cancer. Methods: In these studies we have used a wide range of biochemical and functional methods, including ligand-binding assays, gene reporter assays, gene expression microarrays and cell proliferation assays, in order to try to define an alternative mechanism of action for trilostane in the context of breast cancer. Results: Trilostane does not displace radiolabelled estradiol from estrogen receptor preparations. It inhibits MCF-7 breast cancer cell proliferation at concentrations of 1μM and above. It has direct inhibitory effects on ER function and events mediated through oestrogen response elements (EREs) and activating protein-1 motifs. In a study of the effects of trilostane and tamoxifen, on MCF-7 cells using microarrays, striking differences were found in gene expression profiles. Interestingly, trilostane was found to selectively up-regulate the expression of the beta subtype of estrogen receptor (ERβ), an effect replicated in vivo. This is significant as tamoxifen-resistant breast cancer is often associated with reduced ERβ protein expression and trilostane can inhibit proliferation of tamoxifen-resistant MCF-7 cells in vitro. Conclusions: Our data support previous clinical findings that trilostane can influence the effectiveness of tamoxifen and may subvert tamoxifen resistance, and demonstrates in a wider context that allosteric modulation of receptor function may be a useful therapeutic approach in circumstances in which the receptor ligand binding domain is occupied by its cognate ligand or an antagonist. This concept has potential application in clinical situations in which either nuclear receptors or surface membrane located receptors are implicated in cancer progression.

Elesclomol: A Novel Oxidative Stress Inducer for the Treatment of Metastatic Melanoma BARSOUM J^1, FOLEY KP^1, WILLIAMS A^1, JACOBSON E^1, O’DAY S^2, 4783-03 STUDY GROUP ^1Synta Pharmaceuticals Corp., Lexington, MA, USA; ^2The Angeles Clinic and Research Institute, Santa Monica, CA, USA. Background: Elesclomol (E) is a small molecule drug candidate that stimulates production of reactive oxygen species (ROS) and selectively induces cancer cell apoptosis. The clinical efficacy of E in combination with paclitaxel (P) was investigated in a double-blind, randomized, controlled Phase 2 trial in melanoma. Methods: The in vitro activity of E was assessed in human Hs294T melanoma, Ramos lymphoma and HSB2 leukemia cells. Preclinical efficacy was tested in an M14 melanoma human xenograft model in mice. Stage IV metastatic melanoma patients were randomly assigned to 213 mg/m^2 E plus 80 mg/m^2P (E+P) or 80 mg/m^2 P as a weekly one-hour intravenous infusion, three of every four weeks until disease progression per RECIST or death. The primary efficacy endpoint was progression-free survival (PFS). Results: E rapidly increased ROS production in cancer cells. Sustained oxidative stress led to activation of the intrinsic mitochondrial apoptosis pathway, with cardiolipin oxidation, decreased mitochondrial membrane potential, cytochrome c release and caspase activation. Antioxidants blocked all activities of E. ROS increase and apoptosis preferentially occurred in cancer cells relative to normal cells. E inhibited tumor growth as a single-agent and synergized with P in causing tumor regression in human xenograft tumor models. At 21 US sites, 53 subjects were randomized to E+P and 28 subjects to P. The addition of E to P yielded a doubling of median PFS (112 v 56 days) and a 41.7% risk reduction for disease progression/death (hazard ratio = 0.583, P = 0.035). Respective response rates were 15% v 3%. Median overall survival was 11.9 v 7.8 months. E+P was well tolerated. Conclusions: 1. E increased ROS levels in cancer cells, leading to apoptosis. Cancer cells produce elevated ROS relative to normal cells, and are therefore susceptible to further ROS increases which push the cell beyond its oxidative stress tolerability limit. In this way, E takes advantage of a fundamental hallmark of cancer to selectively kill cancer cells with little effect on normal cells. 2. In a randomized, blinded Phase 2 clinical trial, E+P resulted in a clinically meaningful, statistically significant increase in PFS, with an acceptable toxicity profile and encouraging survival data. A global Phase 3 trial (SYMMETRY^SM) of E+P v P in chemotherapy-naïve stage IV metastatic melanoma patients is ongoing.

Blood-brain barrier P-glycoprotein function in aging and neurological disease: in vivo measurements in humans with [^11C]-verapamil PET. BARTELS AL, WILLEMSEN ATM, BART J, KORTEKAAS R, LEENDERS KL Depts of Neurology and Nuclear Medicine & Molecular Imaging, University Medical Centre Groningen, The Netherlands Introduction: The blood-brain barrier (BBB) possesses a highly concentrated transporter, the P-glycoprotein (P-gp) transporter. It protects the brain from several endogenous and exogenous compounds by acting as an active cell membrane efflux pump. Changes in P-gp function have been implied in several neurological conditions. Upregulation of P-gp has been found to prevent uptake of drugs in brain tumours and in epilepsy, leading to therapy-resistance. Decreased P-gp function has been associated with Amyloid-β transport and progression of Alzheimer’s disease (AD). Also, it has been related to toxin exposure and the development of Parkinson’s disease (PD). Finally, age-associated decline in P-gp function may facilitate the accumulation of toxic substances in the brain. We studied in vivo BBB P-gp function in older healthy subjects and in patients with PD. Methods: BBB P-gp function was studied using positron emission tomography (PET) and[^11C]-verapamil. 17 healthy volunteers with age 18-86, 22 patients with Parkinson’s disease in different disease stages and six subjects with parkinsonism (PSP and MSA) were scanned. Distribution volume (DV) of the tracer in the brain was calculated using Logan analysis. Statistical Parametric Mapping (SPM2) was used to study specific regional differences between the subject groups. Results: Older subjects showed significantly increased brain tracer uptake as compared to the younger healthy subjects, indicating decreased BBB P-gp function. In PD, the more advanced patients showed increased tracer uptake in frontal regions, while de novo patients showed decreased uptake. In patients with parkinsonism, increased uptake in basal ganglia regions was observed. Conclusion: Decreased blood-brain barrier P-gp function with aging could be a mechanism by which age acts as the main risk factor for the development of neurodegenerative disease. It also means that several drugs will reach higher concentrations in the brains of elderly patients. The knowledge that P-gp function may be modulated by drugs is also important, as P-gp modulators may provide opportunities for intervention, not only in treatment resistant cancer or epilepsy but also in the progression of neurodegenerative diseases.

Laser-based Stable Isotope Tracing in Human Breath BARTLOME R and SIGRIST MW ETH Zurich, Zurich, Switzerland Background: Stable isotopes have become widely available at affordable prices. They are increasingly used as tracers in clinical research and diagnosis. In breath analysis, analytical methodology has relied upon expensive and cumbersome instrumentation such as isotope ratio mass spectrometry (IRMS). In the last decade, isotope ratio infrared spectrometry has become a serious competitor to IRMS. Lasers have recently been employed to measure the ^13C/^12C isotope ratio of exhaled carbon dioxide. However, breath samples must be prepared to reduce the water vapor content. In this work, we measured—without sample preparation—the D/H isotope ratio increase in exhaled water vapor following the intake of a deuterated tracer (D2O). Current sensitive trace-gas detection schemes for infrared laser spectroscopy cannot handle condensable vapors. For this purpose, we developed a high-temperature multipass cell. Methods: A healthy volunteer drank 5.14 mL of 99.9 % pure D2O diluted in 200 mL tap water. The D2O intake corresponds to a dosage of 16 mg deuterium/kg body weight, a safe level that is within the dosage range used in clinical studies. Breath samples were then collected at 24-hour intervals in a commercial bag heated above 323 K until original background levels were obtained. The high-temperature multipass cell was filled with a collected breath sample without any preliminary sample preparation. Roto-vibrational spectra were recorded by finely tuning a mid-infrared laser between 2788.60 and 2789.80 cm^-1. Results: The D/H isotope ratio is usually expressed in parts per thousand relative to a standard, using the common notation ^2H = (Rsample-Rstandard)/Rstandard  1000, where R is the ratio of the heavier to the lighter stable isotope of hydrogen. We performed repetitive measurements before D2O intake and determined ^2H with a standard deviation of 23 ‰. Following D2O intake, we measured a significant ^2H increase in exhaled breath of 768 ‰. We will present time-dependent measurements after D2O intake and derive the total body water. Conclusions: Following the ingestion of only 5 mL D2O, an infrared laser spectrometer determines — without sample preparation — the D/H isotope ratio increase in exhaled water vapor for the first time. This opens the door to many clinical applications. The laser spectrometer has a great potential to become a portable and affordable device.

Erythromycin: Magic Bullet for Sore Throat and its Consequences in Children BASNET NB^1,2, BASNET SB^1 ^1Children’s Medical Diagnosis Center (CMDC), Kathmandu, Nepal;^2Durga Bhawani Polyclinic, Kathmandu, Nepal. Background: Most people suffer from sore throat particularly in the first two decades of life. Previous studies have proved that sore throat is a major cause of sickness and a potentially dangerous source of infection in children. We evaluated the clinical situation of sore throat in Nepalese children, and reviewed literature to assess erythromycin as its magic remedy. Methods: We prospectively evaluated the clinical data of children below 19 years who visited Children’s Medical Diagnosis Center and Durga Bhawani Polyclinic from January 2006 to December 2007. All patients were consulted and the final diagnosis was made by a consultant pediatrician and pediatric cardiologist. We also analyzed all the publications indexed in Pub Med upto June 27, 2008 on key words children and sore throat and erythromycin conducted in various studies. Results: Of the total 1175 examined patients 159 (13.53%) had tonsillitis. Amongst 159 patients 63.53% were between 4 to 10 years. The male female sex ratio was 1.48. Our clinical experience supplemented by reported 150 literatures have showed that erythromycin has been used in all age groups and sex, is effectively acceptable, well tolerated orally, and also available in parenteral form. It has been available worldwide after its discovery by McGuire and coworkers in 1952 from a strain of Streptomyces erythreus, originally obtained from soil in the Philippines. It is able to eradicate a broad range of bacteria including streptococci, staphylococci, listeria, legionella, diphtheria, pertussis, tetanus, syphilis and Camphylobacter jejuni without any fatal toxic effect. It exerts effective concentration on tonsilar tissues. Erythromycin has both curative (e.g. sore throat) and prophylactic (e.g. rheumatic fever, rheumatic heart disease, acute glomerulonephritis) benefits in man. It has been proved effective in treating sore throat and other infections thereby decreasing its untoward life threatening cardiac, renal, respiratory and neuronal complications, as well as the cost of treating cardiac consequence, such as valvular surgeries. Conclusions: Erythromycin has reliable chemotherapeutic effect in treating sore throat and preventing its cardiac, respiratory, renal and neuronal consequences in children of developing and industrialized nations thus proving its role as a ‘magic bullet’.

Application of Saffron and Its Ingredients as a Known Pharmacological Herb from Ancient Times and the Mechanisms of Their Action Bathaie SZ^1, Mousavi SZ^2, Hoshyar R^1 and Ashrafi M^1 ^1Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran; ^2Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran. Background: Saffron (Crocus sativus L.), which is named as "red gold" in Iran, has been used not only as a food additive and a dye, but also as a medicinal herb in most parts of the world from ancient times. In the last two decades, it has been reconsidered because of its various biological properties. Chemical analysis has shown the presence of more than 150 components in saffron stigmas. In this presentation we reviewed the historical and medicinal uses of saffron in different parts of the world, especially in Iran where saffron was cultivated for the first time (in addition to the wild type), and where nowadays is the biggest saffron producer (more than 80% of world saffron cultivation). Then, the new findings from our laboratory as well as other research groups, about its medicinal properties and various cellular and molecular mechanisms of action were discussed. Methods: Web of Science and Medline were searched for saffron and its constituents. For ancient uses of saffron, manual searching of Persian books and searching internet resources, among with manual searching of their references were done. In our experiments, after purification of important constituents from Iranian saffron, their application on breast and gastric cancer were studied in model animals; their molecular mechanism of action was also investigated using in vitro experiments. Results: Saffron was known from more than 3000 years ago by Iranians, Assyrians Babylonians and Minoans; not only for its application as a food additive, dye and perfume, but also for its usage as a medicinal herb (alone or in combination with other drugs) to treat a wide range of diseases. Newly, its application in a variety of disorders involving neuronal, cardiovascular and other systems as well as cancer has been investigated. The cellular and molecular mechanisms of its action are also under study. Saffron's more powerful components are carotenoids and monoterpene aldehydes. Structure-function relationship studies show that some properties are related to deglycosylated derivatives, while others belong to more glycosylated ones. Conclusion(s): Saffron as an important medicinal herb is a good candidate to be considered for new drug design.

Ear Surgery – Place for Topical use of Mitomycin C BATTELINO S, ZARGI M University Medical Centre Ljubljana, Ljubljana, EU-Slovenia. Background: Stenosis and atresias of the external auditory canal (EAC) are rare conditions difficult to manage with success. The main reason to operate is severity of the hearing impairment and threatening cholesteatoma of the EAC. Some authors do not find classical surgical methods sufficient and they favor the use of KTP/532 laser, or larger surgical procedures. The proven ability of Mitomycin C (MMC) to inhibit fibroblasts in vitro has stimulated its use it in treatment to prevent stenosis and adhesion, also in otorhinolaryngology. The objective of the study was to evaluate the opening of the external auditory canal in fibrotic atresias (congenitally and secondary) and the hearing improvement, after the surgery and concomitant use of topical MMC. Methods: Ten patients, all together fourteen ears, with fibrotic external auditory canal (EAC) atresias due to chronic external otitis, post-traumatic, post irradiated, or congenital cause were included. During the surgical procedure – meatoplasty only endaural approach was used, and 1 mL of MMC (0.4 mg/mL) was applied for 4 minutes to the EAC. In 5 ears the application of MMC was repeated one to six month later, than 1mg/mL concentration of MMC was used. During the application of MMC the tympanic membrane was protected with a thin layer of dry Gelfoam. Audiometric evaluation included pre and postoperative air-conduction thresholds (AC) and bone-conduction thresholds (BC). Results: Between 9 and 56 months after the surgery and concomitant application of MMC the microscope visual control of opening of EAC was assessed. The hearing improvement was observed by using preoperative and postoperative pure tone threshold audiograms (PTA). In 10 ears (72 %) the ear canals reminded open with a postoperative air-bone gap of 10 dB or less. No sensorineural hearing loss was detected after surgery. Conclusions: 1) MMC used on a limited number of ears during surgery was effective in preventing scaring in fibrosis ending with atresias of EAC. 2) No complications or sensorineural hearing loss were encountered after surgery and MMC application. 3) The results of our study are comparable with the published reports and according to the follow up period our outcome results with a success rate of 72% could be considered as final.

Cefquinome and Amoxicillin in Goats: PK/PD Integration BATZIAS GC Laboratory of Veterinary Pharmacology, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, 54 124 Thessaloniki, Greece Background: Cefquinome (CFQ) and amoxicillin (AMO) are beta-lactam antibiotics, belonging to the groups of cephalosporin and penicillin, respectively. CFQ and AMO are intended for the treatment of respiratory infection diseases, caused by Mannheimia haemolytica and Pasteurella multocida and for the treatment of mastitis in livestock. The aim of this study was to obtain and integrate pharmacokinetic (PK) and pharmacodynamics (PD) data of CFQ and AMO in the goats. Methods: The pharmacokinetic profile of CFQ and AMO in goats was investigated following intravenous (i.v.) and intramuscular (i.m.) administration at the doses of 1 mg/kg and 15 mg/kg, respectively. At the same time, the minimum inhibition (MIC) and minimum bactericidal (MBC) concentrations of CFQ and AMO against two reference strains of Mannheimia haemolytica and Pasteurela multocida were determined in cation-adjusted Mueller-Hinton Broth (MHB) and goat blood plasma. The bactericidal activity of CFQ and AMO was tested against Mannheimia haemolytica and Pasteurella multocida using the time killing method. Results: After i.v. administration of CFQ, the pharmacokinetics of CFQ indicated a small volume of distribution (Vss=0.204±0.02 L), a rapid clearance (2.433±0.59 mL/min) and half-life of 1.36 ± 0.2 h. After i.v. administration of AMO, the short terminal half-life (t1/2z) of 2.0 ± 0.47 h was the net result of ratio of the relatively small volume of distribution to the total clearance. After i.m. dosing absorption of CFQ was complete (F≥100) and rapid and terminal half-life was 1.54±1.4 h. However, after i.m. administration of AMO, the half-life in goats (7.89±2.26 h) was much higher than after i.v. administration of AMO. The difference in half-lives between i.v. and i.m. administration of AMO suggest that the i.m administration of AMO in goats produce a flip-flop phenomenon. In broth MIC and MBC of AMO against M. haemolytica were 0.188 μg mL^-1 and against P. multocida were 0.250 μg mL^-1. MIC and MBC of AMO against M. haemolytica in goat plasma were MIC=MBC=0.188 μg mL^-1 and against P. multocida in blood plasma of goats were MIC=MBC=0.375 μg mL^-1. MIC and MBC of CFQ, when determined in MHB, were 0.047 μg mL^-1, against M. haemolytica and P. multocida. MIC and MBC of CFQ against M. haemolytica in goat plasma were 0.094 µg mL^-1. The respective value against P. multocida was MIC= 0.047 and MBC= 0.094 μg mL^-1 in goat plasma. MIC and MBC data against two reference strains M. haemolytica and P. multocida indicated that the AMO generally exhibited higher MIC and MBC when compared with CFQ. CFQ and AMO were shown to be time dependent bactericidal antibiotics against target pathogens and the killing occurring at a concentration close to the MIC. The rate of killing was not significantly influenced by the increase of antibiotic concentration. Conclusions:Taking into consideration the PK and PD parameter of AMO in goats, the concentration of the drug in the plasma remain above the MIC (=0.375 μg/mL) of M. haemolytica and P. multocida in the goats for 12 hours after i.m. administration, and it can be concluded that a once-daily amoxicillin i.m. administration at the dose of 15 mg/kg b.w, yields therapeutically effective drug levels. Furthermore, according to PK analysis and PD data of CFQ, it can be concluded that, for susceptible bacteria, a twice-daily cefquinome i.m. administration, at the dose of 1 mg/kg b.w., will yields therapeutically effective drug levels.

Cell-free octameric hemoglobin as a blood substitute BAUDIN-CREUZA V^1, VASSEUR C^1, DOMINGUES E^1, HO C^2, MARDEN MC^1 ^1INSERM U779, University of Paris XI, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre, France. ^2Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA. Background: Considerable progress has been made in the development of red blood cell substitutes, in particular with hemoglobin (Hb) based oxygen carriers designed to correct oxygen deficiency. The two major problems are encountered with acellular Hb in the plasma: the optimum oxygen affinity for adequate oxygen delivery to tissues and the dissociation of Hb tetramers (α2β2) into dimers (αβ). Clinical trials with different Hbs obtained either by chemical modification or by protein engineering to prevent tetramer dissociation, have shown a vasoactivity in plasma. Increasing the molecular size of the carrier has been proposed to reduce the undesirable vasoactive properties. In order to eliminate problems related to the small size of acellular oxygen carriers, we have made the next logical step of going from a tetramer to an octameric form of Hb by introducing the cysteine residues oriented towards the exterior of the β-subunits. Methods: The mutation βG83C was introduced by site-directed mutagenesis into the co-expression vecteur pHE7 containing human , β-globin cDNAs and an E. coli methionine aminopeptidase cDNA. The recombinant Hb βG83C (rHb βG83C) have engineered in JM 109 strains of E.coli and purified by ion exchange chromatography. Results: The analysis of purified rHb βG83C by exclusion size chromatography shows the presence of a single molecular species corresponding to a molecular weight of 129 kDa ie a dimer of tetramer. The CO rebinding kinetics after flash-photolysis of this octameric rHb are similar to that of tetrameric Hb with two phases corresponding to two allosteric states (R and T states). The different studies show that this octameric rHb was stable and did not dissociate easily into small molecular species at low concentration. This rHb was resistant toward potential disulfide reducing agents present in fresh human plasma. Moreover these octamers does not interact with haptoglobin, a plasma glycoprotein that binds dimers to participate in the elimination of Hb from blood circulation. Conclusions: This molecule is thus potentially useful as blood substitutes. The objectives are now to evaluate the effects of this rHb on the biology of the endothelial cell.

Inhaled H2S for suspended animation in anesthetized and ventilated mice BAUMGART K^1, WAGNER F^1, SIMKOVA S^1, WEBER S^1, CALZIA E^1, RADERMACHER P^1, SENFTLEBEN U^1, HUBER-LANG M^2, KNÖFERL M^2, GEORGIEFF M^1 ^1Klinik für Anästhesiologie und ^2Unfallchirurgie, Universitätsklinikum, Ulm, Germany Background: Several organisms can reversibly slow down their vital functions in order to sustain otherwise lethal environmental stress, a phenomenon called hibernation. In mice, inhaling hydrogen sulfide (H2S) induced such a hibernation–like state called „suspended animation“ with decreased heart rate, respiratory minute volume and metabolic expenditure, and consecutively hypothermia^1. H2S also is an endogenous gaseous messenger and signaling molecule, and both pro- and antiinflammatory effects were reported. Since in rodents anesthesia per se causes hypothermia, we tested the hypothesis whether inhaled H2S may also induce suspended animation in anesthetized, mechanically ventilated and instrumented mice. Methods: 15 hours after laparatomy animals received 100 ppm H2S or vehicle for 5 hours with core body temperature maintained at 38° or 27°C. Left ventricular pressure-volume loops were assessed using a pressure-conductance catheter. Cytokine (TNF-α, IL-6) and chemokine (MCP-1, MIP-2) formation, myeloperoxidase and NF-kB activation were measured in lung homogenates, mitochondrial respiration was measured in liver biopsies (high-resolution respirometry). Results: In contrast to awake mice, H2S alone did not affect hemodynamics. Within 3 hours, however, both hypothermia alone and combined with inhaled H2S decreased blood pressure, heart rate and thus cardiac output, while stroke volume, ejection fraction and end-diastolic pressure were unchanged. While inhaled H2S and hypothermia alone comparably attenuated lung chemokine tissue levels, only combining hypothermia and H2S significantly decreased tissue IL-6 levels. Strikingly, H2S significantly increased NF-kB activation during normothermia. Combining hypothermia and inhaled H2S attenuated the cytochrome-c-induced stimulation of mitochondrial respiration. Conclusions: 1) In contrast to awake mice anesthesia blunted the hemodynamic effects of inhaled H2S alone. 2) H2S has anti-inflammatory properties beyond hypothermia alone. 3) Reduced cytochrome-c-induced stimulation of mitochondrial respiration suggests attenuated mitochondrial damage. References: 1. Blackstone et al, Science 2005;308:518 Acknowldegement: Supported by the Deutsche Sepsis-Gesellschaft

Light (LM) and electron microscope (EM) examination of the effects of methotrexate (MTX) on the endosalpinx. BAYRAM M^1, OZOGUL C^2, DURSUN A^3, ERCAN ZS^4, ISIK I^3, DILEKOZ E^4 Gazi University, Faculty of Medicine, ^1Department of Gynecology and Obstetrics, ^2Department of Hystology and Embryology, ^3Department of Pathology, ^4Department of Pharmacology Background: To examine the effects of increasing doses of mtx on the fallopian tubes. Methods: The study was carried out on 24 female rats (Albino Wistar type, 250-300 g). Different doses of Mtx were given to the randomly divided 4 groups by IP injection: 1mg/kg to group 1, 5mg/kg to group 2, 10 mg/kg to group 3 and physiological serum to group 4 as the control group. After ten days, the fallopian tubes of the rats were removed for examination separately by LM and EM for comparison. Results: LM showed that in group I the surface epithelial cells were normal and the lamina propria was infiltrated by numerous inflammatory cells with a prevalence of polymorphonuclear leucocytes. Findings in groups 2 and 3 were similar: the lamina propria was infiltrated with granulocytes in one specimen from each of the two groups, and granulocytes were also observed among epithelial cells. In the control group all surface structures were found to be in a normal condition. EM showed cilial loss in the epithelial cells and central crystolysis in mitochondria in all group 1 specimens. Findings in groups 2 and 3 were similar. The cytoplasm of the epithelial cells seemed to be dense, there was prominent crystolysis in the mitochondria, and vacuolisation in the cytoplasm seemed to be augmented. Cilial loss was prominent, and the basal membrane was irregular. Epithelial cell nuclei were in disorder. Lipid granules were observed extensively in epithelial cells. Eosinophils seemed to be dominant in connective tissues below the epithelium. In all control group specimens the epithelium seemed to be normal with all organelles in place; the condition of intercellular junctions, ciliated epithelium and all mitochondria also seemed to be normal, and the basal membrane was observed to be in order. Conclusion: The ultrastructural derangements resulting from administration of Mtx in doses in excess of 1mg/kg can cause a reduction in the surface epithelium's ability to make rhythmic lashing movements and can impair the patency of the fallopian tubes. All these disturbances could be involved to some degree in the causation of infertility and recurrent ectopic pregnancy. Therefore, the dosage of Mtx should be limited to use of the lowest effective dose to avoid these adverse effects.

Gastrointestinal Prokinetic 5-HT4 Agonists; Receptor Selectivity and Benefit-to-Risk Profile BEATTIE DT, SMITH, JAM Theravance, Inc., South San Francisco, CA, USA Background: Selective 5-HT4 receptor agonists (e.g. TD-5108 and prucalopride) appear to produce more robust gastrointestinal (GI) prokinetic activity in patients with chronic constipation than non-selective agonists (e.g. tegaserod or cisapride). Interaction with non-5-HT4 receptors may also result in adverse effects; cisapride and tegaserod are associated with cardiac arrhythmias (via hERG potassium channel blockade) and ischemic events, respectively. In this study, the pharmacological profiles of several 5-HT4 receptor agonists are compared. Methods: Recombinant receptor binding affinities were measured by radioligand techniques. Smooth muscle mechanical activity was studied using guinea pig colon mounted in tissue baths. Colonic transit was measured in conscious guinea pigs (excretion time of dye injected into the colon) as was GI contractility in conscious dogs (implanted with strain gauges). Results: TD-5108, TD-8954, tegaserod and cisapride had high affinity for the human 5-HT4(c) receptor (mean pKi = 7.7, 9.4, 8.4 and 7.1, respectively). TD-5108 and TD-8954 had high selectivity for the 5-HT4(c) receptor over all other 5-HT receptor types (>500- and >2,000-fold, respectively). Tegaserod and cisapride had affinity for other 5-HT receptors (mean pKi = 7.5, 8.4, 7.0, 7.2 and 7.2 for tegaserod at human 5-HT2A, 5-HT2B, 5-HT2C, 5-HT7 and bovine 5-HT1D receptors, and 6.2 and 7.4 for cisapride at 5-HT2A and 5-HT2B receptors, respectively). TD-5108, TD-8954, tegaserod and cisapride contracted the guinea pig colon (pEC50 = 7.9, 8.6, 7.7 and 7.0, respectively); mean intrinsic activities (relative to 5-HT) were 81%, 50%, 47% and 75%, respectively). TD-5108, TD-8954, tegaserod and cisapride (0.03 3 mg/kg sc), increased guinea pig colonic transit; TD-5108 and TD-8954 had the highest potency. In dogs, po dosing with TD-5108 and TD-8954 increased antral, duodenal and jejunal contractility more potently than tegaserod. Conclusions: There remains an unmet medical need for agents to treat patients with GI disorders such as chronic constipation and constipation-predominant irritable bowel syndrome. Future clinical evaluation with selective 5-HT4 receptor agonists such as TD-5108 and TD-8954 should determine whether they are associated with a more acceptable benefit-to-risk profile than older, less selective agents.

Gut Health-Promoting Adhesion Of Enteropathogens To Dietary Polysaccharides Becker PM^1, Galletti S^1,2 ^1Wageningen UR, Lelystad, The Netherlands; ^2University of Milan, Milan, Italy Background: Bacterial adherence to host tissues is regarded as an important initial step for colonisation and infection. Hence, agents that interfere with the ability of pathogens to adhere to gut cells are promising antidotes. Different plant products and food stabilizers were tested in terms of their binding capacity for enteropathogenic bacteria using a miniaturised adhesion test. Method: Bacterial strains were allowed to adhere to fibrous substances (Table 1) supplied as well coatings in microplates (Becker et al., 2007). Adhering bacteria were provided with liquid medium and allowed to grow during incubation in a microplate reader at 37°C. Bacterial growth was recorded as optical density (OD) at 650 nm over time. The more cells were retained, the shorter the detection time of their subsequent growth. Detection times of growth were determined at an OD of 0.05 (tOD=0.05) in triplicate in two independent assays each. Means were compared by Fisher's unprotected least significant difference test. Results: Table 1 shows the performance of various substances in terms of their binding capacity for different E. coli and Salmonella enterica. Table 1. Detection times of growth [h] of different E. coli and Salmonella enterica. Substances with the shortest detection time bound most bacterial cells (Becker and Galletti, 2008). Conclusion: With growth as measuring variable for adhesion, a simple high-throughput method was developed and applied for the screening of large numbers of different food and feed components and bacteria. References: Becker PM et al. (2007) J. Appl. Microbiol. 103, 2686-2696. Becker PM, Galletti S (2008) J. Sci. Food Agricult. 88, 2026-2035. Authors’ acknowledgements: This study was funded by the Dutch Product Board of Animal Feed (PDV) and by SAFEWASTES (EU project no. 513949) in equal shares.

Effect of Inoculum Size on the Antibiotic Susceptibilities of Enterobacteriaceae Producing Shv and Ctx-M Extended-Spectrum Beta-Lactamases BEDENIC B^1,2, VRANES J^1,3, PLECKO V^1,2, MATULIC-BEDENIC I^4, KALENIC S^1,2 ^1School of Medicine, University of Zagreb, Zagreb; ^2Department of Clinical and Molecular Microbiology, Clinical Hospital Center Zagreb^3Zagreb Institute of Public Health, Zagreb; ^4Trg Bana Jelačića 3 (retired) Background and aim: Many extended-spectrum β-lactamases (ESBL) producing isolates of E. coli and K. pneumoniae are susceptible in vitro to amoxycillin-clavulanate (AMC), ceftazidime-clavulanate (CAZ/cl), and piperacillin-tazobactam (TZP) but MICs increase substantially when higher inoculum is applied. The aim of this study was to determine the effect of inoculum size on the susceptibility of E. coli and K. pneumoniae isolates with well characterized ESBLs to amoxycillin (AMX), AMC, ceftazidime (CAZ), CAZ/cl, piperacillin (PIP), TZP, imipenem (IMI) and meropenem (MEM). Material and methods: Minimum inhibitory concentrations (MIC) were determined by broth microdilution method using inocula that differed 100 fold in density according to CLSI. The inocula contained 10^5 CFU/ml and 10^7CFU/ml approximately. The study was performed on the set of K. pneumoniae and E. coli strains producing SHV-2, SHV-5, SHV-12, CTX-M-3 and CTX-M-15 β-lactamases. Results: Inoculum effect for CAZ/cl was detected in 52% of SHV-2 producing K. pneumoniae strains followed by AMC (43%) and TZP (38%). SHV-5 producing K. pneumoniae strains showed the most pronounced inoculum effect with CAZ/cl (57%) and AMC (55%) and to lesser extent with TZP (44%). Inoculum effect was observed for AMC, CAZ/cl and TZP in 71% of SHV-12 producers. E. coli producing SHV-5 β-lactamase showed the most pronounced inoculum effect with AMC (61%) followed by CAZ/cl (51%) and TZP (22%). Strains producing CTX-M β-lactamases had a marked inoculum effect with CAZ/cl (71%), AMC (57%) and TZP (50%). AMC and CAZ/cl were associated with inoculum effect against all type of ESBL producers: SHV-2, SHV-5, SHV-12 and CTX-M. TZP was less affected by the inoculum size then AMC, and CAZ/cl particulary with CTX-M producers. The activity of TZP was mostly compromised in the presence of high density of SHV-5 producing K. pneumoniae. Conclusions: Carbapenems were the most stable compounds to inoculum effect regardless of the type of ESBL.

Synthesis and QSAR Studies of CADA Analogs with CD4 Down-modulating and anti-HIV Activities BELL TW^1, SCHOLS D^2, VERMEIRE K^2 ^1Univ. of Nevada, Reno, NV, USA; ^2Katholieke Univ. Leuven, Belgium. Background: HIV attachment via the CD4 receptor is an important target for developing novel approaches to HIV chemotherapy. Cyclotriazadisulfonamide (CADA, shown at right) inhibits HIV at submicromolar levels by specifically down-modulating cell-surface and intracellular CD4. Aims: 1) To synthesize many CADA analogs having various CD4 down-modulating activities. 2) To develop computational models that can be used to predict activities of novel analogs. 3) To prepare fluorescent analogs for studies in cells. Methods: The five-step synthesis of CADA was modified to produce more than 50 analogs having various groups on the 3 nitrogen atoms, including some bearing the dansyl fluorophore. Testing 30 of these analogs in MT-4 cells showed a wide range of CD4 down-modulating potencies. Three-dimensional quantitative structure-activity relationship (3D-QSAR) models were constructed using the comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) approaches. To generate these computer models, the solid-state structure of CADA was energy minimized and used to generate the structures of the other 29 analogs. Results: A CoMFA model was generated having strong statistical significance (r^2= 0.80, P < 0.001) and internal predictive ability (q^2 = 0.41). Potencies of all 30 compounds are predicted well by this model (within 10-fold of the experimental values). The steric and electrosataic contributions to the model are 59% and 41%, respectively, indicating that the sizes of groups attached to the nitrogens are more important than their polarities. The CoMSIA model also showed the strongest correlation between potency and steric bulk. One of the CADA analogs bearing a dansyl fluorophore is as potent as CADA and has proven to be useful for monitoring uptake and distribution of the drug in T-cells. Conclusions: 1) Effective approaches have been developed for the synthesis of many CADA analogs with a wide range of potencies. 2) Computer models have been developed for predicting potencies of novel analogs. 3) Fluorescent, potent CADA analogs have been developed that may be useful for elucidating the mechanism by which CADA compounds down-modulate CD4.

Pharmacokinetics and Pharmacodynamics of Amphotericin B and its Lipid Formulations BELLMANN R Department of Internal Medicine I, Innsbruck Medical University, Innsbruck, Austria Background: Because of its broad spectrum, the polyene amphotericin B (AMB) has still an important role in treatment of invasive fungal infections. It is eliminated unchanged via the bile and the urine. However, its use is limited by infusion related adverse events and by its nephro-toxicity. Three less toxic lipid formulations are available, displaying different composition of their lipid moieties and thus different particle size and shape. Liposomal AMB (LAMB) comprises small uni-laminar vesicles. Its volume of distributions is small, its half-life is short and high plasma concentrations are achieved. AMB lipid complex (ABLC) forms large ribbon-like structures resulting in a large volume of distribution a half-life of about 1 week whereas only low plasma level are reached. The 3^rd lipid formulation, AMB colloidal dispersion (ABCD) displays intermediate plasma levels, half-life and volume of distribution. Methods: AMB pharmacokinetics was analyzed in 18 critically ill patients on treatment with lipid-formulated AMB (13 requiring hemofiltration). Lipid-associated and liberated AMB were separated by solid phase extraction and quantified by high performance liquid chromatography. AMB tissue distribution was assessed in autopsy sample obtained from 32 patients who had died during therapy with lipid formulated AMB. Results: In critically patients, mean peak plasma levels (Cmax) of liberated AMB amounted to 0.5 µg/ml after infusion of LAMB or ABCD, whereas Cmax of total AMB was 3.4 µg/ml and 0.8 µg after ABCD. The clearance (CL) of liberated AMB was 0.2 l/kg/h (CL of LAMB [lipid-formulated] = 0.08 l/kg/h, CL of ABCD = 3.0 l/kg/h). AMB-pharmacokinetics on and off hemofiltration were similar. Tissue concentrations of 100 µg/g were reached in the liver, 70µg/g in the spleen. Lung concentrations were significantly higher after ABCD than after LAMB treatment (33 vs.12 µg/g). Levels in myocardium and brain were 3 µg/g and 1 µg/g, respectively. Conclusions: 1) The lipid-associated moieties of AMB lipid formulations display considerable differences in their pharmacokinetics whereas pharmacokinetics of the liberated AMB fraction is independent from the preparation administered. 2) No dose adjustment is required during hemofiltration. 3) AMB accumulates in liver and spleen, tissue concentrations in lung and kidneys are intermediate and low in myocardium and brain.

Mitochondrial Respiratory Chain (MRC) and Mitochondrial Permeability Transition (MPT) Effectors against Heavy Metal-induced Cytotoxicity: State-of-the-art and Perspective BELYAEVA EA Sechenov Institute of Evolutionary Physiology and Biochemistry, St.-Petersburg, Russia Background: Heavy metals, such as cadmium, mercury, etc. are strong carcinogenic, genotoxic and cytotoxic agents of high environmental and occupational hazard. Mitochondria are important targets for these pollutants. MRC disturbance and activation of different ionic channels in inner mitochondrial membrane, mainly nonselective MPT pore, are considered to be the key events in different types of cell death. Aims: 1) To find effective protectors against the heavy metal-induced cytotoxity. Methods: To further underscore mechanism(s) and role of mitochondrial dysfunction in toxic action of heavy metals we used rat neuronal cell line PC12, rat ascites hepatoma AS-30D cells and isolated rat liver mitochondria as a model system, as well as flow cytometry, different fluorescent probes and ion-selective electrodes, and swelling technique. Results: We found that against Cd^2+-induced injury not only well-known antioxidants, such as N-acetylcysteine, vitamin E, butylhydroxytoluene, and MPT inhibitors – cyclosporine A, bongkrekic acid, ADP, Mg^2+ and dithiothreitol are effective but also inhibitors of different MRC components, namely of complex I (rotenone), and of complex III (stigmatellin and antimycin A). Interestingly, stigmatellin was shown to be one of the strongest protectors that exhibited its action not only on isolated mitochondria but on both types of the cells. Conclusions: 1) Our data pointing to the direct involvement of the MRC, and especially reactive oxygen species (ROS) production on respiratory complex III in Cd^2+-induced mitochondrial membrane permeabilization and cell death. 2) On the basis of own findings and data in literature, we proposed a hypothetical model of MPT pore structure and/or regulation with the involvement of a mitochondrial supercomplex formed by respiratory complex I (P-site) and III (S-site) which may constitute not only critical Me^2+-binding sites but also main loci for ROS generation that was instrumental in oxidation of thiol groups crucial for MPT pore induction; moreover, depending on conditions and cell type, either one or both respiratory complexes could participate in triggering of the MPT pore assembly and cell death. 3) The data obtained are important for searching new drugs which could become real “magic bullets” in the nearest future. Authors’ disclosure statement: The author thanks the Russian Foundation for Basic Research for financial support (grant No. 07-04-00722).