Magic Microspheres for Joint Treatment. Why Doxycycline? Haerdi-Landerer MC1, Steiner A2, Suter MM2, Gander BA1 1ETH Zurich, Switzerland; 2Vetsuisse-Faculty Berne, Switzerland.
Background: Septic arthritis is common in large animals and local therapy has proven to be favorable over systemic antibiotic treatment. Doxycycline (DOX) was thought to encompass the spectrum against bacteria usually isolated from bovine and equine joints and to provide in addition a chondroprotective effect. DOX was microencapsulated into poly(lactide-co-glycolide) (PLGA) microspheres for prolonged release over 10 to 15 days, which should afford a single injection for a fortnight's treatment.
Methods: In study I 10 calves (5 per treatment, body mass about 80 kg, both sexes) were administered 5 or 10 mg DOX in aqeous solution locally in one antebrachiocarpal joint The contralateral joint served as control, injected with saline. Clinical and pathological examination and laboratory analyses of synovial fluid and blood were evaluated.
In study II DOX release kinetics from PLGA microspheres, DOX stability in the microspheres, joint tissue compatibility, and antimicrobial activity of the newly developed DOX containing microspheres was tested in vitro.
Results: In study I, the general clinical and pathological outcome between treated and control joints did not differ statistically. Only the metalloproteinase activity in synovial fluid of DOX-treated joints was significantly lower than in control joints; No significant differences were found between the 2 dosage groups.
Study II confirmed a triphasic release profile of DOX-microspheres, an initial burst, a 3 day phase of very little elution and a final almost constant release from day 4 to 15. No signs for preterm degradation were found chromatographically irrespective of γ-sterilization of the PLGA microspheres. Tissue compatibility of the DOX-loaded PLGA microspheres in terms of expression of pro-inflammatory cytokines by cultured synoviocytes was excellent and comparable to control cells. Only nitric oxide production was elevated and close to the positive group stimulated with bacterial polysaccharide. Finally, the antimicrobial activity of DOX released from the microspheres was fully maintained.
Conclusions: DOX may be used intra-articularly and has shown a chondroprotective effect. DOX containing microspheres could provide adequate local drug levels over prolonged periods of time. This should solve the problem of repeated injections.However, in vivo and clinical effects remain to be evaluated.
The Chick Embryo as a Tool to Discover and Validate New Therapeutic Targets Against Tumor Angiogenesis JAVERZAT, S1,2, BIKFALVI, A1,2, HAGEDORN M1,2 1 INSERM U920, ELAT (European Laboratory for Angiogenesis and Translational Research), Talence, F-33405, France; Univ Milan, Milan I-20122, Italy; 2 Univ Bordeaux 1, Talence, F-33405, France
Background: Recent technical advances such as Affymetrix chicken GeneChips now make the chick embryo a very attractive tool for gene expression studies in physiological angiogenesis, tumor angiogenesis and response to treatment of experimental tumors.
Methods: We have developed a glioma model on the chorioallantoic membrane (CAM) of chick embryos which recapitulates key steps of human tumor progression on a cellular and molecular level. Given the strong parallels between developmental angiogenesis and immature tumor blood vessels, we have created a detailled time-course gene expression atlas which describes for the first time global transcript changes in a developing vascular organ in vivo.
Results: Genes which are induced in the vascularization phase of growing tumors on the CAM are also overexpressed in highly malignant patient glioma. When experimental glioma on the CAM were treated with angiogenesis inhibitors, they were significantly smaller than controls, but remaining tumor cells upregulated genes which control invasion and other biological phenomena, which could promote tumor cell survival. Accordingly, high expression levels of two of these genes (PI3 and CHI3L1) were strongly associated with poor survival in a cohort of glioblastoma patients.
We determined human orthologs of hundreds of chicken genes regulated during the maturation of the CAM and assigned in silico endothelial cell specificity using novel bioinformatic methods.
Conclusions: 1) The chick embryo allows gene expression studies of physiological and pathological angiogenesis in a defined context, revealing new candidate genes relevant for human disease. 2) The comprehensive molecular map of vascular maturation during developmental angiogenesis constitutes a valuable resource to streamline further research of candidates susceptible to mediate tumor angiogenesis.
Erratum to “Quantification of allantoin in various Zea mays L. hybrids by RP-HPLC with UV detection” [Pharmazie, 59(2004) 524-527] HAGHI G, ARSHI R, SAFAEI A
Research Center of Barij Essence Pharmaceutical Company, Kashan, Iran Background: The Zea mays or corn silk is used in the treatment of urinary troubles such as cystitis, urethritis, enuresis, and prostatitis, specifically for acute or chronic inflammation of the urinary system. Allantoin is an astringent and keratolytic. It is frequently used topically as a vulnerary to stimulate tissue repair in suppurating wounds, resistant ulcers, acne, seborrhea, cold sores, psoriasis, hemorrhoid and other anorectal disorders. Accomplishing satisfactory separation of this compound with similar polar constituents in biological samples by high performance liquid chromatography (HPLC) is difficult due to overlapping of peaks. We intended to find a HPLC method for allantoin analysis in Zea mays. A literature study revealed method above which contained a significant error in the identification of the analyte. We duplicated this method and observed that acetone has been mis-identified as allantoin in both the extract and standard solutions. Therefore, aim of this work was to correct this error and present a new analytical method for quantification of allantoin in Zea mays by HPLC.
Method: A HPLC validated and improved method for the analysis of allantoin in silk and seed of Zea mays has been developed. Allantoin was extracted from sample and separation of crude extract was achieved on a C18 column and phosphate buffer (pH 3.0) as mobile phase at ambient temperature at a flow rate 1.0 mL/min and detected at 210 nm. A comprehensive validation of the method including sensitivity, linearity, repeatability and recovery was conducted.
Results: The calibration curve was linear over the range of 0.2-200 µg/mL with a correlation coefficient (r2›0.999). Limit of detection (LOD, S/N=3) and limit of quantification (LOQ) values of the allantoin were 0.05 and 0.2 µg/mL respectively. Relative standard deviation (RSD) value of the repeatability was reported within 1.2%. The average recovery of allantoin added to samples was 100.6% with RSD 1.5%. The results showed that the amount of allantoin in samples was between 14 and 271 mg per 100 g of dry plant material.
Conclusion: The present study describes a validated and developed method for quantification of analyte in drug sample and can be used for routine analysis of allantoin as one of the bioactive components in the quality assessment of Zea mays.
Studies on new tuberculosis vaccine candidates in animal models HAILE M1, PAWLOWSKI A1, SVENSON SB2.
1Swedish Institute for Infectious Disease Control, Stocholm, Sweden; 2Swedish University for Agricultural Sciences, Uppsala, Sweden.
Background: Tuberculosis (TB) is a major health problem in many countries, especially in low income countries. Globally, TB causes more than 2 million human deaths annually and 1/5 of all adult deaths in developing countries. Most of the world's population is vaccinated with the only available TB vaccine, the Bacillus Calmette-Guérin (BCG) vaccine. The aim of this study is to evaluate the protective efficacy of new TB vaccine candidates.
Methods: New vaccine candidates and adjuvant/delivery systems for mucosal applications were developed and tested in animal models. Lipoarabinomannan (LAM) was purified from a virulent Mtb strain and used to prepare novel oligosaccharide-protein conjugate vaccines. There is a vast number of experimental adjuvants but most of them are intrinsically toxic and only few can be considered for use in man. Aluminum salts are so far the only adjuvants approved for large scale human use. In this study a new adjuvant L3 was investigated. L3 is non-toxic and approved by the Swedish FDA for human phase I/II trials.
Results: The different AM-Prot vaccines and a vaccine based on heat killed whole-cell BCG (H-kBCG) were formulated with L3 and studied for their ability to protect against virulent Mtb challenge in the mouse and guinea pig models. Both types of vaccines, when given nasally, evoked specific and robust cellular and humoral immune responses. Therefore, in this study the role of antibodies in Mtb infection was re-evaluated in passive protection experiments using an AM-specific monoclonal antibodies (MoAb). Mice were infected intravenously with virulent Mtb and the MoAb was added intravenously either prior to or together with the bacteria. The MoAb protected against the infection in terms of a dose-dependent reduction in bacterial load in spleens and lungs, reduced weight loss and, importantly, enhanced long-term survival.
Conclusions: AM-Prot vaccines can, when formulated with the L3 adjuvant and given nasally, provide as good protection as live BCG. The AM-Prot vaccine affords protection both when used as a primary vaccine and as a boost vaccine. H-kBCG vaccine did not protect when administered alone but was protective when given in the L3 adjuvant. The pre-clinical studies of this vaccine candidate are concluded and the vaccine is considered ready for phase I/II trials in man.
The insulin-like growth factor pathway- the key to overcoming resistance in cancer therapy? HALUSKA P Division of Medical Oncology, Mayo Clinic, Rochester, MN
The insulin like growth factor (IGF) pathway is a complex signaling system that has importance in human growth and development. However, dysregulation of this system has been shown to be important in the proliferation and survival in many malignancies including lung, breast and ovarian cancer. Importantly, the IGF signaling has also been implicated as a mechanism of resistance to cytotoxic chemotherapy, hormonal therapy, radiation and biological therapy. Due to the potential of blocking these critical pathways of resistance, novel therapies targeting IGF-1R signaling have been developed and are currently being evaluated clinically. Initial investigations with agents targeting the IGF-1 receptor (IGF-1R) have proven to be very well tolerated and have demonstrated early signs of clinical activity. Among the most common adverse events is hyperglycemia. Recently, demonstration of IGF-1R inhibition having clinical improvement in response to chemotherapy was demonstrated in patients with non-small cell lung cancer. This enhancement of activity was most pronounced in the squamous cell subtype of non-small cell lung cancer, which has a relatively high expression level of IGF-1R. The preclinical and clinical activity of IGF-1R inhibitors will be reviewed. Accumulated data has suggested that crosstalk signaling between the IGF-1R and HER family of receptors (e.g., EGFR, HER2) is responsible for resistance to therapy targeting the individual pathways. Our group has recently demonstrated that the co-inhibition of IGF-1R and HER family of receptors has synergistic activity in multiple models in vitro, through blockade of “crosstalk” signaling. Based on these and other data, we are investigating the clinical activity combined IGF-1R/HER receptor blockade in HER2 positive breast cancer. Through evaluation of phospho-epitopes on circulating tumor cells, we will be investigating whether crosstalk between the IGF-1R and HER2 is clinically apparent and important for resistance to HER2 targeted therapy. These early preclinical and clinical investigations have indicated that inhibition of IGF-1R signaling may be useful in overcoming resistance to many clinically important therapies.
Practice of PK/PD theory with no measuring medicine for new quinolones injectable agents in Japan- As an example of the liver abscess -. HAMADA Y1, IMAIZUMI H1, SUNAKAWA K1, YAGO K1 1Kitasato UniversityHospital, Kanagawa, Japan
Background: In the past, liver abscess was associated with high mortality. However, its mortality has recently been reduced due to improvements in diagnostic imaging, progress in drug therapy, and concomitant use of abscess drainage. In the future, shortening of the period of treatment using more effective therapeutic methods, and reduction of patient burden and medical expenses will be of increasing importance. Pazufloxacin(PZFX) and ciprofloxacin (CPFX) are now being marketed in Japan as new quinolone injectable agents. We evaluated three cases of liver abscess being resolved with PZFX. We experienced those of liver abscess in which PZFX was effective, and this case together with a discussion of PK/PD theory is presented with reference to the literature.
Methods: PZFX was intravenously administered to three patients who had undergone abscess drainage at a dose level of 500 mg × 2/day from 2004 to 2005. We aimed Cmax/MIC≧10 has been established as an efficacy index for new quinolones at Milan University Hospital in Italy.
We compared PZFX and CPFX using tissue concentration and period of drainage referring to the documents published.
Results: Since the present patient had liver abscess, the maximal concentration in blood was replaced by the maximal concentration in bile for calculations of PZFX concentration. Since the peak of PZFX concentration in bile appears 2-3 hours after administration, the mean concentration in bile (mean ± standard deviation) was calculated using the PZFX concentrations in human bile published in a previous report, and a value of 28.88 ± 16.85μg/mL was obtained. On the other hand, in cases reported previously, the MIC of PZFX against K. pneumonia isolated from drainage was <0.5μg/mL, and the finding of concentration in bile/MIC>57.76 was obtained, indicating the usefulness of PK/PD theory.
Conclusions: PZFX therapy thereby allowed the patients to shorten the period of hospital stay. Measurement of blood concentrations in new quinolones during clinical application is not possible at present in Japan. However, we can expect clinical effectiveness with reference to the literature.
The blood-brain barrier and its magic transporters – a pharmacokinetic perspective on how to optimize drug delivery to the brain HAMMARLUND-UDENAES M Division of Pharmacokinetics and Drug Therapy, Uppsala University, Sweden
Background: Drug delivery to the brain is intricate due to the tight junctions and active efflux transporters of the blood-brain barrier (BBB). Recently, also active influx has been identified for some drugs. Thus, it would be possible to utilize efflux or influx transporters for the purpose of delivering less or more drug to the brain. Regarding increasing brain drug delivery through active influx, very little is still known. Therefore much more work is needed to understand and map active influx in relation to physico-chemical properties of drug candidates.
Methods: In order to choose the optimal drug candidates for clinically relevant drug delivery to the brain, the methods used in early discovery and development need to be optimised on the relevant measures, i.e. the unbound, pharmacologically active drug concentrations. Unspecific binding in the brain parenchyma distorts the measures when total brain concentrations are utilised. The latter approach has for too long retarded successful development of drugs aiming at the brain. Also, focusing on the permeability (rate) seems to be of less importance for clinical relevance than focusing on the extent of drug delivery to the brain, although the former focus is by far the most common. The advantages with focusing on unbound concentrations include correlations of pharmacologically active concentration at the target site to in vitro receptor binding properties or other pharmacodynamic assays.
Results and Conclusions: We have therefore proposed that brain drug delivery be divided into three main aspects, the rate (PS, CL,in), the extent (Kp,uu) and the affinity of the drug to brain tissue, described by the unbound volume of distribution in the brain (Vu,brain). In this way the unbound concentrations at the target site can be estimated from total brain concentrations and plasma concentrations after measuring fraction unbound, and be related to unbound plasma concentrations. This approach gives quantitative understanding on the role of active efflux or influx in vivo. Rapid methods to study these three aspects are needed in early drug discovery and development. At the same time, in vivo knowledge is needed to validate the methods. We are presently focusing on optimising methods for this purpose.
Rho-Kinase Inhibitors Augment the Inhibitory Effect of Anesthetic Agents on Rat Airway Smooth Muscle Contraction HANAZAKI M1, YOKOYAMA M1, MORITA K1, KOHJITANI A2, SAKAI Y3, CHIBA Y3, MISAWA M3 1Okayama University, Okayama, Japan; 2Kagoshima University, Kagoshima, Japan; 3Hoshi University, Tokyo, Japan.
Background: Most anesthetic agents relax airway smooth muscle (ASM). ASM contraction is caused by both increasing intracellular Ca2+ ([Ca2+]i) and increasing the force at the same [Ca2+]i (increase in Ca2+ sensitivity). The small G-protein RhoA and Rho-kinase (ROCK) play important roles in regulating Ca2+ sensitivity. In this study, we investigated the effects of selective ROCK inhibitors on ASM contraction and the influence of ROCK inhibitors on anesthetic-induced relaxation in ASM to test the hypothesis; although both anesthetics and ROCK inhibitors relax ASM independently, anesthetic-induced relaxation would be enhanced by addition of a low concentration ROCK inhibitor.
Methods: This study included 50 male Wistar rats (6 weeks, weight 180 - 220g). Ring strips from intrapulmonary bronchus were placed in 400-L organ baths containing Krebs–Henseleit solution. After obtaining stable contraction with 30 M acetylcholine (ACh), isometric forces were measured with the following protocols: A) Y-27632 (0.01 – 300 M), fasudil (0.01 – 100 M), or H-1152 (0.01 - 100 M) were cumulatively applied. B) propofol (1 M – 1 mM), with or without Y-27632, fasudil or H-1152 (0.03, 0.1 M), was cumulatively applied. C) isoflurane (0.5 – 4.0%), with or without Y-27632 (1 M), was cumulatively applied. Statistical significance of difference between groups was determined by Two-way analysis of variance (ANOVA), followed by Bonferroni’s test (p<0.05 was considered significant).
Results: (A) All ROCK inhibitors, especially H-1152, produced concentration-dependent relaxation.(n=5 each) (B) 0.03 M Y-27632 and fasudil did not affect the relaxation by propofol, while 0.1 M both agents significantly shifted concentration-response curves to the left ( p=0.040 (Y-27632), p=0.023 (Fasudil)) (n=5 each). H-1152 (0.03 and 0.1 M) significantly shifted the concentration-response curve to the left (p<0.001). (n=5 each) (C) Y-27632 significantly shifted the concentration-response curve for isoflurane to the left. (P<0.001) (n=5)
Conclusions: 1) ROCK inhibitors augment anesthetics-induced relaxation of rat ASM. 2) Combined use of ROCK inhibitor and anesthetics may be useful for anesthetic managements and the treatment of asthmatic patients.
Polyethylene Glycol Gold Coated Nanoparticles for the Enhancement of the Efficacy of a Specific Nutrient Synergy HARAKEH S1; PARAK W3; SPERLING R3; NIEDZWIECKI A1; RATH M1;
BAYDOUN E2 1 Dr.Rath Research Institute, Santa Calra, CA, USA
2 Biology Department, American University of Beirut, Beirut, Lebanon
3 Philipps University Marburg, Physics Department, Renthof 7, 35037 Marburg, Germany
The effects of nanoparticles on the antiproliferative efficacy of a Specific Nutrient Synergy (SNS) was investigated in both HTLV-1 infected (C91-PL and HuT-102) and non-infected (CEM and Jurkat) malignant T-cells. In all the cell lines tested, the results indicated an enhancement of the efficacy of the SNS in the presence of 0.536 μM polyethylene glycol gold coated nanoparticles using 100 and 200 μg/ml of SNS at 48h and 96h. This effect was molecular weight dependent with maximum effect at 5,000; 10,000 KDa. PEG-coated nanoparticles were more effective than their uncoated counterparts. In addition, the simultaneous or sequential application of SNS and nanoparticles did not affect the overall performance of the SNS. This study suggests a possible role of nanoparticles as a drug delivery vehicle. Future work needs to elucidate the underlying mechanism.
Vaccine Adjuvants for Sexually Transmitted Infections:
Recent Developments and Future Challenges HARANDI AM Department of Microbiology & Immunology, Institute of Biomedicine, University of Gothenburg, Sweden. e-mail: ali.harandi@gu.se
Background: The vast majority of sexually transmitted infections occur through heterosexual contacts. Globally, rates of sexually transmitted infections among women particularly those in their reproductive age continue to rise disproportionately. The failure to date to produce effective vaccines against sexually transmitted chronic infections including HIV and genital herpes infections, using the empirical approaches successful in the case of diseases such as tetanus and hepatitis B has uncovered a need to develop rational vaccines tailored to elicit specific immune response. Further, since sexually transmitted pathogens invade through and cause disease at the mucosal surfaces, there is a drive to develop vaccines or intervention approaches targeted to the mucosal surfaces.
Most vaccines presently under investigation represent recombinant or highly purified subunit components of pathogens and hence lack most of the features of the original pathogens such as the inherent immunostimulatory property. This calls for the development of safe and potent immunologic adjuvants capable of generating a broad and long-lasting immunity to. Recent advances in immunology have revealed the potential of the stimulators of innate immunity, including Toll-like receptor (TLR) agonists and other non TLR targeting immunostimulators hold promise as vaccine adjuvants. Nonetheless, the potential risk of reactogenicity associated with some of the recently developed immunopotentiators requires special attention.
Results: We were the first to show that CpG oligodeoxynucleotide, a TLR9 agonist, can serve as potent inducer of innate protective immunity in the murine female genital tract, and could also document that CpG can work as a potent vaginal adjuvant. We could also recently document that mucosal vaccination (vaginal and rectal) can elicit protective immunity against genital herpes infection in the female genital tract independent of the use of Toll/MYD88 signaling pathway. This led us to the discovery of a non-TLR targeting immunostimulator, namely alfa-galactosyl ceramide, a synthetic NKT cell ligand, as potent mucosal adjuvant to induce potent protective immunity in the female genital tract.
Conclusion: Recent developments in vaccine adjuvants for generation of protective immunity in the female genital tract, including results from our lab provide a new ground work to develop mucosal adjuvants to mount protective immunity in the female genital tract to counter sexually transmitted infections in humans.
Selective inhibition of signal peptide-dependent cotranslational translocation by the cyclopeptolide CAM741 HARANT H, OBERHAUSER B, de VRIES JE, LINDLEY, IJD
Novartis Institutes for BioMedical Research, A-1235 Vienna, Austria
Background: The cyclopeptolide CAM741, a derivative of the natural compound Hun-7293, is a potent and selective inhibitor of vascular cell adhesion molecule 1 (VCAM1) expression on endothelial cells.
Results: Extensive studies on the mechanism of action led to the identification of the signal peptide of VCAM1 as target of the compound action. The inhibitory effect of the compound takes place at the signal peptide-dependent process of cotranslational translocation. While CAM741 does not inhibit targeting of the VCAM1 nascent chains to the Sec61 translocon, it inhibits the translocation of the polypeptide chains to the luminal side of the endoplasmic reticulum. Chemical crosslinking further demonstrated that targeted VCAM1 nascent chains are differently associated with the translocon in the presence of compound, and that also the signal peptide itself shows altered positioning within the Sec61 translocon. During a search for other CAM741-sensitive signal peptides, that of vascular endothelial growth factor-A (VEGF) was identified as another target of the compound. Although both signal peptides are sensitive to CAM741, they do not share any obvious similarities within their primary sequence. However, mutagenesis of both signal peptides demonstrated that by increasing the hydrophobicity and decreasing the flexibility of the signal peptides, sensitivity to CAM741 decreased.
Conclusions: Our current model of the compound action depicts that the efficiency of signal peptide binding to the Sec61 translocon at least contributes to the inhibitory action of CAM741 and that the compound competes with the incoming signal peptide for translocon binding. Further investigations on the mechanism of translocation inhibition and the search for other sensitive signal peptides should help to dissect the mode of action and to understand the process of cotranslational translocation driven by signal peptides. These studies provided the first proof-of-principle that the process of cotranslational translocation can be inhibited in a signal peptide-specific manner without affecting the overall translocation process of other polypeptides.
Rationale for the use of memantine in the treatment of glaucoma and other neurodegenerative disease. HARE WA, WHEELER L
Allergan Inc.; Irvine, CA; USA
Background Activity of NMDA (N-Methyl-D-Aspartate) type glutamatergic ion channels (excitotoxicity) has been implicated in injury to CNS neurons for a wide range of disorders including glaucoma and retinal disease. Memantine (1-amino-3,5-dimethyladamantane), an NMDA channel blocker, has been approved for the treatment of Alzheimer's disease and has been shown to be effective for reduction of neuronal injury in animal models of CNS disease. In the experiments described here, memantine was tested for its ability to reverse excitotoxicity in an isolated retina preparation and also for its efficacy to reduce injury in animal models of chronic glaucoma.
Methods In-vitro recordings of the ERG (electroretinogram) and spiking activity of retinal ganglion cells (RGCs) were made from rabbit retinas. Excitotoxicity was induced either by application of NMDA, perfusion with zero Mg++ medium, or block of glutamate transporters with TBOA (DL-threo-β-Benzyloxyaspartic acid). For in-vivo studies, experimental glaucoma was induced in one eye by laser injury of aqueous drainage vessels, resulting in chronic elevation of intraocular pressure (IOP). Glaucomatous injury was assessed using electrophysiological recordings of the ERG and VEP (visually-evoked cortical potential) in a monkey model and also using histological measures of RGC survival in both rat and monkey models.
Results Experimental excitotoxicity was associated with an increase in tonic RGC spiking activity and a decrease in spike amplitude. Memantine (10 μM) reversed excitotoxicity induced by all three experimental conditions. Applied in isolation, memantine produced dose-dependent delays in ERG and RGC responses. Experimental glaucoma rats treated systemically with memantine (3 mg/kg-day via osmotic pump) lost on average 7% of the RGCs in the glaucomatous eye compared with a 31% loss in vehicle treated animals (p< 0.001). Glaucoma monkeys treated orally with 4 mg/kg-day memantine had better preservation of ERG (p = 0.03) and VEP (p = 0.04) responses compared to vehicle treated animals and also showed better preservation of RGCs in the inferior part of the retina (p = 0.006). Memantine had no effect on histological or functional measures in normotensive eyes.
Conclusions Memantine was effective to reduce neuronal injury at concentrations that have little or no effect on normal function.
Proton Transport Inhibitors (PTI) as Selective Anticancer Agents HARGUINDEY S1, ARRANZ JL1, WAHL ML2, RESHKIN SJ3, ORIVE G4
1Institute of Clinical Biology and Metabolism, c) Postas 13 - 01004 Vitoria, Spain; 2Department of Pathology, DUMC 3712, Duke University, Durham, NC 27710, USA; 3Department of General and Environmental Physiology, University of Bari, 70126 Bari, Italy; 4Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of the Basque Country, c) Paseo de las Universidades 7 - 01006 Vitoria, Spain.
CONFERENCE OUTLINE AND SUMMARY
“We can only cure what we can understand first”. Otto Warburg
A proton [H+]-related mechanism underlying the initiation and progression of the neoplastic process has been recently described by different research groups. Regardless of their origin and genetic background, all cancer cells and tissues have a pivotal energetic and homeostatic disturbance of their metabolism that is completely different from all normal tissues: an aberrant regulation of hydrogen ion dynamics leading to an intracellular pH to extracellular pH (?pHi to ?pHe), gradient reversal of cancer cells and tissues, that leads to an interstitial acid microenvironment secondary to an initial, specific and etiopathogenic intracellular alkalosis. This specific abnormality is increasingly considered as one of the most sensitive and differential hallmarks of cancer. This approach, which focuses on the relationships among the intracellular and the extracellular dynamics of the hydrogen ion permits the creation of a unifying view of several of the most important fields of cancer research, from etiopathogenesis, cancer cell metabolism and neovascularisation, to multiple drug resistance (MDR), selective apoptosis, the metastatic process, cancer chemotherapy and even the spontaneous regression of cancer (SRC). The integral and rational perspective behind these findings is likely to open new pathways towards the development of more selective and less toxic therapeutic measures for all malignant diseases. New therapeutic approaches are advanced.
Hierarchy of Immune Responses behind the Blood-Brain-Barrier (BBB) in the Normal Brain: Implications for the development of CNS Diseases and Treatment HARLING-BERG CJ1, KNOPF PM1 1Brown University, Providence RI 02912, USA
Background: Interactions between the brain and immune system are hierarchical and highly regulated. We have shown that antigens microinfused into the rodent brain reach cervical lymph nodes by outflow pathways for brain fluids along cranial nerves and stimulate a specific response in nodes within days. Tumor cells in the brain activate cytotoxic T lymphocytes (CTL) but do not destroy the brain tumor due to suppression of CTL activity by TGF-ß in cerebrospinal fluid. Delayed type hypersensitivity response to microinfused protein fails to develop while the same dose elicits a robust serum antibody (Ab) response and intrathecal Ab synthesis, without inflammation, that persists for months. Aim: To determine if the presence of Abs behind the BBB can alter brain function if they cross-react with brain antigens.
Methods: Multiple studies (I-IV) were done using a rodent model with normal BBB function. A cannula was stereotaxically implanted into rat brain and 7 days later, dilute sera or immunoglobulins (Igs) with anti-neuronal activity were microinfused (Alzet® pump) through the cannula. Controls received normal sera or Igs. In study I, sera or Igs from children with Tourettes Syndrome (TS) were microinfused bilaterally into caudate nucleus (CN) and rats were assessed for stereotypies and Ig binding to neurons. In studies II-IV, cannula placement was into the region of the subthalamic nucleus (STN) and the turning response to s.c. injection of apomorphine was measured following unilateral microinfusion of: sera from children with Sydenham’s Chorea (SC), Study II; rat anti-sera against rheumatogenic streptococci (rStrep), which contains brain-cross-reactive epitopes, Study III; and rabbit-anti-Igs against a peptide of oligodendrogliocyte myelin glycoprotein, Study IV.
Results: Rats microinfused with TS sera or Igs (I) had significant changes in spontaneous behavior (licking and vocalization p< 0.04)) and TS-Igs were bound to neurons in CN. Rats receiving a microinfusion of anti-neuronal Abs into STN (II-IV) had significantly greater ipsilateral rotational turning behavior in response to the bioassay compared to control rats. The altered turning response persisted for weeks. In Study II, Igs were bound to neurons within the ventral striatum, an area linked to movement.
Conclusion: Antibodies capable of binding brain antigens can affect brain function if they are placed behind the BBB. Affinity of Ehrlich’s magic bullets (Ab) and the brain milieu, supportive of Ab synthesis, may be a double-edged sword if synthesized Abs recognize brain epitopes.
The Development of Tumor-Inhibiting Metal Complexes: (Multinuclear) Metal Complexes and Mode-of-Action Studies HARTINGER CG1,2, NAZAROV AA1,2, MENDOZA-FERRI M-G1, DYSON PJ2, KEPPLER BK1 1University of Vienna, Vienna, Austria; 2EPFL, Lausanne, Switzerland.
Background: Platinum-based drugs are widely used in the therapy of cancer although being only active in a limited number of tumors and often exhibiting serious side effects. Ruthenium compounds are the best developed metal-based non-platinum anticancer agents, in particular with KP1019 and NAMI-A, two ruthenium(III) compounds, undergoing clinical trials. In recent years, organometallic ruthenium(II)-arene compounds moved into the focus of interest and compounds with activity against primary tumors and antimetastatic activity were reported.
Methods: The synthesis, (bio)analytical characterization and in vitro anticancer studies of dinuclear (η6-p-cymene)ruthenium(II) complexes with varying spacer length are reported. The compounds were characterized by NMR spectroscopy and ESI mass spectrometry, and the molecular structure of 1,6-bis{chlorido[3-(oxo-κO)-2-methyl-4-pyridinonato-κO4](η6-p-isopropyltoluene)ruthenium}hexane was determined by X-ray diffraction analysis.
Results: The coupling of two (η6-p-cymene)ruthenium(II) moieties via alkyl-pyridinone spacers (alkyl = propane, hexane, dodecane) resulted in compounds 1–3 with IC50 values in the low micromolar range against the human tumor cell lines A2780 and SW480, whereas the mononuclear analogue 4 is not active. The anticancer activity was found to be dependent of the spacer-length (see Table), which also influences the lipophilicity of the complexes.
Conclusions: Ru(II)–arene metallodrugs with spacer-length-dependent activity in human tumor cell lines were developed. Notably, not only is an additive effect of the analogous mononuclear complexes observed but a synergistic effect in two cell lines was present – in the SW480 cells being of extraordinary dimension for Ru compounds.
Orally Ingested Lactoferrin and Glycine Display in vivo Synergistic Anti-Inflammatory Activity HARTOG A1,2, GARSSEN1,2,
1 Danone Research, Centre for Specialised Nutrition, Wageningen, The Netherlands. 2 Department of Pharmacology & Pathophysiology, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, The Netherlands
Background: There is a growing awareness of the interaction of food constituents with the immune system. The present studies aim to evaluate immunomodulatory effects of two of these nutritional components, i.e. glycine and lactoferrin.
Methods: Mice orally supplemented with glycine, lactoferrin or a combination, were injected intradermally in the ear, with zymosan. Ear swelling (as a measure for inflammation) and the number of TNF-α producing spleen cells were analysed. In a collagen induced arthritis (CIA) model mice were orally supplemented with a combination of glycine and lactoferrin starting after the second collagen booster. Arthritis development was scored and the pro-inflammatory cytokine levels in the serum were detected.
Results: Glycine and lactoferrin were able to decrease the zymosan induced inflammatory response locally (decreased ear swelling) as well as systemically (reduced number of TNF-α producing spleen cells). Glycine effects (20, 50 and 100 mg/mouse/day) were concentration dependent whereas for lactoferrin only the lowest doses (0.1 and 1 mg/mouse/day) inhibited the inflammatory response significantly. Surprisingly higher doses of lactoferrin (5 and 25 mg/mouse/day) failed to influence the inflammatory reaction. A combination of both nutrients (lactoferrin 0.1mg/mouse/day in combination with glycine 20 or 50 mg/mouse/day) inhibited the zymosan induced ear swelling synergistically. In the CIA model the combination of glycine and lactoferrin (lactoferrin 0.1mg/mouse/day with glycine 20 mg/mouse/day) was able to inhibit arthritis development and decrease the level of pro-inflammatory cytokines in the serum.
Conclusions: The present data indicate that the glycine-lactoferrin concept might offer in the near future a powerful nutritional way in modulating chronic inflammatory diseases.
PACAP Signaling: A Promising Drug Target for Neuropsychological Disorders HASHIMOTO H1,2, HASHIMOTO R2, SHINTANI N1, TAKEDA M2, BABA A1 1Grad Sch of Pharmaceutical Sciences; 2Grad Sch of Medicine, Osaka Univ., Suita, Osaka, Japan
Background: Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with pleiotropic activities including neurotransmission, neural plasticity, and neurotrophy. After we cloned a cDNA for PACAP-specific (PAC1) receptor belonging to the Class B GPCR and observed its strong expression in the nervous systems in 1993, we have been focusing on harnessing the therapeutic potential of PACAP signaling.
Methods: To this purpose, we generated knockout mouse models for PACAP and PAC1 receptor by a gene targeting technique and examined their behavioral phenotypes and pharmacological reposponses to various psychotropic drugs. In addition, we performed a case-control association analysis of schizophrenia for PACAP and PACAP receptor genes on a Japanese population comprising 804 cases and 967 control subjects. Intermediate phenotypes that can be linked to the disease were also analysed.
Results: Mice lacking PACAP displayed neuropsychological abnormalities, including altered psychomotor behaviors, sensorimotor gating deficits as determined by prepulse inhibition, hippocampal long-term potentiation and memory deficits, as well as depression-like behavior, most of which were amenable to treatment of the atypical antipsychotic risperidone and intracerebroventricular injection of PACAP. A case-control genetic association study provided evidence that single nucleotide polymorphism (SNP) variants in the PACAP gene and the PAC1 receptor gene were associated with schizophrenia. The overrepresented allele of the PACAP gene in schizophrenia was also associated with poorer memory performance and reduced hippocampal volume.
Conclusions: 1) These convergent data suggest that alterations in PACAP signaling could contribute to the pathogenesis of schizophrenia. 2) As PACAP has pleiotropic actions, e.g. modulation of various signaling systems such as dopamine and glutamate, PACAP signaling pathway can be a target candidate for new therapies.
Role of Endothelial Cells and Treatment with Gentamicin in Septic Shock HASWANI DK1, OETTINGER CW2 and D'SOUZA MJ3 1Patheon Pharmaceuticals, 2110 E Galbraith Road, Cincinnati, OH, United States; 2Dialysis Clinic, Atlanta, GA, United States; 3College of Pharmacy & Health Sciences, Mercer University, Atlanta, GA, United States.
Background: Endothelial cells (EC) play an important role in the pathogenesis of sepsis. It is known that various stimuli such as release of lipopolysaccharide from the bacterial cell wall can trigger the release of large amount of cytokines which in turn can contribute to the microvascular dysfunction and organ injury. The solution form of gentamicin kills the infectious organism in the flowing blood, but is not able to effectively penetrate the cells lining the blood vessels where the bacteria must have been internalized. Aim: To evaluate the uptake of bacteria such as E.Coli by EC and compare the effect of gentamicin in the solution versus the microencapsulated form in targetting the bacteria phagocytosed by the EC.
Methods: 1) Human micro vascular EC were grown until they were 80-90% confluent. Labeled E.Coli were exposed to these cells and evaluated for total uptake. The internalized bacteria were then targetted using either the solution or the microencapsulated form of gentamicin. 2) This study included 12 rats and the rats were injected with a single dose of gentamicin (45mg/kg) either in the solution form or the microsphere suspension form. 3) Microspheres were labeled using 100μg/mL of fluorescamine solution and injected intraperitoneally, various organs were isolated and evaluated for microsphere distribution using fluorescent microscope.
Results: Treatment with gentamicin microsphere showed 80% inhibition in the growth compared to the solution form which showed only 50% inhibition in the growth and upake of E.Coli. Gentamicin solutionshowed ahalf life of 175 minutes whereas the microsphere form had a half life of 1910 minutes. Also the extent of absorption for solution group was 5 times lower than the microsphere group. Liver, lung, heart, spleen and kidney showed a significant distribution of micorspheres
Conclusions: 1) Gentamicin microsphere was more effective in targetting the internalized E.Coli compared to its traditional solution form. 2) The microsphere form showed a prolonged residence time compared to the solution form. 3) Biodistribution studies demonstrated the engulfment of microspheres by various organs, thus proving it to be an effective carrier of drug in targetting the internalized bacteria.
Cancer stem cells - towards a new generation of antineoplastic drugs HATINA J1 1Charles University, Medical Faculty in Pilsen, Institute of Biology Plzen, Czech Republic
Background: There is evidence accumulating that tumour growth is perpetuated by a rare subpopulation of tumour cells, the cancer stem cells. It is increasingly clear that these cells are causally implicated in therapy resistance, tumour relapse and resulting treatment failure. The typical cancer stem cells are slowly cycling, intrinsically chemoresistant due to a constitutive drug efflux and detoxification activities and display a high level of DNA-repair activity.
Methods: We have performed a literature review of publications directly relating cancer therapy resistance and cancer stem cells. We have developed a method of visualisation of cancer stem cells in living cell cultures, based on isolation of doxorubicin resistant bladder carcinoma cell line and a doxorubicine – responsive GFP – reporter gene construct.
Results and Conclusions: Direct relationship between cancer chemoresistance and cancer stem cells was obtained for a range of tumours, including colon cancer, glioblastoma, hepatocellular carcinoma, endometrial carcinoma, nasopharyngeal carcinoma, ovarian cancer, lung cancer, as well as leukaemia. We are able to visualize naturally chemoresistant cells in a bladder carcinoma cell line and we can show that these intrinsically chemoresistant cells are uniquely clonogenic and responsible for growth restoration after terminating the experimental doxorubicin treatment, thus fulfilling the essential criteria for cancer stem cells. Our method of visualization of cancer stem cells might facilitate identification of therapeutic compound specifically targeting cancer stem cells, which might be an attractive way for future cancer chemotherapy. Targeting normal tissue stem cells by such a stem cell – directed therapy would represent an essential limitation of these approaches of anticancer therapy.
Combination with hyperthermia and radiation contributes to the magic of cisplatin in cancer treatment. HAVEMAN J, FRANKEN NAP, STALPERS L
Department of Radiotherapy, Academisch Medisch Centrum, University Hospital Amsterdam, The Netherlands.
Background: It has been shown that hyperthermia hyperthermia killas preferentially tumor cells, but not to an extent that it can be used alone. Combination with other treatment modalities is apparent very favourable as many preclinical studies have shown that hyperthermia improves cell killing by radiation and chemotherapy. Promising chemotherapeutic agents in combination with hyperthermia include cisplatin and derivatives, gemcitabine, melphalan, cyclophosphamide, BCNU, bleomycin, vincristine and mitomycin C.
With the clinical application of hyperthermia temperatures in the range of 40 to at maximum 45 °C are used. Sophisticated and safe equipment is commercially available, not only for induction of hyperthermia locally or regionally in the body, but also for advanced planning of treatment. The thermal dose is generally defined as CEM 43 °C, i.e. cumulative equivalent minutes at 43 °C and this is used in the treatment planning. Although in many cases temperatures in the range of 42 to 43 °C are prescribed, this goal is often not reached.. Therefore we performed preclinical studies with the aim to study effects at the ‘low’ temperature of 41 °C in combination with radiation and cisplatin.
DNA double strand breaks (DSBs) induced by ionizing radiation in mammalian cells, can be repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ) in S and G2 phase cells. The results of several studies suggest that the two repair processes can interact. Therefore, inhibition of one of the pathways might result in the shunting of DNA DSB repair to the other pathway. We investigated the effect of temporary HR inhibition by hyperthermia on the repair of ionizing radiation induced DSBs in G2 phase cells. Moreover we studied effects of cisplatin on NHEJ
Methods: The effect of incubation of cells for 1h at 41C on the accumulation of Rad51, Mre11 and Mdc1 (proteins involved in DNA DSB repair) in ionizing radiation induced foci (IRIF) was studied in two human tumor cell lines. HR efficacy is measured by gene targeting to the Rad54 locus in E14 mouse embryonic stem (ES) cells. The formation of chromosomal fragments (resulting from DNA DSBs) and translocations (resulting from erroneous DNA DSB repair) is measured in G2 phase cells as soon as possible (1h) after irradiation with or without prior incubation of cells at 41C and/or with cisplatin (an inhibitor of NHEJ).
Results: Incubation of cells at 41C leads to a temporary inhibition of Rad51 accumulation in IRIF and HR efficacy. This inhibition of HR was accompanied by an increase in the number of chromosomal translocations which could be prevented by incubation of cells with high ( 10µM) concentrations of cisplatin. Our results suggest that in G2 phase of the cell cycle the decision which pathway to use for repair of IR-induced DSBs is made early after damage induction. After inhibition of HR, DSB repair might be shifted to the error-prone NHEJ pathway resulting in the rapid formation of chromosomal translocations.
Conclusion: These preclinical results point to favourable effects when three modalities are combined, also at relatively low hyperthermic temperatures. Three modality treatment is currently investigated as an experimental therapy in the clinic.
Gold activates mast cells through L- type calcium channels. HAYAMA K1,2, SUZUKI Y1, YOSHIMARU T1, INOUE T1, RA C1 1:Nihon University Graduate School of Medical Science, Tokyo, Japan
2:Department of Dermatology, Nihon university School of Medicine, Tokyo ,Japan
Background:. Xenobiotic heavy metals including mercury, gold and silver have been shown to highly induce allergy and autoimmunity in genetically susceptible humans and/or experimental animals. Mast cells are implicated to play a role in the development of these adverse immunological reactions. Recently, it is suggested that both allergic and autoimmune mechanisms are involved in autoimmune diseases, including multiple sclerosis, a metal-associated disease. We have previously shown that L-type calcium channels (LTCCs) play a critical role in regulating mast cell activation. Here we demonstrate that gold activates mast cells through LTCCs via the production of H2O2.
Methods: Degranulation was determined by β-hexosaminidase and leukotriene C4 (LTC4) secretion was measured using an enzyme-linked immunosorbent assay (ELISA). The production of intracellular H2O2 was mesuered using DCFH-DA by flow cytometer. The cytosolic calcium concentration ([Ca2+]i) was measured using the Fluo3/AM.
Results: Au(Ⅲ) at concentrations of raging from 10 μM to 100 μM dose-dependently induced degranulation and LTC4 secretion with displaying a minimal cytotoxity. In parallel, Au(Ⅲ) stimulated the production of intracellular H2O2 and scavenging the oxidant by the glutathione peroxidase mimetic ebselen blocked [Ca2+]i increase, degranulation, and LTC4 secretion. Subsequent studies revealed that Au(Ⅲ) stimulated LTCC activity, which was activated by H2O2. The effects of Au(Ⅲ) were patially similar to those of Hg(Ⅱ) and Ag(Ⅰ). Further investigations on the role for LTCCs using LTCC gene silencing are underway.
Conclusions: Au(Ⅲ) appears to utilize a unique ROS- and LTCC-dependent mechanism which is partially overlapping with those activated by Hg(Ⅱ) and Ag(Ⅰ). This finding may explain the fact that the three xenobiotic heavy metals induce autoimmunity by similar but not identical mechanisms.
Type II Alveolar Epithelialcells play a role in pulmonary host defense system against infectious disease. Takuma HAYASHI, Xuesong QIAN, Hiroshi KANOH and Kazuo SUGANE
Department of Immunology and Infectious Diseases, Shinshu University Graduate School of Medicine, Matsumoto, Nagano, JAPAN
The mechanism of immune defense against pathogens in the lung, has so far been poorly understood. Here, we show that human type II alveolar epithelial cells play a key role in defense via interactions between B7h, also known as ICOS ligand, and its receptor ICOS expressed on activated T cells. The A 549 alveolar type II cell line abundantly expresses B7-H2, CD40 and B7-1, but not B7-2 or hGL50. TNF- significantly induced B7-H2 and CD40 expression by A549 cells, but had no effect on B7-1 or B7-2 expression. TNF- deficient mice exhibited low B7-H2 expression on alveolar epithelial cells in comparison with wild type mice. Co-culture of TNF- pre-stimulated A549 cells with CD4+ T cells promoted CD154 expression, CD4+ T cell proliferation and cytokine production, especially IFN-. Monocytes-derived TNF- in combination with IFN- and LPS markedly induced B7-H2 expression in A549 cells. Furthermore, in vivo experiments demonstrated TNF--inducibility of B7-H2 in alveolar epithelialtype II cells. B7-H2 might play a key role in pulmonary host defense system against infectious disease, for instance Pneumocystis carinii. This study thus identifies a unique costimulatory pathway via alveolar epithelialtype II cells that preferentially affects T-helper cell function, implying that alveolar epithelialtype II cells play a crucial role in innate immunity in the lung by regulating IFN--synthesis via B7-H2/ICOS interactions.
Increase of fibrin network porosity and the consequent fibrinolysis as an anticoagulant effect of acetylsalicylic acid HE S1+2, BARK N2, WANG HY2, SVENSSON JE2, BLOMBÄCK M1+2 Karolinska Institutet: 1)Dept of Clinical Sciences, Danderyds Hospital; 2)Dept of Molecular Medicine & Surgery /Coagulation Research, Sweden, Stockholm
In patients with stable angina pectoris, the fibrin network permeability shown as the Darcy constant – Ks was significantly enhanced during acetylsalicylic acid (ASA) therapy. Interestingly, a daily intake of 75 mg gave a greater increase in fibrin gel permeability (+92%) than an intake of 320 mg (+5%) in another investigation involving healthy volunteers.
Since above assessments were made in platelet-poor plasma samples, the fibrin formation can be manipulated by numerous pro- and/or anti-coagulation factors in plasma. To confirm whether ASA is able to depress fibrinogen clotting property and thus modify the structure of fibrin network, purified fibrinogen was incubated with different concentrations (0.02 - 2.56 mmol/L) of ASA; a dialysis followed to remove residual drug and its hydrolysis products. The fibrinogen–ASA product was examined with regard to the fibrin network porosity and fibrin resistance to plasmin. Results showed that fibrinogen “clotting time” kept unaffected in all the treated samples. Ks levels were increased and then decreased when ASA levels varied from 0 to 0.08 mmol/L and then to 2.56 mmol/L, respectively. This outcome of fibrin gel permeability was supported by the findings from analysis of 3D-confocal microscopy and from that of fibrin fiber thickness.
Thus, both the studies in vivo and in vitro confirm that fibrinogen clotting property is partially impaired by ASA in a low dose-dependent way. The formed looser network with thicker fibrin fibers favours fibrinolysis, which should be one anticoagulant effect of this therapy.
Cardiotoxicity Plagues Bupivacaine HEAVNER JE TTUHSC, Lubbock, TX, USA
Background: Bupivacaine was introduced into clinical practice as a local anesthetic in 1963. Not until 1979 did serious concerns about bupivacaine-induced cardiotoxicity surface following fetal-maternal deaths when bupivacaine was used for epidural analgesia during childbirth. This led to refinement in assumptions about structure-activity relationships for local anesthetics and investigations that furthered understanding of the biological actions of this class of drugs.
Methods: Literature reports of clinical, translational and fundamental investigations of the cardiotoxic effects of bupivacaine were reviewed. Key observations were summarized.
Results: Resuscitation from bupivacaine induced cardiovascular collapse is difficult. The ratio of bupivacaine central nervous system toxic dose to cardiovascular toxic dose is narrower than that for other local anesthetics. Kinetic difference in binding to sodium channel sites influences relative difficulty in resuscitation from bupivacaine cardiotoxicity vs other local anesthetics. The kinetic differences also influence pro and anti arrhythmic activity. Bupivacaine differs from other local anesthetics with respect to the spectrum of effects it has on voltage gated ion channels and on ligand gated receptor signaling. The S isomer of bupivacaine is more potent and less toxic than the R isomer or racemic mixture.
Conclusion: After being considered a “silver bullet” for producing long lasting local or regional anesthesia for 16 years, bupivacaine now is plagued by cardiotoxic effects that sets it apart from other clinically used local anesthetics.
