Ehrlich II –2nd World Conference on Magic Bullets

A cost-effective Haemophilus Influenzae type b conjugate vaccine in combination with DTwP-Hep B vaccine for developing countries – A perspective YADAV S^1, LALWANI S^2, CHAKRAVARTY A^1, BHARDWAJ S^3,SHARMA H^3 ^1Maulana Azad Medical College & Hospital, New Delhi. ^2Bharti Vidyapeeth Hospital, Pune ^3 Serum Institute of India Ltd., Pune Background: Despite the availability of Hib conjugate vaccines, Hib remains a leading cause of meningitis and pneumonia deaths, worldwide.Advent of combination vaccines has greatly simplified immunization activities and also helped in additional vaccines being incorporated into immunization schedule. IAP reviewed data on combination vaccines and declared that addition of Hib/Hepatitis B vaccines to DTwPw vaccine as safe and effective for primary as well as booster vaccination. This study was conducted to assess the immunogenicity and reactogenicity of DTPw HB + Haemophilus Influenzae type b conjugate vaccine manufactured by SIIL in comparison with Tritanrix HB + Hiberix vaccine of Glaxo Smith Kline (GSK) in Indian Children aged 6-14 weeks. Methods: 304 children aged 6-8 weeks at enrollment were equally randomized to receive three doses of 0.5 ml of either SIIL or GSK vaccine intramuscularly, with a gap of one month between each dose. Pre and Post vaccination IgG antibody titres were assessed by ELISA at an independent laboratory using Good Laboratory Practice (GLP) guidelines. Reactogenicity was assessed from the frequency of adverse events recorded by parents on diary cards. Study was conducted as per International Conference on Harmonization - Good Clinical Practice (ICH-GCP) guidelines and Declaration of Helsinki. Results: Demographic characteristics were comparable in both the groups. Post-vaccination GMTs in SIIL group were 2.78 IU/ml, 50.87 U/ml, 1.34 IU/ml, 616.73 mIU/ml and 7.55 µg/ml for anti Tetanus, anti-Pertussis, anti Diphtheria, Anti HBs and anti-PRP. Corresponding values in GSK group were 2.52 IU/ml, 48.28 U/ml, 0.99 IU/ml, 463.12 mIU/ml and 7.82 µg/ml respectively. Post-vaccination sero-protection was 100% for all components, in both groups except Pertussis component, which was 96.06% in SIIL group and 95.4% in GSK group. Frequency of adverse events was higher with the first dose and thereafter reduced in both the groups. Common adverse events were pain (SIIL-42%, GSK-44%), redness (SIIL-15%,GSK-14%), swelling (SIIL-29%,GSK-30%) and fever (SIIL-41%, GSK-45%). No Serious Adverse Event was reported in the study. Conclusion: 1) DTPw HB + Hib vaccine manufactured by Serum Institute of India Ltd., Pune was found to be safe, immunogenic and non-inferior to GSK vaccine 2) This vaccine is also cost effective and therefore affordable to a larger population in the developing world.	In Vivo Analysis of Brain Muscarinic Receptor (mAChR) Occupancy by Anticholinergic Agents Analyzed via Quantitative Autoradiography (ARG) and Positron Emission Tomography (PET) YAMADA S^1, MARUYAMA S^1, ITO Y^1, OKU N^1, TSUKADA H^2 ^1Sch. of Pharm. Sci., Univ. of Shizuoka, Shizuoka;^2Central Res. Lab., Hamamatsu Photonics K.K., Shizuoka, Japan Background: For anticholinergic therapy for overactive bladder (OAB), it is concerned that chronic administration of these agents in older patients may result in a non-degenerative mild cognitive impairment, depending on the ability to occupy brain mAChR. Aims: 1) To characterize in vivo brain mAChR occupancy by anticholinergic agents for OAB treatment. Methods: At 10 min after iv injection of oxybutynin (Oxy), solifenacin, tolterodine or darifenacin in rats, [^11C](+)3-MPB (selective mAChR radioligand) was injected (iv), followed by removal of brain for ARG. Following oral Oxy, rhesus monkeys were injected (iv) [^11C](+)3-MPB, and PET scan was performed. mAChR occupancy was estimated by distribution volume of each brain region. Plasma concentrations of Oxy and its active metabolite, N-desethyloxybutynin (DEOB) were measured by LC/MS/MS. Results: In ARG study, there was a dose-dependent decrease in in vivo specific [^11C](+)3-MPB binding in rat brain regions after iv injection of anticholinergic agents, indicating brain mAChR occupancy. Dose ratios of anticholinergic agents for brain mAChR occupancy (RO50) to inhibition of carbachol-induced increase in intavesical pressure (ID50), which reflects in vivo bladder selectivity to brain, were significantly greater for solifenacin and tolterodine than Oxy. Darifenacin displayed little occupancy of brain mAChR. In PET study, following oral administration of Oxy (0.1, 0.3 mg/kg) in conscious rhesus monkey, plasma concentrations of Oxy and DEOB were dose-dependently increased, with greater concentration of DEOB. At 1-4 hr after oral Oxy at these doses, mAChR occupancy in brain regions was 40-60%. The plasma concentrations in rhesus monkey after oral Oxy administration were similar to those in humans received clinical dose of Oxy. Conclusions: Oxy occupies brain mAChR under in vivo condition, suggesting a risk of CNS adverse effect under clinical condition. In the treatment of OAB, CNS side effects could be avoided by newer generation of anticholinergic agents with high bladder selectivity. In conclusion, in vivo ARG and PET analysis of brain mAChR occupancy may provide fundamental basis for managing CNS side effects in anticholinergic therapy for OAB.
Noninvasively Evaluation on Human Stress Using Salivary Biomarker YAMAGUCHI M Iwate University, Morioka, Japan Background: Saliva sampling has the advantage that it is noninvasive, making multiple sampling easy and stress free. Salivary amylase activity (SAA) can be a useful index of plasma norepinephrine concentration under a variety of stressful conditions, since it appears that increased sympathetic nervous activity is a major stimulator of amylase secretion. In order to realize a hand-held monitor of the sympathetic nervous system, we fabricated a completely automated analytical system for SAA using a dry-chemistry system. Methods: The monitor consisted of a disposable test-strip and an optical analyser (130 × 87 × 40 mm^3; 190 g), which was incorporated within an automatic saliva transfer device. The test-strip consisted of a collecting paper and a reagent paper containing 2-chloro-4-nitrophenyl-4-O-β-D-galactopyranosylmaltoside (Gal-G2-CNP), a substrate for amylase. The collecting paper is directly inserted into an oral cavity, and approximately 30 l of whole saliva is collected from under the tongue. When Gal-G2-CNP is hydrolyzed by amylase, the hydrolyzed product (CNP) develops a yellow color and the reflectance is measured by an optical device. The definition of one unit activity (U) per mass of enzyme is that this activity produces 1 mol of the reducing sugar, maltose, in 1 min. The SAA by video viewing was observed in 64 healthy adults. The ethical committee approved the study. Results: When this monitor was used, it took 30 s for saliva sampling, 30 s for saliva transfer and measurement, and a total of one minute was enough to measure the SAA. The calibration curve of the monitor was within a range between 10 and 140 kU/l showing a coefficient with R^2 = 0.97. With regard to reproducibility of the measured results for the saliva transfer volume of the same samples, the coefficient variation (CV) was 5.5%. In our time course experiment, the SAA level was increased just after the beginning of stressful video viewing in 63 of 64 subjects, and immediately returned to the pre-sress level just after the end of the video viewing. On the other hand, the SAA level was significantly decreased by smoothing video viewing. Conclusions: It was demonstrated that the manufactured monitor enabled a user to automatically measure the SAA with a high accuracy. The SAA measurement will be powerful tool for psychological research.	Soluble Leishmanial Antigen and Plasmid Expressing Interleukin-12 Protects BALB/c Mice from Leishmania major Infection YAMAKAMI K^1, TADAKUMA T^2 ^1Department of Preventive Medicine and Public Health, National Defense Medical College, Tokorozawa, Saitama, Japan; ^2Faculty of Health Care, Kiryu University, Midori-shi, Gunnma, Japan Background: In murine leishmaniasis, the induction of the T-helper type 1 (Th1) response contributes to infection resistance, whereas the establishment of the Th2 response makes the mice susceptible to infection. Interleukin-12 (IL-12) plays a pivotal role in the diversification of immune responses to the Th1 type. In this study, we tested whether the co-administration of IL-12 expression plasmid and leishmanial antigen will skew highly susceptible BALB/c mice to Th1 response and protect from leishmaniasis. Methods: The BALB/c mice were intradermally injected with the combination of IL-12 plasmid which compose p35 and p40 subunits and soluble leishmanial antigen (SLA) 7 days prior to the challenge with lethal dose (1x10^6) of promastigotes of Leishmania major. The courses of disease and immune responses of the mice were assayed. Results: The mice which received IL-12 expressing plasmid plus SLA completely healed and the parasite burden in the local lymph nodes significantly decreased. The cured mice attained long-term immunity, and were resistant to any subsequent rechallenge of the lethal dose of the parasite. The protective effect was associated with the development of a Th1 response, as demonstrated by the enhanced level of antigen-specific interferon- (IFN-) and dominant production of IgG2a in the serum. In contrast, the administration of empty plasmid plus SLA or IL-12 plasmid alone failed to protect the disease and shape the Th1 response. Furthermore, the protective efficiency induced by the vaccination was clearly prevented by the injection of either neutralizing anti-IL-12 mAb or anti-IFN- mAb. Conclusion: The IL-12 expression plasmid is thus an effective adjuvant for the elicitation of a protective Th1 response against leishmaniasis and is therefore, considered to be appropriate for vaccinations that require the induction of Th1 type immunity.
Antitumor Activity of New Combination Chemotherapy with Iirinotecan Hydrochloride(CPT-11) and Nedaplatin(NDP) against Human Cervical Cancer Cell Lines YAMAMOTO K^1, SHIMAOKA M^1, ABE K^2, HOSHIAI H^2, NODA K^2 ^1 Sakai Hospital,Kinki University , Sakai, Japan; ^2 Kinki University , Osakasayama, Japan Background: Antitumor activity of combination chemotherapy with irinotecan hydrochloride(CPT-11) and nedaplatin was compared to that with CPT-11 and cisplatin. Methods: In vitro cytotoxicity of SN-38 (an active metabolite of CPT-11) in combination with nedaplatin or cisplatin was evaluated using three human cervical cancer cell lines(ME-180,CaSki and SiHa).Interactions between two drugs in combination were investigated using a simultaneous-exposure schedule and analyzed by the IC50-based isobologram method. In vivo antitumor effects of CPT-11 in the combination with each platinum were studied using SiHa xenografts. Results: IC50 values of nedaplatin against these three human cervical cancer cell lines were about 2-fold as high as those of cisplatin, indicating somewhat weak cytotoxic effects of nedaplatin. Simultaneous exposure to SN-38 with each platinum preparation showed synergistic and additive effects against ME-180 and SiHa. While CPT-11, nedaplatin and cisplatin alone hardly showed any antitumor effects even at the maximum tolerated dose (MTD) levels, the combination chemotherapy with CPT-11 and nedaplatin or cisplatin resulted in significant antitumor effects even at three-quarter MTD of CPT-11 combined with two-third MTD of platinum. All treatments were tolerable for mice, indicating that the combinations did not cause significant enhancement in toxicity. Conclusions: In clinical application, nedaplatin causes a lower incidence of nephropathy and does not require the replacement of a large volume of fluid, which is needed for cisplatin administration, facilitating treatment at the outpatient clinic. In addition, the incidences of digestive disorder, peripheral neuropathy and auditory disorder are lower.These findings suggest that the combination chemotherapy with CPT-11 and nedaplatin for squamous cell cancer of uterine cervix is very useful in clinical practice. A dose-finding study should be conducted.	Clinical trial of immunochemotherapy (personalized peptide vaccination and gemcitabine) for metastatic pancreatic cancer patients YANAGIMOTO H^1, SATOI S^1, MINE T^2, YAMAMOTO T^1, HIROOKA S^1, TOYOKAWA H^1, YAMADA A^2, KWON AH^1, ITOH K^2 ^1Department of Surgery, Kansai Medical University ^2Department of Immunology, Kurume University School of Medicine Background: The present multicenter phase I/II study was conducted to confirm the efficacy and toxicity of immunochemotherapy for metastatic pancreatic cancer. Methods: HLA-A2 or A24 positive patients with histologically or cytologically proven pancreatic adenocarcinoma with at least one measurable metastatic lesion were eligible for the study. Other eligibility criteria included: age of 20-80 years, ECOG performance status (PS) of 0 or 1, and adequate organ function. Gemcitabine was given intravenously at a dose of 1,000 mg/m^2 over 30 min once a week for three weeks, followed by 1 week of rest, and three or four peptides that had been positive for pre-existing peptide-specific immune responses in the circulation (personalized peptide vaccine) were injected subcutaneously in the femoral area once a week. The objective response rate was assessed according to RECIST. Results: Twenty patients from 2 institutions were enrolled in this study. The median age was 63 years (range: 41-80 years). The PS was 0 in 10 patients (50%) and 1 in 10 patients (50%). The efficacy and toxicity were analyzed in 20 patients who received at least one course (8 weeks) of immunochemotherapy. Although no complete response was seen, a partial response was achieved in 5 patients, resulting in an overall response rate of 25%. Eleven patients (55%) had stable disease. The median overall survival was 9.0 months with a 1-year survival rate of 33%. The major grade 3-4 toxicities using CTCAE criteria were neutropenia (25%), anemia (20%), thrombocytopenia (20%) and anorexia (5%). All 17 dermatologic reactions at the vaccination site were scored as grade 1 or 2. No treatment-related deaths occurred during the study. Augmentation of peptide-specific CTL responses in the post-vaccination peripheral blood mononuclear cells was observed in 75%, while increased titer of peptide-specific IgG antibodies was observed in the post-vaccination plasma in 80%. Conclusions: This study could be recommended for further stages of clinical trials because of safety, boosting of immune responses, and potential clinical benefits.
Interfere IGF-I pathways to augment sensitivities of colorectal cancer cells to current therapeutic agents YANG SY, SALES KM, FULLER B, SEIFALIAN AM, WINSLET MC Department of Surgery, University College Medical School, University College London, Royal Free Campus, Rowland Hill Street, London NW3 2PF, UK Current chemotherapy agents are not highly effective against disseminated colorectal cancers. One major contributor to the limited effectiveness of the treatment is cancer cell producing high level of IGF-I and IGF receptors which has been proven protecting a broad range of colon cancer cells from a variety of apoptotic challenges caused by chemotherapy agents. The IGF system, therefore, has become an attractive molecular target for anticancer therapies. Based on IGF-I structure we have designed and synthesised a novel IGF type I receptor antagonist. The effect of the antagonist on human colon cancer cell proliferation was examined by a non-radioactive assay; the apoptosis was revealed by determining the activities of cellular caspases 3/7 8, 9. The apoptosis pathways were investigated by examining the level of pro-apoptosis singling protein with Western blotting. The results showed that IGF-I receptor antagonist induce cancer cell apoptosis and inhibits colon cancer cell proliferation. The changes of Caspase 3/7, 8 and 9 activities and different expression level of Bax in cancer cells after treatment with the peptide suggested that the extrinsic pathway may play an important role in IGF-I receptor antagonist induced apoptosis in colon cancer cells. Treating different type of colorectal cancer cells by combination of IGF-I receptor antagonists with current chemotherapeutic agents has shown that IGF-I receptor antagonists significantly augment sensitivities of colorectal cancer cells to current chemotherapeutic agents. All these data suggested that IGF-I receptor antagonists could be developed to a therapeutic strategy for colorectal cancer.	Immunogenicity of mucosally delivered Lactococcus lactis expressing a malaria protein in rabbits YASAWARDENE SG^1, RAMASAMY R^2 ^1Faculty of Medical Sciences, University of Sri Jayewardenepura, Nugegoda, Sri Lanka; ^2Institute of Medicine, University Brunei Darussalam [e-mail] Oro-nasal immunization is preferable to injections from the point of view of safety, ease of administration and compliance. The gram positive food grade bacterium Lactococcus lactis is a potential vehicle for delivering immunogens to the mucosal immune system. We have investigated the immunogenicity in rabbits of the Plasmodium falciparum merozoite surface protein MSA-2 expressed on L. lactis. The 819 bp coding sequence of Plasmodium falciparum MSA-2 was presented on L. lactis in two forms viz. as an intracellular molecule and one covalently anchored to the peptidoglycan layer of the cell wall. The recombinant L. lactis were delivered intranasally or orally for mucosal immunization. Intramuscular immunization was also used as a control. Serum antibody response was investigated by immunofluorescnce assay. High titre serum antibodies that recognize native parasite MSA-2 in immunofluorescence assay were generated on oral and intramuscular immunization. The surface displayed form was more immunogenic than intracellular MSA-2. Either the surface or intracellular form of MSA-2 did not elicit high titres on intranasal immunization. Recombinant L. lactis is a suitably safe vector for subunit vaccines. The strain expressing cell wall anchored MSA-2 elicited higher levels of serum antibodies than strain expressing MSA-2 in the cytoplasm with all three routes of immunization. Systemic IgG antibodies could be generated through mucosal immunisation.
Atorvastatin protects spinal motor neurons from glutamate mediated neurotoxicity YASUO I Department of Neurology Toho University Omori Hospital 6-11-1 Omorinishi Otaku Tokyo JAPAN In the recent few years several studies have demonstrated that statins,in addition to their lipid-lowering effects,have neuroprotective properties. These properties of statins have beneficial effects in neurological disorders. We have analyzed the atorvastatin in a postnatal organotypic spinal cord cultures exposed to glutamate. Cultures were treated for two weeks. Cultures treated with glutamate alone had significant reduction of motor neuron numbers. Cultures treated with glutamate and atorvastatin showed sparing the organotypic morphological appearance. This results showed the atorvastatin plays an important role in the survival and maintenance of spinal motor neurons in their neuroprotection against glutamate induced neurotoxicity. This indicates a potential therapeutic use of atorvastatin in treating diseases that kill motor neurons,such as motor neuropathy and motor neuron disease.	Membrane transport of alkaloids and anticancer drugs in plants: specificity and analogy to mammalian cells YAZAKI K RISH, Kyoto Univ., Uji, Japan Background: Higher plants produce a large number of secondary metabolites, which are classified as alkaloids, terpenoids, phenols, and quinones. Some of them, e.g. plant alkaloids, are utilized as medicines for the purpose of anticancer, antibacterial, analgesic, and so on. They are often accumulated in particular sink organs where they are biosynthesized, while some of them are translocated from source cells to sink organs via long distance transport. Methods: We have been characterizng the function of plant transporters in heterologous system, e.g. Xenopus oocyte, budding yeast, and insect cells depending on the compatibility of the expressin of those membrane proteins. We also used transgenic tobacco to characterize the human ATP-binding cassette (ABC) transporter, MRP1. Results: We identified an ABC transporter CjMDR1 as a primary transporter for an isoquinoline alkaloid berberine in Coptis japonica, in which this transporter molecule mediates the unloading of the alkaloid at the plasma membrane of the rhizome for its accumulation. In contrast, the vacuolar sequestration of berberine in this plant is mediated by a proton gradient-dependent transport mechanism at the tonoplast. In the comparison of behavior of human MRP1, we characterized the membrane localization and transport function in tobacco cells. Surprisingly human MRP1 was localised to vacuolar membrane in tobacco cells, while it could recognize daunorubicin as the substrate. By contrast, etoposide did not seem to be transported by the MRP1 when expressed in tobacco cells. This is probably due to the difference in conjugation systems between plant and mammalian cells, i.e. etoposide is transported as glucuronate in mammalian cells, while glucuronide does not play an important role in plant for the detoxification of drugs. Conclusions: 1) Isoquinoline alkaloid, berberine, is effluxed by P-glycoprotein (ABCB1) in mammalian system, while it is transported in an inward direction in Coptis japonica by an ABCB-type transporter. 2) For the efficient detoxification of the endogenous alkaloid, the berberine-producing cells sequestrate this toxic compound by proton-antiporter at the vacuolar membrane. 3) When expressed in plant, mammalian ABC transporter may show different transport ability due to the altered membrane localization and the different detoxification counterpart in the conjucation reaction.
Vaccines: The Magic Wand of Indian Public Health? YENNAPU M National Institute of Science Technology and Development Studies New Delhi, India Backgroud: Adoption of vaccines as magic bullets in disease prevention is not the same as treating them as the magic wand of public health. In a country like India, where the government bears the entire cost of universal childhood vaccination for 25 million newborns every year, vaccination decisions have to be based on economic prudence and evidence-based policy. As disease patterns keep changing, vaccination decisions must be based on disease surveillance, pathogen variation, incidence levels that qualify for selective vs. universal use, logistics and cost-benefit analysis. Aims: To analyse the changing Indian vaccine policies and practices with reference to 1) the roles of public and private sectors 2) adoption of new vaccines 3) Individual vs. Combination vaccines 4) National priorities and Internationalpressures. Methods: Historical, economic and policy analyses based on various official documents, literature and field surveys. Results: Over the last two decades, Indian vaccine scenario has witnessed: 1) a drift away from the policy objectives of self-reliance; 2) declining role of the government and public sector and increasing role of the private sector in production and policy; 3) widening demand-supply gaps in primary vaccines due to private sector’s emphasis on new and expensive vaccines 4) adoption/promotion of new vaccines without adequate epidemiological proof of their need and cost-benefit analyses, 4) private sector using combination vaccines (combining universal and non-universal vaccines) to gain backdoor entry into universal vaccine markets, 5) emphasis on vaccine ‘coverage’ rather than on the ‘protection’ achieved, 6) overall vaccine strategies drifting along “supply push” rather than “demand pull” arguments and 7) Ignorance or complacency in local government and lack of suitable policy support from international organizations to reverse these trends. Conclusion: 1) Indian vaccine policies and practices are increasingly being driven by the market forces rather than evidence-based decisions, leading to shortage of affordable primary vaccines, proliferation of unnecessary new vaccines or their expensive combinations. 2) Immunization policies must be based on scientific principles and not by market vagaries. 3) The success of vaccination must be determined by protection achieved and not by money spent or ‘coverage’.	New Anti-Endotoxins Agents against Gram-Negative Bacterial Infections YERMAK IM^A., DAVYDOVA VN^A, BARABANOVA AO^A., GORBACH VI^A, KHASINA EI.^B, SOLOV’EVA TF.^A, MALEEV VV^C a-Pacific Institute of Bioorganic Chemistry, FED of RAS, Vladivostok, Russia b-Institute of Marine Biology, FED of RAS, Vladivostok, Russia c-Central Research Institute of Epidemiology, Moscow, Russia Background: Endotoxins (lipopolysaccharides, LPSs) are the major components of the outer membrane of Gram-negative bacteria that at higher endotoxin levels cause toxic effects and a number nonspecific patothysiology changes of organism. The present work reveals an interaction of LPS with natural polysaccharides – chitosan and carrageenans – and opportunity of application of polysaccharides as preparations for treatment of gram-negative the infections connected to accumulation endotoxins in organism. Methods. FTIR, absorbance-, fluorescent-, photon correlation - spectroscopy, microscopy, ultracentrifugation, ELISA, Human embryonal kidney cells transfected with TLR4, patomorphological researchers of bodies and biochemical analyses of blood of mouse, clinical effects of polysaccharides in therapy of 120 patients with food toxic infection on the basis of infectious hospital. Results. LPS interact with the chitosan and produce stable complexes. The interaction is a complicated process and depends of various factors. The conversion of ultra-structure of LPS by action of carrageenans was observed. Acute toxicity of LPS decreases 20-fold and 5-fold in mixture with chitosan and carrageenan, respectively. An activity of the induction of TNF by LPS with polysaccharide was 70% lower than that of the parent LPS. Chitosan-induced secretion of the pro-inflammatory cytokines in macrophages is not dependent on TLR4- receptors. The activation of cells by carrageenan occurs through specific for LPS TLR4 receptors. Chitosan and carrageenans increase non-specific resistance to impact of LPS-induced endotoxinemia in mice. Polysaccharides hampered the involution of thymus, the changes on level of thyroid hormones in serum, the activation of glycogenolysis, peroxidation of lipids in liver. Carrageenan restored system of a hemostatic and corrected parameters of immune system of organism in the course of treatment of patients with food toxic infection of Salmonnela etiology. Conclusion: 1. Marine polysaccharides have the highest affinity to endotoxins and modulate significantly the biological activity of LPS. 2. Chitosan and carrageenan can be useful as preparations of auxiliary therapy in clinical practice at gram-negative infection.