The particle filter - IPS algorithm improves upon standard HCE and OLF stochastic control policies. These results are encouraging, and have specific importance in the context of pharmacokinetic applications, as most current drug dosage designs are either of the HCE (cf., [1]) or OLF (cf., [2,3]) type. A manuscript [4] has been submitted.
Acknowledgements: Supported by NIH Grant GM068968
REFERENCES:
[2] D.S. Bayard, R.W. Jelliffe, A. Schumitzky, M.H. Milman, M. Van Guilder, “Precision drug dosage regimens using multiple model adaptive control: Theory and application to simulated Vancomycin therapy,” in Selected Topics in Mathematical Physics, Prof. R. Vasudevan Memorial Volume, Ed. by R. Sridhar, K. S. Rao, V. Lakshminarayanan, World Scientific Publishing Co., Madras, 1995.
[3] R. Jelliffe, D. Bayard, A. Schumitzky, M. Milman, F. Jiang, S. Leonov, V. Gandhi, A. Gandhi, A. Botnen, “Multiple Model (MM) dosage design: Achieving target goals with maximal precision,” 14th IEEE Symposium on Computer-Based Medical Systems (CMBS'01), July 26-27, 2001.
[4] Bayard DS, Schumitzky A. Implicit Dual Control Based on Particle Filtering and Forward Dynamic Programming. Submitted for publication to the International Journal of Adaptive Control and Signal Processing, 2008.
Therapeutics: New Protocols For Instituting Continuous Intravenous Vancomycin Therapy JELLIFFE R Laboratory of Applied Pharmacokinetics, USC Keck School of Medicine, Los Angeles, CA, USA
Wysocki et al [1] showed that continuous intravenous (CIV) vancomycin therapy did as well as intermittent (IIV) therapy, with similar clinical outcomes and safety. Target serum concentrations were reached in 36 ± 31 hrs for CIV versus 51± 39 hrs for IIV. Ten day treatment cost was $321± 81 for CIV versus $454 ± 137 for IIV. They concluded that CIV may be a cost-effective alternative to IIV.
We were impressed with their approach and began to create treatment protocols for CIV, and to compare them with IIV every 12 yours over 1 hour. For a representative 65 year old man, 70 in tall, 70 kg weight, serum creatinine of 1.0 mg/dL. For a target trough goal of 15 ug/ml, the ideal IIV regimen was a 1929 mg first dose, a 978 mg second dose, tapering down to 831 mg q 12 h for the last four doses. Total 10 day AUC was 5329 ug*hr/ml. Peak concs were 83 ug/ml for the first dose, 56 for the second, tapering down to 50 ug/ml after 60 hours. Trough concs were 15 ug/ml throughout, as shown below. The total daily dose approached 1662 mg/day.
Figure 1. Plot of predicted serum concentrations on IIV therapy q 12 h for 10 days.
In contrast, using the MM-USCPACK clinical software [2,3], for the same 15 ug/ml continuous concentration, the ideal CIV regimen consisted of a loading dose of 540 mg over 3 hours, then 271 mg for the next 3 hours, then 948 mg over the next 18 hours to finish out the first day. Subsequent doses were 1120 mg for day two, tapering down to 1056 mg for the last day. Total 10 day AUC was only 3133 ug*hr/ml, only 59% of the IIV AUC. All serum concs were 15 ug/ml, as shown below. For such a typical patient, this regimen can be approximated as 500 mg for the first 3 hrs, 250 mg for the neat 3 hours, 1000 mg for the next 18 hours. And then 1000 mg daily thereafter. Predicted concentrations at the end of the first 500 mg infusion are 13.9 ug/ml, after the 250 mg infusion are 13.8 ug/ml, after the 18 hour 1000 mg infusion are 15.4 ug/ml, and thereafter are about 14 ug.ml. This is therefore a very practical approximation of the ideal infusion regimen. A plot of these predictions is shown below. Effective concentrations are now achieved after only three hours instead of the longer times found by Wysocki et al [1].
As vancomycin is not a concentration dependent drug, usual target trough goals are about 5-6 times the anticipated MIC of the infecting organism. Above this, bacterial kill plateaus off, and higher peak concentrations are not needed. As shown by Wysocki et al., CIV vancomycin is just as effective, saves drug, and visibly reduces the risk of toxicity. The author has seen two patients with aplastic anemia felt by hematology to be due to vancomycin toxicity. They had received it IIV q 12 h. It is very likely that is they had received it CIV instead, their toxicity would have been visibly less.
Figure 2. Plot of predicted serum concentrations on CIV therapy for 10 days.
CIV vancomycin protocols such as this one, individualized for each patient using the MM-USCPACK software are easy to develop. Monitoring can be done with a sample at 6 hours, then q AM as needed. Dosage adjustment is then simple proportional adjustment to the deviation of the serum concentration from the desired target goal.
ACKNOWLEDGEMENTS: Supported by NIH grant GM005803.
References:
Wysocki M, Delatour F, Faurisson F, et al: Continuous versus Intermittent Infusion of Vancomycin in Severe Staphylococcal Infections: Prospective Multicenter Randomized Study. Antimicrobial Agents and Chemotherapy 45: 2460-2467, 2001.
“The Multiple Model Method of Maximally Precise Individualized Dosage Regimens, in the MM-USCPACK Clinical Collection of Computer Programs”, Pharmacokinetics Section, XI Annual Meeting of “Chelovek i Lekarstvo” (Man and Drugs), Moscow, Russia, April 20, 2004.
Jelliffe R, Schumitzky A, Bayard D, Milman M, Van Guilder M, Wang X, Jiang F, Barbaut X, and Maire P: Model-Based, Goal-Oriented, Individualized Drug Therapy: Linkage of Population Modeling, New "Multiple Model" Dosage Design, Bayesian Feedback, and Individualized Target Goals. Clin. Pharmacokinet. 34: 57-77, 1998.
Therapeutics: Two New Bayesian Methods For Parameter Updating In Individual Patients BAYARD D1,2, SCHUMITZKY A1,3, BOTNEN A1, THOMSON A 4, JELLIFFE R1 1. Laboratory of Applied Pharmacokinetics, USC Keck School of Medicine, Los Angeles CA, USA, 2. Guidance and Control Section, Jet propulsion Laboratory, Pasadena CA USA, 3. Department of Mathematics, USC, 4. Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK
The MM-USCPACK clinical software for dosage individualization uses nonparametric (NP) pharmacokinetic models. It develops maximally precise dosage regimens to hit targets with minimum weighted squared error. It then computes the Bayesian posterior probability of each support point in the NP model. Those points fitting the patient’s data well become more likely, and vice versa. However, the patient’s individual model must be reasonably well represented by support points in the model. If this is not the case, poor parameter updates may be obtained.
1. We have now developed anewhybrid (H) Bayesian approach to such updating. It begins with conventional maximum aposteriori probability (MAP) Bayesian estimation. However, extra support points are then added in the region of the MAP estimator to augment the population model for the data it will now receive. This provides a much richer set of support points in that area. It also provides a richer and safer approach to individualized NP Bayesian parameter estimation and maximally precise dosage design. This new approach is now being implemented in our clinical software.
2.We have also developed a newsequential interacting multiple model (IMM) Bayesian approach to best track and estimate patient parameter distributions as they change in unstable patients who have changing parameter values with their changing clinical status. All current Bayesian updating procedures assume that there is only one set of fixed parameter values that best fit the data. The IMM Bayesian procedure is widely used in aerospace. It permits parameter values to change during the fitting procedure [1].
Results: IMM tracks drug behavior in a simulated changing patient with less than half the error of the MAP or MM procedures. It has been incorporated into the MM-USCPACK clinical software. It now has tracked the behavior of gentamicin and vancomycin significantly better than MAP and MM methods in over 130 unstable post-cardiac surgical patients. Both tools show great promise in optimizing patient care [2].
ACKNOWLEDGEMENTS: Supported by NIH grants GM068968 and EB005803,
References:
Bayard D, and Jelliffe R: A Bayesian Approach to Tracking Patients having Changing Pharmacokinetic Parameters. J. Pharmacokin. Pharmacodyn. 31 (1): 75-107, 2004.
Macdonald I, Staatz C, Jelliffe R, and Thomson A: Evaluation and Comparison of Simple Multiple Model, Richer Data Multiple Model, and Sequential Interacting Multiple Model (IMM) Bayesian Analyses of Gentamicin and Vancomycin Data Collected From Patients Undergoing Cardiothoracic Surgery. Ther. Drug Monit. 30:67–74, 2008.
P2X Purinergic Receptor Modulation Of Excitatory Nociceptive Transmission Involve NMDA Receptors JENNINGS EJ2, WILLIAMS MC2, CHO H-J2, SESSLE BJ1 1University of Toronto, Toronto, ON, Canada
2University of Melbourne, Vic, Australia
Background: The majority of trigeminal small-diameter nociceptive primary afferent fibres innervating craniofacial tissues terminate in trigeminal subnucleus caudalis (also termed the medullary dorsal horn, MDH). We have previously shown in vivo that central sensitisation of nociceptive neurons in deep MDH laminae can be induced by purinergic (P2X) receptor agonists and blocked by P2X antagonists applied in this area (Chiang et al., 2005). Our in vitro studies indicate that P2X agonists act presynaptically to facilitate MDH excitatory neurotransmission (Jennings et al., 2006). This study aimed to test if these P2X receptor processes involve NMDA receptor mechanisms and PKC. These are important as it addresses some of the underlying mechanisms of prolonged neuronal excitation in pain pathways.
Methods: Sprague-Dawley rat pups (9-16 days) were anaesthetised with halothane, decapitated and horizontal slices (250µm) were cut from the caudal brainstem. Whole-cell patch-clamp recordings (voltage clamped at –70mV) were made from MDH neurons.
Results: An increase in excitatory neurotransmission in deep MDH laminae, as reflected in an increase in mEPSC rate, was induced by the ATP analogue ,-methylene-ATP (,-meATP, 30 µM, n=14; P<0.01) but was blocked (no change in mEPSC rate or amplitude; n=7; P>0.1) following superfusion of the NMDA antagonist AP5 (40µM). In other brain areas, phosphorylation of NMDA receptors through PKC has been shown to potentiate glutamatergic neurotransmission, so we tested if ,-meATP activated an intracellular kinase cascade that altered NMDA receptor function in MDH ,-meATP (30µM) applied in the presence of the kinase inhibitor staurosporine (2µM) did not affect mEPSC rate (n=7; P>0.9). Furthermore, application of the phorbol ester (PDBu; 500 nM), which potentiates PKC-induced responses, caused a 294 ± 71 % (n = 6; P < 0.05) increase in mEPSC rate without change in amplitude. However, in the presence of AP5, PDBu caused a smaller increase (50 ± 5 %) in mEPSC rate, suggesting that phosphorylation of NMDA receptors is important.
Conclusion: These results suggest that P2X receptor-mediated central sensitisation in the deep MDH is mediated via NMDA receptors. Furthermore, our preliminary data suggests Protein kinase phosphorylation of NMDA receptors is involved.
Perspective of chromatin structure on Topoisomerase II: Exclusive dynamics between the nucleosome and the drug target in Saccharomyces cerevisiae JEONG KS, SPERLING A, GRUNSTEIN M
Univ. California, Los Angeles, USA
Background: Topoisomerase II (Top2) is a ubiquitous and highly conserved enzyme that serves as a target for anticancer and antibiotic therapy. Top2 introduces DNA double-strand breaks and rejoins the DNA after strand exchange to resolve topological problems. Although Top2 activity in chromosome segregation is essential for cell viability, the mechanisms of replication-independent cell killing remain to be elusive. The DNA-binding of Top2 and the DNA-Top2 covalent complex caused by Top2 inhibitors are the critical steps for its inhibition. Despite the importance of Top2 interaction with naked DNA, very little is known about its relationship with histone proteins which limit access to DNA by forming a nucleosome, a basic unit of chromatin. This study aims to understand the effect of changes in chromatin structure on Top2 activities.
Methods: We determined nucleosome density and the genome-wide distribution of Top2 and RNA polymerase II (Pol II) using high-density oligonucleotide tiling arrays. This study included budding yeast mutants defective in Top2 activity, nucleosome formation and Pol II transcription to determine their relationship. The analysis of DNA sequences was performed with genomic location data of Top2 to compare DNA motifs for Top2 binding with nucleosome positioning sequences.
Results: Top2 is localized in nucleosome-free regions in the S. cerevisiae genome. Mutually exclusive distribution between nucleosome and Top2 is a genome-wide phenomenon determined primarily by relocalization of nucleosome. Actively transcribed genes contain more Top2 than poorly expressed genes. However it is not simply a consequence of Pol II activity leading to an accumulation of excessive DNA supercoils. Nucleosome loss together with increased DNA binding of Top2 is a prerequisite for Pol II recruitment. This exclusive relationship in DNA binding between histones and Top2 in part depends on the anti-phase properties between nucleosome positioning DNA sequences and DNA motifs for Top2 binding.
Conclusions: 1) The binding of Top2 to DNA strictly depends on nucleosome remodeling. 2) The DNA-Top2 interaction, a key step in pharmacological inhibition of Top2, prominently occurs in the promoters of actively transcribed genes. 3) Effectiveness of Top2 inhibitors needs to be considered in the context of DNA-histone interaction.
Effect of Fish Oil with Garlic Oil Supplementation on the Anthropometric Measurements,Serum lipid Profile and Blood Pressure in Women with Hypercholesterolemia and Hypertension JEYARAJ SS, SHIVAJI G
Women’s Christian College, Chennai, India
Background: Elevated levels of low density lipoprotein( LDL),serum trigyceride(TG),serum lipoprotein(a)-[Lp(a)] and low levels of high density lipoprotein(HDL) have been documented as leading risk factors for the development of coronary heart disease(CHD) in Indian women.In view of an increment in LDL levels with fish oil supplementation (Suppl’n) ,the effect of a combined suppl’n of fish oil (MEGA-3) with garlic oil (GARLIC PEARLS) on the anthropometric measurements (AM), serum lipid profile(SLP) and blood pressure(BP) in women with hypercholesterolemia and hypertension were studied. Aims: To determine the effect of fish oil(FO) with garlic oil (GO) suppl’n on the AM like body mass index(BMI) ,waist circumference (WC) and waist:hip ratio(WHR) ,SLP and BP levels of the premenopausal women(Group-I) and postmenopausal women(Group-II) in the test groups and to compare the same with that of the respective control groups (Placebo).
Methods: This study included 60 hypercholesterolemic (>220 mg/dl) and hypertensive (>140/90 mmHg) women,of which, an equal number((n =30) belonged to the premenopausal group(30-45 yrs.) and the postmenopausal group(46-60yrs.). The subjects were further equally sub-divided (n=15 ) within each group as control group and test group.The study was conducted for a period of 90 days and with a withdrawal period of 30 days (Test groups).The dosage of fish oil was 600mg per day and garlic oil was 500mg per day(Test groups).The biochemical parameters were analyzed using the enzymatic kit methods .
Results:Statistically significant reductions were seen in all the AM, SLP(except HDL) and BP levels of the women in the test groups after 90 days of suppl’n compared to that of the respective control groups.After the withdrawal period,the hypolipidemic and hypotensive effect of FO with GO was sustained in both the test groups.Minor reductions were reported in the AM in both the test groups The systolic and diastolic BP reduced by 14.8% and 14.9% respy in Group-I compared to 20.3% and 15.9% in Group-II. The total cholesterol(TC),LDL,Very low density lipoprotein(VLDL), serum TG,TC:HDLratio, TG:HDL ratio and serum Lp(a) reduced by 25.5%,33.3%,24.8%,24.7%,28.2%27.3% and 30.3%.respy in Group-I compared to 21.8%, 27.7%, 18.6%, 18.3%,22.8%, 17.9% and 14.6% respy in Group-II.
Conclusion: The co-administration of garlic oil with fish oil was more effective than placebo in the management of dyslipidemia and hypertension in women.
Development of an Inactivated Rotavirus Vaccine for the Global Immunization Agenda JIANG B1, WANG Y1, GLASS RI1,2 1Centers for Disease Control and Prevention, Atlanta, Georgia and 2National Institutes of Health, Bethesda, Maryland, USA
Background: The efficacy of two live oral rotavirus vaccines (ORV), RotaTeq® and Rotarix®, has been demonstrated for children in developed and middle-income countries but remains to be confirmed for children living in resource-poor countries where rotavirus remains a major killer. We have pursued the development of an inactivated rotavirus vaccine (IRV) as a backup strategy for ORVs. IRVs could be more uniformly reliable when used in the challenging populations. IRVs would not be burdened by the threat of intussusception, a known complication of earlier ORVs and could be tested in smaller clinical trials powered for efficacy (ie. <5000 children) rather than safety against intussusception (>60,000) with ORVs. IRVs could be combined with other parenteral EPI vaccines making them easier to administer as part of routine childhood immunizations. Finally, IRV does not replicate so its efficacy should be free from interference from antibodies obtained through cord blood or breast milk, a problem that may inhibit the immune response to ORVs in children.
Methods: We examined the role of serum antibody in protection against rotavirus infection or disease in a primate model or children with acute diarrhea. We assessed the immunogenicity and protective efficacy of our candidate IRV in mice and gnotobiotic piglets.
Results: We have demonstrated that serum IgG is either an effector or a reliable proxy against rotavirus infection and disease in monkeys and children and established the proof of principle that serum antibodies can protect against rotavirus replication and shedding. We have gone on to develop a candidate IRV from human rotavirus strains and a novel method to effectively inactivate rotavirus while maintain the structural integrity of the viral particles. This candidate IRV adjuvanted with alum and administered intramuscularly to mice elicited strong total antibody and neutralizing antibody responses in serum. This IRV further induced solid protection against rotavirus infection in piglets.
Conclusion: IRV could provide an important insurance policy for the global immunization agenda if ORVs fail to provide adequate protection to those children in low income countries at greatest risk of a fatal infection or if problems of intussusception should again arise from ORV immunization.
Cytochrome P450 Inactivation By Pharmaceuticals And Phytochemicals: Therapeutic Relevance Johnson WW Drug Metabolism and Pharmacokinetics, OSI Pharmaceuticals, Boulder, CO 80301, USA
Background: One of the major clinical concerns is possible drug interactions that can be the result of abrogation of the P450 pathway(s) of metabolism causing toxicity due to elevated exposures of other drugs metabolized by these pathways. When the P450 substrate is catalytically activated to a reactive intermediate, this transient molecule may react with available nucleophilic residues from the enzyme -- thereby resulting in the inactivation of the P450.
Methods: The effects of CYP inactivation on the pharmacokinetics of co-administered drugs or on the inactivator itself depend on complex factors involving the molecular entities, the kinetics of inactivation (KI, kinact), the partition ratio, the zero-order synthesis rate of new enzyme, multiple pathways of metabolism (competing pathways), the dose or exposure, and specific patient characteristics.
Results: This review summarizes the catalytic efficiencies of many inactivator drugs along with any consequent clinical relevance. The chemical agents described have been ranked for the kinetic efficiency of inactivation and contrasted with the known clinically relevant drug interactions.
Conclusions: This will allow judicious consideration of the many factors that influence the importance of CYP inactivation and their relative contribution to systemic clearance of co-administered drugs. This study allows an improved characterization and dissection of potential physiological interactions with various drugs and nutrients. Knowing more about selective inactivation of cytochrome P450 by common xenobiotics, drugs and phytochemicals allows better understanding of expected interactions with chemotherapeutics and other xenobiotics.
Bioconjugated Cytotoxin Prodrugs and Imaging Agents in Anticancer Research JONES GB Department of Chemistry & Chemical Biology, Northeastern University, Boston, MA 02115 USA
Background: The enediynes are a class of potent antitumoral agents isolated from soil bacteria. One of these (Mylotarg®) represents the first monoclonal antibody-cytotoxin conjugate to be approved by the FDA and is currently used for treatment of acute myeloid leukemia (AML).The natural enediynes use a variety of elaborate triggering mechanisms which activate the pharmacophore through cycloaromatization, producing cytotoxic diyl radicals which damage DNA and other cellular and nuclear macromolecules. There has been considerable interest in the preparation of synthetic enediyne prodrugs that can be delivered to specific targets, and we are currently investigating photo-activated variants.
Methods: A family of substituted aryl enediyne templates were synthesized using standard Pd coupling methodology. The building blocks were converted to various PEG conjugates, which allowed subsequent coupling with targeting antibodies via NHS chemistry.The enedynes were also coupled to Au nanoparticles using standard thiolate chemistry, to allow development of heterobifunctional carrier systems.
Results: The PEGylated enediynes and their Mab conjugates undergo rapid photocyclization to produce the cytotoxic radicals on demand. Furthermore, the immunocompetence of the Mab conjugates are preserved, as confirmed by ELISA.Preliminary experiments confirm the selective cytotoxic activity of the Mab conjugates in cellular bioassays.
Conclusions: Readily available aryl enediynes can be prepared and converted to versatile and thermally stable bioconjugates. Subsequent photoactivation of the systems is equally effective in parent and Mab conjugated state, allowing the development of targeted cytotoxins and biochemical reagents. An immediate possibility is the development of heterobifunctional Au nanoparticles which can be selectively loaded with the PEGylated enediyne and Mab’s, and can also accomodate imaging tags for animal studies using PET and SPECT.
Short-term, low dose methotrexate for immune tolerance induction JOSEPH A, MUNROE K, HOUSMAN M, GARMAN R, RICHARDS S
Immunology Laboratory, Biologics R&D, Genzyme Corporation, Framingham, USA
Background: Antibody responses that develop against protein therapeutics can potentially impact patient safety and therapeutic efficacy. In an effort to control therapy-specific antibody responses, various approved immunosuppressive agents were evaluated for their ability to induce immune tolerance to two protein therapeutics, recombinant human -galactosidase (rh-Gal) and recombinant human acid -glucosidase (rhGAA). These include mycophenolate mofetil, cyclosporine A with azathioprine, CTLA4-Ig fusion protein, rapamycin, cyclophosphamide and methotrexate.
Methods: C57Bl/6 wild-type, Balb/c wild-type, 6neo/6neo GAA knock-out mice (GAAKO), -galactosidase knock-out mice and x-linked immunodeficiency mice were immunized with either rhGAA or rh-Gal in the presence and absence of the following immunosuppressive agents: mycophenolate mofetil, cyclosporine A with azathioprine, CTLA4-Ig fusion protein, rapamycin, cyclophosphamide and methotrexate. rhGAA-specific and rh-Gal-specific antibody responses were evaluated by ELISA. The cellular immune response to rhGAA was investigated in C57Bl6 wild-type mice and GAAKO mice using flow cytometry. Immunogenicity studies involved 8-10 animals/group and were repeated three times. Flow cytometry studies involved 5 animals/group and were repeated three times.
Results: The only agent that successfully induced a long-lived reduction in anti-rh-Gal and anti-rhGAA antibody responses following short-term immune suppression was methotrexate. For example, three weekly courses of 0.5mg/kg of methotrexate could significantly reduce rhGAA-specific antibody responses by 47% through at least eight months of weekly rhGAA treatment in GAA knock-out mice. Flow cytometry-based studies characterizing the cellular immune response to rhGAA helped inform the treatment schedule and effective dose of methotrexate.
Conclusion: Short-term, low dose methotrexate can significantly reduce antibody responses to at least two different protein therapeutics. Currently, low-dose methotrexate is under clinical evaluation as part of an immune tolerance induction regimen for rhGAA in Pompe patients. Low dose methotrexate may hold promise in reducing antibody responses against protein therapeutics in general.
Is Cyclosporine The Magic Bullet In Dermatology? JOVANOVIĆ MA, BRKIĆ SB, KARADAGLIĆ DC aFaculty of Medicine, University of Novi Sad, Clinical Centre of Vojvodina, Novi Sad, Serbia;
bFaculty of Medicine, University of Novi Sad, Novi Sad, Serbia;
cPrivate Clinic "Professor Karadaglic", Belgrade, Serbia
The effects of cyclosporine explain the interest in its history. Since the drug of fungal origin with anti-inflammatory properties, low myelotoxicity, and the T-cell immunomodulating effects revolutionized transplantation medicine, it was claimed that serendipity played a significant part in its discovery. Cyclosporine acts primarily during T cell activation by modulating lymphokine production and by reducing the activation and proliferation of T-helper and cytotoxic T cells. These selective effects have been recognized as beneficial in the treatment of dermatologic diseases, and this has particularly revolutionized skin immuno-interfering. The only dermatological indication for cyclosporine approved by the US Food and Drug Administration is psoriasis. The authors discuss excellent therapeutic responses reported in patients with other dermatologic diseases: atopic dermatitis, erosive mucosal lichen planus, Behcet's disease, epidermolysis bullosa acquisita, lichen planus and pyoderma gangrenosum. Cyclosporine A can be safely administered when potential toxicities, dosing (3-5 m/kg/d), and guidelines are known. Also, highly variable results of systemic cyclosporine in the treatment of severe alopecia areata, have been achieved. The authors of this review following the concepts of lower dosages of systemic cyclosporine (2.5 mg/kg/d decreased by 0.5 mg/kg monthly) and the use of low dose prednisone simultaneously (5 mg/d), developed complete long-duration sustained terminal hair re-growth in universal alopecia areata (chances for cure were less than 1%). Thus, in spite of the tremendous progress in immunologic research, practical therapeutic immune-intervention has yet to reach the level of specificity and precision that is needed, i.e. the “immunologic magic bullet”. However, cyclosporine is a good candidate.
Discovery & Development of Antineoplastic Magic Bullets from Natural Products JUNG M Yonsei University, Seoul, Korea
Background: Most clinically useful anticancer drugs were discovered from natural products as examplied in adriamycin, vinblastine, taxol, and camptothecin, etc. Aims: 1) A practical and stereospecifc syntheis of novel derivatives of deoxoartemisinin and daumone, a kind of pheromone. 2) in vitro and in vivo testing of antineoplastic activity of the synthesized compounds. 3) Establishment of strucure and activity relationship(SAR) to design and provide an anticancer drug candidate.
Previously, we discovered a novel pheromone that postpones aging in worms from C. elegans with a laborious, a large-scale purification and 3-dimensional structural determination.
Methods: This study included synthesis, anticancer activity screening and SAR study including proposed biomechanism of anticancer activity. Most of synthetic efforts have been focused in derivatization at C-12 position of artemisinin resulting acetal-type derivatives. To overcome instability in simulated stomach acidic condition, and recently appearing neurotoxicity of artemisinin and its C-12 acetal derivatives such as arteether, artemether, and artelinic acid, we prepared non-acetral type deoxoartemisinin and its derivatives from artemisinin, a natural product isolated from A. annua as potential anticancer drug candidates. Total synthesis of new derivatives of dauer-effect pheromone isolated C. elegance were efficiently achieved.
Results: Non-acetal C-12, 13 derivatives, (+)-deoxoartelinic acid and dimers of (+)-deoxoartemisinin were synthesized either from naturally occuring artemisinic acid or directly from artemisinin via a short and regiospecific process. Some of its novel derivatives show comparable antitumor activities to those of clinically useful drugs.
A stereospecific, 10 step synthesis of pheromone was successfully achieved starting from commercially available rhamnose. Both isolated and synthetic daumone induce the morphological changes that accompany worm hibernation.
The detail of stereospecific synthesis and the antneoplastic activities of (+)-deoxoartemisinin, its C-12 and 13 derivatives, natural plakortolide and novel anticancer derivatives of daumone along with the structure-activity relationship will be presented. In vitro antineoplastic screening of derivatives of deoxoartemisinin and daumone against human cancer cell lins (ovary, lung, brain, colon) showed potential anticancer activities.
Conclusions: 1).The non-acetal type and anticancer derivatives of deoxoartmisinin showed acid stability, and may overcome neurotoxicity.
2). These results may shed light on the discovery of antineoplastic magic bullets derived from natural products and a signaling pathway thought to be similar to those that lead to aging and obesity in humans. 3). Trimers of deoxoartemisinin and daumone against human cancer cell lins showed potential anticancer activities comparable to that of clinically useful anticancer drugs.
Composition Of Essential Oil Of Nigella sativa (L.) Seeds And Inhibition Of Human Neutrophil Elastase KACEM R¹, BUHLING F², WELTE T²
¹Laboratory of Biochemistry, Department of Biological Sciences, Faculty of Sciences, University Ferhat Abbas, Setif, Algeria; ²Institut für Immunologie, Medizinische Fakultät, Otto-Von-Guericke-Universitat Magdeburg, Germany.
Background: Inhibition of Human Neutrophil Elastase (HNE) activity by essential oil extracted from Nigella sativa (L.) seeds was tested in this study.
Methods: Hydrodistillation method was used to extract essential oil and the yield was found to be 0.4%. Chemical analysis of essential oil was performed by GC-MS. Determination of HNE activity was realized by spectrophotometry method using N-Methoxysuccinyl-Ala-Ala-pro-Val p-Nitro-Anilide as elastase substrate. Essential oil (5.8 mg/mL) was a strong inhibitor to HNE.
Results: Aggregate effects of total essential oil caused 100 % Inhibition. testing the inhibitory effects of major constituents of essential oil on HNE activity by microassays revealing that 5-Isopropyl-2-Methyl Phenol was a strong inhibitor to HNE with a very low IC50 (12 μM).
Conclusions: We conclude that 5-Isopropyl-2-Methyl Phenol is a natural antielastase compound that could be used in the treatment of some pathological cases such as COPD and emphysema
The Importance of Appropriate Animal Models for Evaluating Agents for Proliferative Retinopathy: Failure of Combretastatin to inhibit Diabetes-Like Proliferative Retinopathy in the Galactose-Fed Dog. KADOR PF,1,2, BLESSING K1,2, WYMAN M1 1Laboratory of Ocular Therapeutics, National Eye Institute, National Institutes of Health, Bethesda, MD, USA and 2Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE USA
Background: “Magic bullets” for ocular use have primarily focused on the treatment of neovascularization and the prevention of secondary cataracts. Patients with macular degeneration are intravitreally injected with anti-VEGF antibodies to reduce angiogenesis while anti-neoplastic agents are being investigated for the prevention of post-surgical lens cell proliferation. Combretastatin A-4 (CA4P) is a vascular targeting agent that destroys rapidly growing tumor capillaries. In mouse eyes, CA4P also suppresses retinal neovascularization that occurs within days to weeks following laser induced choroidal neovascularization or genetic over expression of retinal VEGF. To determine the clinical potential of CA4P to treat neovascularization in diabetics, we have evaluated CA4P in galactose-fed dogs, an animal model that slowly develops proliferative retinopathy similarly to that observed in diabetics.
Methods: Eight beagles fed 30% galactose diet for 80-104 months and 4 age-matched normal beagles were made aphakic so that retinal changes could be easily doumented. These were divided into 2 groups composed of 4 galactose-fed and 2 control dogs. Each group received CA4P as either sub-Tenon’s or intravitreal injections. Six weeks later, all dogs received systemic (IV) injections of CA4P. Changes in neovascularization and blood flow were clinically monitored at 2-week intervals by fluorescein angiography and fundus photography.
Results: CA4P was well-tolerated in the healthy eyes of normal dogs. In galactosemic dogs, CA4P administration was associated with corneal edema and increased intraocular pressure. All galactose-fed dogs demonstrated retinal neovascular lesions. Sub-Tenon’s, intravitreal or systemic CA4P administration failed to alter these retinal lesions.
Conclusions: Failure of CA4P to ameliorate neovascularization suggests that chronic, long-term administration is required to destroy the slowly growing retinal endothelial cells. This treatment is unlikely to be clinically useful for reducing neovascularization in diabetics.
Antibody Based Protein-Function Analysis On The Ryanodine Receptor And Troponin Isoforms In Caenorhabditis Elegans KAGAWA H1, AMINN MD. Z1, 2, HAMADA, T1, 3 1Okayama Univ., Okayama, Japan; 2Sir Salimullah Med. Col., Dhaka, Bangladesh; 3BBRI,Boston, USA
Background: After genome sequence data are accumulated, understanding of the relation between structure and function of a big molecule or isoform proteins is important to solve biological details. Aims: 1) To compare the bacteria produced fusion proteins and a native ryanodine receptor. 2) To develop antibody based protein analysis that can describe a molecular difference in muscle proteins. 3) To propose a tool for comparing closely related proteins from animals.
Methods: We constructed several expression vectors to generate recombinant proteins of the ryanodine receptor, three body-wall and one pharyngeal troponin I isoforms and other muscle components in Escherichia coli. Protein overlay assays and Western blot analyses were performed using anti-rabbit antibodies. Tissue specific localization of the protein is also performed to know whether raised antibody can detect in situ localization of the isoforms.
Results: Ten region-peptides of the ryanodine receptor of C. elegans (CeRyR) containing 5071 amino acid residues were produced in E. coli. One of eight anti-rabbit anti-sera enabled detection of CeRyR in situ but others didn’t. One peptide had Ca++-binding activity. Using peptides and peptide specific-antibodies analysis we proposed the local structure and function of individual domains within a large molecule. Similar approach in other experiments we demonstrated that pharyngeal TNI-4 interacted with only the pharyngeal isoforms of troponin C/T and tropomyosin in C. elegans. In contrast, the body wall TNI-2 bound both the body wall and pharyngeal isoforms of these components. As is known in other invertebrates, the N-terminus of troponin I contribute to interactions with troponin C. Full-length troponin I was essential for interactions with tropomyosin isoforms. From these molecular interaction results we can estimate evolutional history of tissue specific TN isoforms of muscle proteins.
Conclusions:
1) Region specific peptides of a big molecule keep a local structure of whole protein. 2) Antibody based analysis on molecular interactions allows us more information than those of sequence alignments.
This experimental approach is powerful to solve biological function of many accumulated protein sequences and is applicable for determining epitope in functional regions
Peculiarities of Blood-Brain-Barrier Penetration of Pralidoxime KALASZ H, TEKES, K
Semmelweis University, Budapest, Hungary
Background: Organophosphate poisoning in agriculture (millions of cases) and the threat of chemical terrorist attacks require intensive research on antidotes. Organophosphates cause overabundance of acetylcholine at all kinds of its receptors in the central nervous system, and the periphery. Antidote treatment of victims is possible, the therapy is known by the acronym “A FLOP” (atropine, fluid, oxygen and pralidoxime). Clinical experiments, however, have been disappointing with any one of the presently available aldoximes. Fast and effective disposition and excretion of the organophosphate – aldoxime conjugates (phosphoryloximes, POXs) may have vital importance as POXs play an essential part of anticholinestrase activity, and POXs may be hydrolyzed with endogenous enzymes to give the original organophosphates.
Kuca et al. synthesized a series of very effective bis-pyridinium aldoximes marked with the letter K- and a serial number. Petroianu et al. examining relative risk of death of rats in pesticide organophosphate poisoning found K-27 and K-48 to be the most effective antidotes. When ability of several aldoximes to reactivate tabun-inhibited acetylcholinesterase was evaluated by Musilek et al. and superiority of K-203 was found.
Methods: Lipophilicity of pyridinium aldoximes was determined using on silica (planar chromatography) and in silico (computer assisted lipophilicity calculations) methods. Distribution/penetration of pyridinium aldoximes were determined using reversed-phase chromatography of blood, CSF (cerebrospinal fluid) and brain samples of rats following intramuscular treatments of the animals. Various brain regions were also dissected.
Results: Both thin-layer chromatography and computer-assisted lipophilicity calculations showed highly polar characteristics of pyridinium aldoximes. Even, pralidoxime and the other N-substituted pyridinium aldoximes do enter the brain and the cerebrospinal fluid. Results indicate that low doses of pralidoxime, K-27, K-48 and K-203 doses (below 10 microM/rat) show higher relative brain and CSF penetration. Considering the various brain parts (frontal cortex, hypothalamus, hippocampus, striatum, etc.) pyridinium aldoximes show an even distribution.
Conclusions: For patients who have been poisoned, infusion with low-concentration pyridinium aldoximes may be preferable to one injection with a high dose.
Novel Immuno - Potency Drugs KALAVI K*, MOHAGHEGH HAZRATI S**
* Golestan University of Medical Sciences and Golestan Research Center of Gastroenterology & Hepatology, **Dr. Mohaghegh's foundation researches on Industerial Biotechnology
Background:
The protective role of the immune system in health and the correlations of its failures as a path of pathogenesis are well clarified. Based on recent studies the immunologic concern in the field of health has been improving. The occurrence of diseases like viral, bacterial and fungal diseases, auto- immune diseases and cancer in turn denotes to immunological problems as natural and acquired immune system defects. Because of critical need to support the immune system we proposed that there is need to design kinds of novel generations of drugs to get such a goal. We aimed with this strategy to support the immune system in a way through which it potentiates itself to fight against pathogenic factors, regulate, modulate or reverse the defects as needed.
Methods:
Three different drugs prepared from bacterial and lipid extracts (G2, PC & G2F) that could increase Th-1 cells response depending on the methods of experiments including Delayed type Hypersensitivity (DTH), Lymphocyte Transformation Test (LTT) with PPD and rgp63 cocktails. They prepared in cream and injection forms. The clinical trials Phase I, II performed successfully and also III done in regard with some diseases.
Results:
Based on methods of experiments Th-1 response was significant (P<0.01 to P<0.001). Cytogenetic and mutagenetic studies showed no chromosomal abnormalities .Healing of up to 42% ductal adenocarcinoma of breast cancer in laboratory mice with significant increase in survival lives(P=0.000). Control and cure or prevent after surgery of different cancers in volunteers such as: Leukemia, Breast Cancer, Macroadenoma, Prolacinoma of pituitary gland, colorectal cancer, Hepatoma and other types. These drugs controlled and cured (followed up) of 70 % of adult and up to 90 % of children asthmas respectively.
Complete healing of grade 3-5 of diabetic foot ulcers in some volunteers with resistant to therapy wound types. Newly generated foot ulcers in diabetic patients cured within few days.
Conclusion:
We concluded that the formulations mentioned above are optimized compounds of effectiveness with no side effects reported till now. Because of its effectiveness we suppose that it would be considered as a remedy of next decade. It would be applicable in treatment of chronic diseases, cancers and in all fields required the body to get normal by regulating, modulating and reversing the defects.
Successful Treatment Of Methicillin-Resistant Staphylococcus Aureus Meningitis Using Linezolid Without Removal Of Intrathecal Infusion Pump: Three Years Follow-Up KALLWEIT U1,2, HARZHEIM M2, WIETHOELTER N2, MARKLEIN G3, POEHLAU D2 1 University Hospital Zurich, Department of Neurology, Zurich, Switzerland
2 Kamillus Klinik, Department of Neurology, Asbach, Germany
3 University Hospital Bonn, Department of Microbiology and Immunology, Bonn, Germany
Abstract:
Infection of an intrathecal pump system is a rare but serious complication and usually leads to the removal of the pump. We report the first case of methicillin-resistant Staphylococcus aureus (MRSA) meningitis in a patient with such a pump successfully treated with linezolid without the need for removal of the intrathecal pump. A 77-year old woman with cervical myelopathy underwent implantation of an intrathecal pump system for baclofen administration. Two weeks after the procedure she developed meningitis caused by MRSA as isolated in cerebrospinal fluid (CSF) cultures, blood samples, and serum obtained from the pump pouch. Clinically she presented with meningism,
somnolence, and signs of sepsis. With a combined intravenous antibiotic treatment regimen of vancomycin and rifampicin no clinical improvement occurred. The regimen was then discontinued and linezolid was administered intravenously as monotherapy. Within 3 days clinical and laboratory findings showed significant improvement. After 1 week of linezolid treatment, blood and CSF cultures were sterile. Intravenous treatment was administered for a total of 3 weeks, after which the patient was treated with oral linezolid for 3 months. During 36 months now of follow-up, no signs of infection or any severe adverse events were observed. These results confirm previous reports of the efficacy of linezolid for the treatment of severe infections of the central nervous system caused by multidrug-resistant Gram-positive bacteria, especially postneurosurgical infections.
A Preparation Of NI-Lipid Nanoparticles By Combination Of Roll Mill And High Pressure Homogenization, And Stabilization Of The Nanoparticles By Gel Solidification Method --Aiming At Preparation Methods Of The Nanoparticle Without Using Organic Solvent— KAMIYAS1,2, MIYAGISHIMA A2, SONOBE T2, ITaI S2, ARAKAWA M1 1Univ. Nagasaki International University, City, Sasebo, Country; Japan, 2Univ. University of Shizuoka, City, Shizuoka, Country; Japan
Background: Nanotechnology has recently been attracting attention, and establishment of pharmaceutical technologies to micronize drug particles is extremely expected. However, almost all methods use organic solvents in the process of preparing nanoparticles. Accumulation of residual solvents in the body and environmental pollution by liquid waste are matters of concern regarding the use of organic solvents. Thus, we try to prepare nanoparticles without using organic solvents. In addition, we investigate a method to maintain the dispersed condition of the nanoparticles by adding gelatin and solidifying the suspension. Freeze-drying allowed reproduction of the nanoparticle condition. However, this procedure is inconvenient for oral administration due to requiring preparation at the time of use. Simple method substituting for freeze-drying method was desired.
Methods: A 30:1000 (weight ratio) of Nifedipine (NI)-phospholipid (PL) mixture prepared by roll milling was dispersed in water and premixed. Subsequently, the premixed suspension was applied to high-pressure homogenization (HPH). Physical characteristics of NI-PL mixtures were analyzed by using powder X-ray diffraction and Fourier-transform infrared spectroscopy. Particle size was measured by using light scattering photometer.
Results: The mean particle size of the NI-PL nanoparticles decreased as the pass number increased, and the size after 40 passes were 55nm, indicating that roll mixing was as effective as ethanol treatment. NI-specific diffraction peaks of powder X-ray diffraction appeared at 8.2, 16.2, 24.4, and 25.9°. The peaks of NI-PL mixtures were present at the position of NI crystals, and no peak shift was induced by interaction with PL, showing that NI mostly remained as crystal in the PL.
Conclusions: 1) The NI-PL nanoparticle suspension prepared by combination of roll mill and HPH, indicating that nanoparticles could be prepared without organic solvents. 2) The mean particle sizes were about 55nm before and 24h after the gelatin solidification, suggesting that gel solidification method is helpful.
Synbiotics: An Important Tool For Preventing Enterocolitis And Promoting Physical Growth In Severely Ill Pediatric Patients –10 Years’ Experience In One Institute- KANAMORI Y1), IWANAKA T1), SUGIYAMA M1), YUKI N2), TAKAHASHI T2), MOROTOMI M2), TANAKA R2) 1The University of Tokyo Hospital, Tokyo, Japan, 2Yakult Central Institute for Microbiological Research, Tokyo, Japan
Background: Pediatric surgical patients often suffer from severe enteritis and it causes sepsis, hepatic failure and malnutrition. In order to disclose the etiology of these complications we investigated the intestinal microbiota of the patients and the effects of probiotics.
Methods: We designed a new synbiotic therapy consisted of Bifidobacterium breve Yakult, Lactobacillus casei Shirota, and galactooligosaccharides. Our experience was the first clinical trial of synbiotic therapy in the world. We applied synbiotic therapy to more than 50 pediatric patients who had short bowels, intestinal functional disorders, severe respiratory distress, and liver dysfunctions with abnormal intestinal microbiota (therapeutic use of synbiotics). Intestinal microbiota and nutritional states were followed before and after the therapy. Recently we administered our synbiotics to neonatal surgical patients who had not yet acquired intestinal microbiota (prophylactic use of synbiotics).
Results: Almost all patients had a quite abnormal intestinal microbiota; decrease of anaerobic bacteria and increase of pathogenic microorganisms. After starting the therapy many patients acquired the probiotics dominant intestinal microbiota and it was maintained well during through the treatment course. When the patients recovered from the critically ill states, the intrinsic anaerobes increased in the intestine and probiotics suppressed in its number. The nutritional states improved with synbiotic therapy in many patients.
As to prophylactic use of synbiotics, being still preliminary results, it is very effective for severely ill infants to establish anaerobes dominant intestinal microbiota and maintain good nutritional states.
Conclusions: 1, Our synbiotics are safe and effective for pediatric surgical patients with abnormal intestinal microbiota. Probioitcs were well resided in the intestine and prevented enteritis, improved intestinal functions and patients’ nutritional states (therapeutic use of synbiotics). 2, Early start of synbiotics for neonatal patients is more effective to acquire normal intestinal microbiota and it is recommended for very severe neonatal surgical petients (prophylactic use of synbiotics).
Anti-Infective Properties And Functions Of Betaine (Trimethylglycine) As An Nutritional Agent KANBAK G Eski?ehir Osmangazi University, The Medical School, Department of Biochemistry
Betaine is found in most microorganisms, plants, and marine animals. Its main physiologic functions are as an organic osmolyte to protect cells under stress and as a source of methyl groups needed for many biochemical pathways. Betaine is also found naturally in many foods and is most highly concentrated in beets, spinach, grain, and shellfish. Strains of enterobacteria that cause urinary tract infections are able to grow in urine with high tonicity. The betaine analogues have antibacterial effect against E. coli strains, but only in presence of an osmotic stress. Analogues of gamma-butyrobetaine appear to be prospective drugs for the treatment of circulatory complications of sepsis. Betaine is used by cells to defend against changes in osmolarity. It is suggested that there are relationships among betaine, osmolarity and coccidiosis. The chemotaxis of monocytes toward chemotactic factors released by heterophils was increased by betaine. Increased chemotaxis of monocytes and NO release by macrophages may explain the decreased intestinal pathology but increased leukocyte numbers that were observed when betaine was fed during a Cocci infection. Urine has long been known to inhibit the activity of aminoglycosides against urinary tract pathogens. Glycme betaine which is present in urine confers resistance against high osmolarity to Gram-negative organisms. The betaines in urine permit the expression of increased resistance to aminoglycosides in concentrated urine. Hepatitis C virus (HCV) infection is an important cause of chronic liver disease. Standard therapy, pegylated interferon alpha (pegIFNalpha) combined with ribavirin, results in a sustained response rate in approximately half of patients. It has been shown that treating cells with S-adenosyl-L-methionine (AdoMet) and betaine could restore STAT1 methylation and improve IFNalpha signaling. Furthermore, the antiviral effect of IFNalpha in cell culture could be significantly enhanced by the addition of AdoMet and betaine.. In our study, we investigated the protective effect betaine and prednisolone on the level of nitric oxide in sepsis. As a result, according to hematologic and biochemical assessments, we concluded that betaine administration without prednisolone is more useful and effective in decreasing susceptility to sepsis.Our data in our laboratory and recent studies in literatures showed that betaine (trymethyglycine) and betaine analogues have anti-infective peroperties
Epitopic Peptides With Low-Similarity To The Host Proteome. Towards The Magic Bullets. KANDUC D Department of Biochemistry and Molecular Biology, University of Bari, Italy
A robust set of experimental data suggests that low level of sequence similarity to the host proteome modulates the B cell epitope pool in the humoral immune response. The data appear to be widely supported by structured meta-analyses of scientific literature. Theoretically, proteomic similarity analyses might elucidate the regulatory mechanisms/factors that dictate peptide immunogenicity assessment. From the clinical point of view, low-similarity peptides may have a strong repercussion on the rational development of peptide-based treatments in cancer, infection and autoimmunity. De facto, the most attractive feature of the similarity concept is that it appears a guarantee of highest specificity and lowest cross-reactivity in designing effective, safe and theoretically infallible immuno-therapeutical tools. The Ehrlichian idea of therapeutic agents equipped with high affinity to the causative agent and efficacy in concentrations harmless for the patient, appears feasible. The Ehrlichian therapia magna sterilisans might be at hand.
Ames Test And Gene Expression Analysis For Polyhydroxybutyrate – A Biomaterial KANNAN TP, ABDULAZIZ QA, AZLINA A, SAMSUDIN AR
School of Dental Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia
Background: It is imperative to test new materials for mutagenic or carcinogenic properties before they are approved for medical use. Polyhydroxybutyrate (PHB), is finding many useful applications as an implant material due to its biocompatibility and resorbability.
Aims: 1) To determine the mutagenicity of a locally produced PHB using Salmonella mutagenicity test. 2) To determine whether PHB alters the expression of p53 and c-myc proto-oncogenes and bcl-xl and bcl-xs anti-apoptotic genes in the human fibroblast cell line.
Methods: Different concentrations of PHB (5, 2.5, 1.25, 0.625 and 0.3125 mg/plate) were incubated with special genotypic variants of Salmonella strains (TA1535, TA1537, TA1538, TA98 and TA100) carrying mutations in several genes both with and without metabolic activation system (S9) and the test was assessed based on the number of revertant colonies. Concurrently, both negative (sterile distilled water) and positive controls (sodium azide, 9-aminoaridine hydrochloride monohydrate, 4-nitro-O-phenylenediamine and 2-aminoanthracene) were used. For the gene expression analysis, fibroblast cell line (CCL-171) designated as MRC-5 was used. The cells were treated with five doses of PHB (0.3125, 0.625, 1.25, 2.5 and 5 mg/ml) and incubated for 1, 12, 24 and 48 hours at 37ºC in a CO2 incubator. The total RNA was isolated and reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out for β-actin, p53, c-myc, bcl-xl and bcl-xs genes. The amplified products were run on an agarose gel and then quantified based on the intensity of bands of the different genes.
Results: The average number of revertant colonies per plate treated with PHB was less than double as compared to that of negative control. Also, the PHB did not show over or under expression of the genes studied.
Conclusions: 1) The Salmonella mutagenicity test indicated that the locally produced PHB is non-mutagenic on the variants of Salmonella strains TA1535, TA1537, TA1538, TA98 and TA100. 2) PHB does not alter the expression of the proto-oncogenes (p53 and c-myc) and anti-apoptotic genes (bcl-xl and bcl-xs) in this study under the present test conditions.
Alpha2-Antiplasmin Is A Critical Regulator On Bleomycin-Induced Fibrosis
KANNO Y, MATSUNO H
Doshisha Women’s Collage of Liberal Arts, Kyoto, Japan.
Background: Fibrotic disease occurs in most tissues. It has been known that the transforming growth factor (TGF)- and connective tissue growth factor (CTGF) are the main regulators of fibrosis, and TGF- the expression of CTGF. The fibrinolytic system is considered to play an important role in the degradation of extracellular matrices (ECM). However, the detailed mechanism how this system affects fibrosis remains unclear.
Methods: To verify the effect of the 2-antiplasmin (2AP), which is a potent and specific plasmin inhibitor on fibrosis, we showed a histological examination on bleomycin-induced dermal fibrosis in 2AP +/+ mice and 2AP -/- mice. In addition, we examined that the level of mRNA on bleomycin-induced dermal fibrosis in 2AP +/+ mice and 2AP -/- mice by using RT-PCR. Moreover, to obtain genetic evidence that 2AP functions, we examined the levels of mRNA and protein in the fibroblasts from both 2AP -/- and 2AP +/+ mice by using RT-PCR and ELISA.
Results: We found that the absence of 2AP attenuated bleomycin-induced TGF- synthesis and fibrosis. In addition, the production of TGF- from the explanted fibroblasts of 2AP -/- mice decreased dramatically compared to that in 2AP +/+ mice, and 2AP specifically induced the production of TGF- in fibroblasts. Moreover, we found that CTGF induced the expression of 2AP.
Conclusion: 2AP is involved in the production of TGF-, and CTGF induces the expression of 2AP. This study suggests that 2AP plays a crucial role on the progression of fibrosis. Our findings may hopefully provide new insight into this field which could eventually lead to the development of new clinical therapies for the prevention of fibrosis.
In Vitro Preclinical Studies For A Rational Design Of Cancer Chemotherapy Combinations KANO Y1, AKUTSU MIYUKI1, FURUKAWA Y2 1Division of Hematology, Tochigi Cancer Center, Tochigi, Japan.
2Division of Stem Cell Regulation, Jichi Medical University, Tochigi, Japan.
Background: Combination chemotherapy has been used for the treatment of cancer, based upon theoretical advantages and on proven clinical efficacy. In vitro drug combination studies play an important role in designing and optimising combination protocols. However, the design and analysis of preclinical studies to assess the cytotoxic effects in combinations is complex, and there is no consensus as to the best method, since dose-response curves of anticancer agents are variable. Even the same data are used, the results are often different, depending on the evaluation method used.
Methods: A variety of cancer cell lines were used for drug combination studies. Cells were exposed to two drugs simultaneously (and sequentially). Cell growth inhibition was determined using MTT assay. We used the isobologram method (Steel and Peckham) for studying the cytotoxic effects of drug combinations, since it can cope with a variety of doseresponse curves of anticancer agents. This method is generally stricter for synergism and antagonism than other methods.
Results and Conclusions: 1) Therapeutic benefit was highly dependent on the combination and schedule of administration. 2) The anticancer agents which it was easy to use together (additive or synergistic effects in simultaneous exposure) included alkylating agents, anthracyclines, cisplatin, etoposide, imatinib, irinotecan, and antimetabolites (excluding antifolates). 3) The simultaneous administration of methotrexate and all other agents studied produced antagonistic effects, while methotrexate first followed by most agents produced synergistic effects. 4) Similar tendency was observed in pemetrexate in combination with cisplatin, gemcitabine, irinotecan, or paclitaxel. 5) Paclitaxel had a tendency to act subadditively when administered simultaneously but additively when administered earlier. 6) These findings are useful for designing clinical trials of combination chemotherapy.
Differential Targeting Of Immune Response After Antigen Encounter At Different Mucosal Sites – A Tool For Vaccine Development
KANTELE A Div. of Infect. Dis., Helsinki Univ. Central Hosp. and Univ. of Helsinki, Finland
Antigen encounter results in activation of specific lymphocytes, which then leave the site of antigen encounter and enter the blood as plasmablasts. These cells are found only temporarily in the circulation while on their way to home to tissues, where they settle down as the final end-stage B cells, plasma cells. While representing only 1% of all circulating B cells, plasmablasts are the only active effector B cells in the circulation, often identified as cells producing specific antibodies against the recently encountered antigen. Plasmablasts express homing receptors (HR) and chemokine receptors (CCR) that allow them to leave the circulation through the endothelium and enter the tissues. Even if blood takes the cells everywhere in the body, plasmablasts can only enter those tissues where the ligands for their HR and CCR are found. The tissue-specificity of homing results from a differential expression of chemokines and ligands for HR between the tissues. The selection of HR and CCR is imprinted on the activated lymphocytes already at the original site of antigen encounter by dendritic cells. Investigation of homing-associated molecules, the homing profile, on plasmablasts provides information on the targeting of the immune response in the body.
Almost all pathogens are encountered at mucosal surfaces. These portals of entry are guarded by the local mucosal immune system. Mucosal immunization is the most effective way to induce mucosal immune responses. The different mucosal sites in the body are regarded to be interconnected with one another via circulating lymphocytes thus constituting a sc. Common Mucosal Immune System (CMIS). Antigen encounter at one mucosal site can elicit an immune response at a distant mucosal site. However, lymphocytes don’t migrate equally to all mucosal sites, but there is compartmentalization within the CMIS. Our results show that in humans, antigen encounter in the intestine, urinary, genital, upper and lower respiratory tract each label the circulating plasmablasts with a characteristic homing profile. As the targeting of the immune response depends on the site of antigen encounter, vaccines should be administered to sites from where the immune response is most effectively targeted to that particular site (intestine, genitourinary, respiratory tract), where the immune protection is desired the most.
Infection Site Concentration Of Metronidazole (MTZ) And Meropenem (MER) In Patients With Septic Shock (SS) KARJAGIN J, STARKOPF J
Tartu Univ., Tartu, Estonia
Background: Sepsis is a frequent syndrome and cause of mortality in modern intensive care. The inadequate concentration of antimicrobial drugs in site of infection might be one of the therapeutic failure reasons. Microdialysis (MD) is a sampling technique of tissue fluid, allowing protein-free fluid sampling from different tissues. The main purpose of the present work was to elucidate distribution of MTZ and MERO in clinical conditions such as perioperative prophylaxis, SS and severe peritonitis.
Methods: 18 patients were studied: 6 female patients receiving MTZ in perioperative period, 6 male patients in intensive care unit (ICU) receiving MTZ for the treatment of anaerobic infection and 6 patients in ICU receiving MERO for the treatment of severe peritonitis associated with SS. Both drugs were given intravenously. MTZ sampled from plasma (PL) and muscle tissue (MT) using MD and MERO from PL and peritoneal cavity (PC) using MD. Samples were taken at predetermined time-points before and after administration of drugs. Drugs’ concentration were measure using HPLC with ultraviolet detection (MTZ) and LC-MS/MS (MERO). Pharmacokinetic-pharmacodynamic (PK/PD) modeling were also performed: in vitro with MTZ and using mathematical modeling with MERO.
Results: MTZ mean max concentration (Cmax) in surgical patients and patients with SS was 16.5 and 11.4 mg/L in PL and 7.8 and 8.2 mg/L in MT, respectively. Time over minimal inhibitory concentration (T>MIC) was 23.3 and 36.2 hours for MT of surgical and septic patients, respectively. The ratio of Cmax/MIC was also higher in septic patients being 31.1 and 32.7, respectively. In vitro PK/PD modelling using MT concentration from patients with SS showed that time for kill 99.9% of inoculum is between 1 and 3.5 hours after exposure. MERO mean Cmax in PL and PC was 86.1 and 36.8 mg/L, respectively. T>MIC was at least 87% of interdosing interval for MIC 4 mg/L in both PL and PC and was 55 and 43% for MIC 16 mg/L in PL and PC, respectively.
Conclusions: 1) Distribution of MTZ is not significantly different in healthy patients and SS patients. 2) MTZ demonstrates excellent antimicrobial killing 3) Concentration of MERO in PC in case of severe peritonitis is high enough to produce antimicrobial killing.
Authors’ disclosure statement: Concentration of MERO in microdialysates was determined by Lefevre S., Marchand S and Couet W, University of Poitiers, France. The PK/PD analysis for MERO was performed by prof. Sawchuk RJ, University of Minnesota, USA and prof. Couet W, University of Poitier, France
Toxic Effect Of Homocysteine On Nervous And Immune System KARPOVA L (1,2), BRYUSHKOVA E (2), MAKHRO A (2), MASHKINA A (1,2), SOLENAYA O (2), BULYGINA E (1,2), VLADYCHENSKAYA E (2), STEPANOVA M (1), FEDOROVA T (1), BOLDYREV A(1,2)
1 - Research Center of neurology, Russian Academy of medical sciences, Moscow, Russia, 2 – International Biotechnological Center of M.V. Lomonosov Moscow state University
Homocysteine (HC) is a risk factor of neurodegenerative and cardiovascular diseases. In terms of effect of HC and homocysteic acid (HCA, the product of spontaneous oxidation of HC) on NMDA-receptors (Carpenter et al, 1994) toxic effect of HC and HCA on NMDA-receptors on neuronal and immune competent cells was studied. These compounds were found to activate NMDA-receptors in a dose dependent manner (0,1-1 mM) resulting in calcium and reactive oxygen species signal and subsequent activation of MAP kinase. Over-loading of NMDA-receptors with these ligands induces injury process in both neurons and lymphocytes resulting in apoptosis at lower concentrations and necrosis at higher concentrations. Thus hyperhomocysteinemia induces death of the neuronal and immune competent cells resulting in massive exhaustion of both systems. Prenatal hyperhomocysteinemia induced by over-loading of pregnant rats with dietary methionine (1 g per kg body weight) resulted in modification of properties of NMDA-receptors and memory and behavioral deficiency. Treatment of these animals with carnosine, natural neuromodulator and antioxidant preserved the pups against toxic effect of hyperhomocysteinemia preventing loss of body weight and memory deficiency. We have concluded that carnosine may protect metabolic function of neuronal and immune systems against systemic oxidative stress induced by HC.
Valproate (VPA): Unrecognized Value From An Old Molecule
igure 2. Plot of predicted serum concentrations on CIV therapy for 10 days.
IV vancomycin protocols such as this one, individualized for each patient using the MM-USCPACK software are easy to develop. Monitoring can be done with a sample at 6 hours, then q AM as needed. Dosage adjustment is then simple proportional adjustment to the deviation of the serum concentration from the desired target goal.