Ehrlich II –2nd World Conference on Magic Bullets

Amperometric Biosensor Development And Their Clinical Application NAGY L, NAGY G University of Pecs, Pecs, Hungary Background: Polyamines have important roles in biochemical processes. Their analysis in clinical diagnosis has substantial importance. In our earlier work an amperometric putrescine biosensor was successfully developed for clinical detection of bacterial infection. Elevated polyamine concentrations in human urine and serumappear to be associated with the presence of many types of cancer. The detection of diamines in clinical samples can be important for diagnosis of malignancy and for monitoring the efficiency of treatment. We made efforts to improve measuring range of the sensor. Methods: Chemically modified electrodes and advanced electrochemical work stations were employed. Electrochemical method was used for preparation ultra thin size exclusion membrane to provided selectivity. Novel enzyme immobilization procedure was developed for getting highly active reaction layer. Guinea pig blood and plasma samples served for spiking experiments. In vivo studies were also performed in abdominal tissue of anesthetized Wistar rats. Results: A.) Improved biocompatible amperomertic enzyme sensor was developed. It was made of an of a flat form amperometric microcell fabricated with thin film technology on flexible Kapton® substrate, and of an improved biocatalytic reaction layer. Highly active putrescine oxidese (PUO) in reaction layer catalyzes the reaction: The platinum-working electrode detects the hydrogen peroxide produced. Preparation of the biocatalytic enzyme- and outer protective layers was optimized for improved sensitivity and response time. A detection limit of 50 nM was achieved in pH-adjusted whole blood samples, which is below pathological levels. B.) A novel detection principle, named periodically interrupted amperometry (PIA) that generally can be applied for improving performance of amperometric biosensors has been developed. It involves application of new amperometric working program. Using it the sensitivity of amperometric biosensors like putrescine electrode could be improved by several orders of magnitudes. Conclusions: Two different methods were successfully used for improving the performance of amperometric biosensors. The improvements were experimentally tested measuring low level putrescine that has importance in clinical diagnosis. The measuring procedure is simple, needs small sample size or in vivo application is available with minimal invasion.

Properties Of The Key Reaction Steps In Demechlocycline Biosynthesis Of The Engineered Streptomyces Aureofaciens Strains NAKANO T^1, DAIRI T^2, MIYAKE K^1, KATSUMATA R^3 ^1Kyowa Hakko Bio Co.,Ltd., Tokyo, Japan; ^2Tohoku Univ., Sendai, Japan; ^3Toyama Pref. Univ., Imizu, Japan Background: 6-Demethyl-7-chlortetracycline (demeclocycline; 6-DCT) and 6-demethyltetracycline (6-DMT) are produced by the mutants lacking the 6-methylation step of chlortetracycline (CTC) biosynthesis in Streptomyces species. To understand the pathway metabolism, we focused on two by-products, and investigated the causes of the syntheses. One of the by-products is a melanin-like pigment (MP) shown in both culture broths. Another is tetramid blue (TB) shown in the 6-DMT culture broth. Methods: Mutants defective in the 6-DCT biosynthetic pathway were derived from Streptomyces aureofaciens strains, and classified by metabolites. To identify the causes of the by-product syntheses, genetic complementation by integrative transformation, gene analysis, and bioconversion analysis were performed. Results: MP synthesis is caused by a lack of the 6-methylation step, redirecting the carbon flux from a certain intermediate in the altered CTC biosynthetic pathway to a shunt pathway leading to MP. The enzyme for the penultimate reaction step of CTC biosynthesis, designated ATC oxygenase, also takes part in the synthesis of MP. TB is a shunt product of 6-DMT biosynthesis, branched off from the labile last intermediate in the pathway. The corresponding 7-chlorinated shunt product was not shown in the 6-DCT culture broth, although the tchA mutants defective in the last step of the 6-DCT pathway produced it abundantly. The analysis of the tchA gene revealed that the gene product is a homolog of FbiB, which is known to modify a cofactor 7,8-dedimethyl-8-hydroxy-5-deazariboflavin (FO) to yield F420-5,6 in Mycobacterium, and the tchA homologs are also conserved in some Streptomyces strains. Conclusions: The lower substrate specificity of ATC oxygenase contributes to the diversity of the CTC-related compounds. The reactivity of the final step for adding antibiotic activity to the intermediates is crucial for the differentiations of the yield of demethyltetracyclines in Streptomyces aureofaciens. The tchA gene involved is not a special gene in the CTC biosynthetic gene cluster but a conserved gene in the Streptomyces genome, suggesting that the native products of CTC biosynthesis might not be antibiotics.

Reduced Vancomycin Clearance Despite Unchanged Creatinine Clearance In Patients Treated With Vancomycin For Longer Than 4 Weeks NAKAYAMA H^1, ECHIZEN H^2, SATO M^1, TANAKA M^1, ORII T^1 ^1Kanto Medical Center NTT East Corporation, Tokyo, Japan, ^2Meiji Pharmaceutical University, Kiyose, Japan Background: For patients with certain types of infection such as osteomyelitis and endocarditis, vancomycin may be administrated during long-term beyond 4 weeks. Creatinine clearance-based nomograms are used routinely during the early phase of vancomycin therapy for individualizing doses. We studied whether such nomograms are also valid for patients receiving the drug for an extended period of longer than 4 weeks. Methods: A retrospective analysis was conducted on the therapeutic drug monitoring data obtained from 85 patients who received an intermittent intravenous infusion of vancomycin. The patients were allocated to one of five groups according to the length of drug exposure: Group 1 (4 to 7 days; n = 31), Group 2 (8 to 14 days; n = 22), Group 3 (15 to 21 days; n = 13), Group 4 (22 to 28 days; n = 8) and Group 5 (longer than 29 days; n = 11). Systemic clearance of vancomycin (CLVCM) and estimated creatinine clearance (CLcr) calculated by Cockcroft & Gault’s formula obtained from Groups 2 through 5 were compared to those from Group 1 by Dunnet test. In addition, correlation between CLVCM and the duration of vancomycin therapy was examined by Pearson’s correlation analysis. Results: Patients who had received vancomycin for longer than 4 weeks (Group 5) showed a significant (p < 0.05) reduction in CLVCM by 50% compared to Group 1, whereas CLcr remained unchanged. In addition, a significant negative correlation was found between CLVCM/CLcr ratio and dutation of vancomycin exposure (r=-0.337, p<0.01). In the analysis of patients administrated nephrotoxic agents concomitantly with vancomycin, there were no significant differences in CLcr, CLVCM and CLVCM/CLcr ratio between patients treated for less than 14 days and longer than 14days.. Conclusions:This study demonstrated that prolonged administration of vancomycin for over 4 weeks may result in a more pronounced reduction in CLVCM than CLcr. Our data suggest that CLcr-based nomograms for individualizing vancomycin doses should be used with caution in patients who require substantially prolonged drug exposure, such as those with infective endocarditis. Authors’ disclosure statement Reference Nakayama H, Echizen H et al, Ther Drug Monit 2008; 30:103-107

Central Sympatholytic and Anti-arrhythmic Effects of Serotonin-1A Agonists NALIVAIKO E^1, MASTORCI F^2, SGOIFO A^2 ^1Univ. of Newcastle, Newcastle, NSW, Australia; ^2Univ. of Parma, Parma, Italy Background: Psychological stressors may provoke cardiac effects ranging from mild tachycardia to ventricular arrhythmia, fibrillation and ultimately to sudden cardiac death. At present, the only class of pharmacological agents used for preventing these consequences is -blockers acting directly on the heart. Since these drugs have a number of side effects and counter-indications, the ability to suppress potentially deleterious increase in cardiac sympathetic activity at its origin, in the brain, would be a valuable alternative. In the current study, we tested whether ventricular arrhythmias precipitated by acute stresses could be suppressed by systemic administration of 8-OH-DPAT, a 5-HT1A agonist possessing central sympatholytic properties. Methods: The study was conducted on 33 adult male rats instrumented for telemetric recordings of ECG, body temperature and locomotor activity. In the first experiment, rats were subjected to social defeat after either 8-OH-DPAT (100 µg/kg s.c.) or vehicle. In the second experiment, prior to vehicle/8-OH-DPAT administration, animals were pre-treated with zatebradine (2 mg/kg sc.), a blocker of the pacemaker current. Results: 8-OH-DPAT caused prolongation of basal RR interval (169±6 to 200±5 ms, p<0.01), increase in locomotion (10±2 to 19±2 counts/min, p<0.01) and hypothermia (37.9 to 36.6ºC, p<0.05). Subjecting vehicle-treated animals to social defeat caused shortening in RR interval, increase in locomotor activity and hyperthermia, and provoked the occurrence of ectopic ventricular and supraventricular beats; all these effects were substantially attenuated by 8-OH-DPAT. Zatebradine caused prolongation of RR interval. In zatebradine/vehicle-treated rats, incidence of ventricular and supraventricular ectopic beats during defeat increased 2.5-fold and 3.5-fold, respectively. 8-OH-DPAT administered after zatebradine completely abolished these stress-induced arrhythmias. Conclusions: 1) Pharmacologically induced prolongation of RR interval results in an increased susceptibility to stress-induced cardiac arrhythmias, possibly due to the prolongation of the ventricular diastolic period with restored excitability; 2) Systemic administration of 8-OH-DPAT entirely abolishes these arrhythmic events, likely by suppressing stress-induced cardiac sympathetic outflow.

Immune Refocusing Technology - Improving On Mother Nature’s Immunogenicity NARA P^1,^2, CHAUDURI R^1, LIN G^1,3, TOBIN G^1 ^1Biological Mimetics, Inc., Frederick, MD., USA; ^2Iowa State University, Ames, IA.,USA, ^3 Brigham’s and Women’s Hosp., Boston Mass., USA Background: The “potential“immunogenicity of a protein in nature is critically important to the design of future vaccines and monoclonal antibodies and yet remains a poorly understood phenomenon. Even less understood are technologies for modulating the antigenic properties while keeping the original protein conformation intact. We have found that many pathogens adopted a naturally-occuring, dis-porportionate immunogenicity through evolved immunodominance and genetic instability to mis-direct the host defense systems, a phenomena termed “Deceptive Imprinting“ We have devised a way to modulate the natural antigencity of a molecule and hypothesized that dampening immunodominant subtype-restricted viral epitopes while maintaining complex conformation would allow the immune system to target other potentially more broadly protective epitopes. Methods: To test this hypothesis the immunodominant heirarchy of the major antigenic B-cell eptiopes (in vivo and in silico) were mapped and a panel (n=10) of epitope dampened HA1 hemagglutinin (HA) subunit antigens made. Ten groups of mice (n=6) were immunized with a DNA prime and recombinant HA protein boost of wildtype (HA-wt) or HA-dampened (HA-d) antigens and the sera tested for ELISA, hemagglutination inhibition (HAI) and micro-neutralization activities against homologous and a panel of heterologous H3N2 viruses. Results: Sera from the HA-wt and HA-d animal groups contained quantitatively similar levels of anti-HA antibodies as measured by HA ELISA (~1:300,000), however against different H3 viral subtypes showed significant qualitative HAI differences. Sera from HA-d animals were capble of protective HAI titers (>/=1:40) for viruses in the panel from 4 years prior (Pan/99) to three years ahead of that used in the vaccination. As much as 8.5-fold higher HAI titers were observed for some hetero-typic subtypes (Well.-1:10,240 vs. 1:1280) and Pan.-1:1920 vs. 1:226. Other HA-d muations when combined, demostrated more than additive HAI activity. Conclusions:The study demonstrates that Immune Refocusing Technology is cable of altering the natural immunogenicity and producing a subunit antigen capable of inducing in the host new anti-viral immune responses (e.g. increased breadth and titers of protective antibody).

Optimising High Dose Melphalan In Patients With Multiple Myeloma: Preliminary Results From A Multi-Centre Trial NATH CE^1, SHAW PJ^1,7, TROTMAN J^2,7, ZENG L^1,7 , TILEY C^3, JOSHUA D^4,7, KERRIDGE I^5,7, KWAN Y^2, PRESGRAVE P^6, GURNEY H^5,7, MCLACHLAN AJ^2,7, EARL JW^1 ^1The Children’s Hospital at Westmead, ^2Concord Hospital, ^3Gosford Hospital , ^4Royal Prince Alfred Hospital, ^5Westmead Hospital, ^6Wollongong Hospital, ^7University of Sydney, NSW, Australia Background: The aim of this study is to examine the pharmacokinetics (PK) and pharmacodynamics (PD) of total and unbound melphalan in patients with myeloma. Such studies are essential for determining the optimal dose, to avoid the profound toxicity, including cytopenia and gastrointestinal toxicity, and to ensure a good disease response. Methods: NONMEM VI was used to develop population PK models for total and unbound melphalan from concentration-time data collected from 77 patients (36-73 years) who received a median 191 mg/m^2 dose (range: 140 – 450 mg) on a single occasion. Posterior Bayesian estimates of total and unbound area-under-the-concentration-versus time curve (AUC) were obtained and tested for significant associations with (1) Overall Disease Response, based on % change in paraprotein from diagnosis to post transplant (2) Transplant-Related Disease Response, based on % change in paraprotein from pre to post transplant, (3) the severity of mucositis, vomiting, nausea or diarrhoea (graded using CTC criteria version 3.0) and (4) the duration of hospital admission. Results: Unbound AUC ranged from 0.95 to 6.55 mg/L.h and was significantly higher for patients who had Complete or Very Good Partial Overall Disease Response (p < 0.05, n = 25), severe (≥ Grade 3) mucositis (p < 0.001, n = 9) or nausea (p < 0.05, n =11) and those whose duration of hospital admission was > 22 days (the 75th percentile, p < 0.005, n = 18) using the Mann-Whitney test. Total AUC ranged from 6.4 to 24.6 mg/L.h and was significantly higher in patients who had severe mucositis (p < 0.05) and those whose duration of hospital admission was > 22 days (p <0.01). Total and unbound AUC were not significantly associated with Transplant-Related Disease Response, but some PD data is missing. Conclusions: These preliminary results suggest that melphalan pharmacokinetics (in particular, unbound AUC) is an important determinant of melphalan toxicity and efficacy. We are continuing to recruit patients and collect data (including missing data) and, eventually, we hope to devise an optimal dosing strategy for this patient group

Aminoglycosides: Deadly Bullets In The Hands Of Unexperienced NEEF C^1, CROES S^1,3, KOOP AH^2, van GILS SA^2 ^1Maastricht University Medical Center, Maastricht, The Netherlands; ^2University of Twente, Enschede, The Netherlands.^3presenting author Background: Therapeutic Drug Monitoring (TDM) of aminoglycosides has been a topic during the last thirty years. There is a tendency that – because of the once daily regimen – monitoring is considered not necessary anymore. Using the model we developed for efficacy and toxicity of aminoglycosides we are able to demonstrate that optimal dosing still needs a sophisticated system of TDM. Methods: A numerical program in Matlab is developed to run simulations for suitably chosen parameters and that can be used in TDM. The extended mathematical model that is derived incorporates the effects of aminoglycosides on bacteria, the saturable and active uptake into kidney cells, the reversible nephrotoxicity and the irreversible ototoxicity. For a continuous administration, analytical solutions are calculated for the optimal concentration in the blood for efficacy and the concentration without nephrotoxicity. Results: From a previous model we developed it appears that the efficacy of antibiotics is not only concentration, but also dependent on the exposure time, as long as the concentration is above the MIC. This means that the AUC above the MIC is the pharmacokinetic parameter that correlates best with efficacy. In this model an optimal concentration is defined for tobramycine as a constant factor to the MIC of a specific bacteria. At this concentration (3.2 x MIC) the efficacy is optimal. The model predicts that this effect occurs when the drugs are given using a continuous infusion. This is not possible for aminoglycosides because of their toxicity. Thus aminoglycosides have to be administered intermittently. In the figure it is demonstrated that a dosing regimen leading to adequate plasma levels leading to bacterial death causes severe nephro- and ototoxicity. For a micro-organism with a MIC of 1 mg/L a dose of 480 mg is the optimal dose in terms of efficacy. In a once daily regimen nephrotoxicity can be noticed after 15 days, whereas damage to the ear cells starts after 30 days. Conclusion: The model can be used to design appropriate antibiotic drug regimes to combine maximal efficacy and minimal (acceptable) toxicity.

Synthesis And Biological Evaluation Of Novel Pyrazole And Pyrazolo[3,4-D]Pyrimidines As Anti-Inflammatory Agents ABD-EL-AZIZ AS^1, YOUSSEF AM^2, NEELAND EG^1, KLEGERIS A^1 ^1University of British Columbia Okanagan, Kelowna, British Columbia, Canada, ^2Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt Background: Non-steroidal anti-inflammatory drugs (NSAIDs) are important therapeutic agents for the treatment of pain and inflammation. Their mechanism of action involves inhibition of prostaglandin synthesis by the enzyme cyclooxygenase (COX). It has been reported that pyrazolopyrimidines inhibit COX enzymes, and therefore, may possess anti-inflammatory properties. Recently, we have synthesized novel pyrazole and pyrazolopyrimidine derivatives to investigate their anti-inflammatory effects. Methods: A diketo-dipyrazole compound and an acid chloride of pyrazolo[3,4-d]pyrimidine 4(5H)-one were prepared via the reaction of 5-amino-4-cyano-1-phenylpyrazole with oxalyl chloride in tetrahydrofuran at room temperature. The product compounds were then reacted with a number of amine nucleophiles. The resultant novel compounds were tested in several in vitro assays to assess their anti-inflammatory activity. Their effects on the secretion of pro-inflammatory cytokines and neurotoxins by human THP-1 monocytic cells were studied. In addition, effects of the various compounds on the viability of human cell lines were monitored. Results: The derivatives of pyrazolo[3,4-d]pyrimidine 4(5H)-ones displayed significant differences in their effects on the various parameters studied. Some derivatives induced significant toxic effects at concentrations above 50 M, while other derivatives appeared to be well tolerated by the cultured cells. We identified novel compounds that were able to reduce secretion of pro-inflammatory cytokines by human monocytic cells without causing any toxic effects. These compounds also inhibited monocytic cell toxicity towards human neuronal cells. The anti-inflammatory activity was observed in the low micromolar range. Conclusions: Some of the newly synthesized derivatives displayed anti-inflammatory properties when tested in vitro by using human THP-1 monocytic cells. In several cases significant toxic effects were also observed. The newly synthesized compounds may hold potential as either anti-inflammatory or anti-neoplastic agents.