Ehrlich II –2nd World Conference on Magic Bullets

Background: E_n indicates a Sum of Exponential Terms (SETs) model of n compartments. Using E₁, we prior isolated a normal G predictive formula with exponents at a P = 0.88 proximity to V ^2/3W ^1/4, where V (ml) is volume of distribution and W is mass (kg). This suggested fractal and not compartmental body composition and implied a need for testing SET fits to data.

Methods: Four statistical tests were applied to SET models for studies with 8 or 9 blood samples taken from 10 to 240 min. after injection of ¹⁶⁹Yb-DTPA (pentatate, n=41) and 5 to 180 min. after ^99mTc-DTPA (n=5). Test 1 used bootstrap with 1000 resamplings to determine whether the 4 multiplicative and rate constant parameters of an E₂ SET model were all statistically warranted. Test 2 examined residual structure of E₁ and E₂ SET models fit to subselections of samples and classified non-E₂ fit results. Test 3 examined E₂ goodness of fit with Chi-squared and t testing. Test 4 examined extrapolation. For 46 cases, E₂ SET models were fit to m1 and m blood samples, designated by E₂(m1, t) and E₂(m, t) respectively, and evaluated and compared at the time of the m^th sample.

Results: Test 1 found that E₂ parameter tolerances included 0 for at least 1/4 parameters, 45/46 times using standard error of the mean derived tolerances and 19/46 times using 95% tolerances from 5% range trimming, i.e., the E₂ model was too complex given its performance 41 or 98% of the time, ruling out even more complex E_n>2, for improving fit performance. In Test 2, the estimated variance of E₁ and E₂ fits varied with the sample times chosen and was worse than that expected for a 3% standard deviation sampling error rate. Upon fitting, 8.1% of the E₂requests were non-E₂ models. Test 3, goodness of fit testing, found 1) that E₂ fits failed t testing for mean residual magnitude for 6 of 8 residuals of the 41 cases with 8 samples. And, 2) for 46 cases with all samples, the Chi-squared goodness-of-fit was P<0.039, suggesting very poor fits. Test 4, extrapolation, showed that E₂(m1, t=t_m) significantly underestimated E₂(m, t=t_m) (31/46 times, Wilcoxon signed-rank sum P=0.0035) thus physical area under the concentration curve over time is underestimated and G, overestimated.

Conclusions: SETs failed testing of model fit to data. Replacement of compartmental models with a fractal consistant model is suggested for fitting DTPA concentration in time.

Magic Bullets: Once Fired – How are They Recaptured? On Metabolic Transformation and Environmental Fate of the Veterinary Fluoroquinolone Enrofloxacin WETZSTEIN HG Bayer Animal Health GmbH, Leverkusen, Germany Background: Fluoroquinolones (FQs) are synthetic antibiotics widely used to treat bacterial infections in humans and animals. Hence, drug residues enter the environment via urine and feces, i.e., in wastewater streams and animal waste, respectively, the latter often being spread as fertilizer on agricultural fields and pastures. Due to very tight binding of FQs to feces and soils as well as a fluoro-aromatic structure element not yet found in natural products, concerns have been expressed on FQ biodegradability, long-term persistence, and potential selection of FQ-resistant zoonotic species in the environment. Metabolic transformation of enrofloxacin (ENR), a veterinary FQ, is limited to glucuronidation and partial oxidation of its amine substituent. In the only soil degradation study yet published (1997), ≤0.6% of the ^14C label applied with [2 ^14C]sarafloxacin could be recovered as ^14CO2 after 80 days, prompting our work on the fate of ENR in agricultural matrices. Methods and Results: In samples of pre-rotted wheat straw, cattle dung pats, a manure hill, and an agricultural soil only about 0.5% of ^14CO2 was produced from [4 ^14C]ENR over one year, while ^14CO2 production rates from [piperazine-2,3-^14C]ENR were on the order of 7 to 30%. Similar rates obtained with cipro- and moxifloxacin indicated a limited utility of recording ^14CO2 formation to prove FQ biodegradation. Quantitative biotransformation of ENR in two agricultural soils, a plant-derived compost, and cattle dung (t1/2 = 83 to 113 d) could only be demonstrated, if monitoring of the fate of [4-^14C]ENR was based on chemical analysis of the matrices. Degradation pathways, outlined by 135 metabolites, indicated multiple hydroxylations of the aromatic core and the piperazine moiety of ENR, causing oxidative decarboxylation and defluorination as well as the formation of reactive ortho-aminophenol- and catechol-type intermediates, which - upon further oxidation - provided labile cic,cis-muconic acid-type metabolites (Appl Microbiol Biotechnol 71:90-113; 2006). Seven key metabolites had no or very little residual antibacterial activity. ENR was metabolized by diverse basidiomycetous fungi, indigenous to soils and animal waste. Conclusions: Soil microbes are likely to provide for the inactivation/recapturing of magic bullets such as ENR, with no risk confirmed.	Transfer factors - Magic Bullets for preventing and treating viruses and mycobacteria in the 21st century WHITE A Department of Psychiatry, Division of Medical Psychology, Duke University Medical Center, Durham, NC 27713 USA When the body is exposed to a pathogen, the immune system directs its response down one of two complimentary pathways, denoted as Th2 and Th1. The Th2 response leads to antibody-mediated immunity. That is, immunity that involves the generation of antibodies, which attach to a pathogen floating in the body and disable it. The Th1 response leads to cell-mediated immunity. That is, immunity that involves the identification and destruction of cells in the body that are infected with a virus or mycobacterium. Memories of Th2 immune battles are stored in antibodies and the B-cells that make them. In 1949, immunologist H.S. Lawrence discovered that memories of cell-mediated immune battles are stored in peptides, which he labeled "transfer factors." Like antibodies, transfer factors are passed from mothers to their offspring in colostrum. In the last few decades, researchers have developed protocols for extracting transfer factors from cow colostrum, and also from chicken eggs. By first exposing the cow or chicken to a particular virus or mycobacterium, transfer factors specific to any known pathogen can be generated and extracted. They can be taken orally and produce minimal side effects. Dr. Lawrence labeled them transfer factors because they literally transfer immunity from one host to the next. When taken prior to exposure to a pathogen, transfer factors allow the body to protect itself from infection. When taken after exposure to a pathogen, transfer factors help the body defeat the infection. More than 1000 manuscripts regarding transfer factors have been published in the past 60 years. They represent experimentally verified Magic Bullets in the prevention and treatment of viruses and mycobacteria.
Methotrexate – the past and the present WIELA-HOJENSKA A Medical University, Wroclaw, Poland Background: Methotrexate (MTX) has a long and an unusual history of use in pharmacotherapy. Moreover, it is characterised by several unique features that are responsible for its popularity as a therapeutic agent: it is active against various cancer and immunologic diseases, including rheumatoid arthritis and psoriasis; it has the broadest dose-range; it can be given by many different routes; it is a cytostatic with a special position in anticancer chemotherapy, as the drug to be recommended for routine determination of its concentration in blood serum and cerebrospinal fluid; it is the drug which has an antidote (leucovorin). Efficient and safe therapy requires broad knowledge of its mechanism of action, pharmacokinetics, toxicity, as well as interaction between simultaneously administered drugs. The therapy with this drug is very effective but its adverse effects are the most common reasons for discontinuing the treatment. Many authors have been trying to find risk factors of its toxicity. Methods: The studies were carried out in 226 persons: 112 children and teenagers with acute lymphoblastic leukemia (ALL), non Hodgkin`s lymphoma (NHL) and osteogenic sarcoma; 49 adults with rheumatoid arthritis; 65 healthy adults. MTX concentrations in blood serum were determined by the fluorescence polarization immunoassay applying TDx Abbott analyser. The following pharmacokinetic parameters for methotrexate were calculated: elimination rate constant, biological half-life time, apparent clearance, area under the the serum concentration-time curve. Determination of MTX toxicity was based on analysis of biochemical laboratory tests that characterised the function of the liver, the kidney, hematopoietic system and clinical symptoms. Activity of N-acetyl-beta-D-glucosaminidase (NAG), as an early factor of renal tubules efficiency, was determined in urine by the enzymatic method. The influence of diuresis amount on enzyme activity was eliminated by scalling NAG activity in relation to creatinine concentration in urine. Results: The presented author`s research showed the relationships between MTX concentrations, as well as its pharmacokinetic parameters and biochemical laboratory tests in the children and teenagers with ALL, NHL and osteogenic sarcoma. On the basis of those correlations mathematic equations were formulated for early predicting MTX concentrations, its pharmacokinetic parameters and the values of the biochemical tests connected with the liver and the kidney function. No such correlations was observed in patients with rheumatoid arthritis. In order to predict the drug concentration, the equations allowing the calculation of MTX concentration at 24, 48 and 168 h after administration were formulated using values measured at 1.5 and 24 h after the administration of the drug. In these patients, the mean activity of NAG was significantly increased both before and after treatment with MTX when compared to corresponding values in the control group of healthy subjects. Conclusions: 1. Determination of MTX concentrations is very important for the optimalization of pharmacological treatment in cancer patients. It could lead to making therapy easier in the patients exposed to toxic effects connected with delayed MTX elimination from the organism. 2. MTX monitoring therapy in patients with rheumatoid arthritis does not significantly improve the effectiveness of the treatment, but it can play an important role in increasing the safety of this drug. 3. The necessity of detailed estimation of kidney excretory function before the beginning and after the end of MTX therapy in the rheumatoid arthritis patients exists. That procedure may be helpful for the recognition of the patients at high nephrotoxicity risk, who need special care.	The Anti-Tumor Action of the Hybrid Drug Nitric Oxide-Donating Aspirin Relies on the ‘Passive’ Linker and not on Nitric Oxide Nor on Aspirin: An Overview of an Interesting Twist HULSMAN N^1,2, MEDEMA J P^1, BOS C^1, JONGEJAN A^2, LEURS R^2, SMIT M^2,DE ESCH I^2, RICHEL D^1, WIJTMANS M^2 ^1 University of Amsterdam, Amsterdam, The Netherlands. ^2 Vrije Universiteit Amsterdam, Amsterdam, The Netherlands Background: Hybrid drugs, by design, rely on the combined action of two pharmacologically active units in one molecule. The hybrid drug nitric oxide-donating aspirin (NO-ASA) consists of aspirin (ASA) and a nitrate group connected through a chemical linker. Its design was based on the assumption that NO, released from the nitrate group, would counterbalance the gastrointestinal side-effect of ASA. Benchmark derivatives are meta-NO-ASA and para-NO-ASA, both having a benzyl-type linker. Both meta-NO-ASA and para-NO-ASA proved to be active anti-tumor agents in preclinical models, generating excitement in the hybrid drug community. Our aim was to study the actual contribution of the nitrate and ASA hybrid components in the most active derivative, para-NO-ASA. Methods: Structural analysis of NO-ASA led us to synthesize model compounds in which the nitrate- and/or ASA-moiety in para-NO-ASA were replaced by a ‘dummy’ chloro- and/or acetyl-group, respectively, but with retention of the benzyl-type linker. Organic and computational chemistry as well as spectroscopic analyses were used to study the chemical characteristics of NO-ASA and model compounds. Furthermore, we studied the mode of action of these compounds in colon cancer cells, including the effect on glutathione levels, caspase-3 activation, cyclin D1 expression, DNA fragmentation and cell death. Results: All the synthesized model compounds, i.e. lacking ASA and/or the NO-donating group, retained or improved upon the in vitro anti-tumor activity of NO-ASA in colon cancer cells. They do so by exhibiting similar (bio)chemical effects as para-NO-ASA does. We show that this is a result of a unified mechanism involving formation of a cytotoxic quinone methide from the ‘passive’ benzyl linker through ester hydrolysis and subsequent elimination of an intact nitrate ion. This quinone methide depletes intracellular glutathione by a selective chemical reaction. Ultimately, this leads to apoptosis. Conclusions: Both NO and ASA, paradoxically, are not involved in the in vitro anti-tumor action of para-NO-ASA. Rather, a cytotoxic quinone methide formed from the ‘passive’ linker is the responsible agent.
1Targeted Cytotoxins in Neuroscience: Magic Bullets for Pain Research and Treatment WILEY RG^1, LAPPI DA^2 ^1VA TVHS and Vanderbilt University, Nashville, TN, USA and ^2Advanced Targeting Systems, San Diego, CA, USA Background: Targeted delivery of cytotoxic proteins has been extensively explored as a “Magic Bullet” treatment for cancer with limited success. However, the same approach has proven highly successful in neuroscience, giving birth to “molecular neurosurgery”. Methods: Neuropeptides, such as substance P (SP), dermorphin (mu opiate peptide), neuropeptide Y (NPY) and galanin, can be used to target the ribosome inactivating protein, saporin, to neurons expressing the respective cognate receptors by conjugation through a disulfide bond. The peptide vectors bind to the corresponding receptors and are internalized taking saporin into the target cells. Rats are injected via temporary lumbar intrathecal catheters. After two weeks, the rats undergo nociceptive testing followed by immunohistochemical staining of spinal cord sections for the appropriate peptide receptors to assess loss of target neurons. Results: Lumbar intrathecal injections of SP-sap, dermorphin-sap, NPY-sap and galanin-sap selectively destroy superficial spinal dorsal horn neurons that express the corresponding receptors but have no effect on dorsal root ganglion neurons. SP-sap in rats transiently reduces hotplate reflex responses, but produces long lasting reduction in operant escape responses to aversive heat and cold and nocifensive responses to hindpaw formalin injection while preventing/reversing hyperalgesia and allodynia due to nerve injury or inflammation. Dermorphin-sap destroys lamina II mu opiate receptor-expressing neurons producing an increase in nocifensive responses to hindpaw formalin and reducing analgesic potency of intrathecal and systemic morphine while NPY-sap and galanin-sap both reduce sensitivity on the hotplate and to formalin in rats. In rats and dogs, SP-sap, at doses that produce significant anti-nociceptive effects, produces little or no evidence of non-specific neural or systemic toxicity. Conclusions: Molecular neurosurgery with targeted toxins is a powerful research tool in neuroscience and the “Magic Bullets” SP-sap, NPY-sap and galanin-sap have great promise as a new approach to treatment of chronic pain based on targeted ablation of key populations of spinal dorsal horn nociceptive neurons via receptor-mediated endocytosis of the neuropeptide-saporin conjugates.	Red blood cell omega-3 fatty acids and the risk of ventricular arrhythmias in patients with heart failure WILHELM M Background: Epidemiological studies support the protective effect of omega-3 fatty acids on sudden cardiac death. However, patients with structural heart disease and an implantable cardioverter defibrillator (ICD) showed no effect or even a proarrhythmic response to fish oil supplementation. Animal studies suggest different electrophysiologic effects of circulating and incorporated omega-3 fatty acids. Methods: In 102 ICD patients in New York Health Association functional class II or III, the fatty acid composition of red blood cells was analyzed by gas chromatography. The omega-3 index was calculated from eicosapentaenoic acid and docosahexaenoic acid. Patients were followed for 1 year, and ventricular arrhythmias requiring antitachycardic therapy were analyzed. Twenty-five healthy subjects served as control. Results: In ICD patients, the fatty acid profile was significantly altered and the baseline omega-3 index was significantly elevated, as compared to control subjects (5.12% ± 0.87% vs 4.24% ± 0.96%, P b .001). Kaplan-Meier estimates of probability of ventricular arrhythmias showed significant differences among quartiles of the omega-3 index. Twelve percent of patients in the lowest quartile had ventricular arrhythmias, as compared to 54% of patients in the highest quartile (P = .022). In a multivariate analysis, the omega-3 index was the only independent predictor for ventricular arrhythmias up to 9 months. At 12 months, a reduced ejection fraction was an additional risk predictor. Conclusions: In heart failure patients, the red blood cell fatty acid profile is altered. Omega-3 fatty acids are elevated and predict the risk of ventricular arrhythmias.
Decoding infections - How to use „administrative“ data for clinical quality improvement WILKE MH In many countries vast data collections are existing, which all – more or less – are containing coded patient informations. These data collections are mainly used for administrative purposes especially in the DRG (CaseMix) settings they are used for funding, reimbursement, planning, etc. On the other hand the data contain – at least if the respective country is ‘mature’ in CaseMix – multitudes of clinical and medical information. In some countries even medication information (using ATC-codes) such as France or the U.S. are collected and stored. Surprisingly enough there are comparatively few publications that are using these data collections to reflect on clinical research questions on a broader basis than e.g. in the own hospital settings. About the reasons can only be speculated, a common allegation – at least among clinical researchers – is that the data quality is not eligible for clinical research as it was collected for ‘administrative’ purposes. On the other hand vast data collections are waiting to be exploited and years of workpower for extra double or triple data acquisition for various purposes could be saved. We started a scientific project “decoding infections” where we developed algorithms and a database that allows us, retrieving information about hospital acquired infections out of the German standard DRG – dataset (§21-dataset) which is a mandatory dataset, that all German hospitals have to provide each year to a central institution called InEK Insitute. After processing the data of a hospital, we are able to identify – on the basis of the ICD coding – infections that occurred during a hospital stay. We can analyze clinical outcomes, such as mortality, length of stay, ICU stay, hours of mechanical ventilation as well as economical outcomes such as cost and payments. Via the database we can show clinicians an overview over infections in their hospital and use the data for discussion about antibiotic therapy strategies, hygiene improvement projects. Moreover we are able to draw the discussion towards the economical impact of hospital infections. The goal of the project is to provide the data needed to initiate clinical improvement projects on hospital infections and to enable both clinicians and economists in the hospital to measure effects of those projects over time. In the presentation we’ll show the methods and materials as well as some sample results of our work. As the database is still ‘under construction’ we’ll also show our next goals in development such as setting up a benchmarking opportunity for hospitals.	Investigation of Dibenzyl Trisulphide (Dts) Isolated from Petiveria Alliacea as an Immunomodulator with Cytotoxic/Anti- Proliferation Activity WILLIAMS LAD^1, KRAUS W^2, ROSNER H^3 ^1The Natural Products Unit of the Scientific Research Council, P.O. Box 350, Hope Gardens, Kingston 6, Jamaica, West Indies; The University of Hohenheim, Institutes for Bio-organic Chemistry^2 and Zoology^3, Garbenstr 30, D-70593 Stuttgart, Germany Background: Cancer is considered to be one of the leading cause of death. The need to find effective and safe therapeutic agents for treating cancers has been one of the greatest challenges for mankind. Thus, 70% of all human cancers such as breast, small cell- lung, malignant melanoma, ovary and colon have remained refractory or untreatable. To date, the most effective drugs known for treating malignant diseases are derived from natural products and their associated derivatives. Method: The following molecular biological techniques; cell culture, immunostaining, staining of F-actin, SDS-PAGE and Western blotting, tyrosine phosphorylation, proliferation/viability assay and one-dimensional (1D) NMR interaction studies were conducted according to Rosner et al., (2001). Animal studies using mice, were employed for evaluating the cell biological (cytotoxic/anti-proliferation) and toxic effects of dibenzyl trisulphide (DTS) isolated Petiveria alliacea. Results: Dibenzyl trisulphide increased the weight of the thymus and Peyers patches via cell proliferation. The trisulphide has a dose dependent anti-proliferation effect on the following human cancer cell lines; SH-SY5Y neuroblastoma, MCF-7 mammary carcinoma, IPC-melanoma, A549 small cell lung cancer and 5637 primary bladder carcinoma. The SH-SY5Y neuroblastoma cells were the most susceptible to DTS with an IC50 of 0.43 µM. Binding DTS to serum albumin in buffered saline enhanced its cytotoxic effects by 2500 on the neuroblstoma cells. Dibenzyl trisulphide had little or no toxic effects on the non-cancer cell line, HOFA. The mode of action found for DTS is an attenuation of the dephosphorylation of tyrosyl-residues of mitogen activated protein (MAP) kinases (ERK 1 and ERK 2). Conclusion: The data obtained for DTS suggest that the compound could be an interesting prototype for investigation as an anti-cancer agent. Reference: Rosner H, Williams LAD, Jung A and Kraus W. (2001). Biochimica et Biophysica Acta (BBA), Molecular Cell Res. Vol. 1540, No. 2, pp. 166 – 177.