Ehrlich II –2nd World Conference on Magic Bullets

Serum Alcohol Dehydrogenase: A Sensitive Biomarker of Ongoing Graft Function After Liver Transplantation
SHIMOJIMA N¹, SHIMAZU M¹, KIKUCHI H², KAWACHI S¹, TANABE M¹, HOSHINO K¹, WAKABAYASHI G¹, MORIKAWA Y¹, KITAJIMA M^1
1Department of Surgery and ²Department of Laboratory Medicine, Keio University School of Medicine, Tokyo, Japan
Background: In the postoperative management of liver transplantation, it is essential to monitor the graft status on a real-time basis to ensure the early diagnosis of postoperative complications. Currently, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, lactate, and other markers are used for that purpose. Alcohol dehydrogenase (ADH) is an enzyme specifically located in the cytoplasm of hepatocytes. The purpose of this study was to assess the potential usefulness of serum ADH activity as a biomarker of graft function following liver transplantation.

Methods: Blood samples were obtained from 26 patients who underwent living-donor liver transplantation. Serum ADH activity, ALT, AST, total bilirubin, and lactate were evaluated for 3 weeks after transplantation in each case. In our hospital, the reference range of serum ADH activity is  4 IU/l (37C).

Results: In patients without any postoperative complication, serum ADH activity normalized at 2.9±1.2 days. Values of serum ADH activity were remarkably elevated in patients with vascular complications, whereas they were only slightly elevated or remained within the reference range in patients with acute cellular rejections. In vascular complications, serum ADH activity peaked prior to elevation of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and once the cause of damage was resolved, the values returned to reference range more quickly than did ALT and AST.

Conclusions: ADH has a lower molecular weight and a shorter blood half-life than ALT and AST. This can explain how ADH is released into the blood earlier than ALT and AST and normalizes more rapidly after the cause of liver damage is removed. ADH is abundantly distributed in the centrilobular area in the liver. Therefore, ADH shows extremely high values in those conditions that cause severe damage to the centrilobular area, such as blood flow decrease.

In conclusion, monitoring serum ADH activity in addition to ALT and AST may provide more sensitive ongoing graft status and valuable information for the differential diagnosis of vascular complications and acute cellular rejection.

Authors’ disclosure statement (not counting towards the character count):

This section is NOT required. If authors wish to disclose any relevant information, please include this information in brief here.

A Bevacizumab, An Anti-VEGF Antibody, As A MAGIC BULLET for Retinal Disease? SHIMURA M Department of Ophthalmology, NTT East Japan Tohoku Hospital Background: Recently, many ophthalmologists who specialize in retinal diseases have interests in macular edema (ME) which directly leads visual disturbance. Macular edema was often seen in vasoproliferative and ischemic retinal diseases as diabetic retinopathy (DR) and retinal vein occlusion (RVO), and which pathogenesis are related with VEGF expression, therefore anti-VEGF therapy is expected to reduce ME. In this presentation, the effectiveness of intravitreous injection of bevacizumab, an anti-VEGF antibody against DR- and RVO-associated macular edema was evaluated. Methods: 86 eyes of DR-associated ME and 75 eyes of RVO-associated ME participated in this study. After the initial ocular examination, 1.25 mg of 0.5 ml bevacizumab was injected intravitreously in each eye. ME was evaluated by measuring macular thickness with optical coherence tomography (OCT) for up to 24 weeks. Clinical course of best corrected visual acuity (VA) in each eye was monitored. Results: Before starting this study, averaged macular thickness was 634.6 ± 96.5 m in DR-ME, and 662.1 ± 104.8 m in RVO-ME, which is no statistical difference between two groups. After the injection, averaged macular thickness in both diseases showed prominent decrease to 63.5 ± 14.2 % in DR-ME and 40.7 ± 11.6 % in RVO-ME within 1 month. However 70 eyes of 86 (81.3%) DR-ME and 49 of 75 (65.3%) eyes of RVO-ME showed recurrence of ME by 24 weeks. There is no significant difference of initial VA between both groups. After the injection, VA in both groups showed rapid and temporary improvement and after that it gradually decreased but kept significant improvement at 24 weeks. Temporal regression of VA in RVO-ME eyes was significantly better than that of DR-ME eyes. Conclusions: Intravitreous injection of bevacizumab is possible to reduce macular edema, however, recurrence of the edema was observed in most cases. Although the other therapies including retinal photocoagulation or vitreous surgery must be added, the rapid and prominent reducing effects by intravitreous bevacizumab prevent visual dysfunction which is reflected by continuous macular edema. The effect of bevacizumab against macular edema was more expected in RVO than in DR.	Prospective Magic Bullet Cobalt: Its Effect, Mode of Action and Utility for Hypoxic Study SHRIVASTAVA K *, ANJU B^*, MUSTOORI S^1, LILY G*, GOVINDASWAMY I* *Expt. Biol. Div., DIPAS, DRDO, Lucknow Road, Delhi-110054,India. ^1 Biologicals E Ltd. Hyderabad, India Background: Ascent to high altitude results in the risk of altitude sickness including high-altitude cerebral edema (HACE). HACE is the most severe form and if no intervention is implemented, death may result. Aims: To evaluate effect of cobalt chloride (CoCl2) supplementation on 1) Hypoxic tolerance and facilitation of acclimatization to hypobaric hypoxia (HH), 2) HH induced oxidative stress, 3) HH induced vascular leakage, 4) global gene expression with and without HH in rat brain Methods: Male Sprague Dawley rats supplemented with CoCl2 (12.5mg Co/kg b.w., oral) for 7days were subjected to 32,000 (till gasping) and 25,000 ft (48hrs) for tolerance and acclimatization/oxidative stress studies respectively by monitoring gene expression and various biochemical parameters. Cerebral vascular leakage and edema were monitored by radiometric and histological analysis after subjecting the rats to an altitude of 30,000ft (5 hrs). Global gene expression was studied by microarray. P<0.05 was taken to be significant. Results: CoCl2 improved hypoxic tolerance via increased expression of hypoxia inducible transcription factor (HIF-1) and its regulated genes. Administration of CoCl2 appreciably attenuated the free radical (ROS) generation, oxidation of lipids and proteins and maintained GSH/GSSG ratio after HH similar to that of control rats by induction of HO-1 and MT levels via HIF-1 signaling mechanisms. CoCl2 preconditioning inhibited HH induced vascular leakage by lowering NFκB activity and its regulated pro-inflammatory mediators. This is shown to be mediated by cobalt-induced reduction in ROS/NO and increase in HO-1 and MT. A battery of core genes was identified by microarray; a few genes were confirmed by RT-PCR and immunoblotting. Pathway predictions were also performed using KEGG software highlighting certain pathways affected by these treatments. Conclusion: 1) CoCl2 improves hypoxic tolerance and facilitates acclimatization to HH, 2) It attenuates HH induced oxidative stress, 3) It attenuates vascular leakage induced by HH, 4) A number of endogenous molecular mechanisms studied may explain how preconditioning protects against deleterious effects of hypoxia and may provide novel therapeutic targets for treatment of cerebral edema.
One hundred years of Paul Ehrlich’s Chemotherapia specifica: How to get it for effective and safe cure of cancer patients SHUKLA SK^1, PETRUCCI F^2, S. CAIMI S^2, CUSUMANO R^3, CIPRIANI C^1 ^1S. Eugenio Hospital, Rome, Italy; ^2Istituto Superiore di Sanità, Rome, Italy; ^3Aurelia Hospital, Rome, Italy Background: Both neoadjuvant and adjuvant chemotherapy, being carried out so far with cytotoxic agents, single or combined, are highly toxic to the patient because they distribute in the whole body. The lifelong experience of Paul Ehrlich for finding chemotherapia specifica agents had been based mainly on: i) healthy nutrition; ii) avoiding disease risk factors in life-style; iii) study of the cause of cancer; iv) finding a suitable non toxic pharmaceutical or radiopharmaceutical which can eliminate the cause of cancer. Epidemiological and etiological results for the incidence of different cancers are of great help in finding the cause of cancer. Methods: We determine the total-body distribution of radiopharmaceuticals of known chemical composition, and study trace elements in the disease and also in the neighbouring healthy tissue with sector-field inductively coupled plasma mass spectrometry (SF-ICP-MS). Results: Our study have shown that radiopharmaceuticals for cancer diagnosis and therapy are continuously changing their chemical composition after synthesis. They are therefore of little use for diagnosis and therapy. We are, at present, trying to develop chemotherapia specifica for safe and effective cure of bladder cancer patients. The risk factor for bladder cancer in the order of their decreasing harm are: i) cigarette smoking, ii) red meat consumption, iii) alcohol drinking, iv) coffee drinking. Conclusions: The bladder cancer patients have to avoid these injurious substances as soon as possible when early signs of bladder cancer are observed. We have found how these risk factors work together to cause bladder cancer	Vaccine-derived polioviruses (VDPVs) - a grey cloud around a magic bullet SHULMAN, LM^1,2, MANOR Y^1, ‘SOFER D^1, MENDELSON E^1,3 ^1Central Virology Laboratory, Tel Hashomer, Israel ^2 Univ. Tel Aviv , Tel Aviv, Israel ^2 Univ. Bar Ilan, Ramat Gan, Israel Background Poliomyelitis has nearly been eradicated. The number of endemic countries has been reduced to 4 world-wide since the 1950’s by use of inactivated and oral polio vaccines (IPV and OPV, respectively). Surveillance programs are an essential component of eradication programs that insures that all chains of transmission have in fact ceased. Israel has been poliomyelitis free since 1988. Methods Since 1989, Israel has employed routine monthly sewage surveillance at >15 sites to monitor for presence of non-vaccine poliovirus. Sampling sites cover at least a third of the Israeli population. Results 34 highly diverged [8 to >15%], neurovirulent in a transgenic mouse model, type 2 vaccine derived polioviruses have been periodically isolated from sewage in the greater Tel Aviv area [pop. 1800000] over the past 10 years. Molecular analyses strongly suggest that the isolates represent two major lineages originating from two persistently infected and continuously excreting individuals and or a small group of their contacts. By moving sampling sites up the sewage system, the population that contain each of the excretors has been reduced to 50,000 and 350,000. Conclusions 1. To meet the challenge of eradication, the number of persistently infected individuals globally should be estimated. 2. High vaccination coverage must be maintained as long as persistently infected individuals continue to excrete. 3. OPV should be replaced by a non-live vaccine as soon as feasible to prevent new persistent infections.
When “Magic Bullets” Cause Collateral Damage (SJS/TEN and AGEP) SIDOROFF A^1 for the RegiSCAR Study Group ^1Medical University of Innsbruck, Department of Dermatology and Venereology, Innsbruck, Austria Background: A “magic bullet” drug is supposed to have a desired therapeutic effect ideally without side effects. Yet unwanted reactions to drugs are very common in clinical routine. Licensing procedures – from preclinical data to pase III clinical trials - try to detect the risk of drugs for unwanted effects before they get on the market. Yet some reactions are too rare to be noticed within such trials. Typical examples for this are severe cutaneous adverse reactions like the Stevens-Johnson syndrome (SJS) / toxic epidermal necrolysis (TEN) spectrum (incidence 2-3/Mio inhabitants/year) and acute generalized exanthematous pustulosis (AGEP), Due to their severity (mortlity rate for TEN is still more than 30%) and sequelae they cause a severe burden to patients and the healthcare system. Methods: For more than a decade an international study group collected and investigated a large number of patients with severe cuatneous adverse drug reactions in a series of pharmacoepidemiological studies. In the EuroSCAR study relative risks for different drugs to cause SJS/TEN and AGEP could be calculated in a case control setting. Results: Strong associations with the occurrence of SJS/TEN could be confirmed for anti-infective sulfonamides, allopurinol, carbamazapine, phenobarbital, phenytoin, and oxicam-NSAIDs (allopurinol being the most common cause of these reactions within our network). Among drugs recently introduced into the market, associations were significant for nevirapine and lamotrigine, and weaker associations for sertraline, pantoprazole, and tramadol. For AGEP strongly associated drugs, i.e. drugs with a lower bound of the 95% confidence interval (CI) of the odds ratio (OR) > 5 were pristinamycin, ampicillin ⁄amoxicillin, quinolones, (hydroxy)chloroquine, anti-infective sulphonamides, terbinafine, and diltiazem. Conclusions: Different drugs have a different risk of causing SCARs. The spectrum of drugs is also different for diferent type of unwanted reactions. Due to the rarity of such events their risk i soften not detected in the premarketing phase which emphasizes the importance of pharmacovigilance activities. Apart from survelillance, research has o be focussed on pharmacogenetics to reduce the risk of these life threatening unwanted drug effects in the future.	A New Strategy to Inhibit Foam Cell Formation in Atherosclerosis Using the Soluble Immunoadhesion CD68-Fc DAUB K, SCHOENBERGER T, GAWAZ M, SIEGEL-AXEL D Medical Clinic III, Dept. of Cardiology, Eberhard-Karls-University, Tuebingen, Germany Background: Despite their potent lipid-lowering effects, the systemic use of statins is limited by side effects. To establish a new strategy for the inhibition of foam cell formation in a highly specific manner at the site of plaque formation, we cloned a fusion protein consisting of the extracellular domain of the scavenger receptor CD68 and a human Fc domain (CD68-Fc). The aim of this study was to evaluate the binding properties and the antiatherosclerotic potential of CD68-Fc. Methods: For binding studies, an ELISA assay against the human Fc-fragment was used on human carotid plaque pieces sticking to culture plates. In vivo the binding of radioactively labeled CD68-Fc (^124JCD68-Fc) was studied using ApoE^-/--mice which develop lipid rich-atherosclerotic lesions within 20 weeks. In vitro, effects of CD68-Fc were studied in our atheroscreen model in which cocultivation of human platelets with CD34^+-progenitor cells for 5-10 days induces foam cell formation. Finally, CD68-Fc uptake kinetics of Dil-Ox-LDL was performed at monocyte/macrophages. Results: Stainings and ELISA with Fc-antibodies showed that binding of CD68-Fc at lipid-rich human plaques was significantly higher compared to control-Fc. After intravenous injection of ^124J-CD68-Fc, specific binding could also be detected by autoradiography in contrast to wildtype-mice without atherosclerotic lesions. In vitro, laser scan microscopy showed that the foam cells express the scavenger receptor CD68 and that they take up labelled OxLDL. Cell counting and confocal microscopy demonstrated that foam cell formation could be prevented efficiently by CD68-Fc. Furthermore, CD68-Fc inhibited matrix metalloproteinse-9 activity, an important function of foam cells. Finally, FACS-analysis provided proof that the underlying mechanism of CD68-Fc is a reduction in cellular uptake of modified lipoproteins. Conclusions: This new fusion protein, a soluble form of the human scavenger receptor CD68, specifically bound to foam cells in vitro and to lipid-rich plaques in vivo with high affinity and attenuated foam cell formation. Thus, CD68-Fc seems to be a promising new tool to prevent foam cell formation or to transform vulnerable lipid-rich plaques into stable fibrous plaques with reduced lipid content in patients with advanced atherosclerosis.
Application of the Fluorescence Quenching Method to the Study of the Interaction of the Anti-psychotic Drug Chlorpromazine with Serum Albumin SILVA D, CORTEZ CM, BASTOS JC Rio de Janeiro State University, Rio de Janeiro, Brazil Background: The proper understanding of drugs binding to plasmatic proteins is an important pharmacological parameter determining their distribution, absorption and elimination, with possible undesirable clinical effects The aim of this work is to study the binding of chlorpromazine (CPZ) with human and bovine serum albumin (HSA and BSA). CPZ is a neuroleptic still applied in psychosis treatments and its chronic use may cause severe side effects, such as parkinsonism and tardive diskinesy. Here we present the Stern-Volmer constants, besides discussing the binding process. Methods: Intrinsic fluorescence of BSA and HSA were measured by selectively exciting their tryptophan residues at 290nm. The gradual quenching was observed by the titration of both proteins with CPZ, and the Stern-Volmer graphs were plotted to evaluate the quenching constants. Results: HSA titrated by CPZ yielded a linear Stern Volmer plot, whereas BSA yielded a slightly concave curve upward when CPZ concentration was higher than 10 M. Comparison of experiments with HSA carried out at 35º C and 25^oC revealed an important deviation in the slope of the Stern-Volmer plot. For HSA, the Stern-Volmer constants were 4.1x10^4 (1.0x10^2) M^-1 at 25ºC, and 6.7x10^4 (2.0x10^2) M^-1 at 35°C. The increasing of the angular coefficient caused by the higher temperature suggests the occurrence of dynamic quenching for HSA. The estimated constant for BSA was 3.3x10^4(9.0x10^2) M^-1at 25ºC. Conclusions: (1) CPZ quenches the fluorescence of HSA by collisional quenching, interacting with this protein without forming complex. (2) The primary binding site of CPZ is close to the single tryptophan residue of HSA, at the position 214, in the hydrophobic region of the chain. (3) CPZ binds to BSA near to the tryptophan residue located at the position 212.	The Proarrhythmic Potential of Macrolides: The Role of Interactions. A Review SIMKÓ J1 , LŐRINCZ I2 1Semmelweis Hospital, Health Center of Miskolc, Hungary; 2Medical and Health Science Center, University of Debrecen, Hungary Background: Several antiarrhythmic and non-cardiovascular drug therapy, including antimicrobial agents have been implicated as a cause for QT interval prolongation, torsades de pointes (TdP) ventricular tachycardia and sudden cardiac death. Most of the drugs that have been associated with lenghtening of the QT interval or development of TdP can also block the rapidly activating component of the delayed rectifier potassium current (IKr) in the ventricular cardiomyocytes. Methods: We present a review of the current literature on the QT interval prolonging effect of macrolides and the role of interactions in their proarrhythmogenic effect based on results of in vitro, in vivo studies and case reports. Our observations were derived from Medline database until June 2008 using the key words „QT interval”, „torsades de pointes”, „macrolides”, „macrolide interactions” and „acquired long QT syndrome”. We then reviewed the references of the original articles for additional publications. Results: The most frequently QT interval prolonging macrolides are erythromycin and clarithromycin. Almost every macrolide-associated QT interval prolongation occurs in patients with multiple risk factors of the following: drug interactions, female gender, advanced age, structural heart disease, genetic predisposition, and electrolyte abnormalities. Macrolides can potentiate cardiotoxicity of some other torsadogenic agents in two ways: by a direct effect on IKr (pharmakodynamic interaction) and by an indirect effect on the metabolisation of the other agents (pharmacokinetic interaction). Conclusion: We wish to call the health professionals’ attention to this potentially lethal side effect of macrolides, and discuss the facilities of prevention and therapy. In conclusion, physicians should avoid prescribing antimicrobials having QT-prolonging potential for patients with multiple risk factors. Recognition and appropriate treatment of TdP are also indispensable.
Microdialysis with Moderately Lipophilic Drugs: What Is Essential to Know for Voriconazole and its Drug Product to be applicable in Pre-/Clinical Microdialysis Settings? SIMMEL F, KLOFT C Martin-Luther-Universitaet Halle-Wittenberg, Halle, Germany Introduction: Microdialysis (μD) is regarded as method of choice for the determination of unbound tissue concentrations at target sites. Prior to its in vivo applicability in vitro investigations are crucial. For treatment of severe fungal infections voriconazole (VRC) presents a very potent antifungal. VRC combines several difficult physico-chemical properties for μD application: lipophilicy and low solubility. An i.v. formulation is available containing solubilising sulphobutyl-betacyclodextrine- sodium (SBECD). The aim was to investigate whether unfavourable properties of VRC allow the applicability and conditions of the μD technique in pre- /clinical pharmacokinetic studies. Methods: A reliable and rapid HPLC assay for the quantification of VRC from small μD volumes was developed and validated. For the in vitro μD investigations a robust and easy-to-handle system was developed permitting physiological conditions. Experiments on the influence of flow rate (0.4-10.0 μL/min), VRC concentration (1.0-50.0 μg/mL), steady state conditions and VRC flow direction regarding the membrane on relative recovery (RR) were performed. In addition, the impact of SBECD in the i.v. VRC formulation was assayed under retrodialysis conditions. Results: All validation parameters met the criteria set in the international FDA guideline for bioanalytical methods. In vitro μD experiments revealed no binding of VRC to any part of the μD device and therefore VRC concentrations achieved in the dialysate are considered to be sufficient for in vivo investigation. From the observed flow rate dependency a flow rate of 2 μL/min for optimal recoveries for future in vivo investigations was determined yielding adequate sample volumes >12 μL for HPLC assay. RR was independent of VRC concentration. Steady state investigations were performed for the first time and led to recommendations for the in vivo probe calibration solution concentration to range between 100 μg/mL and 200 μg/mL. An impact concerning recovery enhancement of SBECD in the drug product of VRC could be precluded.