Ehrlich II –2nd World Conference on Magic Bullets

Results: One of the most important findings of the experiment is that learning (CS+UCS) induced increase in numerical density of GAT-1 puncta in hollow barrels of trained row of vibrissae (the experimental side value was on the average 50% higher compare to the control side). In contrast, numerical density of GAT-1 puncta was unchanged in both control groups.

Conclusions: 1) Present data suggested that GAT-1 may be involved in learning-dependent changes in layer IV of the barrel cortex. 2) In any event, these changes in the immunolabeling for GAT-1 in hollow barrels of trained row of vibrissae would cause an increased uptake of GABA. (Supported by MNiSW grant 188).

Potential use of denosumab in the treatment of giant cell tumor of bone SKUBITZ KM Masonic Cancer Center and University of Minnesota, Minneapolis, MN, USA Background: Giant cell tumor (GCT) of bone, or osteoclastoma, is usually a benign but locally aggressive osteolytic neoplasm in which monocytic macrophage/osteoclast precursor cells and multinucleated osteoclast-like giant cells infiltrate the tumor. While the origin of GCT is unknown, the tumor cells of GCT produce chemoattractants that can attract osteoclasts and monocytic osteoclast precursors, such as receptor activator of NF-KB ligand (RANKL). RANKL is over-expressed in the stromal-like tumor cells of GCT, and is a key mediator of osteoclast formation and survival that could recruit osteoclast-like giant cells to the tumor. The potential role for tumor cell-osteoclast interaction/co-dependence, as in a paracrine loop in which the osteoclast-like cells support the stromal tumor cells, in GCT is unknown. New agents that inhibit osteoclastogenesis via the RANK/RANKL pathway, such as denosumab (a fully human monoclonal antibody against RANKL), may be useful in the treatment of GCT. Methods: Gene expression in 8 GCT was done by microarray using the Affy U133 chip set. A multi-center open-label phase II trial, of patients with GCT treated with denosumab, 120 mg monthly, with loading doses on days 8 and 15 of month 1 was reviewed. The primary endpoint was tumor response (elimination of 90% of giant cells or no radiographic progression of the target lesion) at week 25. In an interim report, 24 patients had received denosumab and 15 were evaluable for efficacy. Results: Gene expression suggested an osteoclastogenic environment in GCT, with RANKL overexpression. In ongoing trials, 13 of 15 patients had a tumor response: 9 of 9 had a histologic response, and 4 of 6 had a radiographic response. The 2 patients who did not meet radiographic response criteria had stable disease per investigators. Clinical benefit was reported in 9 patients, including reduced pain and increased range of motion. No treatment related serious adverse events or neutralizing anti denosumab antibodies were reported. Conclusions: In the interim analyses reported to date of a multi-center phase II trial, 13/15 evaluable patients with recurrent or unresectable GCT responded to denosumab treatment. The results suggest a possible trophic effect of the recruited osteoclasts on the primary tumor cells. Further investigation of denosumab as a new therapy for GCT is warranted.	Treosulfan-Containing Regimens – an Option to Overcome Transplant-Related Toxicity in Children with Refractory Leukemia SKVORTSOVA YV, DISHLEVAYA ZM, MASCHAN AA, MASCHAN MA, SKOROBOGATOVA EV Russian Children’s Hospital, Moscow, Russia Background: Allogeneic hematopoietic stem cell transplantation (HSCT) in refractory leukemia patients is associated with high transplant-related morbidity. Treosulfan is a myeloablative alkylating agent that is recently used in conditioning both in children and adults and shows lower non-hematological toxicity, especially comparing to busulfan. We explored treosulfan-containing regimens in children with refractory leukemia. Methods: 24 patients (17 boys and 7 girls) of median age 7 years (range, 2-17) underwent HSCT from allogeneic donors: 6 matched related, 13 matched unrelated, and 5 haploidentical. All children had refractory leukemia (acute myeloid leukemia – 16, juvenile myelomonocytic leukemia – 3, chronic myeloid leukemia – 1, acute lymphoblastic leukemia – 3, acute biphenotypic leukemia –1), 79% of patients (19/24) didn’t achieve any remission to the moment of HSCT. All haploidentical transplants were CD3, CD19 depleted with usage of CliniMACS device. Conditioning regimens consisted of treosulfan 36-42 gr/m^2, fludarabine, antithymocytic globulin ± thiotepa or melphalan. Average number of transplanted cells was 10,2x10^8/kg (range, 1,6-42) for nucleated cells and 6,6x10^6/kg (range, 1-14) for CD34 cells. Results: All except one patient (96%) achieved stable engraftment at a median of 17 days after transplant. Extramedullary toxicity was rather limited: 16 patients (67%) experienced only mild mucositis. Incidence of acute “graft versus host” disease (GVHD) was 61% (14/23), but only 5 patients presented grade 3-4 acute GVHD. 27% of children (4 of 15 survived to Day 100) developed chronic GVHD (only one case was extensive). 15 of 24 patients died (62,5%) due to relapse in 9 cases (60%), infections in 4 (27%), and acute GVHD in 2 (13%). Two children underwent second HSCT; in 8 children donor lymphocyte infusions were used. 9 of 24 patients (37,5%) survive in complete remission. Conclusions: In this group of patients with refractory leukemia and unfavorable prognosis without HSCT, treosulfan-based regimens showed reduced regimen-related toxicity, but intensive myeloablative activity and excellent engraftment achievement. Treosulfan could be recommended as a safety regimen compound in pretreated children with refractory leukemia not eligible for standard myeloablation.
Genetic aspects of tramadol PK and PD SLANAŘ O Institute of Pharmacology, 1st Faculty of Medicine, CharlesUniversity, Albertov 4, Praha 2, 128 00 Czech Republic Background: Tramadol, widely used analgesics, is mainly metabolized into an active metabolite O-demethyltramadol (M1) via polymorphic CYP2D6. Aim of our studies were to objectify PK properties and opioid action of tramadol in subjects with different CYP2D6 and MDR1 genotypes and to evaluate the clinical efficacy of tramadol in acute pain in patients after knee artroscopy. Methods: Three studies with healthy volunteers (n=87) and one study with patients after knee artroscopy (n=100) were conducted. Volunteers received a standard dose of 100 mg slow release tramadol tablet, while patients were treated by on demand regime after knee surgery. Samples for PK analysis were collected from healthy volunteers over 24 h post-dose and drug – induced miosis over 12 h post dose. Tramadol and M1 analysis were done by GC-MS method. Pupillography was performed using monocular infrared pupillograph „Pupilscan II(TM)“ in a quiet and fully darkened room. Visual analogue scale with werbal description, drug consumption, and neccesity to use emergency pain treatment were used to evaluate analgetic efficacy in pateint group. Results: Tramadol displayed phenotypic pharmacokinetics and it was possible to identify CYP2D6 PM subjects with >99% confidence from single blood sample taken between 2.5 and 24 h post-dose. MDR1 genotype affected PK parameters of tramadol mainly in CYP2D6 PM subjects. Mean (SD) differences of basal and 8 hours drug-constricted pupillary diameters were 1,36 (0,68); 0,77 (0,62); and 0,33 (0,34) mm in groups of extensive, intermediate, and poor metabolizers, respectively. A tendency towards better analgetic efficacy in CYP2D6 EM patient group was also observed. Conclusions: Tramadol may be used as a convenient CYP2D6 phenotyping tool and that a pharmacokinetic-pharmacodynamic interaction produces two discernable high and low response phenotypes with respect to PK and pupillary constriction. MDR1 seems to affect PK into a lesser extend than CYP2D6. Genetic predisposition for clinical efficacy need to be confirmed. Acknowledgement: The author would like to thank Michael Friedrich Bőttcher for Pupillscan II. This study was supported by grant No. MSM 0021620820, MSM0021620849 and GACR 305/08/P069.	Chromosome Damage in Peripheral Lymphocytes of Children with Urinary Tract Infection after Antimicrobial Therapy with Nitroheterocyclic Compounds SLAPSYTE G, MIERAUSKIENE J Department of Botany and Genetics, Vilnius University, Lithuania Background: Nitroheterocyclic compounds such as nitrofurantoin [N-(5-nitro-2-furfurylidene)-1-aminohydantoin] and furagin [N-(5-nitro-2-furyl)-allylidene)-1-aminohydantoin] have been used effectively in the therapy of urinary tract infection (UTI) for many years. Since recurrence of UTI is common in susceptible individuals, the objective of the present study was to determine whether nitrofurantoin and furagin, used for long-term low dose antimicrobial prophylaxis of UTI, may induce chromosome aberrations (CAs) and sister chromatid exchanges (SCEs) in lymphocytes of the treated patients. Methods: Cytogenetic analysis was performed in 153 blood samples from 109 children aged from 0.2 to 13 years and suffering from UTI. The treatment consisted of oral administration of nitrofurantoin or furagin at doses of 5-8 mg/kg/day for the first 7 days and at doses of 1-2 mg/kg/day for the rest of the treatment period. Blood was sampled before the start of the therapy and after 1, 3, 6 and 12 months of the therapy. For each subject, three lymphocyte cultures were set up according to conventional techniques. CAs were scored in 100 first mitotic division cells and SCEs in 25-50 second division cells per individual. Results: CA frequency was not affected by age, nitrofurantoin or furagin therapy or previous treatment with antibiotics. The only effect on CAs was a higher frequency of CAs (mainly acentrics) due to urethrocystography that preceded the therapy. Analysis of different types of CAs revealed two statistically significant trends: decrease in the frequency of acentrics (Y) with the time elapsed since urethrocystography (X) (Y1.793-0.062X, r^20.77), and increase of chromatid exchanges (Y) with duration of furagin therapy (X) (Y0.066+0.019X, r^20.83). SCE frequency was not affected by age, antibiotic treatment, urethrocystography and nitrofurantoin therapy. A time-independent significant increase in SCE frequency was found in lymphocytes of children treated with furagin. Conclusions: 1) Furagin induced SCEs and CAs (chromatid exchanges) in lymphocytes of the treated patients. Nitrofurantoin was not genotoxic. 2) Since both nitrofuratoin and furagin are interchangeable in a sense of their antimicrobial efficiency, preferential use of nitrofurantoin in treatment of UTIs might be recommended.
Cancer Immunotherapy with Adenovirus-IFN (TG1042) Slos P, Bataille V, Acres B,  De Meyer M, Pichon C, S. Hermann S, Derbij A, Lusky M Transgene SA, 11, rue de Molsheim, 67082 Strasbourg cedex, France. [e-mail] Background. Primary cutaneous lymphomas comprise a heterogeneous group of lymphoproliferative disorders characterized by clonal accumulation of T-lymphocytes (CTCL) or B-lymphocytes (CBCL) homing to the skin. While a variety of registered therapies, including immunotherapies exist for CTCL, no registered therapy is available for CBCL, apart from radiotherapy and surgery. For the management of relapsing CBCL, after first line radiotherapy or surgery, various off-label therapeutic options are being explored. Presently all current treatments including radiotherapy and surgery do not cure the disease but lead to relapses at various frequencies, indicating further medical need for alternative therapeutics. Systemic administration of recombinant IFN- has shown promising response rates in CTCL, however, therapy with recombinant cytokines is limited by their short half-life and significant side effects upon systemic administration. Transgene SA is developing TG1042 (Ad5IFN-) which is based on a replication- deficient adenovirus type 5 (Ad5) carrying the human IFN- gene. TG1042's anti-tumoral activity relies on the anti-proliferative, immunomodulating and anti-angiogenic activities of the transgene-encoded IFN- cytokine. Methods. The investigational treatment consists of intralesional injections of TG1042 at a dose of 5 x 10^10 viral particles (vp) per lesion into up to six lesions at days 1, 8, 15 of a monthly treatment cycle. Patients are treated for up to 4 cycles. Results. An open-label, multicenter, dose-escalation TG1042.01 phase I/II trial enrolled a total of 39 patients with advanced cutaneous T-cell lymphoma (CTCL) and multilesional cutaneous B-cell lymphoma (CBCL). Intralesional injections of TG1042 included a dose escalation regimen in the phase I part. Treatment with TG1042 induced a high rate of local clinical responses: 19 (57%) of 33 evaluable patients responded to the treatment with 9 complete responses (CR) and 10 partial responses (PR). All five enrolled patients with CBCL responded to the TG1042 treatment (100% response). Concerning safety, the results from this phase I/II trial demonstrated that TG1042 was well tolerated up to the highest dose level (3 x 10^11 vp). Adverse events (AEs) were mostly of grade 1 and grade 2. The most common registered AEs were injection site reactions, fever, chills, and fatigue. A multicenter, open-label TG1042.06 phase II study of intralesional administration of TG1042 in relapsing CBCL patients (PCMZL, PCFCL) is now enrolling patients at centers in Switzerland, France, USA, Serbia, Croatia, and Poland. This is the first clinical trial ever performed in CBCL with a large cohort of 41 patients. Twelve patients are currently enrolled in the study. Interim results of the ongoing phase II study will be presented. Conclusions. Adenovirus-based cytokine transfer may represent a new treatment modality for cutaneous lymphomas and especially for CBCL given the limited number of treatment options and the lack of registered drugs for this indication. TG1042 may become a convenient treatment option for patients who would prefer other treatments options than radiotherapy or surgery.	Pharmacokinetics and Biological Effects of the Recombinant Human Bone Morphogenetic Protein-7 in Vivo SMAJILAGIC A(1), REDZIC A (2), FILIPOVIC S (3) 1. University Clinic Center Sarajevo, Clinic for Maxillofacial Surgery, Sarajevo, Bosnia and Herzegovina; 2. Medical Faculty University in Sarajevo, Institute for Biology and Human Genetic, Sarajevo, Bosnia and Herzegovina; 3. Faculty of Veterinary Medicine University in Sarajevo, Clinic for Surgery, Orthopedic and Opthalmology, Sarajevo, Bosnia and Herzegovina BACKGROUND: The cellular and molecular mechanismsthat determine autonomous hematopoietic competence in the Bone Morphogenetic Protein induced osteoblastogenesis and angiogenesis havenot been characterized yet. AIMS: In the present study we investigated biological activity’s of recombinant human Bone Morphogenetic Protein-7 (rhBMP-7) during osteogenesis and their effect on the autonomous hematopoetic system. MATERIAL AND METHOD: On 14 New Zeealand White Rabbits surgicaly were created full-thickness non-critical size mandible defects. On 7 animals defects were treated with 100 micrograms of the rhBMP-7 in collagen as carrier (ACS) and on other group of 7, defects were treated with autologues iliac crest bone grafts (control). Markers of the osteogenesis function included alkaline phosphates enzyme (ALP) specific activity. Histology analysis were performed on day 7,14,30 and 60 postoperative and C-T scan with Bone Mineral Density analysis done on day 30 postoperative. Standard CBS tests from the periphery blood done preoperative and day 5,14,21,30 postoperative. RESULT: Day 7, histology analysis rhBMP-7 induced new form tissue showed granulation tissue and strong angiogenetic response and day 30 mesenchymal tissue enrichement with osteoblast and plenty of new formed capilars and vessels network. Day 60 mature bone formation with collagen and osteocytes was detected. 5 of total 7 test animals showed completely bridged defect with new formed bone. Osteogenesis was characteristic for intramembraneous ossification, without cartilage involvement. ALP activity was significantly increased on day 21 on test group compared with controul group detected day 30. C-T and Bone Mineral Density showed average density of the new formed tissue between 413 mg/cm3 and 519 mg/cm3, determinate by program as the bone, on all animals. All hematologist values were in physiologic range on both groups. CONCLUSIONS: 1. rhBMP-7/ACS constuct showed stronger osteoinductive capacity then autologue bone graft, gold standard in conventional medicine; 2. Osteoinductive effect rhBMP-7 during osteogenesis, didn’t affect hematopoetic competence determinate by the Er. Le, HCR and Hg; 3. Strong BMP induced osteogenesis effect is prerequisite et least by strong angiogenetic response.
The cytostatic treatment effect on nucleoli of leukaemia cells at the single cell level SMETANA K Institute of Haematology and Blood Transfusion, Prague Background: Nucleoli are multifunctional organelles. Their structure, cytochemistry and size reflect main cell states as proliferation, differentiation, resting state, ageing and death. Methods: Nucleoli and main nucleolar components of leukaemia cells untreated or treated with various cytostatics were visualised by light microscopic cytochemistry for demonstration of DNA, RNA and silver stained. nucleolar organisers (AgNORs). Electron microscopy visualised main nucleolar structural components, i.e. chromatin, RNA containing structures and Fibrillar Centers (FCs) corresponding to AgNORs. Results: Proliferating less differentiated cells are characterised by „active“ RNA transcribing large nucleoli containing multiple AgNORs and FCs. However, large nucleoli were also detected in leukaemia myeloblasts after cytostatic therapy with histone deacetylase inhibitors or laevulinic acid induced photodynamic treatment. In such nucleoli AgNORs were markedly reduced and electron microscopy demonstrated the translocation of FCs to the nucleolar periphery followed by their expulsion from the nucleolus. Ring shaped nucleoli are characterised by a single AgNOR surrounded by a RNA containing peripheral shell. They are in „resting“ cells and reflect the reversible decrease of RNA transcription. They may be produced from „active“ nucleoli by intercalators or imatinib. The presence of „active“ and „resting“ nucleoli in one cell was noted in leukaemia cells resistant to the cytostatic treatment with antimetabolites, alkylating or intercalating drugs, and histone deacetylase inhibitors. The photodynamic treatment also produced that phenomenon. Micronucleoli (nucleoli smaller than 1µ) with a characteristic structure accompanying terminal differentiating and apoptotic state are produced by all types of current cytostatics. However, the effect of cytostatics may differ. In patients with chronic myeloid leukaemia hydroxyurea produced a larger number of myeloblasts with micronucleoli than interferon. Conclusions: The fine structure and cytochemistry might provide useful information on the effect of cytostatics on leukaemia cells at the single cell level with a full respect to cell differentiation and maturation stage. Acknowledgements: The presented studies were supported by the Czech Ministry of Health – Research Project 0002373601.The author would like to express his gratitude to the members of Haematology Departments of the Institute and 2nd Medical Faculty of the Charles University for their continuous support.	[C11]Mirtazapine: Magic Bullet for PET Brain Imaging of Antidepressants SMITH DF Center for Psychiatric Research, Psychiatric Hospital of Aarhus University, Skovagervej 2, 8240 Risskov, Denmark Background: Dr. Paul Ehrlich knew the importance of a close match between spatial features of drugs and biomolecules, and his vision continues to guide research in medicinal chemistry. Today, positron emission tomography (PET) is often used during drug development to explore the inner workings of the body. Methods: Over the years, we have used PET to explore actions of antidepressant drug in the brain in an effort to discover “magic bullets” for assessing neuroreceptors that may mediate causes and cures of psychic depression. Results: Our research has focused in recent years on the antidepressant compound mirtazapine, radiolabelled with C-11 for PET. We have found that the PET radiotracer has suitable properties for neuroimaging of antidepressant binding sites in the brain of laboratory animals and humans. The receptor occupancy obtained by therapeutic doses of mirtazapine can be quantified by [C11]mirtazapine in regions of the living human brain. Studies of the enantiomers of [C11]mirtazapine in vivo and in vitro showed differences in the stereoselectivity of the findings. Conclusion: [C11]Mirtazapine provides a reliable PET-procedure for quantifying receptor sites involved in antidepressant actions.
Resolution of cancer and infection induced wounding is an essential factor in immune enhancement. The answers to why and how revealed SMITH GR Perses Biosystems Ltd, Warwick Science Park, Coventry, UK Background: The lifecycle of Cancer can be simplified into two phases of the disease; the earlier phase where abnormal growth is a prerequisite for carcinogenesis and the later phase, malignancy, where abnormal growth cannot continue without harnessing the body's wound response. Induced wounding in the stroma provides many benefits to Cancer progression including destruction and remodeling of competing healthy tissue, growth of new blood vessels and immune suppression. Methods: Systems Analysis of published journal literature Results: Evidence shows that Cancer induced wounding is critically dependent upon up-regulation of the Angiotensin II type 1 (AT1R) receptor. Classically associated with the vascular system, expression of AT1R is a systemic cellular response to oxidative, hypoxic and physical stresses. Activation of AT1R by Angiotensin II leads to the production of a host of pro-inflammatory mediators including cytokines, chemokines, adhesion molecules and other factors such as TGF-beta, VEGF and Matrix Metalloproteinases. Early clinical studies utilising Angiotensin Receptor Blockade in Cancer and other chronic inflammatory diseases (many of which having, or suspected to have, an infectious component) demonstrate increased patient survival, reduced biological disfunction and pain. Conclusions: Lessons learnt from a systems analysis of the behaviour of Angiotensin in Cancer explains that the induced ‘Wound that never heals’ is an environment that is of vital importance in suppressing the adaptive immune response and that Cancers and other infections utilise chronic inflammation in order to spread and evade learned immune responses. Angiotensin Receptor blockers (ARBs) have a safety profile, with side effects comparable with placebo even at supramaximal dosage. These drugs can be used alone, turning malignant tumours benign, however ARBs would be synergistic with conventional and developing therapies, especially vaccines, greatly improving effectiveness.