Ehrlich II –2nd World Conference on Magic Bullets

Magic Bullets And Vaccines: Learning From The Brain LAMPSON LA Brigham & Women’s Hospital and Harvard Medical School, Boston MA, USA Background: For developing magic bullets and vaccines, the brain is of interest in two different ways: 1) The brain is an important target organ in its own right. 2) The brain’s low background level of immune activity allows general principles to be revealed. Methods: Evolving understanding about delivery of magic bullets and about therapeutic vaccines is reviewed. Examples are taken from rat models that include: local cytokine injection, autoimmune inflammation, implanted tumor, and blood-borne metastases (1-4). Results: “IMMUNE PRIVILEGE.” 1) Immune activity is regulated in the brain – just as in other organs, and the normal baseline is low. However, the brain is not “privileged.” Both beneficial and harmful immune responses can occur. 2) The low baseline affects development of vaccines that target the brain. Most effort has gone to identifying appropriate antigens and initiating a response. It is just as important to enhance the effector phase within the brain. 3) The low baseline makes it possible to see subtle effects that are relevant to all organs. In the brain, as in other organs, individual regulatory molecules have many functions and affect many cell types. The neuropeptide, substance P, and the neurotransmitter, glutamate, also affect immune regulation. BLOOD-BRAIN BARRIER (BBB): its role is often misunderstood. 1) The normal BBB does indeed prevent the passive entry of large proteins and many drugs. 2) The BBB is not normal at tumor masses or other sites of pathology. However, other factors can also impede drug entry. 3) The BBB does not prevent the entry of metabolically-active, migratory cells. However, other factors are important, if cell-mediated responses are to be manipulated. Conclusions: Misconceptions have hindered research in the brain. Increased understanding can aid delivery of new drugs and improve success with vaccines. The brain’s low baseline allows subtle effects, such as the role of local regulatory molecules, to be revealed. 1) McCluskey LP, Local immune regulation in the CNS by substance P vs. glutamate. J. Neuroimmunol. 116: 136-146, 2001; 2) Basic Principles of CNS immunology. Winn, HR, ed., Youman's Neurological Surgery, 5^th ed. Saunders, 2003, pp. 673-688; 3) Antibody-secreting cells to deliver antibody against brain metastases, Journal of Clinical Oncology, 2007. 25(18S): 3047; 4) Targeted therapy for neuro-oncology: Reviewing the menu. Drug Discovery Today, 2008, in press.	Clinical Guidelines For The Use Of Extended Interval Dosage Regimens Of Gentamicin In Neonates LANAO JM^1, CALVO MV^2, MARTÍN-SUÁREZ A^1, MESA JA^2, HERAS MI^3, CARBAJOSA T^3, DOMINGUEZ-GIL A^2 ^1Pharmacy and Pharmaceutical Technology, University of Salamanca, ^2Pharmacy Service, ^3Paediatric Service, University Hospital Background: Development and validation of guidelines for gentamicin dosing in neonates using extended-interval dosage regimens. Methods: With the base of previously obtained population pharmacokinetic parameters (JM Lanao. JAC 48:1038-48,2004), dosing guidelines were designed to achieve serum gentamicin concentrations (SGCs) within the ranges considered therapeutic in adults for extended-interval dosing (peak 15-20 mg/L and trough <0.5 mg/L). These guidelines were adopted as the dosing practice at our Institution. The validation population comprised 81 neonates dosed according to the proposed guidelines, routinely monitored, with the following clinical characteristics: gestational age (GA) 24 - 40 weeks (mean (SD); 33.48 (4.42)), and postnatal ages of 1 - 11 days (2.80 (1.52)). C-reactive protein (CRP) and serum creatinine were measured at the start and end of treatment for the evaluation of efficacy and toxicity of the treatment. Results: In term newborns and premature babies with GA between 31 – 38 weeks, extended-interval dosage regimens with initial gentamicin doses of 10-12 mg/kg and dosage intervals of 36-48 h are recommended. Owing to their high distribution volumes and prolonged half-lives, for premature babies of GA <31 weeks we recommend initial doses of 5 mg/kg and dosage intervals of 36-48 h to reach SGCs between 0.5-10 mg/L. A linear relationship between the individualized dose after SGCs monitoring (ID) and guideline dose (GD) was obtained: ID = 0,9469 GD - 0,909; r^2 = 0,8991. A statistically significant difference (p<0.05) was found between initial and final CRP levels in patients with sepsis (2.45 (1.38) vs 1.29 (1.56)mg/dl) or suspected infections (2.04(1.85) vs 0.87 (0.56) mg/dl). A statistically significant decrease in the serum creatinine concentration was also observed (p<0.01). Conclusions: The pharmacokinetic and clinical validation of the guidelines developed suggests that they are efficient and safe for the initial dosing of gentaminicin in term and premature babies.
Erythropoietin in Cancer Anaemia: Friend or Foe? LAPPIN TRJ Haematology Research Group, Centre for Cancer Research and Cell Biology, Queen’s University, Belfast Erythropoietin (EPO), a glycoprotein hormone produced mainly in the kidney and the liver, binds to the EPO receptor (EPO-R) on erythroid precursor cells in the bone marrow, thereby promoting their survival, proliferation,and differentiation. In adults normal erythropoiesis produces about 2.3 million red blood cells per second, regulated by basal levels of 0.8–4.0 pmoles/L of EPO (5–25U/L) in plasma. Many cancer patients suffer from anaemia, and recombinant humanEPO and other erythropoiesis stimulating agents (ESAs) are widely used therapeutically, to increase haematocrit, lower blood transfusion requirements, and improve qualityof life. However in recent years, several investigators have identified EPO-R expression in numerous cancers and tumour cell lines, raising concerns about the safety of ESA therapy for cancer-associated anaemia. Pharmacological doses of EPO elevate plasma concentrations several fold and potentially could modulate tumour growth. In 2006, a Cochrane Review collated data on over 9000 cancer patientsfrom 57 trials in which recombinant EPO or darbepoetin alfawas given to prevent or treat anaemia. ESA-treated patients had significantlylower blood transfusion requirements. Although there was no significant difference in survival between ESA-and placebo-treated patients, none of the trials includedin the meta-analysis had sufficient statistical power to confidentlydetermine the effects of ESAs on overall survival. The relative risk for thromboembolic events was much higher in ESA-treated patientscompared with controls[1, 2]. Overall, these studies have raised concerns that ESAscould, in certain circumstances, adversely affect survival incancer patients. It has been speculated that these agents may enhance thrombosis, tumour growth, and neovascularization.In 2007, the FDA issued safety warnings alerting clinicians to the potential harm associated with ESA therapy for cancer-associated anaemia. Continued scrutiny of clinical trials, particularly survival data, and current clinical practicepatterns is important to fully understand the risk–benefitratio of ESA treatment in anaemic cancer patients. Bohlius J, Wilson J, Seidenfeld J et al. Erythropoietin or darbepoetin for patients with cancer. Cochrane Database Syst Rev 2006;3:CD003407. Bohlius J, Wilson J, Seidenfeld J et al. Recombinant human erythropoietins and cancer patients: Updated meta-analysis of 57 studies including 9353 patients. J Natl Cancer Inst 2006;98:708–714.	From Classic Autohemotherapy To Autologous Hemoderivative Cancer Vaccine Through A Drug And Drug-Carrier Immunomodulatory Adjuvant System Lasalvia-Prisco E^1,3, Garcia-Giralt E^2, Vázquez J^1,3, Cucchi S^1, Lasalvia- Galante E^1,3, Larrañaga J^1,3 ^1Interdoctors MP, Uruguay; ^2Centre Cancérologie Hartmann, France; ^3Natl. Ins. of Oncology, Uruguay Background: From 1898 to 1940, limited but repeated successful results of autohemotherapy (AH) in several diseases including cancer were described. The interest on AH dimmed after the appearance of antibiotics and chemotherapy. Discovery of serum tumor markers confirmed a cancer AH paradigm, the release of potential antigens from tumor to the blood. Revisiting AH allowed to demonstrate and to potentiate AH immunomodulatory activity. Since 1995, 20 peer review publications reported the development of an Autologous Thermostable Hemoderivative Cancer Vaccine (ATH-CV). In this unpublished study, ATH-CV was used as immunomodulatory pretreatment of chemo in advanced Non Small Cell Lung Cancer (NSCLC), hypothesizing a new strategy with ATH-CV as anti-tolerogenic adjuvant and chemo as endogen vaccination. Methods: Patients (60) in randomized Phase II 2-arm trial (A1: Chemo, A2: Chemo after ATH-CV). Eligibility: NSCLC, stage 4, no previous chemo, PS ≤ 2, available tumor sample to prepare tumor lysate (TL) for immunity tests. Exclusion: Brain metastasis, immunity disease. Assessments: Delayed Type Hypersensitivity elicited by TL (DTH-TL); IFN-γ Elyspot challenged with TL (ELY-TL); immunophenotyping of lymph node cells (IPh-LNC) and peripheral blood mononuclear cells (IPh-PBMC) measuring activated dendritic cells (aDC) as CD1a+CD83+ and T-Regulatory cells (T-Reg) as CD4+CD25+FOXP3+; 30-day Tumor Growth (30-TG); survival curves (Kaplan & Meier); Median Survival Time (MST); 1 Year Overall Survival (1-OS). Statistics: A1, A2 assessments were compared by Student-t and Log-rank tests. Results: At pre-chemotherapy IPh-LNC (% A1, mean ± SD) aDC: A1=100 ± 12, A2=380 ± 16; T-Reg: A1=100 ± 8, A2=32 ± 5. IPh-PBMC (% A1, mean ± SD) T-Reg: A1=100 ± 14, A2=24 ± 4. DTH-TL (% cases+) A1=0%, A2=30%. ELY-TL (spots by 10 ^6 target) A1=24 ± 1.1, A2=108 ± 6.2. 30-TG (%) A1=34 ± 12, A2=16 ± 6. At trial day 120 DTH-TL A1=5%, A2=60%. ELY-TL A1=54 ± 12, A2=325 ± 36. 30-TG A1=21 ± 4, A2=12 ± 3. At trial day 360 MST (wks) A1=28, A2=38. 1-OS (%) A1=30, A2=42. Conclusions: 1) ATH-CV switched IPh-LNC increasing aDC and depleting T-Reg (p<0.02); 2) ATH-CV pre-treatment, increased anti-tumor Chemo effect (p<0.01); 3) Chemo after ATH-CV enhanced anti-tumor immune response (p<0.02).
Temperature, Denaturant And Ligand Effects On Solution Stability And Conformational Properties Of Human Interleukin-1 Receptor Antagonist Latypov RF, Liu D, Jacob J, Raibekas AA, Kleemann GR, Brems DN, Treuheit MJ Amgen, Inc., Thousand Oaks, CA 91320, USA Background: A thorough understanding of solution stability and aggregation of protein therapeutics is important for successful manufacturing and delivery of protein drugs. Aim: To investigate stability and structural properties of a therapeutic anti-inflammatory cytokine, human interleukin-1 receptor antagonist (IL-1ra). Methods: HPLC, optical spectroscopy, high resolution 2D NMR and small angle X-ray scattering (SAXS). Results: The results are consistent with a variety of non-cooperative changes within the folded state ensemble of the protein. In particular, the interface between the first and the second folding motifs was progressively destabilized by increasing urea concentrations. This region outlines an intrinsically labile part of the folded structure which undergoes perturbations with no detectable exposure of hydrophobic surfaces. Additional evidence for non-cooperative unfolding of IL-1ra comes from nonuniform peak intensity variations, weighted average chemical shift differences, as well as bell-shaped denaturation profiles for some of the minor peaks. In addition, binding of 8-anilinonaphthalene-1-sulfonic acid (ANS) to IL-1ra was investigated to address protein aggregation in the presence of this low molecular weight compound. Effects of ambient to elevated temperatures on the affinity and specificity of ANS binding were assessed. Overall, the affinity of ANS was lower at 37 °C compared to 25 °C, but no significant change in the site-specificity of binding was observed from the chemical shift perturbation data. No evidence was found for any partially denatured or aggregated forms of IL-1ra throughout the experimental conditions, consistent with a cooperative and reversible denaturation process. The results support earlier observations on the tendency of ANS to interact with solvent exposed positively charged sites on proteins. Conclusions: 1) Equilibrium unfolding of IL-1ra is associated with accumulation of highly native-like intermediates rather than molten globule-like states. 2) ANS binding occurs within a previously identified aggregation-critical region in the vicinity of the intrinsically labile part of the structure, thus providing an insight into the ligand-dependent aggregation of IL-1ra.	Innovation in Anticoagulation: Discovery and Development of Novel Small-Molecule Coagulation Inhibitors as New Treatment Options for Thromboembolic Diseases LAUX V Bayer Schering Pharma, Wuppertal, Germany Background: Thrombosis, defined as the formation or presence of a clot in a blood vessel leading to ischaemia, infarction, or organ damage, is a leading cause of death and disability in the Western world. Prevention or treatment is presently managed by heparins or vitamin K antagonists (VKAs), although clear drawbacks exist with these treatments, especially in regard to long-term use. Such drawbacks include parenteral administration, unpredictable pharmacology, extensive drug or food interactions, a narrow therapeutic window, and a need for monitoring. However, the discovery of oral, direct inhibitors of thrombin and Factor Xa (FXa) showed that it might be possible to overcome these limitations. In particular, inhibition of FXa is an attractive target for novel, orally available anticoagulants, because FXa occupies a central position in the coagulation cascade and, unlike thrombin, has no other known functions. Methods and Results: Rivaroxaban is an oral, direct FXa inhibitor that has received a positive CHMP recommendation for the prevention of venous thromboembolism (VTE) after elective total hip or total knee replacement surgery (THR/TKR), and is also approved in Canada for this indication. It is also in advanced clinical development for the prevention and treatment of other thromboembolic disorders. Rivaroxaban is a highly selective inhibitor of FXa (Ki of 0.4±0.02 nM) and its antithrombotic effect has been demonstrated in arterial and venous thrombosis animal models. At effective antithrombotic doses, rivaroxaban does not significantly prolong bleeding times, in contrast to the VKAs. In phase II studies, rivaroxaban was effective and well tolerated in the prevention of VTE after THR or TKR, as well as in the treatment of deep vein thrombosis. In phase III studies in TKR and THR (RECORD1–4), various rivaroxaban regimens demonstrated significantly superior efficacy to enoxaparin regimens for thromboprophylaxis, with similar rates of major bleeding. Rivaroxaban is also being assessed for the treatment and secondary prevention of VTE, stroke prevention in atrial fibrillation and secondary prevention in acute coronary syndrome. Conclusion: Rivaroxaban offers the potential to overcome the limitations of current pharmacological agents in the prevention and treatment of thromboembolic disorders.
Vitamin C In Intravenous Nutritive Solution: Double-Edge Effect For Premature Newborn Infants LAVOIE JC, CÔTÉ F, ROULEAU T Departments of pediatrics & nutrition, Sainte-Justine hospital, University of Montréal. Background: Premature infants are at risk of pathological complications related to oxidative phenomenon. Among them, the incidence of broncho-pulmonary dysplasia (BPD) is near of 40% in newborns of < 28 weeks of gestation. Because of the immaturity of their gastro-intestinal system, these children are frequently nourished by intravenous way. Although this parenteral nutrition (PN) contains antioxidant vitamins such as vitamin C, this solution is contaminated by peroxides. This contamination is associated with inadequate proto-protection of nutritive solution. The interaction, catalyzed by photo-exited riboflavin, between vitamin C and oxygen dissolved in solution generates hydrogen peroxide (H2O2). The infusion of PN without photo-protection to newborn guinea pigs induces a lower amount of alveoli in lungs, a characteristic feature of BPD. This observation is not induced by an infusion of H2O2, vitamin C or riboflavin alone, but with the combination of vitamin C + riboflavin. The interaction between dehydroascorbate and H2O2 generated in PN leads to the formation of a new compound, named ascorbylperoxide (2,3-diketo-4-hydoxyperoxyl-5,6-dihydroxyhexanoic acid). We hypothesis that ascorbylperoxide is the active agent leading to a low alveoli development in newborns. Methods: Three days old guinea pig pups received intravenous solution containing increasing concentrations of ascorbylperoxyde. After 4 days, lungs were samples for histological determination of alveoli. Ascorbylperoxide concentrations in PN as well as in urine samples were determined by mass spectrometry. Results: The alveoli count was negatively correlated (r^2 = 0.64; p<0.01) with urinary logarithmic concentration of ascorbylperoxide. The addition of glutathione into PN solution allows the recycling of DHA in ascorbate, thus preventing the generation of ascorbylperoxide and degradation of vitamin C. Conclusion: Results suggest that the interaction of ascorbate with other components present into PN such as riboflavin and oxygen contributes greatly to development of BPD in premature infants. The addition of glutathione to PN could prevent the loss of alveoli and improve the availability of vitamin C. Supported by Canadian Institutes of Health Research (MOP 79403)	Controlled Release Amoxicillin Therapy In Veterinary Medicine LAVY E, FRIEDMAN M, HOFFMAN A School of Veterinary Medicine, Rehovot, Israel. Antibiotics are the most commonly prescribed drugs in small animal medicine that are applied in a variety of microbial infections. The most common veterinary treatment protocol is performed on ‘outpatient’ basis and involves oral treatment with beta-lactam antibiotics, such as ampicillin, amoxicillin, amoxicillin-clavulanic acid, cephalexin and cefuroxime. The short biological half-life of beta-lactam antibiotics and their pharmacodynamic properties that require prolonged exposure of the pathogen to the effective drug concentrations necessitate multiple daily dosing of the beta-lactam drugs throughout the treatment period that usually lasts for 5-7 days. The major drawback of antimicrobial treatment protocols is related to the fact that effective antimicrobial therapy requires multiple daily drug administrations during the treatment period. As a result, one of the common reasons for failure of antimicrobial therapy is the low drug compliance by the pet owners. The prime motives for this poor compliance are: being out of the house during the day, difficulties with restraining the animal, and lack of confidence. We are developing an effective solution to the low drug compliance problem by development of oral dosage form for beta-lactam antibiotics that after a single administration would provide effective drug concentrations throughout the whole treatment period. The main drawback in the design of single oral administration treatment strategies for beta-lactam antibiotics is the fact that these drugs are absorbed only in the small intestine and thus have a narrow ‘absorption window’ with no colonic absorption and the drug effect terminates shortly after the formulation reaches the colon. The approach of a single dose controlled release antibiotic therapy (SCRAT) is based on expandable swelling matrix tablet with prolonged retentivity in the stomach that releases the drug over several days. Thereby, enables continuous input of the beta-lactam drugs to the "absorption window" at the upper parts of the gastrointestinal and ensure treatment of the infection over several days following single administration. Thus, SCRAT provides a means to utilize the major pharmacokinetic and pharmacodynamic advantages of controlled release dosage forms for beta-lactam antibiotics.
Folate-Targeted Chemotherapy LEAMON, C.P., REDDY, J.A., VETZEL, M., DORTON, R., WESTRICK, E., PARKER, N., WANG, Y. and VLAHOV, I.R. Endocyte Inc., West Lafayette, USA Background: The small molecular weight ligand, folic acid, is capable of targeting covalently attached bioactive agents quite specifically to folate receptor (FR)-positive cancers. To date, impressive anti-tumor activity has been observed preclinically with folate conjugates of potent chemotherapeutic molecules, and a few of these agents have recently entered the clinic with more to soon follow. In this present investigation, we explored the possibility of using the folate ligand to target a potent, semi-synthetic analog of the microtubule inhibitor, tubulysin B, to FR-enriched tumors. Methods: EC0305, a folate-tubulysin conjugate, was evaluated in vitro for dose-dependent cytotoxic activity against a panel of FR-positive and negative cells. Cells were pulsed with EC0305 for 2 h in the presence and absence of excess folate, and then chased in fresh medium up to 72 h. nu/nu mice (Balb/c background) were inoculated with FR-positive KB cells, and 74 ± 14 mm^3 tumors were established 11 days later. EC0305 was then administered through the lateral tail vein, and tumors were measured every 2-3 days using a caliper. Tumor volumes were calculated and then compared to untreated controls. Results: EC0305 was found to specifically inhibit the growth of a panel of FR-positive cell lines (IC50 range 1 to 10 nM) in a dose-dependent manner, whereas cells lacking FR expression were unaffected. EC0305’s potency was also confirmed against a human KB xenograft-nu/nu mouse cancer model. Here, a brief three times per week, 2 week regimen yielded remarkable anti-tumor activity (100% tumor-free animals) without causing significant weight loss or major organ tissue degeneration. In contrast, anti-tumor activity was completely abolished in EC0305-treated animals that were co-dosed with an excess of a nontoxic folate-containing analog, thereby confirming that this agent’s antitumor effect was mediated by FRs. The advantage provided by folate conjugation was further proven by the un-targeted free drug, which was found to be completely inactive at both tolerable and highly toxic dose levels.