Ehrlich II –2nd World Conference on Magic Bullets

Staphylococcal Enterotoxin Vaccines: Future Utility for Fighting Staphylococcus aureus?
STILES BG
USAMRIID, Integrated Toxicology Division, Fort Detrick, Maryland, USA
Background: There is little doubt that Staphylococcus aureus is as much today a microbial menace as it was when first discovered by Alexander Ogston in the late 19^th century. With rising rates of antibiotic resistance evident now in the 21^st century, it seems clear that additional measures must be made to counter S. aureus. Past efforts of pacifying this pathogen and its myriad virulence factors have included the generation of recombinantly-attenuated versions of staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin 1 (TSST-1). Naturally, these toxins are superantigens that bind to major histocompatibility complex II and specific T-cell receptors. These protein toxins stimulate T cells that then release unhealthy levels of pro-inflammatory cytokines throughout the host. Ultimately, such events can cause toxic shock. Vaccine constructs, in particular those towards SEB and TSST-1 used in our studies, were devoid of receptor-binding capabilities and thus rendered non-toxic. The major goal of our studies was to show protection against a toxin challenge in various animal models. Hopefully, these studies will translate into human protection against S. aureus-induced toxic shock and thus provide clinicians with additional tools against disease.

Methods: A mouse model was first developed to screen potential vaccine candidates. This model exploits the natural synergy that exists between superantigens and lipopolysaccharide derived from Gram-negative bacteria. After extensive mouse testing, a non-human primate model was also used for establishing vaccine efficacy. Vaccine constructs were evaluated by various routes of administration which include intraperitoneal, intramuscular, and intranasal. Animals were challenged with a lethal dose of toxin administered into either the peritoneal cavity or lungs.

Results: Various animal studies, employing mice and non-human primates, revealed excellent efficacy of these protein vaccines against a lethal toxin challenge.

Conclusions: Recombinantly-attenuated versions of S. aureus superantigens represent worthy, non-toxic targets for vaccination. Multiple studies from various groups suggest promising efficacy of superantigen-based vaccines towards specific toxins and the microbe; however, further studies await results from human clinical trials.

Transdermal Delivery of Naltrexone, In Vitro Testing to Human Volunteers STINCHCOMB AL University of Kentucky College of Pharmacy, Lexington, KY USA; AllTranz Inc., Lexington, KY USA Background: Alcoholism and opiate addiction lead to major health-related problems and societal costs for people around the world. Therefore, research and development for improved pharmacologic treatments for drug and alcohol abuse are very important. Naltrexone, an opioid antagonist, is currently used in oral tablet form to help maintain opioid addicts in a drug-free state. Most recently, naltrexone has been indicated as an adjunct in the treatment of alcohol dependence, as well as reported to reduce alcohol craving in certain alcoholic populations. Transdermal delivery of naltrexone is desirable for opioid addicts and alcoholics in order to help reduce side effects associated with oral therapy and improve compliance. Naltrexone itself does not have the essential physicochemical properties that would allow a therapeutic dose of the drug to cross the human skin barrier. Methods: For the last 12 years we have been designing and synthesizing prodrugs, which are more skin permeable than naltrexone, in order to make a therapeutically successful drug delivery system. We have hypothesized that prodrugs of naltrexone and prodrugs in combination with microneedle treatment will improve the transdermal delivery rate of naltrexone. These prodrugs have made excellent research tools for investigating quantitative structure-permeability relationships (QSPR) for transdermal flux and optimization of flux in combination with microneedle enhancement. Results: These prodrugs/microneedles have improved naltrexone delivery rates across the skin because of optimized physicochemical properties for faster diffusion. Testing has been completed to measure the drugs’ penetration and concurrent bioconversion through human skin in vitro with and without microneedle use, and to examine the pharmacokinetics of the drugs in guinea pigs in vivo with and without microneedle use. Correlation of our in vitro data with the in vivo model allowed us to design and complete a human proof-of-concept study using microneedles and naltrexone hydrochloride. Conclusions: We have been able to improve the flux of naltrexone across the skin up to 10-fold with these methods. The current focus of our studies is to increase the rate of naltrexone transdermal delivery by combining microneedle technology with a prodrug and formulation approach. Funding:NIHR01DA13425.
Extensive Necrosis of Visceral Melanoma Metastases after Immunotherapy STOETER D*^1, DI LIGUORI CARINO N^1, MARSHALL E^2, POSTON GJ^1 and WU A^1 ^1The Department of Hepatobiliary Surgery, Aintree University Hospital NHS Foundation Trust, Lower Lane, Fazakerley, Liverpool, L9 7AL, UK, ^2The Clatterbridge Centre for Oncology NHS Foundation Trust, Clatterbridge Road, Bebington, Wirral, CH63 4JY, UK Background: The prognosis for metastatic melanoma remains poor even with traditional decarbazine or interferon therapy. 5-year survival is markedly higher amongst patients undergoing metastatectomy. Unfortunately not all are suitable for metastatectomy. Alternative agents for systemic therapy have, to date, offered no greater rates of survival beyond traditional therapy. A toll-like receptor 9 agonist, PF-3512676 (formerly known as CPG 7909) is currently being evaluated for its potential. Case presentation: We present the case of a 54-year-old Caucasian male with completely resected metastatic cutaneous melanoma after immunotherapy. The patient initially progressed during adjuvant high-dose interferon, with metastases to the liver, spleen, and pelvic lymph nodes. During an 18-month treatment period with PF-3512676 (formerly known as CPG 7909), a synthetic cytosine-phosphorothioate-guanine rich oligodeoxynucleotide, slow radiologic disease progression was demonstrated at the original disease sites. Subsequent excision of splenic and pelvic nodal metastases was performed, followed by resection of the liver metastases. Histologic examination of both hepatic and splenic melanoma metastases showed extensive necrosis. Subsequent disease-free status was demonstrated by serial positron emission tomography (PET). Conclusions: Existing evidence from phase I/II trials suggests systemic treatment with PF-3512676 is capable of provoking a strong tumor-specific immune response and may account for the prolonged tumor control in this instance.	The Inverse Relationship between Cisplatin and Paclitaxel Resistance: Two ‘Magic Bullets’ are Needed STORDAL B ^1,2, PAVLAKIS N^1, DAVEY R^1 ^1Bill Walsh Cancer Research Laboratories, Department of Medical Oncology, Royal North Shore Hospital. St. Leonards NSW 2065, AUSTRALIA ^2National Institute for Cellular Biotechnology, Dublin City University, Dublin 9, IRELAND Background: Cisplatin and paclitaxel have both been successfully used for the treatment of solid tumours, however the majority of patients will relapse with resistant disease. Work in our laboratory and preliminary reading of the literature suggested that cisplatin-resistant cell lines were often sensitive to paclitaxel and the reverse was also true, paclitaxel-resistant cells were often sensitive to cisplatin. The objective of our systematic review was therefore to 1) To examine the pre-clinical evidence for an inverse resistance relationship between cisplatin and paclitaxel and 2) The clinical evidence for the use of paclitaxel salvage chemotherapy in patients with cisplatin-resistant cancer. Methods: Medline was searched for 1) Cell models of acquired resistance reporting cisplatin and paclitaxel sensitivities and 2) Clinical trials of single agent paclitaxel salvage therapy for cisplatin/carboplatin-resistant ovarian cancer. Results: Our systematic literature review found 137 models of acquired drug resistance. 68.1% of cisplatin-resistant cells were sensitive to paclitaxel and 66.7% of paclitaxel-resistant cells were sensitive to cisplatin. A similar inverse pattern was observed for cisplatin vs. docetaxel, carboplatin vs. paclitaxel and carboplatin vs. docetaxel. These associations were independent of cancer type, agents used to develop resistance and reported mechanisms of resistance. Sixty-five eligible clinical trials of paclitaxel-based salvage after platinum therapy were identified. Studies of single agent paclitaxel in platinum-resistant ovarian cancer where patients had previously received paclitaxel had a pooled response rate of 35.3%, n = 232, compared to 22% in paclitaxel naive patients n = 1918 (p < 0.01, Chi-squared). Suggesting that pre-treatment with paclitaxel may improve the response of salvage paclitaxel therapy. The response rate to paclitaxel/platinum combination regimens in platinum-sensitive ovarian cancer was 79.5%, n = 88 compared to 49.4%, n = 85 for paclitaxel combined with other agents (p < 0.001, Chi-squared), suggesting a positive interaction between taxanes and platinum. Therefore, the inverse relationship between platinum and taxane resistance seen in cell models is mirrored in the clinical response to these agents in ovarian cancer. An understanding of the cellular and molecular mechanisms responsible would be valuable in predicting response to salvage chemotherapy and may identify new therapeutic targets. Conclusions: While neither cisplatin or paclitaxel may be a universal ‘magic bullet’ on their own, the correct sequencing between these two agents may lead to improved response rates particularly in ovarian cancer.
Daptomycin: Old drug, New data, Endless Puzzles HO SW^1, JUNG D^2, BAEK SB^1, SCOTT WRP^1, CALHOUN JR^3, LEAR JD^3, CHENG JTJ^1, HANCOCK REW^2, STRAUS SK^1 ^1Dept. Chemistry, University of British Columbia, Vancouver, BC, CANADA; ^2Dept. of Microbiology and Immunology, University of British Columbia, Vancouver, BC, CANADA; ^3Dept. Biochemistry, University of Pennsylvania, PA, USA Background: Daptomycin is a potent antibiotic that displays rapid bactericidal activity and has recently been approved by the FDA to treat complicated skin infections. It is a cyclic anionic tridecapeptide, with a number of D-amino acids (D-asparagine, D-alanine, and D-serine), 3 uncommon amino acid residues (ornithine, (2S,3R)-3-methyl-glutamic acid and kynurenine), and a N-terminus that is acylated with a n-decanoyl fatty acid side chain. It functions in a calcium-dependent manner. Aim: To understand its mode of action, specifically its interaction with 1) calcium and 2) bacterial model membranes. Methods: To understand the effect of calcium on the structure of daptomycin, we have performed analytical ultracentrifugation experiments, as well as characterized the structure of daptomycin in the presence of Ca^2+ and Mg^2+ by nuclear magnetic resonance (NMR). To determine to what extent daptomycin perturbs membranes, we have performed differential scanning calorimetry, fluorescence, and NMR experiments. Results: Our NMR and other biophysical data suggest that daptomycin forms a micellar structure in solution when Ca^2+ is present in a 1:1 Ca^2+/daptomycin ratio and that this binding event, as well as binding to lipid membranes is not accompanied by as significant a structural change as originally postulated in Jung et al., Chem Biol. 2004 Jul;11(7):949-57. Our data also suggest that the presence of negatively charge phosphatidylglycerol and Ca^2+ are essential for membrane interaction and perturbation (by fusion). Conclusions: The implication of these results for the mechanism of action of daptomycin will be discussed.	Ocular Infections: Impact of Resistance to Fluoroquinolones and Other Antibiotics on Topical Therapies Schlech BA, Stroman DW Alcon Research, Ltd. Ft. Worth, Texas USA Background: Ocular infections of the anterior segment include bacterial conjunctivitis and keratitis. Both of these infections are treated with topical antibiotic drops. Ophthalmic antibiotic drops include aminoglycosides, tetracyclines, chloramphenicol, fusidic acid, combinations of two or more antibiotics and most recently in the last 15 years, fluoroquinolones (FQs). An important question is whether or not acquired resistance to an antibiotic limits the efficacy of the corresponding ophthalmic products. Methods: The clinical and microbiological efficacy was correlated with in vitro susceptibility of the pre-therapy pathogens in Alcon-sponsored clinical studies with FQs conducted during the past decade. Results: Clinical and microbiological efficacy results from more than 3000 conjunctivitis patients revealed that frequency of treatment failures due to a specific pathogen were directly proportional to the frequency of occurrence of the specific pre-therapy bacterial species. Generally, the pre-therapy pathogens from treatment failure patients were susceptible to FQs and did not change their FQ susceptibility as a result of FQ therapy. Conclusions: Antibiotics applied topically that are rapidly bactericidal lead to more rapid resolution of clinical signs and symptoms and eradication of pathogens than antibiotics that are bacteriostatic. FQ ophthalmic products are formulated with very high concentrations (3000 – 5000 mcg/ml) relative to the FQ MICs for pathogens, even those that have become resistant to FQs. Treatment failures due to FQ resistant pathogens did not occur more frequently than treatment failures due to FQ susceptible pathogens indicating equivalent efficacy for FQ susceptible and resistant pathogens. Moreover, in no case did the FQ susceptibility of a pre-therapy pathogen change as a result of FQ therapy.
Fluoride and Aluminum Interactions: AlFx as the Magic Bullets Producing Aberration of G Proteins STRUNECKA A, KLENEROVA V 1st Faculty of Medicine, Charles University, Prague, Czech Republic. Background: The wide use of fluoride (F) and aluminum (Al^3+) in medicine, industry, and agriculture, started the era of supplementation of living environment with these ions as never before. Many effects primarily attributed to F are caused by its interactions with Al^3+. The effects of aluminofluoride complexes (AlFx) - new analogues of phosphate groups - have been demonstrated by many studies with proteins, intact cells, and whole animals. The widespread use of AlFx as a general activator of G proteins provided evidence that AlFx represent Magic Bullets for human body. The warning of the potential danger from the interactions of Al^3+ and F to human health has emerged from research on cell signaling. Methods: Biochemical and immunohistochemical methods were used for in vitro experiments. Meta-analysis techniques for human studies were used because only summary statistics are typically available in the literature. Results: Fluoride affects the activity of numerous enzymes and various biological processes. The effective dose is much lower in the presence of trace amounts of Al^3+. AlFxinteract with all known G protein-activated effector enzymes. Numerous findings demonstrate the positive correlation with the impairment of homeostasis, growth, development, cognition, and behavior. The predictability from in vitro data as well as the correlation with numerous epidemiological, ecological, and clinical studies from fluoridated communities will be presented. AlFx may induce most of the pathological hallmarks of autism and Alzheimer’s disease. Conclusion: 1) The definition of a safe concentration of Ffor humans must consider that the dose is much lower in the presence of Al^3+. 2) It is evident that AlFx is a molecule giving the false information, which is amplified by processes of signal transmission. Pharmacologists estimate that up to 60% of all medicines used today exert their effects through G protein signaling pathways. AlFx are man-made Magic Bullets that produce aberration of G proteins. 3) Assessment of the health risks linked to the F and Al^3+ interactions would contribute significantly to the decline of many several disorders in the 21^st century. Supported by MSM 0021620806 Strunecka et al. Fluoride interactions. Curr. Signal Transd. Therapy 3, 190, 2007.	Strategic Role of Prednisolone, Vincristine and Asparaginase in Pediatric Leukemia as a Paradigm of Success in Oncology: Individualized Tumor Response Testing and Microarray Analysis STYCZYNSKI J Department of Pediatric Hematology and Oncology, Collegium Medicum,Nicolaus Copernicus University, Bydgoszcz, Poland Background: A significant progress has been obtained in pediatric acute lymphoblastic leukemia (ALL). In analysis of last 50 years, the cure rate increased from 1% to over 70%. Recent results showed even up to 90% of cure in subgroups of patients. Aims: The value and role of individualized tumor response testing (ITRT) to prednisolone, vincristine and asparaginase in pediatric ALL. Methods: The in vitro drug resistance profile in 359 children with newly diagnosed ALL was determined by the MTT assay. All patients were stratified into 2 risk groups on the basis of white blood cell (WBC) count: patients with WBC>50G/L (n=77) were treated as high-risk group according to New York protocol, while all others (n=282) with BFM protocol for standard risk group. Patients with early deaths, bcr-abl rearrangement, in vivo prednisolone poor-responder, and infants were excluded from this study. Combined in vitro drug resistance profile to prednisolone, vincristine and asparaginase (PVA score) was determined for each patient. Results: (1) Based on result of ITRT, all patients were divided into 3 groups: those, whose samples underwent spontaneous apoptosis were regarded as sensitive (S), those with PVA score 3-7 were regarded as intermediately sensitive (M) and those with PVA score 8-9 as resistant (R). With median follow-up of 20 months, probability of disease free survival (pDFS) was 0.900.03, 0.820.05 and 0.640.11 (p=0.023) for S, M and R groups, respectively. Within subgroups characterized by initial WBC count similar distribution was observed. (2) In multivariate analysis, two factors were significant: bone marrow response by day 33 (p<0.001) and PVA score (p=0.002) for the group of all patients. When standard-risk patients were analyzed separately, also PVA score (p=0.038) and bone marrow response by day 33 (p=0.031) were the only significant factors for pDFS. (3) Results of ITRT study were recently confirmed (by other groups) using microarray technology. Conclusions: (1) Combined in vitro drug resistance profile to prednisolone, vincristine and asparaginase is a potent prognostic factor in childhood de novo acute lymphoblastic leukemia. (2) ITRT results were confirmed by microarray analysis. Support: Grant MNiSW N407 078 32/2964.
Induction of Complete Tumor Remission by DNA-Directed Alkylating Agents SU TL^1, CHOU TC^2, CHIOU SH^3 ^1Institute of Biomedical Sciences, Academia Sinica, Laboratory of Bioorganic Chemistry, Taipei, Taiwan; ^2Memorial Sloan-Kettering Cancer Center, Molecular Pharmacology & Chemistry Program, NY, NY, USA; ^3Taipei Veterans General Hospital and National Yan-Ming University, Department of Medical and Research and Education, Taipei, Taiwan Background: DNA alkylating agents possess a number of drawbacks including high chemical reactivity and lack of intrinsic DNA binding affinity.To overcome these drawbacks, it is important to design and synthesize chemical stable alkylating agents with potent antitumor efficacy. Aims: 1) To synthesize DNA-directed alkylating agents by linking the phenyl N-mustard pharmacophore to DNA-affinic molecules (i. e., 9-anilinoacridines or quinolines) via a urea or carbamate linker. 2) To study structure-activity relationships. 3) To study the mechanism of action. Methods: A series of DNA-directed alkylating agents, N-mustard-9-anilinoacridine and N-mustard-quinoline conjugates bearing a urea or carbamate linker were synthesized by reacting 4-[N,N-bis(2-chloroethyl)amino]phenyl isocyanate or 4-[bis(2-chloroethyl)amino] phenyl 4-nitro-phenyl ester with appropriate 9-anilinoacridines or 4-aminoquinolines in dry DMF under basic conditions. Results: The newly synthesized derivatives exhibited significant cytotoxicity against human acute lymphoblastic leukemia and various solid tumors with IC50 in submicro molar range and have little or no cross-resistance to either taxol or vinblastine. Several derivatives are able to induce complete tumor remission in nude mice bearing human breast carcinoma MX-1 xenograft and maintain no relapse for a long period of time. Among these conjugates, BO-1051 exhibited potent cytotoxity against various human glioma stem-like cells growth in vitro and induced autophagy in these cells. Moreover, this agent significantly inhibits human glioma U87 xenograft. The new N-mustard derivatives are able to induce DNA interstand cross-linking in tumor cell and have long half-life in rat plasma.	Inhibitory Effects of Demecolcine on NK Cell Functions: Implications for Cancer Therapy SUCK G^1, KOH MBC^1,2 Blood Services Group, Health Sciences Authority^1 and Department for Haematology Singapore General Hospital^2, Singapore Background: The cytostatic property of the microtubule-disrupting agent demecolcine has previously been exploited to some degree in oncology. Neoplasms of NK cells are generally highly aggressive. However, current therapies (chemotherapy and/or radiotherapy e.g.) are suboptimal and overall survival is dismal. Effects of demecolcine on NK functions were studied using the aggressive NK leukemia cell line KHYG-1 as a model. Methods: KHYG-1 was treated with different demecolcine doses, 0.1 to 30 μM, for 12-15 hrs at 37 ^oC/ 5% CO2, washed 3-4X in PBS and compared to untreated cells in subsequent experiments. Microtubules (anti α-tubulin) and cytotoxic granules (anti perforin) were imaged by confocal microscopy. Viability, Annexin V and 7AAD staining, and receptor expression levels were analyzed by flow cytometry and cytotoxicity measured in a flow cytometric assay. Degranulation (anti perforin) after target contact and phosphorylation of the kinase ERK were assessed by immunoblotting.