PRASAD KN, SEN M.
Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India
Background: The World is facing the problems of growing antimicrobial resistance in almost all categories of human pathogens (bacteria, fungi, viruses and parasites), difficult to treat infections and slow drug development. The problem has become more complicated due to increase in immunocompromised and critically ill patients. Infectious complications associated with multidrug resistant (MDR) organisms lead to high morbidity and mortality, and increased health care cost. This update aims to review our experience on the growing problems of multidrug resistant bacteria and their clinical implications.
Methods: This study is data analysis of last 5 years on antibiotic resistance in clinically important bacterial pathogens at our centre and review of literatures. Resistance was determined following standard screening, confirmatory phenotypic and molecular methods.
Results: Isolation of pathogenic bacteria from different anatomic sites in 2003 through 2008 ranged from 26% to 33%. The increasing trends of bacterial resistant to almost all classes of antibiotics were observed. The resistance rate to important bacterial pathogens were as follows: extended spectrum ß-lactamase and AmpC ß-lactamase producing Enterobactariaceae 78%, metallo ß-lactamase producing Pseudomonas and Acinetobacter species 31% and 21% respectively, methicillin resistant Staphylococcus aureus 51%, vancomycin resistant entercocci 11%. Percent change in resistance rate for different pathogens over the period ranged from 6 to 18%. Prior use of antibiotics, presence of invasive devices, prolonged hospitalization, admission in ICUs and ventilatory support were associated with antimicrobial resistance. Mortality rate was reduced by almost 50% and hospital stay by several days in patients who received appropriate antibiotic therapy. Mortality was significantly higher in patients already colonized with MDR bacteria prior to infection.
Conclusions: The trends of bacterial pathogens resistant to almost all classes of antibiotics are increasing with adverse clinical outcome. The key to address this escalating antimicrobial drug resistance problem lies in: 1) judicious use of available antibiotics with the principle of “hit fast and hit hard” with appropriate antibiotics, 2) avoidance of excessive and unnecessary use of antibiotics and 3) adoption of stringent infection control measures.
In vitro hypothesis, in vivo veritas. Success and failure of Imatinib incancer target therapy PRICL S MOSE Laboratory, DICAMP, University of Trieste, Trieste, Italy
Background: Imatinib is currently registered for two major indications: (a) monotherapy in chronic myeloid leukemia (CML) and (b) monotherapy in c-Kit (CD117)-positive unresectable or metastatic gastrointestinal stromal tumors (GISTs). Imatinib mechanism of action is to bind to specific tyrosine kinases (Bcl-Abl in CML and c-Kit in GISTs), thereby blocking the corresponsing signaling for cell growth and proliferation of malignant cells in CML and GIST. Imatinib is an effective drug; nevertheless, resistance developes over time in many patients. Although kinase overexpression and gene amplification have been observed, the most common event in resitance is the occurrence of mutation(s) in the corresponding genes.
Methods: Clinical, biochemical and molecular modeling analyses of some important successful/unsuccessful cases observed during imatinib therapy of GIST patients are reported. Transient transfection experiments with plasmids carrying different patients KIT acquired point mutations were performed along with immunoprecipitation of total protein extracts, derived form imatinib treated and untreated cells. The molecular mechanics/Poisson Boltzmann Surface Area (MM/PBSA) computational techniques were applied to study the interactions of the wild-type and mutated receptors with Imatinib at molecular level.
Results: KIT phosphorylation was detected in cells transfected with vectors carrying the specific mutant genes. Imatinib treatment demonstrated that some mutations were insensitive to the drug at all applied concentrations, while others were inhibited by imatinib, although to different extent. Modelling of the mutated receptors revealed some motations substantially modify the protein binding pocket, thus hampering inhibitor binding, whilst others induce only relatively confined structural changes, still compatible with drug binding.
Conclusions: The results obtained from the clinical/biochemical analysis on mutated receptors testing the actual imatinib inhibitory efficiency coupled with molecular modelling highlighted the streght and weakness of this inhibitor towards c-kit mutated isoforms. Therefore these investigation ensemble could be of help in the design of new drugs and give important information to medical oncologist indicating the most suitable dose for escaping secondary resistance.
The Discovery of New Scaffold Antibacterial Agents PRIMEAU J AstraZeneca R&D Boston
At AstraZeneca a key strategy, used to discover and develop antibacterial agents that will address the growing challenge of bacterial resistance, has focused on the design of new chemical scaffolds for new or under-exploited bacterial targets. The application of a diversity of lead generation techniques such as HTS and fragment based screening (using NMR and high concentration techniques), supported by strong structure based design capabilities has provided a range of novel compound starting points that have yielded novel lead scaffolds for these targets. This presentation will describe some of our efforts to date to transform these novel lead scaffolds into compounds that display excellent on-target potency, antimicrobial activity and in more recent cases, efficacy in animal models bacterial disease.
Novel biologic therapies for psoriasis PROHIC A Department of Dermatovenerology, University Clinical Center, Sarajevo, Bosnia and Herzegovina
Psoriasis is a chronic, inflammatory skin disease that usually necessitates treatment over the course of patient’s lifetime. Although topical medications usually suffice, about 25% patients with moderate-to-severe psoriasis will need additional systemic therapy, phototherapy or both. The long-term continuous use of traditional systemic therapies such as methotrexate, cyclosporine, oral retinoids and phototherapy (PUVA, which is psoralen plus ultraviolet light) is often limited due to potential organ toxicity, myelossuppression and carcinogenicity. The recognition of psoriasis as a T-cell mediated disease has led to the development of the new biologic agents that specifically target key steps of the immune pathways. This therapeutic approach is in contrast to current systemic therapies that act predominantly on hyperkeratinizaton and epidermal infiltrate, or broadly and non-specifically suppress the immune system. Biologics are pharmacologically active proteins extracted from animal tissue or synthesized through recombinant DNA techniques. They are designed to mimic the action of normal human proteins or to interact with circulating proteins or cellular receptors. There are three distinct classes of biologic agents: monoclonal antibodies, fusion proteins and recombinant cytokines.
Several biologics agents are now recognized for the treatment of moderate to severe psoriasis and/or psoriatic arthritis. These agents can be categorized into two broad classes based on their mechanism of action: the T cell inhibitors (alefacept and efalizumab) and TNF-α inhibitors (etanercept, infliximab and adalimumab). The aim of these new therapies is to improve the treatment of psoriasis, particularly moderate to severe psoriasis, with agents that are well tolerated and safe for long-term use. Potential limitations in the use of biologic agents include the high annual costs for treatment, lack of long-term follow up and selective nature of the patient populations thus far.
Application of Prodrug Strategies to Create Magic Bullets for the Treatment of CNS Maladies PROKAI L Department of Molecular Biology & Immunology, University of North Texas Health Science Center at Fort Worth, USA.
Background: Finding pharmacological interventions that would truly meet the definition of magic bullet in treating maladies affecting the central nervous system (CNS) has been challenging. Targets for interventions are generally not specific in the CNS and, in addition, protected from uncontrolled exposure of substances delivered into the systemic circulation by the blood-brain barrier (BBB). Although many drugs cross the BBB and exert therapeutic CNS-effects, their systemic exposure that impacts the rest of the body is unavoidable.
Methods: Strategies that, along with in vivo conversion of an inactive precursor (prodrug) to the active drug, rely on the manipulation of both the influx and efflux of substances across the BBB or, alternatively, site-specific activation of the prodrug may create magic bullets for the CNS. In this presentation, design and in vitro/in vivo evaluation of prodrugs that confine the action of neuropeptides (with focus on thyrotropin-releasing hormone, TRH) and estrogens are featured as respective examples for these strategies.
Results: For hydrophilic compounds such as peptides that practically do not cross the BBB, prodrug design has been the most versatile strategy to target them into the CNS. Simultaneously controlling activity, metabolism, transport and target-site retention via chemical manipulations is the key for creating magic bullets from neuropeptides such as TRH with promise to treat various neurological diseases (motorneuron diseases, various forms of dementia and brain trauma). For lipophilic compounds such as estrogens, profound endocrine responses by off-target peripheral tissue burden therapeutic intervensions for disorders of central origin that could be ameliorated by them (menopausal symptoms, impaired cognition, ischemic stroke, etc.). The success of CNS-selective estrogen therapy by a prodrug approach rests on the proper alteration of BBB-transport properties along with the facilitation of specific metabolic conversion(s) in the target tissue versus systemic bioactivation.
Conclusions: Various ways of creating magic bullets for the treatment of CNS maladies by the prodrug approach have been conceived. Although much remains to be learned about their merits, progress in the field has been steady, which clearly warrants continued exploration and development.
Ackowledgements: Supported by the National Institute of Health (grant numbers MH59360 and NS044765) and an endowed professorship (BK-0031) to L.P. from the Robert A. Welch Foundation.
Cellular Immune Toxicity of Alcohol and Cocaine: Medical Practice Based on the Evidence vs. Evidence-Based Medical Practice PROLO P1,CHIAPPELLI F2 1. Dip. Sanità e Socialità Canton Ticino, CH & Div. Oral Biol & Med, UCLA, 2. Division Oral Biology & Medicine, UCLA, Los Angeles, CA USA (fchiappelli@dentistry.ucla.edu)
In 1906, Prof. Ehrlich shared the Nobel Prize in medicine for his pioneering research on the immune system. Largely based on his seminal work, we now realize that immune responses are complex biological processes modulated by intertwined epigenetic, genomic, molecular, biochemical, cellular and physiologic regulatory mechanisms. When acting in concert and appropriately, these processes converge toward efficient, rapid and complete removal of the invading pathogen. Immune events are delivered by several cell populations that work in conjunction with soluble factors, including antibody species that recognize and target individual pathogens. Prof. Ehrlich termed this remarkably specific process the "magic bullet". Our understanding of how immunity works has improved significantly over the past decades, and the "magic bullet" theory is today the prevalent model for treatment intervention in infectious diseases, cancer, oral pathologies, and even certain neurodegenerative pathologies (e.g., Alzheimer's disease). Illicit drugs of abuse, often co-abused with alcoholic beverages, interfere significantly with magic bullet-driven treatment interventions. Reports describe the many benefits of detoxification programs, but do not concur with respect to immune ameliorations.
The contemporary model of clinical intervention is grounded on the systematic evaluation of the best available evidence, and the judicious integration of appropriately revised clinical practice guidelines in the evidence-based treatment decision-making process. We present a systematic review of the available evidence with respect to the immunotoxicity of alcohol and joint alcohol/cocaine abuse, and of the immune benefits of detox programs, followed by level of evidence analysis, acceptable sampling analysis and meta-analysis. We demonstrate the practical usefulness of our findings in the context of the novel evidence-based model for XXI Century medical practice.
Discovery of the HCV NS3/4A Protease Inhibitor, Boceprevir (SCH503034). Key Steps in Structure-Based Optimization.
PRONGAY AJ Schering Plough Research Institute, Cambridge, United States of America.
The Hepatitis C Virus (HCV) infects about 200 million people worldwide. The current combination therapy of pegylated-interferon- and ribavirin gives a 70-80% sustained virological response against most genotypes, but only 40% against genotype 1. Finding a more effective anti-HCV treatment has been a major objective of pharmaceutical companies for the past decade. In an effort to find a “Magic Bullet”, a number of the enzyme activities of HCV have been targeted. The approach undertaken at Schering-Plough Research Institute has been to use structural information to assist the more traditional assay-based methods to move compounds that inhibit viral enzyme activity from a “Hit” to a “Lead” state, then through optimization into a clinical candidate. This research paradigm has resulted in an inhibitor of the HCV NS3 protease entering Phase III Clinical studies. An overview of this Structure-Based Drug Design process will be presented.
The Potential of Orally Presented Mistletoe Lectins in Cancer Therapy. PRYME IF University of Bergen, N-5009 Bergen, Norway.
Background: Hitherto mistletoe preparations have been essentially administered by subcutaneous (s.c.) injection. This route, however, has clear limitations. Evidence now shows that a preparation containing mistletoe lectins (MLs) can be presented orally achieving the same results as s.c. administration. Being heavily glycosylated MLs can be taken orally since they are both resistant to low pH in the stomach and are unaffected by proteolytic enzymes.
Methods: Experiments were performed on female NMRI mice. Mice were initially maintained on a standard pellet diet with free access to water. Groups of five mice were prefed for 3 days on a Lactalbumin based, semi-synthetic diet (La). All mice were then injected s.c. with 2 x 106 Krebs II Non-Hodgkin´s Lymphoma (NHL) tumour cells. One group was switched to an ML-1 diet (lectin concentration : 1.7mg/ kg diet) a second (control) group was continued on the La diet. After 11 days animals were sacrificed and solid s.c. tumours were excised, weighed and prepared for histological examination.
Results: Following binding to specific receptors ML-1 induces a biological response. Immunomodulatory effects are initiated resulting in both activation and an increase in the population of natural killer cells. Many NHL cells enter the apoptotic pathway. An anti-angiogenic response severely curtails tumour growth. An interesting property of ML-1 is modulation of tumour characteristics seen as an increased level of differentiation.
Conclusions: In addition to the effects mentioned in the Results section ML can exert a cytotoxic effect on sensitive cells (e.g. cancer cells) bearing surface receptors to which the B-chain can bind. On internalization of the A-chain, and its activation, a RIP effect (ribosome-inactivating protein) is exerted leading to an induction of apoptosis, cumulating in tumour cell death. An advantage of using the oral route is that MLs can come into direct contact with tumours that are localized in the oral cavity, oesophagus or gastro-intestinal tract and thus be able to exert a direct cytotoxic effect on tumour cells. A further advantage of using the oral route is that injection is avoided and the involvement of health personnel is thus unecessary. It is proposed that the use of an oral mistletoe lectin-containing preparation would provide an excellent alternative, or supplement, to conventional forms of cancer treatment.
Virus-Like Particles As Magic Bullets For Immune System PUMPENS P, GRENS E
Latvian Biomedical Research and Study Centre, Riga, Latvia.
The development of genetic engineering techniques in the 1970s offered a broad range of applications, which were immediately followed by the expression of viral and non-viral genes in efficient heterologous expression systems, first of all, in bacteria and yeast. Special attention has been devoted to the synthesis of viral structural proteins as constituents of viral capsids and envelopes with their subsequent spontaneous self-assembly into correctly organized virus-like particles (VLPs). Food and Drug Administration’s (FDA)-approved vaccines against hepatitis B and human papilloma viruses represent genetically engineered VLPs generated in heterologous expression systems. VLP-based vaccines are also being developed against malaria, HIV/AIDS, hepatitis C, human and avian influenza, as well as against many other infectious and non-infectious diseases. Moreover, VLPs from almost all classes of viruses are being evaluated now or have just been adopted to use as a scaffold for presentation of foreign immunological epitopes on their surface. VLP technologies possess obvious advantages for generation of safe and efficacious prophylactic and therapeutic vaccines. First, the repetitive antigenic structure of VLPs makes them highly immunogenic. Second, chimeric VLPs are lacking viral genomes or genes and are non-infectious, although they are mimicking infectious viruses in their structural and immunological features. Third, VLPs are generated by highly efficient heterologous expression of the cloned viral structural genes with subsequent quantitative in vivo or in vitro self-assembly of their products. Fourth, VLPs can be obtained by simple and efficient purification procedures. A broad range of viral structural proteins is able to form autologous VLPs consisting solely of structural protein(s) of the target virus. Many of them have been tested successfully for the construction of chimeric VLPs retaining their VLP-forming ability, but carrying foreign epitopes. VLP technologies allow the generation of (1) uniform chimeric VLPs consisting of identical fusion protein subunits, (2) mosaic VLPs consisting of carrier and fusion protein subunits, and (3) pseudotyped VLPs consisting of non-fused autologous and foreign proteins. VLPs can be used for a broad range of applications, including nanotechnology, but first of all for vaccine development, as magic bullets for immune system.
Elucidating Inhibitor-enzyme Interaction of Highly Potent Non-nucleoside Reverse Transcriptase Inhibitors Active Against Wild-type and Mutant HIV-1 Strains: Computer-aided Molecular Design Approaches PUNGPO P1, PUNKVANG A1, SAPARPAKORN P2, WOLSCHANN P3,HANNONGBUA S2 1Faculty of Science, Ubonratchathani University, Warinchamrap, Ubonratchathani, Thailand; 2Faculty of Science, Kasetsart University, Bangkok ,Thailand;3Institute for Theoretical Chemistry, University of Vienna, Vienna ,Austria
Background: Because of the drug resistance, the treatment for AIDS still remains the worldwide medical problem. Efavirenz, a second-generation NNRTI, has recently been approved to treat HIV-1 infection with the high inhibitory activity. However, the efficacy of efavirenz is significantly diminished by the rapid drug-resistance mutations of HIV-1 RT, in particular the K103N. Thus, it is necessary to find new and more effective inhibitors remaining active across these virus mutations. To gain an insight into the potential binding orientation and the interaction of efavirenz derivatives with HIV-1 RT, docking studies, 3D-QSAR and quantum chemical calculations (QCC) were performed for efavirenz derivatives in the WT and K103N HIV-1 RT binding pocket.
Methods: The starting geometry of efavirenz was taken from X-ray crystallographic data. Efavirenz compounds were built and fully optimized at HF/3-21G level. Docking studies were carried out for efavirenz derivatives in the WT and K103N HIV-1 RT binding pocket. Based on the docked binding conformations, 3D-QSAR methods using CoMFA and CoMSIA were applied. The individual interaction between some derivatives and the surrounding amino acid in the WT and K103N binding pocket were investigated using QCC at single point calculation of MP2/6-31G(d) levels of theory.
Results: The potential binding orientation of the inhibitors in the binding pockets could be identified, by using docking studies. The docking results provide additional insight into essential inhibitor-enzyme interactions for different types of wild type and mutant type of HIV-1 RT. Based on the docking conformations, the reliable and predictive CoMFA and CoMSIA models of efavirenz derivatives for the WT and K103N RT inhibition were derived. The models are successfully used to discriminate between the structural requirements for WT and K103N inhibitory activities. Moreover, the interaction energy trend calculated from QCC of the inhibitors and individual amino acid residues in the binding pockets is informative to highlight particular ligand-receptor interaction in molecular level. The results derived from all approaches validate each other and agree well with the ligand-receptor complex interaction derived from the X-ray crystallographic data.
Conclusions: The molecular docking calculations, 3D-QSAR analyses and QCC were successfully combined to investigate the interaction and the relationship between structural requirements of efavirenz derivatives for WT and K103N HIV-1 RT. Consequently, the obtained results enable to provide beneficial guidelines to design novel compounds with higher anti-HIV–1 RT activities against WT and K103N RT.
Molecularly Imprinted Polymers (MIPs) for the Drug Targeting PUOCI F Università della Calabria, Arcavacata di Rende (CS), Italia;
Background: Specific molecular recognition is a fundamental requirement of living systems and, through millions of years and countless rounds of evolutionary optimization, biology has become a master of the art.
At the cellular and sub-cellular level the fundamental processes of life, information transfer and reaction catalysis, rely on the specific interaction of low molecular weight molecules with macromolecular hosts. In the majority of such events the macromolecule is a protein.
Processes as diverse as neural transmittance, respiration, immune defence, cellular differentiation and nutrition all rely on the basic principle of specific molecular recognition.
It is therefore not surprising that scientists have invested huge amounts of time and effort, initially into harnessing the potential of biological molecular recognition, antibodies and enzymes, and more recently in trying to mimic these properties in synthetic materials. One of the most promising of these ones is molecular imprinting.
Molecular imprinting is a very useful technique for incorporating specific substrate recognition sites into polymers. The molecular recognition characteristics of these polymers are attributed to the complementary size, shape, and binding sites imparted to the polymers by the template molecules
The concept of molecular imprinting has a long history dating back to the early 1930s. However, the preparation of organic polymers with molecular recognition was first reported only in 1972 ,initiating the molecular imprinting technology as it is known today. Molecular imprinting has now become an established method and has also been applied in the areas of synthetic chemistry and analytical chemistry. MIPs have been used as chromatographic stationary phases for enantiomeric separations , and for solid-phase extraction , catalysis and sensor design, as well as for protein separation, as receptor, antibody and enzyme mimics and recently Drug Delivery Systems too .
But it is perhaps in the area of drug delivery, in particular “intelligent drug release” and “magic bullet” drug targeting, that significant future opportunities lie.
Although relatively few studies have been reported in the literature about the intelligent drug delivery and targeting, they represent an important starting point for the development of new generations of intelligent and selfregulated drug delivery systems.
