Targeted Therapeutics: From Magic Bullets To Multifunctional Nanoparticles MOHAPATRA SS, MOHAPATRA S, LOCKEY RF
Joy McCann Culverhouse Airway Disease and Nanomedicine Research Center, VA Hospital and USF College of Medicine, Tampa, FL, USA
Background: The first ‘magic bullet’, arsphenamine, a synthetic organic arsenic compound, was discovered by Paul Ehrlich in 1909 and proved to be the best cure for syphilis at that time. The world of medicine has changed unimaginably since then, with the discovery of DNA as the genetic material, the evolution of biotechnology, and more recently the promising new dimensions of nanobiotechnology. This presentation intends to review how the state-of-the-art in therapeutics, irrespective of whether they are small molecular drugs or genes, deploy the multifunctional nanoparticles to achieve specific targeting of drugs.
Methods: A plethora of technological advances in genomics, including the sequencing of the entire human genome with its revelation of disease-related genes and the discovery of small interfering RNAs and micro RNAs have led to novel targets. Also, substantial progress has been made on the nanotechnology and nanoscience front in applying new knowledge to targeted gene and drug delivery strategies.
Results: A diverse array of human-friendly polymeric biomaterials has been developed into multifunctional nanoparticles, which can not only carry the drug payload but also specific targeting and imaging moieties. A myriad of cell targeting approaches have been tested including not only antibodies, but also small molecular ligands, peptides and other biologics. Examples of successful applications of combined genomics and nanotechnology will be presented.
Conclusion: Thus, multifunctional nanoparticles provide a platform and new vistas in cell-specific targeting of drugs and biologics and represents rebirth of ‘magic bullet’.
References: Mohapatra SS and Vergara H.S. Designer monoclonal Antibodies as drugs, the-state-of the-art. Expert Rev. Clin. Immunol 4(3), 305-307, 2008.
Wang X, Xu W, Mohapatra S et al. Prevention of airway inflammation with topical cream containing imiquimod and small interfering RNA for natriuretic peptide receptor.Genet Vaccines Ther. 15;6:7, 2008.
Kong X, Wang X, Xu W et al. Natriuretic peptide receptor a as a novel anticancer target.Cancer Res. Jan 1;68(1):249-56, 2008.
Zhang W, Yang H, Kong X et al. nhibition of respiratory syncytial virus infection with intranasal siRNA nanoparticles targeting the viral NS1 gene. Nat Med. Jan;11(1):56-62, 2005.
Methemoglobin As The Biomarker Of Environmental Oxidants And Precursor Of Adverse Effects Of Oxidative Sress On Mother And Fetus - Reasons For Its Early Detection And Therapy MOHOROVIC L1, MATTURRI L2 1Obstetric and Gynecological Primary Care, Labin, Croatia, 2Institute of Pathology, University of Milan, Italy
Background: Methemoglobin (MetHb) and its catabolic products are prooxidant. In the first blood circulation stage, the inhalation of nitrogen oxides transforms the hemoglobin into its pathological MetHb. Methemoglobinemia symptoms in “maternal preeclampsia“ are also common in severe anemia, preeclampsia and eclampsia, suggesting them to be a precursor for these conditions. The oxidants crossing the placental barrier cause “fetal preeclampsia”. The levels of MetHb and its catabolic products as bilirubin-biliverdin, CO and toxic Fe (III) with paramagnetic nature probably go over to BBB and provoke adverse effects on brain development.
Objective: The objective is to confirm the anatomo-pathological and genetic alterations that underlie sudden unexpected and unexplained perinatal and infant death and their correlation with environmental factors.
Methods and results: The MetHb level was determined from the samples that were taken three times, with a one-month pause between each test, for each pregnant woman (N=122) in the exposure period of power plant operation and in the control period when the power plant was closed (N=138). The significant positive correlation between the level of MetHb and the daily ground level concentration of SO2 was found (r=0.72, p<0.01). The reproductive loss was significant between the »control« (N=4) and »exposure« periods (N=10) (p=0.0369) and the frequency of stillbirths with the amount of MetHb >1.5 g/L in the exposure period was also statistically significant (p=0.0336). The test of chromosome aberration (SCI) was not significant in newborns whose mothers’ MetHb level was >1.5 g/L (N=36). We observed them by collecting data from hospitals, preschool and school services at health centers until they were eighteen years of age, and we found that the incidences of neonatal jaundice (p=0.034), later heart murmur (p=0.011) and dyslalia and learning memory mpairments (p=0.002) were significantly higher than in children born by control mothers (n=19).
Conclusion: The MetHb level is a useful biomarker but also has a degree of predictive validity. Our current research intends to study the brain morphological aspects of the development of both the autonomic nervous system and the cardiac conduction system and to individuate the environmental interactions resulting in the perinatal unexplained death, as well as to help find therapy, especially in underdeveloped countries with high maternal and perinatal morbidity and mortality.
Plant Alkamides Bioactive Molecules On A Wide Range Of Organisms MOLINA-TORRES J1, LOPEZ-BUCIO J2, RAMÍREZ-CHÁVEZ E1, HERRERA-ESTRELLA L3 1Cinvestav U, Irapuato, Irapuato, Gto, México. 2Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo., Ciudad Universitaria. Morelia, Mich, México. 3LaNGeBio, Cinvestav-Guanajuato Libramiento Norte, Irapuato, Gto, México
Along human history, different civilizations have traditionally used many plant species both as herbal medicines and as stimulants. The molecular activities that are responsible for these effects typically result from metabolites present in plant tissues. In many of these herbal remedies alkamides are present as bioactive components.
In recent years, it has become apparent that there are novel signaling molecules, such as N-acylethanolamides (NAEs), alkamides, glutamate and nitric oxide, which might play important roles in the regulation of morphogenetic and adaptive processes. Recent work with A. thaliana seedlings has provided evidence of the potent biological activities of some alkamides, such as affinin widest distributed alkamide in plants, and NAEs in seedling growth and root development.
In animals, NAEs acting as endogenous signaling molecules, and alkamides interact with cannabinoid receptors, which are coupled to signal. Hence, there is a possibility that cannabinoid signaling represents an evolutionary conserved pathway that modulates cellular and physiological processes in eukaryotes. In plants the formation of NAEs, structurally related to alkamides and to anandamide, was initially associated with germination and responses to pathogen attack.
Studies in our lab showed that alkamides in lower concentrations, but not NAEs, greatly stimulate roots and aerial parts development, however both groups of compounds in higher concentrations inhibit plants development. Two affinin-derived alkamides (N-isobutyl-2E-decenamide and N-isobutyl-decanamide) were found to be even more active than affinin in stimulating cell responses.
Unsaturated alkamides have been shown to be fungistatic and bacteriostatic. Additionally when N-isobutyl-decanamide is applied to A. thaliana seedlings cultured in vitro, an increased expression of defense response genes occurred in parallel with accumulation of salicylic acid and jasmonic acid.
Cell Cycle Kinases As Molecular Targets In Anticancer Therapy CARPINELLI C, MONTAGNOLI A, VALSASSINA B, BERIA I, FANCELLI D, PULICI M, VANOTTI E, MOLL J Nerviano Medical Sciences Srl., Nerviano (Mi), Italy
Methods: Small molecule kinase inhibitors were identified by screening a proprietary kinase targeted library of approximately 50.000 compounds using recombinant kinases in a biochemical screening format. After reconfirmation, hits were screened for cellular activity and mechanism of action in vitro and in vivo. Efficacy and PK/PD modeling of lead compounds were assessed in different tumor animal models. Toxicity was assessed in primary and secondary non-rodent species.
Results:Upon screening of our chemical library for Aurora, Polo and CDC-7 kinases we have identified potent and selective hits. These hits were optimized and SARs were developed. Upon lead optimization, small molecules were identified for all targets and were found to be highly efficient in vivo and are well tolerated. The most advanced compound, PHA-739358, was the first Aurora kinase inhibitor to enter clinical trials and is currently in phase II. An additional clinical opportunity is based on its cross-reactivity with wild-type and T315I mutant BCR-Abl, which will be shown and discussed.
Conclusions: Small molecules inhibiting different cell cycle kinases have potent anti-tumor activity, however the cellular responses and mechanisms of action are distinct for inhibitors of Aurora, Polo and CDC-7 kinases. This difference might be important for treatment of different indications or subsets of patients. It is expected that small molecules inhibiting these targets will add to the future armory of available anti-cancer agents.
All authors are full-time employees of Nerviano Medical Sciences Srl.
Expression Of MAGE-A12 In Oral Squamous Cell Carcinoma MOLLAOGLU N1,2, VAIRAKTARIS E3, NKENKE E1, FRIEDRICH WN1, RIES J1 1Friedrich- Alexander University of Erlangen/Nuremberg, Germany; 2Gazi University, School of Dentistry, Ankara, Turkey; 3University of Athens Medical School, Attikon Hospital, Athens, Greece
Background: Melanoma associated-A antigens (MAGE-A) are silent in normal tissues except testis. However, they are activated in a variety of different tumors. Thus, their expression is highly specific to cancer cells. Reverse transcription-nested polymerase chain reaction (RT-nPCR) is a highly sensitive technique that has been used successfully for the detection ofMAGE genes in tissue samples. Aim: To analyze the expression rate of MAGE-A12 in oral squamous cell carcinoma (OSCC) using a high sensitive RT-nPCR.
Methods: Total of 57 tissue samples obtained from patients with OSCC and 20 normal oral mucosal (NOM) probes of otherwise healthy volunteers were included to this study.
Results: No expression of MAGE-A12 was observed in the non-neoplastic NOM tissues. MAGE-A12 was expressed in 49.1% of the investigated tumor samples. The correlation between malignant lesion and MAGE-A12 detection was significant (p < 0.001).
Conclusions: 1) Results of this study may indicate MAGE-A12 as a useful additional diagnostic marker especially for the early detection of OSCC distinguishing neoplastic transformation and detection of occult and/or rare disseminated cancer cells. 2) MAGE-A12 expression in OSCC may also determine a new immunotherapeutic target and might be warranted to develop vaccine for OSCC.
Trans-Cinnamaldehyde From Cinnamomum Zeylanicum Bark Essential Oil Reduces The Clindamycin Resistance Of Clostridium Difficile In Vitro Shahverdi AR1, Monsef-esfahani HR2, Tavasoli F1, Zaheri A1, Mirjani R2, Mollazadeh Moghaddam K1 1Department of Pharmaceutical Biotechnology and Pharmaceutical Sciences Research Center and 2Department of Pharmacognosy,, Faculty of Pharmacy, Medical Sciences/University of Tehran, Tehran, Iran
Background: Therapy with antimicrobial drugs, such as clindamycin, that perturb the intestinal flora but fail to inhibit growth of other microorganisms can permit the proliferation of Clostridium difficile and the elaboration of exotoxin. Therefore, there has been increasing interest in the use of inhibitors of antibiotic resistance for use in combination therapy. The essential oil of Cinnamomum zeylanicum bark enhanced the bactericidal activity of clindamycin and decreased the minimum inhibitory concentration of clindamycin required for a toxicogenic strain of Clostridium difficile.
Methods: Thin-layer chromatography (TLC) analysis of the essential oil separated a fraction (Rf = 0.54) that was the most effective at enhancing the clindamycin antimicrobial activity. Using gas liquid chromatography and known standards, the active fraction was identified as trans-cinnamaldehyde (3-phenyl-2-Propenal). Combinations of clindamycin and trans-cinnamaldehyde were tested to determine the fractional inhibitory concentration (FIC) index by conventional checkerboard titration. The FIC index for Clostridium difficile was found to be 0.312, which confirmed the synergistic actions of clindamycin and trans-innamaldehyde.
Results: The presence of 20 µg/mL of trans-cinnamaldehyde decreased the MIC of clindamycin for Clostridium difficile sixteen-fold, from 4.0 to 0.25µg/mL.
Conclusion: low concentrations of trans-cinnamaldehyde elevate the antimicrobial action of clindamycin, suggesting a possible clinical benefit for utilizing these natural products for combination therapy against Clostridium difficile.
Development of SERCA Inhibitors Targeted towards Prostate Cancer Cells MØLLER JV1, BRØGGER CHRISTENSEN S3, OLESEN C1, WINTER AML2, SØHOEL H3, LIU H3, NISSEN P2 1Institute of Physiology and Biophysics, 2Department of Molecular Biology, PUMPKIN Research Center, Aarhus University; 3Department of Medicinal Chemistry, University of Copenhagen
The ability to produce well ordered crystals of sarcoplasmic reticulum (SERCA) Ca2+-ATPase has resulted in the elucidation of a vast number of the structures of this protein at atomic resolution, corresponding to its various phosphorylated / non-phosphorylated and inhibitor bound states. Thus the specific high-affinity binding site of the sesquiterpene lactone thapsigargin (TG), isolated from the mediterrenan plant Thapsia garganica, to the E2 conformations of SERCA 1a has been found in a preformed notch, located at the lipid-protein interface between the M3, M5, and M7 transmembrane segments of the Ca2+-ATPase (Olesen et al. 2004, 2007). Although the inhibition of SERCA pumps in both normal and cancer cells precludes the use of TG as an antineoplastic agent , this difficulty can be addressed in the case of prostate cancer by targeting these cells with an inactive prodrug derivative of thapsigargin where the butanoyl group of the guaianolide ring has been replaced by 12-aminododecanoyl conjugated to a BOC- peptide (Søhoel et al. 2006). After specific cleavage of the peptide moiety by the PSA (prostate surface antigen) protease, the liberated hydrophobic thapsigargin derivative can cross the plasma membrane to exert apoptosis on the prostate cells (Denmeade et al. 2003). Cocrystallization of the TG derivative with rabbit SERCA 1a at 3.3 Å resolution shows that the long flexible hydrocarbon chain of the inhibitor penetrates the transmembrane region of the protein, reaching towards one of the Ca2+ binding sites (Søhoel et al. 2006), confirming the potential of this strategy to kill prostate cancer cells. However, due to the strong hydrophobic properties the mentioned prodrug is not ideal for therapeutic use. At the meeting I will report on progress why TG is such a specific and high affinity inhibitor of Ca2+-ATPase, and also on our efforts towards development of a rational approach to synthetize more soluble and equipotent derivatives, suitable as candidates for prodrugs in the treatment of slowly growing and hormone insensitive prostate cancers.
References:Denmeade, S. R. et al. (2003) J. Natl. Cancer Inst.95, 990-1000
Olesen, C., Sørensen, T. L., Nielsen, R. C., Møller, J V. and Nissen, P. (2004) Science306, 2251-2255. Olesen, C. et al. (2007) Nature450, 1036-1042. Søhoel, H. et al. (2006) Bioorg. & Medic. Chem. 14, 2810-2815.
Inhibition Of Drug Resistance Of Bacteria And Cancer Cells MOLNÁR J, MÁNDI Y, ENGI H, SCHELZ ZS AND MUCSI I
Department of Medical Microbiology and Immunobiology, University of Szeged, Hungary
Background: Chemotherapy started with Ehrlich’s “Magic Bullets” and large number of antibiotics were developed on the basis of his ideas. Resistance to antibacterial and anticancer drugs appeared soon after the introduction of antibiotics and chemotherapeutics, resulting in non-treatable infections and malignancies. This paper reports on the reversal of the multidrug resistance (MDR) of bacteria and tumor cells.
Methods: Plasmid replication, partition and conjugal transfer were inhibited simultaneously in various bacterial species carrying plasmids encoding antibiotic resistance in the presence of phenothiazines and disiloxanes in vitro. The inhibition of the ABC transporter of the cancer cells was measured in the presence of phenothiazines and patented disiloxanes by rhodamine accumulation in flow cytometry in human MDR1 gene-transfected mouse lymphoma, human breast and colon cancer cell lines.
Results: Antiplasmid effects of substituted phenothiazines and structurally related compounds were demonstrated on Gram- positive and negative bacteria. Elimination of the antibiotic resistance of R plasmids of E. coli and other bacterial species was induced by phenothiazines. The effects of antiplasmid compounds are based upon intercalation into the superhelical form of plasmid DNA and the simultaneous inhibition of conjugal plasmid transfer. The phenothiazine promethazine synergized the antibacterial effect of gentamycin in chronic recurrent pyelonephritis in vivo. On the basis of structure-activity correlations, anthril-derivatives with antiplasmid activity were synthetized.
MDR is the main reason for the failure of cancer chemotherapy. The MDR1 activity of human MDR1 gene-transfected mouse lymphoma, human breast and colon cancer cells was inhibited in the presence of resistance modifier phenothiazines and disiloxanes. The disiloxane resistance modifiers synergized the antiproliferative effect of doxorubicin and taxol in vitro. In addition the disiloxane reduced the growth rate of cancer cells co-cultivated with normal cells (unpublished results).
Conclusions: The results of model experiments on the synergistic interactions between antibiotics, chemotherapeutics and resistance modifiers on bacteria and cancer cells can be exploited in rational drug design for combination chemotherapy.
