Conclusions: In the investigantion, all the isolated compounds were the first example of clerodane diterpenes isolated from a plant source. Allthough the cytotoxic activities of three novel clerodane diterpenes [1-3] were appreciable, none of them showed selectivity for any cell line, indicating that the compounds were functioning as a gerneral cytotoxic agents. This finding suggest that the cytotoxic diterpenes from Casearia genera of Australian Flacourtiaceae would be a good source of antineoplastice agents. Therefore, more extensive work should be carry out to isolated bioactive compounds from Australian Flacourtiaceae and to understand their machanism of action.
The Unexpected Hidden Face Of The Cephalosporin Antibiotic Ceftazidime: From Biological To Chemical And Physical Activities Against Oxidant Species Produced By Phagocytes
MOUITHYS-MICKALAD A1, DEBY-DUPONT G1,2, MATHY-HARTERT1 M, LAMY M1,2, SERTEYN D1,3, DEBY C1
1Centre for oxygen, R&D (CORD), Institute of Chemistry B6a 2Department of Anesthesiology and Intensive Care Medicine, 3Equine Clinic, University of Liège, Liège, Belgium
Background: Over several decades the fight against gram negative bacteria infections is a main challenge, and ceftazidime (CAZ), a cephalosporin’s family compound, is largely used in intensive care units to treat severe sepsis. Here in, we demonstrated that, beside its antibiotic effect, CAZ has unexpected antioxidant properties, and tried to better understand its mechanism of action.
Methods: Five in vitro (cell-free systems) and ex-vivo experimental models were designed to assess the capability of CAZ to protect 1) the trypsin (1.4 µg) inhibitory activity of alpha2-macroglobulin (2M) submitted to an oxidant inactivation by stimulated phagocytes (PMNs) 2) human endothelial cells in culture against the toxicity induced by myeloperoxidase (MPO) or stimulated PMNs 3) alveolar cells (A549, 1010 cells/ml; adherent or in suspension) against the oxidant damage induced by anoxia/reoxygenation, using oxymetry and EPR-spin trapping techniques 4) linoleate from lipoperoxidation induced by -irradiation, Fe2+/ascorbate system or ferryl species; 5) against hydroxyl radical or singlet oxygen (1O2) produced respectively by the Fenton (Fe2+/H2O2) and the Mallet (H2O2/NaOCl) reactions. All experiments were done at least 5 times.
Results: CAZ at 10-3M had a significant protective effect on 2M against oxidative stress; CAZ had a significant dose-dependent (from 10-5 to 10-3M) inhibitory capacity against MPO toxicity on endothelial cells and protected A549 cells from an excessive production of free radicals during anoxia/reoxygenation. In cell-free systems, CAZ at 10-4 M significantly protected linoleate from lipoperoxidation and had a unique 1O2–deactivating activity by scavenging 1O2 energy. CAZ was less active on ferryl species, acting by a chelating activity on iron.
Conclusions: Overall results indicate that: 1) CAZ, beside its antibiotic activity, has a protective activity against oxidant species 2) due to these unexpected properties, the clinical use of CAZ, which is essential to fight against bacteria, may have beneficial side effects in inflammatory and anoxia/reoxygenation situations where excessive activation of leukocytes and increased production of ROS are described.
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Pharmacogenetic and pharmacokinetic approaches after kidney and liver transplantation
MOURAD M
Department of Surgery, Surgery and Abdominal Transplantation Division, Université Catholique de Louvain, Cliniques Universitaires Saint Luc, Avenue Hippocrate 10, 1200 Brussels, Belgium. Michel.Mourad@chir.ucl.ac.be
Immunosuppressive drugs such as cyclosporine (CsA), tacrolimus (Tac) and sirolimus (Sir) are characterized by a narrow therapeutic index and display wide interindividual variability in the oral dose, to which many factors contribute. Special interest in polymorphisms in genes encoding biotransformation enzymes and drug transporters has offered new insights towards a better understanding of this variability. Several single nucleotide polymorphisms (SNPs) have been identified in both CYP3A4 and CYP3A5. A particular SNP within intron 3 of the CYP3A5 gene results in expression of the active CYP3A5 enzyme when associated with the wild-type CYP3A51 allele, and in production of a truncated protein when associated with the CYP3A53 allele. CYP3A5 SNP has been shown to influence the pharmacokinetics of calcineurin inhibitors (CsA, Tac) and Sir after solid organ transplantation. Other SNPs have also been identified in the ABCB1 gene encoding P-glycoprotein (P-gp), and those in exons 11, 12, 21 and 26 have been extensively studied in organ transplantation. However, the association between calcineurin inhibitor (CNI) pharmacokinetic parameters and the ABCB1 genotype/haplotype is still a matter of debate.
We observed that post-transplantation renal function could be affected in the long term by genetic polymorphisms of the donor in patients treated with CNIs, which has enhanced our understanding of factors affecting long-term graft survival. After liver transplantation, Tac concentrations in hepatic tissue showed a significantly better correlation with the severity of organ rejection than pre-dose blood levels. In addition, ABCB1 polymorphisms were found to significantly influence Tac hepatic concentrations.
In conclusion, CYP3A5 polymorphism is undoubtedly closely associated with Tac disposition. P-gp activity in the renal parenchyma appears to be a relevant factor linked to CNI nephrotoxicity, and ABCB1 genotyping of donors and recipients may serve as a marker for CNI toxicity risk assessment. However, a causal relationship between ABCB1 SNPs, P-gp activity, intrarenal CNI concentrations and drug-related nephrotoxicity still needs to be confirmed by further studies. The contribution of a genetic approach to pharmacokinetic modeling programs may thus optimize dosing strategies after organ transplantation.
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Role Of Ultrasound Waves In Enhancing The Effect Of Antitumor Drug
MOUSTAFA MM, FIKRY NM, ABDELKHALK MM
Univ. Alexandria, Alexandria, Egypt
Background: Chemical activation of drugs by ultrasound energy for treatment of cancers is a new field recently termed “sonodynamic therapy”. The combination of a drug and ultrasound has a killing effect on the cell . Despite this fact very little is known about ultrastructural changes of tumor cells after treatment with anticancer drug in combination with ultrasonocation. Also, the mechanism of sonodynamic action in tumor cells is poorly investigated. Aims: 1) To evaluate the role of ultrasonic waves in enhancing the effect of 5-fluorouracil (5-FU) as anticancer drug. 2) To study the physical parameters, which enhance ultrasonic effect on anticancer drug transportation through biological membrane.. 3) To propose optimized dosage regimens.
Methods: To evaluate the role of ultrasonic waves in enhancing the effect of 5-fluorouracil (5-FU) as anticancer drug, tumor growth, cell ultrastructure and temperature rises of ascites Ehrlich tumor implanted in mice were investigated at a frequency of 0.8 MHz. Different conditions of ultrasonic intensity (1, 2 and 3 W/cm2), sonocation time (1, 3 and 5 min) and waveform (continuous and pulsed) were studied. A total of 10 mg/kg body weight of 5-FU was IV injected into the mice bearing Ehrlich tumors on days of 1, 3, 5, 8, 10 and 12 of therapy. After 24 h of each injection with 5-FU, tumor sites were sonocated at room temperature of 22°C. Tumor volumes were monitored by using ultrasonic imaging system during treatment just before each 5-FU injection, and on the 15th day of treatment. Density and ultrasonic attenuation of excised tumor tissues were measured in vitro and used to estimate the temperature rises due to ultrasonic absorption.
Results: Tumor volumes were monitored by using ultrasonic imaging system during treatment just before each 5-FU injection, and on the 15th day of treatment. Density and ultrasonic attenuation of excised tumor tissues were measured in vitro and used to estimate the temperature rises due to ultrasonic absorption. Density and attenuation coefficients of excised tumor tissues were found to be dependent on the treatment regimen. The estimated rate of temperature rise and equilibrium temperature, and the characteristic time to reach equilibrium are given for each group. Results obtained indicate that tumor growth decreases with increasing of ultrasonic intensity and sonocation time. Tumor growth was delayed 4 to 6 days by combined treatment of 5- fluorouracil and ultrasound (US). Ultrastructure investigations of tumor cells showed severe damage in cytoplasmic organelles and cytoplasmic vacuoles that increased with increasing ultrasonic intensity and sonocation time. This damage appears as prominent crowded vacuoles among swollen ruptured organelles, chromatin fragments and severe increase in numbers of pyknotic and apoptotic cells.
Conclusions: The combination of 5-FU and ultrasound produced significantly greater antitumor activity than ultrasound or 5-FU alone.
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Investigations Into Sulfur Nanoparticles As Drug Carriers
MUELLER RS
Westfälische Wilhelms-Universitaet, Muenster, Germany
Background: Inorganic nanoparticles are used in the field of in vivo diagnostic nuclear medicine practice. E.g. 99mTc labeled sulfur colloid dispersions are used in sentinel node detection. After injection the nanoparticles are taken up by the lymphatic system. This uptake mechanism could also be used in the field of drug targeting. It is the aim of our studies to perform basic investigations into the potential of inorganic colloidal particles as drug carriers.
Methods: Sulfur colloids were prepared by redispersion of colloidal sulfur (method M1) or by synproportionation of sodium sulfide and sodium sulfite in a solution containing sulfuric acid (M2). The formed particles were used as reference. Caffeine was added during the particle formation process (M2). PCS (photon correlation spectroscopy) measurements were performed (Malvern Autosizer IIc) after filtration (1.2 resp. 0.45 µm cellulose nitrate). SEM (scanning electron microscopy) (Cam Scan, Electron Optics LTD) was used to visualize the nanoparticles, which had been collected on 0.1 µm filters (polyether sulfonate). The caffeine concentration was determined UV spectroscopically (Specord S100, Analytik Jena).
Results: Sulfur nanoparticles in colloidal range were formed. The size distribution was not small according to PCS results. The particles were stable enough to be collected on filters and examined using SEM. The formation of the sulfur nanoparticles was influenced by the presence of the model drug caffeine. Particles with a mean diameter of 73 ± 6 nm were measured (PCS, n=6). The polydispersity index is smaller compared to the value calculated for drug-free sulfur nanoparticles. The nanoparticle dispersions containing caffeine were further investigated (n=6). By using filters (0.45µm and 0.1 µm), fractions of nanoparticles were separated, the remaining particle dispersions were investigated. About 4.7 % (w/w) of the total caffeine amount was assigned to the colloidal fraction. To get information if caffeine interacts with the surface of the sulfur particles, caffeine was added to colloidal sulfur. Even after 2 hours, the absorption of drug was negligible.
Conclusions: The presence of the model drug caffeine has an influence on the particle formation of sulfur nanoparticles, which are manufactured by synproportionation. Surface adsorption as mechanism of interaction between caffeine and sulfur nanoparticles is not relevant.
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Teaching An Old Dog New Tricks: Intralesional Bleomycin Injection (IBI) Treatment Of Vascular Birthmarks
MUIR T1, SAINSBURY D , KESSELL G , FALL A , GUHAN A , MILLER V
James Cook University Hospital, Middlesbrough, United Kingdom
Background: We have provided a novel use to an old chemotherapeutic agent as a scar less injection treatment of vascular birthmarks for the last three years. 82 of 300 patients seen in our centre received intralesional Bleomycin injection treatment.
Methods: Clinical response, administered dose, amount of sessions and complications is recorded. The following pathologies were treated thus far: Hemangioma 18 pts, venous malformation 49, cystic hygroma 2, lymphatic malformation 6, Arterio-venous malformation 2, and mixed malformations 5.
52 of the 82 patients completed their treatment. Respiratory surveillance is provided by an adult and paediatric pulomonologist utilising the locally agreed Cleveland malformation surveillance protocol.
Results: 19 children and 36 adults completed treatment with a mean of 4.2 injection sessions. Treatment lasted for an average of 106 days. Complete resolution occurred in 56%, with an overall response rate of 96%. Skin ulceration occurred in 1 patient, blistering in 5, infection 1, swelling 1, headache 1, bruising 1, rash 1 and skin pigmentation occurred in 3 patients. One patient presented with a major complication needing ITU admission and ventilation following treatment of a pan-facial and thoracic lymphatic malformation. The maximum administered dose was 3mg/kg.
Conclusion: As a single site under the auspices of a multi-disciplinary team, the Cleveland Vascular Malformation Group, we have successfully treated complex and recurrent vascular anomalies. The data and outcome results provide a reference and experience of a national single site in treatment of these challenging lesions.
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Indocyanine Green: Revisiting An Old Friend
MUKHERJEE S, ROGERS MA, BUNIAK B
Liver Gallbladder Pancreas Center, Section of Gastroenterology and Hepatology, Department of Medicine, State University of New York Upstate Medical University, Syracuse, New York, USA
Background: Indocyanine green clearance, measured by percentage disappearance rate, detects alterations in liver function and may be used as a non-invasive determinant of hepatic reserve. The aims of this study were to compare liver histology and Child's-Pugh score with percentage disappearance rate (PDR) and determine which variables correlated with PDR.
Methods: Child's-Pugh score, liver function tests, liver biopsies and indocyanine green testing (0.5mg/kg) were performed in 102 consecutive patients with cirrhosis of diverse etiologies. Indocyanine green concentration was determined using spectrophotometric analysis (806nm) and plotted logarithmically with Michaelis-Menten kinetics to calculate the PDR. Liver biopsies were graded using the modified Knodell score to obtain a histological activity index.
Results: In multivariable analysis, percentage disappearance rate significantly correlated with Child's-Pugh score, albumin, bilirubin, prothrombin time and histological activity index. Albumin, prothrombin time and histological activity index were independent predictors of percentage disappearance rate in the final model (albumin p<0.01, prothrombin time p<0.046, histological activity index p=0.033), accounting for 46.2% of variability in percentage disappearance rate measurements.
Conclusion: Percentage disappearance rates correlated with Child's-Pugh scores in this series of cirrhotic patients. However, 46.2% of its variability was accounted for by albumin, prothrombin time and histological activity index.
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A Novel Biomimetical Transformation To Construct Natural Product Frameworks
MULJATI T, KALESSE M
Institute for Organic Chemistry, Leibniz University of Hannover, Hannover, Germany
The chemical synthesis of natural product and their derivates has become an integrated part of developing pharmaceutical drugs. Its role is especially important in producing a scarce, but biologically intriguing, natural product in larger quantities for further biological investigations and medicinal applications. [1] During the last century, the synthetic approach has been strived for to apply the biogenesis consideration, aiming at the simulation of biogenetic key step which mimic nature in its elegance and efficiency. [2] Such challenge has motivated our group to involve a biomimetic transformation in our synthetic plan of a potent anti-tumor agent hexacyclinic acid 1. [3,4,5]
Central to the strategy is the biomimetic Michael-Prins reaction to construct the C ring and to deliver the tertiary alcohol, resulting from water addition to carbenium ion intermediate.
Scheme 1. Biomimetic route utilizing Michael-Prins reaction in hexacyclinic acid 1 synthesis
Through our investigation, we were able to conduct the above reaction which provides not only a new C-C coupling reaction but also a Lewis acid-catalyzed reaction that could be carried out in aqueous media under a mild condition with a high level of catalyst recovery. This could give chance for pharmaceutical companies to synthesize efficiently and deliver more environmentally friendly reactions.
[1] K.C. Nicolaou, Proc. Natl. Acad. Sci. U S A. 2004, 33, 11928. [2] M. Torre, M. Sierra, Angew. Chem. Int. Ed. 2004, 43, 160. [3] R. Höfs, M. Walker, A. Zeeck, Angew. Chem., Int. Ed. 2000, 39, 3258. [4] T. Stellfeld, U. Bhatt, M. Kalesse, Org. Lett. 2004, 6, 3889. [5] A. Stelmakh, T. Stellfeld, M. Kalesse, Org. Lett. 2006, 8, 3485.
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Cell Penetrating Antibody Delivery To Intracellular Targets
MULLER S, KOHLER H, ZHAO, Y
Univ. of Kentucky, Lexington, KY USA
Background: A limitation of developing therapeutic antibodies specific for intracellular structures is to allow antibodies to enter cells without loosing cell viability. The objective of this project was to create antibodies for intra-cellular targets to control cell proliferation.
Methods:. Trans-membrane antibodies were generated by photo-affinity cross-linking a 17mer peptide derived from human sarkosi sarcoma virus. Targeting of antigens inside live cells is demonstrated by confocal microsopy, and functionally by inhibition of apoptosis.
Results: Trans-membrane penetrating (TMP) antibodies stained in live cells specifically actin and paxillin while naked antibodies did not. TMP antibodies also did not affect cell growth in culture. Furthermore, TMP modified anti-caspase-3 antibodies were shown to penetrate human T cell lymphoma cells, and rapidly inhibit actinomycin D-induced apoptosis.
Conclusions: Membrane penetrating peptide-modified antibodies are endowed with a potency of targeting intra-cellular antigens in living cells and tissues, suggesting the clinical potential of immuno-therapeutic TMP-antibody delivery by cell-penetration.
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