Predicting Optimal Endocrine Treatment Of Hormone-Dependent Breast Cancer Through The Right Bullets And Targets MICHALIDES R, ZWART W, NEEFJES J, KOK M, LINN S
The Netherlands Cancer Institute, Amsterdam
Functioning of the estrogen receptor (ER) is a prerequisite for outgrowth of hormone-dependent breast cancer. Anti-estrogens such as tamoxifen, inhibit ER functioning by inducing a structural alteration of the ER, which results in a reduced interaction with the transcription factor complex, ultimately leading to cell death.
We found that modification of the ER by phosphorylation prevents the structural alterations that are the result of binding of anti-estrogens to ER. This converts the anti-estrogen tamoxifen into an agonist. Each different anti-estrogen demands for its specific modification(s) to render ER resistant, providing a resistance “signature” (the target) for each anti-estrogen (the bullet).
We generated an antibody that detects one of these modifications; the ER phosphorylated at Serine 305 by Protein kinase A, and could identify resistance to tamoxifen in a group of breast cancer patients. Modifications in ER by phosphorylation provide a diagnostic tool for personalized breast cancer treatment, since alternative anti-estrogens are at hand for the signature-positive subgroup. They also guide optimal development of novel anti-estrogens.
Supported by TIPharma and KWF.
Analysis Of Linezolid Chiral And Achiral Impurities By Capillary Electrophoresis MICHALSKA K1, TYSKI S1,2 1Department of Antibiotics and Microbiology, National Medicines Institute, Warsaw, Poland; 2Department of Pharmaceutical Microbiology, Medical University of Warsaw, Warsaw, Poland
Rapid growth of pharmaceutical industry as well as the appearance on the market of large number of new medicinal products, combined with growing knowledge about drugs efficiency and safety, cause the necessity of introducing and developing new analytical techniques. Capillary electrophoresis (CE) is a relatively modern analytical technique which enjoyed for the tremendous interest in the last years.
Linezolid is the first available oxazolidinone antibacterial agent, showing strong activity against Gram-positive pathogens, including multidrug-resistant strains. Linezolid contains a centre of asymmetry at C-5 of the oxazolidinone ring imparting chirality in the molecule. Separation of enantiomers is vital in the pharmaceutical chemistry. The stereochemistry of such compounds can affect their biological activities, therefore developing of method for their purification and separation is crucial. The separation of chiral compounds can be achieved in CE by addition of appropriate chiral selector to the background electrolyte (BGE).
The first stage on linezolid study was to elaborate principles of CE enantioseparation of S-linezolid (biologically active form) from its impurity (R-isomer) and to use this method for quantitative analysis of R isomer only. A simple, fast, accurate, precise chiral CE method was elaborated using 27.5 mM heptakis-(2,3-diacetyl-6-sulfato)-ß-cyclodextrin (HDAS-ß-CD) dissolved in 50 mM borate buffer, pH 9.0 and CE in 15°C, normal polarity, uncoated capillary. Effect of HDAS-ß-CD concentration in BGE low pH was also studied.
NMR study and molecular modeling was performed to improve the level of understanding of the chiral recognition process occurring between linezolid and applied HDAS-ß-CD. NMR spectrometry allowed to estimate the 1:1 complex stoichiometry and to determine the binding constants.
Besides, NMR and molecular modeling were applied for investigations of the host-guest complexation of R- or S-linezolid with HDAS-ß-CD.
The next step of the study was to elaborate simplest and reliable method for determination of linezolid and its 14 achiral impurities.
The application of a sweeping preconcentration enabled fast, simple, accurate, and precise separation of linezolid from all its achiral impurities by CE using UV absorption detection method. Satisfactory separation was possible after less than 19 min of electrophoresis.
Capillary electrophoresis proved to be suitable tool for chiral and achiral determination of linezolid in medicinal products.
Recombinant Factor VIIa (rFVIIa) For The Treatment Of Bleeding In Abdominal Surgery And Cardiac Surgery MICHALSKA GM, HAMERA I
University Hospital nr1, Szczecin, Poland
Background: Factor VIIa plays a key role in hemostasis.The aim of this study is clinical evaluation of efficacy of rFVIIa in the treatment of bleeding during and after abdominal and cardiac surgery.
Methods: Meta-analyses of case series studies (n==67) on the treatment of bleeding with rFVIIa with regarding reduction or cessation of bleeding and mortlality. We used the questioners of Novo Nordisk to asses the indications and effectiveness of treatment. We compared the amount of blood loss within 12 hours(hrs) before and within 12 hrs after giving rFVIIa, the dynamic of bleeding. In 10 patients the bleeding started after surgery intraoperatively, in 57 patients severy hemorrhage developed in the postoperative period. We also compared full blood count and laboratory coagulation profile parameters before treatment, 2 and 12 hrs after treatment. In cardiac surgery rFVIIa was administered in 5 to 49 minutes after neutralization of heparin with protamine sulfate. The dosage of rFVIIa was 39.23 ±20.70 μ/kg We used students t-test for statistical analysis.
Results: After administration of first median dose (14.45-81.35μg/kg) rFVIIa bleeding stopped in 47 patients. Markedly decreased in 15patients. 5 patients who didn´t benefit from initial rFVIIa administration received additional drug in dose 38.25 µg/kg with good results.
The average blood loss within 12 hrs before treatment was 2510.00 mL ±1642.07 mL and the average blood loss within 12 hrs after treatment was 1057.75±810.67mL The average dynamic of bleeding before treatment was 216.35±138.82 mL/h and 87.90±67.82mL/h after treatment. Transfusion requirements were reduced for PRBC, FFP, plateletes, and crystalloid and/or colloids. Reduction in transfusion requirements was statistically significant (p≤0,05).
Conclusions: The meta-analysis of series cases showed that in a mean of 85% patients rFVIIa achieved at least a reduction of bleeding and reducing the need for hemotransfusions.
Novel Anti-Cancer Peptides That Cause Reversion, Lysis And Necrosis Of Tumor Cells But Have No Effect On Normal Cells MICHL J, YAZDI ES, ADLER V, SOOKRAJ K, ZENILMAN M, BOWNE W, PINCUS MR
Departments of Pathology, Anatomy and Cell Biology, Microbiology and Immunology, and Surgery, SUNY Downstate Medical Center, Brooklyn, NY; Departments of Clinical Laboratory Medicine and Surgery, VA Harbor View Medical Center, Brooklyn, NY, USA
During the last several years an increasing number of peptide-based therapeutics have been developed and studied for their efficacy in the treatment of cancer. Many of these peptides are directed against molecules that play central roles in cell growth such as ras-p21 protein and p53. In cancer, the genes coding for these two proteins are most frequently subjected to mutations, deletions or inactivation, events that are at the center of the affected cells’ malignant behavior. Our interest is in the identification of peptide domains in each of these two proteins that lead to the alteration of each protein’s conformation and, consequently, its biological function in response to oncogenic amino acid (AA) substitutions. We have employed computer-based modeling to design peptides from the ras-p21 and p53 protein that block proliferation of cancer cells. By first generating and comparing low energy average structures for oncogenic and wild-type proteins using conformational energy calculations we identified peptide domains from the molecules regulatory sites and that were synthesized for the studies described in this presentation. To accomplish trans-membrane transport, the peptides were linked by their carboxyl terminus to a leader sequence, penetratin, from the antennapedia protein.
We have synthesized two ras-p21 peptide domains, PNC-7 (AA 35-47) and PNC-2 (AA 96-110). The PNC-7 domain of ras-p21 is known to participate in the binding of SOS-guanidine nucleotide exchange protein, GAP, PI3K and RBD of raf-p74. The PNC-2 peptide domain participates in the binding of ras-p21 to the jun-protein and/or the jun-N-terminal kinase. We also synthesized two p53 peptide domains, PNC-27 (AA 12-26) and PNC-28 (AA 17-26) which include the AA region that is directly involved in the binding of p53 to hdm2 which prepares p53 for ubiquitination, cytoplasmic delivery and degradation. Each of the four synthetic peptides was linked to penetratin. Similarly, control peptides including scrambled AA sequences of the selected peptide domains were linked to penetratin. We have tested these peptides’ efficacy in vitro and in vivo against rodent and human cancer cells as well as against normal untransformed cells. The results showed that both PNC-7 and PNC-2 induce phenotypic reversion to their untransformed phenotypes of ras-p21val12-transformed BMRPA1.TUC3 pancreatic cancer cells and of ras-transformed HT1080 human fibrosarcoma cells. Furthermore, both peptides were potently cytotoxic to human MIA-PaCa-2 pancreatic cancer and U-251 astrocytoma cells while they had no effect on the growth of untransformed normal cells. Control peptides showed no effect on tumor cell growth or survival. We then tested the two p53-derived peptides PNC-27 and PNC-28, that contain the p53 hdm2-binding domain, and observed that they kill effectively a variety of cancer cells (cell lines) and primary cancer cells in vitro. We have also shown that PNC-28 is cytotoxic to the metastatic pancreatic cancer cell line BMRPA1.TUC3 in vivo without adverse side-effects on the host. Most remarkably, neither peptide has any effect on the viability or growth of untransformed normal cells including hematopoietic stem cells. Several control peptides including PNC-29 (pepide from cytochrome p450), scrambled PNC-27 and the penetratin sequence itself had no effect on cancer cells. We further showed that PNC-27 and PNC-28 induce rapid and complete tumor cell necrosis, and not apoptosis, as shown by the early (1-8h) release of LDH by PNC-27- and, respectively, PNC-28-treated cancer cells in the absence of induction of caspase(s) and of DNA laddering. Pursuing the unique specificity of the PNC-27 and PNC-28 effect on tumor versus normal cells, we identified by immunoblotting hdm2/mdm2 antigen in the plasma membrane of human and rodent cancer but not of normal cells. Moreover, confocal immuno-fluorescence microscopy showed green-(FITC)-labeled anti-PNC-27 antibody (Ab) on the plasma membrane tumor cells co-localized with red-(TRITC)-labeled andi-hdm2 Ab. This finding may help explain the selectivity of PNC-peptides for cancer cells. Ultrastructural studies using gold-conjugated hdm2 and PNC-27 Abs confirmed the co-localization of two proteins suggesting the formation of an oligomeric complex of consisting of several hdm2 molecules. Further examination showed that within minutes of drug treatment the disruption of tumor cell plasma membrane and of mitochondria was evident, whereas the nuclear membranes remained intact supporting the notion of cancer cell death by a necrotic-cytotoxic process. Using spinning-disc confocal microscopy with propidium iodide for membrane leakage and nuclear staining and mitochondrion-specific MitoTracker dyes we have been able to record the rapid membrane- and mitochondrial-destructive events that follow the exposure of cancer cells to PNC-27 but not of untransformed normal cells. Taken together these findings let us propose that these novel peptides, PNC-7, PNC-2, PNC -27 and PNC-28, individually and combined, will help arm the oncologist with a new class of a therapeutics that are selectively and potently cytotoxic to cancer cells.
Blockade Of IL-6 Signalling With A Humanized Anti-IL-6 Receptor Antibody, Tocilizumab, For The Treatment Of Rheumatoid Arthritis OHSUGI Y, MIHARA M Chugai Pharmaceutical Co, Ltd., Tokyo, Japan
Background: IL-6 is a proinflammatory cytokine and is known to play important roles in the pathogenesis of rheumatoid arthritis (RA). Several clinical studies have demonstrated that blockade of IL-6 signalling by a humanized anti-IL-6 receptor antibody is highly effective in the treatment of patients with active RA who are inadequately responsive to traditional anti-rheumatic drugs. IL-6 has a variety of biological activities that match many of the symptoms observed in RA patients. The precise mechanisms of action of tocilizumab are still not fully understood, however.
Methods: We reviewed possible mechanisms of action of tocilizumab, based on recently published papers.
Results: The data from Phase III clinical studies confirmed that tocilizumab can improve symptoms (including the number of swollen joints, the number of tender joints, fever, fatigue, anaemia and anorexia) in moderate to severe active RA. It has also been reported that tocilizumab prevented the radiographic progression of joint destruction. Tocilizumab has generally been well tolerated. Interestingly and importantly, serum IL-6 levels gradually decreased during long-term treatment, even though tocilizumab does not directly inhibit the synthesis of IL-6. This might be explained by the finding, in an animal model, that blockade of IL-6 signalling suppressed the induction of Th17 cells, which play a pathogenic role in the development of autoimmune diseases. In addition, we recently found that tocilizumab inhibited IL-6-induced RANK ligand expression on synovial cells obtained from RA patients, resulting in the inhibition of osteoclast formation. Tocilizumab also inhibited the gene expression of vascular endothelial growth factor, which causes neovascularisation that increases the supply of oxygen and nutrition to growing synovial tissues.
Conclusion: Clinical studies have demonstrated that targeting the IL-6 signalling pathway with tocilizumab could be an attractive and innovative therapeutic option for RA. It is highlighted that high efficacy was achieved consistently in several studies, and this adds to the evidence for the deep involvement of IL-6 in the pathogenesis of RA.
In addition, blockade of IL-6 signalling inhibited the induction of Th17 cells and inhibited angiogenesis and bone destruction.
Bile Acid Derivatives As BBB Modifiers MIKOV M1, TUCKER I1, YANG L1, VASOVIC V2, GOLOCORBIN-KON S2, KUHAJDA K2, KEVRESAN S2, POSHA M2, JAKOVLJEVIC V2 1School of Pharmacy, Univ. of Otago, Dunedin, New Zealand; 2Department of Pharmacology and Toxicology, Medical Faculty, Univ. of Novi Sad, Serbia
Background: The aim of this study was to test the efficacy of the sodium salt 3α,7α-dihydroxy-12-oxo-5β-cholanate (MKC-Na) and methyl ester of 3α,7α-dihydroxy-12-oxo-5β-cholanate (MKC-Me) as a blood-brain barrier (BBB) permeator by examining its effect on quinine uptake into the central nervous system in rats. In our previous studies we have shown MKC-Na antidiabetic effect and potentiation effect on morphine analgesia.
Methods: Experiments were carried out on Wistar rats. Thirty minutes before injecting quinine to the right a. axillaris, the animals of the test group were given s.c. solution of MKC-Na in a dose of 2 mg/kg; the second group received s..c MKC-Me in a dose of 2 mg/kg; the animals of control group received s.c. saline solution. Animals of all groups were given quinine at a dose of 25 mg/kg by the retrograde bolus injection to the right a. axillaris. Before the decapitation the brain was washed with 5 ml of saline. The animals were decapitated 30, 60, 150, and 240 s after quinine injection. The brain was divided into: cerebrum, brain stem and cerebellum. After their weighing, the particular brain parts were homogenized. The quinine was extracted and analyzed in the homogenates by the method of Cram’er and Isaksson.
Results: Given with MKC-Na, quinine uptake by the cerebrum was increased 2.3 times, by brain stem 1.5 times and by cerebellum 1.7 times in the comparison to the control. Given with MKC-Me, quinine uptake by the cerebrum was decreased to 0.41, by brain stem to 0.39 and by cerebellum 0.32 in the comparison to the control.
Each value in the table represents the mean±SD (n=6)
Group
Control
MKC-Na
MKC-Me
CNS compartment
Cmax (pmol/g)
Cmax (pmol/g)
Cmax (pmol/g)
Cerebrum
3.39±0.31
7.80±0.65*
1.39±0.09*#
Brain stem
4.28±0.37
6.20±0.49*
2.44±0.15*#
Cerebellum
5.67±0.43
9.62±0.65*
3.11±0.25*#
*p<0.01 to the control;# p<0.01 to the MKC-Na group
Conclusions: The results indicate that MKC-Na is a potent enhancer of the BBB permeation, with the highest effect at the level of cerebrum. MKC-Me exibits an opposite effect in the test of quinine uptake and suggests a specific character of its action, depending on the bile acid derivative structure. The promotion of quinine uptake with MKC-Na may be the result of a mollecular agregates which does not occur with MKC-Me.
Challenges And Potential Solutions To Innovative Vaccine Development In Developing Countries MILSTIEN JB University of Maryland School of Medicine, Center for Vaccine Development, Department of Geographic Medicine, Baltimore, MD USA
Background: One of the barriers to meeting Millennium Development Goal Number 4 is access for large populations of developing countries to vaccines against their most serious and common diseases. Innovative vaccines have been more readily available at affordable prices once more producers enter the market, but this means that there will be a delay in access until these manufacturers can access the technology. Several solutions to enhance access have been proposed; none so far is optimal.
Methods: The factors relating to competition in the market and an affordable price were examined. Historical price data to countries were reviewed. Various explanations for lack of competition were also analyzed, including unequal access to financing, lack of optimal research and development capacity, barriers to technology transfer, issues in accessibility to intellectual property.
Results: Differences in vaccine scale up, know-how, GMP practices and regulatory oversight are decreasing between emerging suppliers and established multinationals. Developing country vaccine prices are at about the same levels for all manufacturers, but the level of vaccine development is lower for emerging suppliers who have few high priced markets to offset investments. There appear to be three major differences between multinational manufacturers and emerging suppliers:
(1) limited access to research results that lead to new vaccine constructs;
(3) inability to spread the investments that would be incurred in addressing issues (1) and (2) over a large enough financial base.
In terms of pricing, experience with early adopting countries has shown that other factors may overcome the perceived pricing barrier: that is, willingness to pay appears not to correlate with country wealth.
Conclusions: Based on these analyses it appears that the interventions of the international community might be better directed to achieve vaccine access.
Enigmatic Eosinophil As Magic Bullet: Eosinophil-Induced Prognosis Improvement Of Solid Tumors Could Be Enabled By Their Vesicle-Mediated Barrier Permeability Induction MINGOMATAJ EÇ Mother Theresa School of Medicine, Tirana, Albania
Background: Eosinophils are multifunctional cells, which contain and produce many biologically active substances. Generally, eosinophilia is associated with parasitic infections or allergic disorders, while according to recent studies eosinophil infiltration is present also in target tissues of both physiological and pathological processes. With respect to solid tumors, the eosinophilic infiltration is associated with a better prognosis. In this review, the aim was to create a suitable hypothesis about this relationship.
Methods: The relevant abstracts of PubMed publications are used in this review. Apart from abstracts reporting data about the relationship between mentioned tumors and eosinophils, to create the hypothesis are used abstract which describe eosinophil functions or abilities in diverse physiologic or patho-physiologic situations.
Results: Reflecting on prognosis improvement in the case of solid tumors after eosinophilic infiltration of their capsules, it could be hypothesized that eosinophils are not tumoricidal per se; rather they can perforate such barriers through their vesicles’ content, whereas the tumoricidal cytokines such as interleukin 4 (IL-4) fulfill the tumor necrosis. This scenario can be supported by the fact that IL-4 originated from macrophages and lymphocytes fails to mediate tumor necrosis in vitro conditions in absence of eosinophils. In analogy with solid tumors, the requirement of eosinophil-mediated increasing permeability among diverse biologic barriers and tissues may explain the eosinophils’ introduction in capsules of cists, mucosal membrane of respiratory and gastroenteric systems, hemato-encephalic barrier, in embryos, as well as in bacterial and parasitic membranes.
Conclusions: In some situations such as solid tumors, rather than being multifunctional effectors per se, eosinophils, due to induction of target barrier dysfunction, may assure the host-required action, mediated by various kinds of leucocytes and their biologic effectors. Consequently, a better understanding of physiology and pathophysiology of this enigmatic cell will lead to new clinical strategies.
