Conclusions: These results collectively show that a potent anti-proliferative tubulysin compound can be specifically delivered to FR-positive tumors to provide substantial therapeutic benefit using well-tolerable dosing regimens.
Hydrophilic hexapeptides – a new class of ATF – dependent transport proteins of multiple drug resistance
LEBEDEV VV
Central Research Institute of Epidemiology, Russia, Moscow.
Multiple drug resistance (MDR) appears as a result of shart rise in pumping out medical products from a cell into extracellular space by ATF – dependent transport proteins. At present no transport proteins effective inhibitor is available that could be used for the MDR overcoming. The effect of three hydrophilic hexapeptides, including cyclic hexapeptide on activity and amount of transport proteins responsible for MDR formation has been investigated. Cell lines of human throat cancer, Hep2, human oral cavity carcinoma, KB 8-5, and human prostate cancer, PC – 3, have been used. The examination of specified hexapeptides effects was performed. The examination of specific hexapeptides effects was performed by comparison of Rh 123 output intensivity and transport proteins and their genes expression values. It has been shown that hydrophilic hexapeptides inhibit substrate output from a cell into extracellular space in concentrations of 10-10 M.
However, the effect of Hydrophilic hexapeptides on the MDR modulation is carried out by different mechanisms of action. It was unknown so far the tumor multidrug resistance inhibitors, which showed activity in such low concentration.
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Effects of Central Penicillin Administration on Neuronal Response of the Nucleus Reticularis Gigantocellularis
LEBEDEVA MA
Institute of General Pathology and Pathophysiology, Russian Academy of Medical Sciences, Moscow, Russia.
Background: Reticular neurons of medulla oblongata participate in a large variety of sensory, motor and autonomic functions. The gigantocellular reticular nucleus is a part of brainstem neuronal network with a high density of GABAergic neurons. This structure plays the regulatory, integrative and coordinative role in cardiovascular and respiratory control. The aim of this paper was to study the neuronal responses of bulbar reticular units after disturbance of inhibitory processes.
Methods: Penicillin was used as a convulsant agent, which is able to non-selectively block GABAA-mediated synaptic inhibition. Benzyl penicillin (50 U/μl) was locally applied by pressure microinjection into the central part of reticular gigantocellular nucleus of 65 anesthetized or paralyzed Wistar rat. Effects of penicillin administration were examined on the firing rate, discharge pattern and interspike intervals of reticular units.
Results: Penicillin caused enhancement of the number of active firing neurons (138%) and reorganization the spatial neuronal architecture of the examined structure. In gigantocellular nucleus penicillin application induced excitatory responses in 78% of extracellularly recorded units and inhibition in 15% of them. 18% of recorded neurons exhibited high-frequency firing activity. These facts support data which showed that this reticular region includes functionally labile cells capable of transforming tonic activity into burst pattern. Penicillin microinjection increased the mean firing rate of reticular neurons from 9.63±1.24Hz to 12.11±1.59Hz (p<0.05).The peak and mode of interspike intervals histograms were shifted toward shorter intervals and their amplitude was increased after penicillin microinjection. Distributions of interspike intervals histograms were transformed (unimodal to exponential, multimodal to unimodal). In no case did vehicle administration produce the significant changes in neuronal activity.
Conclusion: We concluded that neurons of reticular gigantocellular nucleus are very sensitive to the local blockage of synaptic inhibition by penicillin within of medulla oblongata. Results suggest a considerable physiological role of medullary inhibitory mechanisms in homeostasis maintenance.
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The Role Of Central Vagal Control In The Action Of Lipophilic And Hydrophilic Beta-Adrenobloсkers
LEBEDEVA MA1, KAVERINA NV2, LYSKOVTSEV VV2
1Institute of General Pathology and Pathophysiology, Russian Academy of Medical Sciences, Moscow, Russia, 2Zakusov Institute of Pharmacology, Russian Academy of Medical Sciences, Moscow, Russia.
Background: The effects of systemic administration of beta-adrenoblockers on the chronotropic heart functions are well known, however little is known about its influences to the heart rate variability and central effects of these drugs. In the present study we aimed to compare the efficacy of lipophilic beta-adrenobloker Propranolol to that of hydrophilic beta-adrenoblocker Sotalol by various methods of administrations.
Methods: Experiments were performed in anesthetized and artificially ventilated 51 white outbred rats. The heart rhythm disturbances caused by occlusion of the left coronary artery. ECG was recorded in three standard leads. To reveal the central effects of the drugs intracisternal injections were made (Propranolol at dose to 10 µg per animal, Sotalol - to 250 µg) throw the occipital membrane, to assess the peripheral effects the drugs were administrated intraperitoneally prior coronary occlusion (Propranolol at dose 1mg/kg, Sotalol - 5 mg/kg).
Results: In the control experiments coronary occlusion leaded to polymorphous cardiac arrhythmias and ventricular fibrillation was developed. Propranolol produced the reduction of all cardiac disturbances observed after coronary occlusion irrespective of the methods of administrations. Its intracisternal injections led to increase of RR interval (161.35±3.4 ms to 181.5±4.6 ms, p<0.01), Sotalol had effect only by introperitoneally injections. Propranolol caused the enhancement of total spectral power of the heart rate variability and all spectral components. The pronounced effect Propranolol of the HF component was found. The bulbar brain stem structures participate in regulation of cardiac functions. The HF component may reflect mainly the fluctuating activity of vagal center. Thus Propranolol contrary to Sotalol manifests the central effect possibly to its lipophylic properties.
Conclusion: Results allow concluding that more pronounced activity of Propranolol is probably associated with its lipophylic profile, and it is also likely to depend on Propranolol-induced activation of the central vagal path from medulla oblongata to the heart.
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Fentanyl: How Delivery System Can Modify Clinical Properties Of Molecule
LECYBYL R
Royal Free Hospital, London, UK.
Fentanyl (N-phenyl-N-(1-phenethyl-4-piperidinyl)propanamide) was first synthesized by Paul Janssen in 1959. Fentanyl was introduced into medical practice in the 1960s. It has long been established in anesthetics and ITU practice due to potent analgesic action, intravenous administration, very rapid onset, short half-life and low incidence of histamine.
Fentanyl administrated orally has low bioavability due to first pass metabolism. Intravenous route of administration have limited significantly fentanyl application outside anesthesia and ITU until “magic bullets” was applied. Fentanyl “magic bullets” have form of different delivery systems, which significantly modify clinical properties of molecule.
Development of transdermal fentanyl patches allows avoiding intravenous route of administration offers very long opioid analgesia (up to 72 hours) and could be used for patients with swallow difficulties. Transdermal fentanyl patches are widely used for background pain.
Utilization of transbuccal route of administration creates very fast rescue medication for breakthrough pain. Transbuccal preparation is available as lozenges and effervescence tablet.
Electrophoretic fentanyl patch offers postoperative patient controlled analgesia system, which eliminates need of syringe driver and intravenous (or subcutaneous) contact. Fentanyl is administrated on patient request (pressing button) by transdermal electrophoresis.
Application of “magic bullets” (delivery systems) transformed simple molecule into number of different systems, which could be used widely in cancer and non-cancer pain management.
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COXEN: A New Strategy For Predicting The Chemosensitivity Of Human Cancers And Its Application To Drug Discovery
LEE JK
University of Virginia School of Medicine, Charlottesville, USA.
The U.S. National Cancer Institute has used a panel of 60 diverse human cancer cell lines (the NCI-60) to screen >100,000 chemical compounds for anticancer activity. However, not all important cancer types are included on the panel nor are drug responses on the panel predictive of clinical efficacy in patients. We thus asked whether it would be possible to identify common chemosensitivity biomarkers from that rich database to predict drug activity in cell types not included in the NCI-60 panel or, even further, clinical responses in patients with tumors. We address that challenge by developing a novel pharmacogenomic approach "Co-eXpression ExtrapolatioN" (COXEN), which can effectively identify concordant genomic chemosensitivity biomarkers between two independent expression profiling data sets, here extrapolating the genomic expression patterns of NCI-60 biomarkers with those of clinical tumors. Applying our COXEN approach in a prospective fashion, we predicted anticancer drug activities on completely independent bladder cancer, which is not included in the NCI-60 panel, and chemotherapeutic responses and survival of breast, bladder, and ovarian cancer patients treated with commonly used single and multi-agent chemotherapies. We also used COXEN for in silico screening of 45,545 compounds and identify a novel agent with superior growth inhibition activity against human bladder cancer.
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Erythropoietin-Binding Protein And Its Antibodies For Possible Clinical Application
LEE MS, LEE JS, LEE JY
Northwestern Univ. School of Medicine, Chicago, USA, Univ of MN School of Medicine, Minneapolis, USA.
Background: Hypertension (H), prevalent worldwide, is associated with high morbidity and mortality. Many investigators believe that genetic components are important in the etiology of H. In embryo transfer studies, genetic factors dominantly affected blood pressure (BP) (P < 0.0001) and its fluctuations ((P < 0.025), when we transferred one-cell homozygous embryos into hypertensive or normotensive rats' oviduct, and pups were cross-suckled at birth. Erythropoietin (Epo) induced H is the most serious complication in Epo treatments (Rx). Uncontrollable BP rise in 1/3 of patients, end-organ damage and even death have resulted in the U.S. Food and Drug Administration “re-evaluating'' the safety of Epo.
Method & Result: Human Epo-binding protein (Epo-bp) and anti-Epo-bp antibodies (Epo-bp) were genetically engineered to test the adversity of Epoetin. Epo Rx increased hematocrit (Ht) markedly overall when compared to saline (S), Epo-bp, and Epo-bp Rx (0.62 vs.0.43, 0.44, and 0.44, respectively) and at each of the 6 test times (all P < 0.0001). They had almost no effect on the Epo-induced Ht increase. Circadian BPs for Epo vs. S, Epo-bp, and Epo-bp Rx were 136 ± 2 vs. 116 ± 2, 118 ± 2 and 117 ± 2 mm Hg, respectively (each P < 0.0001). Splenomegaly characterized each rat in the Epo Rx: in grams 1.58 in Epo vs. 0.86 in S, 0.89 in Epo-bp, and 0.85 in Epo-bp (each P < 0.0001). Ligand-binding sites were detected using fluorescein-labeled Epo-bp & Epo-bp in various blood progenitors. We developed diagnostic kits to detect Epo, Epo-bp and their antibodies to differentiate Epo- from EpoR-related diseases; Epo levels in serum & plasma: 25.4 ± 2; 24.2 ± 2; Epo-bp: 24.2 ± 2; 25.0 ± 1 mU/ml, respectively. Cell membrane proteins play a key role in cell-cell communications. Thus, exploring membrane protein polymorphisms and hormonal interactions may expand our knowledge of the normal and abnormal physiological process, and lead to the development of a new strategy in those Rx.
Conclusion: Epo-bp and Epo-bp effectively eliminate Epo-induced H without affecting Ht. They are predicted to be therapeutic agents for hematopoietic malignancy, and used as diagnostic tools at test sites and quick detection of the athletic abuse of Epo as a doping agent, and as research tools, not only for Epo-EpoR-, but also for many other circulatory, vessel and tissue-related malignancies. Clinical implications of our materials are enormous and diverse, and provide hope for the effective Rx of those problems without damaging adversity, the perfect concept for the Magic Bullet envisioned by Dr. Ehrlich.
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Systematic Discovery Of Novel Multi-Target Therapeutics: Finding The New Magic Bullets
LEE MS, RICKLES R, JOHANSEN L, AVERY W, GIORDANO T
CombinatoRx, Incorporated, Cambridge, MA, USA.
Background: Biological pathways fundamental to disease are highly complex and perturbation of a single molecular target often has little or no effect on a disease process due to redundancy and buffering of complex biological systems. Conversely, the perturbation of multiple targets in a critical disease pathway can simultaneously attack disease pathology on multiple fronts enabling treatment of disease through entirely new mechanisms of action. The rationale for a multi-targeted approach is especially applicable in oncology where pathogenesis often results from the complex intersection of many biological pathways.
Methods: To systematically discover multi-target mechanisms we generate a concentration matrix for each compound combination, capturing the combined activity of two compounds over a broad range of single agent concentrations. Analysis and quantitative scoring of concentration response matrices using multiple mathematical models allows insight into the biological mechanism of action of a drug combination and the discovery of novel therapeutic applications. Novel synergistic combination therapeutics are validated in secondary disease relevant in vitro and in vivo model systems and rapidly advanced to clinical proof of concept studies.
Results: We have discovered multiple unexpected synergistic combinations in oncology indications. In one example, the combination of an anti-parasitic agent, pentamidine and a phenothiazine anti-psychotic, chlorpromazine exerts an anti-proliferative effect through synergistic action on the mitotic targets KSP/Eg5 and PRL phosphatase. This combination synergizes in vitro and in vivo with the microtubule binding agents paclitaxel and vinorelbine, supporting a model where dual inhibition of mitotic kinesins and PRL phosphatases synergize in mitosis to inhibit tumor cell growth. In a second example, a multi-target mechanism screen in multiple myeloma cell lines has revealed novel and highly selective synergistic interactions between molecular pathways not previously known in multiple myeloma.
Conclusions: The systematic survey of multi-target mechanisms enables the discovery of interesting new biology, the definition of specific target pairs for therapeutic development and demonstrates the power of combination biology for drug discovery.
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Magic Activity Of Beta-Lactam Antibiotics Against Intracellular Methicillin-Resistant S. Aureus (MRSA): Role Of Acidic pH
LEMAIRE S, VAN BAMBEKE F TULKENS PM
Université Catholique de Louvain, Brussels, Belgium.
Background. Early studies showed that MRSA strains become susceptible to -lactams when they are exposed to acidic pH (pH ≤ 5.5) (Sabath et al., AAC, 1972). Because S. aureus survives in the phagolysosomes of macrophages (where the pH is in the acidic range), we have examined the intracellular susceptibility of MRSA strains to cloxacillin and meropenem.
Methods. Intracellular activity was determined against S. aureus phagocytosed by human THP-1 macrophages or skin keratinocytes. Expression of mecA and its regulatory genes was determined by RT-PCR. Cloning, purification of PBP2a, and Bocillin FL – PBP2a binding assay were performed as described earlier (Fuda et al, JBC, 2006; Lemaire et al, JBC, 2008).
Results and conclusions. MRSA ATCC 33591 phagocytosed by human THP-1 macrophages shows complete restoration of susceptibility to cloxacillin and meropenem, becoming indistinguishable from MSSA ATCC 25923 due to the acidic pH prevailing in the phagolysosomes (Lemaire et al, AAC, 2007). This influence of acidic pH was first ascribed to a diminished copy numbers of PBP2a (Hartman and Tomasz, J. Bacteriol., 1984), a unique transpeptidase that is poorly inhibited by beta-lactam antibiotics because of a closed conformation of its active site. However, we showed that growing bacteria at acidic pH (i) alters neither the expression of the PBP2a-encoding gene (mecA) nor that of its regulatory genes. We also found that MRSA grown and exposed to a radioactive penicillin ([14C]penicillin) at acidic pH show a larger retention of radioactivity than if the bacteria has been grown at neutral pH (Lemaire et al, AAC, 2007). Concentrating our effort on a purified PBP2a, we showed that, at lower pH, PBP2a bind more avidly -lactams and undergoes a conformational change (which is a crucial step for the opening of the active site) (Lemaire et al, JBC, 2008). In terms of mechanistic consequences, these variations were quite similar than those recently reported for ceftobiprole (a novel anti-MRSA cephalosporin inhibiting more efficiently PBP2a). Therefore, these observations argue that PBP2a is most likely evolved for its physiological function at pH 7.0 owing to its closed conformation, which is not maintained at acidic pH.
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Dendritic Cell Immunotherapy Of Malignant Gliomas
LEPLINA O1, STUPAK VV2, PENDURIN IV2, NIKONOV SD1, OSTANIN AA1, CERNYKH ER1
1Institute of Clinical Immunology Siberian Branch of the Russian Academy of Medical Sciences, 2Institute of traumatology and orthopaedy, Novosibirsk, Russia.
Background: Despite resent advances in radio-, chemotherapy and surgical treatment, the prognosis for patients with malignant glioma is still very poor. Therefore, the development of new treatments, such as immunotherapy is very important. Dendritic cells (DCs) are antigen presenting cells that play a central role in the initiation and modulation of immune response. In this study we investigated the safety and immunologic and clinical response of tumor lysate-pulsed DC therapy for patients with malignant glioma.
Methods: Thirty nine patients with anaplstic astrocytoma (AA, n=24) and glioblastoma (GB, n=15) were enrolled in this study. Controls represented the population of 80 patients (AA=47 and GB=33) who received surgical resection and radiation therapy. The patient’s peripheral blood DC were generated with granulocyte macrophage colony-stimulating factor plus interferon-alpha during 4-5 days and pulsed with an autologous tumor lysate. DCs were used for the generation of CTL (that were inoculated in the cavity of rejected tumor) and the course of 4-6 biweekly subcutaneous vaccinations after radiation therapy.
Results: The protocol was well tolerated and was not associated with an evidence of toxicity or serious adverse effects. Sixty five percent of patients developed systemic immune response according to proliferation and positive delayed-type hypersensitivity skin test to autologous tumor lysate. Antigen (Ag)-specific response after 6 vaccinations increased in 10 folds (table). The level of 1- 2- and 3-year survival in this group was significantly higher in compare with controls (74 vs 52,5%, 61 vs 27,5 and 50 vs 19%, respectively). The most effect was observed in GB patients. A median survival in this group was 14 months vs 8 months in controls (p=0,003).
Conclusion: Thus, our results showed the safety and clinical response of autologous tumor lysate-pulsed dendritic cell therapy for malignant glioma patients and the accumulation of antigen-specific immune response.
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Exploring Anti-Microbial Herbs Of Chinese Medicine
LEUNG PC1, SREEDHAR B1, FUNG KP1, YIP M2
Institute of Chinese Medicine, 1& Department of Microbiology, 2 The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong.
Background: The global increase in resistance to antimicrobial drugs, including the emergence of bacterial strains that are resistant to antibacterial agents, has created a public health problem of potentially crisis proportions. Thus, there is an urgent need for new antibacterial agents that are able to overcome multidrug-resistant mechanisms.
Aim: To identify herbs and their active components exhibiting inhibitory effect either on the growth or the bacterial resistance mechanism.
Methods: Traditional Chinese Medicinal (TCM) herbs, which are commonly employed in the treatment of bacterial infections, were identified. Potential herbs were then extracted using a standardized protocol. Assays for screening were prepared using a Biomek 3000 (Beckman Coulter) liquid handling system, and microtitration plates. Five µl of extract with desired concentration was added to each well of the 96 square-well plate containing Mueller-Hinton medium and 106 CFU/ml of the bacteria. Plates were then transferred to the integrated DTX 880 Multimode detector for incubation (37°C) and growth was monitored for 48 hours by measuring the absorbance under O.D. 600nm. The extract was considered very active if there was no bacterial growth after 24hrs incubation and as active if bacterial growth was more than 10% of the negative control.
Results: A total of 10 TCM herbs were subjected to preliminary antibacterial screening against 3 clinical and pathogenic bacterial strains of Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923) and Methicillin-resistant S. aureus (ATCC BAA- 43). The results indicated that out of 10 herbs screened, ethanol extract (100µg/ml) of Caulis Sargentodoxae (Sargentodoxa cuneata) and Coretx Lycii (Lycii chinensis) showed broad-spectrum antibacterial activity against both S. aureus and MRSA compared to aqueous extract. Percentage inhibition of bacterial growth was 88% - 90% and 90 – 100% for both C. Sargentodoxae and C. Lycii respectively (p<0.001). However, extracts from these two herbs exhibited little or no activity, against E. coli.
Conclusions: We have validated the protocol for screening bioassay for evaluating the antibacterial properties of herbal extracts. Our preliminary results suggest that the ethanolic extracts of Caulis Sargentodoxae and Coretx Lycii have antibacterial activity. Although further studies are needed to determine safety and clinical efficacy, these effective extracts may prove to be clinically useful in the treatment of multidrug resistant bacterial strains.
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