Conclusion: The results present the essential prerequisites for further VRC investigations especially for the use of clinically approved Vfend® for determination of relative recovery in pre-/clinical trials.
How Prolonged Exposure to Caffeine Can Affect Dopaminergic Transmission: Evidence from Rats Subchronically Treated with Caffeine and Possible Implications
SIMOLA N1, TRONCI E1, PINNA A1,2, SEEMAN P3, MORELLI M1,2
1Univ. Cagliari, Cagliari, Italy; 2CNR Neuroscience Institute, Cagliari, Italy; 3Univ. Toronto, Toronto, ON, Canada
Background: Caffeine is a widely consumed psychostimulant, whose effects are mediated by blockade of adenosine A1 and A2A receptors; an influence of dopaminergic transmission in caffeine-mediated psychostimulation is however acknowledged. The nature of caffeine/dopamine interactions was here investigated by using a rodent experimental paradigm of long-term caffeine exposure.
Methods: Male Sprague-Dawley rats (180-200 g, N = 70) received subchronic-intermittent caffeine (15 mg/kg i.p.) or vehicle (i.p.) every other day for 14 days. Three days after treatment cessation, rats were challenged with either caffeine (15 mg/kg i.p.) or amphetamine (0.5 mg/kg s.c.) to disclose long-term neuroplastic changes. Behavioral evaluation was performed in combination with neurochemical techniques (in situ hybridization, microdialysis, binding). Differences between caffeine- and vehicle-treated rats were evaluated by one- or two-ANOVA. Significance was set at P<0.05.
Results: Subchronic-intermittent caffeine elicited sensitization to its motor stimulant effects (which reflects the occurrence of neuroplastic changes in dopaminergic transmission) paired with a decrease in the levels of both the mRNA for adenosine A2A receptors (which deeply interact with dopamine receptors) and the mRNA for the early gene zif-268 (which belongs to the dopamine receptors signaling pathway) after caffeine (15 mg/kg i.p.) challenge. Moreover, caffeine-sensitized rats displayed cross motor sensitization with amphetamine (0.5 mg/kg, s.c.), paired with an increase in the levels of zif-268 mRNA, and, finally, an elevation in high-affinity dopamine D2 receptors (D2High). All the above neurochemical changes were observed in the rats’ corpus striatum at three days after subchronic caffeine cessation and were not paired with modifications in dopamine release.
Conclusions: 1) Prolonged caffeine exposure elicits enduring neuroadaptations involving striatal dopaminergic transmission by acting at the post-synaptic level 2) This finding may elucidate the mechanisms underlying the interactions between caffeine and drugs acting on dopamine transmission, including addictive psychostimulants such as amphetamines.
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H2S-induced suspended animation during porcine aortic occlusion-induced ischemia reperfusion injury
SIMON F1,2, WAGNER F1, BAUMGART K1, HAUSER B1, GRÖGER M1, CALZIA E1, RADERMACHER P1, SCHELZIG H2, GEORGIEFF M1
1Klinik für Anästhesiologie und 2Thorax- und Gefäßchirurgie, Universitätsklinikum, Ulm, Germany
Background: In mice, inhaled hydrogen sulfide (H2S) produced a “suspended animation”-like metabolic status characterized by hypometabolism and hypothermia, which protected against otherwise lethal hypoxia and hemorrhage1,2. The large surface area / mass ratio, however, facilitates cooling in rodents3, and controversial data were reported on the effects of H2S in large animals4. Therefore we investigated whether H2S may induce a similar metabolic response and thus be organ-protective during aortic occlusion-induced ischemia/reperfusion (I/R) injury.
Methods: In a first series, 16 pigs undergoing 30 min of aortic occlusion received the i.v. H2S-donor Na2S or vehicle started 2 h before ischemia and continued during the whole reperfusion period. During aortic occlusion mean arterial pressure (MAP) was maintained at 80-120% of baseline with esmolol, nitroglycerine and ATP, during reperfusion noradrenaline was titrated to keep MAP at baseline levels. In the second series, 19 pigs underwent prolonged (90 min) aortic occlusion to produce acute renal failure. In both studies the reperfusion period was 8 h.
Results: In the first study Na2S significantly reduced heart rate and cardiac output without affecting stroke volume, significantly decreased the dose of noradrenaline required to maintain hemodynamic targets, and caused a significant drop in O2 uptake, CO2 production, and, consecutively, core temperature. These results were confirmed in the second study. Moreover, Na2S was reno-protective as demonstrated by a significantly higher creatinine clearance and a lower serum creatinine level at the end of the reperfusion period, which coincided with a reduced oxidative DNA-damage in the kidney tissue as assessed by the tail moment in the alkaline comet-assay.
Conclusions: I.v. Na2S i) reduces metabolism and energy expenditure in anesthetized large-animals, ii) improves noradrenaline responsiveness during reperfusion after aortic occlusion, and iii) protects the kidney against I/R injury.
References: 1. Blackstone et al, Shock 2007;27:370; 2. Morrison et al, J Trauma 2008;65:183; 3. Leslie, Science 2008;320:155 ; 4. Tisherman & Draben, Pediatr Crit Care Med 2008;9:129
Acknowledgement: Supported by the DFG (SCHE 899/2-3)
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Neuropeptide FF receptors: a novel target for pain treatment
SIMONIN F
Institut Gilbert Laustriat, UMR 7175-LC1 CNRS-ULP, Illkirch, FRANCE
Background: morphine and related compounds are well recognized as unsurpassed analgesics for relieving acute and chronic pain. However, their use is limited by the development of tolerance to their analgesic effects that accrues following repeated exposure. It has been proposed that opioid receptor desensitization is at the origin of this phenomenon. A challenging hypothesis is that stimulation of opioid receptors triggers activation of anti-opioid systems that in turns produce hyperalgesia thus diminishing the net analgesic effect of the opioid agonist. This process has been evidenced in vivo both in rats and in man where acute and prolonged opioid treatments induce a long lasting hyperalgesia. Anti-opioid receptor antagonists could therefore represent a promising strategy for opposing to pain hypersensitivity associated with chronic pain especially when an opioid treatment is used. Several neuromodulator systems have been shown to display anti-opioid properties including neuropeptide FF (NPFF) system. However, the lack of pharmacological tools for NPFF receptors has severely limited our comprehension of the in vivo functions of this system.
Methods: in order to identify pharmacological tools for studying the cellular and in vivo functions of NPFF receptor system, we have generated a chemical library of Arg-Phe-NH2 di-peptide derivatives. This library was screened in competition experiments and a selected compound was evaluated for its ability to prevent the development of opioid-induced hyperalgesia and associated tolerance in rats.
Results: We identified a small potent and selective Neuropeptide FF receptor antagonist, RF9, which can be administered systemically. This compound does not show any effect by itself but can block efficiently the increase in blood pressure and heart rate evoked by Neuropeptide FF thus confirming its antagonist properties in vivo. When chronically co-injected with heroin, RF9 prevents the development of tolerance to opioid analgesia and completely blocks the delayed and long lasting paradoxical opioid induced-hyperalgesia.
Conclusion: Our data indicate that Neuropeptide FF receptors are part of a bona fide anti-opioid system and that selective antagonists of these receptors could represent useful therapeutic agents to avoid the development of secondary hyperalgesia and tolerance induced by opioid treatment.
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Multifunctionality of Native and Recombined Proteins
of Honeybee Royal Jelly: Assumptions for Application in Pharmacy
Šimúth J, BÍLIKOVÁ K
Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia
Background: Royal jelly (RJ) is the principal food for the honeybee queen and has been demonstrated to possess several pharmacological activities. The substantial parts of RJ are major proteins, designated as apalbumins (Apa) and belongs to a protein family consisting of nine members with Mr of 49-87 kDa. Because the extreme complexity of protein complement of RJ limits an evaluation of physiological functions of RJ we applied molecular-biological approach to the characterization of Apa and antimicrobial petides with the aims to show if postranslation modification affects production of tumor necrosis factorα (TNFα).
Methods: The native apa1 and apa2 were obtained by ultracentrifugation of RJ at 350 000xg followed by ion exchahge cromatography. The recombinant apalbumins (rApa) and antimicrobial peptides with cluster of 6xHis tag were expressed in bacterial or plant hosts and purified by metal affinity chromatography. The level of TNFα produced by murine macrophage (MM) was monitored by ELISA.
Results: Apa1 is oligomeric 420 kDa glycoprotein built up from 55 kDa monomers subunits with ability to create various regular self assembled noncovalent structures similar to those occurring also in RJ. Stimulation MM with the native Apa1, Apa2 and rApa1 (without postranslation modification) reached production TNFα on level in range 900 pg/ml. The oligomeric Apa1 and antifungal and antimicrobial RJ peptide apisimin (5.1 kDa) were less effective as Apa1 in monomeric form. The N-terminal fragment of rApa1 (13.56 kDa) was the most effective elicitor of TNFa release (1414.8 ± 112.2 pg/ml). Other authors showed that C-terminal of Apa1 is precursor of antimicrobial peptides. Our findings indicate that (1) the stimulating effect of Apa1 is derived from its protein domain and it is not affected by postranslation modifications (2) the stimulating effect of honey is based on Apa1, the dominant protein of honey, and (3) Apa1 is regular component of honey and honeybee pollen.
Conclusions. The experimental data on molecular and multifunctional properties of RJ proteins present them as immunomodulators, suppressors of allergic reactions, antibiotic and anti-hypertensive agents, and stimulators of proliferation, and opened real possibility for their application to pharmacy.
Authors’ disclosure statement: A honeybee colony as one of the best organized social entities in the nature is armed against pathogens with very efficient exogenous defense system based on multifunctionality of nutritive proteins and antimicrobial peptides. Such a system might become a core of new research program, which would unite activities dispersed till now in various institutions, many of which do not dispose of precisely defined honeybee proteins. We would like to invite pharmaceutical scientific community to collaborate on a new proposed EU research project named as ApiPharm based on (1) purification of native honeybee proteins, (2) preparation of recombinant one biotechnologically by heterologous expression, and (3) determination of their physiological properties in specialized laboratories oriented pharmaco-medically and apidologically. It is a contribution to objective evaluation of therapeutic effects of honeybee proteins before their application on pharmaceutical market.
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Surface Modified Nanostructured Carriers of Nevirapine for Brain Targeting
SINGH KK, SHEGOKAR R
C U Shah College of Pharmacy, S N D T Women’s University, Juhu Road, Santacruz West
Mumbai, India
Background: AIDS is one of the most frightening syndromes worldwide with 5 million new HIV infections a year. Around 14000 people are becoming infected each day & 68 million will die of AIDS BY 2020. HIV potentially harbors in different reservoirs like CD4+T lymphocytes, CNS, lymph nodes, GALT, genital organs, kidney, liver, eye & lungs. Antiretroviral drugs lack target potential to HIV reservoirs. Specific engineering of nanosystems with right drug candidate promote transport across biological barriers. The main objective of present research project was to formulation & evaluation of surface modified nanoparticulate drug delivery systems of Nevirapine, Nevirapine is a non-nucleoside reverse transcriptase inhibitor (NNRTI) with activity against human immunodeficiency virus type 1 for brain targeting.
Methods: Nanosuspensions of Nevirapine were fabricated by high pressure homogenization technique applying 32 factorial design for intravenous application. Further Surface modification of nanoparticles was done using PEG 300,400,1000, Poloxamer 188, Dextran 60 and bovine serum albumin by covalent conjugation. The developed nanodispersions were characterized for physiochemical parameters & stability. The nanoparticles were sterilized & converted into reconstituable form. Formulations were tested for their anti-HIV potency, cytotoxicity and phagocytic uptake in J744.A1 cell line. The in-vivo tissue distribution was carried out in Wister rats using gamma Scintigraphy.
Result and discussions: Nevirapine nanodispersions were successfully prepared. Homogenization parameters and concentration of surfactant had marked effect on particle size distribution. The nanoparticles with mean particle size of 482 nm and drug release with t50% of 0.22 h and t90% of 3.4h were obtained. AFM confirmed surface modification with increase in particle size. Among cryoprotectants, trehalose gave the desired particle size after reconstitution. Developed nanoparticles crossed BBB within 30 min and remained there up to 8h.
Conclusion: Thus we have successfully developed Nevirapine surface modified nanocarriers for brain targeting
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Lipid Nanocarriers for delivery of antimalarial drug Primaquine
SINGH KK, VINGKAR S
C.U.Shah College of Pharmacy,SNDT Women’s University,Mumbai, India
Background: Primaquine(PQ), is the only available antimalarial that acts specifically on the pre-erythrocytic schizonts of plasmodium, which reside and multiply in live and is considered to be a crucial weapon against resurgent vivax malaria. PQ has limited bioavailability because of pre-systemic metabolism and excretion and is characterized by severe tissue toxicity when required in high doses to treat severe cases. Targeting of the drug to the site of action, liver, would possibly reduce the dose and overcome the toxicity problem.
Methods: Lipid nanoemulsion and nanoparticles of PQ were prepared using highly specialized short and medium chain triglycerides with combinations of various hydrophilic and lipophilic surfactants & optimized by applying 32 factorial designs. Antimalarial activity was investigated using Peter’s four-day suppressive test using swiss albino mice. Parasitic culture of Plasmodium bergheii yoelii was injected intraperitonially to induce malaria. After 4h of infection, the formulations were administered at 4 different dose levels, both by oral and parenteral route. From the 5th day onwards, blood smears were collected and observed for the presence of parasites. Parasitemia(%) and mean survival time(MST) was recorded.Bio-distribution and pharmacokinetic studies were performed in Wistar rats. Lipid nanocarriers were administered orally and intravenously and the animals were sacrificed at the stipulated time intervals and drug content in various organs was determined
Results: The nanocarrier systems showed a narrow particle size distribution with mean particle size ranging between 91-117nm, high drug entrapment and 100% drug release within 6-7 h. Control groups showed invasion & ruptured RBCs with 90% parasitemia by day 10 and no animal survival after day 11. The developed nanocarriers showed 3.5 times better suppression of parasitemia and higher MST at 25% lower dose as compared to conventional formulation. PQ showed improved oral bioavailability and higher levels in the liver after incorporation into lipid nanocarriers. Kidney showed much lower concentration of unmetabolized drug, indicating reduced nephrotoxicity with the developed formulations.
Conclusions: Thus lipid nanocarriers have high potential for the delivery of primaquine to liver with enhanced antimalarial efficacy and reduced toxicity.
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Targeting HIV Reservoir & Sanctuary Sites Using Peptide Backbone Polyethylene Glycol (PEG) Nanocarriers
SINKO PJ*, STEIN S, WAN L, POOYAN S, ZHANG X, SAMIZADEH M, CHEN P
Ernest Mario School of Pharmacy, Rutgers University, Piscataway, New Jersey USA 08854
Background: Despite the wide variety of highly potent anti-HIV drugs that have been developed and brought to the clinic over the years, eradication of HIV infection has not been achieved. We have focused our efforts towards the design and development of PEG-nanocarriers for macrophage targeting. Macrophage targeting represents a key challenge in HIV therapy, since they are not only the primary target of HIV infection, but along with CD4+ T-lymphocytes, they are an important source of HIV persistence. Aim: To develop, characterize and evaluate macrophage targeted peptide-based PEG nanocarriers exploiting the formyl peptide (fMLF) and mannose receptors.
Methods: A modular PEGylated peptide [(acetyl-Cys--Ala--Ala-Lys)n-PEG5k] nanocarrier was designed and developed incorporating two and four copies of fMLFK(fluorescein)C. Two aspects were investigated: fMLF copy number required for optimal binding and optimal PEG size for macrophage uptake. One, two, and four-arm PEG scaffold of molecular weights 5, 10, 20, and 40 kDa were used to conjugate up to four copies of fMLFK(fluorescein)C. Nanocarrriers were characterized using amino acid analysis, MALDI-TOF mass spectrometry, and Size-Exclusion chromatography. Receptor expression was confirmed using RT-PCR and Western Blot analysis. Macrophage-like differentiated human U937 cell-specific binding and cellular uptake studies were performed. Binding, avidity, and uptake were evaluated.
Results: Nanocarrrier uptake was found to be energy dependant and mediated by the fMLF receptor. fMLF copy number was found to influence the binding and uptake behavior. Increasing the number of fMLF moieties from one to two resulted in enhanced uptake of 4 fold, but increasing fMLF copy number to four led only to a modest increase. Molecular size was also found to influence the uptake behavior as increasing the PEG molecular weight from 5 to 20 kDa resulted into an increase in the uptake but further increase to 40 kDa led to decreased uptake.
Conclusions: Receptor-mediated endocytotic uptake of nanocarriers was size dependant with optimal size requirement of ~25 nm or <50 nm. Thus, two copies of fMLF along with a molecular size of 20 kDa PEG appears to be a prerequisite for optimum macrophage targeting.
Support: NIH AI 33789
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The Application of Early Bactericidal Activity (EBA) studies in Assessing Antituberculosis Drugs
SIRGEL FA
Medical Research Council, Cape Town, South Africa
Background: Alternative therapeutic regimens with new faster-acting drugs are needed to stop the spread of tuberculosis (TB). Quantitative and reproducible testing methods are vital to formulate appropriate treatments. Studies of the early bactericidal activity (EBA) were established as an initial step to assess new antituberculosis drugs. It measures the ability of individual antimicrobials to kill active growing bacilli in cavities of patients with pulmonary tuberculosis at the beginning of therapy. Objectives: To retrospectively review the value of EBA studies to determine: 1) whether a drug has activity against bacilli in cavities excreted in sputum. 2) The dose size that is just ineffective and the maximum dose above which no improvement in EBA is found (therapeutic margins).
Methods: A minimum of ten treatment-naïve patients with smear-positive pulmonary TB were randomly allocated to standard daily doses of first line drugs for 2 to 5 days with either: isoniazid (H); rifampicin (R) pyrazinamide (Z); ethambutol (E) and HRZ in combination. Nil groups were included for a maximum of 2 days. EBAs for Ofloxacin (O) given daily and rifapentine (RPE) administered once at the beginning of a 5-day study period are also included. Dose titrations were done on H, R and RPE and therapeutic margins were estimated for H and R. EBA measures the decrease in log10 colony forming units (CFU) of M. tuberculosis per ml sputum per day. Sixteen hour sputum collections were homogenised, digested with sputasol, serial dilutions were plated onto selective 7H11 agar plates, incubated at 37 ºC for 3 weeks for CFU counts.
Results: The 0-2 day EBAs (and standard deviation) were: H 6 mg/kg, 0.50–0.64 (0.16–0.30) in 5 studies; R 12 mg/kg, 0.17–0.22 (0.05-0.25) in 3 studies; Z 40 mg/kg, 0.003 (0.04); E 25 mg/kg, 0.25 (0.14); HRZ in combination 0.558 (0.16). Succeeding 2-5 day EBAs were: H 0.06 (0.12); R 0.20-0.30 (0.11-0.18). Ofloxacin had a 0-2 day EBA of 0.39 (0.19) and a 3-5 day EBA of 0.17 (0.20). The 0-2 day EBAs for RPE at 600, 900 and 1200 mg were 0.244 (0.189), 0.364 (0.186) and 0.234 (0.199), respectively and the 2-5 day EBAs 0.193 (0.145) 0.251 (0.116) and 0.241 (0.112), respectively. The therapeutic margins for H was 300/15 = 20 and for R 600/150 ≥ 4.
Conclusion: The EBA procedure requires small numbers of patients and can be carried out at relatively low costs to rapidly determine the therapeutic value, dosage and role of new drugs.
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Associative learning induces selective changes in the quantitative distribution of GAT-1, a high-affinity γ-aminobutyric acid transporter, in adult mice barrel cortex
SIUCINSKA E
Nencki Institute, Warsaw, Poland
Background: The increase of functional cortical representation of vibrissae in the mouse brain occurs as a result of classical conditioning where stimulation of selected vibrissae (CS, conditioned stimulus) is coupled with aversive reinforcement (UCS, unconditioned stimulus). The plastic change was demonstrated by labeling with 2-deoxyglucose (2 DG) in layer IV of the barrel cortex. We have also shown that functional reorganization of barrel cortex is accompanied by increased density of small GABAergic cells, GAD67 mRNA, and GAD67-positive puncta in the hollows of barrels of the “trained row”. The aim of this study was to determine whether GAT-1, a high affinity, GABA plasma membrane transporter is affected by learning.
Methods: Unbiased optical disector counting was applied to sections from the mouse barrel cortex that had been immunostained using a polyclonal antibody raised against GAT-1 C-terminal sequence with standard avidin-biotin complex (ABC) method. Quantification of numerical density of GAT-1 positive puncta was performed for “trained” (CS+UCS n=5), and control groups: which received only stimulation of vibrissae without the unconditioned stimulus (CS n=5) and naive mice (n=5).
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