Ehrlich II –2nd World Conference on Magic Bullets

Central glutamatergic dysfunction as an explanation of resistance and refractoriness in Obsessive-Compulsive Disorder PASQUINI M, BERARDELLI I Department of Psychiatry and Psychological Medicine SAPIENZA University of ROME About 50% of patients affected by Obsessive-compulsive Disorder (OCD) do not respond to pharmacological treatment with selective serotonin re-uptake inhibitors (SSRI) and with clomipramine, drugs considered the gold standard treatment for OCD. Resistant patients are those who participate in a trial with any first-line therapy and do not have a satisfactory response, while the refractory patients are those who do not respond appropriately to several treatments administered in an adequate manner. In patients who do not respond to the above treatments, therapeutical strategies include switching and augmentation. A number of studies have shown that switching strategies can determine an improvement of OCD symptoms in about 40% of refractory OCD patients. Other studies have demonstrated that clinical improvement can be obtained adding drugs acting on dopaminergic system (typical and atypical antipsychotic drugs). Recently, it has been shown that drugs acting on systems other than dopamine, in particular glutamatergic drugs can ameliorate clinical OCD symptoms. The glutamatergic system was included in the physiopathology of the OCD after the observation of an increase in the glutamate concentration in the caudate nucleus of children with OCD and normal glutamate levels after treatment with paroxetine. Increased glutamate levels in the LCR of OCD patients was also described. Drugs that modulate the glutamatergic system, as riluzole, d-cycloserine and memantine, were recently used for refractory OCD patients. Memantine blocks the N-methyl-d-aspartate (NMDA) receptor-associated ion channel, and acts as an uncompetitive, low-affinity, open-channel blocker that enters the receptor channel preferentially when it is excessively open. The NMDA receptor is normally activated by the binding of glutamate, the major excitatory neurotransmitter in the central nervous system. It is believed that increased influx of calcium ions from the excessive activation of this channel may lead to excitotoxic damage to neurons in the brain. Neurotoxicity due to central glutamatergic hyperactivity may explain refractoriness to serotoninergic agents. There are still few data on the efficacy of the use of agents that affect the glutamatergic system of OCD patients however, the preliminary results seem to be promising.	Nitrendipine as a putative probe for CYP3A phenotyping PASTERA J, ZOULOVÁ J, KOPECKÝ J and KVĚTINA J Institute of Experimental Biopharmaceutics, Joint Research Center of PRO.MED.CS Praha a.s. and Academy of Sciences of the Czech Republic, Heyrovského 1207, 500 03 Hradec Králové, Czech Republic Hepatic and small bowel CYP3A3/4 content and activity is known to vary 10-20-fold among individuals. Though such a wide interindividual variability has been explained by ethnic and/or diet differences, newer investigations make also genetic contribution evident. Our aim was to develop a simple method for sorting out the extreme metabolisers (both rapid and slow) of nitrendipine (metabolised also by CYP3A3/4) and to use this method in the bioequivalence study of two nitrendipine formulations. The peak plasma concentrations (cmax) of nitrendipine found after a single dose (20 mg) administration of nitrendipine tablet (Baypress) were evaluated. In a pilot study with 18 healthy volunteers, the cmax acquired values from 1,4 to 51,3 ng/ml (median 6,95 ng/ml). As the time to reach cmax (tmax) was usually 1 or 2 hours in the pilot study, we decided to include into pivotal bioequivalence study of two nitrendipine formulations only volunteers who showed: a) 1 or 2 hours after the “diagnostic 20 mg single dose” the nitrendipine plasma concentrations in the range 5-40 ng/ml (i.e. less then one order), and b)neither in 1 hour nor in 2 hour after administration these concentrations exceeding 40 ng/ml. Of 45 examined volunteers, 9 were extreme metabolisers (8 rapid and 1 slow). Remaining 36 healthy volunteers entered the bioequivalence study (open, randomized, cross-over) which proved bioequivalence of two compared oral tablet nitrendipine formulations conclusively (the 90% confidence intervals for AUCs and cmax lie within an interval of 0,80-1,25).
Retinoic Acid Regulates the Expression of the Anti-Apoptotic Protein PKCδVIII. Patel NA ^1,2, Cooper DR ^1, 2 James A. Haley Veterans Hospital^1 and College of Medicine, Department of Molecular Medicine^2, University of South Florida, Tampa, FL Background: The human teratocarcinoma cell line, NT2 cells differentiate into hNT neurons upon treatment with retinoic acid (RA). This is a widely accepted model to investigate the expression of genes involved in neurogenesis, neuronal differentiation and early development of nervous system. Protein kinase C (PKC) δ plays an important role in the regulation of cell apoptosis. We have recently described a novel PKCδ isozyme- PKCVIII that is expressed in human NT2 cells upon RA treatment. PKCδI and PKCδVIII are the alternatively spliced variants expressed in human cells.Expression of PKCδVIII peaks at day 1 of RA treatment and then declines by 7 days. RT-PCR analysis and sequencing data revealed that this isozyme is generated via utilization of a downstream alternate 5 splice site of exon 10 which results in an insertion of 93 bp in the caspase-3 recognition sequence within the V3 domain. We have shown that PKCδVIII is resistant to caspase-3 cleavage and that PKCVIII regulates anti-apoptotic effects in these cells. Methods: RNA isolations of NT2 cells treated with retinoic acid were performed using RNA Bee (Tel Test, Inc.) RT-PCR was performed using primers that detect both PKCδ isoforms as well as primers specific for PKCδVIII. Results: We have identified the nuclear serine-arginine rich splicing factor SC35 (i.e. SRp30b) which promotes the expression of PKCδVIII mRNA via utilization of the alternative 5′ splice site II on PKCδ exon 10. Western blot analysis demonstrates that the expression of SC35 increased with RA treatment concurrent with the increase in PKCδVIII expression. Overexpression of SC35 in NT2 cells promotes the expression of PKCδVIII. To further decipher the mechanism of alternative splice site selection we have designed and cloned a minigene which includes PKCδ exon 10 and its flanking introns in the pSPL3 splicing vector. We show that this minigene is responsive to retinoic acid. Further, co-transfection of SC35 with PKCδ minigene promotes selection of 5′ splice site II. Transfection of cells with SC35 siRNA along with PKCδ minigene results in a decline of PKCδVIII expression. Conclusions: (1) PKCδVIII plays a role in development of nervous system (2) RA regulates expression of PKCδVIII via SC35.	Differential roles of physiological and physicochemical parameters on low and variable bioavailability of Saquinavir- hurdles of effective drug treatment PATHAK SM, MUSMADE P, UDUPA N Department of pharmaceutical Quality Assurance, Manipal College of pharmaceutical Sciences Manipal-University, Manipal-576104, INDIA Background: Saquinavir (SQV), the first of the Human Immunodeficiency Virus (HIV) protease inhibitors to reach the market, remains one of the most widely prescribed agent which has markedly improved morbidity and mortality in HIV-infected patients. Inclusion of SQV in ‘highly active anti-retroviral therapy’ has substantially improved the clinical outcomes of AIDS patients. However, the complete therapeutic potential of this class of drugs is yet to be exploited due to number of limitations related to their poor and variable transport across important biological membranes, especially gastrointestinal tract. The pharmacokinetic profile of SQV being characterized by its low and variable bioavailability is primarily attributed to metabolism by cytochrome P-450 3A4. Moreover, there is increasing understanding that membrane transporters (P-gp, MRP-2) contribute significantly to the biopharmaceutical characteristics of SQV and this entire class of drugs. However, the relative contributions of these eliminating organs to the first-pass effect and the significance of incomplete absorption of SQV have not been explored. Methods: In this study, our objective was to determine the various factors governing the bio-availability of SQV. In order to understand the contributions of i) first pass metabolism in gut and liver and ii) solubility and permeability of SQV across the lumen, towards its low bioavaibility, in-vitro, in-situ and in-vivo study results in rat model were compared. Though, the first-pass intestinal and hepatic metabolism has been shown to be a major determinant in the oral clearance of SQV, we found that physicochemical characteristics may also have an important role in determining the oral absorption and disposition properties of this drug. To best of our knowledge, we are the first to report a double-peak phenomenon in plasma concentration time profiles of SQV via oral administration. Although the double peaks in the plasma concentration-time profiles after p.o. doses could be attributed to a gradient expression of transporter proteins along the gastrointestinal tract, we hypothesize that the phenomenon is due to differential solubility profile of SQV across the intestine. In-vitro solubility data clearly indicates the pH dependent solubility profile of this drug. SQV has pKa values of 1.1 and 7.1, corresponding to the quinoline and octa-hydroisoquinoline nitrogens, respectively. A base pka of the drug just above pH 7.0, so the solubility would be expected to decrease significantly as the drug moves from duodenum (fasting pH about 6) to distal ileum (at about pH 7.8), and then increase again in cecum (at about pH 5). The time of the second peak (4-5 hours) coincides with the expected arrival time in rat cecum which was confirmed with intestinal motility test using charcoal. To relate bioavailability to molecular transport characteristics, we, speculated that an efflux (counter transport) mechanism might contribute to the low and variable bioavailability of SQV. Single pass in-situ absorption method was employed for the determination of the permeability (Peff) of SQV in various segments of rat intestine, viz. duodenum, jejunum ileum and colon. The data reveals that the Peff of SQV through rat duodenum (2.91 × 10^-5 cm/s) is higher than jejunum (1.86 × 10^-5 cm/s) or ileum (2.11 × 10^-5 cm/sec), which is in line with the higher extent of absorption of the drug in the duodenum. On the other hand, Peff values of the drug in jejunum, ileum and colon were found almost similar. The results hence clearly suggest that SQV is a low permeable drug, apart from being poorly soluble at intestinal pH. Conclusion: Because P-gp is found lower down the gut than is CYP3A, the exposure of SQV to P-gp is high because of its low solubility, and this could explain the very long period over which SQV is absorbed. Absorption, rather than elimination, controls the pharmacokinetics of SQV, and its very slowness is probably responsible for the low and variable kinetic profile of this drug. In conclusion, the bioavailability of SQV is controlled by a combination of solubility in the gut lumen, p-glycoprotein mediated efflux in the gut-wall, and first-pass intestinal and hepatic metabolism by CYP3A4. Given the differential and complex roles of physiological and physicochemical characteristics in SQV oral absorption, the optimization of AIDS boosting regimens requires careful consideration in order to avoid therapy limiting drug-drug transporter and enzyme interactions.
(-)-α-Bisabolol – a Specific Ergosterol Biosynthesis Inhibitor ? PAULI A^1, SCHILCHER H^2 ^1ReviewScience, Zirndorf, Germany; ^2Immenstadt, Germany Background: At the level of zymosterol in the ergosterol biosynthesis all subsequent intermediates are solely produced by fungi. The recognition of a specifically acting inhibitor within this part of ergosterol biosynthesis would leed to a new class of antifungal drugs. To obtain information on the existence of compounds having both, structural similarities to fungi specific ergosterol precursors plus antifungal properties, previous findings in literature were analyzed in view of this aspect. Methods: A chemical (sub)structure and MIC searchable computer database on antimicrobials (Amicbase) was used. In the data query antifungal compounds were searched, which include one of four different side chains types as they occur in the section from zymosterol to ergosterol. Results: Among antifungals the side chain of zymosterol was most frequently found. Addition of oxygen and limiting to Candida albicans resulted in 19 different compounds, whose MIC increased with molecular volume (r = 0.77). Like zymosterol, (-)-α-bisabolol contains a cyclohexene ring plus a second double bond in similar interatomic distance. This may point to an inhibition of zymosterol conversion into fecosterol by (-)-α-bisabolol, which would offer the opportunity to disturb specifically fungal biochemistry. (-)-α-Bisabolol from chamomile has antiinflammatory, wound-healing, anticancer, antispasmodic properties and inhibits fungi (3-100 µg/ml), gram-(+) (32-500), but less or not gram-(-) bacteria. Due to its low toxicity (monkeys tolerated oral 15 ml/kg bw) and the lack of reports on allergic reactions in its cosmetic use, the compound was taken to treat fungal and bacterial infections. Own case reports will be given. Conclusions: The antifungal mechanism of (-)-α-bisabolol is worthwile for further investigation due to its safety and its unique pharmacological profile.	Discovery and Use of the Magic Bullets in Human Taeniosis (Niclosamide, Praziquantel) PAWLOWSKI ZS Medical University, Poznan, Poland Background: Taeniosis/cysticercosis is a serious public health problem in several countries of Latin America, Africa and Asia. Approximately 2 500 000 people carry a Taenia solium tapeworm. Globally, conservative estimates calculate 50 000 deaths from neurocysticercosis every year. Older taenicides: Several more or less toxic natural remedies, such as male fern extract, Kosso flowers, areca nuts, pomegranates have been used for centuries. In early 20^th century in Germany among 22 000 cases of taeniosis treated with male fern extract 18 patients died and 71 others have lost vision. Several synthetic drugs were tried as taenicides in 20^th century e.g. thymol (1912), carbon tetrachloride (1931), hexylresorcinol (1932), mepacrine (1947), dichlorophen (1956), bithionol (1962), paromomycine (1967) and mebendazole (1975). In 1950s my patients with T.saginata taeniosis were treated with pumpkin seeds (cure rate 65% among 163 treated), atabrine (respectively 42%, 44), acranil (88%, 89) or metallic tin compounds (88%, 226). Many of these drugs were unsafe or poorly tolerated, although some were rather effective. Modern taenicides: The first magic bullet was - niclosamide, introduced in 1959. As a barely absorbed substance it is safe and well tolerated. However, it’s early original version (and still some generic products) has lost the efficacy during storage due to polymerization of its active particles. The efficacy of a single dose of niclosamide in human taeniosis is about 85%. Since 1972 niclosamide has been gradually replaced by the second magic bullet - praziquantel, being more stable taenicide, more efficacious (95%) and much cheaper (10 US cents for a dose). It has been used widely in the control of schistosomoses. Due to autoinfection more than 10% of T.solium tapeworm carriers develop neurocysticercosis. Therefore, the use of praziquantel in control of taeniosis is questioned, as this drug may damage existing brain cysticerci and change asymptomatic neurocysticercosis into a symptomatic one. The problem is partly solved by a reduction of the dose of praziquantel in taeniosis to 5-10mg/kg b.w. in a single dose. Still some uncertainty exists whether it is worth to risk such a rare side effect at a mass-treatment in T.solium endemic areas. Conclusions: 1)There is still a place for another magic bullet in taeniosis/cysticercosis. 2)Nitazoxanide, a broad spectrum antiparasitic drug, is waiting in a row.
Metallic gold reduce TNFα expression, oxidative DNA damage and pro-apoptotic signals in brain injured mice. PEDERSEN MØ, LARSEN A, PEDERSEN DS, PENKOWA M The University of Copenhagen, Section of Neuroprotection Background: Traumatic brain injury (TBI) continues to represent a major global health problem, and represents a leading cause of morbidity and mortality in young individuals. There is therefore an imperative need for neuroprotective treatments limiting the neurologic impairment following such injury. Gold compounds have been used for treating the inflammatory condition rheumatoid arthritis for over 50 years. Due to the well-established anti-inflammatory effects of gold compounds in the treatment of arthritis and the known role of inflammation in fueling cell death in the injured brain, we investigated the potential of metallic gold in alleviating inflammation, oxidative stress and apoptotic markers of cell death following experimental traumatic brain injury in mice. Methods: C57BL6 mice were subjected to a unilateral traumatic cryo(freeze)-lesion with concomitant injection of 25-45 µm gold particles suspended in hyaluronic acid near the lesion. Placebo-treated mice served as controls. Immunohistochemical markers of inflammation, oxidative stress and apoptosis were then compared in the gold- and placebo-treated animals at 1 and 2 weeks post-lesion. Results: The study revealed a statistically significant decrease in tumor necrosis factor alpha (TNFα) and oxidative stress (as judged by immunohistochemical staining for 8-oxoguanine) in gold-treated animals, as compared to controls. Moreover, gold-treatment resulted in a statistically significant decrease in the release of cytochrome c from the mitochondria and in the activation of caspase-3. Conclusion: We have previously shown that intracerebral deposition of metallic gold liberates gold ions that reduce microglial activation and neuronal apoptosis, while increasing reactive astrogliosis and neuronal stem cell response following focal brain injury. In this study we showed that bio-liberated gold ions result in marked anti-inflammatory, anti-oxidative and anti-apoptotic responses in the injured brain, and could have clinical potential for treating traumatic brain injuries. The findings thus support the role of metallic gold as a neuroprotective compound. We further hypothesize that this local gold implant application results in negligible systemic disseminations of gold ions, hereby limiting the risk of severe adverse effects, such as nephrotoxicity. We therefore propose that metallic gold implants may be useful as an enhancer of neuronal protection and regeneration following both TBI and perhaps in many other neurodegenerative conditions.	New Calcipotriol Analogs, their Toxicity and Antitumor Activity in vivo in Comparison to the Affinity with VDR and DBP PELCZYNSKA M^1, FILIP B^2, MILCZAREK M^2, KUDUK-JAWORSKA J^3, KUTNER A^4, WIETRZYK J^2 1. Karkonosze College, Higher Professional State School, Jelenia Góra, Poland 2. Polish Academy of Sciences, Institute of Immunology and Experimental Therapy (IITD), Wrocław Poland 3. Wrocław University, Wrocław Poland 4. Pharmaceutical Research Institute, Warszawa, Poland Background:Calcitriol has been proven to be a potent antiproliferative agent against various normal and neoplastic cells. Moreover, calcitriol and other vitamin D analogs revealed the ability to induce differentiation of many human cancer cells. Biological activity of these compounds is mediated by the nuclear vitamin D receptor (nVDR). Calcitriol is carried with plasma Vitamin D Binding Protein (DBP). The affinity with DBP could be responsible for the toxicity and bioavailability. Such biological properties suggest the potential therapeutic application for these agents, including antitumor therapy. Two of the promising calcipotriol analogs: PRI-2202, PRI-2205 and the paternal compound have been the objects of our intensive studies. Methods: In this work, we present results of the affinity of different analogs of vitamin D with VDR or with DBP using Molecular Operating Environment (MOE) program. Furthermore, the toxicity, calcemic activity and antitumor activity of these analogs in the LLC mice tumor model were tested. Results: PRI-2205 analog exhibited the highest affinity with VDR and DBP. PRI-2205 analog exhibitad both the low toxicity and calcemic activity and the highest antitumor activity with comparison to other derivatives. Conclusion: 1.Based on these results, we could formulate the hypothesis about the positive correlation between the antitumor activity of new calcioptriol analog and its affinity with VDR. 2.Its lower toxicity and calcemic activity seems to be correlated with its higher affinity with DBP. The authors are grateful to Chemical Computing Group Inc for making the MOE program available for free testing work.
Modifying cytoplasmic protein complexome involved in energy metabolism as a trategy of Escherichia coli against ceftriaxone Xuan-Xian PENG^1, JIAN-YI PAN^2, SAN-YING WANG^2, HUI LI^1, BIN JIANG^1, BAO-CHENG WANG^1, WEN-JIAO XU^1 ^1Center for Proteomics, State key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, University City, Guangzhou, 510006, China; ^2School of Life Sciences, Xiamen University, Xiamen, 361005, China Background: Recently, altered proteins or proteomes in response to antibiotic resistance are systematically investigated, but information regarding to a protein complexome in the resistance is not available. Aims: 1) To establish a cytoplasmic protein complexome involved in ceftriaxone (CRO) resistance by combined 2-D native/SDS PAGE and protein-protein interaction approaches, 2) To investigate functional characteristics of the complexome using genetic modified strains of the gene deletion of the proteins involved. Methods: Combined 2-D native/SDS PAGE and Co-IP, far-Western blotting or His-tag pull down assays were utilized for the cytoplasmic protein complexome of E. coli involved in CRO resistance. Antimicrobial susceptibility and carbohydrate metabolism of the complexome was investigated by minimun inhibitory concentrations (MIC) and survival capability assays of genetic modified strains with gene deletion of the proteins. Results: MalP, AtpD, PflB, LysS, MalE and CysK were found to be down-regulated, and SucC and SucD were up-regulated in the CRO-resistant E. coli strain. They respectively belonged to seven protein complexes, MalP homodimer, AtpD homodimer, PflB-TnaA, LysS homodimer, MalE-TalB, CysK-SodB and SucC-SucD. Most of them were involved in carbohydrate metabolism. MalP homodimer, SucC-SucD, LysS homodimer and CysK-SodB may play more important roles in the control of CRO resistance than other complexes identified. On the other hand, CRO-R and these mutants grew normally as the same as CRO-R-O in LB medium but most of them showed growth-combating in M9 medium with fructose, D-mannitol, maltose, glycerol or glucose. Importantly, up-regulated SucC and SucD in 2-DE gels respectively played negative and positive role in regulation of TCA circle and CRO resistance. All complexes but SucD-SucC were contributed to down-regulated the classical carbohydrate pathways as a strategy against CRO.