Ehrlich II –2nd World Conference on Magic Bullets

KHAVINSON VKH, MALININ VV

Saint Petersburg Institute of Bioregulation and Gerontology of the NW Branch of the Russian Academy of Medical Sciences

Background: Ageing is characterized by desorganization of peptidergic system of regulation of organism functions. The development and study of peptide geroprotectors is very prospective.

Methods: The level of expression of various genes, intensity of protein synthesis in the cells, indices of immune and endocrine system, metabolism and antioxidant defense were studied in animals and humans.

Results: Peptides regulated gene expression and protein synthesis in the cells. This was largely conditioned by immunomodulating, oncomodifying and stress-protection properties of peptides. Peptides contribute to DNA structure restoration and decreased incidence of chromosome aberrations in the cells induced by radiation, chemical effects and hypokinesia. Mechanisms of their geroprotective action are related to activation of chromatin in blood lymphocytes of old patients. Peptides show opioid activity and produce modulating effect on the content of biogenic amines (noradrenalin, dopamine, 5-oxyindolacetic acid, serotonin, histamine) in brain cortex and blood serum of animals, due to their effect on the central and peripheral regulatory mechanisms of stress and inflammation. Geroprotective action of peptides is also related to their influence on the mechanisms of hormone regulation and antioxidant defense. Administration of thymus and pineal peptides to mice and rats of different strains promoted reliable increase in an average life span by 30-40% and depressed growth of spontaneous, induced and transplanted tumors in animals. Animals revealed restoration of melatonin level, antioxidant defense enzymes and normalization of some components of mitochondria respiratory chain. Administration of pineal peptides to old monkeys promoted reliable restoration of melatonin, cortisol and glucose in the blood to the level in young animals.

Application of thymus and pineal peptides in elderly and old patients resulted in restoration of melatonin level, indices of antioxidant defense, immune, endocrine and cardio-vascular systems, brain functions. It was accompanied by a 2-fold decrease in the mortality rate in these patients during 8-12 randomized clinical studies.

Conclusions: The results of studies evidence prospects for application of peptide geroprotectos for prevention of premature ageing, age-related pathology and an increase in the period of active longevity.

New Steroidal Hormones Promise to Become a Multi-Purpose “Magic Bullet” KHRIPACH VA^1, ZHABINSKII VN^1, ALTSIVANOVICH KK^2, SAMUSEVICH MP^2, ZAVADSKAYA MI^1, TARAZEVICH EV^3, SEMENOV AP^3. ^1Inst. Bioorganic Chemistry, Minsk; ^2Mikonik Technologies Ltd, Minsk; Fish Industry Inst, Minsk, Belarus. Background: Discovery of plant steroid hormones called brassinosteroids (BS) showed that steroids are versatile hormonal regulators, characteristic to most organisms inhabiting the Earth. BS demonstrate wide spectrum of regulatory effects on plant growth and development. Their important feature is the ability to enhance plant resistance to unfavorable factors (diseases, stresses, pollutants, etc.) and to improve plant productivity together with the quality of crops. BS protective action is a result of multiple changes at molecular and cell level including activation of protein and nucleic acids’ biosynthesis, changes in hormonal balance, activity of enzymes, in composition and properties of membranes. As obligatory constituents of plants, BS have been and are consumed by mammals with food over all the evolution, but till recently there were practically no attempts to investigate their specific effects in higher animals except toxicological studies. Methods: This paper reports our results in studies of effect of 24-epibrassinolide (EBl - one of the typical and most active BS) on the serum cholesterol levels, data on its anti-HIV activity in vitro and some other data reflecting its action on immune and hormonal system of animals. Effects on the serum cholesterol levels were studied in rats and human volunteers. Anti-HIV activity was studied using Formazan assay, Supravital cell staining by the trypan blue assay and Indirect immunofluorescence assay. Some effects of EBl on immune, hormonal and reproductive systems were investigated in mice, in rats and in chickens and in fishes. Results: The study showed a high efficacy of EBl as a cholesterol-lowering agent in mammals for a wide range of doses. Tests on anti-HIV activity showed that EBl is efficient as anti-viral agent at average concentration of 10^-7 mol/L. Effect on hormonal balance and stimulation of immune and reproductive system were registered. Conclusions: The obtained data provide evidence that BS possess different activities in animals, and these activities are similar to a certain extent to those we know in plants both in respect to profile and active doses. This finding allows looking at BS from a new point of view: they are promising multi-purpose agents for human and veterinary medicine.	From 1-4 Weeks Of Treatment Down To A Single Application: A Novel Terbinafine Topical Treatment Of Tinea Pedis KIENZLER JL^1, de CHAUVIN MF^2, ORTONNE JP^3 ^1Novartis, Nyon, Switzerland; ^2Hôpital Saint-Louis, Paris, France; ^3Hôpital l’Archet 2, Nice, France, Background: Tinea pedis is a common dermatophytosis requiring topical antifungals for at least 1-4 weeks (W). This has a negative impact on compliance and outcomes. A novel topical solution of terbinafine (film-forming-solution - FFS) was developed to allow single application Methods: 1- The stratum corneum (SC) pharmacokinetics (PK) of terbinafine following single application was investigated in three PK trials on healthy volunteers (n = 6, 12 and 18). Drugs were applied to the back and skin strips were taken from defined areas at baseline and from 1 to 312 h after application. Samples were analysed using validated liquid chromatography/mass spectrometry. 2- Dose-finding and efficacy trials: 344 and 273 tinae pedis outpatients confirmed by mycological examination were evaluated for efficacy of 10% and 5% (dose-finding trial only) and 1% FFS in randomised double blind vehicle controlled parallel group trials. Evaluations were carried out at baseline, 1 and 6 W. Effective treatment rate (ETR) based on negative mycology and minimal symptoms was measured at W 6. In the efficacy trial, recurrence (positive cultures at W 12) was also assessed. Results: 1- The residence time of the film on the skin was up to 72 h. 30% of the total amount of drug delivered into the SC occurred during the first 2 h, 31% from 2-12 h, and 39% thereafter. The maximum concentration was observed as early as 1.5 h. Fungicidal SC terbinafine levels were still detected after 13 days (24 ng/cm^2). 2- ETR at W 6 with 10%, 5% and 1% FFS were 61%, 70%, 66% compared to 18% with the vehicle. All three active treatments were significantly superior to the vehicle (P<0.001). 1% and 5% FFS were non-inferior to 10% FFS. In the efficacy trial, ETR was 63% in the 1% FFS group and 17% for the vehicle (P≤0.0001). Recurrence occurred in 12.5% of the effectively treated patients at W 6. 1% FFS was well tolerated. Conclusions: This novel formulation delivers high amounts of terbinafine to the SC for a prolonged time. 1% FFS was the minimal effective dose. Effectiveness of 1% FFS was confirmed by the efficacy trial which also showed a similar relapse/re-infection rate to that previously demonstrated with terbinafine 1 % cream for 1 week. This novel product represents a significant advance in the treatment of tinea pedis with the enhanced compliance and convenience that it offers.
Tumor Necrosis Factor (TNF)- Inhibitors Effectively Treat Asthma KIM J, REMICK DG Department of Pathology and Lab Medicine, Boston University School of Medicine Boston, USA The morbidity and mortality from asthma in the Western world have profoundly increased in the past two decades. Recent studies showed that sensitization and exposure to cockroach allergens strongly correlated with increased asthma morbidity and severity for children, especially among inner city children. As a unique form of chronic airways inflammation, asthma is characterized by reversible airway obstruction, airway hyperresponsiveness, and the production of multiple inflammatory mediators. Local activation of both immune and nonimmune cells in the lung triggers the release of these immunomodulator proteins including tumor necrosis factor (TNF) – . TNF-, as a multipotent pro-inflammatory cytokine, has been postulated to be a critical mediator directly contributing to the bronchopulmonary inflammation and airway hyper-reactivity in asthma. The successful treatment of various chronic inflammatory diseases such as rheumatoid arthritis, Crohn’s disease, and psoriasis provides great potential that inhibition of TNF- activity may have application for the treatment of asthma. Recently we have shown that airway expression of TNF- peaked shortly after allergen challenge in a mouse model of asthma induced by a house dust extract that contains high level of cockroach allergen and endotoxin. TNF neutralization with a specific antibody significantly reduce the pulmonary inflammation and airway hyperresponsiveness. Recent developments in clinical trials in patients with severe asthma provide strong support for the concept that blocking TNF- activity represents a new approach in asthma therapy.	Effect Of A Subtoxic Dose Of Acetaminophen On The Toxicity Of Chemicals That Are Metabolically Activated Kim YC, Park HK, Kim SJ, Kwon DY Seoul National University, College of Pharmacy, Seoul, Korea Background: While numerous studies describe the toxic consequences resulting from an excessively large dose of acetaminophen (APAP), a widely used analgesic-antipyretic, its potential adverse effects at a lower or therapeutic dose have hardly been explored. The present study was aimed to examine the effects of prior exposure to a subtoxic dose of APAP on the metabolic disposition and toxicity of a following dose of this drug. Methods: In a preliminary experiment an APAP dose of 500 mg/kg, ip, was shown to be non-toxic to female Sprague Dawley rats used in this study. At 18 hr after administration of APAP at this dose, rats were challenged with an identical dose of APAP and the elimination of APAP from blood was determined. Serum enzyme activities were measured 24 hr after the challenging dose of APAP to estimate the liver injury. Also the hepatic microsomal drug metabolizing enzyme activities and their expression were measured in rats treated with a single dose of APAP 18 hr prior to sacrifice. Results: APAP and APAP-glucuronide concentrations in plasma were unaltered by APAP pretreatment. APAP-sulfate concentrations were decreased, while APAP-cysteine concentrations were elevated significantly. The elevation of serum hepatotoxic parameters was also enhanced by APAP pretreatment. In rats treated with a single dose of APAP 18 hr prior to sacrifice, hepatic microsomal chlorozoxazone 6-hydroxylase, p-nitrophenol hydroxylase, p-nitroanisole O-demethylase, and aminopyrine N-demethylase activities were all increased to 173 %, 151 %, 158 %, and 116 % of normal control, respectively. Immunoblotting analysis indicated that expression of CYP2E1, 3A, and 1A was also induced significantly. Neither hepatic glutathione contents nor glutathione S-transferase activity was changed by the single dose of APAP. Conclusions: 1) A subtoxic dose of APAP may increase the CYP2E1, 3A, and 1A expression and their metabolizing activities. 2) The altered CYP contents and activities may actually influence the metabolism and resulting toxicity of a repeated dose of APAP. 3) Considering the wide use of APAP as an analgesic-antipyretic, it is suggested that a greater concern should be expressed regarding the effects of acute or repeated dosing of this drug even at a therapeutic level, especially when used in combination with other medications.
Quinolone Resistance In Campylobacter Isolates Originating From Chicken In Senegal. KINANA AD^1, CARDINALE E^2, TALL F^3, BAHSOUN I^1, SIRE JM^1, BREUREC S^1, GARIN B^1, BOYE CSB^4, PERRIER-GROS-CLAUDE JD^5. ^1 Institut Pasteur, Dakar, Sénégal; ^2 CIRAD, Montpellier, France; ^3 ISRA, Dakar, Sénégal; ^4 Université Cheikh Anta Diop, Dakar, Sénégal; ^5 Institut Pasteur, Casablanca, Maroc. Background: In Senegal, fluoroquinolones (norfloxacin and enrofloxacin) were first used in poultry production in 1996 to treat respiratory and intestinal diseases. It is known, however, that ciprofloxacin resistance in Campylobacter isolates derived from commercial chickens reached 40%. Aims: 1) To investigate the genetic basis of quinolone resistance in Campylobacter strains isolated from chicken in Senegal. 2) To determine the relationship between quinolone resistance and sequence type (ST). Methods: This study included 54 Campylobacter jejuni and 33 Campylobacter coli isolates obtained from 14 dispersed collection sites over a 3-year period. The susceptibility of isolates to nalidixic acid and ciprofloxacin was determined by E-test and the agar dilution method. The quinolone resistance-determining regions (QRDR) of gyrA and gyrB genes were sequenced. Multilocus sequence typing (MLST) was used to study the clonality of isolates. Results: Among the 27 ciprofloxacin-resistant C. jejnui isolates (MICs 8 to >32µg/ml), 18 exhibited the Thr-86-Ile substitution, 4 had the Thr-86-Ala substitution and 5 showed no mutation in the GyrA QRDR. However, two isolates susceptible to ciprofloxacin but intermediate to nalidixic acid (MIC 16 µg/ml) had also the Thr-86-Ala substitution in the GyrA protein. Two additional substitutions (Asn-203-Ser and Ala-206-Thr) were identified regardless of quinolone susceptibility. For C. coli, among the 14 ciprofloxacin-resistant isolates, 12 displayed the Thr-86-Ile sustitution, and 2 had no substitution within the GyrA QRDR. The sequencing of the gyrB QRDR from quinolone-resistant isolates revealed no substitution. MLST showed that the resistance phenotype varied for the same ST and within the same lineage. Conclusions: 1) The Thr-86-Ile substitution in the GyrA protein was the predominant mechanism of quinolone resistance. However, some isolates displayed an unusual mechanism of resistance to quinolones. 2) There was no link between quinolone resistance and ST, and that the emergence of quinolone resistance is not related to the diffusion of a unique clone.	Opioid Agonist or Opioid Antagonist: Magic Bullets in the Treatment of Opioid Addiction KING VL, KIDORF MS, BROONER RK Johns Hopkins University School of Medicine, Baltimore, MD, USA Background: Methadone maintenance treatment for chronic opioid addiction was envisioned as a “magic bullet” to eliminate opioid withdrawal and craving to make meaningful rehabilitation possible. Methadone maintenance over long periods was undesirable to some patients, yet discontinuation of methadone maintenance often led to heroin relapse even after long periods of abstinence. So an alternate orally active “magic bullet,” naltrexone, was developed to improve overall acceptability of opioid addiction treatment. It blocks the opioid receptor and prevents opioid physical dependence. Unfortunately, this highly efficacious treatment is poorly effective because patient adherence is worse than that for methadone maintenance. The effectiveness of both medications is significantly limited by patient non-adherence, so systems of care must be developed to ensure delivery of the medication and other services needed for optimal rehabilitation. This “magic gun” for the “magic bullet” is key to the treatment process, but is an element often lacking in treatment of chronic medical problems. Methods: We developed Motivated Stepped Care (MSC) as a "magic gun” to improve the effectiveness of methadone maintenance by using the behaviorally reinforcing properties of methadone to motivate improved adherence to a stepped care, patient-treatment matching paradigm. Patients enter MSC in low intensity counseling care and are referred to higher, discrete intensities (“steps”) based on objective indicators of current treatment response. Once stabilized, they are returned to lower steps of care in an efficient and cost effective manner. A randomized, controlled trial of 127 new admissions was used to evaluate the effectiveness of MSC. Results: Patients randomly assigned to MSC (n = 65) had lower rates of poor treatment response (46% vs 79%, p < .001), and improved counseling attendance (83% vs 44%, p < .001) compared to a standard treatment condition (n = 62). MSC was well tolerated and associated with excellent attendance across varying treatment schedule intensities. Conclusions: 1) Treatment adherence and response to MSC is superior to standard methadone maintenance treatment. 2) This treatment approach has broad theoretical and practical application in treatment of addiction and other chronic behavioral problems that share the common problem of poor treatment adherence.
Exploiting Plant Sources for Potential Drugs. Alpinumisoflavones in Perspective KINGSFORD-ADABOH R,^1 HARRISON JJEK^1, DITTRICH B^2, HUEBSCHLE CB^2, GBEWONYO SKW^1, TABUCHI Y^3,OKAMOTO H^3, KIMURAE M^3, ISHIDA H^3 ^1University of Ghana, Accra, Ghana; ^2Free University of Berlin, Berlin, Germany; ^3Okayama University, Okayama, Japan. Background: Isoflavones/flavones have been known to have an array of medicinal properties and currently several medicines in allopathic medical practice are Isoflavones/flavones and their derivatives. Scientific investigations have established a myriad of biological activities justifying their concomitant use in folklore medicine several of which have been catalogued. In Franco West Africa, the bark infusion is used as a laxative for children, in Nigeria however, the pulverized root bark decoction is drunk as a relief for dysmenorrhea, as a de-wormer and blood purifier while the leaf extract is used as a cure for dysentery and diarrhea (Irvine, 1961; Abbiw, 1990). The leaf juice is reported to be lethal to Bulinus snail (Abbiw, 1990; Maillard et al., 1993), a water snail carrying the microorganisms that cause that cause schistosomiasis-bilharzias, a parasitic disease endemic throughout South America, Africa and the Far East. Our recent investigation into the structural and bioactive properties of selected alpinumisoflavones reveals a very strong brine shrimp lethality of these compounds (Kingsford-Adaboh et al., 2006). Brine shrimp lethality tests have been used for assaying potential anticancer candidates (McLaughlin JL et al., 1993). The discovery of the therapeutic potential of inhibitors of Monoamineoxidase-B in aging –related neurodegradative diseases such as the Parkinson’s and Alzheimer’s diseases has increased tremendously the interest in the search for compounds which have this therapeutic potential. It has been suggested elsewhere that the fruits of Cudrania tricuspidata, containing potent Monoamineoxidase-B (MAO-B) inhibitory prenylated Isoflavones, could be possible therapeutic candidates for the Parkinson’s and Alzheimer’s diseases. The chemical constituents of this plant which exhibited this high inhibition of MAO enzyme in concentration depended manner included 4-O-Methylalpinumisoflvone and Alpinumisoflavone which also occur in Milletia thonningii and other plants (Han XH et al., 2005). The potential antioxidant and anticancer properties of prenylated isoflavone have also been commented on (Comte et al., 2001). In confirmation of the folkloric use of the West African Legume Milletia thonningii in Ghana and other parts of the world as anthelmintic and a purgative (Irvine, 1961; Abbiw, 1990), Perrett et al., (1995) found that the chloroform extract of the seed of Milletia thonningii which contain predominantly alpinumisoflavones elicited molluscicidal and cercaricidal activity when topically applied to mouse skin 2 and 24h prior to exposure to Schistosoma mansoni cercariae. The presence of the Milletia thonningii extract components on the skin appeared to be effective in preventing subsequent establishment of infection. This anti-schistosomal activity bioactivity has been corroborated (Maillard et al., 1995 and Lyddiard et al., 2002). Lyddiard and Whitfield mentioned and inhibitory effect of the crude extracts on site I mitochondrial electron transport system (Lyddiard and Whitfield, 2001). While these bioactivities have been unambiguously established, one is still at sea as to which of the many isoflavones are responsible for these bioactivities and how the presence, absence or relative positions of functional groups as well as differences in the molecular conformation are linked to these bioactivities. As part of our multidisciplinary approach towards the study and systematic characterization of the crystals of this plant, seven compounds have been studied some of which were isolated from the dichloromethane extracts of the rootbark and the seeds (Kingsford-Adaboh et al., 2001, 2006; Harrison et al., 2008) with the hope that the crystal structure, molecular and electronic properties can deepen our understanding of their observed bioactivities. The scheme below shows the compounds so far studied. Methods: Sample preparation and crystallization Seeds of M. thonningii collected from the University of Ghana Botanical Gardens were air dried. These were ground into powder which was continuously soxhlet-extracted in methanol. Column chromatography and preparative thin layer chromatography yielded compounds (II) and (III), O,Odimethylalpinumisoflavone and 5-O-methyl-4-O-(3-methylbut-2-en-1-yl)alpinumisoflavone, respectively. Compound (I),4-O-methylalpinumisoflavone, which hitherto had been difficult to crystallize for X-ray diffraction purposes, was obtained from demethylating a product of (II) in a mixture of cold BCl3 and chloroform. IR and NMR spectra confirmed the methylated product. These were crystallized from ethanol. For compounds (IV)- (VII) which were obtained from the demethylation, products obtained was divided into four and each recrystallized using acetonitrile, methanol, ethanol and water and the melting points of the compounds determined. The solid-solid phase transition temperatures between the solvent included compounds and the efflorescent crystals were measured by differential scanning calorimetry apparatus. X-ray diffraction experiments X-ray data of the compounds(IV,V,VI and VII) were collected on a Rigaku R-AXIS RAPIDII imaging plate diffractometer with graphite monochromated Mo Kα radiation (λ = 0.71075 Å). The absorption corrections were carried out using multi-scan (ABSCOR; Higashi, 1995). Cell refinement was carried out using PROCESS-AUTO and the data reduced by using Crystal Structure (Rigaku/MSC, 2004). The structures were however solved using SHELXS97 (Sheldrick, 2008) and the structures refined with SHELXL97 by full-matrix least-squares on F^2 against ALL reflections. Toxicity measurement of samples (I),(II) and III) Toxicity of the isolated compounds to brine shrimp Brine shrimp, Artemia salina Leach, has been successfully used as a bench-top assay to determine the toxicity levels of natural products from plants (Meyer et al., 1982), as brine shrimp larvae are sensitive to small doses of biologically active chemicals. Eggs of brine shrimp (Brine Shrimp Direct, California, USA) were hatched in sea salt water (3.8%) and were allowed to hatch and mature for 48 h, before nauplii were used for a bioassay,in which dilutions of the compounds ranging between 0.01 and 100 mg ml^_1 were used. [Regrettably not enough space on this page.]