Methotrexate in Dermatology through 50 Years ZACHARIAE H University of Aarhus, Denmark
Background: Historical evaluation. Methotrexate (MTX) is a cytostatic, well established in treatment of malignancies. It is the oldest of drugs used today for systemic therapy of psoriasis. 1958 was the start of MTX in dermatology. Originally it was prescribed daily for 5-10 days followed by a rest period of a few days. Since 1970 weekly oral or parental doses became the established way and together came trials of combination therapy. In 1978 it was found that MTX lead to more than 75% improvement in 90% of psoriatic patients.
Methods: Studies of mechanisms of action and establishment of side-effects. Despite many years use of MTX, there are uncertainties concerning mechanisms of action. Among the many are cytokine inhibition (Interleukin 1 and 6, and tumor necrosis factor (TNF) alpha) and inhibition of neutrophil leukotriene and platelet activity factor (PAF). Besides inhibition of adhesion, immune-modulation, and aspects of anti-inflammatory properties, a main effect is induction of apoptosis in activated T-cells. In mid nineteen-seventies it was well established, that long-term usage of MTX could induce liver damage, which in some cases led to fibrosis or even cirrhosis. Decline in popularity of MTX followed due to a “liver scare”, as it did due to the recommendations that liver biopsies be performed to monitor treatment. We do not recommend a pre-treatment liver biopsy unless one is dealing with a high risk factor for liver disease. Measuring aminoterminal propeptide of type III procollagen (PIIINP) is preferable. This is a non-invasive marker of fibrogenesis, that can show that as long as PIIINP is normal, no significant development of fibrosis is taking place. Dermatologists are also in increasing numbers using MTX in diseases like dermatomyositis, cutaneous sarcoidosis, scleroderma, pemphigoid and pemphigus, Behcet’s disease, impetigo herpetiformis and mycosis fungoides. This will often be given in combination therapy as in psoriasis. Among the latter can be mentioned combinations of MTX with the TNF-alpha inhibitors etanercept or infliximab, where a large number of patients over several years have benefited.
Results and Conclusion:If the rules for accepted control are adhered to, the risk/benefit ratio for MTX should be considered one of the most valuable treatments in dermatology. The biologics are generally, but not always of higher dimension in relation to clearing.
New View of Retroviruses; Consequences in Aids Diagnostics and Therapy ZAJAC V1, MEGO M2, MATELOVA L1, SCHREINER A3, STEVURKOVA V1, STANEKOVA D4 1Cancer Research Institute, Vlarska 7, 83391 Bratislava, Slovakia
2National Cancer Hospital, Bratislava, Slovakia
3Ardeypharm, Herdecke, Germany
4Slovak Medical University, National HIV/AIDS Centre, Bratislava, Slovakia
Despite unquestionable success in diagnostics and therapy of AIDS, it is still not possible to stop the worldwide pervasion of this infection. This situation is challenging for us to fight against this disease more complexly and overcome all taboos and dogmas (HIV=AIDS) established in diagnostics and therapy of this disease. The fundamental, still unanswered question is the origin of the HIV. The dogma, which originated HIV by transferring from African apes/monkeys to human population some decades ago, doesn’t explain tremendous increase of AIDS incidence, mainly in Asia.
Our untraditional approach is based on original detection of the HIV-like sequences in intestinal bacteria of American and Slovak HIV/AIDS patients by colony and dot blot hybridization assay. Using specific PCR primers for gag, pol and env HIV-1 genes, were synthesized PCR products on bacterial template, which were found in more than 90% homologous to the corresponding HIV-1 sequences. The hight capacity of intestinal bacteria isolated from HIV-positive patients to enter into HL-60 cells and normal human lymphocytes was confirmed by gentamicine protection assay. The reduction of bacteria bearing HIV-like sequences was performed by per oral application of probiotics bacteria Escherichia coli strain Nissle 1917 to AIDS patients. After three months of probiotics treatment, the remission of the viral load was found in 61% of tested patients.
The achieved results allowed us to: 1. predicate that in the AIDS process intestinal bacteria play a basic or even the key role; 2. create a new hypothesis dealing the induction of immunodeficiency with new therapeutic possibilities.
Structural and Functional Diversity of Endogenous Antimicrobial Oligopeptides ZAMYATNIN AA1,2 1A.N.Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia; 2Universidad Tecnica Federico Santa Maria, Valparaiso, Chile
Background: Chemical substances named as oligopeptides consist of 2 to 50 amino acid residues. Natural oligopeptides may regulate nearly all vital processes. To date, primary structures of more than 7000 oligopeptides have been identified. Aims: 1) To elucidate all known natural antimicrobial oligopeptides; 2) To describe their structural-functional diversity; 3) To point their usage in medicine, veterinary, and food conservation.
Methods:This study was performed using our EROP-Moscow (Endogenous Regulatory OligoPeptides) database (http://erop.inbi.ras.ru/). It contains complete information on natural oligopeptides.
Results: It have been shown that chemical structures of natural oligopeptides have been identified from more than 1000 different species representing all the biological kingdoms. More than 1500 oligopeptides possess antimicrobial functions. They are active against bacteria, fungi, viruses, stimulate antiviral and antitumoral resistance. These substances were found out in animals (1211), bacteria (109), fungi (162), plants (162), and viruses (2). It is known 121 human oligopeptide structures. Primary structures of antimicrobial oligopeptides are characterized as having widely diverse sequences. Many of them display a net positive charge, ranging from +2 to +18. Nearly all antimicrobial oligopeptides form amphipathic structures upon interaction with target membranes and exhibit antimicrobial activity against a wide variety of micro-organisms. Antimicrobial oligopeptide structure and function offers hope for discovery and development of improved agents to prevent or treat infectious diseases caused by pathogens that resist conventional antimicrobial agents. Various human oligopeptides show a potent effect on pathogenic micro-organisms including antibiotic-resistant bacteria. Moreover, human defensins indicate that these oligopeptides are involved in various biological processes associated primarily with defensive and regulatory responses to infections by pathological agents.
Conclusions: Mammalian antimicrobial oligopeptides without any dangerous side effects due to their natural origin 1) can be used in therapy and veterinary; 2) can be considered as natural preservatives of food products. 3) are promising for hygiene and cosmetics.
Portal Haemodynamics and Plasma Transaminase Levels before and after Prostacyclin Analog Administration in Patients with Chronic Viral Hepatitis ZARDI EM1, VECILE E2, BARTOLOZZI F1, AFELTRA A1, DOBRINA A2 1Area of Clinical Medicine “Campus Bio-Medico”, Rome, Italy
2Department of Physiology and Pathology, Trieste University, Italy
Background: Under physiological conditions, hepatic stellate cells produce prostacyclin to regulate the haemodynamics of portal microcirculation; in chronic liver disease, there is a progressive dysfunction of hepatic stellate cells and a reduction in prostacyclin production. The administration of a prostacyclin analog might optimize the regulation of hepatic stellate cell function in patients with chronic liver disease. Aims: 1) To evaluate the hepatic haemodynamics and 2) to monitor the inflammatory indexes before and after a prostacyclin analog infusion. Methods: 11 patients (6 males and 5 females; mean age 60.7±8.7 years) with histological diagnosis of chronic viral hepatitis were enrolled in the study after the informed consent document was signed by each-one. In each patient, before and after 3 days of physiological solution infusion (placebo) and before and after 3 days of prostacyclin analog infusion (at a dosage of 2 ng/Kg/min for 6 hours/day), serum levels of liver enzymes (GOT, GPT) were evaluated and, with color Doppler sonography, portal flow velocity (cm/s) and portal diameter (cm) were measured to calculate portal flow volume (PFV) and congestion index (CI).
Results:After prostacyclin analog infusion, patients had an increase in PFV (p<0.01) and a decrease in CI and GOT serum levels (p=0.01 and p=0.05, respectively), as compared with pre-prostacyclin analog infusion. No statistically significant difference was observed in GPT serum levels.
Conclusions: After 3 days of prostacyclin analog infusion, a significant amelioration of the hepatic haemodynamics (as demonstrated by the increase in PFV and the decrease in CI) and of the liver inflammatory indexes (as demonstrated by the decrease in GOT serum levels) was achieved. 1) All patients had a better portal liver perfusion. 2) 5 patients had an excellent decrease in liver enzymes while in only one patient their levels worsened. 3) The mean GOT transaminase serum levels were significantly lower relative to those of pre-prostacyclin analog infusion; 4) the liver function seems to benefit from prostacyclin analog infusion, but prospective studies on a larger population are needed.
Neuropeptides in Stress-Related Disorders: Possible Interaction of Vasopressin and Corticotropin Releasing Hormone ZELENA D1, PINTÉR O1, MERGL Zs1, BARNA I1, MAKARA GB1 1Institute of Experimental Medicine, Hungarian Acaemy of Sciences, Budapest, Hungary
Background: Despite the great importance the treatment of affective disorders is not solved, yet. New drugs target neuropeptides, especially vasopressin (AVP) and corticotropin releasing hormone (CRH) as important regulatory molecules of the stress axis. Aims: 1) Summarize the available data on the role of AVP and CRH in affective disorders based upon their stress axis regulation. 2) Discuss the possible interaction of the two molecules.
Methods: This study will present our own data from AVP-deficient Brattleboro rats in scope of the literature. Behavioral studies (anxiety on elevated plus maze, depression-like behavior in the forced swim test etc.) are combined with analysis of the stress axis by plasma hormone content measurements (radioimmunoassay) and molecular biological methods (e.g. in situ hybridization).
Results: AVP-deficient animals are less anxious and show lower prevalence of depressive-like behavior in behavioral tests. These behavioral changes do not go parallel with changes in hormone levels. The CRH mRNA levels in the nucleus paraventricularis hypothalami are higher in AVP-deficient rats. Our results are supported by studies on high anxiety rats as well as by human data. The contribution of V1a or V1b receptors seems to be controversial. Moreover, several studies suggest that CRH rather than AVP is the key molecule of the process.
Conclusions: 1) AVP seems to be causally involved in the development of anxiety and depression. 2) The participating brain AVPerg circuits are distinct from those regulating the stress axis (different receptor subtypes should be targetted), however their activations may go parallel. 3) Complex intervention on both AVP and CRH system may lead to better results in therapy.
Nasal Vaccination against Bacterial Toxins ZENG M University of Rochester School of Medicine and Dentistry, Rochester, New York, USA
Background: Toxins produced by infectious bacteria often cause fatal diseases. Vaccines against bacterial toxins have been demonstrated as effective countermeasures against infectious diseases. However, with traditional immunizations by needle injection, there remain concerns about the availability of medical service and social acceptability. Our research aims to develop highly efficient and easily administered nasal vaccines against bacterial toxins.
Methods: This study tested a prototypical nasal vaccine against botulinum neurotoxin type C (BoNT/C). A replication-incompetent adenoviral vector encoding a human codon-optimized heavy chain C-fragment (HC50) of BoNT/C was produced in the laboratory. It was evaluated as a mucosal vaccine against BoNT/C in a mouse model. Groups of mice were intranasally administered with single doses of adenoviral vector Ad/opt-BoNT/C-HC50, ranging from 105 to 2 × 107 plaque forming units (pfu). Serum and mucosal Anti-HC50 antibody responses were measured by ELISA. The neutralization capacity of anti-sera from mice vaccinated with Ad/opt-BoNT/C-HC50 was determined by in vitro toxin neutralization assay. The protective efficacy of the nasal vaccine was assessed by challenge with up to 104 × MLD50 of active BoNT/C.
Results: Single intranasal inoculation of the Ad vector elicited a high level of HC50-specific IgG, IgG1, and IgG2a in sera and IgA in mucosal secretions as early as 2 weeks after vaccination. The antigen-specific serum antibodies were maintained at a high level at least until the 27th week. Immune sera showed high potency in neutralizing BoNT/C, as indicated by in vitro toxin neutralization assay. The mice that received a single dose of 2 × 107 pfu of Ad vector were completely protected against challenge with up to 104 × MLD50 of BoNT/C. The protective immunity showed vaccine dose-dependence from 105 to 2 × 107 pfu of adenoviral vector. In addition, animals that received a single intranasal dose of 2 × 107 pfu Ad vector could be protected against 100 × MLD50 27 weeks after vaccination. Animals with preexisting immunity to adenoviral vector could also be vaccinated intranasally and protected against lethal challenge with BoNT/C.
Conclusions: These results suggest that the adenoviral vector is a highly effective gene-based mucosal vaccine against BoNT/C. This study has set up a platform to develop needle-free nasal vaccines against bacterial toxins.
Authors’ disclosure statement:This work was supported by the US Public Service research grant AI055946 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health.
New Development of Drug Solubilization ZHANG H1, MAROJU R1, MENGER F2 1Mercer University, Atlanta, GA USA; 2Emory University, Atlanta, GA USA
Background: Approximately one of every two new drug candidates identified by high-throughput methods suffers from a very low water solubility, which usually gives rise to poor or erratic bioavailability, and consequently these compounds are frequently not taken forward for further development. Therefore, water-solubilization of poorly soluble drugs is a considerable challenge for drug discovery and development. The commercially available polyoxyethylene-based nonionic surfactants, such as Cremophor EL and Span 80, are used as drug solubilizers, but their applications are limited by their relatively low solubilizing capacity and serious side effects. Amides in conjunction with a hydrophobic chain were examined here because 1) amides should have greater solubilizing power than alcohols, ethers, etc. 2) amides are enzymatically hydrolyzable (yet chemically stable); and 3) amides, being both hydrogen-bond acceptors and donors, may improve the solubilization capacity by interacting with drug substances having hydrogen-bonding sites.
Methods: Twelve amide-based surfactants (“peptoads”) were synthesized through multi-step sequences, and their physicochemical properties were studied by X-ray analysis, solubility studies, surface tensiometry, and molecular dynamics simulations. One peptoad (namely peptoad G) was investigated for its capacity to solubilize eleven poorly water-soluble drugs by the shake-flask method, and the solubilizing mechanism was explored by molecular dynamic simulations. Additionally, the in-vitro toxicity of peptoads was evaluated on rat macrophage cell lines (RAW 264.7) by MTT assay.
Results: Peptoads possessing short carbon chains (seven carbons) are highly surface-active. Peptoad G can solubilize poorly water-soluble drugs very efficiently, by enhancing the water solubility ranging from about 20 to 1100-fold for eleven drugs tested. Molecular dynamic simulations on solubilizing paclitaxel by peptoad G showed that peptoad molecules, hydrogen-bonded to the drug, surround the drug with hydrocarbon chains that embed themselves into the interior of peptoad “clumps”. It is by this mechanism that paclitaxel is solubilized. Additionally, the in-vitro toxicity of peptoad may be comparable to Cremophor EL and Tween 80.
Conclusions: The colloidal structure of peptoads, along with their solubilizing capacity on poorly water-soluble drugs, their unique solubilizing mechanism and their low toxicity, makes them promising drug-solubilizing agents.
The combined use of HPLC, Gel-LC-MS/MS and microarray in monitoring product quality and safety for gene therapy ZHAO Y, KEATING K, WHEELER J, DOLMAN C, THORPE R
Biotherapeutics Group, National Institute for Biological Standards and Control (NIBSC), Blanch Lane, Herts, UK
Gene therapy has a unique potential for treating genetic diseases and is now regarded as an indispensable addition in conventional medicines. Quality and safety assessment of gene therapy products presents a great challenge, because they are cell products as well as multi-gene products, including viral and therapeutic proteins and modified cells. In this presentation, we will review our collective experience of using a Gel-LC-MS-MS/MS analysis and the gene-array technology to evaluate quality of HPLC purified products and, to assess the contribution of a range of potential components presented in a final production in product potency and safety in vitro and in vivo systems. The identification of protein biomarkers, transcriptional and toxicogenomic markers using the advance Gel-LC-MS-MS/MS analysis and gene-array supports the development of robust and product-specific assays for monitoring the quality, potency and safety of complex products for gene therapy.
Competition of Sulindac with Specific Markers for the Major Binding Sites on Human Serum Albumin Studied by High Performance Liquid Affinity Chromatography ZHIVKOVA Z, RUSSEVA V Faculty of Pharmacy, Medical University, Sofia, Bulgaria
Background: The binding of drugs to human serum albumin (HSA) is extensively studied because of the clinical significance of binding and displacement interactions. To predict the binding behavior of one drug in the presence of others and to enable their safe use it is necessary to have knowledge about the nature of drug-protein interactions. Aims: 1) Quantitative characterization of sulindac (SUL) binding to HSA. 2) Study of the competition of SUL with drugs, specific markers for both major binding sites on HSA: phenylbutazone (PBZ) and warfarin (R-WAR and S-WAR) for Site I, and diazepam (DAZ) for Site II. 3) Clarification of the molecular basis of the binding process using thermodynamic approach.
Method: The experimental technique is based on displacement chromatography on HSA-immobilised column. The drug is injected onto the column, and its capacity factor k’ is a measure of the binding to HSA. It is influenced by adding a cobinding agent to the mobile phase. A new mathematical approach is used to calculate the affinity constants at several temperatures. The major thermodynamic parameters (free energy change G0, free enthalpy change H0, and free entropy change S0) are calculated.
Results: SUL binds to HSA with high affinity constant ~106 M-1. The cobinding of SUL and PBZ does not affect the binding parameters. Both H0 and S0 are negative. R- and S-WAR influence differently the binding of SUL. In the presence of R-WAR the affinity constants of SUL are lower. The complexation is accompanied by small negative H0 and positive S0 suggesting different kinds of interactions. The cobinding of SUL and S-WAR does not affect the binding behavior. The cobinding of SUL and DAZ results in competition for two types of binding sites: high affinity ~ 106 M-1 and low affinity ~ 104 M-1.
Conclusions: 1) SUL binding to HSA is dominated by hydrogen bonds. The high negative H0 is the driving force for SUL binding. 2) The cobinding of SUL and PBZ is simply competitive. 3) The binding affinity of SUL is significantly decreased by R-WAR. Conformational changes in HSA may be assumed resulting in the alteration of the binding mechanism. 4) The binding of SUL and S-WAR is simply competitive involving the same kind of interactions. 5) DAZ significantly affects SUL binding developing another type of binding site, dominated by hydrophobic interactions.
Pituitary Resistance To Thyroid Hormone ZIELENIEWSKI W, JAGODZINSKA A
Department of Endocrinology, Medical University of Lodz, Sterlinga 3, 91425 Lodz, Poland, e-mail: wz1964@wp.pl Background: Ultrasensitive arrays for determination of TSH remains the milestone for the assessment of thyroid function. However, there are several situations when TSH concentration does not predict the real function of the thyroid. Thyrotropinoma and resistance to thyroid hormone remains the most common causes of the “Inappropriate TSH secretion”.
Aim of study: Differential diagnosis between thyrotropinoma and resistance to thyroid hormone.
Results: We present a case of a 24-year-old woman who suffered from mild thyrotoxicosis and diffuse goiter for several years. She had elevated fT3 and fT4 with slightly elevated TSH concentration. Pituitary adenoma was excluded as MRI showed normal pituitary gland, alpha-subunit was normal and TSH concentration raised after TRH administration. Sonography revealed normoechogenic, enlarged thyroid gland. Previously, she was given thiamazole, but without any significant amelioration. Thus, the diagnosis of the syndrome of pituitary resistance to thyroid hormone was established. The patient was given bromocriptine at a dose of 10 mg per day. After 2 months of treatment she achieved a state of euthyreosis.
Conclusions: A single TSH concentration measurement may be unreliable. Bromocriptine is efficient in patients with pituitary resistance to thyroid hormone.
Proline Specific Peptidases as New Targets for Drug Development. Pharmacology and Pathophysiology ZOLOTOV NN, GARIBOVA TL, LEBEDEVA MA, VORONINA TA, SEREDENIN SB
Zakusov Institute of Pharmacology, Russian Academy of Medical Sciences, Moscow, Russia
Background: Enzymes hydrolyzing peptide bonds involving proline are of particular interest because of the significance of this imino acids in the determining the conformation of peptide chains. Recent studies have shown that prolyl endopeptidase (PE) and dipeptidylpeptidase IV (DP-IV) participate in the metabolism of proline-containing neuropeptides related to pathogenesis of Alzheimer's disease, Parkinsonian syndrome, muscular dystrophies and other denervating diseases. The aim of our study was to investigate the antidepressive, antiamnestic and antihypoxic properties of PE and DP-IV inhibitors.
Methods: Forced swimming test was used for antidepressive activity studies. The investigation of age related mental decline, motor behavior and estimation of PE levels in different brain regions (cortex, hippocampus, hypothalamus) was carried out in Wistar rats of different age (3 - 24 month old). Hypobaric and acute hypoxic hypoxies were used for study DP-IV inhibitors.
Results: The administration of the PE inhibitors reduces the duration of immobility and potentiates apomorphine-induced stereotyped behaviors. Strong correlation between Ki values and antidepressive and antiamnestic activities of tested inhibitors were revealed. PE inhibitors mimicked the effects of antidepressants. PE level significantly rose in a brain rats 18- and 24 month old. A reduction of learning ability in the active avoidance test and a decline of performance in the reflex of passive avoidance by rats 16-24 month old was shown. Strong correlation between cognitive impairments, neurological deficiency and brain PE level was found. All tested PE inhibitors protected animals from scopolamine-induced amnesia in a rat passive avoidance test. DP-IV inhibitor reduced animal mortality during hypobaric hypoxia, and injected intracisternally changed ventilatory response to acute hypoxia.
Conclusions: 1) The pharmacological activities of PE and DP-IV inhibitors appears to be mediated via possible accumulation of neuropeptides in the brain. 2) The biochemical and pharmacological investigations of PE and DP-IV inhibitors may lead to appearance of a new class of psychotropic drugs with antidepressive, antiamnestic and antihypoxic properties.
Paroxetine-induced CYP2D6 Phenotype Conversion and Clinical Outcomes Žourková Aa, Juřica Jb aDepartment of Psychiatry,bDepartment of Pharmacology, Faculty of Medicine, Masaryk University, Brno, Czech republic
Background: Selective serotonin reuptake inhibitor paroxetine is often prescribed antidepressant in depressive and/or anxiety disorders. Metabolised largely by the polymorphic cytochrome P4502D6, paroxetine exhibits substantial variation in activity among patients. Paroxetine has an inhibitory effect on isoenzyme CYP2D6, this can result in a phenotype conversion to the poor metabolic (PM) phenotype. Paroxetine therapy may be associated with incidences of adverse effects due to an inhibition of its own metabolism. Aims: effect on drug toleration and drug efficacy during paroxetine therapy.
Methods: Subjects: Part A: Influence of paroxetine treatment on phenotype conversion from extensive metabolisers (EM) to PM (n=55). Part B: Influence of CYP2D6 phenotype on clinical response in acute paroxetine treatment (n=16). Part C: Influence of phenotype on the efficacy and the adverse effects in the maintenance paroxetine treatment (n=55). Dextromethorphan test was used for the determination of the metabolic activity of CYP2D6. Genotype determination of patients is based on the detection of polymorphisms in CYP2D6 gene( exons *3,*4,*5,*6). The Hamilton Anxiety Scale (HAMA) and Clinical Global Impression-Severity of Illness Scale (CGI S) were used for clinical response. The Arizona Sexual Experiences Scale (ASEX) was used for sexual dysfunction examination. UKU scale (Utvalg for Kliniske Undersøgelser) was used for examination of adverse effects.
Results:Part A: The effect of paroxetine treatment on CYP2D6 phenotype: in 55 patients sample 34 patients with homozygote EM genotype were found. In 20 of them, PM phenotype was assessed. Part B: In general, earlier onset of the effect of therapy in the acute study (n=16) was observed. Part C: No variation in therapeutic efficacy and adverse effects between PM and EM was found in the maintenance treatment (n=55). One unique output of the study is the variance in the incidence of sexual dysfunction depending on CYP2D6 activity in the females.
Conclusions: 1) PM phenotype patients are more vulnerable to the paroxetine treatment. 2) CYP2D6 activity testing is recommended in all cases with an unexpected therapeutic response. 3) Predicting sexual dysfunction based on the metabolic phenotype and gender during treatment with CYP2D6 inhibitors could be recommended.
