1Universidade Federal de Mato Grosso do Sul, Campo Grande, MS, Brasil
2Universidade Federal de Goiás, Goiânia, GO, Brasil
Background:This study investigated the seropositivity for hepatitis B virus (HBV), the vaccination index, and the vaccine response index in dentists from Campo Grande, MS.
Methods: Blood samples from 474 dentists (63.7% women and 36.3% men), with a mean age of 38.5 +/- 10.5 years were analyzed by enzyme-linked immunosorbent assay to detect the serological markers: HBsAg, anti-HBs, and anti-HBc. The HBsAg positive samples were tested for anti-HBc IgM, HBeAg, and anti-HBe. Viral DNA was detected by polymerase chain reaction in HBV seropositive samples.
Results: A total of 51 (10.8%) dentists showed seropositivity for HBV. Three (0.6%) were HBsAg/anti-HBc/anti-HBe positive, 43 (9.1%) were anti-HBc/anti-HBs positive, and 5 (1.1%) had only anti-HBc. Viral DNA was detected in 9 (17.6%) out of 51 HBV seropositive samples. A vaccination index of 96.6% (458/474) was observed, although 73.1% (335/458) completed the three-dose schedule. Excluding 46 HBV seropositive individuals from 458 that reported vaccination, 412 were analyzed for vaccine response index. It was observed that 74.5% (307/412) were anti-HBs positive; this percentage increased to 79.1% when three doses were administered.
Conclusions: The results showed a high vaccination index and a good rate of vaccine response; however, the failure in completing the three-dose schedule and the occurrence of HBV infection reinforce the need for more effective prevention strategies.
Blocking the Peptide Tunnel- Is the Magic Still There? FERNANDES P1,MATSA S1,ROMERO A2,BAKER W3,ICHIKAWA Y2,
PEREIRA D1 1Cempra Pharmaceuticals, Chapel Hill, NC 27517. USA. 2Optimer Pharmaceuticals, San Diego, USA. 3Advisor, Cempra Pharmaceuticals
Background: The bacterial ribosome peptide tunnel is the target of many antibiotics, including diverse classes of natural products such as macrolides, chloramphenicol, clindamycin and streptgramins, and the synthetically-derived agent, linezolid. These antibiotics bind to different yet closely located sites creating a "traffic jam" and thereby preventing new peptide synthesis. Bacteria, in the mean time, have gained resistance by methylation of these sites to prevent those antibiotics from binding. Can we improve upon these antibiotics to make new ones?
Methods: Structure-activity relationships within each class of antibiotic, as well as across antibiotic classes that bind to the peptide tunnel have been useful in delineating and mapping the critical regions on the ribosomes that must be blocked to produce new antibiotics with properties that are not seen with any single class of known antibiotic. Novel synthetic chemistry techniques coupled with knowledge obtained from recent structural data on the bacterial ribosome and co-crystal structures were used to design new molecules.
Results: A-60667 and a series of 11-12 carbamate erythromycins were first reported in 1989 to gain activity against bacteria with inducible and constitutive macrolide resistance. This concept was extended by the development of a novel ketolide class, which includes telithromycin, where additional activity was gained by removal of the 3-cladinose sugar and oxidation of the resulting 3-OH group. CEM-101, a ketolide that has a metabolically stable side chain, is more active than telithromycin and is active against macrolide-resistant bacteria. Novel chemistry at the 5-position that allows replacement of the desosamine sugar is leading to molecules that take even further advantage of lessons learned from different classes of peptide tunnel binding antibiotics.
Conclusions: CEM-101 is a new antibiotic that is in clinical development to address infections caused by macrolide-resistant bacteria. Further exploration of the peptide tunnel could provide macrolides with the next big leap in activity and expanded spectrum.
Authors’ disclosure statement The authors are employees or advisors of Cempra Pharmaceuticals, Inc.
HIV/AIDS and Malarial/Toxoplasmosis Co-Infections: a Magic Bullet Approach to Drug Development FIALHO AM1 and CHAKRABARTY AM 2 1 Instituto Superior Tecnico, Lisbon, Portugal;
2 University of Illinois, Chicago, USA
Background: AIDS, caused by the virus HIV-1, is not only deadly, but becomes deadlier when the patient gets co-infected with another pathogen such as the malarial parasite Plasmodiumfalciparum, or Toxoplasma gondii, the causative agent of toxoplasmosis. The industry approaches to anti-retroviral drug development consist of targeting specific steps in viral entry or replication/maturation. Unfortunately, HIV-1 can quickly mutate to change such target sites, thereby becoming drug resistant. A cocktail of such individual drugs has been more effective, but does not address the problems of co-infections with unrelated pathogens. An ideal drug will be a magic bullet, that will not only simultaneously attack targets in HIV-1, P.falciparum and T. gondii, but also host factors that act as receptors of viral/parasite entry and growth.
Methods: Two bacterial proteins, azurin and Laz, members of the family called cupredoxins, showed both antiviral and antiparasitic activities. Assays of HIV-1 growth suppression, inhibition of P. falciparum parasitemia, Toxoplasma adhesion/invasion as well as protein-protein interaction studies have shown the ability of azurin and Laz to inhibit the growth of HIV-1 and malaria/toxoplasmosis-causing parasites, thus acting as a magic bullet.
Results: We demonstrate that azurin and Laz can avidly bind key envelope/surface proteins of HIV-1, P. falciparum or T. gondii, thereby interfering in their invasion of host cells and growth. Additionally, azurin/Laz strongly binds the host receptor CD4, or the dendritic cell surface protein DC-SIGN that contribute to HIV-1 transport and entry to T cells. Azurin and Laz demonstrate structural features similar to immunoglobulins, thereby implying the possibility of a common evolutionary origin of cupredoxins and immunoglobulin folds. The elucidation of broad target specificity of azurin or Laz towards viruses and parasites appears to suggest that this cupredoxin is used by the producing bacterium as a weapon targeted to other intruders in human body.
Conclusion: Our data show that azurin/Laz acts as a promising magic bullet drug candidate as conceived by Paul Ehrlich, by interfering in several essential steps in HIV-1 entry/growth and also in preventing co-infections by other human parasites.
It’s not only genes - The many dimensions of personalized medicine FIERZ W1 1LogoLab AG, Kilchberg, Switzerland
Background: The concept of Personalized Medicine is strongly associated with pharmacogenomics. In fact, the term ‘personalized medicine’ is not occurring in the headings of articles before the advent of pharmacogenomics. Most articles published so far on personalized medicine also contain the term ‘pharmacogenomics or -genetics’ in their text. However, there is more to it than gene polymorphism.
Methods: Here, it is looked for factors that shape the idiosyncratic way of how health and disease develops in individual human beings. What are the main influences that make us distinct in developing disease and responding to treatment? How should healthcare respond to these individual characteristics in order to make medicine really personal?
Results: On top of the genetic background, three main factors form our health-related individuality: the individual environment, the individual immune system, and our personal lifestyle. It is mainly the microbiological environment that challenges our health, but also nutrition and toxic products of the environment play their roles. The immune system learns how to cope with the microbial challenges and our personal lifestyle strongly influences the spectrum of environmental threats we encounter. These factors - genes, environment, immune system, and lifestyle - are not independent but highly intermingled.
For healthcare to successfully respond to these challenges it has first and foremost to be aware of them, and then to find personal solutions. Both require an individual information management that brings together patient-specific and knowledge-based information in order to make the right personal decisions. Finding the most effective personal treatment with minimal side-effects is an important target of personalized medicine, but genetic counseling and personal patient education is equally important on the long term in order to prevent further disease. Also privacy, protection of minorities, and prevention of discrimination have to be considered.
Conclusions: Personalized medicine is multi-dimensional. Since these dimensions are intermingled, only integrated healthcare based on effective information management will be successful. Prevention, diagnosis, treatment, and care will have to be integrated in order to leverage personalized medicine. The goal is magnificent: personal health planning, early diagnosis, the right drug for the right patient, and predictable side effects.
The Pharmacokinetics (PK) and Pharmacodynamics (PD) of Platinum (Pt) Analogs in Birds FILIPPICH LJ, CHARLES BG
Univ. of Queensland, St. Lucia, Australia.
Background: Chemotherapy as a treatment modality is increasingly being used in avian oncology. However, most chemotherapeutic agents, like Pt analogs, can only be used empirically as PK data in birds is lacking. Aims: 1) To study the PK/PD of cisplatin and carboplatin in cockatoos (Cacatua galerita) so that clinical trials can begin.
Methods: This study was done on 2 groups of 6 healthy cockatoos. One group received a single 1 h intravenous (IV) infusion of cisplatin (1 mg/kg), while the other group received a single 3 min IV or intraosseus (IO) infusion of carboplatin (5 mg/kg). For cisplatin, birds were hydrated before and 2 h after the infusion. Serial blood samples were collected for 96 h and urine samples were collected during the 2 h hydration period. Tissue samples from 10 organs were obtained at necropsy (96h). Total and filterable Pt in plasma, and urine and tissue Pt were assayed by inductively coupled plasma–mass spectrometry. A noncompartmental pharmacokinetic analysis was performed on the data.
Results: For cisplatin, the respective mean systemic clearances (Cl) for total and filterable Pt were 373 and 699 mL/h/kg, the steady state volumes of distribution (Vss) were 4.19 and 0.356 L/kg, and the mean residence times were 111 and 0.512 h. Total plasma Pt displayed a bi-exponential decay profile with average half-lives (T1/2) of 0.398 and 79.0 h, while filterable Pt had a monoexponential decay with mean T1/2 of 0.413 h. The renal clearance was 0.167 L/h/kg. For carboplatin, the maximum plasma filterable Pt concentration of 27.3 mg/L occurred at the end of the infusion, thenceforth declining exponentially over the next 6 h. The terminal T1/2 was 1.0 h, Cl was 330 mL/h/kg and the Vss was 0.378 L/kg. Tissue Pt distribution was similar for both Pt analogs with the greatest accumulation occurring in the kidney.
Conclusions: Cisplatin was well tolerated while mild alimentary tract signs were seen with carboplatin. Plasma Pt concentrations with cisplatin were similar to those of carboplatin and to those measured during treatment of solid tumors in human patients. Filterable plasma Pt concentrations for carboplatin persisted longer than for cisplatin, due mainly to the difference in Cl. Despite anatomical, physiological and biochemical differences among animal species, the PK disposition of Pt in cockatoos shares some features with the kinetics reported previously in rodents, dogs and human beings.
Rasagiline, a Selective Suicide Inhibitor of Monoamine Oxidase B, Increases Striatal Extracellular Fluid Dopamine Levels and Locomotor Stimulation Following L-dopa Without a Corresponding Increase in Dyskinetic Movements in a Parkinsonian Rat Model. FINBERG JPM, LOBODA L, SADER-MAZBAR O
Rappaport Faculty of Medicine, Technion, Haifa, Israel
Background: Rasagiline is a new selective monoamine oxidase B (MAO-B) inhibitor used in the treatment of Parkinson's disease alone or together with L-dopa. In rat brain tissue, the IC50 for inhibition of MAO-B in vitro is 4.4 ± 0.92 nM, with a selectivity ratio for inhibition of MAO-B:MAO-A of 1:94. Following oral administration to rats, the selectivity ratio for MAO-B:MAO-A inhibition ex vivo is 1:65 (based on ED50 values). In the striatum of rats with extensive unilateral nigro-striatal lesions induced by local injection of 6-hydroxydopamine (6-OHDA), dopamine (DA) behaves as a MAO-A substrate in vivo, however when the DA lesion is combined with a serotonergic lesion, inhibition of MAO-B produces an increase in extracellular levels of L-dopa-derived DA (DAec).
Methods: Rats were lesioned by injection of 6-OHDA to the left medial forebrain bundle (single lesion, SL), or by 6-OHDA together with 5,7-dihydroxytryptamine intracerebroventricularly (double lesion, DL). Rats were screened for DA lesion extent with apomorphine and then, commencing 3 weeks after the apomorphine test, were treated daily with rasagiline (0.05mg/kg s.c.) or saline. On the 14th day, microdialysates were collected from the striatum following L-dopa/carbidopa injection (25/6 mg/kg i.p.) and assayed by EC-HPLC. Another group of lesioned rats received L-dopa/carbidopa for two weeks daily with rasagiline or saline and the rotational behavior and dyskinetic movements following L-dopa i.p. were measured every two days.
Results: Rasagiline increased maximal DAec to a greater extent in DL than in SL rats (by 2.4 and 1.4 fold respectively) and with greater duration. In the DL rats, rasagiline treatment also significantly prolonged the duration of L-dopa-induced turning (T50 for turning duration = 131 ± 13.4 vs 91 ± 3.4 min, P<0.05), without a significant increase in total dyskinetic movement score (24.2 ± 12 vs 24.2 ± 11); n=5 for each treatment.
Conclusion:
1) MAO-B inhibition becomes important in the maintenance of L-dopa-derived DA levels in the striatum following combined DA and 5-HT denervation.
2) Locomotion is related to striatal DAec but dyskinesia is dependent on additional factors other than striatal DA release.
Recombinant Immunotoxins for the Treatment of Cancer. FitzGerald DJ, Pastrana DV, Hassan R, Kreitman RJ and Pastan I
Lab of Molecular Biology, CCR, National Cancer Institute, NIH. Bethesda, MD.
Background: recombinant immunotoxins are single chain antibodies genetically fused to protein toxins. The design of these agents dictates that the antibody portion binds a surface antigen on cancer cells and the toxin portion kills the targeted cells. Immunotoxins composed of a truncated version of Pseudomonas exotoxin (PE38) have been evaluated as therapeutic agents in clinical trials targeted to antigens on both hematologic and non-hematologic tumors.
Methods: immunotoxins were expressed in E coli. Clinical trials were conducted with IRB and FDA approval. For tissue culture experiments, cell lines were grown at low or high density and evaluated for immunotoxin killing using WST1 or assays for apoptosis.
Results: The best clinical results were achieved with the immunotoxin (BL22) targeted to CD22 on Hairy Cell Leukemia cells, where a 65% complete remission rate was reported. In contrast, targeting to mesothelin, an antigen expressed on mesotheliomas and ovarian cancers with the immunotoxin, SS1P, produced some partial responses and stabilization but no complete remissions. Comparing the two treatments reveals several differences: Hairy Cell Leukemia patients rarely make anti-PE38 antibodies in the first cycles of treatment (allowing retreatments) whereas ~90% of patients treated with SS1P do, (precluding retreatments). Also, leukemic cells are readily accessible to immunotoxin whereas target cells within solid tumors (such as ovarian and mesotheliomas) are presumably more difficult to reach. Other factors have also been investigated to explain the differential response, including shedding of target antigens and the possibility that tumor cells growing at high density are resistant to killing by immunotoxins. We have investigated the effect of cell density and found that a number of cancer cell lines exhibit a density-dependent resistance to killing by immunotoxins. Resistance was not due to failure of toxin delivery to the cell cytosol, as cells grown at high or low density were equally affected by a reduction in protein synthesis (PE38 inhibits protein synthesis). The basis for high-density resistance is currently being characterized.
Conclusions: Solid tumors are difficult to treat with immunotoxins. The reasons for this include poor access to tumor cells within tumor masses and possibly a novel resistance mechanism related to high density growth.
Jasmonates Kill Cancer Cells Selectively by Dissociating Hexokinase from Mitochondrial VDAC Flescher E, Goldin N
Tel Aviv University, Tel Aviv, Israel
Background: Cellular bio-energetic metabolism and mitochondria are recognized as potential targets for anticancer agents, due to the numerous relevant peculiarities cancer cells exhibit. Jasmonates are anticancer agents that interact directly with mitochondria. Many types of cancer cells exhibit overexpression of the key glycolytic enzyme, hexokinase, and its excessive binding to mitochondria. These characteristics are considered to play a pivotal role in cancer cell growth rate and survival. The aim of this study was to identify mitochondrial molecular targets of jasmonates.
Methods: Binding and detachment of hexokinase from mitochondria were determined by hexokinase immunochemical and activity determinations, surface plasmon resonance analysis and planar lipid bilayer voltage dependent anion channel (VDAC)-activity analysis. Hexokinase expression was modified using hexokinase-overexpressing transfectants and its mitochondrial association.
Results:. We report that jasmonates bind to hexokinase and detach it from the mitochondria and its mitochondrial anchor— VDAC. Jasmonate-induced detachment from mitochondria occurs in various types of cancer cells including leukemia and solid tumors. Furthermore, the susceptibility of cancer cells and mitochondria to jasmonates is dependent on the expression of hexokinase, supporting a cause and effect relationship between jasmonate-induced hexokinase detachment and cell death.
Conclusions: 1) Our findings provide an explanation for the selective effects of jasmonates on cancer cells. 2) This is the first demonstration of a cytotoxic mechanism based on direct interaction between an anticancer agent and hexokinase. 3) The proposed mechanism can serve to guide development of a novel class of small anticancer compounds that kill cancer cells selectively by inhibiting the hexokinase–VDAC interaction.
Pharmacokinetics and pharmacodynamics of meloxicam in rats and humans FLORES-MURRIETA FJ1,2, AGUILAR-MARISCAL H3, RODRÍGUEZ-SILVERIO J1,2, CARRASCO-PORTUGAL MC1 1Unidad de Investigación en Farmacología, INER, Mexico City, Mexico; 2Escuela Superior de Medicina del IPN, Mexico City, Mexico; 3Universidad Juárez Autónoma de Tabasco, Villahermosa Tabasco, Mexico.
Background: Meloxicam is a drug endowed of analgesic and anti-inflammatory activities. Although it is widely used in therapeutics, limited information concerning the pharmacokinetic-pharmacodynamic relationship is available. Aims of this study were to establish pharmacokinetic-pharmacodynamic modeling for antihyperalgesic and anti-inflammatory effects of meloxicam in the rat and to evaluate if levels are comparable to those reached in humans after administration of therapeutic doses.
Methods: Pharmacokinetic and pharmacodynamic evaluations were carried out in groups of rats using the paw thermal hyperalgesia and inflammation of the paw models. Measurements of effect and concentration of meloxicam were carried out at selected times for 6 hours. On the other hand oral pharmacokinetics of meloxicam in humans was evaluated in 24 healthy volunteers after an oral 7.5 mg dose and plasma concentrations were measured at selected times during 24 hours. Pharmacokinetic parameters were obtained by non-compartmental approach and effects against blood concentrations were fitted to the sigmoidal Emax model.
Results: Antihyperalgesic and anti-inflammatory effects against blood concentrations of meloxicam were fitted to the sigmoidal Emax model. EC50 for antihyperalgesic and anti-inflammatory effects were 15.1 ± 2.5 µg/ml and 10.01 ± 1.18 µg/ml, respectively; and Emax was in both cases about 75%. On the other hand, pharmacokinetic parameters obtained in humans were: Cmax 0.70 ± 0.03 µg/ml, tmax 4.8 ± 0.65 h, AUC 27.7 ± 1.48 µg.h/ml and t1/2 24.5 ± 1.2 h.
Conclusions: A direct relationship between concentration and antihyperalgesic and anti-inflammatory activities of meloxicam was found, but the effect in rats is produced at much higher levels than the reached in humans at therapeutic doses.
Association between Polymorphisms of Vitamin D Metabolizing Enzymes and Colorectal Cancer Incidence FLÜGGE J1,2, KRUSEKOPF S1, GOLDAMMER M1, KLARE S3, MALZAHN U1, ROOTS I1 1Institute of Clinical Pharmacology and Toxicology, Charité – Universitätsmedizin
Berlin, Berlin, Germany; 2Institute of Pharmacy, Freie Universität Berlin, Berlin, Germany 3CENiMED GmbH, Center for Individualized Medicine – Clinical Pharmacogenomics, Berlin, Germany
Background: Vitamin D (1,25(OH)2D3) plasma levels are inversely associated with colorectal cancer incidence. Vitamin D levels are modified by the metabolizing enzymes 1,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1), vitamin D3 25-hydroxylase (CYP27A1) and 25-hydroxyvitamin D3-1-alpha-hydroxylase (CYP27B1) and by the vitamin D receptor (VDR). The association between polymorphisms of these vitamin D metabolism-related genes and colorectal cancer incidence has been investigated in a German population.
Methods: 18 common single nucleotide polymorphisms (SNPs) of CYP24A1, CYP27A1, CYP27B1 and VDR were determined in a series of 928 caucasian subjects (452 colorectal cancer patients and 508 healthy controls) from Germany using SNPlexTM technology (Applied Biosystems), PCR/RFLP, sequencing and GeneScan® genotyping assays. Allele frequencies and complex genoytpes were determined. Haplotype analysis (Phase 2.0) was applied to the received genotypes. Linkage disequilibria were analyzed with Haploview 3.32.
Results: SNP frequencies correspond to previous studies in Caucasian populations. Allele frequencies were without any characteristic variations regarding the case-control-classification. The polyA microsatellite analysis revealed a bimodal distribution of the alleles. No variant showed any significant deviation from the Hardy-Weinberg equilibrium. CDX-2 was found differently distributed between cases and controls (p=0.037). However, this association and a trend for CYP27B1_-1077 were no longer significant after adjusting for multiple testing by Bonferroni correction. Haplotype analysis and tests of complex genotypes and pairs of variants revealed no significant differences between cases and controls. We found a strong linkage disequilibrium between A2978T, PolyA, C1905A, TaqI, Tru9I and BsmI of VDR and between all three CYP27B1 variants (p<0.001).
Conclusions: The results of this study contribute to our knowledge about the relation between vitamin D metabolism-related genes and colorectal cancer incidence in Caucasians.
5-HT2a antagonists as a new treatment for JCV-associated progressive multifocal leukoencephalopathy FOCOSI D1, MAGGI F1, KAST RE2, GALIMBERTI S1, CECCHERINI-NELLI L1, PETRINI M1.
1Division of Hematology, University of Pisa, Italy
2Division of Psychiatry, University of Vermont, USA
Progressive multifocal leukoencephalopathy (PML) was first described as a complication of immune suppression fifty years ago. The prognosis has remained dismal since then, with discouraging results from clinical trials of various therapeutic approaches. PML is caused by reactivation of latent JC virus, and 5-HT_2a receptors have been identified as the main entry point for JC virus into glial cells. Since then, encouraging case reports have suggested several 5-HT_2a inhibitors (currently approved for other indications) may be effective in the treatment of PML. We discuss here preclinical data and perform a systematic review of case reports.
The study of the adhesion molecules in Non Small Cell Lung Cancer (NSCLC) treated with epidermal growth factor (EGF) can generate a new drugs targets and developing new approaches for systemic treatment in lung cancer FONSECA FLA1,2, AZZALIS LA3, NOGOCEKE E2 1 Oncology/Hematology Department, ABC Medical School, Santo André, Brazil; 2Roche Center for Medical Genomics, Basel, Switzerland; 3 Anhembi Morumbi University, São Paulo, Brazil.
Background: Lung cancer is the leading cause of cancer death in developed countries. At least, six important alterations were catalogued in defined molecular events that are common to all malignant cells: Recently, several investigators attributed some six events to an important group of molecules that are important component for adherence junctions between epithelial cells (adhesion cell-cell) called cadherins and other group which is responsible for adhesion of epithelial cells to extra matrix protein (ECM) called integrins. The purpose of this study was to assess the interactions of cell adhesion molecules (CAM) in cell lines from lung cancer, where 2 of these cell lines were non-metastatic (H-358, H441) and the other two which were metastatic cells (H1299, H292).
Methods: H358 bronchioalveolar cells, H441 lung adenocarcinoma cells, H1299 and H292 lung carcinoma cells were maintained in RPMI 1640 modified medium. The cell lines were treated with epidermal growth factor (EGF) for 30 minutes. Extraction of proteins from cultured cells was performed with denaturing buffer. Protein immunodetection was done by eletrophoretic transfer of SDS-PAGE, separation of proteins on nitrocellulose, incubation with antibody, and chemiluminescent second-step detection. In total of 20 proteins were performed from adhesion cell-cell and cell-extra matrix cellular (cadherins and integrins pathways).
Results: The results of cell-cell adhesion were not influenced by the treatment with EGF for 30 min. However we verified differences between the description types of cells. Otherwise, EGF could modulate the signaling pathways of the integrins and it we have show when different NSCLC cell lines are treated with EGF. We also observed antagonist functions between these proteins (PYK2 and FAK).
Conclusion: 1) The expressions patterns of adhesion cell-cell were not affect by EGF treatment, 2) The treatment can affect and may modulated the adhesion cell-ECM. 3) The antagonist effects can explain the crosstalk between EGFR and integrins pathways. Some proteins searched in this study may be a key of methastic, circulation and proliferation process in lung cancer and this may use to improve the therapeutical approaching of the NSLC.
Pain Relief Without Side Effects: Methylnaltrexone (MNTX) and Alvimopan (ALV) are Silver Bullets In the Use of Opioids for Analgesia FOSS JF Cleveland Clinic, Cleveland, OH, USA
Background: In 1978 Leon Goldberg noted that drugs acting on the opioid receptors of the gut and not crossing into the brain were available. Could we find an analogous opioid receptor antagonist?
Russell et. al (1982) first reported that the gastrointestinal (GI) effects of the morphine (MSO4) could be prevented by MNTX without affecting analgesia and this was later confirmed in volunteers and subjects on chronic opioids. Subjects given placebo (pbo) had transit of 104 min. MSO4/pbo increased transit to 163 min and MSO4/MNTX saw a return to 106 min, with no effect on analgesia.
ALV, while chemically distinct from MNTX, has a similar profile.
Efficacy: MNTX is approved for the treatment of opioid-induced constipation (OIC) in patients with advanced medical illness (AMI). Thomas et al. (2008) presented a RCT of MNTX administered 0.15 mg/kg, or pbo every other day for 2 wk in adults who were receiving opioids and had OIC in spite of laxative use. MNTX significantly increased laxation (48% vs. 15%), both through the 2-wk period and an extension.
ALV is approved for the management of post-operative ileus (POI). In a phase 2 RCT, patients with abdominal surgery given 6-mg capsule of ALV 2 hr before surgery and twice daily post-op had a significantly shorter median time to their first bowel movement (BM) vs. pbo (70 vs. 111 hours).
In bowel resection (BR) or abdominal hysterectomy (TAH), ALV significantly decreased the number of nasogastric tube insertions, time to GI recovery and time to hospital discharge (DC). Results were better with 12 mg than with 6 mg, and better in BR than in TAH.
Safety: MNTX in chronic opioid users and ALV in p-op trials show no reversal of analgesia or change in opioid use.
MNTX is associated with abdominal cramping, gas and discomfort. Orthostatic hypotension in volunteers was dose limiting in volunteers.
In clinical trials of POI ALV has shown a similar side-effect profile to pbo. Trials of ALV for chronic use in opioid-induced bowel dysfunction (OBD) were briefly on hold due to non-statistically higher rates of cardiovascular events, some malignancies and bone fractures, but have been restarted.
Higher doses of MNTX or ALV in tolerant subjects, while not inducing classical opioid withdrawal, will produce GI distress.
Conclusions: MNTX and ALV are new tools for the management of the side effects associated with opioids.
Authors’ disclosure statement:
Dr. Foss is a patent holder for MNTX and receives royalties from the sales of the drug.
Omalizumab: The First Real Magic Bullet for the Treatment of Allergic Asthma? FOX H Novartis Horsham Research Centre, Horsham, West Sussex, UK
Severe, uncontrolled asthma can be fatal and is associated with high levels of hospitalization and urgent care visits. Moreover, it has been estimated that up to 90% of asthma cases have an allergic component. Allergen exposure causes B cells to differentiate into plasma cells that produce and release immunoglobulin E (IgE) antibodies into the circulation. IgE then binds to high-affinity IgE receptors (FceRI) on the surface of tissue mast cells or peripheral-blood basophils that, in the presence of allergen, results in rapid release of inflammatory mediators. These inflammatory molecules can result in bronchoconstriction leading to an asthma exacerbation. Moreover, there is a strong correlation between FceRI receptor expression and fatal asthma. As such, there is a clear rationale for the development of asthma treatments that target IgE.
Omalizumab is a humanized monoclonal antibody that inhibits the binding of IgE to the FceRI receptor. Reduction in surface-bound IgE on FceRI-bearing cells limits the release of allergic response mediators. Omalizumab also reduces the number of FceRI receptors on basophils in atopic patients. In patients with severe persistent allergic asthma that remains inadequately controlled despite available therapy, the addition of omalizumab leads to significant reductions in the incidence of clinically significant asthma exacerbations and emergency visit rates compared with placebo. Furthermore, omalizumab significantly improves asthma-related quality of life, morning peak expiratory flow and asthma symptom scores compared with placebo. Reduction in free IgE levels correlates well with improvements in clinical outcomes achieved with omalizumab. Following cessation of omalizumab, IgE returns to baseline levels and, after a small delay, signs and symptoms of asthma resume. Observed significant reductions in eosinophil counts compared with placebo support the anti-inflammatory effects of omalizumab. Real-life experience confirms the therapeutic benefits of omalizumab observed in clinical studies.
A targeted anti-IgE approach to therapy with omalizumab has revolutionized the treatment of severe persistent allergic asthma and vastly improved clinical outcomes in some patients (12 years old). Ongoing research aims to identify the full potential of this magic bullet in IgE-mediated disease, including paediatric allergic asthma.
Ixostatin, a Novel Tick Salivary Protein that Specifically Binds to the Somatomedin B Domain of Vitronectin and Prevents its Interaction with Integrin alphavbeta3 and Urokinase Receptor FRANCISCHETTI I National Institutes of Health, Bethesda, USA.
Saliva from blood-sucking arthropods is a rich source of modulators of vascular biology. In this report we describe the cloning, expression and mechanism of action of Ixostatin, a novel family of 10 kDa cysteine-rich peptide from Ixodes scapularis salivary gland. Recombinant Ixostatin was expressed in insect cells and was produced in an active form. Surface plasmon resonance experiments show that Ixostatin interacts with monomeric or multimeric human vitronectin with a KD ~ 0.5 nM, but does not bind to other extracellular matrix proteins. Notably, the high-affinity binding site for Ixostatin was identified as the somatomedin B domain (SMTB) of vitronectin. In addition, Ixostatin at nanomolar concentrations inhibits integrin alphavbeta3- and urokinase receptor-mediated cell adhesion to vitronectin, but display negligible effects in fibrinolysis in vitro. It is concluded that the most prominent biological property of Ixostatin is to negatively modulate cell adhesion to the extracellular matrix. Ixostatin is the first ligand from an exogenous source that specifically targets the SMTB domain of vitronectin. It also represents a novel mechanism by which tick saliva manipulates vector-host interactions, and may therefore have potential medical applications.
Are the tropical forests the new frontier for antibiotics discovery? Novel peptidomics insights on the screening of plant antimicrobial peptides. FRANCO OL1, MOREIRA JS1, PELEGRINI PB1, COSTA FT1, NETO SM1, LIMA TB1 1Centro de Análises Proteômicas e Bioquímicas, Pós-Graduação em Ciências Genômicas e Biotecnologia, UCB, Brasília-DF, Brazil.
Background: During human history, microbial infections directly affect World population in several areas causing economical, social, agricultural and health problems. This situation has only been controlled with the development of antibiotics. Nevertheless, the inadequate use of available compounds leaded to microorganism’s resistance. In order to control this problem, different sources of antimicrobial peptides (AMPs) have been screened, including plants, microorganisms, amphibian, sea animals and several others. These small peptides show different and special abilities. They are able to inhibit digestive enzymes or act against bacteria and/or fungi.
Methods: Once those common sources have been deeply explored, in this report AMPs were screened from flowers and fruits collected from Brazilian tropical forest. In both cases were used a combo of classical strategies, which included HPLC chromatography’s and bioassays against human pathogens such as Klebsiella sp., Proteus sp. and Aspergillus fumigatus. All MICs were calculated. Futhermore novel peptidomics strategies such as liquid isoelectric focusing (pI 3-11) associated to LC-MS techniques were also utilized in order to identify antimicrobial peptides in large scale.
Results: Flowers and fruits showed AMPs from novel classes with different structures, evaluated by molecular modeling and dynamics. Among them was observed glycine-rich peptides and peptides pertaining to unpublished classes. Moreover, all of them were able to cause a remarkable reduction on gram-negative and gram-positive bacteria. Additionally, peptidomics techniques showed that it was possible to identify, by liquid IEF followed by LC-MS, at least 30 different peptides with similar properties to antimicrobial peptides, such as cationic and hydrophobic peptides.
Conclusions: Current research in this area here focused, particularly aims to control pathogenic microorganisms, showing that antimicrobial peptides could be extracted and further commercialized in a near future as a common drug. Furthermore, this research also shows that tropical forests culd provide new classes of antimicrobial peptides, helping to solve the infections problem.
Limitations of Non-magic Bullets Compounds in Bioequivalence Assessment. How Can this Enhance Knowledge Towards the Development of Generic Products. The Mycophenolate Mofetil Case. FRANCO SPÍNOLA AC1, ALMEIDA S1, FILIPE A1, NEVES R1 1Grupo Tecnimede, Sintra, Portugal.
Background: Immunosuppressive drugs such as mycophenolate mofetil have been used as prophylaxis of acute transplant rejection in patients receiving allogeneic renal, cardiac or hepatic transplants. Undesirable effects oppose to its clinical efficacy and therein detach it from the magic bullet concept, when seen in clinical and therapeutic perspectives only. When looking into its pharmacokinetic profiling mycophenolate mofetil fits into a standard bioequivalence approach where no major obstacles are foreseen in the clinical trial design. Aims: 1) To compare the rate and extent of absorption of mycophenolate mofetil 500 mg tablet versus reference product. 2) To optimize clinical trial design.
Methods: This study included 12 healthy adult male and female volunteers to whom test and reference formulations were administered as a single-dose, 1x500 mg tablet, in a randomized, open-label, 2-way crossover fasting design. Blood samples were collected until 12.0 hours post-dose and up to 48.00 (±0.5) hours post-dose for mycophenolate mofetil and mycophenolic acid, respectively. Pharmacokinetic parameters AUC0-t, AUC0-inf, Cmax, residual area, Tmax, T1/2el and Kel were assessed following statistical analysis: parametric ANOVA on AUC0-t, AUC0-inf, Cmax, geometric confidence intervals (CI) for AUC0-t, AUC0-inf, Cmax and non-parametric test (Wilcoxon) for Tmax. Ln-transformed bioequivalence standard parameters, AUC0-t, AUC0-inf, Cmax were looked into bearing in mind the 80% to 125% acceptance criteria.
Results: Mycophenolate mofetil AUC0-t, AUC0-inf, Cmax intra-subject coefficient of variation were 23.32%, 24.19% and 49.03% and mycophenolic acid’s 6.16%, 5.23% and 48.33%, respectively.
Conclusions: 1) High unexpected intra-subject CV for bioequivalence parameters may imply sample size readjustment for a fullscale study and design fit. 2-way crossover design may not be the adequate. 2) Regulatory standpoint for highly variable drugs urges to best optimize bioequivalence process.
Population Pharmacokinetics for Nevirapine in Black Newborns to Prevent HIV Transmission FRANK M1, KUNZ A2, HARMS G2, KLOFT C1 1Martin-Luther-Universitaet Halle-Wittenberg, Halle, Germany; 2Institute of Tropical Medicine, Charité, Berlin, Germany.
Background: A prevention of mother-to-child transmission program to reduce risk of HIV transmission was carried out in Uganda. 62 HIV-positiv pregnant women and their newborns participated. Nevirapine (NVP), a non-nucleoside reverse transcriptase inhibitor, was administered as single oral dose to all participants. Population pharmacokinetic (PK) models for mothers and newborns were developed to describe NVP concentration-time profiles for different individuals.
Methods: After a single oral 200 mg dosing to mothers during labour and a 2 mg/kg NVP dosage to newborns after birth, 113 plasma, 95 breast milk samples of mothers and 113 plasma samples of newborns were available. Population PK analysis for mother and newborn data were performed using the nonlinear-mixed-effect modeling approach implemented in NONMEMTM (ADVAN6, TOL5; FOCE INTERACTION estimation method). Final PK models were used for simulating entire concentration-time profiles for different percentiles (P5-95) of individual PK parameter distribution.
Results: Due to sparse data, absorption rate constant was fixed to 1.66 h-1 [1]. A 2 compartment PK model was developed for mother plasma and milk data. V/F (105.4 L) and CL/F (1.5 L/h) resulted in a long half-life of 49 h. Intercompatmental clearance was high (115 L/h). Interindividual variability (IIV) was implemented in CL1/CL2 (28% CV). For newborn data a PK model with ‘multiple input’ was developed. Different input routes from mothers were combined in a ‘bioavailability’ factor (18%). Plasma/placenta-plasma/milk transfer rate constant, V2/F and CL/F were estimated to be 5.1 h-1, 25.8 L and 0.30 L/h, respectively, resulting in a half-life of 61 h. IIV was implemented in F’ (59% CV) and V2/F (29% CV). Simulated concentration-time profiles revealed long-term exposures for mothers and newborns with NVP >IC90 for10-24 d and 12-22 d for different indivduals, respectively.
Conclusions: Population PK models for mother and newborn data were successfully developed to guide single dose NVP prevention strategies of HIV transmission from mother-to-child.
[1] Kappelhoff et al., Antivir. Ther., 10: 145-155 (2005).
Novel BINOL Derivatives as Photoactivatable Carriers of DNA-Targeted Potent Cytotoxic Agents FRECCERO M1,DI ANTONIO M2, PALUMBO, M2; DORIA F1, VERGA D1 1Univ. Pavia, Pavia, Italy; 2Univ. Padova, Padova, Italy.
Background: Several strategies have been developed for a selective and mild activation of DNA-modifying agents, but limited examples of photochemically activated DNA-alkylating agents have been reported. In fact, to date, psoralens are the only well-established class of drugs known to induce DNA or RNA cross-linking upon photoactivation. Recently, our research has been focused on another class of reagents that similarly express a triggerable ability to alkylate DNA.
Methods: This study included: 1) synthesis of a small library of BINOL-derivatives (3-9); 2) mechanistic insights from time-dependent product distribution analysis and laser flash photolysis (LFP); 3) evaluation of the DNA cross-linking by compounds 3-9, using a super coiled plasmid DNA (pBR322) in an alkaline agarose gel assay; 4) DNA alkylation in the cellular environment, using LoVo cells performed by alkaline comet assay.
Results: Photoactivation at 360-450 nm of the BINOL-derivatives 1-9 yielded a short-lived, high energy intermediate undergoing alkylation and DNA cross-linking with high photoefficiency and superior cytotoxicity. Detection of the transient, by laser flash photolysis (LFP), suggests that BINOL-quinone methides (QMs) are key intermediates in the process. QMs trapping experiments, monitored in a time-dependent product distribution analysis, demonstrated that the phototriggered reactivity of these BINOL-derivatives as bis-alkylating agents is the result of a two-steps process involving sequential generation of monoalkylating QMs. Light activation of the BINOL-L-amino esters produced cytotoxic QMs very effective against human tumor LoVo cells with EC50 in the 130–230 nM range.
Conclusions: Trimethylpsoralen (PS) is about 4 times less potent than our newly tested compounds. The BINOL-L-proline methyl ester (5) showed notable photoselectivity, since it displayed cytotoxic effects upon irradiation only and was able to efficiently reach the target DNA inside the cells, where it form both alkylated and cross-linked adducts.
Circular dichroism spectroscopy in stereochemical studies of -lactam antibiotic analogues FRELEK J Institute of Organic Chemistry of the Polish Academy of Sciences, Kasprzaka 44/52 01-224 Warszawa, Poland. E-mail: frelek@icho.edu.pl
Since the introduction of penicillin onto the market in 1940, the -lactam antibiotics are amongst the most frequently prescribed pharmaceutical products to cure many diseases. However, the extensive use of penicillins and cephalosporins has created an increasing number of resistant strains of bacteria. The continuing growth of the bacterial resistance has prompted the search for new structural variants of -lactam antibiotics with enhanced and/or novel biological profiles. Clavams and oxacephams, representing oxaanalogues of penicillins and cephalosporins, respectively, have demonstrated that the high biological activity of -lactam antibiotics is not dependent on the presence of the sulfur atom. Some oxa- and carbaanalogs are more active than natural congeners and exhibit high activity in both of the enantiomeric forms.1 In this context, significant biological activity of -lactam derivatives, closely related to the stereostructure, calls for methods which allow an unequivocal and reliable determination of an absolute configuration. Circular dichroism spectroscopy has been used successfully for this purpose, and it appears to be a convenient, sensitive and fast technique for the stereochemical assignment of azetidinones and their polycyclic derivatives.2
In view of the foregoing the relationship of chiroptical properties and molecular structures of penicillins, cephalosporins in respect of their oxa- and carbaanalogues together with of monobactams will be examined. The applicability of the helicity rule, previously established by us,2 which correlates structure to CD spectra in respect of all classis of -lactam derivatives will be tested. It will be demonstrated that the rule obeys a variety of oxacephams, clavams and their carbaanalogues.
Despite the appealing simplicity and the apparent success of the helicity rule, the underlying assumptions of conformational rigidity and electronic decoupling of the amide chromophore require further validation. Therefore, for the representative -lactam derivatives the ECD spectra computed by means of time-dependent density functional theory (TDDFT)3 will be compared to the experimental curves. These model compounds cover the therapeutically important classes of cephams, oxacephams, carbacephams, and their non-classical analogues. The obtained results pointed to a surprisingly high sensitivity of the CD to the molecular conformation. Nevertheless, the helicity rule may provide a good first guess of the absolute configuration of cepham analogues. For a more definite assignment of the absolute configuration, however, the good corroboration of the predictions made by the helicity rule by the computational results is recommended.3
Organic Chemistry of -Lactams; Georg, G.I., Ed.; VCH: New York, 1993.
R. Łysek, K. Borsuk, M. Chmielewski, Z. Kałuża, Z. Urbańczyk-Lipkowska, A. Klimek, J. Frelek, J. Org. Chem, 2002, 67(5), 1472-1479; J. Frelek, R. Łysek, K. Borsuk, J. Jagodziński, B. Furman, A. Klimek, M. Chmielewski, Enantiomer, 2002, 7, 107-114.
J. Frelek, P. Kowalska, M. Masnyk, A. Kazimierski, A. Korda, M. Woźnica, M. Chmielewski, F. FurcheChemistry – Eur. J. 2007, 13(23), 6732-6744.
