Targeted Pulmonary Delivery of Aerosolized PGE1: A “Magic Bullet” for Neonatal Pulmonary Hypertension? SOOD BG1, MADDIPATI KR1, SHEN Y1, LATIF Z1, JOSHI A1, SLOVIS TL1, HAACKE EM1
1Wayne State University, Detroit, MI, USA
Background: Extensive experience with inhaled nitric oxide in neonatal persistent pulmonary hypertension (PPHN) shows lack of improvement in 30-40% of patients. Inhaled PGE1 (IPGE1) is a potential selective pulmonary vasodilator. Our aim is to characterize the stability, emitted dose (ED) and the aerodynamic particle size distribution (APSD) of PGE1 aerosol generated by the MiniHeart jet nebulizer. An additional goal is to evaluate pulmonary drug delivery and toxicity.
Methods: Chemical stability and ED of PGE1 solution were evaluated during jet nebulization in a neonatal conventional (CMV) and high frequency (HFV) ventilator circuit by High Performance Liquid Chromatography - Mass Spectrometry. A six-stage cascade impactor was used to determine APSD. Pulmonary deposition was assessed with MRI in seven ventilated piglets using T1 weighted spin-echo sequence. Toxicity was evaluated in ten ventilated piglets receiving either high dose IPGE1 or nebulized saline continuously for 24 hours.
Results: There was no significant degradation of PGE1 following nebulization. The ED of PGE1 was 32-40% during CMV and 0.1% during HFV. The PGE1 aerosol had a mass median aerodynamic diameter (MMAD) of 1.4 µm and geometric standard deviation of 2.9 with 90% of particles being < 4.0 µm in size. On MRI, a significant increase in signal intensity (SI) was observed in the lungs 10 min after start of aerosol. At the end of 90 min, the SI increased by 98%. There was no evidence of adverse cardio-respiratory effects related to IPGE1 in ventilated piglets. Histomorphological changes included moderate to severe focal ulceration, flattening of the bronchial epithelium and loss of cilia in the trachea and bronchi.
Conclusions: 1) Nebulization of PGE1 during neonatal CMV or HFV is efficient and results in rapid nebulization without altering the chemical structure. 2) On the basis of the low MMAD, and large proportion (90%) of particles <4.0µm, one can predict predominantly alveolar deposition. 3) This was validated on MRI, with evidence of effective pulmonary aerosol delivery within 10 minutes of contrast nebulization. 4) Inhalation of high dose IPGE1 was not associated with adverse cardiorespiratory effects and produced minimal signs of pulmonary toxicity even after 24 hours. 5) Thus, IPGE1 may be a safe and effective selective pulmonary vasodilator in PPHN.
Oxidative stress in periodontal diseases and associated neoplasias: a role for antioxidants? SOORY M King’s College London Dental Institute, UK
Background: Studies indicate that plaque-associated inflammatory loading in periodontal diseases leading to tooth loss may have associations with developing cancer. There is documentation of a significant association between cancer of the lung, kidney, pancreas, haematological cancers and periodontal diseases. The development of oxidative stress with malignant progression of a tumour has been reported, with evidence of efficacy of antioxidants as anticancer agents. For example, dehydroascorbic acid reacts with homocysteine thiolactone found in cancer cells resulting in the formation of the toxic compound 3-mercaptopropionaldehyde which kills cancer cells. Other effective agents reported in this context are green tea polyphenols, melatonin and vitamin D also reported to be beneficial in periodontal disease outcome. Green tea polyphenols are able to induce apoptosis in various tumour cell systems. This apoptotic mechanism has been shown to be targeted at mitochondria and executed by caspase 3. This has been demonstrated by subjecting tumour cells which had either a caspase 3 deletion or expression of wild type caspase 3 to increasing concentrations of green tea polyphenols indicating mitochondrial targeting. The development of oxidative stress with malignant progression of a tumour has been reported. Drug resistant cell populations can emerge in response to a milieu of oxidative stress. Cellular adaptation is likely to be multifactorial, coordinating factors that induce hypoxia, nuclear factor kappaB (NF-kappaB) and their targets downstream that are linked to resistance mechanisms. This resistance can be overcome by treating the cells with NO mimetic agents to restore their sensitivity to cytotoxic agents both in vivo and in vitro. Mechanisms involved include vascular changes, tumour oxygenation and antioxidant effects, down regulation of the glutathione detoxification / redox buffering system, inhibition of key transcription factors and DNA repair systems.
Conclusions: It is relevant that periodontal diseases are associated with a small but significant overall cancer risk which persists in non-smokers. Periodontal disease may be a useful marker of a susceptible immune system, or directly affect cancer risk as a result of inflammatory loading. Formulation of effective therapeutic dosing of agents shown to have efficacy in this context is a challenge.
Betulonic acid and its alanine amide derivatives - a new multy-target agents for tumor chemotherapy SOROKINA IV, TOLSTIKOVA TG, ZHUKOVA NA, BAEV DS, SCHULTZ EE
Novosibirsk Institute of Organic Chemistry of Siberian Branch of Russian Academy of Science, Novosibirsk, Russia
Background: A multy-target agents based on natural pentacyclic triterpenoid platforms present a new approach to drug design. They may be use to both prevention and therapy of cancer as well as to be synergistic with standard anti-cancer treatments. Aims: 1) To development a multy-target agents using betulonic acid (BA) as new triterpenoid platform. 2) To study in vivo theirs anti-oxidant, anti-inflammatory, antitumor and antimetastasis activities. 3) To study the effect of agents on potency and tissues damage of standard chemotherapy.
Methods: This study included 80 male mice CBA, 220 female mice C57Bl/6, 160 outbreed male mice. Transplantable tumors were Lewis lung carcinoma (LLC) and mice RLS lymphoma. Agents were administered orally at single (250, 500 mg/kg) or daily (50 mg/kg/day during 7 or 14 days) doses. A standard poly-chemotherapy ACOP (adriamycin, cyclophosfamide, oncovin, prednisolone) was carried out 24 hours before dosing. It was estimated: tumor growth, volume areas of metastasis, dystrophic and necrotic changes in some tissues (morphometry using light microscopy), a thiobarbituric acid-reactive substances in serum, anti-inflammatory activity (histamine- and carrageenane-induced mice paw edema models).
Results: It was synthesized 4 derivatives BA with d,l--ala or -ala substitution at C-28 and their methyl esters. All agents at single dose reduce tumor growth (by 10-20%) and inhibit metastasis CLL (by 40-50% as to control). All agents possess anti-oxidant (40-60%) and anti-inflammatory (30%) activities (Р<0,05). After 7 days of dosing (50 mg/kg/day) only two acid derivatives have antitumor activity (12-17%), one with -ala significantly decreases LLC metastasis in lung (by 9 times, Р<0,05) and RLS metastasis in kidneys (by 1,7 times). Treatment of mice with this agents after ACOP leads to rise its antitumor (to 1,3 times, Р<0,05) and antimetastasis (up to 2 times, Р<0,05) potency. The acid derivative with -ala decreases a necrosis (to 42% as to ACOP, Р<0,05) and increases a dystrophic changes (to 28%) in the liver and kidneys. The agent induces a glycogen synthesis in the liver.
Conclusions: Among alanine amide derivatives BA it is found an agent that has high antimetastasis activity, rises a poly-chemotherapy potency and decreases tissues damage.
Lithium and vestibular function in Bipolar Disorder SOZA RIED AM 1, 2 CERTANEC B2 , REYES J2 1Universidad de los Andes, San Carlos de Apoquindo 2200, Santiago, Chile.2ChileanAerospace Medicine Centre Background: Bipolar disorders characterizes by mood changes between mania and depression. Despite lithium is a useful drug for stabilizing mood, the mechanism underlying it is still unknown.
Methods: We studied 2 bipolar I disorder patients during successive depressive and maniac states using electronystagmographic registers after vestibular rotatory stimulation. Slow phase velocity(SPV) of nystagmus was calculated for quantify right and left vestibular activity. Right/Left SPV ratio was assessed for appreciating vestibular lateralization differences between depression and mania. The actual treatment with lithium was inquired in all the mood states.
Results: We recorded 4 different episodes of depression and 4 of mania. In depression we found a 41% diminution of right vestibular activity, and 17% elevation of left. In Mania we found 35.2% diminution of right vestibular activity and 37% diminution of left . Right/Left SPV ratio mean in depression was 0.7+0.1 (mean +SD, 4 records), significantly lower than 1.4+0.1 found in maniac phases (4 records) (p< 0.05, two tailed, Mann-Whitney test). We observed that in 2 cases the vestibular pattern characteristic of maniac state was preceded by the suspention of lithium treatment, and in 2 cases it was reversed after the reposition of the treatment.
Conclusions: We found fluctuations of vestibular dysfunction depending on mood state in Bipolar Disorder patients. Left vestibular activity was the parameter that varied the most between mood states, for this reason we suggest that the stabilizing efect of lithium depends on the capacity for modulating left vestibular nuclei directly or through the modulation of left side vestibular nuclei aferents.
Metalloporphyrins are Versatile and Powerful Therapeutics:
Biomimetics of SOD, Peroxyredoxin, and cyt P450 SPASOJEVIC I1, SHENG H2, WARNER DS2, BATINIC-HABERLE I3 Departments of Medicine1, Anaesthesiology2, andRadiation Oncology3
Duke University Medical Center, Durham, NC 27710, USA, spaso001@mc.duke.edu
Background: Mn-porphyrins with proper substituents possess high superoxide and peroxynitrite scavenging activity, and can modulate redox-based cellular signaling pathways thereby exerting protection in radiation, cancer, diabetes, Alzheimer’s, stroke, cerebral palsy etc. Fe- more than Mn- analogues are efficacious cyt P450 mimics, while Zn-porphyrins are potent photosensitizers.
Mimicing cyt P450
Methods: We compared different Fe- and Mn-porphyrins in their efficacy to catalyze hydroxylation (activation) of cyclophosphamide (CP) to 4-OH-CP, the active anticancer metabolite, under biologically relevant conditions. Thus 1 mM CP was incubated with 10 M Mn-porphyrins in the presence of 2 mM ascorbic acid in PBS at pH 7.4 and 5.5 at 37 oC under aerobic conditions (0.26 mM O2).
Results: The most effective cyt P450-like catalyst was the highly electron-deficient Fe(III) mesotetrakis(2,6-difluoro-3-sulfonatophenyl)porphyrin (4.6% yield). All porphyrins were more effective at pH 5.5 than at pH 7.4.
Conclusions: Fe-porphyrin can hydroxylate (activate) CP at similar efficiency as cyt P450 does (2.6% yield). The observed pH effect could be of importance in terms of specificity since tumor extracellular pH was reported to be as low as 5.2 and near the surface of macrophages to be as low as 3.6.
Antioxidant potency:
Methods: MnTnHex-2-PyP is a lipophilic (hexyl-) analogue of MnTE-2-PyP (ethyl-, highly potent SOD mimetic) which we tested for its efficacy in stroke model: 5 min after 90 min of middle cerebral artery occlusion, rats were given 75 or 225 g/kg subcutaneous bolus of MnTnHex-2-PyP, followed by twice daily injections for 7 days. MnTnHex-2-PyP levels were determined in plasma and brain by LC/MS/MS.
Results: MnTnHex-2-PyP was found at 8:1 ratio in plasma:brain, while it was 100:1 for hydrophilic MnTE-2-PyP. Neurologic scores were dose dependently improved. High dose was without adverse effects. Rats in that group gain on average 14 g while they lost 17 g in saline group. Total infarct size was reduced significantly in high-dose group (30%, p=0.02).
Conclusion: Observed dose-dependent protective effect of MnTnHex-2-PyP in stroke is an important result as there are still no effective treatments for this ailment.
Acknowledgement: IS thanks NIH/NCI Duke University Comprehensive Cancer Center Core Grant (5-P30-CA14236-29)
Inter-kingdom cell-to-cell inhibitors: a novel target for antimicrobial drugs RASKO DA1, MOREIRA CG1, LI DR1, READING NC1, RITCHIE JM2, WALDOR MK2, WILLIAMS N1, TAUSSIG R1, WEI S1, ROTH M1, HUGHES DT1, HUNTLEY J1, FINA M1, FALCK J1, SPERANDIO V1 1UT Southwestern Medical Center, Dallas, USA. 2 Harvard Medical School, Boston, USA
Background: The worldwide challenge of antimicrobial resistance and the paucity of novel antibiotics underscore the urgent need for innovative therapeutics. The increasing understanding of bacterial pathogenesis and inter-cellular communication, when combined with contemporary drug discovery tools and technologies, provides a powerful platform for translating basic science into therapeutic applications to combat bacterial infections. Inter-kingdom chemical signaling bridges the communication between bacteria and their hosts. Many bacterial pathogens rely on a conserved membrane sensor, QseC, to sense and respond to host adrenergic signaling molecules and to bacterial signals to promote expression of virulence factors.
Methods: We used a combination of high throughput screen of small molecules, qRT-PCR, and virulence tests to identify QseC inhibitors.
Results: Here we show that small molecule inhibitors of QseC-mediated signaling markedly inhibit the virulence of several pathogens in vitro and in vivo in animal models. We identified a potent small molecule, LED209, which inhibits binding of signals to QseC, preventing QseC’s autophosphorylation, and consequently inhibiting QseC-mediated activation of virulence gene expression in enterohemorrhagic E.coli (EHEC), Salmonella typhimurium and Francisella tularensis. LED209 also prevented formation of lesions on epithelial cells by EHEC, and F.tularensis survival within macrophages. Remarkably, LED209 treatment protected mice from lethal S.typhimurium and F.tularensis infection. LED209 is not toxic and does not inhibit pathogen growth.
Conclusions: Inhibition of microbial virulence without inhibition of growth may engender less selective pressure to promote the generation of resistance. As demonstrated herein, inhibition of inter-kingdom inter-cellular signaling constitutes a novel and highly effective strategy for the development of a new generation of broad spectrum antimicrobial agents.
The Uncertain Future or the End of Modern Medicine?: A Clinicians View of Antibiotic Resistance-Way to Prevent the Inevitable SPIGELMAN M Visiting Professor Centre for Infectious Diseases and International Health, Department of Infection,Windeyer Institute of Medical Sciences, UCL, 46, Cleveland Street, London W1T 4JF
As a clinitian I am rapidly approaching a time when informed consent to a young healthy adult about to have a minor operation I will have to say –"you may get infected with an organism for which there is no treatment and which may kill you"
We got to this state in three simple ways:
1-antibiotic overuse
2-antibiotic misuse
3-antibiotic abuse
This led to work that resulted in a paper for debate viz: M Spigelman 2005. MRSA Why treat the symptoms and not the disease? Annals of The Royal College of Surgeons of England 87: 6: 452 – 453). This was followed up by 3 articles detailing some of the research we are doing and the ideas we have developed to try to control this modern scourge.
The tackling of the increasing spiral of microbial resistance has been to ignore the real cause and try to limit infections-by various measures based on containment.
Our rearch shows certain areas where improvements can be made to contain cross infection paricularly in one case where we believe probiotics will be of use both as a cleaning disinfactant material and as a soap.
My presentation will highlight areas where we can make possible improvements to contain the development and spread of microbial resistance both in the community and in hospitals. How use in selected cases of pro-biotics can be beneficial in this as we have to learn how to handle bacteria from the source. As well the abuses of antibiotics in the community and food industry which has to be tackled with urgent priority.
Search for the Perfect Synthetic Isoprenoid-Glycolipid Vaccine Adjuvant SPROTT GD1, WHITFIELD DM2 1,2National Research Council, Ottawa, Canada
Background: Subunit vaccines capable of providing protective immunity against the intracellular pathogens and cancers that kill millions of people annually require an adjuvant capable of directing a sufficiently potent cytotoxic T lymphocyte response to purified antigens, without toxicity issues. Archaeosome lipid vesicles, prepared from isoprenoid lipids extracted from archaea, are one such adjuvant in development. Here, the stability of an archaeal core lipid 2,3-di-O-phytanyl-sn-glycerol (archaeol) is used to advantage to synthesize a series of disaccharide-archaeols and show that subtle variations in the carbohydrate head group alters the type and potency of immune responses mounted in a mammal.
Methods: The archaeal core lipid 2,3-di-O-phytanyl-sn-glycerol (archaeol) was prepared from Halobacterium salinarum lipids, and used as a precurser to synthesize various glycoarchaeols. These lipids were combined with phospholipid to form antigen-containing archaeosomes to test for adjuvant activity in animal trials. Mice (6/group) were injected twice subcutaneously. Cytotoxic T cell activity in splenic cells was quantified 8 weeks from first injection.
Results: Critically, a glycosylarchaeol was required in the archaeosome formulation to elicit high cytotoxic CD8+T cell activity in mice, with highest responses to antigen entrapped in archaeosomes containing disaccharides of glucose in β or α 1-6 linkage (β-gentiobiose, β-isomaltose), or of β-lactose.
Conclusions: 1) This first study on synthetic archaeal lipid adjuvants reveals potential for this class of regulatory friendly, easily scalable, inexpensive and potent glyco-adjuvant. 2) The structural detail of the glycosyl head group altered adjuvant activity. 3) The synthesis strategy using biosynthetic archaeol and carbohydrate precursors was simple. 4)The structural possibilities of archaeal glycolipids for synthesis and adjuvant testing are numerous, but may ultimately lead to design of effective and synthetic adjuvants tailered to provide an optimal immune response to protect against specific pathogens.
Penetration of Bioactive Proteins and Peptides across Stratified Mucosae in a Porcine Ex-Vivo Model SQUIER CA, KREMER M
Institute for Dental Research, University of Iowa, Iowa City, IA 52242, USA
Background: The non-keratinized stratified mucosa lining the oral cavity, esophagus and vagina provide relatively permeable surfaces to deliver therapeutic agents to treat local and systemic diseases. On the other hand, if micro-organisms and their toxins present at these surfaces penetrate the tissues they can lead to pathological conditions ranging from periodontal disease to toxic shock syndrome and AIDS. This presentation describes the use of a porcine ex-vivo model to study mucosal permeability and tissue pathogenesis.
Methods: Pig mucosal tissue provides an excellent model for human for drug delivery and permeability studies (Squier C.A., et al., J Pharm. Sci. 97:9-21, 2008). Specimens of pig buccal and vaginal mucosa were removed at slaughter and placed in thermostatted continuous flow mucosal perfusion chambers for up to 12 hours (Squier C.A., et al. J Pharm Sci 86:82-84, 1997). Our studies examined the flux of a bioactive peptide, I125 labeled transforming growth factor β (TGFβ) across buccal mucosa in the presence of phosphate buffered saline (PBS) or the mucoadhesive polymer, chitosan. In separate experiments, the flux of S35 labeled staphylococcal toxic shock syndrome toxin (TSST-1) across vaginal mucosa was determined in keratinocyte serum-free medium (KFSM) medium alone or with the addition of 50µg/ml of a staphylococcal virulence factor, α-hemolysin. Experiments utilized at least 7 replicates for each of the conditions.
Results: Flux of TGFβ was significantly (p<0.05) increased in the presence of chitosan (241±96 dpm/cm2/min) compared to PBS (36±18 dpm/cm2/min) suggesting that the mucoadhesive may provide higher local concentration of compound at the mucosal surface than does a solution. The flux of TSST-1 across the vaginal mucosa in the presence of α-hemolysin (2.7±0.8) was significantly (p<0.05) greater than that in medium alone (1.3± 0.4 ng/cm2/min) and was associated with marked histological changes in the epithelium, including massive lymphocytic infiltration and separation of epithelium from connective tissue, suggesting a synergistic role for virulence factors in the penetration of bacterial toxins.
Conclusions: the use of porcine tissue ex-vivo provides a realistic model for studying the kinetics and pathogenesis of tissue penetration by bioactive peptides and pathogenic toxins in real time for up to 12 hours.
Significant financial support:this work was supported by an unrestricted grant from the Procter and Gamble Company, Cincinnati OH.
17-year Survey of Triclosan Efficacy on Supragingival Plaque HARASZTHY V1, ZAMBON JJ1, SREENIVASAN PK2, DE VIZIO W2 1University at Buffalo, NY, USA, 2Colgate Palmolive Company, Piscataway, NJ, USA
Background: Dentifrices and other oral hygiene preparations maybe formulated with triclosan (TCN), a broad-spectrum antimicrobial agent. The broad spectrum activity of TCN on a wide range of oral organisms including pathogenic species of oral bacteria has been demonstrated in laboratory studies. In clinical studies, subjects using TCN dentifrice demonstrate improved oral health due to significant reductions in dental plaque and gingivitis. This study examined the antimicrobial efficacy of TCN on human supragingival plaque over 17 years.
Methods: Adults who had not received dental treatment or systemic antimicrobial therapy for the previous 30 days were included. Data obtained from 105 adults over 12 separate study periods are presented in this report. Supragingival plaque was collected from the entire dentition and aliquots of the bacterial suspension were distributed onto solid media containing 0, 7.5 or 25 µg/ml TCN. Following incubation at 37oC for 5-7 days, the number of colony forming units (CFU's) was enumerated.
