To investigate the mechanism of polyamine- and acetylpolyamine-induced vasodilation, aortic rings from anesthetized New Zealand white rabbits (2.0-2.5 kg) were incubated in modified Krebs-Henseleit buffer, precontracted with phenylephrine (PE), and isometric tension measured. Concentration-response curves were constructed for polyamines (putrescine, spermidine, and spermine) and acetylpolyamines ($N\sp1$-acetylputrescine, $N\sp1$-acetylspermidine, $N\sp8$-acetylspermidine, and $N\sp1$-acetylspermine) in both endothelium-intact and -denuded rings. In both types of rings, all polyamines and acetylpolyamines except $N\sp1$-acetylputrescine produced concentration-dependent relaxation (potency, spermine $>$ spermidine $>$ putrescine for polyamines; $N\sp1$-acetylspermine $>\ N\sp1$-acetylspermidine $>$ $N\sp8$-acetylspermidine for acetylpolyamines). The inhibition of endothelium-derived relaxing factor (EDRF)/nitric oxide (NO) by reduced hemoglobin and $N\sp\omega$-nitro- sc L-arginine methyl ester (sc L-NAME), and the inhibition of soluble guanylate cyclase by methylene blue did not affect the ability of polyamines or acetylpolyamines to relax vascular smooth muscle with and without endothelium, respectively. Indomethacin had no effect on polyamine- or acetylpolyamine-induced vasodilation in endothelium-intact aortic rings. In endothelium-denuded rings, Ca$\sp{2+}$ agonist, Bay K 8644, induced concentration-dependent contraction in segments of rabbit aorta, partially depolarized with 15 mM KCl. This was blocked by Ca$\sp{2+}$ antagonists, nifedipine and verapamil, and polyamines and acetylpolyamines in a concentration-dependent manner, shifting the concentration-response curve of Bay K 8644 to the right. Polyamines and acetylpolyamines as nifedipine and verapamil shifted concentration-response curves of K$\sp+$ and PE to the right in a concentration-dependent manner. Polyamines and acetylpolyamines also decreased contractions invoked by the Ca$\sp{2+}$ ionophore A23187. The concentration-dependent contraction curve for exogenous Ca$\sp{2+}$ in K$\sp+$-depolarization medium (K$\sp+$ = 120 mM) was shifted to the right by polyamines and acetylpolyamines. Both polyamines and acetylpolyamines also reduced the potentiation of K$\sp+$-induced contraction and Ca$\sp{2+}$ concentration-dependent contraction induced by Bay K 8644. The results indicate that polyamines and acetylpolyamines, as endogenous vasodilators, dilate vascular smooth muscle independent of EDRF/NO, vasodilatory prostaglandins, and by activation of soluble guanylate cyclase. Furthermore, these results suggest that polyamines and acetylpolyamines may relax vascular smooth muscle at the plasma membrane level by a mechanism that involves Ca$\sp{2+}$ influx, although may other mechanism may be possible. Further studies are needed to determine if polyamines and acetylpolyamines have calcium antagonistic properties that are involved in the mechanism of vasodilation of rabbit aortic vascular smooth muscle.

The bZIP transcription factors make up a family of long α-helical proteins that dimerize based on a pattern of hydrophobic residues and bind to DNA through a region of basic residues. Because binding specificity is a particular topic of interest, the dimerization interaction is attractive as a possible candidate to better understand protein quaternary structure. Use of the Knob-Socket (KS) model for determination of packing structure provides a novel approach to analyze protein-protein interactions. A KS analysis of the protein-protein interface provides unique insight into the specificity of the classical leucine zipper pseudo-7mer repeat. From an analysis of the KS packing maps, this research provides evidence of a general framework for defining the specificity between coiled-coils. The KS maps show how hydrophobic specificity is defined in the coiled-coil interface, where knobs are centralized in the middle of the socket packing, while the peripheral socket residues are hydrophilic. Based on this KS analysis, the KS model will be used to design proteins that mimic the leucine zipper region of bZIP proteins. The proteins will be purified into E. coli and its 2º structure will be confirmed through circular dichroism. Binding specificity will be studied through mutations of the designed proteins and compared using the BACTH (bacterial adenylate cyclase two-hybrid) system.

Peptides have been used as targeting ligands in targeted drug delivery. Conjugating peptides to cytotoxicity agents via a linker to build peptide-drug conjugate (PDC) is a promising targeting strategy. The binding affinity of the peptide ligand and the receptor plays a crucial role in the PDC targeted drug delivery. Although the ligand binding which can be used in targeted drug delivery has been established conceptually, the quantitative or semi-quantitative contribution of binding affinity in targeting efficiency has not been fully explored. The optimal range of binding affinity of the peptide for targeted delivery remains unknown. Therefore, there is a lack of knowledge on the relationship between the peptide binding affinity and targeted drug delivery efficiency. The major steps in peptide drug delivery include cellular binding, cellular internalization, and tumor cells killing. In this study, three EGFR-targeting peptides with binding affinity levels ranging from 22 nM to 1.25 μM were selected to study their targeted drug delivery efficiency. The cellular binding study of FITC labeled peptides showed that peptide GE11 with the highest binding affinity had the highest cellular binding among three peptides. PEP11 peptide showed enhanced cellular binding compared to the L1 peptide. Moreover, GE11 also showed the selectivity of cellular binding between EGFR-positive cells and EGFR-negative cells. The cellular distribution showed that GE11-FITC could be successfully internalized into cells. The uptake mechanism studies demonstrated that the cellular uptake of GE11-FITC was based on receptor-mediated endocytosis, meaning that the cellular binding of GE11 was able to trigger the endocytosis. MMAE, a non-selective anticancer agent, was conjugated to the peptides through a protease-sensitive linker. The cytotoxicity assay showed that GE11-MMAE had the highest drug delivery efficiency and selectivity of three peptides, with 200 folds lower IC50 value than MMAE in EGFR-positive cells and 1000 times lower in EGFR-negative cells. PEP11-MMAE also showed an enhanced drug delivery than MMAE and L1-MMAE. L1-MMAE failed to show a significant difference with MMAE. Cellular binding kinetics results revealed that GE11-FITC had a higher rate of cellular uptake than PEP11-FITC. In conclusion, in the range from micromolar to the nanomolar, higher binding affinity of peptide ligand will contribute to higher cellular binding, targeted drug delivery efficiency, and cellular uptake rate. These results suggest that in EGFR-targeting delivery, the nanomolar level binding affinity is necessary for peptides to be used as targeting moiety in the targeted drug delivery. This study provides a starting point for further quantitative probing of the optimal binding affinity for designing and developing peptide ligand-based targeted delivery.

The end of the human chromosome is protected by telomeres which contain a special tandem guanine-rich DNA sequence, 5’-TTAGGG. The length of telomeres is shortened during cell replications, and its length limits the replication capacity of cells. Telomerase is over-expressed in 85–90% of cancer cells, responsible for extending the telomere length in cancer cells. Guanine-rich DNA sequence can self-assemble into unique G-quadruplex structures that interfere with the extension of telomeres by telomerase. Therefore, DNA G-quadruplex has recently received much attention because of its important regulatory functions in telomerase-mediated cancerization. The formation of G-quadruplex requires monovalent cations (Na+ and K+) or small molecules known as G-quadruplex ligands. In the present work, we developed a serial of G-quadruplex ligands by tethering side-chains to two core structures: 1,10-phenanthroline (Phen) and thiazole orange (TO). Biophysical studies including DNA thermal denaturation monitored by fluorescence orcircular dichroism, fluorometric titration, and ESI-MS spectrometry reveal that the binding of the synthesized ligands to G-quadruplex is side-chain dependent. The arylsulfanyl side chains significantly improve the binding affinity and selectivity of 1,10-phenanthroline towards G-quadruplex over duplex DNA. The polyamine side chains are a suitable structural motif for remarkable G-quadruplex binding affinity based on the results from both Phen and TO derivatives. These ligands greatly inhibit the telomerase activity in vitro, determined by a modified telomeric repeat amplification protocol (TRAP) assay. Amongst these promising telomerase inhibitors, a thiazole orange derivative containing a side chain of spermine shows an outstanding telomerase inhibition effect at nanomolar concentrations, which is comparable to the most effective synthetic telomerase inhibitors, BRACO-19.

Evaluation of the Pulmonary Ambulatory Care Clinic at David Grant Medical Center was an attempt to explore several questions that surround the care of patients with chronic obstructive pulmonary disease (COPD) and to assess the role and impact of pharmacists providing pharmaceutical care in an Air Force Medical Center. Four experimental designs were implemented. Thirty-eight ambulatory patients with COPD were randomized into a control group or into a program where pharmacists resolved drug-related problems (DRPs) and educated patients about optimal drug use and management of exacerbations. The groups were evaluated prospectively at baseline, three months and six months for the presence of DRPs, quality of life (QOL) scores, and health care resource utilization (HCRU) parameters. Significant improvements in Health Status Questionnaire scores of study group were at three (F = 4.56, p = 0.04) and six months (F = 3.84, p = 0.05). Chronic Respiratory Disease Index questionnaire scores significantly improved in study group at three months in fatigue category (F = 5.35, p = 0.02). Disease State Knowledge Test scores significantly improved at three (F = 3.90, p = 0.04) and six months (F = 13.37, p = 0.001) in study group. Twenty-two DRPs were resolved. The study group realized significant reductions in mean numbers of physician visits (F = 8.12, p $<$ 0.05), and emergency room visits (F = 5.41, p $<$ 0.05) at three and six months respectively. Mean number of physician visits in study group during six-month period was significantly lower prospectively than retrospectively (T = 3.56, p = 0.003) in the single subject design study. Retrospective study of seventy patients showed that mean numbers of chronic medications (F = 6.57, p $<$ 0.05) and pulmonary hospitalizations (F = 4.08, p $<$ 0.05) were significantly lower in mild patients than severe patients. These results of this four-part study design, showed that patient education and resolution of DRPs improved patients' QOL and increased their knowledge about their disease states. Teaching patients how to manage exacerbation resulted in a decrease in health care visits in this patient population. Categorization of severity by pulmonologist coincided with patients' utilization of HCRs.

Bakgrund Influensa är en akut respiratorisk infektion som orsakas av ett negativt RNA-virus. Insjuknade i influensa sker ofta under vintertid. Influensa finns i fyra olika typer, A-D. A är den vanligaste sorten under influensasäsong och påverkar både människor och djur, medan D påverkar främst grisar och nötkreatur. Influensa kännetecknas av att det börjar med en frossa och med en snabbt stigande feber (över 40℃) samt med muskelvärk och med en märkbar sjukdomskänsla. Efteråt sprids infektionen till luftvägarna, där det uppkommer symtom i form av torrhosta. Influensa sprids genom luftburna aerosoler eller genom dropp- och kontaktsmitta. Influensaviruset tar sig in i cellen via sina ytproteiner hemagglutinin (HA) och neuraminidas (NA) och sedan tar det sig in till värdcellens kärna, vilket leder till replikation av viruset. Vid influensasäsongen ges vaccinationer till personer som är över 65 år, och till är gravida och till personer som har någon underliggande kronisk sjukdom. Detta görs för att förhindra allvarlig sjukdom och för att kunna minska antalet komplikationer och dödsfall under influensasäsongen. Neuraminidashämmare som exempelvis oseltamivir och zanamivir kan ges för smärtlindring under influensan i fem dagar. Växten Echinacea purpurea och dess extrakt används som behandling av luftvägsinfektioner exempelvis hosta och vid inflammatoriska tillstånd, som exempelvis bronkit. Studier på olika extrakt av Echinacea purpurea har visat ökade halter av cytokiner som exempelvis TNF-α, IL-1 och IFN- β. Det har även visat en ökad aktivitet av fagocyterna och makrofager. En ökad mobilitet har också setts hos både leukocyter och aktivering av NK-celler. Syfte Syftet med arbetet var att undersöka om Echinacea purpurea har en symtomlindrande effekt på influensa A, speciellt på symtom som feber och hosta. Metod Publicerade vetenskapliga artiklar söktes i databasen Pubmed och OneSearch. Sökorden för artiklarna var “Influenza AND Echinacea purpurea” eller “Echinacea AND flu” I OneSearch användes sökorden “Influenza AND Echinacea purpurea”.ResultatStudierna visar att symtomen hosta och feber reducerades vid användning av Echinacea purpurea extrakt under behandling av influensa A. Behandlingen med Echinacea purpurea hade en bättre verkan vid användningen av en högre dos av extraktet (2000 mg) och om det användes några dagar innan influensasymtom uppträdde eller vid början av influensasymtom. Slutsats Utifrån de publicerade resultaten av de fyra studierna verkar Echinacea purpurea ha en mildrande effekt på hosta och feber under akuta luftvägsinfektioner. Det visade att dosen som hade mest effekt i dessa studier, var 2000 mg av växtextrakt från Echinacea purpurea. Dessa resultat erhölls vid behandling av i övrigt friskaindivider som endast hade förkylningssymtom eller influensasymtom, vilket gör att Echinacea purpurea extrakt kan ha andra effekter om det används på kroniskt sjuka individer. Flera äldre vaccinerar sig mot influensa, vilket gör att Echinacea purpurea extrakt har mer gynnsam effekt på yngre personer som har drabbats av influensa t.ex. barn eller unga vuxna. / Background Influenza is an acute respiratory infection which is caused by a negative stranded RNA-virus. Infection usually happens during the winter season. There are four different types of influenza, A-D. Type A is the most common one and affects both humans and animals, while type D has only been detected in pigs and cattle. Influenza starts with chills with a quickly rising fever (over 40℃) together with muscle pains. Afterwards the infection spreads to the airways, where the symptoms of dry cough arise. Influenzaspreads through airborne aerosols or through contact. The influenza virus enters the hostcell through the surface proteins HA and NA, while later entering the nucleus where the replication of the virus occurs. Before the flu season begins, people over the age of 65, pregnant women and patients with a chronic disease are vaccinated. This is done to prevent serious diseases that may occur during the flu and to lower the rate of secondary infections and also to prevent death. Medications such as oseltamivir and zanamivir can be given to treat the infection; however, they are mostly used to relieve symptoms. Echinacea purpurea is a traditionally used medicinal plant which has been used to treat respiratory tract infections like respiratory infections and inflammatory conditions such as bronchitis. Studies on different extracts of Echinacea purpurea have shown that they increase levels of TNF-ɑ, IL-1 and IFN-β as well as an increase of macrophages and other phagocytes after treatment with Echinacea extract. Similarly, the extract has shown an increased mobility of leukocytes and activation of Natural Killer-cells. Aim The aim of this study was to examine if treatment with medications containing extracts of Echinacea purpurea relieved symptoms such as, cough and fever, when used in the treatment of influenza A. Method The previously published articles were searched for by using the databases Pubmed and OneSearch. The words used in the search for published articles were “Influenza AND Echinacea purpurea” or ”Echinacea AND flu” in Pubmed. In the database, OneSearch the words “Influenza AND Echinacea purpurea'' were used. Results The selected studies showed that the symptoms such as cough and fever, were reduced in studied patients that used Echinacea purpurea extracts during the treatment of influenza A. Infection was verified through nasal swabs that then were screened. The results showed that the treatment was more effective when using a dose of 2000 mg of Echinacea purpurea extract. Moreover the treatment was effective when used a few days before symptoms occurred or at the beginning of a respiratory tract infection. Conclusion The results from the four studies suggested that the extract from Echinacea purpureareduces cough and fever during acute respiratory infections. The dosage of 2000 mg Echinacea purpurea extract had the most effect. However the extracts were tested on healthy people that only had common cold symptoms or influenza symptoms, thus different results could have been obtained if the effects were tested on unhealthy individuals.

No description available.

Acquired immune deficiency syndrome (AIDS) was first discovered in the 1980s, since then, human immunodeficiency virus (HIV) infection and AIDS have become global health, social, and economic concerns. HIV was identified as the cause of AIDS in 1985, and this launched a wide-reaching effort to understand its biology. The knowledge acquired from these vast research efforts contributed to the development of modern therapeutic and preventative treatment strategies. According to recent data from the United Nations Program on HIV/AIDS (UNAIDS), the ratio of infected people to AIDS- related deaths has decreased because of the expanding access to antiretroviral drugs (ARVs). The application of ARVs to HIV+ patients increases patients’ lifespans and improves the quality of life. Remaining as an incurable disease, expanding access to antiretroviral drugs and using prevention strategies are the best options to control the HIV pandemic for now. Treatment strategies with ARVs, however, are not sufficient to adequately address the HIV pandemic. Traditional combinational antiretroviral therapies (cART) for HIV treatment are limited by multiple drawbacks such as possible toxicity, limited drug concentrations, drug resistance, and viral rebound. Additionally, inadequate physicochemical properties of ARVs, such as poor solubility, permeability, and bioavailability, lead to limited absorption and biodistribution, resulting in poor clinical outcomes. Patient compliance and suboptimal efficacy lead to the development of resistant viruses and viral reservoirs. The presence of HIV reservoirs would cause viral rebound two to four weeks after terminating treatments. The complexity of reservoir structure, prolonged cell half-life, and the latent HIV viruses complicate HIV treatments iii targeting viral reservoirs. cART exhibits insufficient efficacy towards reservoir sites because of biological barriers and poor physicochemical properties. These problems highlight an urgent need for novel treatment strategies that are safe and effective to address HIV reservoirs. Innovative and improved delivery systems have been proposed over the years, especially lipid-formulations. Lipid formulations have emerged as promising vehicles owing to their ability to encapsulate molecules with poor solubility and bioavailability, improve active targeting, prolong circulation time, and sustain drug release. Cell-mediated delivery strategy have posed the obstacles of insufficient drug transport and safety. Macrophages, the very same cells that carry the HIV virus, could reach tissues that would otherwise have little or no drug penetration. Macrophages can protect drugs from metabolic degradation with large quantities of drugs for delivery. Activated macrophages express the folate receptor, a potential targeting moiety. In this study, I intended to develop a novel folate-decorated nanoemulsion (FA- NE) for the delivery of ARVs to HIV infected macrophages. To reach the goal, I focused on two goals: (1) construction of a nanocarrier capable of encapsulating ARV drugs with physiological properties suitable for use in drug delivery and (2) enhancement of delivery to HIV infected macrophages. In Chapter 2, I discuss the rationale for nanoART for HIV treatments. I introduce current HIV treatments and their drawbacks, notably the viral rebound due to limited drug concentration in viral reservoirs. Then I explain why nanotechnology would be a promising strategy for HIV treatment and provide examples of nanomedicine. In all iv cases, however, cell uptake and drug release were limited or complicated by toxicity, which is a significant issue for a validated delivery system that are safe and effective. In chapter 3, I introduce the design and development of the FA-NE. This system includes (1) an oil core to encapsulate antiretroviral drugs that are highly hydrophobic, (2) a lipid monolayer to protect the oil core and to form nanoemulsion (3) folate for target. The system was prepared using the emulsification solvent evaporation method, developed and optimized based on physical properties, including size, PDI, zeta potential, and other in vitro characterizations, such as encapsulation efficacy, drug loading, stability, and drug release. Chapter 4 is a continuation of the work done in Chapter 3 and focuses on the enhancement of cellular uptake with folate overexpression cell models. A lipopolysaccharide (LPS) activated macrophages was built and utilized for intracellular drug release and retention evaluations. In Chapter 5, cytotoxicity and antiretroviral efficacy studies are described. With the conclusion drawn in Chapter 4, I was curious if the enhanced cellular uptake can be translated into improved efficacy. As a result, collaborated with Dr. Kamel Khalili, School of Medicine, Temple University, we evaluated antiretroviral efficacy with an HIV indicator cell and monocyte-derived-macrophages from human donors. Furthermore, I performed cytotoxicity assay to evaluate this nanoemulsion system safety profile. Chapter 6 summarizes the highlights and conclusions of this project and provides suggestions for the future. / Pharmaceutical Sciences

Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Although five mAChR subtypes (M1-M5) share a high degree of homology, they display different physiological effects including controlling smooth muscle tone to neurotransmitter release in the CNS. Hence these receptor subtypes have been investigated as potential therapeutic targets for agents capable of treating Alzheimer's Disease, Parkinson's Disease, peptic ulcer disease, COPD, urinary incontinence, and muscle spasms. Our interest in the development of subtype selective muscarinic ligands led to previous reports detailing the identification of substituted lactones as lead muscarinic compounds. Later work involved molecular modifications of those leads that included the addition of aromatic groups with a variety of substitution patterns. These efforts led to an increase in receptor affinity and produced a lactone-based muscarinic ligand with an IC50 of 340nM. As a continuation of that work, additional novel ligands were designed based on the general pharmacophoric elements proposed for the lactone-based ligands. In that model, the lactone oxygens serve as H-bond acceptor moieties while different nitrogen containing heterocycles provide the requisite cationic group. These groups may be separated by linker groups of varying sizes. In order to synthesize the lactone-based ligands mentioned above, efficient synthetic routes are required for key precursors. These include but are not limited to: 1. A novel high yield synthesis of the hydroxyethyl-lactone precursor was designed using a carefully controlled Prins reaction. The method readily quenches a cationic intermediate and simultaneously protects hydroxyl groups in a single step. A mechanism for the new route to the precursor is proposed and its use in the preparation of the target compounds is presented 2. Microwave-assisted synthesis of various sterically hindered N-aryl piperazines has been developed allowing quick access to structurally diverse muscarinic ligands These synthesis along with other newly developed routes enabled ready access to 59 novel muscarinic ligands. The ligands were tested in a general muscarinic binding assay. The result was analyzed and SAR study was performed to direct ligand design. As a result of this work, ligand affinity was improved by over 100 folds compare to the lead molecules. Several promising compounds were selected and selectivity tested. / Pharmaceutical Sciences

RNase P is a bacterial ribozyme that catalyzes the maturation of tRNA and is conserved across Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). RNase P consists of a RNA component and a protein component, RnpA. In Gram-positive bacteria, RnpA itself possesses ribonuclease activity. The Dunman group demonstrated that inhibition of RnpA activity alone or as part of the RNase P complex was sufficient to inhibit RNA degradation and exert antimicrobial activity in MRSA. Because of its low amino acid homology to mammalian homologs, RnpA may represent a novel, selective antimicrobial target for MRSA. A high throughput screen by the Dunman group identified a number of compounds which inhibit RnpA activity, including RNPA1000. However, RNPA1000 demonstrated cytotoxic effects at higher concentrations and required a high dose to achieve efficacy in a murine model of MRSA infection. We therefore selected another “hit” from the screen (RNPA2000), which contains metabotoxic hydrazide, thiourea and furan moieties, as the starting point for hit to lead activities. We sought to replace these groups, as well as the isopropylphenoxy group, to provide enhanced inhibitory potency against RNase P and RnpA, as well as lowered MIC values against MRSA1000. We designed and synthesized analogs posessing bioisosteres for these moieties and evaluated their effects in an RNase P assay as well as a RNA degradation assay. Compounds with acceptable results in both assays were tested for their antimicrobial effects in MRSA cultures. As a result of this work, several compounds with improved potency for RnpA inhibition were identified, although improved MIC was not seen. Two compounds demonstrated synergy with mupirocin, an isoleucyl-tRNA synthase inhibitor, which may represent a potential way to re-sensitize resistant bacteria to mupirocin. / Pharmaceutical Sciences