Control of lipid microstructures by molecular design and its implications

An overview of the current trends in the lipid design for specific applications has been presented. Lipids with different surface charge and hydrophobic backbone undergo aggregation to produce lamellae or bilayer and multilayer vesicles in aqueous media. Various aspects of present development of chiral superstructures and enzyme-mimics have been discussed. Utility of these molecules for potential applications in immunomodulation and sustained drug-delivery systems is also summarized.

Effective drug dosage design for treatment of infectious diseases using dynamic inversion

A generic nonlinear mathematical model describing the human immunological dynamics is used to design an effective automatic drug administration scheme. Even though the model describes the effects of various drugs on the dynamic system, this work is confined to the drugs that kill the invading pathogen and heal the affected organ. From a system theoretic point of view, the drug inputs can be interpreted as control inputs, which can be designed based on control theoretic concepts. The controller is designed based on the principle of dynamic inversion and is found to be effective in curing the �nominal model patient� by killing the invading microbes and healing the damaged organ. A major advantage of this technique is that it leads to a closed-form state feedback form of control. It is also proved from a rigorous mathematical analysis that the internal dynamics of the system remains stable when the proposed controller is applied. A robustness study is also carried out for testing the effectiveness of the drug administration scheme for parameter uncertainties. It is observed from simulation studies that the technique has adequate robustness for many �realistic model patients� having off-nominal parameter values as well.

Characterization and Performance Study of Packaged Micropump for Drug Delivery

In this work, we present the characterization and performance studies of self-priming peristaltic pump for drug delivery application. Conventional materials and methods have been used to fabricate single cam mechanism based peristaltic micropump. To control the fluid flow precisely in micro liter range, a single cam mechanism has been used instead of conventional roller mechanism. The fabricated pump is suitable for liquid, gas and foam. Using water as a fluid medium, a flow rate of 12.5 mu l/rpm is achieved using a flexible silicone tube of inner diameter 1.5 mm and outer diameter 2.5 mm. Other than water, higher viscosity fluids showed a decrease in the flow rate. The designed micropump exhibits a linear dependence of flow rate in the voltage range of 2.5V to 5V. Drug delivery using micropump demands that the micropump has to pump against the blood pressure (maximum of 25kPa) with constant flow rate. Here the designed pump is able to pump the liquid with a constant flow rate of 500 mu l/min (water) up to a backpressure of 40kPa. It was observed that, by increasing the backpressure above 40kPa, flow rate of the pump gradually decreased to 125 mu l/min at 120kPa. In addition, Micropump based drug delivery demands that the micropump should be normally in closed condition in all the positions to avoid drug leakage and bleeding. Hence, micropump has been characterized for normally closed condition in all positions (0 degrees to 360 degrees). However, a minute leak of 0.14 % was found for an inlet pressure of 140kPa. Also, the normally closed region with no leak is observed up to 60kPa of pressure in all positions (0 degrees to 360 degrees).

Suramin is a potent and selective inhibitor of Mycobacterium tuberculosis RecA protein and the SOS response: RecA as a potential target for antibacterial drug discovery

In eubacteria, RecA is essential for recombinational DNA repair and for stalled replication forks to resume DNA synthesis. Recent work has implicated a role for RecA in the development of antibiotic resistance in pathogenic bacteria. Consequently, our goal is to identify and characterize small-molecule inhibitors that target RecA both in vitro and in vivo. We employed ATPase, DNA strand exchange and LexA cleavage assays to elucidate the inhibitory effects of suramin on Mycobacterium tuberculosis RecA. To gain insights into the mechanism of suramin action, we directly visualized the structure of RecA nucleoprotein filaments by atomic force microscopy. To determine the specificity of suramin action in vivo, we investigated its effect on the SOS response by pull-down and western blot assays as well as for its antibacterial activity. We show that suramin is a potent inhibitor of DNA strand exchange and ATPase activities of bacterial RecA proteins with IC50 values in the low micromolar range. Additional evidence shows that suramin inhibits RecA-catalysed proteolytic cleavage of the LexA repressor. The mechanism underlying such inhibitory actions of suramin involves its ability to disassemble RecA-single-stranded DNA filaments. Notably, suramin abolished ciprofloxacin-induced recA gene expression and the SOS response and augmented the bactericidal action of ciprofloxacin. Our findings suggest a strategy to chemically disrupt the vital processes controlled by RecA and hence the promise of small molecules for use against drug-susceptible as well as drug-resistant strains of M. tuberculosis for better infection control and the development of new therapies.

Heat shock protein 90 as a potential drug target against surra

Trypanosomiasis is caused by Trypanosoma species which affect both human and animal populations and pose a major threat to developing countries. The incidence of animal trypanosomiasis is on the rise. Surra is a type of animal trypanosomiasis, caused by Trypanosoma evansi, and has been included in priority list B of significant diseases by the World Organization of Animal Health (OIE). Control of surra has been a challenge due to the lack of effective drugs and vaccines and emergence of resistance towards existing drugs. Our laboratory has previously implicated Heat shock protein 90 (Hsp90) from protozoan parasites as a potential drug target and successfully demonstrated efficacy of an Hsp90 inhibitor in cell culture as well as a pre-clinical mouse model of trypanosomiasis. This article explores the role of Hsp90 in the Trypanosoma life cycle and its potential as a drug target. It appears plausible that the repertoire of Hsp90 inhibitors available in academia and industry may have value for treatment of surra and other animal trypanosomiasis.

Exploration of new drug delivery pathways: I. Mechanism of folate uptake in cultured human cells. II. Role of nuclear localization signal peptides in the delivery of oligonucleotide into reconstituted nuclei

The roles of the folate receptor and an anion carrier in the uptake of 5- methyltetrahydrofolate (5-MeH_4folate) were studied in cultured human (KB) cells using radioactive 5-MeH_4folate. Binding of the 5-MeH_4folate was inhibited by folic acid, but not by probenecid, an anion carrier inhibitor. The internalization of 5-MeH_4folate was inhibited by low temperature, folic acid, probenecid and methotrexate. Prolonged incubation of cells in the presence of high concentrations of probenecid appeared to inhibit endocytosis of folatereceptors as well as the anion carrier. The V_(max) and K_M values for the carrier were 8.65 ± 0.55 pmol/min/mg cell protein and 3.74 ± 0.54µM, respectively. The transport of 5-MeH4folate was competitively inhibited by folic acid, probenecid and methotrexate. The carrier dissociation constants for folic acid, probenecid and methotreate were 641 µM, 2.23 mM and 13.8 µM, respectively. Kinetic analysis suggests that 5-MeH_4folate at physiological concentration is transported through an anion carrier with the characteristics of the reduced-folate carrier after 5-MeH_4folate is endocytosed by folate receptors in KB cells. Our data with KB cells suggest that folate receptors and probenecid-sensitive carriers work in tandem to transport 5-MeH_4folate to the cytoplasm of cells, based upon the assumption that 1 mM probenecid does not interfere with the acidification of the vesicle where the folate receptors are endocytosed.

Oligodeoxynucleotides designed to hybridize to specific mRNA sequences (antisense oligonucleotides) or double stranded DNA sequences have been used to inhibit the synthesis of a number of cellular and viral proteins (Crooke, S. T. (1993) FASEB J. 7, 533-539; Carter, G. and Lemoine, N. R. (1993) Br. J. Cacer 67, 869-876; Stein, C. A. and cohen, J. S. (1988) Cancer Res. 48, 2659-2668). However, the distribution of the delivered oligonucleotides in the cell, i.e., in the cytoplasm or in the nucleus has not been clearly defined. We studied the kinetics of oligonucleotide transport into the cell nucleus using reconstituted cell nuclei as a model system. We present evidences here that oligonucleotides can freely diffuse into reconstituted nuclei. Our results are consistent with the reports by Leonetti et al. (Proc. Natl. Acad. Sci. USA, Vol. 88, pp. 2702-2706, April 1991), which were published while we were carrying this research independently. We also investigated whether a synthetic nuclear localization signal (NLS) peptide of SV40 T antigen could be used for the nuclear targeting of oligonucleotides. We synthesized a nuclear localization signal peptide-conjugated oligonucleotide to see if a nuclear localization signal peptide can enhance the uptake of oligonucleotides into reconstituted nuclei of Xenopus. Uptake of the NLS peptide-conjugated oligonucleotide was comparable to the control oligonucleotide at similar concentrations, suggesting that the NLS signal peptide does not significantly enhance the nuclear accumulation of oligonucleotides. This result is probably due to the small size of the oligonucleotide.

M-type channels selectively control bursting in rat dopaminergic neurons

Midbrain dopaminergic neurons in the substantia nigra, pars compacta and ventral tegmental area are critically important in many physiological functions. These neurons exhibit firing patterns that include tonic slow pacemaking, irregular firing and bursting, and the amount of dopamine that is present in the synaptic cleft is much increased during bursting. The mechanisms responsible for the switch between these spiking patterns remain unclear. Using both in-vivo recordings combined with microiontophoretic or intraperitoneal drug applications and in-vitro experiments, we have found that M-type channels, which are present in midbrain dopaminergic cells, modulate the firing during bursting without affecting the background low-frequency pacemaker firing. Thus, a selective blocker of these channels, 10,10-bis(4-pyridinylmethyl)-9(10H)- anthracenone dihydrochloride, specifically potentiated burst firing. Computer modeling of the dopamine neuron confirmed the possibility of a differential influence of M-type channels on excitability during various firing patterns. Therefore, these channels may provide a novel target for the treatment of dopamine-related diseases, including Parkinson's disease and drug addiction. Moreover, our results demonstrate that the influence of M-type channels on the excitability of these slow pacemaker neurons is conditional upon their firing pattern. © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

Fingerprinting quality control of Qianghuo by high-performance liquid chromatography-photodiode array detection

A novel, simple and accurate fingerprint method was developed using high-performance liquid chromatography-photodiode array detection (HPLC-DAD) for the quality control of Qianghuo, a Tibetan folk and Chinese herbal medicine used as a diaphoretic, an antifebrile and an anodyne. For the first time, the feasibility and advantages of employing chromatographic fingerprint were investigated and demonstrated for the evaluation of Qianghuo by systematically comparing chromatograms of aqueous extracts with the professional analytical software recommended by State Food and Drug Administration (SFDA). Our results revealed that the chromatographic fingerprint combing similarity evaluation could efficiently identify and distinguish raw herbs of Qianghno from different sources and different species. The effects on Notopterygium forbesii Boiss (Apiaceae) chromatographic fingerprints resulted from collecting locations, harvesting time were also examined. (c) 2006 Elsevier lrelanc Ltd. All rights reserved.

Development of an in vitro multicellular tumor spheroid model using microencapsulation and its application in anticancer drug screening and testing

In this study, an in vitro multicellular tumor spheroid model was developed using microencapsulation, and the feasibility of using the microencapsulated. multicellular tumor spheroid (MMTS) to test the effect of chemotherapeutic drugs was investigated. Human MCF-7 breast cancer cells were encapsulated in alginate-poly-L-lysine-alginate (APA) microcapsules, and a single multicellular spheroid 150 mu m in diameter was formed in the microcapsule after 5 days of cultivation. The cell morphology, proliferation, and viability of the MMTS were characterized using phase contrast microscopy, BrdU-Iabeling, MTT stain, calcein AM/ED-2 stain, and H&E stain. It demonstrated that the MMTS was viable and that the proliferating cells were mainly localized to the periphery of the cell spheroid and the apoptotic cells were in the core. The MCF-7 MMTS was treated with mitomycin C (MC) at a concentration of 0.1, 1, or 10 times that of peak plasma concentration (ppc) for up to 72 h. The cytotoxicity was demonstrated. clearly by the reduction in cell spheroid size and the decrease in cell viability. The MMTS was further used to screen the anticancer effect of chemotherapeutic drugs, treated with MC, adriamycin (ADM) and 5-fluorouracil (5-FU) at concentrations of 0.1, 1, and 10 ppc for 24, 48, and 72 h. MCF-7 monolayer culture was used as control. Similar to monolayer culture, the cell viability of MMTS was reduced after treatment with anticancer drugs. However, the inhibition rate of cell viability in MMTS was much lower than that in monolayer culture. The MMTS was more resistant to anticancer drugs than monolayer culture. The inhibition rates of cell viability were 68.1%, 45.1%, and 46.8% in MMTS and 95.1%, 86.8%, and 91.6% in monolayer culture treated with MC, ADM, and 5-FU at 10 ppc for 72 h, respectively. MC showed the strongest cytotoxicity in both MMTS and monolayer, followed by 5-FU and ADM. It demonstrated that the MMTS has the potential to be a rapid and valid in vitro model to screen chemotherapeutic drugs with a feature to mimic in vivo three-dimensional (3-D) cell growth pattern.

Development of process control methodology for tracking the quality and safety of pain, agitation and sedation management in critical care units

Objective: To develop sedation, pain, and agitation quality measures using process control methodology and evaluate their properties in clinical practice. Design: A Sedation Quality Assessment Tool was developed and validated to capture data for 12-hour periods of nursing care. Domains included pain/discomfort and sedation-agitation behaviors; sedative, analgesic, and neuromuscular blocking drug administration; ventilation status; and conditions potentially justifying deep sedation. Predefined sedation-related adverse events were recorded daily. Using an iterative process, algorithms were developed to describe the proportion of care periods with poor limb relaxation, poor ventilator synchronization, unnecessary deep sedation, agitation, and an overall optimum sedation metric. Proportion charts described processes over time (2 monthly intervals) for each ICU. The numbers of patients treated between sedation-related adverse events were described with G charts. Automated algorithms generated charts for 12 months of sequential data. Mean values for each process were calculated, and variation within and between ICUs explored qualitatively. Setting: Eight Scottish ICUs over a 12-month period. Patients: Mechanically ventilated patients. Interventions: None. Measurements and Main Results: The Sedation Quality Assessment Tool agitation-sedation domains correlated with the Richmond Sedation Agitation Scale score (Spearman [rho] = 0.75) and were reliable in clinician-clinician (weighted kappa; [kappa] = 0.66) and clinician-researcher ([kappa] = 0.82) comparisons. The limb movement domain had fair correlation with Behavioral Pain Scale ([rho] = 0.24) and was reliable in clinician-clinician ([kappa] = 0.58) and clinician-researcher ([kappa] = 0.45) comparisons. Ventilator synchronization correlated with Behavioral Pain Scale ([rho] = 0.54), and reliability in clinician-clinician ([kappa] = 0.29) and clinician-researcher ([kappa] = 0.42) comparisons was fair-moderate. Eight hundred twenty-five patients were enrolled (range, 59-235 across ICUs), providing 12,385 care periods for evaluation (range 655-3,481 across ICUs). The mean proportion of care periods with each quality metric varied between ICUs: excessive sedation 12-38%; agitation 4-17%; poor relaxation 13-21%; poor ventilator synchronization 8-17%; and overall optimum sedation 45-70%. Mean adverse event intervals ranged from 1.5 to 10.3 patients treated. The quality measures appeared relatively stable during the observation period. Conclusions: Process control methodology can be used to simultaneously monitor multiple aspects of pain-sedation-agitation management within ICUs. Variation within and between ICUs could be used as triggers to explore practice variation, improve quality, and monitor this over time

Renal failure and the neural control of the kidney

Renal failure (RF) is associated with an over activation of the sympathetic nervous system. The aim of this thesis was to investigate the hypothesis that as the kidney progresses into RF there is an inappropriate and sustained activation of renal afferent nerves which results in a dysregulation of basal RSNA and reflexly controlled RSNA by the high and low pressure baroreceptors. Baroreflex gain curves for both RSNA and HR were generated in control and RF rats. This study clearly showed a blunted high-pressure baroreflex in RF rats, an impairment which was almost completely corrected by bilateral renal denervation. The integrity of the low-pressure cardiopulmonary receptors to inhibit RSNA was investigated using acute saline volume. Again, a blunted reflex sympatho-inhibition of RSNA was observed, which was corrected by renal denervation. Finally a functional study to examine how the renal excretory response to volume expansion differed in RF was carried out. This study revealed an impairment of the low-pressure baroreflex control of the sympathetic outflow. The result of these studies suggest that cisplatin induced RF initiates a neural signal from within the kidney, which over rides the normal reflex regulation of RSNA by the high and low – pressure baroreceptors and that this impairment in function can be normalised by renal denervation. This raises further questions as to the mechanisms involved in the afferent over activation arising from the diseased kidneys.

Strategies to prevent potentially inappropriate prescribing and adverse drug reactions in older patients

INTRODUCTION: Potentially inappropriate prescribing (PIP) is a major contributor to adverse drug reactions and overall increased healthcare costs. The aim of this thesis was to identify, develop and implement strategies with the potential to prevent PIP and ADRs in older patients. METHODS: A systematic review of the qualitative literature (Meta-synthesis); A qualitative study in Irish hospitals; A randomised controlled trial (RCT) to assess the impact of an online educational module on doctors’ prescribing knowledge and confidence; Exploration of the potential for a frailty index score to enable doctors identify patients at increased risk of PIP and ADRs; Exploration of the potential for the SHiM tool to enable doctors to optimise prescribing for older patients. RESULTS: The meta-synthesis identified four key concepts: (i) Desire to please the patient; (ii) Feeling of being forced to prescribe; (iii) Tension between experience and guidelines; (iv) Prescriber fear. Similar themes also emerged from the qualitative study. In the RCT, the online educational module resulted in a highly significant 22% difference in test scores between intervention and control groups. The studies exploring the frailty index score showed a significant positive relationship between a patient’s frailty status and their likelihood of experiencing PIP/ADRs. Patients above a frailty threshold of 0.16 were at least twice as likely to experience PIP/ADRs. SHiM was found to be a useful tool in terms of reconciling patients’ medications. However, the evidence for it being capable of preventing clinically relevant adverse events was poor. CONCLUSION:Qualitative research in this thesis has proposed novel theories relating to the causative factors of PIP in older patients. In doing so, it has identified several areas for intervention and laid down a road map for future research.

Promising results with exemestane in the first-line treatment of metastatic breast cancer: a randomized phase II EORTC trial with a tamoxifen control.

Because tamoxifen (TAM), a nonsteroidal antiestrogen, is routinely used in the adjuvant setting, other hormone therapies are needed as alternatives for first-line treatment of metastatic breast cancer (MBC). Currently, exemestane (EXE) and other antiaromatase agents are indicated for use in patients who experience failure of TAM. In this multicenter, randomized, open-label, TAM-controlled (20 mg/day), phase II trial, we examined the activity and tolerability of EXE 25 mg/day for the first-line treatment of MBC in postmenopausal women. Exemestane was well tolerated and demonstrated substantial first-line antitumor activity based on intent-to-treat analysis of peer-reviewed responses. In the EXE arm, values for complete, partial, and objective response, clinical benefit, and time to tumor progression (TTP) exceeded those reported for TAM although no statistical comparison was made. Based on these encouraging results, a phase III trial will compare EXE and TAM.

Evaluation of tumor response, disease control, progression-free survival, and time to progression as potential surrogate end points in metastatic breast cancer.

PURPOSE: Overall survival (OS) can be observed only after prolonged follow-up, and any potential effect of first-line therapies on OS may be confounded by the effects of subsequent therapy. We investigated whether tumor response, disease control, progression-free survival (PFS), or time to progression (TTP) could be considered a valid surrogate for OS to assess the benefits of first-line therapies for patients with metastatic breast cancer. PATIENTS AND METHODS: Individual patient data were collected on 3,953 patients in 11 randomized trials that compared an anthracycline (alone or in combination) with a taxane (alone or in combination with an anthracycline). Surrogacy was assessed through the correlation between the end points as well as through the correlation between the treatment effects on the end points. RESULTS: Tumor response (survival odds ratio [OR], 6.2; 95% CI, 5.3 to 7.0) and disease control (survival OR, 5.5; 95% CI, 4.8 to 6.3) were strongly associated with OS. PFS (rank correlation coefficient, 0.688; 95% CI, 0.686 to 0.690) and TTP (rank correlation coefficient, 0.682; 95% CI, 0.680 to 0.684) were moderately associated with OS. Response log ORs were strongly correlated with PFS log hazard ratios (linear coefficient [rho], 0.96; 95% CI, 0.73 to 1.19). Response and disease control log ORs and PFS and TTP log hazard ratios were poorly correlated with log hazard ratios for OS, but the confidence limits of rho were too wide to be informative. CONCLUSION: No end point could be demonstrated as a good surrogate for OS in these trials. Tumor response may be an acceptable surrogate for PFS.

PKC signaling regulates drug resistance of the fungal pathogen Candida albicans via circuitry comprised of Mkc1, calcineurin, and Hsp90.

Fungal pathogens exploit diverse mechanisms to survive exposure to antifungal drugs. This poses concern given the limited number of clinically useful antifungals and the growing population of immunocompromised individuals vulnerable to life-threatening fungal infection. To identify molecules that abrogate resistance to the most widely deployed class of antifungals, the azoles, we conducted a screen of 1,280 pharmacologically active compounds. Three out of seven hits that abolished azole resistance of a resistant mutant of the model yeast Saccharomyces cerevisiae and a clinical isolate of the leading human fungal pathogen Candida albicans were inhibitors of protein kinase C (PKC), which regulates cell wall integrity during growth, morphogenesis, and response to cell wall stress. Pharmacological or genetic impairment of Pkc1 conferred hypersensitivity to multiple drugs that target synthesis of the key cell membrane sterol ergosterol, including azoles, allylamines, and morpholines. Pkc1 enabled survival of cell membrane stress at least in part via the mitogen activated protein kinase (MAPK) cascade in both species, though through distinct downstream effectors. Strikingly, inhibition of Pkc1 phenocopied inhibition of the molecular chaperone Hsp90 or its client protein calcineurin. PKC signaling was required for calcineurin activation in response to drug exposure in S. cerevisiae. In contrast, Pkc1 and calcineurin independently regulate drug resistance via a common target in C. albicans. We identified an additional level of regulatory control in the C. albicans circuitry linking PKC signaling, Hsp90, and calcineurin as genetic reduction of Hsp90 led to depletion of the terminal MAPK, Mkc1. Deletion of C. albicans PKC1 rendered fungistatic ergosterol biosynthesis inhibitors fungicidal and attenuated virulence in a murine model of systemic candidiasis. This work establishes a new role for PKC signaling in drug resistance, novel circuitry through which Hsp90 regulates drug resistance, and that targeting stress response signaling provides a promising strategy for treating life-threatening fungal infections.