Delivery of phosphonoformate prodrugs

AIDS dementia complex is a common neurological syndrome thought to result from the invasion of the CNS by HIV. Phosphonoformate has anti-HIV activity but due to its charged nature is excluded from the CNS by the blood-brain barrier. Lipophilic triesters of phosphonoformate designed to improve transport properties are unsuitable prodrugs due to their rapid and complicated hydrolysis, involving competitive P-O and P-C bond cleavage. Diesters, though hydrolytically stable, are considered too polar to passively diffuse into the CNS. Hydrophilic drugs mimicking endogenous nutrients are known to be actively transported across the blood-brain barrier. In this thesis the possibility that diesters of phosphonoformate may be actively transported is investigated. Triesters of phosphonoformate with labile aryl carboxyl esterrs were synthesised and their hydrolysis followed by 31P NMR spectroscopy. The triesters were found to undergo rapid hydrolysis via P-C bond cleavage to the phosphite. Phosphonoformate diesters designed to be analogues of actively transported -keto acids have been synthesised and fully characterised. Tyrosine-phosphonoformate and lipid-phosphonoformate conjugates have also been synthesised and characterised. An in vitro model of the blood-brain barrier utilising confluent monolayers of porcine brain microvessel endothelial cells grown on a permeable support has been established. The presence of enzyme and antigen markers specific to the blood-brain barrier has been demonstrated for the endothelial cells and the diffusional properties of the model investigated with hydrophilic and lipophilic compounds. Active transport systems for -keto acids and large amino acids have been identified in the endothelial cell monolayers using 14C-pyruvate and 3H-L-tyrosine respectively. Temperature and concentration dependence of the two systems have been demonstrated and transport constants calculated. Competition with 14C-pyruvate transport was shown with other monocarboxylic acids including the anti-epileptic drug valproate. Stereospecificity was shown in that L-lactate inhibited pyruvate transport while D-lactate did not. Sodium methyl methoxycarbonylphosphonate, a phosphonoformate diester was shown not to compete for 14C-pyruvate transport indicating that this compound has no affinity for the carrier. Competition with 3H-L-tyrosine transport was shown with other large amino acids, including the anti-Parkinsonian agent L-dopa. Stereospecificity was shown using L- and D-tyrosine and L- and D-dopa. The tyrosine-phosphonoformate conjugate, which was stable under the experimental conditions, was shown to compete with 3H-Ltyrosine transport indicating that it may be actively transported at the blood-brain barrier. Thirty two triesters, diesters and monoesters of phosphonoformate, showed no activity in an anti-HIV screen above that attributable to hydrolysis to the parent compound.

Development and validation of an HPLC method for the rapid and simultaneous determination of 6-mercaptopurine and four of its metabolites in plasma and red blood cells

An HPLC method has been developed and validated for the rapid determination of mercaptopurine and four of its metabolites; thioguanine, thiouric acid, thioxanthine and methylmercaptopurine in plasma and red blood cells. The method involves a simple treatment procedure based on deproteinisation by perchloric acid followed by acid hydrolysis and heating for 45min at 100 degrees C. The developed method was linear over the concentration range studied with a correlation coefficient >0.994 for all compounds in both plasma and erythrocytes. The lower limits of quantification were 13, 14, 3, 2, 95pmol/8 x 10(8) RBCs and 2, 5, 2, 3, 20ng/ml plasma for thioguanine, thiouric acid, mercaptopurine, thioxanthine and methylmercaptopurine, respectively. The method described is selective and sensitive enough to analyse the different metabolites in a single run under isocratic conditions. Furthermore, it has been shown to be applicable for monitoring these metabolites in paediatric patients due to the low volume requirement (200microl of plasma or erythrocytes) and has been successfully applied for investigating population pharmacokinetics, pharmacogenetics and non-adherence to therapy in these patients.

A simple bioanalytical method for the quantification of antiepileptic drugs in dried blood spots

An increasing number of publications on the dried blood spot (DBS) sampling approach for the quantification of drugs and metabolites have been spurred on by the inherent advantages of this sampling technique. In the present research, a selective and sensitive high-performance liquid chromatography method for the concurrent determination of multiple antiepileptic drugs (AEDs) [levetiracetam (LVT), lamotrigine (LTG), phenobarbital (PHB)], carbamazepine (CBZ) and its active metabolite carbamazepine-10,11 epoxide (CBZE)] in a single DBS has been developed and validated. Whole blood was spotted onto Guthrie cards and dried. Using a standard punch (6 mm diameter), a circular disc was punched from the card and extracted with methanol: acetonitrile (3:1, v/v) containing hexobarbital (Internal Standard) and sonicated prior to evaporation. The extract was then dissolved in water and vortex mixed before undergoing solid phase extraction using HLB cartridges. Chromatographic separation of the AEDs was achieved using Waters XBridge™ C18 column with a gradient system. The developed method was linear over the concentration ranges studied with r ≥ 0.995 for all compounds. The lower limits of quantification (LLOQs) were 2, 1, 2, 0.5 and 1 μg/mL for LVT, LTG, PHB, CBZE and CBZ, respectively. Accuracy (%RE) and precision (%CV) values for within and between day were <20% at the LLOQs and <15% at all other concentrations tested. This method was successfully applied to the analysis of the AEDs in DBS samples taken from children with epilepsy for the assessment of their adherence to prescribed treatments.

Oxidised LDL lipids, statins and a blood-brain barrier

Elevated cholesterol in mid-life has been associated with increased risk of dementia in later life. We have previously shown that low density lipoprotein (LDL) is more oxidised in the plasma of dementia patients although total cholesterol levels remained unchanged. Increased systemic oxidative modification (oxLDL) and nitration is also observed during hypercholesterolemia. We have investigated the hypothesis that disruption of blood brain barrier (BBB) function by oxLDL and their lipids may increase risk of neurodegeneration in later life and that statin intervention can mitigate the effects of hyperlipidaemia in mid-life. LDL isolated from statin-naïve hypercholesterolaemic subjects had higher mobility by agarose gel electrophoresis (Rf;0.53±0.06) and 8-isoprostane F2α concentration (43.5±8.42pg/ml) compared to control subjects (Rf; 0.46±0.05 and 24.2±5.37pg/ml respectively; p<0.05). Compared to HMVEC treatment with the LDL-lipids (5μM) from normolipidaemic subjects, LDL-lipids from hypercholesterolaemic subjects increased barrier permeability (103.4±12.5 Ωcm2 v 66.7±7.3 Ωcm2,P<0.01) and decreased cellular glutathione levels (18.5nmol/mg v 12.3nmol/mg) compared to untreated cells (26.2±3.6nmol/mg). LDL-lipids isolated from normolipidaemic subjects shows reduced risk to damage a BBB model compared with LDL-lipids from hypercholesterolaemic subjects. Moreover, a three month statin-intervention reduced the propensity for LDL-lipids from subjects with hyperlipidaemia to damage HMVEC. Post-statin treatment the cytotoxic and pro-inflammatory effects of LDL lipids disappeared. These data support the hypothesis that in vivo intervention with statins modifies LDL lipid oxidation, exerting a protective effect against in microvascular damage independent of cholesterol concentration.

An investigation of the relationship between antemortem and postmortem drug concentrations in blood

In the field of postmortem toxicology, principles from pharmacology and toxicology are combined in order to determine if exogenous substances contributed to ones death. In order to make this determination postmortem and (whenever available) antemortem blood samples may be analyzed. This project focused on evaluating the relationship between postmortem and antemortem blood drug levels, in order to better define an interpretive framework for postmortem toxicology. To do this, it was imperative to evaluate the differences in antemortem and postmortem drug concentrations, determine the role microbial activity and evaluate drug stability. Microbial studies determined that the bacteria Escherichia coli and Pseudomonas aeruginosa could use the carbon structures of drugs as a source of food. This would suggest prior to sample collection, microbial activity could potentially affect drug levels. This process however would stop before toxicologic evaluation, as at autopsy blood samples are stored in tubes containing the antimicrobial agent sodium fluoride. Analysis of preserved blood determined that under the current storage conditions sodium fluoride effectively inhibited microbial growth. Nonetheless, in many instances inconsistent drug concentrations were identified. When comparing antemortem to postmortem results, diphenhydramine, morphine, codeine and methadone, all showed significantly increased postmortem drug levels. In many instances, increased postmortem concentrations correlated with extended postmortem intervals. Other drugs, such as alprazolam, were likely to have concentration discrepancies when short antemortem to death intervals were coupled with extended postmortem intervals. While still others, such as midazolam followed the expected pattern of metabolism and elimination, which often resulted in decreased postmortem concentrations. The importance of drug stability was displayed when reviewing the clonazepam/ 7-aminoclonazepam data, as the parent drug commonly converted to its metabolite even when stored in the presence of a preservative. In instances of decreasing postmortem drug concentrations the effect of refrigerated storage could not be ruled out. A stability experiment, which contained codeine, produced data that indicated concentrations could continue to decline under the current storage conditions. The cumulative data gathered for this experiment was used to identify concentration trends, which subsequently aided in the development of interpretive considerations for the specific analytes examined in the study.

Multiparameter flow cytometric analysis of C -reactive protein binding to human-derived cell lines and peripheral blood monocytes

C-reactive protein (CRP), a normally occurring human plasma protein may become elevated as much as 1,000 fold during disease states involving acute inflammation or tissue damage. Through its binding to phosphorylcholine in the presence of calcium, CRP has been shown to potentiate the activation of complement, stimulate phagocytosis and opsonize certain microorganisms. Utilizing a flow cytometric functional ligand binding assay I have demonstrated that a monocyte population in human peripheral blood and specific human-derived myelomonocytic cell lines reproducibly bind an evolutionarily conserved conformational pentraxin epitope on human CRP through a mechanism that does not involve its ligand, phosphorylcholine. ^ A variety of cell lines at different stages of differentiation were examined. The monocytic cell line, THP-1, bound the most CRP followed by U937 and KG-1a cells. The HL-60 cell line was induced towards either the granulocyte or monocyte pathway with DMSO or PMA, respectively. Untreated HL-60 cells or DMSO-treated cells did not bind CRP while cells treated with PMA showed increased binding of CRP, similar to U-937 cells. T cell and B-cell derived lines were negative. ^ Inhibition studies with Limulin and human SAP demonstrated that the binding site is a conserved pentraxin epitope. The calcium requirement necessary for binding to occur indicated that the cells recognize a conformational form of CRP. Phosphorylcholine did not inhibit the reaction therefore the possibility that CRP had bound to damaged membranes with exposed PC sites was discounted. ^ A study of 81 normal donors using flow cytometry demonstrated that a majority of peripheral blood monocytes (67.9 ± 1.3, mean ± sem) bound CRP. The percentage of binding was normally distributed and not affected by gender, age or ethnicity. Whole blood obtained from donors representing a variety of disease states showed a significant reduction in the level of CRP bound by monocytes in those donors classified with infection, inflammation or cancer. This reduction in monocyte populations binding CRP did not correlate with the concentration of plasma CRP. ^ The ability of monocytes to specifically bind CRP combined with the binding reactivity of the protein itself to a variety of phosphorylcholine containing substances may represent an important bridge between innate and adaptive immunity. ^

The dual effect of normobaric hypoxia on heart rate variability and substrate partitioning following interval cycling

Recent studies have shown the importance of the beat-by-beat changes in heart rate influenced by the autonomic nervous system (ANS), or heart rate variability (HRV). The purpose of this study was to examine the lasting effects of hypoxic exercise on HRV, and its influences on substrate usage. Results from this study could lead an increased understanding on this topic. Eight active healthy males (age: 31±11 years; height: 180±7 cm; weight: 83±8 kg; VO₂max (maximal oxygen consumption): 4.4±0.6 L•min⁻¹) underwent normoxic and hypoxic (FᵢO₂= 0.15) conditions during high-intensity interval (HIIT) cycling (70%-high interval, 35%-rest interval). Cycling intensity was determined by a peak power output cycling test. Each experimental session consisted of a basal metabolic rate determination, up to 45-minutes of HIIT cycling, and three 30-minute post-exercise metabolic rate measurements (spanning 3 hours and 15 minutes after exercise). During exercise, RPE was higher (p<0.01) and LAC (lactate) increased (p=0.001) at each point of time in hypoxia, with no change in normoxia. After hypoxic exercise, the SNS/PNS ratio (overall ANS activity) was significantly higher (p<0.01) and significantly decreased through time in both conditions (p<0.01). In addition, a significant interaction between time and conditions (p<0.02) showed a decrease in LAC concentration through time post-hypoxic exercise. The findings showed that a single bout of hypoxic exercise alters ANS activity post-exercise along with shifting substrate partitioning from glycolytic to lipolytic energy production. The significant decrease in LAC concentration post-hypoxic exercise supports the notion that hypoxic HIIT induces a greater muscle glycogen depletion leading to increased fat oxidation to sustain glycogenesis and gluconeogenesis to maintain blood glucose level during recovery.

Efeito do exercício físico na estabilidade de membrana de eritrócitos

CHAPTER II - This study evaluated the effects of two different types of acute aerobic exercise on the osmotic stability of human erythrocyte membrane and on different hematological and biochemical variables that are associated with this membrane property. The study population consisted of 20 healthy and active men. Participants performed single sessions of two types of exercise. The first session consisted of 60 min of moderate-intensity continuous exercise (MICE). The second session, executed a week later, consisted of high-intensity interval exercise (HIIE) until exhaustion. The osmotic stability of the erythrocyte membrane was represented by the inverse of the salt concentration (1/H50) at the midpoint of the sigmoidal curve of dependence between the absorbance of hemoglobin and the NaCl concentration. The values of 1/H50 changed from 2.29 ± 0.1 to 2.33 ± 0.09 after MICE and from 2.30 ± 0.08 to 2.23 ± 0.12 after HIIE. In MICE has occurred an increase in the mean corpuscular volume, probably due to in vivo lysis of older erythrocytes, with preservation of cells that were larger and more resistant to in vitro lysis. The study showed that a single bout of acute exercise affected the erythrocyte osmotic stability, which increased after MICE and decreased after HIIE.

Body measurements and blood plasma lipid, PCB and OH-PCB content of polar bear (U. maritimus) mothers and cubs, Svalbard

The aim of this study was to examine the plasma concentrations and prevalence of polychlorinated biphenyls (PCBs) and hydroxylated PCB-metabolites (OH-PCBs) in polar bear (Ursus maritimus) mothers (n = 26) and their 4 months old cubs-of-the-year (n = 38) from Svalbard to gain insight into the mother-cub transfer, biotransformation and to evaluate the health risk associated with the exposure to these contaminants. As samplings were performed in 1997/1998 and 2008, we further investigated the differences in levels and pattern of PCBs between the two sampling years. The plasma concentrations of Sum(21)PCBs (1997/1998: 5710 ± 3090 ng/g lipid weight [lw], 2008: 2560±1500 ng/g lw) and Sum(6)OH-PCBs (1997/1998: 228 ± 60 ng/g wet weight [ww], 2008: 80 ± 38 ng/g ww) in mothers were significantly lower in 2008 compared to in 1997/1998. In cubs, the plasma concentrations of Sum(21)PCBs (1997/1998: 14680 ± 5350 ng/g lw, 2008: 6070 ± 2590 ng/g lw) and Sum(6)OH-PCBs (1997/1998: 98 ± 23 ng/g ww, 2008: 49 ± 21 ng/g ww) were also significantly lower in 2008 than in 1997/1998. Sum(21)PCBs in cubs was 2.7 ± 0.7 times higher than in their mothers. This is due to a significant maternal transfer of these contaminants. In contrast, Sum(6)OH-PCBs in cubs were approximately 0.53 ± 0.16 times the concentration in their mothers. This indicates a lower maternal transfer of OH-PCBs compared to PCBs. The majority of the metabolite/precursor-ratios were lower in cubs compared to mothers. This may indicate that cubs have a lower endogenous capacity to biotransform PCBs to OH-PCBs than polar bear mothers. Exposure to PCBs and OH-PCBs is a potential health risk for polar bears, and the levels of PCBs and OH-PCBs in cubs from 2008 were still above levels associated with health effects in humans and wildlife.

Size, blood organochlorine pesticide concentrations and biochemical indicators measured in hawksbill sea turtles (Eretmochelys imbricata) nesting at Punta Xen (Campeche, Mexico) in August 2010

The impact of environmental pollution on the homeostasis of sea turtles remains scarce, particularly in the southern Gulf of Mexico. As many municipalities do not rely on a waste treatment plant along the coastline of the Yucatan Peninsula, the vulnerability of these specimens could results enhanced. We searched for relationships between presence of organochlorine pesticides (OCP) and the level of several oxidative and pollutant stress indicators of the hawksbill sea turtle (Eretmochelys imbricata) during the egg-laying period 2010 at Punta Xen (Campeche, Mexico). Endosulfans, aldrin related (aldrin, endrin, dieldrin, endrin ketone, endrin aldehyde) and dichlorodiphenyldichloroethylene (DDT) families were detected in 17, 21 and 26 of the 30 sampled sea turtles, respectively. Significant correlation existed between the size of sea turtles with the concentration of methoxychlor, cholinesterase activity in plasma and heptachlors family, and catalase activity and hexachlorohexane family. Cholinesterase activity in washed erythrocytes and lipid peroxidation were positively correlated with glutathione reductase activity. Antioxidant enzyme actions seem adequate as no lipids damages were correlated with any OCPs. Future studies are necessary to evaluate the effect of OCPs on males of the area because of the significant detection of methoxychlor that target endocrine functioning and increase its concentration with size of the sea turtles.

Haematological and biochemical blood study in baby alpaca with diarrhoea

The aim of this study was to determine the haematological value and biochemical blood in baby alpacas with enteric disorder. A total of 30 blood and serum samples were collected from alpacas of 1 month old with diarrhoea and 5 blood samples of clinically healthy baby alpacas (controls). The animals were from communities in the central Andes from Peru. About haematology were determined haematocrit, haemoglobin concentration, red blood count and white blood count that were not significantly different between control animals and animals with diarrhoea. Moreover, biochemical blood parameters as total protein, albumin and calcium decrease significantly (p<0.05). We conclude that our results could be considered as factors in the mortality of baby alpaca by infectious diarrhoea.

Effect of cinnamon tea on postprandial glucose concentration

"Glycaemic control, in particular at postprandial period, has a key role in prevention of different diseases, including diabetes and cardiovascular events. Previous studies suggest that postprandial high blood glucose levels (BGL) can lead to an oxidative stress status, which is associated with metabolic alterations. Cinnamon powder has demonstrated a beneficial effect on postprandial glucose homeostasis in animals and human models. The purpose of this study is to investigate the effect of cinnamon tea (C. burmannii) on postprandial capillary blood glucose level on nondiabetic adults. Participants were given oral glucose tolerance test either with or without cinnamon tea in a randomized clinical trial. The data revealed that cinnamon tea administration slightly decreased postprandial BGL. Cinnamon tea ingestion also results in a significantly lower postprandial maximum glucose concentration and variation ofmaximum glucose concentration (p < 0.05). Chemical analysis showed that cinnamon tea has a high antioxidant capacity, whichmay be due to its polyphenol content. The present study provides evidence that cinnamon tea, obtained from C. burmannii, could be beneficial for controlling glucose metabolism in nondiabetic adults during postprandial period."

Plasma D-lactate Levels in Necrotizing Enterocolitis in Premature Infants

Background: D-Lactate is normally present in the blood of humans at nanomolar concentrations due to methylglyoxal metabolism; millimolar D-lactate concentrations can arise due to excess gastrointestinal microbial production. Objectives: To examine the levels of plasma D-lactate in the necrotizing enterocolitis in premature infants. Patients and Methods: 128 premature infants were divided into control (group I, n = 69), feeding intolerance (group II, n = 42) and NEC (group III, n = 27) groups. Plasma D-lactate levels were measured at the onset of feeding intolerance or NEC and at weeks 2-3 in control infants (group I) by ELISA. Data were analyzed using descriptive statistics, non-parametric tests and Student’s t-test. Results: In groups I, II, III, median birth weights were 1845.7 ± 267.5 g, 1913.1 ± 306.5 g, and 1898.4 ± 285.3 g, median gestational ages were 34.3 ± 1.7 weeks, 33.9 ± 2.2 weeks and 35.1 ± 2.6 weeks, ages of sampling were 12.3 ± 2.9 days, 14.6 ± 3.7 days and 15.1 ± 1.8 days, respectively. The differences of median birth weights, median gestational ages and ages of sampling were not statistically significant (P > 0.05). The plasma D-lactate levels in groups I, II, III were 3.6 ± 1.9 μg/mL, 12.7 ± 8.3 μg/mL, and 35.4 ± 29.1 μg/mL, respectively, group III had higher plasma D-lactate level than groups I, II, and the difference among these groups was significant (x2 = 21.6, P < 0.01). Conclusions: Plasma D-lactate significantly increased early in NEC. Plasma D-lactate levels were associated with extensive disease in NEC infants. Therefore, it could be used as a diagnosis indicator in the early stage of NEC.