The stabilisation of purified, reconstituted P-glycoprotein by freeze drying with disaccharides

The drug efflux pump P-glycoprotein (P-gp) (ABCB1) confers multidrug resistance, a major cause of failure in the chemotherapy of tumours, exacerbated by a shortage of potent and selective inhibitors. A high throughput assay using purified P-gp to screen and characterise potential inhibitors would greatly accelerate their development. However, long-term stability of purified reconstituted ABCB1 can only be reliably achieved with storage at -80 °C. For example, at 20 °C, the activity of ABCB1 was abrogated with a half-life of <1 day. The aim of this investigation was to stabilise purified, reconstituted ABCB1 to enable storage at higher temperatures and thereby enable design of a high throughput assay system. The ABCB1 purification procedure was optimised to allow successful freeze drying by substitution of glycerol with the disaccharides trehalose or maltose. Addition of disaccharides resulted in ATPase activity being retained immediately following lyophilisation with no significant difference between the two disaccharides. However, during storage trehalose preserved ATPase activity for several months regardless of the temperature (e.g. 60% retention at 150 days), whereas ATPase activity in maltose purified P-gp was affected by both storage time and temperature. The data provide an effective mechanism for the production of resilient purified, reconstituted ABCB1.

Pharmacokinetics of melatonin in preterm infants

Aims - Preterm infants are deprived of the normal intra-uterine exposure to maternal melatonin and may benefit from replacement therapy. We conducted a pharmacokinetic study to guide potential therapeutic trials. Methods - Melatonin was administered to 18 preterm infants in doses ranging from 0.04–0.6 μg kg−1 over 0.5–6 h. Pharmacokinetic profiles were analyzed individually and by population methods. Results - Baseline melatonin was largely undetectable. Infants receiving melatonin at 0.1 μg kg−1 h−1 for 2 h showed a median half-life of 15.82 h and median maximum plasma concentration of 203.3 pg ml−1. On population pharmacokinetics, clearance was 0.045 l h−1, volume of distribution 1.098 l and elimination half-life 16.91 h with gender (P = 0.047) and race (P < 0.0001) as significant covariates. Conclusions - A 2 h infusion of 0.1 μg kg−1 h−1 increased blood melatonin from undetectable to approximately peak adult concentrations. Slow clearance makes replacement of a typical maternal circadian rhythm problematic. The pharmacokinetic profile of melatonin in preterm infants differs from that of adults so dosage of melatonin for preterm infants cannot be extrapolated from adult studies. Data from this study can be used to guide therapeutic clinical trials of melatonin in preterm infants.

Population pharmacokinetic model of canrenone after intravenous administration of potassium canrenoate to paediatric patients

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • Little is known about the pharmacokinetics of potassium canrenoate/canrenone in paediatric patients WHAT THIS STUDY ADDS • A population pharmacokinetic model has been developed to evaluate the pharmacokinetics of canrenone in paediatric patients who received potassium canrenoate as part of their therapy in the intensive care unit. AIMS To characterize the population pharmacokinetics of canrenone following administration of potassium canrenoate to paediatric patients. METHODS Data were collected prospectively from 23 paediatric patients (2 days to 10 years of age; median weight 4 kg, range 2.16–28.0 kg) who received intravenous potassium canrenoate (K-canrenoate) as part of their intensive care therapy for removal of retained fluids, e.g. in pulmonary oedema due to chronic lung disease and for the management of congestive heart failure. Plasma samples were analyzed by HPLC for determination of canrenone (the major metabolite and pharmacologically active moiety) and the data subjected to pharmacokinetic analysis using NONMEM. RESULTS A one compartment model best described the data. The only significant covariate was weight (WT). The final population models for canrenone clearance (CL/F) and volume of distribution (V/F) were CL/F (l h−1) = 11.4 × (WT/70.0)0.75 and V/F (l) = 374.2 × (WT/70) where WT is in kg. The values of CL/F and V/F in a 4 kg child would be 1.33 l h−1 and 21.4 l, respectively, resulting in an elimination half-life of 11.2 h. CONCLUSIONS The range of estimated CL/F in the study population was 0.67–7.38 l h−1. The data suggest that adjustment of K-canrenoate dosage according to body weight is appropriate in paediatric patients.

Development of an enantiomer selective microsampling assay for the quantification of ketorolac suitable for paediatric pharmacokinetic studies

Background: Ketorolac, a potent nonsteroidal anti-inflammatory drug used for pain control in children, exists as a racemate of inactive R (+) and active S (-) enantiomers. Aim: To develop a microsampling assay for the enantioselective analysis of ketorolac in children. Methods: Ketorolac enantiomers were extracted from 50 µl of plasma by liquid–liquid extraction and separated on a ChiralPak AD-RH. Detection was by a TSQ quantum triple quadrupole mass spectrometer with an electrospray ionisation source operating in a positive ion mode. Five children (age 13.8 (1.6) years, weight 52.7 (7.2) kg), were administered intravenous ketorolac 0.5 mg/kg (maximum 10 mg) and blood samples were taken at 0, 0.25, 0.5, 1, 2, 4, 6, 8 and 12 h post administration. CL, VD and t1/2 were calculated based on non-compartmental methods. Results: The standard curves for R (+) and S (-) ketorolac were linear in the range 0–2000 ng/ml. The LLOQs of the method were 0.15 ng on column and 0.31 ng on column for R (+) and S (-) ketorolac, respectively. The median (range) VD and CL of R (+) and S (-) ketorolac were 0.12 l/kg (0.07–0.17), 0.017 l/h/kg (0.12–0.29) and 0.17 (0.09–0.31) l/kg, 0.049 (0.02–0.1) l/h/kg, p = 0.043), respectively. The median (range) elimination half-life (t1/2) of the R (+) and S (-) ketorolac was 5.0 h (2.5–5.8) and 3.1 h (1.8–4.4), p = 0.043), respectively. Conclusion: The development of a simple, rapid and reliable ketorolac assay suitable for paediatric PK studies is reported. Copyright © 2013 John Wiley & Sons, Ltd.

Potassium canrenoate treatment in paediatric patients:a population pharmacokinetic study using novel dried blood spot sampling

Objective: To characterize the population pharmacokinetics of canrenone following administration of potassium canrenoate (K-canrenoate) in paediatric patients. Methods: Data were collected prospectively from 37 paediatric patients (median weight 2.9 kg, age range 2 days–0.85 years) who received intravenous K-canrenoate for management of retained fluids, for example in heart failure and chronic lung disease. Dried blood spot (DBS) samples (n = 213) from these were analysed for canrenone content and the data subjected to pharmacokinetic analysis using nonlinear mixed-effects modelling. Another group of patients (n = 16) who had 71 matching plasma and DBS samples was analysed separately to compare canrenone pharmacokinetic parameters obtained using the two different matrices. Results: A one-compartment model best described the DBS data. Significant covariates were weight, postmenstrual age (PMA) and gestational age. The final population models for canrenone clearance (CL/F) and volume of distribution (V/F) in DBS were CL/F (l/h) = 12.86 ×  (WT/70.0)0.75 × e [0.066 ×  (PMA - 40]) and V/F (l) = 603.30 ×  (WT/70) × (GA/40)1.89 where weight is in kilograms. The corresponding values of CL/F and V/F in a patient with a median weight of 2.9 kg are 1.11 l/h and 20.48 l, respectively. Estimated half-life of canrenone based on DBS concentrations was similar to that based on matched plasma concentrations (19.99 and 19.37 h, respectively, in 70 kg patient). Conclusion: The range of estimated CL/F in DBS for the study population was 0.12–9.62 l/h; hence, bodyweight-based dosage adjustment of K-canrenoate appears necessary. However, a dosing scheme that takes into consideration both weight and age (PMA/gestational age) of paediatric patients seems more appropriate.

N-terminal His7-modification of glucagon-like peptide-1(7-36) amide generates dipeptidyl peptidase IV-stable analogues with potent antihyperglycaemic activity

Glucagon-like peptide-1(7-36)amide (GLP-1) possesses several unique and beneficial effects for the potential treatment of type 2 diabetes. However, the rapid inactivation of GLP-1 by dipeptidyl peptidase IV (DPP IV) results in a short half-life in vivo (less than 2 min) hindering therapeutic development. In the present study, a novel His7-modified analogue of GLP-1, N-pyroglutamyl-GLP-1 as well as N-acetyl-GLP-1 were synthesised and tested for DPP IV stability and biological activity. Incubation of GLP-1 with either DPP IV or human plasma resulted in rapid degradation of native GLP-1 to GLP-1(9-36)amide, while N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 were completely resistant to degradation. N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 bound to the GLP-1 receptor but had reduced affinities (IC50 values 32.9 and 6.7 nM, respectively) compared with native GLP-1 (IC50-37 nM). Similarly, both analogues stimulated cAMP production with EC50 values of 16.3 and 27 nM respectively compared with GLP-1 (EC50 4.7 nM). However, N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 exhibited potent insulinotropic activity in vitro at 5.6 mM glucose (P< 0.05 to P< 0.001) similar to native GLP-1. Both analogues (25 nM/kg body weight) lowered plasma glucose and increased plasma insulin levels when administered in conjunction with glucose (18 nM/kg body weight) to adult obese diabetic (ob/ob) mice. N-pyroglutamyl-GLP-1 was substantially better at lowering plasma glucose compared with the native peptide, while N-acetyl-GLP-1 was significantly more potent at stimulating insulin secretion. These studies indicate that N-terminal modification of GLP-1 results in DPP IV-resistant and biologically potent forms of GLP-1. The particularly powerful antihyperglycaemic action of N-pyroglutamyl-GLP-1 shows potential for the treatment of type 2 diabetes. © 2004 Society for Endocrinology.

Pharmacotherapies to manage diabesity:an update

Several pharmacotherapies have recently become available for addition to lifestyle measures to assist the management of coexistent type 2 diabetes and obesity. These are mostly administered as add-on to metformin or as alternative therapies if metformin is not appropriate. The sodium–glucose cotransporter 2 inhibitors (dapagliflozin, canagliflozin and empagliflozin) act by eliminating excess glucose in the urine. These agents provide a non-insulin-dependent mechanism to reduce hyperglycaemia and facilitate weight loss without causing frank hypoglycaemia. Their efficacy requires the individual to have adequate renal function. The glucagon-like peptide-1 (GLP-1) receptor agonists (exenatide, liraglutide, lixisenatide, dulaglutide and albiglutide [the last at the pre-launch stage at the time of writing]) are injected subcutaneously. Different members of the class offer different time courses for their onset and duration of action. Each potentiates insulin secretion and reduces glucagon secretion in a glucose-dependent manner to address prandial glycaemic excursions while avoiding interprandial hypoglycaemia. A satiety effect of these agents assists weight reduction, but delayed gastric emptying can cause initial nausea. The dipeptidyl peptidase-4 inhibitor class now comprises sitagliptin, vildagliptin, saxagliptin, linagliptin and alogliptin. These agents offer similar glucose-lowering efficacy without weight gain or hypoglycaemia by boosting the half-life of endogenous incretins, particularly GLP-1. A fixed-ratio injected combination of insulin degludec with liraglutide (IDegLira) has recently been launched and further agents to address hyperglycaemia and assist weight loss are advancing in development.

Poly lactic-co-glycolic acid controlled delivery of Disulfiram to target liver cancer stem-like cells

Disulfiram (DS), an anti-alcoholism drug, shows very strong cytotoxicity in many cancer types. However its clinical application in cancer treatment is limited by the very short half-life in the bloodstream. In this study, we developed a poly lactic-co-glycolic acid (PLGA)-encapsulated DS protecting DS from the degradation in the bloodstream. The newly developed DS-PLGA was characterized. The DS-PLGA has very satisfactory encapsulation efficiency, drug-loading content and controlled release rate in vitro. PLGA encapsulation extended the half-life of DS from shorter than 2 minutes to 7 hours in serum. In combination with copper, DS-PLGA significantly inhibited the liver cancer stem cell population. CI-isobologram showed a remarkable synergistic cytotoxicity between DS-PLGA and 5-FU or Sorafenib. It also demonstrated very promising anticancer efficacy and antimetastatic effect in liver cancer mouse model. Both DS and PLGA are FDA approved products for clinical application. Our study may lead to repositioning of DS into liver cancer treatment.

Occurrence and environmental fate of Irgarol 1051, a new antifouling agent

The purpose of this study is to characterize the degradation products of Irgarol 1051(2-methylthio-4-tertbutylamino-6-cyclopropylamino- s-triazine), a compound recently developed for use as an antifouling agent on boat hulls. The photolytic fate of this compound in different natural waters will be used in the development of a monitoring program designed to survey the occurrence of this compound and its degradation products in South Florida marinas, the Miami River and surrounding coastal areas. ^ The transformation of Irgarol 1051 and degradation rate constants were characterized in a photo-reactor under simulated natural conditions. The degradation pathway in the UVB-UVA region (300nm to 350nm) closely resembled the transformations under natural conditions in the pond, showing that both direct photolysis and the presence of natural sensitizers play an important role in the abiotic transformation of this compound. Irgarol 1051 has an average environmental half-life of 10 days in surface waters. Average concentrations from samples around Biscayne Bay and the Miami River increased from 1–5 ng/L during 1999 and increased to between 28 and 38 ng/L in 2001, respectively. Irgarol concentrations showed a strong correlation with concentrations of its major transformation product, M1, from samples collected as part of the study ([M1]/[Irgarol] = 0.247, R2 = 0.9165, n = 125). ^

Mangrove tannins in aquatic ecosystems: Their fate and possible influence on dissolved organic carbon and nitrogen cycling

We describe the fate of mangrove leaf tannins in aquatic ecosystems and their possible influence on dissolved organic nitrogen (DON) cycling. Tannins were extracted and purified from senescent yellow leaves of the red mangrove (Rhizophora mangle) and used for a series of model experiments to investigate their physical and chemical reactivity in natural environments. Physical processes investigated included aggregation, adsorption to organic matter-rich sediments, and co-aggregation with DON in natural waters. Chemical reactions included structural change, which was determined by excitation–emission matrix fluorescence spectra, and the release of proteins from tannin–protein complexes under solar-simulated light exposure. A large portion of tannins can be physically eliminated from aquatic environments by precipitation in saline water and also by binding to sediments. A portion of DON in natural water can coprecipitate with tannins, indicating that mangrove swamps can influence DON cycling in estuarine environments. The chemical reactivity of tannins in natural waters was also very high, with a half-life of less than 1 d. Proteins were released gradually from tannin–protein complexes incubated under light conditions but not under dark conditions, indicating a potentially buffering role of tannin– protein complexes on DON recycling in mangrove estuaries. Although tannins are not detected at a significant level in natural waters, they play an important ecological role by preserving nitrogen and buffering its cycling in estuarine ecosystems through the prevention of rapid DON export/loss from mangrove fringe areas and/or from rapid microbial mineralization.

Early diagenesis of plant-derived dissolved organic matter along a wetland, mangrove, estuary ecotone

We studied the role of photochemical and microbial processes in contributing to the transformation of dissolved organic matter (DOM) derived from various plants that dominate the Florida Everglades. Plant-derived DOM leachate samples were exposed to photochemical and microbial degradation and the optical, chemical, and molecular weight characteristics measured over time. Optical parameters such as the synchronous fluorescence intensity between 270 and 290 nm (Fnpeak I), a strong indicator of protein and/or polyphenol content, decreased exponentially in all plant leachate samples, with microbial decay constants ranging from 21.0 d21 for seagrass to 20.11 d21 for mangrove (half-life [t1/2] 5 0.7–6.3 d). Similar decreases in polyphenol content and dissolved organic carbon (DOC) concentration also occurred but were generally an order of magnitude lower or did not change significantly over time. The initial molecular weight composition was reflected in the rate of Fnpeak I decay and suggests that plantderived DOM with a large proportion of high molecular weight structures, such as seagrass derived DOM, contain high concentrations of easily microbially degradable proteinaceous components. For samples exposed to extended simulated solar radiation, polyphenol and Fnpeak I photochemical decay constants were on average 20.7 d21 (t1/2 1.0 d). Our data suggest that polyphenol structures of plant-derived DOM are particularly sensitive to photolysis, whereas high molecular weight protein-like structures are degraded primarily through physical–chemical and microbial processes. Furthermore, microbial and physical processes initiated the formation of recalcitrant, highly colored high molecular weight polymeric structures in mangrove-derived DOM. Thus, partial, biogeochemical transformation of plant-derived DOM from coastal areas is rapid and is likely to influence carbon and nutrient cycling, especially in areas dominated by seagrass and mangrove forests.

Multifunctional nanoparticles in cancer: In vitro characterization, in vivo distribution, and cellular response after laser-NIR dye-induced heating

A novel biocompatible and biodegradable polymer, termed poly(Glycerol malate co-dodecanedioate) (PGMD), was prepared by thermal condensation method and used for fabrication of nanoparticles (NPs). PGMD NPs were prepared using the single oil emulsion technique and loaded with an imaging/hyperthermia agent (IR820) and a chemotherapeutic agent (doxorubicin, DOX). The size of the void PGMD NPs, IR820-PGMD NPs and DOX-IR820-PGMD NPs were approximately 90 nm, 110 nm, and 125 nm respectively. An acidic environment (pH=5.0) induced higher DOX and IR820 release compared to pH=7.4. DOX release was also enhanced by exposure to laser, which increased the temperature to 42°C. Cytotoxicity of DOX-IR820-PGMD NPs was comparable in MES-SA but was higher in Dx5 cells compared to free DOX plus IR820 (p<0.05). The combination of hyperthermia (HT) and chemotherapy improved cytotoxicity in both cell lines. We also explored the cellular response after rapid, short-term and low thermal dose (laser/Dye/NP) induced-heating, and compared it to slow, long-term and high thermal dose cell incubator heating by investigating the reactive oxygen species (ROS) level, hypoxia-inducible factor-1&agr; (HIF-1&agr;) and vascular endothelial growth factor (VEGF) expression. The cytotoxicity of IR820-PGMD NPs after laser/Dye/NP HT resulted in higher cancer cell killing compared to incubator HT. ROS level, HIF-1&agr; and VEGF expression were elevated under incubator HT, while maintained at the baseline level under the laser/Dye/NP HT. In vivo mouse studies showed that NP formulation significantly improved the plasma half-life of IR820 after tail vein injection. Significant lower IR820 content was observed in kidney in DOX-IR820-PGMD NP treatment as compared to free IR820 treatment in our biodistribution studies (p<0.05). In conclusion, both IR820-PGMD NPs and DOX-IR820-PGMD NPs were successfully developed and used for both imaging and therapeutic purposes. Rapid and short-term laser/Dye/NP HT, with a low thermal dose, did not up-regulate HIF-1&agr; and VEGF expression, whereas slow and long-term incubator HT, with a high thermal dose, can enhance expression of both HIF-1&agr; and VEGF.^

Microspheres for liver radiomicrospheres therapy and planning

Liver cancer accounts for nearly 10% of all cancers in the US. Intrahepatic Arterial Radiomicrosphere Therapy (RMT), also known as Selective Internal Radiation Treatment (SIRT), is one of the evolving treatment modalities. Successful patient clinical outcomes require suitable treatment planning followed by delivery of the microspheres for therapy. The production and in vitro evaluation of various polymers (PGCD, CHS and CHSg) microspheres for a RMT and RMT planning are described. Microparticles with a 30±10 µm size distribution were prepared by emulsion method. The in vitro half-life of the particles was determined in PBS buffer and porcine plasma and their potential application (treatment or treatment planning) established. Further, the fast degrading microspheres (≤ 48 hours in vitro half-life) were labeled with 68Ga and/or 99mTc as they are suitable for the imaging component of treatment planning, which is the primary emphasis of this dissertation. Labeling kinetics demonstrated that 68Ga-PGCD, 68Ga-CHSg and 68Ga-NOTA-CHSg can be labeled with more than 95% yield in 15 minutes; 99mTc-PGCD and 99mTc-CHSg can also be labeled with high yield within 15-30 minutes. In vitro stability after four hours was more than 90% in saline and PBS buffer for all of them. Experiments in reconstituted hemoglobin lysate were also performed. Two successful imaging (RMT planning) agents were found: 99mTc-CHSg and 68Ga-NOTA-CHSg. For the 99mTc-PGCD a successful perfusion image was obtained after 10 minutes, however the in vivo degradation was very fast (half-life), releasing the 99mTc from the lungs. Slow degrading CHS microparticles (> 21 days half-life) were modified with p-SCN-b-DOTA and labeled with 90 Y for production of 90Y-DOTA-CHS. Radiochemical purity was evaluated in vitro and in vivo showing more than 90% stability after 72 and 24 hours respectively. All agents were compared to their respective gold standards (99mTc-MAA for 68Ga-NOTA-CHSg and 99m Tc-CHSg; 90Y-SirTEX for 90Y-DOTA-CHS) showing superior in vivo stability. RMT and RMT planning agents (Therapy, PET and SPECT imaging) were designed and successfully evaluated in vitro and in vivo.

Consumo privado de ansiolíticos benzodiazepínicos e sua correlação com indicadores sociodemográficos nas capitais brasileiras

This work aims to analyze the distribution of anxiolytics and their frequency of consumption in the period of 2010 to 2012, on the Federal District and at other Brazilian cities, as well as evaluating the correlation between such consumption and its demographic, epidemiological, economic and social characteristics for each region of this study. Into the analysis, it was observed that social, economic and cultural factors seem to influence the over-consumption of these drugs in many countries. Based on this, the benzodiazepines (BDZs) have achieved great popularity among members of the medical community and among the general population, mainly because of its safety and effectiveness in controlling symptoms of anxiety, insomnia and convulsions. Concerning the methodology of this work, an ecological study was performed having as sampling Brazilian capitals and as data source the 2010 Population Census, as well as information from IBGE, DATASUS and ANVISA. Still in the case of the methodological procedure, a multiple linear regression was used. Through descriptive analysis, it was demonstrated that the Northern region has the lowest average on consumption of these drugs (being 0.24 DHD in Manaus); meanwhile in the capitals of the Southeast, higher means were identified (reaching 7.29 DHD in Belo Horizonte), with a national average of 3.04 DHD. Among the drugs analyzed, it was found that Alprazolam is the most dispensed by pharmacies and private drugstores, averaging 2.00 DHD for Brazilian capitals. A análise de regressão linear múltipla mostrou que 76% da variação no consumo foi explicada pela variação da densidade populacional (β = 0,310 p = 0,045) e pela percentagem de médicos (β = 0,507 p = 0,016). Therefore, it was concluded that the consumption of anxiolytics of short half-life has been increasing over the years, mainly in the cities of greater population density and with the highest concentration of doctors

Polonium-210 and Lead-210 activities measured on 17 water bottle profiles and 50 surface water samples during POLARSTERN cruise ARK-XXII/2

Polonium-210 and Lead-210 have been measured in the water column and on suspended particulate matter during the POLARSTERN cruise ARK-XXII/2. The data have been submitted to Pangaea following a Polonium-Lead intercalibration exercise organized by GEOTRACES, where the AWI lab results range within the data standard deviation from 10 participating labs. Polonium-210 and Lead-210 in the ocean can be used to identify the sources and sinks of suspended matter. In seawater, Polonium-210 (210Po) and Lead-210 (210Pb) are produced by stepwise radioactive decay of Uranium-238. 210Po (138 days half life) and 210Pb (22.3 years half life) have high affinities for suspended particles. Those radionuclides are present in dissolved form and adsorbed onto particles. Following adsorption onto particle surfaces, 210Po especially is transported into the interior of cells where it bonds to proteins. In this way, 210Po also accumulates in the food chain. 210Po is therefore considered to be a good tracer for POC, and traces particle export over a timescale of month. 210Pb (22.3 years half life) adsorbs preferably onto structural components of cells, biogenic silica and lithogenic particles, and is therefore a better tracer more rapidly sinking matter. Our goal during ARK XXII/2 was to trace pathways of particulate and dissolved matter leaving the Siberian Shelf. The pathways of particulate and dissolved matter will be followed by the combined use of 210Po and 234Th as a tracer pair (and perhaps 210Pb) for particle flux (Cai, P.; Rutgers van der Loeff, MM (2008) doi:10.1594/PANGAEA.708354). This information gathered from the water column will be complemented with the results of the 210Po-210Pb study in sea ice (Camara-Mor, P, Instituto de Ciencias del Mar-SCIC, Barcelona, Spain) to provide a more thorough picture of particle transport from the shelf to the open sea and from surface to depth.