The care center influences the management of lymphoma patients in a universal health care system: an observational cohort study.

BACKGROUND: Healthcare providers-related disparities in adherence to the treatment plan among lymphoma patients are found even in a universal healthcare system, but the mechanism remains unclear. We investigated the association between the type of care center and the relative dose intensity and determined whether it persists after adjustment for patients' recruitment differences. METHODS: Prospective observational cohort study of 294 patients treated with standard protocols for diffuse large B-cell lymphoma (DLBCL) in teaching or community public hospitals or in private centers in the French Midi-Pyrénées region from 2006-2013. To test our assumptions, we used multinomial and mixed-effect logistic models progressively adjusted for patients' biomedical characteristics, socio-spatial characteristics and treatment-related toxicity events. RESULTS: Patients treated using standard protocols in the teaching hospital had more advanced stage and poorer initial prognosis without limitation regarding the distance from the residence to the care center. Patients' recruitment profile across the different types of care center failed to explain the difference in relative dose intensity. Low relative dose intensity was less often observed in teaching hospital than elsewhere. CONCLUSION: We showed that even in a universal healthcare system, disparities in the management of DLBCL patients' do exist according to the types of care center. A main issue may be to find and diffuse the reasons of this benefit in cancer management in the teaching hospital to the other centers.

The influence of extreme river discharge conditions on the quality of suspended particulate matter in Rivers Meuse and Rhine (The Netherlands).

As a consequence of climate change, increased precipitation in winter and longer periods of decreased precipitation in summer are expected to cause more frequent episodes of very high or very low river discharge in the Netherlands. To study the impact of such extreme river discharge conditions on water quality, toxicity profiles and pollutant profiles were determined of suspended particulate matter (SPM) collected from Rivers Meuse and Rhine. Archived (1993-2003) and fresh (2009-2011) SPM samples were selected from the Dutch annual monitoring program of the national water bodies (MWTL), representing episodes with river discharge conditions ranging from very low to regular to very high. SPM extracts were tested in a battery of in vitro bioassays for their potency to interact with the androgen receptor (AR), the estrogen receptor (ER), the arylhydrocarbon receptor (AhR), and the thyroid hormone transporter protein transthyretin (TTR). SPM extracts were further tested for their mutagenic potency (Ames assay) and their potency to inhibit bacterial respiration (Vibrio fischeri bioluminescence assay). Target-analyzed pollutant concentrations of the SPM samples and additional sample information were retrieved from a public database of MWTL results. In vitro toxicity profiles and pollutant profiles were analyzed in relation to discharge conditions and in relation to each other using correlation analysis and multivariate statistics. Compared to regular discharge conditions, composition of SPM during very high River Meuse and Rhine discharges shifted to more coarse, sandy, organic carbon (OC) poor particles. On the contrary, very low discharge led to a shift to more fine, OC rich material, probably dominated by algae. This shift was most evident in River Meuse, which is characterized by almost stagnant water conditions during episodes of drought. During such episodes, SPM extracts from River Meuse demonstrated increased potencies to inhibit bacterial respiration and to compete with thyroid hormone to bind to TTR, possibly due to the presence of fycotoxins. Meanwhile concentrations of polychlorobiphenyls (PCBs) in SPM were also increased. Very high River Meuse discharges on the other hand corresponded to increased androgenic and AhR agoniztic responses, which coincided with increased PAH levels and PAH-related in vivo risk estimates (i.e. multi-substance potentially affected fraction of species; msPAF). In River Rhine, very high discharges also corresponded to increasing androgenic potencies in SPM. Concentrations and corresponding msPAF values of PAHs (and metals), however, decreased with very high discharges in River Rhine in contrast to River Meuse. Mutagenicity was observed for SPM extracts from River Rhine collected during all discharge conditions, except during regular discharge. Aggregated toxicity index values, which were useful to identify toxicity profiles deviating from the generally observed pattern, did not correlate with river discharges, probably due to opposite effects of discharge conditions on different bioassay responses. In conclusion, SPM quality and related in vivo risk estimates changed during very low or very high discharge conditions but the changes were specific for the different toxic endpoints and pollutants in the different rivers. Moreover, bioassay responses to a series of consecutively collected samples from River Rhine during the Christmas flood of 1993 indicated that SPM quality is variable within a single episode of extreme discharge.

Tracing glacial refugia of Triturus newts based on mitochondrial DNA phylogeography and species distribution modeling.

INTRODUCTION: The major climatic oscillations during the Quaternary Ice Age heavily influenced the distribution of species and left their mark on intraspecific genetic diversity. Past range shifts can be reconstructed with the aid of species distribution modeling and phylogeographical analyses. We test the responses of the different members of the genus Triturus (i.e. the marbled and crested newts) as the climate shifted from the previous glacial period (the Last Glacial Maximum, ~21 Ka) to the current interglacial. RESULTS: We present the results of a dense mitochondrial DNA phylogeography (visualizing genetic diversity within and divergence among populations) and species distribution modeling (using two different climate simulations) for the nine Triturus species on composite maps. CONCLUSIONS: The combined use of species distribution modeling and mitochondrial phylogeography provides insight in the glacial contraction and postglacial expansion of Triturus. The combined use of the two independent techniques yields a more complete understanding of the historical biogeography of Triturus than both approaches would on their own. Triturus newts generally conform to the 'southern richness and northern purity' paradigm, but we also find more intricate patterns, such as the absence of genetic variation and suitable area at the Last Glacial Maximum (T. dobrogicus), an 'extra-Mediterranean' refugium in the Carpathian Basin (T. cristatus), and areas where species displaced one another postglacially (e.g. T. macedonicus and western T. karelinii). We provide a biogeographical scenario for Triturus, showing the positions of glacial refugia, the regions that were postglacially colonized and the areas where species displaced one another as they shifted their ranges.

Liquid-based cervical cytology using ThinPrep technology: weighing the pros and cons in a cost-effectiveness analysis.

PURPOSE: Cervical cancer screening with liquid-based cytology (LBC) has been developed as an alternative to the conventional Papanicolaou (CP) smear. Cost-effectiveness is one of the issues when evaluating LBC. Based on the results of a Dutch randomised controlled trial, we conducted cost-effectiveness threshold analyses to investigate under what circumstances manually screened ThinPrep LBC is cost-effective for screening. METHODS: The MISCAN-Cervix microsimulation model and data from the Dutch NETHCON trial (including 89,784 women) were used to estimate the costs and (quality-adjusted) life years ((QA)LYs) gained for EU screening schedules, varying cost-effectiveness threshold values. Screening strategies were primary cytological screening with LBC or CP, and triage with human papillomavirus (HPV) testing. RESULTS: Threshold analyses showed that screening with LBC as a primary test can be cost-effective if LBC is less than 3.2 more costly per test than CP, if the sensitivity of LBC is at least 3-5 % points higher than CP, if the quality of life for women in triage follow-up is only 0.39, or if the rate of inadequate CP smears is at least 16.2 %. CONCLUSIONS: Regarding test characteristics and costs of LBC and CP, only under certain conditions will a change from CP to manually screened ThinPrep LBC be cost-effective. If none of these conditions are met, implementation of manually screened ThinPrep LBC seems warranted only if there are advantages other than cost-effectiveness. Further research is needed to establish whether other LBC systems will be more favorable with regard to cost-effectiveness.

Patients with cervical cancer: why did screening not prevent these cases?

OBJECTIVE: The objective of the study was to assess the screening history of women with cervical cancer and review normal cervical smears 5 years preceding the diagnosis. STUDY DESIGN: Cytological and histological results of 401 women treated for invasive cervical cancer between 1991 and 2008 at the Radboud University Nijmegen Medical Center were studied. Ninety-eight normal smears were reviewed. RESULTS: Of the 401 women, 269 (67%) received at least 1 invitation for the national screening program for cervical cancer (NCSP). One- third fell outside the target age of the NCSP. Seventeen percent never responded to the invitation(s). Twenty-one percent had 1 or more normal smears within 5 years preceding the diagnosis. After review, only 39% of those smears were reviewed as a normal smear. CONCLUSION: Half of the women with cervical cancer were never screened because of the limited target age range or nonattendance. Twenty-one percent had a normal smear within 5 years preceding the diagnosis, caused by interpretation and/or sampling errors.

Comparison of liquid-based cytology with conventional cytology for detection of cervical cancer precursors: a randomized controlled trial.

CONTEXT: Liquid-based cytology has been developed as an alternative for conventional cervical cytology. Despite numerous studies and systematic reviews, controversy remains about its diagnostic accuracy. OBJECTIVE: To assess the performance of liquid-based cytology compared with conventional cytology in terms of detection of histologically confirmed cervical intraepithelial neoplasia (CIN). DESIGN, SETTING, AND PARTICIPANTS: Cluster randomized controlled trial involving 89,784 women aged 30 to 60 years participating in the Dutch cervical screening program at 246 family practices. One hundred twenty-two practices were assigned to use liquid-based cytology and screened 49,222 patients and 124 practices were assigned to use the conventional Papanicolaou (Pap) test and screened 40,562 patients between April 2004 and July 1, 2006. Patients were followed up for 18 months through January 31, 2008. INTERVENTION: Screening for CIN using liquid-based cytology or conventional papanicolaou (Pap) test and the blinded review of all follow-up of screen-positive women (blinded to the type of cytology and the initial result). MAIN OUTCOME MEASURES: Intention-to-treat and per-protocol analysis of the detection rates of and positive predictive values for histologically verified CIN in both cytology systems. Outcomes are presented as crude and adjusted rate ratios (adjustment for age, urbanization, study site, and period). RESULTS: The adjusted detection rate ratios for CIN grade 1+ was 1.01 (95% confidence interval [CI], 0.85-1.19); for CIN grade 2+, 1.00 (95% CI, 0.84-1.20); for CIN grade 3+, 1.05 (95% CI, 0.86-1.29); and for carcinoma, 1.69 (95% CI, 0.96-2.99). The adjusted positive predictive value (PPV) ratios, considered at several cytological cutoffs and for various outcomes of CIN did not differ significantly from unity. CONCLUSION: This study indicates that liquid-based cytology does not perform better than conventional Pap tests in terms of relative sensitivity and PPV for detection of cervical cancer precursors. TRIAL REGISTRATION: trialregister.nl Identifier: NTR1032.

Liquid compared with conventional cervical cytology: a systematic review and meta-analysis.

OBJECTIVE: To compare test performance characteristics of conventional Pap tests and liquid-based cervical cytology samples. DATA SOURCES: Eligible studies, published between 1991 and 2007, were retrieved through PubMed/EmBase searching and completed by consultation of other sources. METHODS OF STUDY SELECTION: Studies were selected if a conventional and a liquid-based sample were prepared from the same woman or when one or the other type of sample was taken from a separate but similar cohort. The current systematic review and meta-analysis is restricted to studies where all subjects were submitted to gold standard verification, based on colposcopy and histology of colposcopy-targeted biopsies, allowing computation of absolute and relative test validity for cervical intraepithelial neoplasia grade 2 or worse. Randomized trials were selected as well if all test-positive cases were verified with the same gold standard, allowing computation of the relative sensitivity. Impact of study characteristics on accuracy was assessed by subgroup meta-analyses, meta-regression, and summary receiver operating characteristic curve regression. TABULATION, INTEGRATION, AND RESULTS: The relative sensitivity, pooled from eight studies, with complete gold standard verification and from one randomized clinical trial, did not differ significantly from unity. Also, the specificity, considering high-grade and low-grade squamous intraepithelial lesions as cutoff, was similar in conventional and liquid cytology. However, a lower pooled specificity was found for liquid-based cytology when presence of atypical squamous cells of undetermined significance was the cutoff (ratio 0.91, 95% confidence interval 0.84-0.98). Differences in study characteristics did not explain interstudy heterogeneity. CONCLUSION: Liquid-based cervical cytology is neither more sensitive nor more specific for detection of high-grade cervical intraepithelial neoplasia compared with the conventional Pap test.

Cytologic detection of cervical abnormalities using liquid-based compared with conventional cytology: a randomized controlled trial.

OBJECTIVE: To compare test positivity rates of liquid-based and conventional cytology. METHODS: This study was a cluster randomized controlled trial with family practice as the unit of randomization, performed within the Dutch national cervical screening program. Women aged 30-60 years (n=89,784) recruited from 246 family practices were included. One-hundred twenty-two practices (49,222 individuals) were randomly assigned to the experimental arm, and 124 practices (40,562 participants), to the conventional arm. Inclusion was performed during a 3-year period between April 2003 and July 2006. Cytologic test positivity rates of liquid-based compared with conventional cytology was compared in terms of crude and adjusted odds ratios, applying a per-protocol analysis. RESULTS: Crude ratios of the odds of test positivity rates of liquid-based compared with conventional cytology for atypical squamous cells of undetermined significance or more severe, low-grade squamous intraepithelial lesion or more severe, and high-grade squamous intraepithelial lesion or more severe were 0.95 (95% confidence interval [CI] 0.82-1.10), 1.00 (95% CI 0.83-1.20), and 0.97 (95% CI 0.77-1.22), respectively. Liquid-based cytology resulted in fewer unsatisfactory tests (odds ratio 0.30, 95% CI 0.23-0.38). The results did not change when the odds ratios were adjusted for age, study site, study period, and urbanization level. Of 128 women screened with liquid-based cytology, one unsatisfactory preparation is avoided. CONCLUSION: This study found no statistically significant difference in cytologic test positivity rates between liquid-based and conventional cytology. However, liquid-based cytology resulted in significantly fewer unsatisfactory tests. CLINICAL TRIAL REGISTRATION: Nederlands Trial Register, www.trialregister.nl, NTR1032 LEVEL OF EVIDENCE: I.

2B-Trans technology: targeted drug delivery across the blood-brain barrier.

Drug delivery across the blood-brain barrier (BBB) is a major obstacle for the development of effective treatments of many central nervous system disorders. Sophisticated cell culture models of the BBB have helped us to identify, characterize, and validate a novel targeted drug delivery technology, designated 2B-Trans, for the receptor-mediated uptake and transport of drugs across the BBB. This paper describes in great detail how such a BBB cell culture model should be prepared and handled, and applied for the use of targeted drug delivery across the BBB.

Identification of Griffon Vulture's Flight Types Using High-Resolution Tracking Data.

Being one of the most frequently killed raptors by collision with wind turbines, little is known about the Griffon vulture's flight strategies and behaviour in a fine scale. In this study, we used high-resolution tracking data to differentiate between the most frequently observed flight types of the Griffon, and evaluated the performance of our proposed approach by an independent observation during a period of 4 weeks of fieldwork. Five passive flight types including three types of soaring and two types of gliding were discriminated using the patterns of measured GPS locations. Of all flight patterns, gliding was classified precisely (precision = 88%), followed by linear and thermal soaring with precision of 83 and 75%, respectively. The overall accuracy of our classification was 70%. Our study contributes a baseline technique using high-resolution tracking data for the classification of flight types, and is one step forward towards the collision management of this species.

Strategies to improve drug delivery across the blood-brain barrier.

The blood-brain barrier (BBB), together with the blood-cerebrospinal-fluid barrier, protects and regulates the homeostasis of the brain. However, these barriers also limit the transport of small-molecule and, particularly, biopharmaceutical drugs such as proteins, genes and interference RNA to the brain, thereby limiting the treatment of many brain diseases. As a result, various drug delivery and targeting strategies are currently being developed to enhance the transport and distribution of drugs into the brain. In this review, we discuss briefly the biology and physiology of the BBB as the most important barrier for drug transport to the brain and, in more detail, the possibilities for delivering large-molecule drugs, particularly genes, by receptor-mediated nonviral drug delivery to the (human) brain. In addition, the systemic and intracellular pharmacokinetics of nonviral gene delivery, together with targeted brain imaging, are reviewed briefly.

Objective assessment of cancer biomarkers using semi-rare event detection.

Objective and reproducible assessment of cancer biomarkers may be performed using rare event detection systems. Because many biomarkers are not true 'rare events', in this study a semi-rare event detection system was developed. The system is capable of assigning a discriminant score to detected positive cells, expressing the extent and intensity of the immunocytochemical staining. A gallery image is constructed showing the diagnostically most interesting cells as well as quantitative data expressing the biomarker staining pattern. To increase scanning speed, an adaptive scanning strategy is studied in which scanning is aborted when a sufficient number of positive cells has been identified. System performance was evaluated using liquid based cervical smears, stained with an antibody directed against p16(INK4a) tumor suppressor protein. Overexpression of p16(INK4a) in cervix is related to high-risk HPV infection, which is associated with carcinogenesis. Reproducibility of the system was tested on specimens containing limited positivity. Quantitative analysis was evaluated using 10 cases within normal limits and 10 high grade lesions. The system was highly reproducible in detecting positive cells and in calculating discriminant scores (average CV 0.7%). Quantitative features were significantly increased in high grade lesions (p<0.001). Adaptive scanning decreased scanning time with only minor impact on scanning results. The system is capable of automated, objective and reproducible assessment of biomarker expression and may be useful for a variety of applications.

Pharmacokinetic/pharmacodynamic modelling of the anti-hyperalgesic and anti-nociceptive effect of adenosine A1 receptor partial agonists in neuropathic pain.

The objective of this investigation was to characterise the pharmacokinetic-pharmacodynamic correlation of adenosine A1 receptor partial agonists in the chronic constriction injury model of neuropathic pain. Following intravenous administration of 8-methylamino-N6-cyclopentyl-adenosine (MCPA; 10 mg/kg) and 2'deoxyribose-N6-cyclopentyl-adenosine (2'dCPA; 20 mg/kg), the time course of the effect on the mechanical paw pressure threshold was determined in conjunction with plasma concentrations. Population pharmacokinetic/pharmacodynamic analysis was applied to derive individual concentration-effect relationships. A composite model consisting of an E(max) model for the anti-hyperalgesic effect in combination with a linear model for the anti-nociceptive effect accurately described the concentration-effect relationship. For both compounds, a full anti-hyperalgesic effect was observed. The values of the EC50 for the anti-hyperalgesic effect were (mean+/-S.D.): 3170+/-1460 and 2660+/-1200 ng/ml for MCPA and 2'dCPA versus 178+/-51 ng/ml for the reference full agonist 5'deoxyribose-N6-cyclopentyl-adenosine (5'dCPA). The values of the slope for the anti-nociceptive effect were 1.9+/-0.30 and 1.2+/-0.20 g.microl/ng, respectively, versus 55+/-8 g microl/ng for 5'dCPA. Adenosine A1 receptor partial agonists behave as full agonists with regard to the anti-hyperalgesic effect in neuropathic pain, but the anti-nociceptive effect is diminished.

Targeted delivery across the blood-brain barrier.

The safest and most effective way of targeting drugs to the entire brain is via delivery systems directed at endogenous receptor-mediated uptake mechanisms present at the cerebral capillaries. Such systems have been shown to be effective in animal models including primates, but no clinical trials have been performed so far. This review focuses on the well-characterised transferrin and insulin receptor-targeted systems, as well as on the more recently described systems that use the low-density lipoprotein-related protein 1 receptor, the low-density lipoprotein-related protein 2 receptor (also known as megalin and glycoprotein 330) or the diphtheria toxin receptor (which is the membrane-bound precursor of heparin-binding epidermal growth factor-like growth factor). The possibilities and limitations of these systems are compared and their future for human application is discussed.

Targeting liposomes with protein drugs to the blood-brain barrier in vitro.

In this study, we aim to target pegylated liposomes loaded with horseradish peroxidase (HRP) and tagged with transferrin (Tf) to the BBB in vitro. Liposomes were prepared with the post-insertion technique: micelles of polyethylene glycol (PEG) and PEG-Tf were inserted into pre-formed liposomes containing HRP. Tf was measured indirectly by measuring iron via atomic absorption spectroscopy. All liposomes were around 100 nm in diameter, contained 5-13 microg HRP per mumol phospholipid and 63-74 Tf molecules per liposome (lipo Tf) or no Tf (lipo C). Brain capillary endothelial cells (BCEC) were incubated with liposomes at 4 degrees C (to determine binding) or at 37 degrees C (to determine association, i.e. binding+endocytosis) and the HRP activity, rather than the HRP amount was determined in cell lysates. Association of lipo Tf was two- to three-fold higher than association of lipo C. Surprisingly, the binding of lipo Tf at 4 degrees C was four-fold higher than the association of at 37 degrees C. Most likely this high binding and low endocytosis is explained by intracellular degradation of endocytosed HRP. In conclusion, we have shown targeting of liposomes loaded with protein or peptide drugs to the BCEC and more specifically to the lysosomes. This is an advantage for the treatment of lysosomal storage disease. However, drug targeting to other intracellular targets also results in intracellular degradation of the drug. Our experiments suggest that liposomes release some of their content within the BBB, making targeting of liposomes to the TfR on BCEC an attractive approach for brain drug delivery.

Brain penetration of synthetic adenosine A1 receptor agonists in situ: role of the rENT1 nucleoside transporter and binding to blood constituents.

The blood-brain barrier (BBB) transport of synthetic A(1) receptor agonists was studied in an in situ brain perfusion model in the presence and absence of the selective nucleoside transport inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBTI). For 8-methylamino-N(6)cyclopentyladenosine (MCPA), N(6)-cyclopentyladenosine (CPA), 2'deoxy-N(6)-cyclopentyladenosine (2'dCPA) and 5'deoxy-N(6)-cyclopentyl adenosine (5'dCPA) the brain uptake clearance was low with values of 0.0045+/-0.0012, 0.018+/-0.0020, 0.022+/-0.0028 and 0.12+/-0.054 ml min(-1)g(-1), respectively. In the presence of an average NBTI plasma concentration of 2.6+/-0.3 microg ml(-1) (NBTI dose: 3 mg kg(-1) i.v.) the values of the brain uptake clearance were 0.0062+/-0.0012, 0.013+/-0.0017, 0.014+/-0.0030 and 0.13+/-0.066 ml min(-1)g(-1), respectively and not significantly different from the values in the absence of NBTI. In a separate experiment the brain uptake of MCPA from phosphate buffered saline (PBS) and whole blood were compared. The brain uptake clearance from whole blood (0.0012+/-0.001 ml min(-1)g(-1)) was significantly lower than from PBS (0.0045+/-0.0012 ml min(-1)g(-1)). The results of these studies show that the rENT1 nucleoside transporter does not contribute significantly to the transport of synthetic A(1) receptor agonists across the BBB and that binding to blood constituents restricts the brain uptake.

Using Poaching Levels and Elephant Distribution to Assess the Conservation Efficacy of Private, Communal and Government Land in Northern Kenya.

Efforts to curb elephant poaching have focused on reducing demand, confiscating ivory and boosting security patrols in elephant range. Where land is under multiple uses and ownership, determining the local poaching dynamics is important for identifying successful conservation models. Using 2,403 verified elephant, Loxodonta africana, mortality records collected from 2002 to 2012 and the results of aerial total counts of elephants conducted in 2002, 2008 and 2012 for the Laikipia-Samburu ecosystem of northern Kenya, we sought to determine the influence of land ownership and use on diurnal elephant distribution and on poaching levels. We show that the annual proportions of illegally killed (i.e., poached) elephants increased over the 11 years of the study, peaking at 70% of all recorded deaths in 2012. The type of land use was more strongly related to levels of poaching than was the type of ownership. Private ranches, comprising only 13% of land area, hosted almost half of the elephant population and had significantly lower levels of poaching than other land use types except for the officially designated national reserves (covering only 1.6% of elephant range in the ecosystem). Communal grazing lands hosted significantly fewer elephants than expected, but community areas set aside for wildlife demonstrated significantly higher numbers of elephants and lower illegal killing levels relative to non-designated community lands. While private lands had lower illegal killing levels than community conservancies, the success of the latter relative to other community-held lands shows the importance of this model of land use for conservation. This work highlights the relationship between illegal killing and various land ownership and use models, which can help focus anti-poaching activities.

Population pharmacokinetic-pharmacodynamic modelling of the anti-hyperalgesic effect of 5'deoxy-N6-cylopentyladenosine in the mononeuropathic rat.

The objective of this investigation was to characterise the pharmacokinetic-pharmacodynamic correlation of 5'-deoxy-N6-cyclopentyl-adenosine (5'dCPA) in the chronic constriction injury model of neuropathic pain. Following intravenous administration of 5'dCPA (0.30 or 0.75 mg kg(-1)), the time course of the drug concentration in plasma was determined in conjunction with the effect on (1) the mechanical paw pressure and (2) the Von Frey Hair monofilament withdrawal threshold. Population pharmacokinetic-pharmacodynamic analysis was applied to derive individual concentration-effect relationships. For mechanical paw pressure a composite model consisting of an Emax model for the anti-hyperalgesic effect in combination with a linear model for the anti-nociceptive effect accurately described the data. The EC50 for the anti-hyperalgesic effect was 178+/-51 ng ml(-1) and the slope of the anti-nociceptive effect 0.055+/-0.008 g ml ng(-1). For the Von Frey Hair monofilament withdrawal threshold responders and non-responders were observed. Typically, in responders, full pain relief was observed at concentrations exceeding 100 ng ml(-1). The high plasma concentrations required for the anti-hyperalgesic effect relative to the receptor affinity are consistent with restricted transport of 5'dCPA to the site of action in the spinal cord and/or the brain.

Coupling of metal containing homing devices to liposomes via a maleimide linker: use of TCEP to stabilize thiol-groups without scavenging metals.

Liposomes for drug delivery are often prepared with maleimide groups on the distal end of PEG to enable coupling of homing devices, such as antibodies, or other proteins. EDTA is used to stabilize the thiol group in the homing device for attachment to the maleimide. However, when using a homing device that contains a metal, EDTA inactivates this by scavenging of the metal. Holo-transferrin (Tf) containing two iron atoms (Fe(3+)), has a much higher affinity for the Tf receptor than apo-Tf (which does not contain any Fe(3+)). To couple Tf to a liposome, the introduction of a thiol group is necessary. During this process, by using N-succinimidyl S-acetylthioacetate (SATA), followed by 2-3 h coupling to the liposomes, Fe(3+) is scavenged by EDTA. This causes a decreased affinity of Tf for its receptor, resulting in a decreased targeting efficiency of the liposomes. Tris(2-carboxyethyl)phosphine (TCEP) hydrochloride is a sulfhydryl reductant that is often used in protein biochemistry. We found that TCEP (0.01 mM) does not scavenge Fe(3+) from Tf and is able to protect thiol groups for the coupling to maleimide. Furthermore, TCEP does not interfere with the maleimide coupling itself. In this communication, we describe the preparation of liposomes, focussing on the coupling of Tf to the maleimide linker at the distal end of PEG, without loosing Fe(3+) from Tf. This method can be applied to other metal-containing homing devices as well.

Validation of the transferrin receptor for drug targeting to brain capillary endothelial cells in vitro.

Recently, we have shown that transferrin (Tf) is actively endocytosed by the Tf R on primary cultured bovine brain capillary endothelial cells (BCEC). The objective of this investigation is to determine whether the Tf R can facilitate endocytosis of a (protein) model drug, using Tf as a targeting vector. Secondly, the mechanism of endocytosis was investigated. Horseradish peroxidase (HRP, 40 kDa) was chosen as a model drug, since it normally does not cross the blood-brain barrier (BBB) and its concentration in biological media can be easily quantified. Tf-HRP conjugates (1:1) are actively and specifically endocytosed by BCEC in vitro in a concentration and time-dependent manner. At an applied concentration of 3 microg/ml, association (a combination of binding and endocytosis) of Tf-HRP reached equilibrium at a concentration of 2 ng/mg cell protein after 1 h of incubation at 37 degree C. This was approximately 3-fold higher compared to binding at 4 degree C (0.6 ng/mg cell protein). Association of Tf-HRP was compared to BSA-HRP. After 2 h of incubation at 37 degree C association levels were 5.2 and 2.5 ng/mg cell protein, for Tf-HRP and BSA-HRP, respectively. Under those conditions, association of Tf-HRP could be inhibited to approximately 30% of total association by an excess of non-conjugated Tf, but not with BSA, while association of BSA-HRP could be inhibited by both proteins. Furthermore, by using specific inhibitors of endocytotic processes, it was shown that association of Tf-HRP is via clathrin-coated vesicles. Association of Tf-HRP is inhibited by phenylarsine oxide (an inhibitor of clathrin-mediated endocytosis) to 0.4 ng/mg cell protein, but not by indomethacin, which inhibits formation of caveolae. Finally, following iron scavenging by deferoxamine mesylate (DFO, resulting in a higher Tf R expression) a 5-fold increase in association of Tf-HRP to 15.8 ng/mg cell protein was observed. In conclusion, the Tf R is potentially suitable for targeting of a (protein) cargo to the BBB and to facilitate its endocytosis by the BCEC.