RGD-based strategies for selective delivery of therapeutics and imaging agents to the tumour vasculature

During the past decade, RGD-peptides have become a popular tool for the targeting of drugs and imaging agents to alphavbeta3-integrin expressing tumour vasculature. RGD-peptides have been introduced by recombinant means into therapeutic proteins and viruses. Chemical means have been applied to couple RGD-peptides and RGD-mimetics to liposomes, polymers, peptides, small molecule drugs and radiotracers. Some of these products show impressive results in preclinical animal models and a RGD targeted radiotracer has already successfully been tested in humans for the visualization of alphavbeta3-integrin, which demonstrates the feasibility of this approach. This review will summarize the structural requirements for RGD-peptides and RGD-mimetics as ligands for alphavbeta3. We will show how they have been introduced in the various types of constructs by chemical and recombinant techniques. The importance of multivalent RGD-constructs for high affinity binding and internalization will be highlighted. Furthermore the in vitro and in vivo efficacy of RGD-targeted therapeutics and diagnostics reported in recent years will be reviewed.     

Testing bronchial hyper-responsiveness: provocation or peak expiratory flow variability?

BACKGROUND: Assessing bronchial hyper-responsiveness (BHR) is a main diagnostic criterion of asthma. Provocation testing is not readily available in general practice, but peak expiratory flow (PEF) is. Several guidelines promote the use of PEF variability as a diagnostic tool for BHR. This study tested the agreement between histamine challenge testing and PEF variability, and the consequences for diagnosing asthma. AIM: To investigate the possibility of assessing BHR by PEF variability, using a histamine provocation test as a reference. METHOD: Subjects with signs of symptoms indicating asthma (persistent or recurrent respiratory symptoms or signs of reversible bronchial obstruction) (n = 323) were studied. They had been identified in a population screening for asthma. A histamine provocation test and PEF variability were assessed over a three-week period. Asthma was defined as signs or symptoms together with a reversible airflow obstruction or BHR to the histamine challenge test. BHR was defined as a PC20 histamine of < or = 8 mg/ml or a PEF variability of > or = 15%. Overall correlation between PC20 and PEF variability was calculated using Spearman's rho. Furthermore, a decision tree was constructed to clarify the role of BHR in diagnosing asthma. RESULTS: Thirty-two patients had a reversibility in forced expiratory volume in 1 second (FEV1) of > or = 9% predicted, 131 patients showed a PC20 of < or = 8 and 11 patients had a PEF variability of > or = 15%. Overall correlation was poor at only -0.27 (P < 0.0001). One hundred and fourteen of the 131 patients diagnosed as having asthma when the histamine challenge test was used were not diagnosed by PEF variability. CONCLUSION: PEF variability cannot replace bronchial provocation testing in assessing BHR. This indicates that PEF variability and bronchial provocation do not measure the same aspects of BHR. If BHR testing is required in diagnosing asthma, a bronchial provocation test has to be used in general practice as well.     

VCAM-1 specific PEGylated SAINT-based lipoplexes deliver siRNA to activated endothelium in vivo but do not attenuate target gene expression

In recent years much research in RNA nanotechnology has been directed to develop an efficient and clinically suitable delivery system for short interfering RNA (siRNA). The current study describes the in vivo siRNA delivery using PEGylated antibody-targeted SAINT-based-lipoplexes (referred to as antibody-SAINTPEGarg/PEG2%), which showed superior siRNA delivery capacity and effective down-regulation of VE-cadherin gene expression in vitro in inflammation-activated primary endothelial cells of different vascular origins. PEGylation of antibody-SAINTPEGarg resulted in more desirable pharmacokinetic behavior than that of non-PEGylated antibody-SAINTPEGarg. To create specificity for inflammation-activated endothelial cells, antibodies against vascular cell adhesion molecule-1 (VCAM-1) were employed. In TNFα-challenged mice, these intravenously administered anti-VCAM-1-SAINTPEGarg/PEG2% homed to VCAM-1 protein expressing vasculature. Confocal laser scanning microscopy revealed that anti-VCAM-1-SAINTPEGarg/PEG2% co-localized with endothelial cells in lung postcapillary venules. Furthermore, they did not exert any liver and kidney toxicity. Yet, lack of in vivo gene silencing as assessed in whole lung and in laser microdissected lung microvascular segments indicates that in vivo internalization and/or intracellular trafficking of the delivery system and its cargo in the target cells are not sufficient, and needs further attention, emphasizing the essence of evaluating siRNA delivery systems in an appropriate in vivo animal model at an early stage in their development.     

The course of inhalation profiles during an exacerbation of obstructive lung disease

BACKGROUND: Acute exacerbations of asthma and chronic obstructive pulmonary disease (COPD) are associated with increased airflow Limitation, hyperinflation and respiratory muscle fatigue. It is unclear, whether patients are able to perform adequate inhalations through various inhalation devices with different orfices during an exacerbation. The aim of this study was to examine the evolution of inhalation profiles of patients inhaling through Diskus, Turbuhaler, pressurized metered dose inhaler (pMDI) and Volumatic and consequently the appropriateness of using the various devices during an exacerbation. MEASUREMENTS: 15 hospitalized patients participated in this randomized comparison of inhalation profiles through the four placebo-devices. For each device, triplicate inhalation profiles were recorded during day 1-9 of admission and in stable phase (day 50). RESULTS: The mean percentage of patients performing optimum inhalation profiles was 100% for Diskus, 60% for Turbuhaler, 14% for pMDI and 87% for Volumatic over the interval of day 1-9 and day 50. Patients with an inspiratory muscle strength (MIP) of less than 6kPa were generally unable to generate the optimum flow through the Turbuhaler (>60 l/min). CONCLUSION: The Diskus and Volumatic can be used effectively in the acute phase of an exacerbation of asthma or COPD. The Turbuhaler could be optimally used after the fifth day of convalescence. The pMDI is rather unsuitable during an exacerbation.     

A comparison of six different ways of expressing the bronchodilating response in asthma and COPD; reproducibility and dependence of prebronchodilator FEV1.

Various indices are used to express the bronchodilating response. It is unclear, however, which index is most informative. The aim of this study was to compare six expressions of the bronchodilating response and to examine: 1) the independence of the prebronchodilator forced expiratory volume in one second (FEV1); and 2) the reproducibility of the bronchodilating response. Bronchodilating responses (increases in FEV1 60 min after salbutamol 400 micrograms and ipratropium bromide 80 micrograms) on six test occasions, during two years, of 183 patients (72 asthma, 111 chronic obstructive pulmonary disease (COPD)) from a large bronchodilator intervention study were used. The dependence of the prebronchodilator FEV1 was investigated both between patients (cross-sectional analysis) and within patients (longitudinal analysis) by means of linear regression analysis. The reproducibility of the bronchodilating response was calculated by means of the coefficients of variation (CVs) of the six bronchodilating responses during two years. The CVs of the six expression indices were compared by analysis of variance (ANOVA). No index was independent of the prebronchodilator FEV1. However, some indices were significantly more dependent on the prebronchodilator lung function and, therefore, less reproducible than others. The "% initial" index (change as a percentage of the prebronchodilator value) was the most dependent on the prebronchodilator lung function and had the worst reproducibility (CV ranged from 50-61%). The "% possible" (change as a percentage of the predicted minus prebronchodilator value) and "% achievable" (change as a percentage of the maximal postbronchodilator minus prebronchodilator value) indices were the least dependent on the prebronchodilator value and had the highest reproducibility (CV ranging from 34-53%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Validity of an automated telephonic system to assess COPD exacerbation rates

Current tools for recording chronic obstructive pulmonary disease (COPD) exacerbations are limited and often lack validity testing. We assessed the validity of an automated telephonic exacerbation assessment system (TEXAS) and compared its outcomes with existing tools. Over 12 months, 86 COPD patients (22.1% females; mean age 66.5 yrs; mean post-bronchodilator forced expiratory volume in 1 s 53.4% predicted) were called once every 2 weeks by TEXAS to record changes in respiratory symptoms, unscheduled healthcare utilisation and use of respiratory medication. The responses to TEXAS were validated against exacerbation-related information collected by observations made by trained research assistants during home visits. No care assistance was provided in any way. Diagnostic test characteristics were estimated using commonly used definitions of exacerbation. Detection rates, compliance and patient preference were assessed, and compared with paper diary cards and medical record review. A total of 1,824 successful calls were recorded, of which 292 were verified by home visits (median four calls per patient, interquartile range three to five calls per patient). Independent of the exacerbation definition used, validity was high, with sensitivities and specificities between 66% and 98%. Detection rates and compliance differed extensively between the different tools, but were highest with TEXAS. Patient preference did not differ. TEXAS is a valid tool to assess COPD exacerbation rates in prospective clinical studies. Using different tools to record exacerbations strongly affects exacerbation occurrence rates.     

Platelet-derived microparticle count and surface molecule expression differ between subjects with and without type 2 diabetes,independently of obesity status

This study investigated the impact of either type 2 diabetes or obesity, separately or in combination, on the absolute amounts of microparticles (MP) and the pathways by which these are associated with either condition. The concentrations of circulating MP derived from platelets (PMP), leukocytes (LMP) and monocytes (MMP), together with their specific activation markers, were compared in 30 subjects who were characterised across 4 cohorts as obese or type 2 diabetes. The subjects with type 2 diabetes had elevated concentrations of total PMP (P = 0.003), and PMP that were fibrinogen-positive (P = 0.04), tissue factor-positive (P < 0.001), P-selectin-positive (P = 0.03). Type 2 diabetes did not alter either total or activated LMP or MMP. Obesity per se did not impact on any MP measurement. Elevated concentrations of plasma PMP occurred in subjects with type 2 diabetes, whether they were obese or non-obese. In contrast, obesity in the absence of type 2 diabetes had no effect. The increased concentrations of specific marker-positive PMP in the subjects with diabetes might reflect potential pathways by which PMP may contribute to the pathogenesis of atherosclerosis and type 2 diabetes.     

The Antiviral Protein Human Lactoferrin Is Distributed in the Body to Cytomegalovirus (CMV) Infection-Prone Cells and Tissues

Purpose. Lactoferrin has anti-Cytomegalovirus (CMV) and -HIV properties in vitro. However, the pharmacokinetic behavior of the 80-kD protein has not been well defined. We, therefore, assessed the plasma decay and body distribution of lactoferrin after intravenous administration to freely moving rats. Furthermore, the systemic availability of lactoferrin after intraperitoneal dosing was determined. Methods and Results. After intravenous injection, human lactoferrin (hLF) was rapidly cleared from the plasma, but higher doses resulted in prolonged plasma levels. Immunohistochemical analysis revealed a pronounced distribution of hLF to endothelial cells in the liver whereas diffuse staining in hepatocytes indicated the presence of considerable amounts in this large cell population. This endothelial association, which also was found in other organ/tissues, including blood vessels, was confirmed by in vitro cell-binding studies. In addition, leukocytes in plasma that were infiltrated in various organs showed binding of hLF. A small fraction of hLF was transported into the lymphatic system. Western blot analysis revealed that hLF, present in the various organs, mainly consisted of an 80-kD protein. After intraperitoneal administration, small amounts of 80-kD hLF distributed to the general circulation. The bioavailability was 0.6% but increased to 3.6% after multiple administrations. Conclusions. The affinity of hLF for endothelial cells and leukocytes, and its penetration into the lymphatic system, indicates that this protein reaches target cells and body compartments that are crucial for CMV and HIV replication. The ability to reach the blood compartment after intraperitoneal dosing offers opportunities for parenteral administration of the protein in future studies on its antiviral effects in vivo.     

The influence of charge clustering on the anti-HIV-1 activity and in vivo distribution of negatively charged albumins

The substitution of human serum albumin with negatively charged molecules, such as succinic acid (Suc-HSA) or aconitic acid (Aco-HSA), resulted in proteins with potent anti-HIV activities, by binding to viral gp120 (V3 loop). The aim of the present study was to investigate whether the distribution of negative charges on the albumin backbone influences the anti-HIV activity. Therefore, we prepared albumins with clusters of negatively charged groups by coupling of heparin. The effects of this substitution on anti-HIV activity, in vivo distribution and the protein structure as compared to random succinylation were assessed. In vitro studies indicated that HSA-modified with heparin 6 or 13 kD displayed anti-HIV activity (IC50=660 and 37 nM, respectively) and exhibited affinity for gp120-V3 loop, although the activity was lower than that of Suc-HSA. Combined derivatization of HSA with heparin 13 kD and aconitic acid groups resulted in significantly increased inhibitory actions (IC50=2.8 nM). Structural analysis showed that modification of HSA with heparin did not lead to extensive unfolding of the protein, meaning that these modified proteins were still globular in structure. In contrast, succinylation of HSA resulted in a highly randomly coiled conformation. Dynamic light scattering experiments revealed that, at neutral pH, the heparin fragments attached to the protein were wrapped around the molecule rather than sticking out into the solution. In conclusion, coupling of sufficient clustered negative charges, by coupling of Hep-fragments, on HSA resulted in a clear anti-HIV activity of the protein. Yet, random distribution of anionic groups in the albumin seemed more optimal for in vitro anti-HIV activity. The higher plasma and lymphatic concentrations of Hep-HSA compared to Suc-HSA seemed more favorable for an anti-HIV activity in vivo.