Objectives EU directives can lend themselves to evaluation through natural experiments because of the freedom regarding their implementation within individual countries. Natural experiments may be criticised because of the lack of direct casual evidence for a relationship between the intervention and the exposure or disease outcome. Such experimental designs could be strengthened by an a priori estimate of the impact of an intervention. Here we aim to predict the impact of EU Directive 2002/44/EC on VWF, CTS and sensorineural symptoms (HAVS). Methods Previously we described the development of a Markov chain Monte Carlo accelerated failure time model to predict the incidence and prevalence of HAVS. The model was developed using longitudinal data from Italian workers and validated using published data for Swedish and UK workers, and compensation data from the Czech Republic. For the next step we have used the exposure data from the Eurofound European Working Conditions Survey from 2000 to 2015 and population level demographic data to predict the impact of the directive on HAVS in Europe. Results The model predicted that a reduction in vibration exposure to 5 m/s2 (i.e. complete success of the directive) would result in a reduction in lifetime prevalence of VWF of around 25% in the UK. Predictions of the variation in impact according to differing reductions in exposure and across different age groups will be presented for the UK and other countries. Conclusion Future work will compare these estimates of the impact of the directive using routinely collected data in European countries.

PURPOSE:Determining metabolic ratio from single-point plasma is potentially a good phenotyping method of CYP2D6 to reduce the required time interval and increase the reliability of data. It is difficult to conduct large sample size clinical trials to evaluate this phenotyping method for multiple plasma points. A physiologically based pharmacokinetic (PBPK) model can be developed to do simulations based on the large virtual Chinese population and evaluate single-point plasma phenotyping method of CYP2D6. METHODS:Pharmacokinetic data of dextromethorphan (DM) and its metabolite dextrorphan (DX) after oral administration were used for model development. The SimCYP® model incorporating Chinese demographic, physiological, and enzyme data was used to simulate DM and DX pharmacokinetics in different phenotype groups. RESULTS:The ratios of the simulated to the observed mean AUC and Cmax of DM were 1.01 and 0.81 for extensive metabolizers (EMs), 0.90 and 0.81 for intermediate metabolizers (IMs), and 1.12 and 0.84 for poor metabolizers (PMs). The ratios of the simulated to the observed mean AUC and Cmax of DX were 1.12 and 0.89 for EMs, 0.66 and 0.62 for IMs. All ratios were within the predefined criterion of 0.5-2. The simulations of DM and DX pharmacokinetic profiles in 1000 virtual Chinese subjects with reported frequencies of different phenotypes indicated that statistically significant correlations were found between metabolic ratio of DM to DX (MRDM/DX) from AUC and from single-point plasma from 1 to 30h (all p-values <0.001). CONCLUSION:MRDM/DX from single-point plasma from 1 to 30h after the administration of 30mg controlled-release DM could predict the MRDM/DX from AUC well and could be used as the phenotyping method of CYP2D6 for EMs, IMs, and PMs.

Background Regular exposure to hand-arm vibration can lead to hand-arm vibration (HAVS) and carpal tunnel (CTS) syndromes. EU Directive 2002/44/EC, which aims to improve surveillance and reduce exposure to vibration at work, defines exposure limits for hand-arm-vibration. The aim is to investigate whether the staged implementation of the directive since 2005 has been associated with a change in the incidence of HAVS or CTS in European countries. To model the range of potential outcomes of the intervention, we sought to define an exposure-risk relationship based on the best available prospective longitudinal data for exposed workers. Methods In a series of studies, which have been subject of previous publications,1,2 forestry and stone workers were monitored at intervals of 1 to 5 years, on 1 to 4 occasions. Health status, annual hours of exposure and magnitude of vibration were recorded. In the present study, a novel analytical approach was applied to define a parametric survival function describing the incidence of HAVS as a function of various exposure metrics. Results Incidence of the vascular condition vibration-induced white finger was well described by a Weibull distribution with an increasing hazard rate. The resulting parametric equation reveals the role of duration of exposure and the vibration magnitude (m s-2) relative to a threshold magnitude. Conclusion The exposure-risk relationship, which differs in some respects from the exposure relationship described in current standards (e.g. ISO 5349-1, 2001), has a simple, mechanistically based form that can be easily applied to simulate the consequences of workplace interventions. References Bovenzi M. Int Arch Occup Environ Health 2008;81:401–408. doi:10.1007/s00420-007-0225-9 Bovenzi M. Int Arch Occup Environ Health 2010;83:259–272. doi:10.1007/s00420-009-0461-2

Using a previously-characterised solid tumour model we demonstrate that a mixture of pH sensitive peptide and liposome nanoparticles (PLNs) can be used to induce payload release in response to small changes in tumour acidity. The classical pH sensitive GALA peptide was modified at two different positions to help tether with preformed liposomes and cause release of calcein marker in response to a less than 1.0 unit drop in pH from the physiological value. The circular dichroic spectra of the peptide showed that, unlike the GALA sequence, its helical structure was unusually independent of pH suggesting a different mechanism of action for calcein release. Intravenously-administered PLNs accumulated in tumour tissue in mice, and subjecting the excised tumour tissue slices to low pH buffer confirmed that these nanoparticles remained pH responsive. Quantitative in vivo imaging of tumours implanted in mouse dorsal skin allowed real time measurements of nanoparticle accumulation, monitored through DiI labelling of the liposome membrane, and intra-tumour pH dependent release, monitored by calcein release, to be followed. The release of calcein in the live tumours at their natural acidity of pH 6.8 was further enhanced with MIBG glucose treatment to induce tumour acidification to approximately pH 6.5. Following accumulation a substantially greater and more rapid calcein release was observed in tumours administered with PLNs and subjected to pH-lowering using MIBG treatment, compared to control tumours administered with liposomes lacking the peptide or tumours not treated with MIBG. This work highlights that while PLNs were relatively unresponsive to intrinsic tumour pH, they did have the ability to respond rapidly to extrinsically-induced small changes in pH near the patho-physiological range.

Tat (Transactivating transcriptional activator) peptide, derived from HIV type-1 Tat protein, is the most commonly studied cell penetrating peptide (CPP) as a peptide or as a gene carrier. As a gene carrier (GDS), its low transfection efficiency remains a disadvantage when compared with the viral vectors. As it may be due to a loss of cell penetrating activity upon binding to DNA, improved efficiency was sought by coupling membrane active peptides e.g. LK15 to Tat peptide. While it has been shown that the resulting Tat-LK15 is orders of magnitude more efficient than Tat at mediating transfection in cells its trafficking mechanism as well as the influence of the amphipathic peptide upon binding remain unclear thus its limits refinement. Saleh et al (ref) have shown that Tat-LK15 DNA complexes enter cells by endocytosis but could not elucidate the cellular trafficking in particular the endocytosis route remained unclear and needed a more detailed approach. Based on this we hypothesised that (i) the endocytosis route may depend on the size and the concentration of the GDS and (ii) blocking of one endocytosis route may stimulate or induce another pathway to compensate the effect consequently. The initial phase consisted in assessing the concentration and of incubation times of specific inhibitors to observe a significant shift by FACS. The chosen inhibitors are chloropromazin, filipin and N-ethyl isopropyl amiloride (EIPA) which inhibit clathrin mediated, caveolae mediated endocytosis and macropinocytosis respectively. To test the effect of the inhibitors, specific marker s were used i.e. transferrin, albumin and dextran for clathrin mediated-, caveolae mediated endocytosis and macropinocytosis respectively and the effect of these inhibitors [chloropromazin, filipin and (EIPA)]. In parallel, Tat-LK15 has been labelled with TAMRA and purified to follow the fate of the peptide during cellular trafficking in later experiments.

Quantitative fluorescence microscopy provides valuable insight into drug delivery and pharmacokinetics. The technique is based on analysis of statistical fluctuations in fluorescence that arises as fluorophores pass through a small volume illuminated by a focused laser beam, and has been applied to measure particle motion and binding interactions in solutions, on surfaces and inside the cells. We examined the use of fluorescence correlation spectroscopy combined with a microscope (FCSM) to assess the transport of fluorescent beads and macromolecules in aqueous solutions, gels and living biological tissue. Obstructed diffusion of fluorescent beads in gels of various densities was tested to get a sensible estimate of diffusion in the interstitial tissue matrix consistent with previous reports. Fluorescently labelled liposomes as an artificial drug or gene carrying vehicles were used for pharmacokinetic tests of drug delivery in living tissue. The results indicate that FCS is an accurate and valuable tool for measuring the physical properties of gene vectors in vitro and for characterizing interactions with tissue in vivo.

Fluorescence correlation spectroscopy combined with microscopy (FCSM) is used to study the mobility of DNA fragments in aqueous solution and tissue models on the single molecule level. The effective hydrodynamic radius was measured for various lengths of ds-DNA chains and obeyed the theoretically inveterate [DNA length](0.5) relationship. Hindered diffusion of ds-DNA through the gel matrix of various densities is thought of as an extension of Kramer's problem for a flexible polymer chain. With increasing DNA length the average barrier crossing time rises as [DNA length](2) and this agrees with theory predictions for polymer molecules surmounting an entropic barrier.

The possibilities of the method of fluorescence correlation spectroscopy for studying the molecular dynamics in living cells are demonstrated. The method provides point measurements of extremely low concentrations of fluorescent molecules and their diffusion coefficients with a high time resolution in a microscopic volume, which is especially important in pharmacological investigations. A biological model of the interaction of liposomes with a cellular membrane is considered. The diffusion coefficients of fluorescent molecules are measured directly in the living cell cytoplasm.

The tumour extracellular matrix acts as a barrier to the delivery of therapeutic agents. To test the hypothesis that extracellular matrix composition governs the penetration rate of macromolecules in tumour tissue, we measured the diffusion coefficient of nonspecific IgG in three rhabdomyosarcoma subclones growing as multicellular spheroids in vitro or as subcutaneous tumours in dorsal windows in vivo . In subcutaneous tumours, the diffusion coefficient decreased with increasing content of collagen and sulphated glycosaminoglycans. When grown as multicellular spheroids, no differences in either extracellular matrix composition or diffusion coefficient were found. Comparison of in vitro vs in vivo results suggests an over-riding role of host stromal cells in extracellular matrix production subjected to modulation by tumour cells. Penetration of therapeutic macromolecules through tumour extracellular matrix might thus be largely determined by the host organ. Hence, caution must be exercised in extrapolating drug penetrability from spheroids and multilayer cellular sandwiches consisting of only tumour cells to tumours in vivo .

Two-photon correlation spectroscopy enables the deeper insight into the living tissue in comparison to single photon spectroscopy and visualise the biophysical processes occurring there. We present the basic features of an experimental set-up based on a research Nikon microscope and preliminary experimental results of suitable dye with two-photon excitation for pharmacokinetic studies. Despite some technical problems the proposed design differs by its simplicity, requires relatively low-cost optics and has a reasonably low dispersion on the optical elements.

The large size of many novel therapeutics impairs their transport through the tumor extracellular matrix and thus limits their therapeutic effectiveness. We propose that extracellular matrix composition, structure, and distribution determine the transport properties in tumors. Furthermore, because the characteristics of the extracellular matrix largely depend on the tumor–host interactions, we postulate that diffusion of macromolecules will vary with tumor type as well as anatomical location. Diffusion coefficients of macromolecules and liposomes in tumors growing in cranial windows (CWs) and dorsal chambers (DCs) were measured by fluorescence recovery after photobleaching. For the same tumor types, diffusion of large molecules was significantly faster in CW than in DC tumors. The greater diffusional hindrance in DC tumors was correlated with higher levels of collagen type I and its organization into fibrils. For molecules with diameters comparable to the interfibrillar space the diffusion was 5- to 10-fold slower in DC than in CW tumors. The slower diffusion in DC tumors was associated with a higher density of host stromal cells that synthesize and organize collagen type I. Our results point to the necessity of developing site-specific drug carriers to improve the delivery of molecular medicine to solid tumors.

Quantitative fluorescence microscopy methods can provide valuable insight into drug delivery and pharmacokinetics. We are investigating the use of single photon fluorescence correlation spectroscopy (FCS) to measure particle concentration and mobility in living tissue. In this study we examined whether a relatively large illumination volume (laser spot radius of similar to 20 mum) could be used to probe the state of macromolecules in free solution and in tissue. The FCS set-up is based upon an upright research microscope, diode laser with 635 nn wavelength an avalanche photodiode/single photon counting module, and PC based correlation electronics. Diffusion coefficients were extracted from measured autocorrelation functions. We used fluorescent monodisperse beads with diameter 20 and 200 nm to calibrate the excitation volume. Particle diffusion coefficients measured by FCS were compared with conventional light-scattering measurements. We then applied the technique to measure fluorescently labelled liposome distribution in tissue and tissue models. We found that the difference in quantum brightness and diffusion times of liposomes and free dye may be used to detect changes due to liposome interaction with living cancer cells.