Vandetanib-eluting radiopaque beads for chemoembolization: physicochemical evaluation and biological activity of vandetanib in hypoxia.

Vandetanib-eluting radiopaque beads (VERB) have been developed for use in transarterial chemoembolization of liver tumours, with the goal of combining embolization with local delivery of antiangiogenic therapy. The objective of this study was to investigate how embolization-induced hypoxia may affect antitumoural activity of vandetanib, an inhibitor of vascular endothelial growth factor receptor (VEGFR) and epidermal growth factor receptor (EGFR), in the context of hepatocellular carcinoma (HCC) treatment. We studied the effect of vandetanib on proliferation, cell cycle and apoptosis of HCC cells, in hypoxic conditions, as well as the direct effects of the beads on 3D HCC spheroids. Vandetanib suppressed proliferation and induced apoptosis of HCC cells in vitro and was equipotent in hypoxic and normoxic conditions. High degrees of apoptosis were observed among cell lines in which vandetanib suppressed ERK1/2 phosphorylation and upregulated the proapoptotic protein Bim, but this did not appear essential for vandetanib-induced cell death in all cell lines. Vandetanib also suppressed the hypoxia-induced secretion of VEGF from HCC cells and inhibited proliferation of endothelial cells. Incubation of tumour spheroids with VERB led to sustained growth inhibition equivalent to the effect of free drug. We conclude that vandetanib has both antiangiogenic and direct anticancer activity against HCC cells even in hypoxic conditions, warranting the further evaluation of VERB as novel anticancer agents.

Hypoxia as a target for drug combination therapy of liver cancer.

Hepatocellular carcinoma (HCC) is the third most frequent cause of cancer deaths worldwide. The standard of care for intermediate HCC is transarterial chemoembolization, which combines tumour embolization with locoregional delivery of the chemotherapeutic doxorubicin. Embolization therapies induce hypoxia, leading to the escape and proliferation of hypoxia-adapted cancer cells. The transcription factor that orchestrates responses to hypoxia is hypoxia-inducible factor 1 (HIF-1). The aim of this work is to show that targeting HIF-1 with combined drug therapy presents an opportunity for improving outcomes for HCC treatment. HepG2 cells were cultured under normoxic and hypoxic conditions exposed to doxorubicin, rapamycin and combinations thereof, and analyzed for viability and the expression of hypoxia-induced HIF-1α in response to these treatments. A pilot study was carried out to evaluate the antitumour effects of these drug combinations delivered from drug-eluting beads in vivo using an ectopic xenograft murine model of HCC. A therapeutic doxorubicin concentration that inhibits the viability of normoxic and hypoxic HepG2 cells and above which hypoxic cells are chemoresistant was identified, together with the lowest effective dose of rapamycin against normoxic and hypoxic HepG2 cells. It was shown that combinations of rapamycin and doxorubicin are more effective than doxorubicin alone. Western Blotting indicated that both doxorubicin and rapamycin inhibit hypoxia-induced accumulation of HIF-1α. Combination treatments were more effective in vivo than either treatment alone. mTOR inhibition can improve outcomes of doxorubicin treatment in HCC.

Carboxybetaine-modified succinylated chitosan-based beads encourage pancreatic ß-cells (Min-6) to form islet-like spheroids under in vitro conditions.

In vitro, pancreatic ß-cells tend to reduce their ability to aggregate into islets and lose insulin-producing ability, likely due to insufficient cell-cell and cell-matrix interactions that are essential for ß-cell retention, viability and functionality. In response to these needs, surfaces of succinylated chitosan-based beads (NSC) were modified with zwitterionic carboxy-betaine (CB) moieties, a compatible osmolyte known to regulate cellular hydration state, and used to promote the formation of ß-cell spheroids using a conventional 2D cell culture technique. The NSC were synthesised by ionic gelation and surface-functionalised with CB using carbodiimide chemistry. Scanning electron microscopy (SEM), dynamic laser scattering (DLS) and Fourier transform infrared spectroscopy (FTIR) were employed as characterisation tools to confirm the successful modification of the succinylated chitosan material into spherical beads with rough surfaces and a diameter of 0.4 µm. NSC with and without CB were re-suspended at concentrations of 0.1, 0.3 and 0.6 mg/mL in saline medium and tested in vitro with MIN6 murine pancreatic ß-cell line. Results showed that a concentration of 0.3 mg/mL, NSC-CB encouraged pancreatic MIN6 cells to proliferate and form spheroids via E-cadherin and Pdx-1 activation within 48 h in culture. These spheroids, with a size of approximately 80 µm, exhibited high cell viability and enhanced insulin protein expression and secretion when compared to cells organised by the non-modified beads.

Synthesis and characterisation of cationic quaternary ammonium-modified polyvinyl alcohol hydrogel beads as a drug delivery embolisation system.

To extend the platform of clinically utilised chemoembolic agents based on ion-exchange systems to support the delivery of anionic drugs, a series of PVA-based beads was produced with different levels of (3-acrylamidopropyl)trimethylammonium chloride (APTA) in their formulation. The beads were characterised to confirm composition and the effect of formulation variation on physical properties was assessed. Suspension polymerisation was shown to successfully produce uniformly spherical copolymer beads with APTA content up to 60 wt%. Equilibrium water content and resistance to compression both increased with increasing APTA content in the formulation. Confocal laser scanning microscopy was used with model drugs to demonstrate that by increasing APTA content, compounds between the molecular weight range 70-250 kDa could permeate the microsphere structures. Interaction with anionic drugs was modelled using multivalent dyes. Dyes with multi-binding sites had increased interaction with the polymer, slowing the release and also demonstrating a reduced rate of elution from beads with higher charge density. The model drug release studies demonstrate that these systems can be engineered for different potential anionic drugs for local therapeutic delivery in the clinic.

Nanoprecipitation of polymeric nanoparticle micelles based on 2-methacryloyloxyethyl phosphorylcholine (MPC) with 2-(diisopropylamino)ethyl methacrylate (DPA), for intracellular delivery applications.

Biodistribution of nanoparticle-based intracellular delivery systems is mediated primarily by particle size and physicochemical properties. As such, overcoming the rapid removal of these by the reticuloendothelial system remains a significant challenge. To date, a number of copolymer nanoparticle systems based on 2-methacryloyloxyethyl phosphorylcholine (MPC) with 2-(diisopropylamino)ethyl methacrylate (DPA), displaying biomimetic and pH responsive properties, have been published, however these have been predominately polymersome based, whilst micelle systems have remained relatively unexplored. This study utilised nanoprecipitation to investigate the effects of solvent and buffer choice upon micelle size and polydispersity, and found using methanol produced monodisperse micelles of circa 70 nm diameter, whilst ethanol produced polydisperse systems with nanoparticles of circa 128 nm diameter. The choice of aqueous buffer, dialysis of the systems, extended storage, and exposure to a wide temperature range (5-70 °C) had no significant effect on micelle size, and the systems were highly resistant to dilution, indicating excellent colloidal stability. Optimisation of the nanoprecipitation process, post precipitation, was investigated, and model drugs successfully loaded whilst maintaining system stability. Subsequent in vitro studies suggested that the micelles were of negligible cellular toxicity, and an apparent cellular uptake was observed via confocal laser scanning microscopy. This paper presents the first report of an optimised nanoprecipitation methodology for the formation of MPC-DPA nanoparticle micelles, and in doing so achieved monodisperse systems with the size and physicochemical characteristics seen as desirable for long circulating therapeutic delivery vehicles.

An adsorbent monolith device to augment the removal of uraemic toxins during haemodialysis.

Adsorbents designed with porosity which allows the removal of protein bound and high molecular weight uraemic toxins may improve the effectiveness of haemodialysis treatment of chronic kidney disease (CKD). A nanoporous activated carbon monolith prototype designed for direct blood contact was first assessed for its capacity to remove albumin bound marker toxins indoxyl sulphate (IS), p-cresyl sulphate (p-CS) and high molecular weight cytokine interleukin-6 in spiked healthy donor studies. Haemodialysis patient blood samples were then used to measure the presence of these markers in pre- and post-dialysis blood and their removal by adsorbent recirculation of post-dialysis blood samples. Nanopores (20-100 nm) were necessary for marker uraemic toxin removal during in vitro studies. Limited removal of IS and p-CS occurred during haemodialysis, whereas almost complete removal occurred following perfusion through the carbon monoliths suggesting a key role for such adsorbent therapies in CKD patient care.

Development of a combination drug-eluting bead: towards enhanced efficacy for locoregional tumour therapies.

Drug-eluting beads (DEBs) are becoming a mainstay locoregional therapy for hepatic malignancies but are currently loaded with single drugs alone. Here, we wished to prepare DEB containing different drug combinations, to screen their efficacy using an in-vitro cell culture assay and to include any promising combinations that demonstrate additive efficacy in an in-vivo model of locoregional tumour treatment. A modified in-vitro assay was used based upon the use of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) with either HepG2 liver cancer or PSN1 pancreatic cancer cell lines. The comparative cytotoxicity of DEB combinations prepared containing doxorubicin, irinotecan, topotecan and rapamycin was evaluated. Those combinations that demonstrated an additive cytotoxicity effect were investigated in vivo using a nude mouse xenograft model of pancreatic cancer. Although many of the DEB combinations showed either no effect or a slight antagonistic effect, the combination of doxorubicin and rapamycin DEBs demonstrated synergistic activity. On the basis of these findings, a method was developed to prepare a doxorubicin/rapamycin dual-loaded DEB, which was shown to possess the same drug-loading capacities, drug elution properties and HepG2 cell cytotoxicity synergy as the single drug-loaded DEB combination. Evaluation of this dual-loaded combination DEB versus the respective single drug-loaded DEBs in a mouse xenograft model of pancreatic cancer showed an equivalent tumour volume reduction as the doxorubicin DEB, but with less toxicity than the rapamycin DEB. The doxorubicin/rapamycin combination DEB offers great potential for enhanced efficacy in the locoregional treatment of malignant tumours.

Activation-dependent adsorption of cytokines and toxins related to liver failure to carbon beads.

In the course of severe pathological conditions, such as acute liver failure and sepsis, toxic metabolites and mediators of inflammation are released into the patient's circulation. One option for the supportive treatment of these conditions is plasmapheresis, in which plasma, after being separated from the cellular components of the blood, is cleansed by adsorption of harmful molecules on polymers or activated carbon. In this work, the adsorption characteristics of activated carbon beads with levels of activation ranging from 0 to 86% were assessed for both hydrophobic compounds accumulating in liver failure (bilirubin, cholic acid, phenol and tryptophan) and cytokines (tumor necrosis factor α and interleukin-6). Progressive activation resulted in significant gradual reduction of both bulk density and mean particle size, in an increase in the specific surface area, and to changes in pore size distribution with progressive broadening of micropores. These structural changes went hand in hand with enhanced adsorption of small adsorbates, such as IL-6 and cholic acid and, to a lesser extent, also of large molecules, such as TNF-α.

Interactions between endothelial cells and epithelial cells in a combined cell model of airway mucosa: effects on tight junction permeability.

Environmental particulates impact first on airway epithelium, whereas circulating infiltrating cells are recruited through the underlying endothelium. An effective cellular immune response requires coordination between endothelium and epithelium. The authors have developed a bilayer culture model consisting of human bronchial epithelial derived cells (16HBE 14o-) and human umbilical vein endothelial cells (HUVECs) cultured as confluent layers on either side of a porous membrane. Confocal microscopy with epithelial and endothelial-specific antibodies showed segregated cell layers. By scanning and transmission electron microscopy, both cell types are polarized and tight junctions formed at the apical interface between cells. Epithelial cells grown in a bilayer showed significantly increased transepithelial resistance (TER) of 2260 +/- 64 Omega.cm(2) compared to epithelial or endothelial monolayers alone (1400 +/- 70 or 80 +/- 12 Omega.cm(2), respectively). This reflected decreased permeability and was unrelated to cell density or height. Increased TER coincided with increased occludin mRNA and protein in the epithelial cell layer as determined by polymerase chain reaction (PCR) and immunoblotting. Conditioned medium showed that decreased permeability was mediated by soluble endothelial-derived factor(s). This model reflects the in vivo relationship of human airway endothelial cells and epithelial cells. Altered tight junction permeability in cocultures indicates that these cells can work together as an active part of the mucosal barrier.

Characterisation of physico-mechanical properties and degradation potential of calcium alginate beads for use in embolisation.

High molecular weight alginate beads with 59% mannuronic acid content or 68% guluronic acid were prepared using a droplet generator and crosslinked in calcium chloride. The alginate beads were compared to current embolisation microspheres for compressibility and monitored over 12 weeks for size and weight change at 37 degrees C in low volumes of ringers solutions. A sheep uterine model was used to analyse bead degradation and inflammatory response over 12 weeks. Both the in vitro and in vivo data show good delivery, with a compressibility similar to current embolic beads. In vitro, swelling was noted almost immediately and after 12 weeks the first signs of degradation were noted. No difference was noted in vivo. This study has shown that high molecular weight alginate gel beads were well tolerated by the body, but beads associated with induced thrombi were susceptible to inflammatory cell infiltration. The beads were shown to be easy to handle and were still observable after 3 months in vivo. The beads were robust enough to be delivered through a 2.7 Fr microcatheter. This study has demonstrated that high molecular weight, high purity alginate bead can be considered as semi-permanent embolisation beads, with the potential to bioresorb over time.

Comparison of DC Bead-irinotecan and DC Bead-topotecan drug eluting beads for use in locoregional drug delivery to treat pancreatic cancer.

DC Bead is a drug delivery embolisation system that can be loaded with doxorubicin or irinotecan for the treatment of a variety of liver cancers. In this study we demonstrate that the topoisomerase I inhibitor topotecan hydrochloride can be successfully loaded into the DC Bead sulfonate-modified polyvinyl alcohol hydrogel matrix, resulting in a sustained-release drug eluting bead (DEBTOP) useful for therapeutic purposes. The in vitro drug loading capacity, elution characteristics and the effects on mechanical properties of the beads are described with reference to our previous work with irinotecan hydrochloride (DEBIRI). Results showed that drug loading was faster when the solution was agitated compared to static loading and a maximum loading of ca. 40-45 mg topotecan in 1 ml hydrated beads was achievable. Loading the drug into the beads altered the size, compressibility moduli and colour of the bead. Elution was shown to be reliant on the presence of ions to perform the necessary exchange with the electrostatically bound topotecan molecules. Topotecan was shown by MTS assay to have an IC(50) for human pancreatic adenocarcinoma cells (PSN-1) of 0.22 and 0.27 microM compared to 28.1 and 19.2 microM for irinotecan at 48 and 72 h, respectively. The cytotoxic efficacy of DEBTOP on PSN-1 was compared to DEBIRI. DEPTOP loaded at 6 & 30 mg ml(-1), like its free drug form, was shown to be more potent than DEBIRI of comparable doses at 24, 48 & 72 h using a slightly modified MTS assay. Using a PSN-1 mouse xenograft model, DEBIRI doses of 3.3-6.6 mg were shown to be well-tolerated (even with repeat administration) and effective in reducing the tumour size. DEBTOP however, was lethal after 6 days at doses of 0.83-1.2 mg but demonstrated reasonable efficacy and tolerability (again with repeat injection possible) at 0.2-0.4 mg doses. Care must therefore be taken when selecting the dose of topotecan to be loaded into DC Bead given its greater potency and potential toxicity.

Inflammatory cytokine removal by an activated carbon device in a flowing system.

A prototype in-line filtration/adsorption device has been developed using novel synthetic pyrolysed carbon monoliths with controlled mesoporous domains of 2-50nm. Porosity was characterized by SEM and porosimetry. Removal of inflammatory cytokines TNF, IL-6, IL-1beta and IL-8 was assessed by filtering cytokine spiked human plasma through the walls of the carbon modules under pressure. The effect of carbon filtration on plasma clotting response and total plasma protein concentration was also assessed. Significant removal of the cytokines IL-6, IL-1beta and IL-8 was observed. Initially marked TNF removal diminished over time. The coagulation studies indicated that the carbon device does not exacerbate the propensity of blood plasma to clot. The total plasma protein concentration remained constant. The device offers a broader approach to the treatment of systemic inflammatory response syndrome (SIRS) by the removal of inflammatory mediators central to its progression.

Inhalation toxicology of ricin preparations: animal models, prophylactic and therapeutic approaches to protection.

Ricin is a toxin and seed protein produced by the castor oil plant, Ricinus communis. The toxin is a dimeric protein consisting of an enzymic A chain and a B chain with lectin properties aiding the uptake of the whole molecule into cells. Ricin has been considered a possible military threat for several decades and is now also of some concern as a terrorist agent. The inhalation route is of primary concern in these situations, although previous attacks with ricin have used other approaches. Medical countermeasures against ricin are urgently required and the strategy adopted has been first to understand the nature of the problem, in this case the inhalation toxicology of ricin, followed by the preparation of vaccine antigens. Toxoided ricin and modified recombinant A chain components have been examined in terms of efficacy as potential vaccine candidates in protection of animal models against inhaled ricin, primarily in laboratories both in the United Kingdom and in the United States. One recombinant A chain vaccine has been taken through to clinical trials in the United States and should become commercially available in the next few years. Toxoided ricin has also been used as an antigen to prepare antitoxin antibodies for therapeutic treatment following poisoning. In this review, a synopsis of the inhalation toxicology of ricin and approaches to medical prophylaxis and therapy of poisoning is given, based on work conducted at our laboratory and at other research institutes.

Preparation and characterisation of vandetanib-eluting radiopaque beads for locoregional treatment of hepatic malignancies.

Since their introduction around a decade ago, embolic drug-eluting beads (DEBs) have become a well-established treatment option for the locoregional transarterial treatment of hepatic malignancies. Despite this success, the therapy is seen to be limited by the choice of drug and more effective options are therefore being sought. These include the small molecule multi-tyrosine kinase inhibitors (MTKi), which exert an anti-angiogenic and anti-proliferative effect that could be highly beneficial in combating some of the unwanted downstream consequences of embolization. Vandetanib is an MTKi which acts against such targets as vascular endothelial growth factor receptor (VEGFR) and epithelial growth factor receptor (EGFR) and has demonstrated modest activity against hepatocellular carcinoma (HCC), albeit with some dose-limiting cardiac toxicity. This makes this compound an interesting candidate for DEB-based locoregional delivery. In this study we describe the preparation and characterisation of vandetanib DEBs made from DC Bead™ and its radiopaque counterpart, DC Bead LUMI™. Drug loading was shown to be dependent upon the pH of the drug loading solution, as vandetanib has multiple sites for protonation, with the bead platform also having a fundamental influence due to differences in binding capacities and bead shrinkage effects. Fourier transform infrared (FTIR) spectroscopy and energy dispersive X-ray (EDX) Spectroscopy confirmed drug interaction is by ionic interaction, and in the case of the radiopaque DEB, the drug is distributed uniformly inside the bead and contributes slightly to the overall radiopacity by virtue of a bromine atom on the vandetanib structure. Drug release from both bead platforms is controlled and sustained, with a slightly slower rate of release from the radiopaque bead due to its more hydrophobic nature. Vandetanib DEBs therefore have suitable characteristics for intra-arterial delivery and site-specific sustained release of drug into liver tumours.

Towards Hypoxia-responsive Drug-eluting Embolization Beads.

Drug release from chemoembolization microspheres stimulated by the presence of a chemically reducing environment may provide benefits for targeting drug resistant and metastatic hypoxic tumours. A water-soluble disulfide-based bifunctional cross-linker bis(acryloyl)-(l)-cystine (BALC) was synthesised, characterised and incorporated into a modified poly(vinyl) alcohol (PVA) hydrogel beads at varying concentrations using reverse suspension polymerisation. The beads were characterised to confirm the amount of cross-linker within each formulation and its effects on the bead properties. Elemental and UV/visible spectroscopic analysis confirmed the incorporation of BALC within the beads and sizing studies showed that in the presence of a reducing agent, all bead formulations increased in mean diameter. The BALC beads could be loaded with doxorubicin hydrochloride and amounts in excess of 300mg of drug per mL of hydrated beads could be achieved but required conversion of the carboxylic acid groups of the BALC to their sodium carboxylate salt forms. Elution of doxorubicin from the beads demonstrated a controlled release via ionic exchange. Some formulations exhibited an increase in size and release of drug in the presence of a reducing agent, and therefore demonstrated the ability to respond to an in vitro reducing environment.

Mesoporous carbide-derived carbon with porosity tuned for efficient adsorption of cytokines.

Porous carbons can be used for the purification of various bio-fluids, including the cleansing blood of inflammatory mediators in conditions such as sepsis or auto-immune diseases. Here we show that the control of pore size in carbons is a key factor to achieving efficient removal of cytokines. In particular, the surface area accessible by the protein governs the rate and effectiveness of the adsorption process. We demonstrate that novel mesoporous carbon materials synthesized from ternary MAX-phase carbides can be optimized for efficient adsorption of large inflammatory proteins. The synthesized carbons, having tunable pore size with a large volume of slit-shaped mesopores, outperformed all other materials or methods in terms of efficiency of TNF-alpha removal and the results are comparable only with highly specific antibody-antigen interactions.

The in vitro adsorption of cytokines by polymer-pyrolysed carbon.

This study investigated a range of phenol-formaldehyde-aniline-based pyrolysed carbon matrices and their component materials, for their ability to adsorb a range of inflammatory cytokines crucial to the progression of sepsis. The efficiency of adsorption of the target molecules from human plasma was assessed and compared to that of Adsorba 300C, a commercially available cellulose-coated activated charcoal. Results indicate that a number of the primary carbon/resin materials demonstrate efficient adsorption of the cytokines studied here (TNF, IL-6 and IL-8), comparable to other adsorbents under clinical investigation. Our findings also illustrate that these adsorbent capabilities are retained when the primary particles are combined to form a pyrolysed carbon matrix. This capability will enable the engineering of the carbon matrix porosity allowing a blend of carbonised particle combinations to be tailored for maximum adsorption of inflammatory cytokines. The present findings support further investigation of this carbon material as a combined carbon-based filtration/adsorbent device for direct blood purification.

Assessing the in vitro biocompatibility of a novel carbon device for the treatment of sepsis.

The aim of the present study was to conduct a preliminary investigation into the blood biocompatibility of a novel, uncoated carbon for use in a filtration/adsorption device for the treatment of sepsis. Carbon well prototypes were manufactured from phenol-formaldehyde-aniline-based pyrolysed carbons using monolithic polymer technology. Inflammatory blood cell and plasma protein mediation of the inflammatory response were evaluated using the novel carbon prototypes and compared with dialyser membrane and tissue culture plate controls. Assays determining monocyte and granulocyte adhesion, platelet adhesion and activation, granulocyte activation and complement activation were performed. Preliminary findings suggest an adsorptive but passivating carbon surface. Moderate levels of monocyte and granulocytes adhesion were seen in conjunction with adsorption of plasma proteins to the carbon surface. Activation of granulocyte and adherent platelets was not detected and the complement cascade was not activated by the carbons, indicating a surface compatible with blood contact. The results support the further development of the proposed carbon-based device for the treatment of sepsis.

Novel biocompatible phosphorylcholine-based self-assembled nanoparticles for drug delivery.

Major challenges associated with nano-sized drug delivery systems include removal from systemic circulation by phagocytic cells and controlling appropriate drug release at target sites. 2-methacryloyloxyethyl phosphorylcholine (MPC) has been copolymerised in turn with two pH responsive comonomers (2-(diethylamino)ethyl methacrylate (DEA) and 2-(diisopropylamino)ethyl methacrylate (DPA), to develop novel biocompatible drug delivery vehicles. Micelles were prepared from a series of copolymers with varying block compositions and their colloidal stability and dimensions were assessed over a range of solution pH using photon correlation spectroscopy. The drug loading capacities of these micelles were evaluated using Orange OT dye as a model compound. The cytotoxicity of the micelles was assessed using an in vitro assay. The MPC-DEA diblock copolymers formed micelles at around pH 8 and longer DEA block lengths allowed higher drug loadings. However, these micelles were not stable at physiological pH. In contrast, MPC-DPA diblock copolymers formed micelles of circa 30 nm diameter at physiological pH. In vitro assays indicated that these MPC-DPA diblock copolymers had negligible cytotoxicities. Thus novel non-toxic biocompatible micelles of appropriate size and good colloidal stability with pH-modulated drug uptake and release can be readily produced using MPC-DPA diblock copolymers.

Differentiating midazolam over-sedation from neurological damage in the intensive care unit.

INTRODUCTION: Midazolam is used routinely to sedate patients in the intensive care unit (ICU). We suspected that midazolam over-sedation was occurring in the ICU of the Guy's and St. Thomas' Trust and that it could be difficult to differentiate this from underlying neurological damage. A sensitive assay for detecting midazolam and 1-hydroxymidazolam glucuronide (1-OHMG) in serum was developed and applied in the clinical setting. METHODS: In the present study we evaluated a series of cases managed in a mixed medical, surgical and trauma ICU. Serum was collected from 26 patients who received midazolam, were 'slow to wake' and in whom there was suspicion of neurological damage. Patient outcome was followed in terms of mortality, neurological recovery and neurological damage on discharge. RESULTS: Out of 26 patients, 13 had detectable serum levels of midazolam and/or 1-OHMG after a median of 67 hours (range 36-146 hours) from midazolam cessation. Of these 13 patients in whom midazolam/1-OHMG was detectable, 10 made a full neurological recovery. Of the remaining 13 patients with no detectable midazolam/1-OHMG, three made a full neurological recovery; 10 patients were subsequently found to have suffered neurological damage (P < 0.002), eight of whom died and two were discharged from the ICU with profound neurological damage. CONCLUSION: These findings confirm that prolonged sedation after midazolam therapy should be considered in the differential diagnosis of neurological damage in the ICU. This can be reliably detected by the assay method described. The effects of midazolam/1-OHMG persist days after administration of midazolam has ceased. After prolonged sedation has been excluded in this patient group, it is highly likely that neurological damage has occurred.