Stoffenmanager Nano version 1.0: a web-based tool for risk prioritization of airborne manufactured nano objects.

Stoffenmanager Nano (version 1.0) is a risk-banding tool developed for employers and employees to prioritize health risks occurring as a result of exposure to manufactured nano objects (MNOs) for a broad range of worker scenarios and to assist implementation of control measures to reduce exposure levels. In order to prioritize the health risks, the Stoffenmanager Nano combines the available hazard information of a substance with a qualitative estimate of potential for inhalation exposure. The development of the Stoffenmanager Nano started with a review of the available literature on control banding. Input parameters for the hazard assessment of MNOs were selected based on the availability of these parameters in, for instance, Safety Data Sheets or product information sheets. The conceptual exposure model described by Schneider et al. (2011) was used as the starting point for exposure banding. During the development of the Stoffenmanager Nano tool, the precautionary principle was applied to deal with the uncertainty regarding hazard and exposure assessment of MNOs. Subsequently, the model was converted into an online tool (http://nano.stoffenmanager.nl), tested, and reviewed by a number of companies. In this paper, we describe the Stoffenmanager Nano. This tool offers a practical approach for risk prioritization in exposure situations where quantitative risk assessment is currently not possible. Updates of this first version are anticipated as more data become available in the future.

Enriching lipid nanovesicles with short-chain glucosylceramide improves doxorubicin delivery and efficacy in solid tumors.

For amphiphilic anticancer drugs, such as the anthracyclin doxorubicin (Dox), uptake by tumor cells involves slow diffusion across the plasma membrane, a limiting factor in clinical oncology. Previously, we discovered that preinsertion of short-chain sphingolipids such as N-octanoyl-glucosylceramide (GC) in the tumor cell membrane enhances cellular Dox uptake. In the present study, we apply this strategy in vitro and in vivo by coadministering GC and Dox in a lipid nanovesicle (LNV). GC enrichment of Dox-LNVs strongly enhanced in vitro cytotoxicity toward B16 melanoma and A431 carcinoma, as evidenced by 6-fold decreased IC(50) values compared with Dox-LNVs. This correlated with enhanced cellular Dox uptake observed by confocal microscopy. Intravital optical imaging in window chamber-bearing mice with orthotopically implanted B16 melanoma demonstrated enhanced GC-mediated Dox delivery to tumor cells. Treatment of nude mice bearing human A431 xenografts with 6 mg/kg GC-Dox-LNVs almost doubled the tumor growth delay compared with Dox-LNVs. A second administration of 5 mg/kg after 3 d induced even 3-fold delay in tumor growth, while no systemic toxicity was found. GC-enriched Dox-LNVs displayed superior in vitro and in vivo antitumor activity, without systemic toxicity. This new drug delivery concept, aiming at increased membrane permeability for amphiphilic drugs, provides an opportunity to improve cancer chemotherapy.

Alkylphospholipids inhibit capillary-like endothelial tube formation in vitro: antiangiogenic properties of a new class of antitumor agents.

Synthetic alkylphospholipids (APLs), such as edelfosine, miltefosine and perifosine, constitute a new class of antineoplastic compounds with various clinical applications. Here we have evaluated the antiangiogenic properties of APLs. The sensitivity of three types of vascular endothelial cells (ECs) (bovine aortic ECs, human umbilical vein ECs and human microvascular ECs) to APL-induced apoptosis was dependent on the proliferative status of these cells and correlated with the cellular drug incorporation. Although confluent, nondividing ECs failed to undergo apoptosis, proliferating ECs showed a 3-4-fold higher uptake and significant levels of apoptosis after APL treatment. These findings raised the question of whether APLs interfere with new blood vessel formation. To test the antiangiogenic properties in vitro, we studied the effect of APLs using two different experimental models. The first one tested the ability of human microvascular ECs to invade a three-dimensional human fibrin matrix and form capillary-like tubular networks. In the second model, bovine aortic ECs were grown in a collagen gel sandwich to allow tube formation. We found that all three APLs interfered with endothelial tube formation in a dose-dependent manner, with a more than 50% reduction at 25 micromol/l. Interference with the angiogenic process represents a novel mode of action of APLs and might significantly contribute to the antitumor effect of these compounds.

Lipid rafts and metabolic energy differentially determine uptake of anti-cancer alkylphospholipids in lymphoma versus carcinoma cells.

Perifosine is a member of the class of synthetic alkylphospholipids (APLs) and is being evaluated as anti-cancer agent in several clinical trials. These single-chain APLs accumulate in cellular membranes and disturb lipid-dependent signal transduction, ultimately causing apoptosis in a variety of tumor cells. The APL prototype edelfosine was previously found to be endocytosed by S49 mouse lymphoma cells via lipid rafts. An edelfosine-resistant cell variant, S49(AR), was found to be cross-resistant to other APLs, including perifosine. This resistance was due to defective synthesis of the raft constituent sphingomyelin, which abrogated APL cellular uptake. Sensitivity of S49 cells to edelfosine was higher than perifosine, which correlated with a relatively higher uptake. Human KB epidermal carcinoma cells were much more sensitive to APLs than S49 cells. Their much higher APL uptake was highly dependent on intracellular ATP and ambient temperature, and was blocked by chlorpromazine, independent of canonical endocytic pathways. We found no prominent role of lipid rafts for APL uptake in these KB cells; contrary to S49(AR) cells, perifosine-resistant KBr cells display normal sphingomyelin synthesis, whereas APL uptake by the responsive KB cells was insensitive to treatment with methyl-beta-cyclodextrin, a cholesterol-sequestrator and inhibitor of raft-mediated endocytosis. In conclusion, different mechanisms determine APL uptake and consequent apoptotic toxicity in lymphoma versus carcinoma cells. In the latter cells, APL uptake is mainly determined by a raft- and endocytosis-independent process, but metabolic energy-dependent process, possibly by a lipid transporter.

A new class of anticancer alkylphospholipids uses lipid rafts as membrane gateways to induce apoptosis in lymphoma cells.

Single-chain alkylphospholipids, unlike conventional chemotherapeutic drugs, act on cell membranes to induce apoptosis in tumor cells. We tested four different alkylphospholipids, i.e., edelfosine, perifosine, erucylphosphocholine, and compound D-21805, as inducers of apoptosis in the mouse lymphoma cell line S49. We compared their mechanism of cellular entry and their potency to induce apoptosis through inhibition of de novo biosynthesis of phosphatidylcholine at the endoplasmic reticulum. Alkylphospholipid potency closely correlated with the degree of phosphatidylcholine synthesis inhibition in the order edelfosine > D-21805 > erucylphosphocholine > perifosine. In all cases, exogenous lysophosphatidylcholine, an alternative source for cellular phosphatidylcholine production, could partly rescue cells from alkylphospholipid-induced apoptosis, suggesting that phosphatidylcholine biosynthesis is a direct target for apoptosis induction. Cellular uptake of each alkylphospholipid was dependent on lipid rafts because pretreatment of cells with the raft-disrupting agents, methyl-beta-cyclodextrin, filipin, or bacterial sphingomyelinase, reduced alkylphospholipid uptake and/or apoptosis induction and alleviated the inhibition of phosphatidylcholine synthesis. Uptake of all alkylphospholipids was inhibited by small interfering RNA (siRNA)-mediated blockage of sphingomyelin synthase (SMS1), which was previously shown to block raft-dependent endocytosis. Similar to edelfosine, perifosine accumulated in (isolated) lipid rafts independent on raft sphingomyelin content per se. However, perifosine was more susceptible than edelfosine to back-extraction by fatty acid-free serum albumin, suggesting a more peripheral location in the cell due to less effective internalization. Overall, our results suggest that lipid rafts are critical membrane portals for cellular entry of alkylphospholipids depending on SMS1 activity and, therefore, are potential targets for alkylphospholipid anticancer therapy.

Rationale and clinical application of alkylphospholipid analogues in combination with radiotherapy.

Concurrent treatment with radiotherapy and chemotherapy has emerged as an effective strategy to improve clinical outcome of cancer. In addition to combining radiation with classical anticancer agents, several new biological response modifiers are under investigation in pre-clinical and clinical studies. Synthetic alkylphospholipids are anticancer agents that in contrast to most anticancer drugs, do not target DNA, but insert in the plasma membrane and subsequently induce a broad range of biological effects, ultimately leading to cell death. Alkylphospholipids kill tumor cells directly by induction of both apoptotic and non-apoptotic cell death, and indirectly by interference with critical signal transduction pathways involved in phospholipid metabolism and survival. Due to their distinct mode of action, these drugs are considered as attractive candidates to combine with radiotherapy. In this review, we will discuss several alkylphospholipids that reached clinical application. These include first-generation alkyl-lysophospholipids edelfosine and ilmofosine, second-generation alkylphosphocholine-prototype miltefosine and more recently developed analogues perifosine and erucylphosphocholine. We focus on mechanisms of action and the rationale to combine these agents with radiotherapy. The preclinical results on molecular targeting underlying this approach will be reviewed, concluded with first clinical data on combined treatment of radiotherapy with perifosine.

Phase I and pharmacokinetic study of combined treatment with perifosine and radiation in patients with advanced solid tumours.

PURPOSE: Perifosine is an orally applicable, membrane-targeted alkylphosphocholine analogue with antitumour activity and radiosensitising properties in preclinical models. The purpose of this phase I study was to determine the feasibility and tolerability of concurrent daily perifosine and radiation in patients with advanced cancer. PATIENTS AND METHODS: Starting dose of perifosine was 50 mg/day; dose escalation was in steps of 50mg. Daily administration commenced 2 days before radiotherapy and was continued throughout the radiation treatment. At least three patients were entered at each dose level; at the 150 mg/day level 10 patients were included. Pharmacokinetic sampling was performed weekly pre-dosing. Twenty-one patients were entered. Tumour types included NSCLC (n=17), prostate, oesophageal, colon and bladder cancer. Most patients (16/21) had received prior chemotherapy; none radiotherapy. Median number of daily perifosine administrations was 31 (range 24-53). Mean radiation dose (BED(10)) was 59.8 Gy (range 50.7-87.5 Gy in 13-28 fractions). RESULTS: Major drug-related toxicities according to CTC criteria were nausea in 57%, fatigue in 48%, vomiting in 38%, diarrhoea in 38% and anorexia in 19%. No bone marrow toxicity was observed. DLT (nausea/vomiting) was encountered in two of five patients at the 200mg/day dose level. Dose-dependent steady-state plasma levels were reached after 1 week. Major radiotherapy-related acute toxicity consisted of dysphagia in 38% and pneumonitis in 29%. CONCLUSION: Perifosine can be safely combined with fractionated radiotherapy. A dosage of 150 mg/day, to be started at least 1 week prior to radiotherapy, is recommended for phase II evaluation.

TRAIL enhances efficacy of radiotherapy in a p53 mutant, Bcl-2 overexpressing lymphoid malignancy.

BACKGROUND AND PURPOSE: Resistance to apoptosis is a contributing factor in the response to radiotherapy. Aim of this study was to evaluate whether TRAIL--in a soluble isoleucine zippered form--enhances the cytotoxic effect of irradiation on tumour cells with a blockade in the mitochondrial apoptosis route and/or a dysfunctional p53 pathway. MATERIALS AND METHODS: The p53 mutant human T acute lymphoblastic leukemia line Jurkat transduced with the Bcl-2 gene was used as model system in vitro and in a subcutaneous transplant setting in immunodeficient mice. Sensitivity to single and combined treatment was read out by apoptosis hallmarks and clonogenic survival in vitro, and by bioluminescence and palpation in vivo. RESULTS: Jurkat cells overexpressing Bcl-2 did not undergo apoptosis after irradiation, but the combination with TRAIL synergistically induced apoptosis without breaking mitochondrial resistance. TRAIL also reduced clonogenic survival after irradiation. In vivo, radiotherapy or TRAIL alone delayed tumour outgrowth, but combination treatment had the most profound effect. CONCLUSIONS: Isoleucine zippered TRAIL can strongly enhance the efficacy of tumour therapy with ionising radiation in an unfavourable setting of p53 mutation and Bcl-2 overexpression.

Radiosensitization of squamous cell carcinoma by the alkylphospholipid perifosine in cell culture and xenografts.

PURPOSE: Combined modality treatment has improved outcome in various solid tumors. Besides classic anticancer drugs, a new generation of biological response modifiers has emerged that increases the efficacy of radiation. Here, we have investigated whether perifosine, an orally applicable, membrane-targeted alkylphospholipid, enhances the antitumor effect of radiation in vitro and in vivo. EXPERIMENTAL DESIGN: Several long-term and short-term in vitro assays (clonogenic survival, sulforhodamine B cytotoxicity, apoptosis, and cell cycle analysis) were used to assess the cytotoxic effect of perifosine in combination with radiation. In vivo, the response of human KB squamous cell carcinoma xenografts was measured after treatment with perifosine, irradiation, and the combination. Radiolabeled perifosine was used to determine drug disposition in tumor and normal tissues. At various intervals after treatment, tumor specimens were collected to document histopathologic changes. RESULTS: In vitro, perifosine reduced clonogenic survival, enhanced apoptosis, and increased cell cycle arrest after radiation. In vivo, radiation and perifosine alone induced a dose-dependent tumor growth delay. When combining multiple perifosine administrations with single or split doses of radiation, complete and sustained tumor regression was observed. Histopathologic analysis of tumor specimens revealed a prominent apoptotic response after combined treatment with radiation and perifosine. Radiation-enhanced tumor response was observed at clinically relevant plasma perifosine concentrations and accumulating drug disposition of >100 microg/g in tumor tissue. CONCLUSIONS: Perifosine enhances radiation-induced cytotoxicity, as evidenced by reduced clonogenic survival and increased apoptosis induction in vitro and by complete tumor regression in vivo. These data provide strong support for further development of this combination in clinical studies.

Coformulated N-octanoyl-glucosylceramide improves cellular delivery and cytotoxicity of liposomal doxorubicin.

The anticancer agent doxorubicin is in certain cases administered as a long-circulating liposomal formulation. Due to angiogenesis-related structural abnormalities in the endothelial lining of many neoplasms, these complexes tend to extravasate and accumulate in the tumor stroma. However, delivery of doxorubicin is still not optimal since liposomes are not taken up directly by tumor cells. Instead, doxorubicin is gradually released into the interstitial space, and the subsequent uptake by surrounding cells is a limiting step in the delivery process. We recently demonstrated that plasma membrane-inserted short-chain sphingomyelin facilitates the cellular uptake of free doxorubicin. Here, we report that N-octanoyl-glucosylceramide acts equally potent but is itself less toxic. When coformulated with liposomal doxorubicin, this short-chain glycosphingolipid administered to cultured A431 epidermoid carcinoma cells led to superior (up to 4-fold) cellular doxorubicin accumulation and cytotoxicity, compared with control doxorubicin liposomes. These results were fully reproducible when N-octanoyl-glucosylceramide was postinserted into Caelyx, a commercial liposomal doxorubicin preparation. The doxorubicin-potentiating effect of N-octanoyl-glucosylceramide-enriched liposomes proved relatively insensitive to high serum concentrations, indicating that in vivo application is a feasible option. N-Octanoyl-glucosylceramide enrichment might thus represent a major improvement of conventional liposomal doxorubicin formulations.

Tumor and normal tissue pharmacokinetics of perifosine, an oral anti-cancer alkylphospholipid.

Clinical use of anti-cancer alkylphospholipids is limited by gastrointestinal toxicity. However, new interest has emerged since it was shown that these drugs enhance the cytotoxic effect of conventional chemotherapy and radiotherapy in preclinical models. The aim of this study was to characterize the pharmacokinetic profile of perifosine, an oral analog of alkylphosphocholine (APC), and to compare in vitro drug uptake with in vivo drug accumulation in three human-derived squamous cell carcinomas (A431, HNXOE and KB). In vitro, KB cells showed a remarkably high uptake and sensitivity for perifosine compared with A431 and HNXOE cells. In vivo, perifosine reached a clinically relevant plasma concentration in mice after a single oral dose of 40 mg/kg. Perifosine was not metabolized and displayed slow elimination, with a terminal half-life of 137 (+/- 20) hours and an apparent volume of distribution of 11.3 l/kg. Comparable tumor accumulation was observed for A431 and HNXOE tumors, whereas perifosine uptake by KB xenografts was substantially higher. Tissue distribution occurred throughout the whole body reaching high perifosine levels in the gastro-intestinal tract, while heart and brain tissue contained relatively low levels. Based on its stability and relatively high tumor uptake in vivo, perifosine is an attractive candidate for further evaluation, e.g. as radiosensitizer.