Zebrafish embryotoxicity test for developmental (neuro)toxicity: Demo case of an integrated screening approach system using anti-epileptic drugs.

To improve the predictability of the zebrafish embryotoxicity test (ZET) for developmental (neuro)toxicity screening, we used a multiple-endpoints strategy, including morphology, motor activity (MA), histopathology and kinetics. The model compounds used were antiepileptic drugs (AEDs): valproic acid (VPA), carbamazepine (CBZ), ethosuximide (ETH) and levetiracetam (LEV). For VPA, histopathology was the most sensitive parameter, showing effects already at 60µM. For CBZ, morphology and MA were the most sensitive parameters, showing effects at 180µM. For ETH, all endpoints showed similar sensitivity (6.6mM), whereas MA was the most sensitive parameter for LEV (40mM). Inclusion of kinetics did not alter the absolute ranking of the compounds, but the relative potency was changed considerably. Taking all together, this demo-case study showed that inclusion of multiple-endpoints in ZET may increase the sensitivity of the assay, contribute to the elucidation of the mode of toxic action and to a better definition of the applicability domain of ZET.

A category approach to predicting the developmental (neuro) toxicity of organotin compounds: the value of the zebrafish (Danio rerio) embryotoxicity test (ZET).

Zebrafish embryos were exposed to different organotin compounds during very early development (<100h post fertilization). Morphology, histopathology and swimming activity (in a motor activity test) were the endpoints analyzed. DBTC was, by far, the most embryotoxic compound at all time points and endpoints studied. In fact, we observed a clear concordance between the effects observed in our zebrafish embryo model, and those observed with these compounds in full rodent in vivo studies. All organotin compounds classified as developmental (neuro) toxicants in vivo, were correctly classified in the present assay. Together, our results support the ZET model as a valuable tool for providing biological verification for a grouping and a read-across approach to developmental (neuro) toxicity.

The bovine oocyte in vitro maturation model: a potential tool for reproductive toxicology screening.

Reproductive toxicity testing according to the present guidelines requires a high number of animals. Therefore, the development of alternative in vitro methods is urgently required. The aim of the present study was to investigate the applicability domain of the bovine oocyte in vitro maturation assay (bIVM) to study female reproductive toxicology. Therefore, bovine oocytes were exposed to a broad set of chemicals of two distinct biological function groups: (a) affecting female fertility and (b) affecting embryonic development and having a broad range of physical and chemical properties. The endpoints evaluated were the oocyte nuclear maturation (progression of meiosis) and general cytotoxicity. The oocyte nuclear maturation was negatively affected by all compounds tested and the effect was observed at concentrations lower than the cytotoxic ones. The bIVM assay correctly predicted the classification of compounds between those predefined groups. Additionally, the bIVM model contributes significantly for the 3R principle, since no test animals are used in this assay. In conclusion, the bIVM is a sensitive and valuable alternative assay to identify potential chemical hazard on female fertility.

Potentially increased sensitivity of pregnant and lactating female rats to immunotoxic agents.

Characteristic susceptibility to environmental and pharmaceutical exposure may occur during periods in life of marked histophysiological changes of the immune system. Perinatal development is such a period; pregnancy followed by lactation is potentially another one. Here, we explored the influence of pregnancy and lactation on the model immunotoxic compound di-n-octyltin dichloride (DOTC) in rats using clinical and histopathological parameters. Female rats were exposed to 0, 3, 10, or 30 mg DOTC/kg feed during pregnancy and up to 20 (at weaning) or 56 days after delivery. Age-matched nonmated females were exposed during the same time periods. DOTC at the level of 10 and 30 mg/kg decreased thymus weight and affected thymus morphology in the lactating rats. In addition, DOTC decreased the numbers of neutrophils in the lactating rats. These effects were no longer apparent at day 56 despite continuous exposure to DOTC. This explorative study indicates that the innate and adaptive immune system may be especially sensitive to immunotoxicants during pregnancy and lactation.

Efficacy of pulmonary insulin delivery in diabetic rats: use of a model-based approach in the evaluation of insulin powder formulations.

The potential of N-trimethyl chitosan (TMC) with two degrees of quaternization (DQ), TMC20 (DQ 20%, as a mucoadhesive) and TMC60 (DQ 60%, as a mucoadhesive and a permeation enhancer), and dextran (as a non-mucoadhesive and non-permeation enhancer) microparticles as carriers for pulmonary delivery of insulin was studied in diabetic rats. The impact of the powder formulation on insulin bioavailability and its pharmacological effect was evaluated using a population pharmacokinetic-pharmacodynamic (PKPD) model. Insulin-loaded microparticles were prepared by a supercritical fluid (SCF) drying technique. They had a median volume diameter and median volume aerodynamic diameter of about 6-10 microm and 4 microm, respectively. The PK of insulin in the diabetic rats was analyzed by a one-compartment disposition model and the PD was described by the minimal model of glucose disappearance. The bioavailability of the pulmonarily administered dextran-, TMC20- and TMC60-insulin microparticles relative to subcutaneously (SC) administered insulin, was 0.48, 0.59 and 0.95, respectively. Histological examinations of the rats' lungs did not show any local adverse reactions after single administration of insulin powders. The pharmacodynamic model could describe the insulin-glucose relationship and pharmacodynamic efficiency of insulin formulations, which was about 0.6(*)10(-5) ml/microU, irrespective of the formulations. The current findings suggest that TMC microparticles are a promising vehicle for pulmonary delivery of insulin.

Plasmid CpG depletion improves degree and duration of tumor gene expression after intravenous administration of polyplexes.

PURPOSE: Tumor gene expression after the intravenous (i.v.) administration of current polymer-based gene delivery systems is generally low and short-lived. Immune stimulatory CpG dinucleotides, present within the plasmid DNA of the polyplexes are likely to contribute to this. The effect of CpG replacement on the levels of transgene expression was studied, after the i.v. administration of polyethylenimine (PEI) polyplexes. METHODS: Tumor transfection and immune stimulation of PEI polyplexes containing plasmid DNA encoding for luciferase and rich in CpG motifs was monitored and compared to polyplexes containing the same gene but devoid of CpG motifs. Lipoplexes based on 1,2-dioleyl-3-trimethylammonium-propane/dioleoylphosphatidylethanolamine liposomes were included as a control. RESULTS: The replacement of CpGrich DNA by CpGfree DNA did neither affect the physical properties of the DNA complexes nor did it affect their in vitro transfection activity or cytotoxicity. The immune stimulation (interleukin-12) after i.v. administration of the PEI DNA complexes was low and unaffected by the presence of CpG motifs. The absence of CpG motifs within the different DNA complexes improved the degree and the duration of organ and tumor gene expression. CONCLUSION: The depletion of CpG dinucleotides within the plasmid DNA of polyplexes enhances the degree and duration of in vivo transgene expression.

Biodegradable, cationic methacrylamide-based polymers for gene delivery to ovarian cancer cells in mice.

A series of cationic, methacrylamide polymers was tested for use as a biodegradable gene carrier in ovarian cancer. Tumor transfection activity of polyplexes consisting of a reporter gene and different methacrylamide polymers was assessed, after intraperitoneal injection in mice bearing an ovarian cancer xenograft. In this model, polyplexes based on poly(HPMA-DMAE) showed transfection activity similar to polyplexes based on the nondegradable and rather toxic polyethylenimine (PEI22). The tumor transfection activity of the pHPMA-DMAE polyplexes was remarkable considering their poor transfection activity in in vitro assays. Polyplexes based on pHPMA-DMAE were devoid of any cytotoxicity and mediated highest transfection activity at the highest N/P ratio investigated. Tumor cell gene expression after a single administration of these polyplexes rapidly declined within time, at a similar rate to that observed after injection with polyplexes based on PEI22. Incubation of the polyplexes with hyaluronic acid (HA), a polyanion accumulating in the ascitic fluid of ovarian cancer bearing mice, changed the physical characteristics of the pHPMA-DMAE and PEI22 polyplexes. The transfection activity of PEI22-based polyplexes, but not that of pHPMA-DMAE based polyplexes, was strongly impaired by HA. Differences in HA sensitivity might have contributed to the in vivo gene expression activities of pHPMA-DMAE- and PEI22-based polyplexes. pHPMA-DMAE-based polyplexes have potential for use in ovarian cancer therapy due to their considerable transfection activity, their low cytotoxicity, and their HA resistance.

Hydrolysable core-crosslinked thermosensitive polymeric micelles: synthesis, characterisation and in vivo studies.

In this study, core-crosslinked (CCL) biodegradable thermosensitive micelles based on mPEG(5000) and N-(2-hydroxyethyl)methacrylamide)-oligolactates (mPEG-b-p(HEMAm-Lac(n))) were synthesised and their properties investigated. Rapidly heating aqueous solutions of partially methacrylated block copolymers to above their critical micelle temperature (CMT), followed by illumination in presence of a photoinitiator yielded almost monodisperse CCL micelles with a size of 68+/-7 nm. Either below the CMT or after addition of sodium dodecyl sulphate, the non-crosslinked (NCL) micelles rapidly disintegrated whereas the CCL micelles kept their integrity. NCL micelles fell apart after 5h in pH 7.4 at 37 degrees C as a result of the hydrolysis of lactate side chains, whereas the CCL micelles had a much higher stability and only degraded after cleavage of the ester bonds in the crosslinks. The circulation kinetics and biodistribution of CCL micelles were considerably better than those of NCL micelles, i.e., 58% of the injected dose (ID) of CCL versus 6% of NCL micelles was recovered in the circulation 4h post-injection. Furthermore, the liver uptake of the CCL micelles (10% ID) was much lower than that of the NCL micelles (24% ID) 4h after administration, while tumour accumulation was almost 6 times higher for the CCL micelles. Likely, NCL micelles dissociated after i.v. administration and/or were opsonised and captured by macrophages while the dense PEG shell of CCL micelles made them less prone towards opsonisation. The excellent physical stability of these degradable CCL micelles and very favourable biodistribution profile renders them very suitable for drug targeting purposes.

Effect of liposome characteristics and dose on the pharmacokinetics of liposomes coated with poly(amino acid)s.

Long-circulating liposomes, such as PEG-liposomes, are frequently studied for drug delivery and diagnostic purposes. In our group, poly(amino acid) (PAA)-based coatings for long-circulating liposomes have been developed. These coatings provide liposomes with similar circulation times as compared to PEG-liposomes, but have the advantage of being enzymatically degradable. For PEG-liposomes it has been reported that circulation times are relatively independent of their physicochemical characteristics. In this study, the influence of factors such as PAA grafting density, cholesterol inclusion, surface charge, particle size, and lipid dose on the circulation kinetics of PAA-liposomes was evaluated after intravenous administration in rats. Prolonged circulation kinetics of PAA-liposomes can be maintained upon variation of liposome characteristics and the lipid dose given. However, the use of relatively high amounts of strongly charge-inducing lipids and a too large mean size is to be avoided. In conclusion, PAA-liposomes represent a versatile drug carrier system for a wide variety of applications.

Biodegradable poly(2-dimethylamino ethylamino)phosphazene for in vivo gene delivery to tumor cells. Effect of polymer molecular weight.

PURPOSE: Previously, we have shown that complexes of plasmid DNA with the biodegradable polymer poly(2-dimethylamino ethylamino)phosphazene (p(DMAEA)-ppz) mediated tumor selective gene expression after intravenous administration in mice. In this study, we investigated the effect of p(DMAEA)-ppz molecular weight on both in vitro and in vivo tumor transfection, as well as on complex induced toxicity. MATERIALS AND METHODS: p(DMAEA)-ppz with a broad molar mass distribution was fractionated by preparative size exclusion chromatography. Polyplexes consisting of plasmid DNA and the collected polymer fractions were tested for biophysical properties, (cyto)toxicity and transfection activity. RESULTS: Four p(DMAEA)-ppz fractions were collected with weight average molecular weights ranging from 130 to 950 kDa, and with narrow molecular mass distributions (Mw/Mn from 1.1 to 1.3). At polymer-to-DNA (N/P) ratios above 6, polyplexes based on these polymers were all positively charged (zeta potential 25-29 mV), and had a size of 80-90 nm. The in vitro cytotoxicity of the polyplexes positively correlated with polymer molecular weight. The in vitro transfection activity of the different polyplexes depended on their N/P ratio, and was affected by the degree of cytotoxicity, as well as the colloidal stability of the different polyplexes. Intravenous administration of polyplexes based on the high molecular weight polymers led to apparent toxicity, as a result of polyplex-induced erythrocyte aggregation. On the other hand, administration of polyplexes based on low molecular weight p(DMAEA)-ppz's (Mw 130 kDa) did not show signs of toxicity and resulted in tumor selective gene expression. CONCLUSION: Polymer molecular weight fractionation enabled us to optimize the transfection efficiency/toxicity ratio of p(DMAEA)-ppz polyplexes for in vitro and in vivo tumor transfection.

Pharmacokinetics of poly(hydroxyethyl-l-asparagine)-coated liposomes is superior over that of PEG-coated liposomes at low lipid dose and upon repeated administration.

'Stealth' liposomes with a poly(ethylene glycol) (PEG) coating are frequently studied for drug delivery and diagnostic purposes because of their prolonged blood circulation kinetics. However, several recent reports have demonstrated that PEG-liposomes are rapidly cleared at single low lipid doses (<1 micromol/kg) and upon repeated administration (time interval between the injections 5 days-4 weeks). Recently, poly(amino acid)-based stealth liposome coatings have been developed as alternative to the PEG-coating. In this study, the pharmacokinetic behavior of liposomes coated with the poly(amino acid) poly(hydroxyethyl-l-asparagine) (PHEA) was evaluated at low lipid doses and upon repeated administration in rats. Blood circulation times and hepatosplenic localization of PHEA-liposomes were assessed after intravenous injection. When administered at a dose of 0.25 micromol/kg or less, PHEA-liposomes showed significantly longer blood circulation times than PEG-liposomes. A second dose of PHEA-liposomes 1 week after the first injection was less rapidly cleared from the circulation than a second dose of PEG-liposomes. Although the mechanisms behind these observations are still not clear yet, the use of PHEA-liposomes appears beneficial when single low lipid doses and/or repeated dosing schedules are being applied.

Poly(amino acid)s: promising enzymatically degradable stealth coatings for liposomes.

Poly(amino acid)s (PAAs) were evaluated as coating polymers for long-circulating liposomes. The pharmacokinetics of PAA-coated liposomes were assessed in rats. Prolonged circulation times were obtained, comparable to those reported for poly(ethylene glycol) (PEG)-liposomes. Besides, the enzymatic degradability of PAAs was studied. PAAs - in free as well as liposome-associated form - are degradable by proteases, which is beneficial for reducing the risks of accumulation in vivo. Furthermore, complement activation by PAA-liposomes was evaluated in vitro and in vivo. Like other liposome types, they appear to activate the complement system. However, a role of endotoxin contamination of the PAA-liposome formulations used cannot be excluded in our complement activation studies.

Effect of cationic carriers on the pharmacokinetics and tumor localization of nucleic acids after intravenous administration.

Nucleic acid based therapeutics are currently being studied for their application in cancer therapy. In this study, the effect of different cationic delivery systems on the circulation kinetics, tumor localization, and tissue distribution of short interfering RNA (siRNA) and plasmid DNA (pDNA) was examined, after intravenous administration in mice bearing a s.c. Neuro 2A tumor. Nanosized particles were formed upon complexation of siRNA with the cationic liposome formulation DOTAP/DOPE and the targeted, cationic polymer RGD-PEG-PEI. Both the circulation kinetics and the overall tumor localization of the siRNA complexes were similar to non-complexed siRNA. Importantly, the different carriers changed the intratumoral distribution of siRNA within the tumor. pDNA was effectively condensed with linear polyethylenimine (PEI), PEGylated linear PEI (PEG-PEI) or poly(2-dimethylamino ethylamino)phosphazene. Only PEG-PEI was able to improve the pDNA circulation kinetics. All pDNA complexes yielded similar pDNA tumor localization (1% of the injected dose, 60 min after administration). We conclude that the level of nucleic acid tumor localization is independent on the type of formulation used in this study. Therefore, the value of carrier systems for the intravenous delivery of nucleic acids cannot be solely attributed to benefits relevant during the transport towards the tumor. Rather, the benefits are arising from carrier-induced changes in the intratumoral fate of the nucleic acids.

Application of poly(2-(dimethylamino)ethyl methacrylate)-based polyplexes for gene transfer into human ovarian carcinoma cells.

Previously, attempts were made in our laboratory to transfect human ovarian cancer (OVCAR-3) cells, growing in the peritoneal cavity of nude mice, by intraperitoneal administration of poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA)-based polyplexes. However, hardly any transfection of the OVCAR-3 cells was observed. The aim of the present study was to examine whether pDMAEMA-polyplexes can transfect OVCAR-3 cells in vivo at DNA doses much higher than used previously [J. Gene Med. 1 (1999) 156-158]. We also explored a specific targeting strategy based on the use of folic acid (FA) as a targeting ligand directed against the folate receptor overexpressed on OVCAR-3 cells. Luciferase expression by OVCAR-3 cells mediated by pDMAEMA-based polyplexes was evaluated in the mouse i.p. OVCAR-3 xenograft model of ovarian cancer. By virtue of new formulation options, we were able to administer polyplex dispersions into OVCAR-3 bearing mice at much larger doses (75-120 microg DNA) than used previously (15 microg). The feasibility of folate-mediated targeting of the polyplexes was studied after coupling of FA to preformed polyplexes with poly(ethylene glycol) (PEG) as a spacer. Intraperitoneal administration of naked pLuc plasmid did not result in significant gene expression by the tumor cells. Administration of uncoated, positively charged pDMAEMA-based polyplexes at a DNA dose of 75-120 microg yielded significant transfection activity. However, also considerable gene expression was observed in non-target cells. To avoid transfection of non-target cells, an active targeting strategy based on the use of FA was studied. At a dose of 75 microg DNA (N/P 5), the folate-targeting approach yielded about 10-fold lower luciferase transfection levels in organs lined by the mesenthelial layer. This beneficial site-avoidance effect was achieved without compromising the degree of tumor cell transfection. Successful transfection of OVCAR-3 cells growing in the peritoneal cavity of nude mice can be achieved by i.p. administration of polyplexes at doses between 75 and 120 microg DNA. It was further demonstrated that active targeting of polyplexes to OVCAR-3 cells growing in the peritoneal cavity of mice is a realistic possibility to avoid transfection of non-target cells.

In vivo tumor transfection mediated by polyplexes based on biodegradable poly(DMAEA)-phosphazene.

In recent years, increasing interest is being paid to the design of transfectants based on non-toxic and biodegradable polymers for gene therapy purposes. We recently reported on a novel, biodegradable polymer, poly(2-dimethylamino ethylamino)phosphazene (p(DMAEA)-ppz) for use in non-viral gene delivery. In this study, the biodistribution and in vivo transfection efficiency of polyplexes composed of plasmid DNA and p(DMAEA)-ppz were investigated after intravenous administration in tumor bearing mice. Data were compared with those of polyplexes based on the non-biodegradable polyethylenimine (PEI 22kDa). Both polyplex systems were rapidly cleared from the circulation (<7% ID, at 60 min after administration) and showed considerable disposition in the liver and the lung, all in line with earlier work on cationic polyplex systems. The lung disposition is attributed to aggregates formed by interaction of the polyplexes with blood constituents. Redistribution of the polyplexes from the lung was observed for both polyplex formulations. Importantly, both polyplex systems showed a substantial tumor accumulation of 5% and 8% ID/g for p(DMAEA)-ppz and PEI22 polyplexes, respectively, at 240 min after administration. The tumor disposition of the p(DMAEA)-ppz and PEI22 polyplexes was associated with considerable expression levels of the reporter gene. In contrast to PEI22 polyplexes, p(DMAEA)-ppz polyplexes did not display substantial gene expression in the lung or other organs (organ gene expression<1/100 of tumor gene expression). The observed preferential tumor gene expression mediated by the p(DMAEA)-ppz polyplexes enables the application of this polymer to deliver therapeutic genes to tumors.

A novel family of L-amino acid-based biodegradable polymer-lipid conjugates for the development of long-circulating liposomes with effective drug-targeting capacity.

The objective of this study was to develop biodegradable polypeptide-lipid conjugates for the design of polymer-coated long-circulating liposomes (LCL). Lipid conjugates of poly(hydroxyalkyl L-asparagine/L-glutamine) were synthesized and incorporated into 0.15 microm dipalmitoyl phosphatidylcholine (DPPC)-cholesterol liposomes. Circulation times and biodistribution were assessed in rats using a radioactive lipid marker. Evaluation of the therapeutic activity of prednisolone phosphate loaded in 0.1 microm PHEA-DPPC-cholesterol liposomes in a rat experimental arthritis model was performed to demonstrate the drug-targeting potential of the polymer-coated liposomes. Coating of liposomes with poly(hydroxyethyl L-asparagine) (PHEA) and poly(hydroxyethyl L-glutamine) (PHEG) extended the circulation half-life to a similar extent as poly(ethylene glycol) (PEG), which is normally used for the preparation of LCL. Glutamine polymers with a hydroxypropyl or a hydroxybutyl group instead of hydroxyethyl group also yield prolonged circulation, however, not to the same extent as PHEA/G. The pharmacokinetic properties of PHEA-liposomes were independent of the lipid dose even at very low lipid doses of around 50 nmol per rat. PLP was successfully entrapped in PHEA-liposomes. These liposomes were shown to be stable in the circulation and equally effective in rat experimental arthritis as PLP encapsulated in PEG-liposomes. PHEA and PHEG are attractive alternative polymers for the design of LCL: their performance is similar to that of PEG-liposomes but they have the advantage of being biodegradable.