Liposomal polyethyleneglycol and polyethyleneglycol-peptide combinations for active targeting to liver in vivo

This report describes the development and evaluation of a range of polyethyleneglycol and polyethyleneglycol-peptide liposome formulations that effectively target liver in vivo. A 19-amino-acid sequence from the N-terminal region of the circumsporozoite protein of Plasmodium berghei was attached to the distal end of di22:1-aminopropane-polyethyleneglycol₃₄₀₀, and incorporated into liposomes containing di22:1-phosphatidylcholine and di22:1-phosphatidylethanolamine-polyethyleneglycol₅₀₀₀. By systematically varying the mole fractions of both the lipid-polyethyleneglycol and the lipid-polyethyleneglycol-peptide conjugates, and screening for serum-induced aggregation in vitro, a serum-stable range of formulations was established. These stable formulations were tested for binding to Hepa 1-6 liver cells in culture, and from these results three formulations were prepared for intravenous administration in mice. All three formulations exhibited effective liposome targeting to the liver, with approximately 80% of the total injected dose recovered in the liver within 15 min. Uptake by liver cells was more than 600-fold higher than uptake by those in the heart, and more than 200-fold higher than uptake by lung or kidney cells. Effective targeting to liver in vivo was successful after repeated (up to three) administrations to the host at 14-day intervals. All formulations prepared for in vivo administration were stable in the presence of serum, as measured by complete retention of entrapped calcein dye. The formulation with the lowest mole fractions of peptide and polyethyleneglycol was the most cost-effective in terms of encapsulation efficiency and minimal use of peptide and polymer compounds. The in vitro biophysical screening, followed by cell culture testing, reduced the number of animals required to develop an effective set of targeted liposome formulations for in vivo application.     

Depletion of glutathione by the hepatotoxins paracetamol and bromobenzene, and their non-hepatotoxic analogues, in a fortified liver microsomal system

The proposal that liver microsome mixed-function oxidase (MFO)-mediated generation of reactive metabolites may be detected by depletion of reduced glutathione (GSH) added to a fortified microsome incubation containing substrate has been investigated. Paracetamol, 3-hydroxyacetanilide, bromobenzene and p-bromophenol were used as substrates for a MFO-mediated depletion of GSH in vitro. Studies involving modulation of the extent of GSH depletion in vitro and comparison with published findings indicated that metabolism-mediated depletion of GSH in a fortified liver microsome incubation correlated with covalent binding of reactive metabolites but did not correlate well with the ability of compounds to cause GSH depletion in vivo or liver damage.     

Study of the effects of cardiovascular drugs in heart cell cultures

Compounds that produce myocardial pathology in vivo can be separated into two main classes—those that are directly toxic to the myocardium and those that are considered to act by way of an indirect vascular or neurologically based mechanism. An in vitro model of myocardium without nervous or systemic influences can be used to differentiate between direct myocardial cytotoxic effects and indirect cardiac pathology arising in vivo from exaggerated vascular or neural pharmacological effects of a number of drugs. In this study direct-acting cardiotoxic compounds are distinguished from those causing cardiac pathology by indirect mechanisms by their different pattern of effects in chick embryonic myocardial myocyte reaggregates (MMRs) cultures. The toxicity of the direct-acting cardiotoxic drugs allylamine (positive control, 50 μ ) and doxorubicin were compared with digoxin and isoprenaline, which show both direct and indirect mechanisms in vivo, and the indirectly acting hydralazine and pinacidil. Changes in spontaneous beating activity (SBA), leakage of lactate dehydrogenase (LDH) and cell morphology by light and transmission electron microscopy were used to assess toxicity. The MMRs were cultured for up to 24 hr in a series of concentrations of the five compounds in the range 0.1 to 10,000 μ . Allylamine, doxorubicin, digoxin and, to a lesser extent, isoprenaline were highly toxic to the MMRs, as shown by alterations in SBA, LDH leakage and cellular morphology. In contrast, hydralazine showed a very mild degree of toxicity at the highest concentrations in the absence of LDH leakage; treatment with pinacidil did not show any evidence of morphological degeneration but did cause a dose-related inhibition of SBA. These results are consistent with the view that doxorubicin and digoxin are directly toxic to myocardial cells and also suggests that this is an important mechanism in vivo for isoprenaline. The absence of a significant degree of toxicity with hydralazine and pinacidil is consistent with an indirect toxic mechanism.     

复方麝香黄芪滴丸的体外及体内血清化学成分分析

目的:应用超高效液相色谱-四级杆/静电场轨道阱高分辨质谱(UHPLC-Q-Orbitrap HRMS)对复方麝香黄芪滴丸的体外及体内血清化学成分进行快速分析鉴别.方法:采用Accucore C18(100 mm×2.1 mm,2.6 μm)色谱柱,以0.1％甲酸水溶液-甲醇梯度洗脱,流速0.2mL·min-1,进样量5...     

Effects of model inducers on thyroxine UDP-glucuronosyl-transferase activity in vitro in rat and mouse hepatocyte cultures.

Thyroxine (T₄)-UDP-glucuronosyltransferase (UGT) activity was measured directly in cultured male Sprague–Dawley rat and OF-1 mouse hepatocyte monolayers. The activity of T₄-UGT (pmol/min/g liver) in vitro in hepatocyte cultures was, after 24 hr in culture, equivalent to that previously measured in vivo in rat and mouse liver microsomes (Viollon-Abadie et al., 1999). A progressive decline in T₄-UGT activity occurred over time in both rat and mouse hepatocyte cultures. Treatment of cultures with various model inducers such as phenobarbital (PB), β-naphthoflavone (NF) and clofibric acid (CLO) induced a strong increase in T₄-UGT activity in rat hepatocyte monolayers. In addition, and as expected from available in vivo data, treatment of rat hepatocyte cultures with NF also increased p-nitrophenol (PNP)-UGT activity and treatment with PB or CLO increased bilirubin (Bili)-UGT activity. In contrast, T₄-UGT activity in mouse hepatocyte monolayers was not affected by the treatments, neither were PNP- and Bili- UGT activities. These in vitro data confirm our previous in vivo observations that these inducers increase rat but not mouse liver T₄-UGT activities (Viollon-Abadie et al., 1999). The present study thus demonstrates that hepatocyte monolayers are appropriated for the evaluation and inter-species comparison of the effects of xenobiotics on T₄-UGT activities.     

In vitro cytotoxicity tests for the prediction of acute toxicity in vivo

Investigations of the use of in vitro cytotoxicity tests for the prediction of acute toxicity in vivo have been reviewed with particular emphasis on those studies that have been published during the past 5 years. Numerous cell types, endpoints and exposure periods have been used in cytotoxicity tests, although these appear generally to have little effect on the resulting correlation between in vitro IC₅₀ values and in vivo LD₅₀ values. The in vitro data correlate better with rodent parenteral (ip or iv) LD₅₀ values than with oral LD₅₀ values due to kinetic considerations. For certain groups of related chemicals (e.g. antitumour compounds, metal salts), and for some sets of unrelated chemicals, the in vitro data correlate very well with LD₅₀ values. However, while cytotoxicity tests are useful for screening chemicals for their intrinsic and relative toxicities, it is impossible to tell whether predictions based on cytotoxicity data alone would be sufficiently accurate for labelling and classifying a new chemical according to its likely acute toxicity in vivo. The in vitro endpoints need to be of greater relevance to the possible mechanisms of chemically-induced acute toxicity in vivo than most of those that are used at present.     

Cadmium-induced metallothionein synthesis in the rat liver slice system

In vivo exposure of a rat to cadmium results in elevation of the hepatic metal-binding protein, metallothionein (MT). The present work describes the induction of MT in the in vitro liver slice system. Incubation of rat liver slices with CdCl₂ resulted in a dose-dependent elevation of tissue MT. The cadmium-binding protein was increased by 140 and 220% in the presence of 10 and 20 μ CdCl₂, respectively. A lower level (5 μ ) of the inducer had only a slight effect. A time-course study showed a gradual increase in the MT level following incubation of the liver slices for 4 and 6 hr with 10 μ -cadmium. Characterization of the metal-binding protein by Sephadex G-75 gel filtration revealed that it is composed of MT and also of a high-molecular-weight fraction that might be either a polymerized or aggregated form of MT, or another type of cadmium-binding protein. These findings indicate that the response of the liver slice system to toxic agents is similar to that of the intact animal. These are encouraging results which need to be extended before the system can be introduced into the routine screening of hepatotoxic agents.     

An interlaboratory assessment of the Eytex system

Seventeen raw materials and chemical formulations were evaluated in the Eytex System to determine the ability of this assay in vitro to predict eye irritation potential in vivo. All the test samples, which represented a wide range of chemical types and eye irritancy potential in vivo, were provided by one of the participating laboratories. Historical data from tests in vivo were available for each of the test samples, so testing in vivo specifically for this study was not necessary. Samples were evaluated by both the membrane partition assay (MPA) and the rapid membrane assay (RMA). The sensitivity, specificity, predictivity and equivalence of the Eytex assay were determined by comparison with the rabbit eye irritation data, using each of the different Eytex Draize Equivalent (EDE) classification schemes. Regardless of the classification scheme used, the correlation between the scores in vivo and in vitro was poor. The Eytex System consistently overclassified materials of low irritancy in vivo and underclassified those test materials of moderate irritancy or above. On the basis of the results from the 17 materials tested in this study, the Eytex System appears unsuitable as a replacement in vitro for ocular irritancy testing of all types of chemical. However, Eytex may have a place as a pre-screening method used as part of a test battery.     

Irreversible in vivo and in vitro binding of thiabendazole to tissue protein of pregnant mice

In vivo experiments showed that [¹⁴C]thiabendazole is irreversibly bound to protein of the liver and embryo in pregnant mice, but not to DNA and RNA in these tissues. In experiments carried out in vitro, the binding of [¹⁴C]thiabendazole to microsomal protein of the liver was proportional to time and protein concentration, was dependent upon the presence of oxygen and NADPH and was inhibited by carbon monoxide or SKF-525A, indicating that it was mediated by the cytochrome P-450-dependent monooxygenase system. The binding capacity of microsomes in various tissues was examined, and that of the liver microsomes was found to be more pronounced than that of other tissues.     

Development of an in vitro test system with human skin cells for evaluation of phototoxicity

Photosensitizing compounds may cause severe skin reactions after appropriate light exposure. On the cellular level this cutaneous in vivo response can be demonstrated by an in vitro model system with adequate target cells of human skin. Normal keratinocytes and fibroblasts were used to establish a predictive assay for phototoxic compounds. To develop a sensitive, specific and easy-to-handle screening system we determined different viability and proliferation parameters, like neutral red (NR) uptake, MTT-reduction, kenacid blue (KB) test, [³H]thymidine incorporation and a fluorometric assay in response to UVA light after treatment with different classes of compounds. These experiments resulted in the selection of a final test pattern, which we used to screen 26 substances of different phototoxic potential in vivo. We found a qualitative correlation between the phototoxic response in vitro and phototoxicity reported in different test systems in vivo.     

Preparation and pharmacokinetics of labeled derivatives of apamin

Apamin, the specific, centrally acting peptide neurotoxin from bee venom, was radiolabeled by different methods in order to study its in vitro and in vivo fate.

Iodination of the histidyl residue resulted in a derivative of diminished toxicity whose label was partially lost within 1 week. Furthermore, it was adsorbed on to Sephadex G-25 gel in a non-specific manner.

Acetylation with ³H-acetic anhydride yielded a slightly less toxic and, according to its behaviour on carboxymethyl cellulose, less basic derivative as compared with the starting material. Acetyl apamin was degraded in vitro by homogenates from liver, kidney, and brain, but not by serum, yielding ³H-acetic acid. In vivo it was degraded to tritiated water.

Reductive alkylation with formaldehyde and sodium borohydride resulted in methylated apamin possessing the basicity and about half the toxicity of the starting peptide. It was also degraded in vitro and in vivo, although to a lesser degree than acetyl apamin. Reductive alkylation appears to be the most favourable approach towards biologically active, labeled apamin.

The radiological or metabolic instability of the different apamin derivatives prevented a quantitative pharmacokinetic approach towards the in vivo fate of intact apamin. However, it is already evident that its acetyl or N-methyl derivatives are enriched not in the pharmacodynamic target organ, i.e. the central nervous system, but in the kidney.     

Nitrofurazone—Genotoxicity studies in mammalian cells in vitro and in vivo

Nitrofurazone has been examined for genetic effects in mammalian cells in vitro and in vivo. In the in vitro studies in Chinese hamster ovary (CHO) cells, metaphase analysis was carried out at different sampling times after nitrofurazone treatment and gene mutation leading to hypoxanthine-guanine phosphoribosyltransferase deficiency was also investigated. Both assays were carried out in the presence and absence of a metabolic activation system (S-9 mix). Metaphase analysis was also carried out on bone-marrow cells at various sampling times after treatment of rats with a single oral dose of nitrofurazone and at one sampling time after administration of five daily oral doses. In the in vitro studies a dose-related increase in aberrant metaphases was observed after nitrofurazone treatment both with and without metabolic activation, but the gene mutation assay was negative. In vivo, despite the use of high doses of nitrofurazone (up to 400 mg/kg body weight) resulting in evidence of toxicity and a reduction in the mitotic index of bone-marrow cells, there was no evidence that nitrofurazone increased chromosomal damage. It is concluded that although the compound has the capacity to cause chromosomal damage in mammalian cells in vitro, it is detoxified by the metabolic system of the animal. These results, together with existing in vivo data, do not suggest that nitrofurazone is likely to give rise to any genotoxic hazard for man.     

Eye irritation: Reference chemicals data bank

A list of 55 chemicals has been developed for which comprehensive in vivo rabbit eye irritation data are available. No new in vivo testing has been carried out to qualify a chemical for inclusion in the list. The 55 chemicals selected are available at high and consistent purity and are expected to be stable on storage. They have been tested undiluted in in vivo studies, except those chemicals where high concentrations of the substance could be expected to cause severe effects. The in vivo data have been generated since 1981 in studies carried out according to OECD Test Guideline 405 and following the principles of Good Laboratory Practice. The data were obtained from tests normally using at least three rabbits evaluated at the same time, involving instillation of 0.1 ml (or equivalent weight) into the conjunctival sac, and in which observations were made at least at 24, 48 and 72 hr after instillation. The chemicals represent a range of chemical classes (acetates, acids, alcohols, alkalis, aromatics, hydrocarbons, inorganics and surfactants) and different degrees of irritancy. They are ranked for eye irritation potential on the basis of a ‘modified maximum average score’. The reference data bank should be of use in validation tests for promising alternatives to the in vivo rabbit eye irritation test. This is an essential step in the progression to regulatory acceptance of alternative procedures.     

Development of a QSAR for worst case estimates of acute toxicity of chemically reactive compounds

Future EU legislations enforce a fast hazard and risk assessment of thousands of existing chemicals. If conducted by means of present data requirements, this assessment will use a huge number of test animals and will be neither cost nor time effective. The purpose of the current research was to develop methods to increase the acceptability of in vitro data for classification and labelling regarding acute toxicity. For this purpose, a large existing database containing in vitro and in vivo data was analysed. For more than 300 compounds in the database, relations between in vitro cytotoxicity and rat or mouse intravenous and oral in vivo LD50 values were re-evaluated and the possibilities for definition of mechanism based chemical subclasses were investigated.

A high in vitro–in vivo correlation was found for chemicals classified as irritants. This can be explained by a shared unspecific cytotoxicity of these compounds which will act as the predominant mode of action for both endpoints, irritation and acute toxicity. For this subclass, which covered almost 40% of all compounds in the database, the LD50 values after intravenous dosing could be predicted with high accuracy. A somewhat lower accuracy was found for the prediction of oral LD50 values based on in vitro cytotoxicity data.

Based on this successful correlation, a classification and labelling scheme was developed, that includes a hazard based definition of the applicability domain (irritants) and a prediction of the labelling of compounds for their acute iv and oral toxicity. The scheme was tested by an external validation.     

In vivo and in vitro microdialysis sampling of free fatty acids

Microdialysis is a technique that allows continuous sampling of compounds from the interstitial fluid of different tissues with minimal influence on surrounding tissues and/or whole body function. In the present study, the feasibility of using microdialysis as a technique to sample free fatty acids (FFA) was investigated both in vitro and in vivo, by use of a high molecular weight (MW) cut-off membrane (3 MDa) and a push–pull system to avoid loss of perfusion fluid through the dialysis membrane. The relative recovery was examined in vitro for three different concentrations of radiolabelled oleic acid-BSA solutions (oleic acid:BSA molar ratio 1:1) and for various temperatures and flow rates. The recovery of oleic acid was found to be dependent on the concentration of analyte in the medium surrounding the membrane (17.3%, 29.0% and 30.6% for 50, 100 and 200 μM oleic acid-BSA solutions, respectively). Addition of 0.25% BSA to the perfusion fluid resulted, however, in a concentration-independent recovery of 31.4%, 28.1% and 28.1% for the 50, 100 and 200 μM solutions, respectively.

The capability of the method to measure FFA together with glycerol was investigated in vivo in visceral adipose tissue of rats, before and after lipolytic treatment with the β₃-adrenergic agent, BRL37344. BRL37344 caused an increase in both dialysate FFA and glycerol, although the increase was markedly higher for glycerol, amounting to 24.5% and 329.2% increase from baseline, respectively. Subsequent in vitro test of probe performance revealed a decrease in the dialysing properties with regard to FFA, but not glycerol. This suggests that clogging of the membrane pores after 110 min prevented the measurement of the full FFA response in vivo.     

Validation of whole chick embryo cultures, whole rat embryo cultures and aggregating embryonic brain cell cultures using six pairs of coded compounds

A comparative study was performed to assess the effects of six pairs of coded compounds using cultures of whole chick and rat embryos as well as aggregating brain cell cultures. Developed originally for basic studies in developmental biology, these three culture systems have been adapted for the screening of chemicals in the field of prenatal toxicology. Chick and rat embryos were cultured for 2 days during the early stages of organogenesis. Aggregating cell cultures were prepared from early foetal rat telecephalon and grown for 14 days in a chemically defined medium. Concentration-response relationships were established by treating whole embryos in vitro for 2 days, and aggregating brain cell cultures for 9 days. After decoding the compounds, the results showed that, in the three test systems, specific effects were induced at comparable concentration levels. Similar compound-related malformations could be observed in both chick and rat whole embryo cultures. In aggregating brain cell cultures, neuron- and glia-specific effects could be distinguished. Based on the results obtained in the three in vitro systems, the following concentration ranges were determined for the teratogenic/toxic potencies of the test compounds (in mol/litre): <10⁻⁶: retinoids (Ro 13-6307, Ro 1-5488), 6-aminonicotinamide, ketoconazole; 10⁻⁶−10⁻³: 4-hydroxypyridine, sulfadiazine, sulfanilamide, caffeine, theophylline, metronidazole, methoxyacetic acid; >10⁻³: methoxyethanol. In general, the three in vitro test systems were found to provide concordant and complementary data on the toxicity and teratogenicity of a given compound. These data were also comparable with those available from in vivo studies. It is therefore concluded that such a test battery could contribute significantly to risk assessment and to the reduction of in vivo experimentation in reproductive toxicology.     

Determination of the hepatocellularity number for human, dog, rabbit, rat and mouse livers from protein concentration measurements

Biologically based scaling factors have to be used to predict in vivo metabolic clearance of xenobiotics from data obtained in vitro. Although standard values for the hepatocellularity numbers for different species are used in the literature, detailed information on the determination of these values has only been presented for humans and rats, and somewhat different results have been obtained in different studies. The present work was undertaken in order to determine the number of hepatocytes per gram of liver for human, dog, rabbit, rat and mouse livers. Hepatocellularity numbers were calculated from the ratio between the liver protein concentration and the protein concentration in the corresponding hepatocyte suspension. For human, rabbit, rat and mouse livers, the hepatocellular values were in the same range, more precisely 139 ± 25, 114 ± 20, 117 ± 30 and 135 ± 10 million cells per gram of liver, respectively. However, for the dog liver, the corresponding value was as high as 215 ± 45 million cells per gram. These values should be of importance during the scaling process of intrinsic clearance for xenobiotics in hepatocytes to in vivo hepatic clearance.     

Lipid microsphere formulation containing rifampicin targets alveolar macrophages

The study demonstrated that lipid microspheres (LM) containing rifampicin (LM-RFP) could deliver the drug to alveolar macrophages in vitro and in vivo, and that intranasal administration to animals could achieve preferential accumulation in the lungs with less effect on the liver. The LM-RFP particles had a mean diameter of 247.2 ± 75.7 nm, and their size remained stable when stored at 4°C or 25°C for at least 4 weeks. In vitro uptake of [³H]LM-RFP by alveolar macrophages was over 4 times higher than that of unencapsulated [³H]RFP, whereas the in vivo uptake was 30 times higher. Flow cytometric analysis and confocal laser scanning microscopy confirmed that LM could deliver the encapsulated drug effectively to alveolar macrophages in vitro and in vivo. Intranasal administration of [³H]LM-RFP to normal mice resulted in preferential pulmonary uptake of the drug and lower levels in the blood and liver compared with administration of unencapsulated [³H]RFP. In conclusion, LM-RFP could be a promising preparation for delivery via the respiratory tract to tuberculosis (TB) and TB/HIV patients.