Identification of candidate synovial membrane biomarkers after Achyranthes aspera treatment for rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disease whose main symptom is a heightened inflammatory response in synovial tissues. To verify the anti-arthritic activities of Achyranthes aspera and its possible therapy-related factors on the pathogenesis of RA, the saponins in A. aspera root were isolated and identified to treat the collagen-induced arthritis (CIA) rats. Phytochemical analysis isolated and identified methyl caffeate, 25-S-inokosterone, 25-S-inokosterone ß-D-glucopyranosyl 3-(O-ß-D-glucopyranosyloxy)-oleanolate, and ß-D-glucopyranosyl 3-(O-ß-D-galactopyranosyl (1→2)(O-ß-D-glucopyranosyloxy)-oleanolate as main compounds in the root of A. aspera. Proteomics was performed to determine the differentially expressed proteins in either inflamed or drug-treated synovium of CIA rats. Treatment resulted in dramatically decreased paw swelling, proliferation of inflammatory cells, and bone degradation. Fibrinogen, procollagen, protein disulfide-isomerase A3, and apolipoprotein A-I were all increased in inflamed synovial tissues and were found to decrease when administered drug therapy. Furthermore, Alpha-1-antiproteinase and manganese superoxide dismutase were both increased in drug-treated synovial tissues. The inhibition of RA progression shows that A. aspera is a promising candidate for future treatment of human arthritis. Importantly, the total saponins found within A. aspera are the active component. Finally, autoantigens such as fibrinogen and collagen could act as inducers of RA due to their aggravation of inflammation. Given this, it is possible that the vimentin and PDIA3 could be the candidate biomarkers specific to Achyranthes saponin therapy for rheumatoid arthritis in synovial membrane.

Efficient targeting to alveolar macrophages by intratracheal administration of mannosylated liposomes in rats.

The success of targeting systems to alveolar macrophages critically depends on internalization into these cells for pharmacological intervention. Direct respiratory delivery via inhalation of mannose modified liposomal carriers to alveolar macrophages is of great interest. To evaluate the targeting efficiency to alveolar macrophages by intratracheal administration of mannosylated liposomes (Man-liposomes), Man-liposomes with various ratio of mannosylated cholesterol derivatives, cholesten-5-yloxy-N-(4-((1-imino-2-D-thiomannosylethyl)amino)alkyl)formamide (Man-C4-Chol) as mannose receptor ligand were investigated with regard to their in vitro uptake in primary cultured alveolar macrophages and in vivo intratracheal administration in rats. The in vitro uptake of Man-liposomes took place in a concentration-dependent manner. The internalization of Man-liposomes with 7.5% (Man-7.5-liposomes) and 5.0% (Man-5.0-liposomes) Man-C4-Chol was considerably higher than that of Man-liposomes with 2.5% of Man-C4-Chol (Man-2.5-liposomes) and Bare-liposomes and significantly inhibited by an excess of mannan, suggesting mannose receptor-mediated endocytosis. After intratracheal administration of Man-7.5 and Man-5.0-liposomes in rats, a significantly high internalization and selective targeting to alveolar macrophages was observed. The enhanced cellular uptake in alveolar macrophages related to the mannose density of Man-liposomes was also confirmed both in vitro and in vivo confocal microscopy studies. These results demonstrate the efficient targeting to alveolar macrophages by the intratracheally administered Man-liposomes via mannose receptor-mediated endocytosis.

[Liver targeting of cationic liposomes modified with soybean-derived sterylglucoside in vitro].

AIM: To construct a liposomal liver targeting delivery system by adding soybean-derived sterylglucoside (SG) to the cationic liposomes. METHODS: The physico-chemical properties of SG modified cationic lipsomes were investigated using fluorescein sodium (FS) as a model drug, as well as the interaction of SG modified liposomes with HepG2 2. 2. 15 cells in the point of involvement of asialoglycoprotein receptor (ASGP-R) mediated transfection. Liver targeting of modified cationic liposomes were also investigated using liver perfusing technique, and hepatocytes and non-hepatocytes were separated and examined after perfusing. RESULTS: All the formula yielded high incorporation efficiency (83.12% - 91.74%), small particle size (93.0 - 124.4 nm). The zeta potential of blank liposomes all showed positive values. The transfection efficiency of FS entrapped in SG-liposomes with HepG2 2.2. 15 was significantly higher than that of liposomes without modification. The transfection of SG-liposomes were reduced significantly by the 20/30 mmol galactose as a competitor of ASGP-R. All the cationic liposomes showed high level of liver uptake of FS. Compared with the uptake of non-hepatocytes of each respectively, only SG/Brij-35 liposomes showed difference in FS uptake by hepatocytes (P < 0.05). CONCLUSION: It showed that SG/Brij-35 modified cationic liposomes are potentially useful drug carrier to liver but may be affected by different modification.

Application of sterylglucoside-containing particles for drug delivery.

Recent advances in biotechnology have promoted biomolecular targeting of drugs, peptides and genes in the treatment and management of major diseases and infections. Therapeutic development of drugs and delivery systems may have various objectives: Systemic drugs require optimal delivery and uptake at target sites; peptide drugs require alternative routes of administration, such as nasal or intestinal absorption; gene medicines need to be delivered efficiently, safely and selectively to diseased areas. The propensity of ligand-modified liposomes to carry drugs and genes to desirable sites has been extensively examined and current reports show considerable progress in this field. Sterylglucoside (SG) is a novel absorption-enhancer of peptide drugs across nasal and intestinal mucosae. Physico-chemical properties and biodistribution of liposomes incorporating SG were studied and compared against the profiles of aglycon and sitosterol derivatives of SG. It was shown that SG particles aided colon drug delivery and increased bioavailability of peptide drugs after nasal and intestinal administration. In addition, they were able to enhance anticancer effects in liver cancer chemotherapy. Biological fate and interaction of SG with hepatocytes support the novel proposition of liver-targeting SG-liposomes.

[Biodistribution and hepatocytes targeting of cationic liposomes surface-modified with sterylglucoside and golyethylene glycol].

AIM: To investigate the biodistribution and the hepatocytes targeting of cationic liposome containing 3beta[N-( N',N'-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) and surface-modified liposomes with sterylglucoside (SG) and polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE). METHODS: Cationic liposomes (CL) composed of DC-Chol and dipalmitoylphosphatidylcholine (DPPC), SG/PEG modified cationic liposome (SG/PEG-CL), both contained trace 3H-cholesterol (3H-Chol) as radiolabel, were prepared. The liposomes encapsulating 125I-labled antisense oligodeoxynucleotide (125I-asODN) (SG/PEG-CL-asODN) were also prepared. The biodistribution of CL, SG/PEG-CL, SG/PEG-C2-asODN as well as 125I-asODN solution, were studied. The radioactivities in hepatocytes and non-hepatocytes after administration of CL and SG/PEG-CL were determined by infuseing method. RESULTS: CL and SG/PEG CL significantly aggregated in liver. The distribution of SG/PEG CL was significantly higher in hepatocytes (P < 0.01) and lower in non-hepatocytes (P < 0.01) than that of CL. The concentrations of SG/PEG-CL-asODN in liver and spleen were significantly higher than that of asODN solution (P < 0.01). CONCLUSION: Cationic liposome modified with SG/PEG changed the distribution of asODN. Cationic liposome can target hepatocytes more effective after being modified with SG.

Chromosomal abnormality rate in human pre-embryos derived from in vitro fertilization cycles cultured in the presence of Follicular-Fluid Meiosis Activating Sterol (FF-MAS).

BACKGROUND: The objective of the study was to investigate the effect of Follicular-Fluid Meiosis Activating Sterol (FF-MAS) when added to the culture media on the incidence of chromosomal abnormalities and pre-embryo development in human pre-embryos. METHODS: 243 women undergoing IVF/ICSI treatment donated 353 oocytes in a multicentre, prospective, randomized, double blind, four-arm, controlled trial performed at Danish and Swedish public and private IVF centers. Metaphase II oocytes were randomly assigned to: FF-MAS 5 microM, FF-MAS 20 microM, ethanol 0.2% (vehicle control) or water for injection (inert control). The exposure regimen of FF-MAS to the human oocytes was 4 h prior to fertilization by ICSI and 20 h exposure post ICSI. The primary endpoint was the incidence of numerical chromosomal abnormalities. Secondary endpoints were cleavage rate and pre-embryo quality. RESULT: On the pre-embryo level, no significant differences in chromosomal abnormality rate were observed among the four groups. However, the percentage of uniformly normal pre-embryos was significantly lower in the pooled FF-MAS group (5 microM: 12% and 20 microM: 17%) than in the pooled control group (inert control 32% and vehicle control 42%). A high level of mosaicism (41-60%) was found in all groups. At the blastomere level, the percentage of blastomeres categorized as normal was significantly lower in the FF-MAS 5 microM group (41%) and the FF-MAS 20 microM (29%) group versus the inert (52%) and the vehicle (61%) groups. Significantly reduced cleavage and good quality pre-embryo rates were found in both FF-MAS groups. CONCLUSION: FF-MAS increased the rate of aneuploidy and had detrimental effects on cleavage and pre-embryo development, when exposed both before and after fertilization.

Evaluation of the anti-inflammatory activity of zhankuic acids isolated from the fruiting bodies of Antrodia camphorata.

We have previously shown that a concentrated ethanol extract of the fruiting bodies of Antrodia camphorata exhibited immunomodulating effects in human leukocytes and fourteen compounds including zhankuic acids A, B, C, and antcin K were identified in the extract. In this study, an acute cellular model in isolated peripheral human neutrophils was established to elucidate the anti-inflammatory effects of these compounds. Reactive oxygen species (ROS) production and firm adhesion by neutrophils display two important responses during inflammation. To evaluate whether these compounds could prevent inflammatory responses by neutrophils, their effects on N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate-13-acetate (PMA)-activated peripheral human neutrophils were examined. Pretreatment with 1 - 25 microM of zhankuic acids A, B, C, or antcin K concentration-dependently diminished fMLP- or PMA-induced ROS production, as measured by a lucigenin-amplified chemiluminescence, with IC (50) (microM) around 5 - 20 microM. Zhankuic acids A, B, C, or antcin K also effectively inhibited the fMLP- or PMA-induced firm adhesion without interfering with the up-expression of surface Mac-1 (CD11b/CD18), a beta2 integrin mediating the firm adhesion of neutrophils to endothelium. The anti-inflammatory actions of these drugs were not due to cytotoxic effects because no significant difference in cell viability was observed compared to vehicle control. These data suggest that inhibition of both ROS production and firm adhesion by neutrophils has no significant cytotoxic effect that could give these drugs the potential to be anti-inflammatory agents for the clinical treatment.

Effects on M5076-hepatic metastasis of retinoic acid and N-(4-hydroxyphenyl) retinamide, fenretinide entrapped in SG-liposomes.

Retinoic acid (RA), a potent inducer of cell differentiation, and N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide), a potent inducer of apoptosis, are well known as anticancer agents that are administered orally to patients for leukemia, breast and prostate cancer, respectively. However, it has not been studied whether both retinoids are effective on metastatic cancer. In mice implanted with M5076 cells, murine reticulum cell sarcoma survival times were prolonged by i.v. treatment of RA and 4-HPR entrapped in liposomes containing soybean-derived sterylglucoside mixture (SG), which accumulates in liver. In contrast, free RA and 4-HPR were inactive. These results indicate that RA and 4-HPR in SG-liposomes exhibit anticancer efficacy on metastatic cancers, and may have great potential for clinical use in the treatment of various cancers.

Stability and antitumor effects of all-trans retinoic acid-loaded liposomes contained sterylglucoside mixture.

The aim of this study was to develop an intravenous delivery of all-trans retinoic acid (ATRA) for the treatment of cancer. Two kinds of liposomes composed of dipalmitoylphosphatidylcholine and cholesterol with sterylglucoside (SG) mixture (SG liposomes) or without SG (non-SG liposomes) were prepared. A limited amount of ATRA was incorporated into liposomes approximately 3mol%. ATRA-loaded SG liposomes were more stable at 4 degrees C with light protection for 24 days than non-SG liposomes; maintaining 83.1% ATRA and the average diameter 198.5 nm (36 days), and in 80% rat serum for 120 min. SG seems to increase the ATRA-loaded efficiency in liposomes and stability of liposomes compared with cholesterol. The mean survival time of mice given SG liposomes (18.2 days) indicated a greater antitumor activity than saline (14.1 days) (P<0.001) without change of mean body weight. It is concluded that the current ATRA-loaded SG liposomes are potentially applicable for hepatic metastasis of M5076 tumor.

Physiology of meiosis-activating sterol: endogenous formation and mode of action.

BACKGROUND: In the context of mammalian oocyte maturation, it has been suggested that intermediates of cholesterol biosynthesis may represent the physiological signal that instructs the oocyte to reinitiate meiosis. METHODS: Endogenous levels of follicular fluid meiosis-activating sterol (FF-MAS) were monitored in rabbit ovarian tissue, and the influence of exogenous gonadotrophins on sterol formation was assessed. The involvement of cAMP in FF-MAS-induced versus spontaneous oocyte maturation in vitro in mice was also investigated, as was the direct microinjection of FF-MAS into mouse oocytes. RESULTS: Levels of FF-MAS in rabbit ovaries were significantly elevated 1 h after hCG/LH induction and remained so for 4 and 12 h after induction. In naked oocytes undergoing spontaneous maturation, a significant decrease in cAMP was detected after 30 min of culture. However, FF-MAS-mediated induction of oocyte maturation in hypoxanthine-arrested naked oocytes was not associated with any detectable decrease in intracellular cAMP levels. Microinjected FF-MAS failed to induce any noticeable meiosis. CONCLUSIONS: A rapid increase in FF-MAS level occurred in vivo in the rabbit ovary in response to LH, and clear differences were seen in the cAMP pattern during spontaneous and induced oocyte maturation in mice.

Efficiency of liposomes surface-modified with soybean-derived sterylglucoside as a liver targeting carrier in HepG2 cells.

We investigated the interaction of liposomes surface-modified with soybean-derived sterylglucoside (SG) (SG-liposomes) with HepG2 cells in the point of involvement of asialoglycoprotein receptor (ASGP-R) mediated endocytosis and examined the efficiency of SG-liposomes as drug carriers using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) as a maker of liposome, carboxylated polystyrene microspheres (Fluoresbrite) as a model drug not taken up in cells and doxorubicin (DXR). SG-liposomes were composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol (Ch) and SG (DPPC/Ch/SG=6:3:1, molar ratio) and DiI, Fluoresbrite and DXR were entrapped in SG-liposomes, respectively. Each SG-liposome was incubated with HepG2 cells at 4 or 37 degrees C, and co-incubated with asialofetuin (AF) as a competitor of ASGP-R. The association of DiI, Fluoresbrite or DXR entrapped in SG-liposomes with HepG2 cells at 37 degrees C was significantly higher than that in liposomes containing no SG. That of DiI and Fluoresbrite was reduced significantly by the incubation with AF, but that of DXR was not affected. These findings suggest that Fluoresbrite behaves like the lipid component of SG-liposomes, but DXR in SG-liposomes does not behave similar to the lipid component of SG-liposomes, thus, its drug behavior released from liposomes may be due to its physicochemical properties. SG-liposomes are potentially useful drug carriers to the liver, because the glucose residue may work as a kind of ligand for ASGP-R.

Meiosis-activating sterol and the maturation of isolated mouse oocytes.

This study was carried out to examine the effects of the meiosis-activating C(29) sterol, 4,4-dimethyl-5 alpha-cholesta-8,14, 24-trien-3 beta-ol (FF-MAS), on mouse oocyte maturation in vitro. Cumulus cell-enclosed oocytes (CEO) and denuded oocytes (DO) from hormonally primed, immature mice were cultured 17-18 h in minimum essential medium (MEM) containing 4 mM hypoxanthine plus increasing concentrations of FF-MAS. The sterol induced maturation in DO with an optimal concentration of 3 microg/ml but was without effect in CEO, even at concentrations as high as 10 microg/ml. Some stimulation of maturation in hypoxanthine-arrested CEO was observed when MEM was replaced by MEMalpha. Interestingly, the sterol suppressed the maturation of hypoxanthine-arrested CEO in MEM upon removal of glucose from the medium. FF-MAS also failed to induce maturation in DO when meiotic arrest was maintained with dibutyryl cAMP (dbcAMP). The rate of maturation in FF-MAS-stimulated, hypoxanthine-arrested DO was slow, as more than 6 h of culture elapsed before significant meiotic induction was observed, and this response required the continued presence of the sterol. Although the oocyte took up radiolabeled lanosterol, such accumulation was restricted by the presence of cumulus cells. In addition, lanosterol failed to augment FSH-induced maturation and was even inhibitory at a high concentration. Moreover, the downstream metabolite, cholesterol, augmented the inhibitory action of dbcAMP on maturation in both CEO and DO. Two inhibitors of 14 alpha-demethylase, ketoconazole, and 14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta, 15 alpha-diol that can suppress FF-MAS production from lanosterol failed to block consistently FSH-induced maturation. These results confirm the stimulatory action of FF-MAS on hypoxanthine-arrested DO but do not support a universal meiosis-inducing function for this sterol.

The relationship between the rigidity of the liposomal membrane and the absorption of insulin after nasal administration of liposomes modified with an enhancer containing insulin in rabbits.

The relationship between the rigidity of the liposomal membrane and the absorption of insulin after nasal administration of liposomes modified with an enhancer containing insulin was investigated for the nasal delivery of peptide drugs in rabbits. The rigid liposomal membrane makes liposomes stable, protecting insulin from enzymatic degradation. Soybean-derived sterol (SS) or its sterylglucoside (SG) was used as an enhancer. Dipalmitoylphosphatidylcholine (DPPC) liposomes modified with SG had increased fluidity of the hydrophobic group of the liposome bilayer compared with the liposomes modified with cholesterol (Ch) or SS, as shown by measurements of the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5,-hexatriene (DPH); however, the fluidity of the polar group of the liposome bilayer was decreased according to measurements of steady-state fluorescence anisotropy of dansylhexadecylamine (DSHA) at 37 degrees C. These findings suggest that the fluidity of the hydrophobic group of the liposome bilayer is responsible for the increase of liposomal leakage and instability of the liposomes. When insulin was administered nasally to rabbits as a solution, no hypoglycemic effect was observed. The administration of insulin contained in DPPC/SG (7/4, mole) liposomes with high fluidity caused a high glucose reduction of long duration (8 hr). DPPC/SS and DPPC/Ch (7/4) liposomes with low fluidity caused low glucose reductions. These results demonstrated that liposomes modified with SG can be useful as carriers of insulin administered nasally.

3 beta-Chlorosteroids: artefacts and possible contaminants in food and feed--toxicological effects in mice.

3 beta-Chlorosteroids, such as cholesteryl beta-chloride and sitosteryl beta-chloride, are formed during the production of protein hydrolysates, which are useful flavour enhancers. These chlorinated steroids may also attract attention as environmental contaminants if they are released from liquid crystal display devices. The effects of orally administered 3 beta-chlorosteroids were tested in female NMRI mice. The animals were fed cholesteryl beta-chloride or sitosteryl beta-chloride at doses of 1 mg and 10 mg/animal/day, that is, 33 mg and 330 mg/body weight/day, over a period of 3 months. Feed intake, body weight and organ weights of the animals, as well as concentration of 3 beta-chlorosteroids in faeces and various organs and tissues showed that cholesteryl beta-chloride and sitosteryl beta-chloride are not acutely toxic compounds. However, chronic toxicity cannot be excluded because small amounts of 3 beta-chlorosteroids, in particular cholesteryl beta-chloride, were absorbed by the intestinal tract and accumulated in adipose tissue. Histopathological examination of sections of organs and tissues showed no indication of irreversible cell damage in the stomach, duodenum, liver, kidneys and spleen caused by the chlorinated steroids.

Studies on intestinal absorption, distribution and metabolism of 3 beta-chloro[4-14C]cholest-5-ene and 3 beta-chloro[4-14C]stigmast-5-ene in mice.

A method for the analysis of 3 beta-chloro steroids by high-performance liquid chromatography is described. These compounds are known to occur in commercial protein hydrolysates. The gastro-intestinal absorption, distribution and metabolism of chlorinated steroids were studied after their intragastric application to mice. At 2 hr after stomach intubation of 3 beta-chloro[4-14C]cholest-5-ene and 3 beta-chloro-[4-14C]stigmast-5-ene, large proportions of radioactivity had passed through the small intestine and were found to be concentrated in the contents of the caecum and colon. Very small amounts of 3 beta-chlorocholest-5-ene were absorbed by the intestinal mucosa and distributed to organs and tissues outside the alimentary canal, whereas intestinal permeability of 3 beta-chlorostigmast-5-ene was negligible. After administration of labelled 3 beta-chlorocholest-5-ene, the highest value of radioactivity, 120 Bq/g tissue, outside the intestinal tract was detected in liver. Altogether, less than 0.5% of the total radioactivity applied to the animals was found to be transported through the intestinal wall and less than 0.5% of the total radioactivity was detected in various metabolites. In general, 3 beta-chlorostigmast-5-ene was transported in smaller proportions and metabolized to a lesser extent than the corresponding cholesterol derivative. Moreover, metabolites of the two radioactive substrates formed by enzymatic attack of enteric micro-organisms were not detected in the contents of the caecum and colon. It appears that 3 beta-chlorinated steroids are fairly stable products that are metabolized poorly both by the cells of the intestinal mucosa and by enteric micro-organisms of mice.