A LC-MS/MS method for the determination of stachyose in rat plasma and its application to a pharmacokinetic study.

A sensitive, simple and rapid analytical method based on a liquid chromatography-tandem mass spetrometry (LC-MS/MS) has been established and validated for the determination of stachyose in rat plasma. Plasma samples were prepared by protein precipitation with acetonitrile. Separation of stachyose and nystose (internal standard, IS) was achieved using acetonitrile-water (55:45, v/v) as the mobile phase at a flow rate of 1 ml/min for 6 min on an Asahipak NH2P-50 4E column with an Asahipak NH2P-50G 4A guard column. Detection and quantification were conducted by LC-MS/MS method in the negative ion mode using multiple reaction monitoring (MRM) transitions at m/z [M-H](-) 665.4→383.1 for stachyose and 665.5→485.0 for IS, respectively. The method was linear over the concentration ranges of 100-30000 ng/ml with a lower limit of quantification (LLOQ) of 100 ng/ml. The intra- and inter- day precision were all within 8.7% and the accuracy ranged from 97.2-108.4% and 98.3-102.4%, respectively. Stability studies indicated that stachyose was stable under short-term, long-term and three freeze-thaw storage conditions. The method was successfully applied to a pharmacokinetic study involving pulmonary administration of micronized Rehmannia glutinosa oligosaccharides (RGOS) to rats.

Preparation of co-spray dried cushioning agent containing stearic acid for protecting pellet coatings when compressed.

This study investigated the applicability of stearic acid as a co-adjuvant in cushioning agent formulated to prevent coat damage when compressing coated pellets. The co-processed and physical blended fillers were prepared by spray drying and physically blending, respectively, with filler ingredients consisting of stearic acid, microcrystalline cellulose, fully gelatinized starch, and corn starch. Pellets containing drug were produced by coating onto non-pariels a drug layer of metformin followed by a sustained-release layer. Drug release from tablets composed of co-processed or physical blended fillers (0, 1, 5, and 10% stearic acid levels) and coated drug containing pellets were analyzed using similarity factor F2. Under the same force and the stearic acid level, co-processed fillers produced pellet containing tablets which showed higher F2 or t50 values and tensile strengths as well as lower yield pressures as compared with tablets containing physical blended fillers. It was shown that the destructive degree of pellet coating was significantly reduced after being co-processed by homogenization and the incorporation of stearic acid in the cushioning agents, as shown by the improved F2 and t50 values. In addition, disintegrate times of tablets containing co-processed agents decreased despite the hydrophobic stearic acid. In conclusion, the inclusion of stearic acid in co-processed cushioning agents was effective at protecting compacted coated pellets from compression-induced damage without compromising disintegratability. The findings could serve as a step towards resolving the technical challenges of balancing the drug release profiles, tablet tensile strength, and disintegration time of compacting coated pellets into multi-particulate-sustained release tablets.

A review of experimental research on herbal compounds in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease worldwide, leading to progressive muscle atrophy and paralysis. The limited success of conventional treatment for ALS has prompted investigations into complementary and alternative therapies. Herbal remedies provide good prospects of ALS prevention and treatment, with advantages such as multiple targets, multiple links, and few side effects. Studies in vitro and in vivo have shown that herbs have a great potential for treatment of ALS, with therapeutic effects against oxidative stress, excitatory amino acid toxicity, neuroinflammation, and calcium cytotoxicity. Active monomers or ingredients extracted from herbs are considered promising candidates for ALS. Therefore, we review recent experimental research on monomers and compounds isolated from herbal remedies for preventing and treating ALS.

[Application of factor analysis to evaluate deformation behaviors of frequently-used tablet excipients].

The present work is to investigate the correlation between physical properties and deformation behaviors of tablet excipients, and rank them according to their plastic performances during compaction. The excipients selected were compacted using Korsch XP1 after measuring their physical properties where the compression parameters for evaluating deformation behaviors were Heckle equation, compression work and elastic stretch in die. The correlations between compaction descriptors and physical parameters were analyzed by canonical correlation analysis, and factor analysis was simultaneously employed to synthetically assess deformation behaviors for all our samples. The canonical variables show that true density (Pa) correlated negatively with plastic coefficient (PL) and positively with yield pressure (YP); compression degree (Cp) correlated negatively with fast elastic stretch (FES) as well as YP and positively with PL. When factor scores were used in combination with original data, the plasticity of our samples was sorted and ranked as high (-0.56 < F' < 0.21), intermediate (-0.16 < F' < 0.36), or low (0.38 < F' < 0.84), which are in accord with plasticity rankings previously reported in literature. This study indicates factor analysis can be an approach to evaluate deformation behaviors of pharmaceutical powders.

[Discrimination of traditional Chinese medicinal materials with different tastes based on electronic tongue].

OBJECTIVE: To investigate the use of the electronic tongue in the evaluation of traditional Chinese medicinal materials with sour, bitter, sweet or salty tastes, and assess the possible application of the sensor in the evaluation of different tastes of traditional Chinese medicinal materials. METHOD: Aqueous extracts of 22 traditional Chinese medicinal materials were measured by the electronic tongue. The data collected with the tongue was evaluated for discrimination of the samples with multivariate statistical methods: principal component analysis (PCA) and discriminant factor analysis (DFA). RESULT: The electronic tongue was capable of discriminating between samples with different taste modalities and could also distinguish different samples eliciting the same basic taste. Twenty-two traditional Chinese medicinal materials could be classified into five clusters based on PCA. These differences seem to derive from the different tastes. DFA was applied to construct a model to discriminate traditional Chinese medicinal materials with different tastes. And the samples yielded about 88.2% accuracy for cross-validation. CONCLUSION: We confirmed that the electronic tongue may provide an analytical procedure for classification of the samples with respect to tastes of the traditional Chinese medicinal materials.

[Characterization of flowability of pharmaceutical powders based on multivariate analysis method].

The main methods of characterizing the flowability of pharmaceutical powders include repose angle method, HR method, Carr's index method, Jenike flow function method, fractal dimension method, and mass flow rate method, etc. Regarding powders with different flowabilities as the research subject, comprehensive features of pharmaceutical materials were investigated and characterized. The multivariate analysis method was employed to evaluate and analyze flowability values of the tested pharmaceutical materials. Comparing with the method of the mass flow rate, it was feasible to use multivariate analysis method to evaluate the flowability of powders. Simultaneously, the flowability of pharmaceutical materials could be ranked and definitely quantified, and critical values be determined according to the actual production, which has promoted the previous methods dependent only on the single parameter, i.e. repose angle and compression degree methods. A relatively objective standard method of evaluating flowability of powders is formed.

Comparison of the effects of fungal endophyte Gilmaniella sp. and its elicitor on Atractylodes lancea plantlets.

The effects of the endophytic fungus Gilmaniella sp. and its elicitor on the defense and metabolic responses of host plants Atractylodes lancea were investigated, in order to understand how to utilize endophytic fungi and their elicitor resources better. The results showed that the promotion effect of the fungus on the growth of host plantlets was much better than that of its elicitor. Both fungus and elicitor enhanced defense-related enzyme activities. In fungus-inoculated groups, phenylalanine ammonia lyase and polyphenol oxidase activities increased slowly, and reached a maximum level during the later stages, whereas peroxidase activity peaked in the first few days. Additionally, the activities of chitinase and ß-1,3-glucanase were significantly higher than those of the control plants. In elicitor-treated groups, however, most of the enzymes were activated during the early stage, and their highest levels were generally lower than those of the fungus-inoculated groups. Compared with the elicitor, fungal infection improved the photosynthetic rate of the host, and increased carbohydrate levels as well as chlorophyll content in host leaves. The total content of the four main components of volatile oil was also increased in elicitor-treated groups, but there was no particular pattern in this increase. Meanwhile, in the fungus-inoculated groups, the content of atractylone significantly increased with time, while the content of ß-eudesmol decreased. These results indicated that fungal elicitor could substantially improve the total content of volatile oil, while the fungus could more effectively enhance the quality of herbal medicines.

MDG-1, a polysaccharide from Ophiopogon japonicus exerts hypoglycemic effects through the PI3K/Akt pathway in a diabetic KKAy mouse model.

ETHNOPHARMACOLOGICAL RELEVANCE: Ophiopogon japonicus is a traditional Chinese medicine that might be helpful for the treatment of type 2 diabetes. Recent studies have confirmed its beneficial properties, but not the mechanism of action. AIM OF STUDY: In this study, we examined the effects of a water-soluble ß-d-fructan (MDG-1) from O. japonicus on type 2 diabetes through the PI3K/Akt pathway in a diabetic KKAy mouse model. MATERIALS AND METHODS: MDG-1 was extracted from the tube root of O. japonicus and purified as described previously (Xu et al., 2005). The KKAy mice were gavaged once daily with either distilled water, MDG-1or rosiglitazonefor 8 weeks. Blood glucose levels were tested regularly for the fed and fasted mice. In order to evaluate the effect of MDG-1 on disease progression, the proteins of InsR/IRS-1/PI3K/Akt/GSK-3/Glut-4 were detected by Western blotting and serum TG, TC, HDL-C, LDL-C were also dertermined. RESULTS: MDG-1 reduced the hyperglycemia, hyperinsulinemia and hyperlipidemia in the KKAy mice. The oral glucose tolerance test (OGTT) and the level of insulin in the serun showed that insulin resistance in KKAy mice was ameliorated after MDG-1 treated. After 8 weeks treatment with 300mg/kg MDG-1, the content of triglycerides (TG) and low density lipoprotein cholesterol (LDL-C) the serum decreased significantly. Meanwhile high density lipoprotein cholesterol (HDL-C) content increased notably. MDG-1 did not have any effect on total cholesterol (TC) content in the serum, whereas rosiglitazone significantly decreased the TC content. In addition, MDG-1 upregulates the phosphoinositide 3-kinase p85 subunit, Akt, insulin receptor (InsR), insulin receptor substrate-1 (IRS-1) and Glut-4 expression, but downregulates glycogen synthase kinase 3ß expression. CONCLUSIONS: These data indicate that MDG-1 has remarkable anti-diabetic activity through the InsR/IRS-1/PI3K/Akt/GSK-3/Glut-4 signaling pathway. We believe that MDG-1 is a promising anti-diabetic compound that will be helpful for the treatment of T2DM.

[Multivariate analysis of relationships between material properties and hygroscopicity of Chinese medicine raw materials].

Material properties and hygroscopicity were determined. Principal component analysis and partial least squares regression were applied to evaluate relationships between material properties and hygroscopicity of Chinese medicine raw materials. The results showed that hygroscopicity was correlated with water content, particle size distribution, water soluble characteristic and cohesion. Balanced moisture content was positively correlated with water content, particle size distribution, water soluble characteristic and cohesion. Moisture absorption velocity was negatively correlated with particle size distribution, protruding degree and positively correlated with water soluble characteristic and cohesion. Moisture absorption acceleration was positively correlated with water content, particle size distribution and negatively correlated with water soluble characteristic and cohesion. Hygroscopicity of Chinese medicine raw materials is interpreted in terms of physics.

Pharmacokinetics, tissue distribution and metabolism of senkyunolide I, a major bioactive component in Ligusticum chuanxiong Hort. (Umbelliferae).

ETHNOPHARMACOLOGICAL RELEVANCE: Ligusticum chuanxiong Hort. (Umbelliferae) is widely prescribed for treatment of cardiovascular diseases in China for centuries. One of the major bioactive components in L. chuanxiong is senkyunolide I (SEI), which shows pharmacological activities in anti-migraine and anti-oxidative damage. MATERIALS AND METHODS: The aim of this study was to investigate in vivo pharmacokinetics, tissue distribution and metabolism of SEI in rats. The concentrations of SEI in plasma and tissues were determined by a high performance liquid chromatography (HPLC) method, and the pharmacokinetic parameters were calculated using and non-compartmental analysis. The metabolites were identified using high performance liquid chromatography tandem mass (HPLC-ESI-MS/MS) method. RESULTS: After oral and intravenous administration, SEI was quickly eliminated from plasma and its oral bioavailability (BA) was about 37.25%, which was smaller than intraportal BA (81.17%), but similar to intraduodenal BA (36.91%), suggesting that gastric first-pass effect of SEI is negligible, and hepatic first-pass effect was approximately 18.83%. After oral administration, SEI could penetrate blood brain barrier and extensively distribute in tested tissues, with the descending order of AUC being kidney, liver, lung, muscle, brain, heart, thymus, and spleen in rat. The parent compound and nine metabolites were found and identified in rat bile after oral administration of SEI (36 mg/kg). The metabolic mechanism of SEI in rat mainly involves methylation, glucuronidation and glutathione conjugation during the phase II biotransformation pathway in rats. CONCLUSIONS: The information gained here may provide a meaningful basis for clinical application of such a bioactive compound of herbal medicines.

Characterization using LC/MS of the absorption compounds and metabolites in rat plasma after oral administration of a single or mixed decoction of Shaoyao and Gancao.

Shaoyao-Gancao decoction (SGD), a traditional Chinese formulation containing Paeoniae Radix (SY) and Glycyrrhizae Radix (GC), is commonly used to relieve abdominal pain. However, the absorption and metabolites of the characteristic constituents of the two herbs in vivo have been reported rarely. The purpose of this study was to investigate the compatibility rationality and the mechanism of the enhanced efficiency of SGD. A single or a mixed decoction (SG and S+G, respectively) was orally administered to rats. Blood samples were collected at different intervals following treatment and analyzed by liquid LC/MS. A total of fifteen ingredients (denoted as M1 to M15) were found in both rat plasma after treatment with the two decoctions. Furthermore, the proposed structures of the remained twelve ingredients were obtained except M9, M10 and M15. The quality of the ingredients in the rat plasma showed no significant difference between the two decoctions. However, the quantity of twelve ingredients differed greatly, indicating that the absorption of SG was greater than that of S+G except M7, M12 and M15. As the compositions associated with the efficacy of SG and S+G were inconsistent, the degree of the absorption of the 15 ingredients by the gastrointestinal tract were different, which caused a significantly enhanced efficacy of certain ingredients. This study presents an exploration of the mechanism behind the improved efficacy of individual components in traditional Chinese medicine therapies through combination with other components.

Comparative pharmacokinetics of senkyunolide I in a rat model of migraine versus normal controls.

Senkyunolide I is an active ingredient of Rhizoma Chuanxiong, a Chinese medicinal herb commonly used for the treatment of cardiovascular ailments. In the present paper, we describe the isolation and elucidation of senkyunolide I from the ethanol extract of Rhizoma Chuanxiong and its pharmacokinetic behavior after intravenous and oral administration to normal and migrainous rats. After intravenous administration, senkyunolide I was rapidly distributed (V ( z )/F 2.07 ± 0.43 L/kg) and eliminated from the plasma (CL( z ) 2.56 ± 0.29 L/h/kg and t (1/2z ) 0.56 ± 0.13 h). After administration orally to normal rats at two dosages (20 and 72 mg/kg), the pharmacokinetic parameters of senkyunolide I were as follows: T (max) 0.25 ± 0.06 and 0.38 ± 0.11 h, C (max) 5,236.3 ± 802.8 and 22,071.9 ± 3,456.1 mg/L, Area under the curve(AUC)((0-t)) 5,217.5 ± 1,029.5 and 21,480.2 ± 3,003.1 µg h/L, respectively. Its oral absolute bioavailability at the two dosages was 67.2 and 76.9%, respectively. Intriguingly, migraine caused some significant changes in its pharmacokinetic parameter. For example, when compared with its pharmacokinetic behavior in normal rats at the two dosages, on average, its clearance decreased by 68% and volume of distribution increased by 342% in migrainous rats, both of which contributed to its several-fold increase in t (1/2z) and AUC. C (max) and AUC of senkyunolide I increased almost proportionally with dose between 20 and 72 mg/kg and the pharmacokinetics fit linear kinetic feature. The pharmacokinetic parameters of senkyunolide I were significantly different in normal and migrainous rats, which should be taken into account during the design of a clinical dosage regimen for senkyunolide I.

Pharmacokinetics of characteristic effective ingredients from individual and combination Shaoyao and Gancao treatment in rats using HPLC fingerprinting.

Shaoyao-Gancao decoction, a traditional Chinese formulation composed of Paeoniae Radix and Glycyrrhizae Radix, is commonly used to relieve abdominal pain. In this paper, the compatibility rationality of this decoction was investigated. Shaoyao-Gancao decoction, Shaoyao decoction and Gancao decoction were orally administered to rats, respectively. Blood samples were collected at pre-determined times after administration and analyzed by high-performance liquid chromatography (HPLC). The pharmacokinetic parameters of characteristic peaks were analyzed and the statistical significance of the obtained parameters was determined. Paeoniflorin (12.0 min) and compounds at retention times of 4.7 and 5.2 min were all significantly higher in the Shaoyao-Gancao decoction than in the Shaoyao decoction (P < 0.05). In contrast, in the Gancao decoction, the compound at a retention time of 14.6 min was significantly lower than in the Shaoyao-Gancao decoction (P < 0.01). However, the compounds at retention times of 17.1 and 18.1 min were significantly higher in the Gancao decoction than in the Shaoyao-Gancao decoction (P < 0.05). These results indicate that poor compatibility of the compounds in the Shaoyao-Gancao decoction could result in poor absorption. The compatibility of the component compounds of the Shaoyao-Gancao decoction was revealed in the pharmacokinetic characteristics of the decoction. Generally, the absorption of Shaoyao components was increased in the Shaoyao-Gancao decoction, while the absorption of Gancao components was time dependent. In the Shaoyao-Gancao decoction, the increased absorption of some Shaoyao components may be related to a reduction in absorption of some Gancao components.

Magnetic resonance imaging in assessment of treatment response of gamma knife for brain tumors.

OBJECTIVE: To review the applications of magnetic resonance imaging (MRI) techniques in assessing treatment response to gamma knife radiosurgery for brain tumors. DATA SOURCES: Published articles about assessing treatment response to gamma knife radiosurgery for brain tumors were selected using PubMed. The search terms were "MRI", "gamma knife" and "brain tumors". STUDY SELECTION: Articles regarding the MRI techniques using for early assessment of treatment response of gamma knife were selected. RESULTS: MRI techniques, especially diffusion weighted imaging, perfusion weighted imaging, magnetic resonance spectroscopy, are useful for early assessment of treatment response of gamma knife by detecting the hemodynamic, metabolic, and cellular alterations. Moreover, they can also provide important information on prognosis. CONCLUSIONS: Diffusion weighted imaging, perfusion weighted imaging and magnetic resonance spectroscopy can provide early assessment of treatment response of gamma knife for brain tumors, and also information of tumor progression or recurrence earlier than conventional MRI. But there are still many questions to be answered which should be based on the development and advancement of MRI and related disciplines.

Comparison of tissue distribution of a PEGylated Radix Ophiopogonis polysaccharide in mice with normal and ischemic myocardium.

PEGylation was found to be a promising approach to improve the anti-myocardial ischemic activity of Radix Ophiopogonis polysaccharide (ROP) by prolonging its retention in plasma. To fully evaluate the effectiveness and safety of this strategy, the tissue distribution of PEGylated ROP was investigated in this study. A long-circulating and bioactive PEGylated ROP with 1.04 mol 20-kDa mPEG per mol ROP ((1.04)P(20k)-R) was prepared by a moderate coupling reaction between the hydroxyl-activated ROP and the amino-terminated mPEG. Its tissue distribution in mice with normal and ischemic myocardium was studied and compared with ROP. The results show that the descending order of tissue distribution of (1.04)P(20k)-R ranked by AUC was kidney, lung, heart, liver, and brain in normal mice and kidney ≈ lung ≈ heart, liver and brain in mice with myocardial ischemia. With the exception of the heart, myocardial ischemia did not cause obvious changes in the distribution of (1.04)P(20k)-R in the other tissues studied. Owing to the enhanced permeability and retention effect caused by ischemia, the AUC of (1.04)P(20k)-R in ischemic hearts was approximately 1.6-fold greater than in normal hearts. Compared with ROP in rats, the distribution tendency of (1.04)P(20k)-R in mouse kidney, brain, and lung was reduced by approximately 42, 1.6, and 1.3 times, respectively, whereas it was increased by approximately 1.3-fold in the liver. The results of this study are highly instructive for the further pharmaceutical development of PEGylated ROP.

Hypoglycemic effects of MDG-1, a polysaccharide derived from Ophiopogon japonicas, in the ob/ob mouse model of type 2 diabetes mellitus.

Ophiopogon japonicus is a traditional Chinese medicine used to treat diabetes mellitus. We investigate the anti-ischemic properties of a water-soluble ß-D-fructan (MDG-1) from O. japonicus, and assess the antidiabetic effects of MDG-1. In the study, ob/ob mice were treated with 150 mg/kg or 300 mg/kg MDG-1 by gavage for 23 d. Blood glucose levels were measured regularly. An oral glucose tolerance test (OGTT) was preformed on day 21. The levels of insulin, total cholesterol and triglyceride in the serum were measured at the end of administration. The liver triglyceride content and tissue weights were also determined. Results show that MDG-1 (300 mg/kg) was demonstrated to exert acute and long-term hypoglycemic effects on fed blood glucose in ob/ob mice. However, only a marginal hypoglycemic effect on fasting blood glucose levels was observed. MDG-1 (300 mg/kg) improved oral glucose tolerance and reduced serum insulin levels and triglyceride content in the liver in ob/ob mice. Furthermore, a reduction in body weight gain and the weight of subcutaneous fat were observed following treatment with MDG-1 (150 mg/kg) compared with the control group. MDG-1 had no significant effects on the total cholesterol and triglyceride levels, food intake and other adipose and organ tissues. These data suggest that MDG-1 exhibits hypoglycemic activity and reduces insulin resistance.

[Pharmacokinetics study on characteristic ingredients of different-dose herbs of shaoyao-gancao decoction].

The paper is to report the observation of pharmacokinetic changes of the characteristic ingredients in the combinations of different-dose herbs of Shaoyao-Gancao decoction. After the establishment of HPLC analytical method of plasma effective constituents, rats were orally administered with different-dose herbs of Shaoyao-Gancao decoction. Blood samples at different times after administering these decoctions were collected, and then were analyzed by HPLC fingerprints technology. Pharmacokinetic parameters of characteristic peaks were analyzed by SPSS 15.0 software and DAS 2.0. At last, we looked for the correlation of those pharmacokinetic parameters and the dosage of Gancao. The best dose of Shaoyao-Gancao decoction was at the ratio of 4 to 4, which was consistent with the dose commonly used in ancient times. The absorption of characteristic peaks from Shaoyao-Gancao decoction was related with the dosage of Gancao, and there existed interaction between each characteristic ingredients. There existed the right dose-ratio of Shaoyao and Gancao to get the best effect. The absorptions of effective constitutents were mutual waxing and waning in order to increase biological effects together. It's demonstrated the compatibility connotation at a right dose-ratio of Shaoyao-Gancao decoction through the angle of pharmacokinetics.

Effect and mechanism of senkyunolide I as an anti-migraine compound from Ligusticum chuanxiong.

OBJECTIVE: To evaluate the analgesic and anti-migraine activities of senkyunolide I from Ligusticum chuanxiong. METHODS: Mice were orally administered various doses of senkyunolide I, and their pain levels were assessed in a hot-plate test and by application of acetic acid. The levels of 5-hydroxytryptamine (5-HT), 5-hydroxytryptophan (5-HTP), 5-hydroxyindoleacetic acid (5-HIAA), norepinephrine (NE) and dopamine (DA) in plasma and brain were assessed, and the monoamine turnover rates (5-HT/5-HTP, 5-HIAA/5-HT and NE/DA) were also calculated. RESULTS: Mice given senkyunolide I at 16 and 32 mg/kg had significantly elevated pain thresholds in the hot-plate test, and a dose of 32 mg/kg also reduced the number of abdominal writhing responses caused by acetic acid. Significant improvements were observed in the neurotransmitter levels of the drug-treated rats compared with the saline-administered controls. Compared to the rats with nitroglycerin-induced migraines, the levels of nitric oxide in the plasma and whole brain of rats given senkyunolide I were lower. CONCLUSIONS: The present study suggests that senkyunolide I may be an active component of L. chuanxiong, traditionally used to treat migraine. The mechanism of pain relief in migraine model rats may be through adjusting the levels of monoamine neurotransmitters and their turnover rates, as well as decreasing nitric oxide levels in the blood and brain. Therefore, senkyunolide I may be developed as a potential treatment for migraine pain.

[Pharmacokinetics of a long-circulating PEGylated Radix Ophiopogonis polysaccharide].

The pharmacokinetics of a long-circulating PEGylated Radix Ophiopogonis polysaccharide (ROP) was investigated in rats following i.v. or s.c. administration at three dose levels (9, 20, 50 mg x kg(-1)). A moderate coupling reaction between the hydroxyl-activated ROP and the amino-terminated mPEG was chosen to produce PEGylate ROP. The grafting degree of the prepared conjugate was 1.03, and the molecular mass of mPEG used was 20 kDa. High-performance gel permeation chromatorgraphy with fluorescein isothiocyanate prelabeling was established to determine levels of the conjugate in plasma. The results showed that the elimination half-life of the conjugate following s.c. administration was basically identical to that after iv administration. An accurate linear correlation was observed between administration doses and areas under the curve of plasma conjugate level vs. time profile, regardless of the administration route. The absolute bioavailability of the conjugate following sc administration was approximately 56%, and the mean in vivo residence time was 52.1 h, increased 2.4 times compared to those of iv administration. In general, linear pharmacokinetics was observed for the conjugate within the dose range studied, and sc should be a promising administration route for the conjugate.

Poly(ethylene glycol)-radix Ophiopogonis polysaccharide conjugates: preparation, characterization, pharmacokinetics and in vitro bioactivity.

Radix Ophiopogonis polysaccharide (ROP), a natural graminan-type fructan with Mw of ∼5kDa, had been found to have an excellent anti-myocardial ischemic activity. However, its rapid renal excretion following administration remarkably limits its efficacy and clinical use, which makes necessary the development of an effective delivery system. In this article, the feasibility of PEGylation to solve this problem was examined. A moderate coupling reaction between the hydroxyl-activated ROP and the amino-terminated mPEG was chosen to PEGylate ROP. Five different mPEG-ROP conjugates (with mPEG of molecular mass 2, 5 or 20kDa) were prepared, purified, characterized and evaluated in pharmacokinetics and in vitro bioactivity. Results showed that only when the apparent molecular weight of the conjugate approached to a certain value, would its plasma elimination reduce abruptly. In general, the conjugation caused the reduction in the bioactivity of ROP; however, well-preserved bioactivity was observed when the grafting degree of the conjugate was lower. Among the five conjugates studied, the one with an average 1.3 mPEG (20kDa) residues per single ROP was found to be satisfactory both in plasma retention and in bioactivity. It had a 47.4-fold increased elimination half-life and preserved approximately 74% of the bioactivity of ROP; moreover, the decrease in bioactivity is not significant. These findings demonstrate that PEGylation would be a promising approach for improving the clinical efficacy of ROP by prolonged retention in plasma.