IBI310 (anti-CTLA-4 antibody) monotherapy or in combination with sintilimab in advanced melanoma or urothelial carcinoma.

IBI310 is a recombinant fully human IgG1 antibody against cytotoxic T lymphocyte antigen 4. This study was conducted to evaluate IBI310 monotherapy or combination therapy with sintilimab in the patients with advanced melanoma or urothelial carcinoma (UC). Patients in phase 1a received IBI310 at 0.3/1/2/3 mg/kg intravenously (IV) every 3 weeks (Q3W) following the accelerated titration and 3 + 3 escalation design. Patients in phase 1b received IBI310 (1/2/3 mg/kg IV, Q3W) plus sintilimab (200 mg IV, Q3W) for four cycles, followed by sintilimab maintenance therapy. The phase 1b expansion of IBI310 plus sintilimab was performed in patients with advanced melanoma or UC. Overall, 53 patients were enrolled, including 10 patients with melanoma in phase 1a, 34 with melanoma, and 9 with UC in phase 1b. Overall, 94.3% of patients (50/53) experienced at least one treatment-related adverse event (TRAE) with most being grade 1-2; 26.4% of patients (14/53) experienced grade 3 or higher TRAEs. In phase 1a, the disease control rate (DCR) was 50.0% (95% confidence interval [CI], 18.7%-81.3%). In phase 1b, the objective response rate (ORR) and DCR were 17.6% (95% CI, 6.8%-34.5%) and 44.1% (95% CI, 27.2%-62.1%), respectively, for melanoma, and were 22.2% (95% CI, 2.8%-60.0%) and 66.7% (95% CI, 29.9%-92.5%), respectively, for UC. IBI310 monotherapy or combination therapy with sintilimab was well tolerated with favorable antitumor activity across patients with advanced melanoma and UC.

Pharmacokinetics, pharmacodynamics and efficacy of pemigatinib (a selective inhibitor of fibroblast growth factor receptor 1-3) monotherapy in Chinese patients with advanced solid tumors: a phase i clinical trial.

Pemigatinib is a selective fibroblast growth factor receptor (FGFR)1-3 inhibitor and has demonstrated acceptable tolerability and clinical activity in advanced solid tumors in Western population. This phase I trial evaluated pharmacokinetics/pharmacodynamics (PK/PD) characteristics, preliminary safety and efficacy of pemigatinib in Chinese patients with advanced, solid tumors. Patients with unresectable advanced or metastatic solid tumors bearing FGF/FGFR1-3 alterations received oral pemigatinib at 13.5 mg once daily (QD) on a 2-weeks-on/1-week-off schedule. The primary endpoint was PK/PD characteristics; secondary endpoints were safety and efficacy. Twelve patients were enrolled (median age: 61 years, 58.3% males). PK data demonstrated pemigatinib (13.5 mg QD) was rapidly absorbed with a geometric mean elimination half-life of 11.3 h. The geometric mean values of maximum serum concentration and area under the plasma concentration-time curve from 0 to 24 h at steady state were 215.1 nmol/L and 2636.9 h·nmol/L, respectively. The mean clearance adjusted by bioavailability at steady state was low (11.8 L/h), and the apparent oral volume of distribution was moderate (170.5 L). The PD marker, serum phosphate level, increased on days 8 and 15 of cycle 1 (mean: 2.25 mg/dL, CV% [percent coefficient of variation]: 31.3%) and decreased to baseline post 1 week off. Three (25.0%) patients experienced grade ≥ 3 treatment-emergent adverse events. Partial response was confirmed in one patient with FGFR1-mutant esophageal carcinoma and one with FGFR2-mutant cholagiocarcinoma. Pemigatinib had similar PK/PD characteristics to Western population and demonstrated an acceptable safety profile and potential anti-cancer benefit in Chinese patients with FGF/FGFR1-3 altered, advanced, solid tumor. (ClinicalTrials.gov: NCT04258527 [prospectively registered February 6, 2020]).

Pharmacokinetic/LDL-C and exposure-response analysis of tafolecimab in Chinese hypercholesterolemia patients: Results from phase I, II, and III studies.

Tafolecimab, a novel fully human monoclonal antibody targeting PCSK9, has been assessed in Chinese healthy volunteers and patients with hypercholesterolemia. This analysis is to develop and qualify a population pharmacokinetics (PopPKs)/LDL-C model to characterize tafolecimab PK and LDL-C profiles, evaluate the impact of potential covariates on tafolecimab, estimate individual predicted exposure, and LDL-C decreasing, furthermore, explore exposure-response relationship to support clinical use. Data from six clinical trials in China were used to develop the PopPK/LDL-C model. A Michaelis-Menten approximation of the target-mediated drug disposition (TMDD) model was used to describe PK data and indirect response (IDR) model was developed to estimate the LDL-C profile. A stochastic approximation expectation maximization algorithm was applied to estimate PopPK/LDL-C parameters. The PK/LDL-C time course data for tafolecimab were well described by TMDD/IDR model. Baseline covariates resulting in statistically significant changes in PK/LDL-C parameters included: body weight and sex on absorption rate constant; body weight, sex, and unbound PCSK9 on central volume; body weight and sex on clearance; baseline LDL-C on first-order rate constants for the removal of an effect); and disease and sex on maximum effect. However, the magnitudes of changes associated with these covariates do not necessitate dose adjustment. Exposure-efficacy relationship indicated that the nadir of LDL-C reduction achieved with the steady-state trough plasma concentration (Ctrough ) of tafolecimab at 5 µg/mL, and no further LDL-C decreasing with the increasing Ctrough . There was no exposure dependency observed in exposure-safety exploration. The PopPK/LDL-C model was successfully developed, validated, and predicted tafolecimab/LDL-C concentrations and individual exposures.

Comparability strategy and demonstration for post-approval production cell line change of a bevacizumab biosimilar IBI305.

High-producing cell line could improve the affordability and availability of biotherapeutic products. A post-approval production cell line change, low-titer CHO-K1S to high-titer CHO-K1SV GS-KO, was performed for a China marketed bevacizumab biosimilar IBI305. Currently, there is no regulatory guideline specifically addressing the requirements for comparability study of post-approval cell line change, which is generally regarded as the most complex process change for biological products. Following the quality by design principle and risk assessment, an extensive analytical characterization and three-way comparison was performed by using a panel of advanced analytical methods. Orthogonal and state-of-the-art techniques including nuclear magnetic resonance and high-resolution mass spectrometry were applied to mitigate the potential uncertainties of higher-order structures and to exclude any new sequence variants, scrambled disulfide bonds, glycan moiety and undesired process-related impurities such as host cell proteins. Nonclinical and clinical pharmacokinetics (PK) studies were conducted subsequently to further confirm the comparability. The results demonstrated that the post-change IBI305 was analytically comparable to the pre-change one and similar to the reference product in physicochemical and biological properties, as well as the degradation behaviors in accelerated stability and forced degradation studies. The comparability was further confirmed by comparable PK, pharmacodynamics, toxicological and immunogenicity profiles of nonclinical and clinical studies. The comparability strategy presented here might extend to cell line changes of other post-approval biological products, and particularly set a precedent in China for post-approval cell line change of commercialized biosimilars.

The Prmt5-Vasa module is essential for spermatogenesis in Bombyx mori.

In lepidopteran insects, dichotomous spermatogenesis produces eupyrene spermatozoa, which are nucleated, and apyrene spermatozoa, which are anucleated. Both sperm morphs are essential for fertilization, as eupyrene sperm fertilize the egg, and apyrene sperm is necessary for the migration of eupyrene sperm. In Drosophila, Prmt5 acts as a type II arginine methyltransferase that catalyzes the symmetrical dimethylation of arginine residues in the RNA helicase Vasa. Prmt5 is critical for the regulation of spermatogenesis, but Vasa is not. To date, functional genetic studies of spermatogenesis in the lepidopteran model Bombyx mori has been limited. In this study, we engineered mutations in BmPrmt5 and BmVasa through CRISPR/Cas9-based gene editing. Both BmPrmt5 and BmVasa loss-of-function mutants had similar male and female sterility phenotypes. Through immunofluorescence staining analysis, we found that the morphs of sperm from both BmPrmt5 and BmVasa mutants have severe defects, indicating essential roles for both BmPrmt5 and BmVasa in the regulation of spermatogenesis. Mass spectrometry results identified that R35, R54, and R56 of BmVasa were dimethylated in WT while unmethylated in BmPrmt5 mutants. RNA-seq analyses indicate that the defects in spermatogenesis in mutants resulted from reduced expression of the spermatogenesis-related genes, including BmSxl, implying that BmSxl acts downstream of BmPrmt5 and BmVasa to regulate apyrene sperm development. These findings indicate that BmPrmt5 and BmVasa constitute an integral regulatory module essential for spermatogenesis in B. mori.

Drosophila transition fibers are essential for IFT-dependent ciliary elongation but not basal body docking and ciliary budding.

Cilia are highly conserved organelles critical for animal development and perception. Dysfunction of cilia has been linked to a wide spectrum of human genetic diseases, termed ciliopathies.1,2 Transition fibers (TFs) are striking ciliary base structures essential for cilia assembly. Vertebrates' TFs that originate from centriole distal appendages (DAs) mediate basal body docking to ciliary vesicles to initiate ciliogenesis and regulate the entry of ciliary proteins for axoneme assembly via intraflagellar transport (IFT) machinery.3 Although no distal appendages can be observed on Drosophila centrioles,4,5 three key TF proteins, FBF1, CEP164, and CEP89, have obvious homologs in Drosophila. We aimed to compare their functions with their mammalian counterparts in Drosophila ciliogenesis. Here, we show that all three proteins are localized like TF proteins at the ciliary base in both sensory neurons and spermatocytes, the only two types of ciliated cells in flies. Fbf1 and Cep89 are essential for the formation of IFT-dependent neuronal cilia, but Cep164 is dispensable for ciliogenesis in flies. Strikingly, none are required for basal body docking and transition zone (TZ) assembly in IFT-dependent neuronal cilia or IFT-independent spermatocyte cilia. Furthermore, we demonstrate that Unc is essential to recruit all three TF proteins and establish a hierarchical order, with Cep89 acting on Fbf1. Collectively, our results not only demonstrate that TF proteins are required for IFT-dependent ciliogenesis in Drosophila, in agreement with an evolutionarily conserved function of these proteins in regulating ciliary protein entry, but also that the basal body docking function of TFs has diverged during evolution.

BmHen1 is essential for eupyrene sperm development in Bombyx mori but PIWI proteins are not.

In lepidopteran insects, sperm dimorphism is a remarkable feature, in which males exhibit two different types of sperms. Both sperm morphs are essential for fertilization: Eupyrene sperm carry DNA and fertilize eggs, whereas apyrene sperm, which do not have nuclei, are necessary for transport of eupyrene sperm into eggs. In this study, we showed that the gene BmHen1, which encodes a methyltransferase that modifies piRNAs, is necessary for eupyrene sperm development in the lepidopteran model insect, Bombyx mori. Loss-of-function mutants of BmHen1 of both sexes were sterile. BmHen1 female mutants laid fewer eggs than wild-type females, and the eggs laid had morphological defects. Immunofluorescence analysis of BmHen1 male mutants revealed that nuclei formation in the eupyrene sperm was defective, whereas apyrene sperm were normal. In mice, worms, and flies, the components in piRNA biogenesis pathway play an important role in gonad development; therefore, we constructed mutations in genes encoding core elements in the piRNA biogenesis pathway, Siwi, and BmAgo3. To our surprise, no obvious phenotypes were observed in the male reproduction system in the Siwi and BmAgo3 mutants, which demonstrated that sperm development in B. mori does not depend on piRNAs. As the sperm development phenotype in BmHen1 mutants mimics the phenotype of the BmPnldc1 mutants, we then performed RNA sequencing analysis of sperm bundles from both mutants. We found that the defects in eupyrene sperm resulted from dysregulation of the expression of genes involved in energy metabolism. Taken together, our findings demonstrate the crucial functions of BmHen1 in the development of eupyrene sperm and provide evidence that spermatogenesis in B. mori is PIWI-independent. Our results suggest potential targets for lepidopteran pest control and broaden our knowledge of the reproduction in this order of insects.

A phase I, randomized, double-blinded, single-dose study evaluating the pharmacokinetic equivalence of the biosimilar IBI305 and bevacizumab in healthy male subjects
.

OBJECTIVE: To compare the pharmacokinetic (PK) profiles, immunogenicity, and safety of the proposed biosimilar IBI305 with those of bevacizumab in healthy male subjects. DESIGN: A phase I, randomized, double-blinded, two-arm, parallel-group study. SETTINGS: The study was conducted in The First Hospital of Jilin University, Changchun, China, from March 2017 to November 2017. PARTICIPANTS: A total of 100 healthy male subjects were enrolled, with 48 in the IBI305 group and 50 in bevacizumab group included in the final analysis. INTERVENTION: In a 16-week study course, participants were randomized at a 1:1 ratio to receive intravenous administration of either a single dose of 3 mg/kg IBI305 (n = 50) or bevacizumab (n = 50). OUTCOME MEASURES: The primary endpoints were area under the concentration-time curve from zero to the time of the last measurable concentration (AUC0-t) and AUC curve from zero to infinity (AUC0-∞). The secondary endpoints include the other PK parameters, immunogenicity, and safety measurements. RESULTS: AUC0-t, AUC0-∞, maximum concentration observed (Cmax), half-life (T1/2), drug clearance, and volume of distribution were similar between IBI305- and bevacizumab-treated subjects. For AUC0-∞, AUC0-t, and Cmax, the 90% confidence intervals for the ratios of geometric means were fully within the range 0.80 - 1.25, confirming the bioequivalence of the two investigational agents. Furthermore, no apparent difference in adverse events was found between the two groups. CONCLUSION: This study demonstrated the similarity of PK, immunogenicity, and safety profiles of IBI305 to those of bevacizumab.

Development of a method for protonema proliferation of peat moss (Sphagnum squarrosum) through regeneration analysis.

The moss Sphagnum (peat moss) is ecologically and economically important. There is a paucity of physiological and developmental studies on Sphagnum because of the lack of an axenic culture system for its whole life cycle. A culture system has been established for the Sphagnum gametophore, but not the protonema (juvenile vegetative stage after spore germination). Therefore, the aim of this study was to develop a protonema culture system for Sphagnum. Sphagnum squarrosum gametophore tissue was disrupted and then cultured in liquid Knop medium. The regeneration of protonemata from the gametophore fragments was analyzed in detail by microscopy. We observed a developmental balance between filamentous and thalloid protonemata, and growth competition between the thalloid protonema and the gametophore. On the basis of these findings, we established a relatively stable peat moss protonema proliferation method. Using this method, all the developmental stages of peat moss vegetative growth could be obtained through differentiation or regeneration. The method can provide abundant homogeneous Sphagnum materials at desired stages for physiological and developmental studies, and will be useful for large-scale Sphagnum vegetative proliferation. The regeneration analysis method will be useful for establishing protonema proliferation systems for other mosses.

IBI303, a biosimilar to adalimumab, for the treatment of patients with ankylosing spondylitis in China: a randomised, double-blind, phase 3 equivalence trial.

BACKGROUND: China approved adalimumab for the treatment of ankylosing spondylitis in 2013. However, the cost of the standard dose regimen exceeds ¥15 000 (around US$2250) per month, which is well beyond affordability for most Chinese patients. No biosimilars of adalimumab are available in China; IBI303 is a monoclonal antibody against TNFα that is currently in development. This study aimed to assess the clinical equivalence of IBI303 to adalimumab in patients with ankylosing spondylitis. METHODS: This phase 3, multicenter, double-blind, parallel, randomised controlled equivalence trial was done in 20 centers across China. Patients were randomly assigned in a 1:1 ratio to receive either 40 mg of IBI303 or 40 mg of adalimumab as a subcutaneous injection every 2 weeks until week 22. Patients were eligible for inclusion if they were between 18 and 65 years old, fulfilled the 1984 Modified New York Criteria for ankylosing spondylitis, were non-responders, inadequate responders, or intolerant to treatment with NSAIDs for 4 or more weeks, and had active ankylosing spondylitis defined by two or more indicators of disease severity. The investigators, site staff, patients, sponsors, and the contract research organisation were masked to treatment allocation. The primary outcome was the proportion of patients who met the Assessment of SpondyloArthritis international Society (ASAS) Response Criteria for a 20% improvement (ASAS20) at week 24 after treatment. Equivalence was established if the 95% CI of the difference in responses between groups was between -15% and 15%. Efficacy analyses were done in the full analysis population and in the per-protocol population. Safety analyses were done in all randomly assigned patients who received at least one drug dose. This trial is registered with ClinicalTrials.gov, number NCT02893254. FINDINGS: Between Sept 22, 2016, and May 11, 2018, 438 patients were randomly allocated either to the biosimilar IBI303 group (n=220) or the adalimumab group (n=218). In the full analysis population, 165 (75%) of 220 patients in the IBI303 group (95% CI 68·7-80·6) and 158 (72%) of 218 patients in the adalimumab group (66·0-78·3) reached the primary outcome of ASAS20 at week 24. The difference between the two groups was 2·3% with a 95% CI of -5·9 to 10·6, which fell within the pre-specified equivalence boundaries at week 24 (-15 to 15). In the per-protocol population, 163 (80%) of 203 patients in the IBI303 group reached ASAS20 at week 24 (95% CI 74·1-85·5), compared with 150 (80%) of 188 patients in the adalimumab group (73·3-85·3%). The difference between the groups was 0·6% with a 95% CI of -7·4 to 8·6%, which also fell within the pre-specified equivalence boundaries at week 24. Safety and tolerability profiles were similar between the two groups; 174 (79%) of 220 patients in the IBI303 group and 178 (82%) of 218 patients in the adalimumab group had treatment-emergent adverse events. INTERPRETATION: This trial showed therapeutic equivalence of IBI303 and adalimumab in the treatment of ankylosing spondylitis. The efficacy, safety, and immunogenicity of both drugs are highly similar. IBI303 could be an alternative treatment option for patients with ankylosing spondylitis in China. FUNDING: Innovent Biologics, National Major Scientific and Technological Special Project for "Significant New Drugs Development".

Platinum(II)-Oligonucleotide Coordination Based Aptasensor for Simple and Selective Detection of Platinum Compounds.

Wide use of platinum-based chemotherapeutic regimens for the treatment for carcinoma calls for a simple and selective detection of platinum compound in biological samples. On the basis of the platinum(II)-base pair coordination, a novel type of aptameric platform for platinum detection has been introduced. This chemiluminescence (CL) aptasensor consists of a designed streptavidin (SA) aptamer sequence in which several base pairs were replaced by G-G mismatches. Only in the presence of platinum, coordination occurs between the platinum and G-G base pairs as opposed to the hydrogen-bonded G-C base pairs, which leads to SA aptamer sequence activation, resulting in their binding to SA coated magnetic beads. These Pt-DNA coordination events were monitored by a simple and direct luminol-peroxide CL reaction through horseradish peroxidase (HRP) catalysis with a strong chemiluminescence emission. The validated ranges of quantification were 0.12-240 µM with a limit of detection of 60 nM and selectivity over other metal ions. This assay was also successfully used in urine sample determination. It will be a promising candidate for the detection of platinum in biomedical and environmental samples.

Simple and rapid determination of unsaturated fatty acids in 1 µl of rat plasma by LC-MS/MS.

BACKGROUND: Deficiency or imbalance of unsaturated fatty acids will promote the pathogenesis of many diseases. In order to monitor the exposure of unsaturated fatty acids, the method based on LC-MS/MS was developed. RESULTS: Standard calibration curves for α-linolenic acid, linoleic acid, palmitoleic acid and oleic acid were linear (r ≥0.99). The intra-and interbatch accuracy (RE%) ranged from -4.5 to 8.6%, while the intra- and interbatch precisions (RSD%) were ≤8.7%. The extraction recovery varied from 85.4 to 99.6%, and no obvious matrix effect was observed. CONCLUSION: The method offers a simple approach for measuring 4 unsaturated fatty acids in 1 µl rat plasma within 3.95 min.

Metabolism of flavonoids in human: a comprehensive review.

Flavonoids are naturally occurring polyphenols, which are widely taken in diets, supplements and herbal medicines. Epidemiological studies have shown a flavonoid-rich diet is associated with the decrease in incidence of a range of diseases. Pharmacological evidences also reveal flavonoids display anti-oxidant, anti-allergic, anti-cancer, anti-inflammatory, anti-microbial and anti-diarrheal activities. Therefore, it is critical to study the biotransformation and disposition of flavonoids in human. This review summarizes the major metabolism pathways of flavonoids in human. First, lactase-phlorizin hydrolase (LPH) and human intestinal microflora mediate the hydrolysis of flavonoid glycosides, which is recognized as the first and determinant step in the absorption of flavonoids. Second, phase II metabolic enzymes (UGTs, SULTs and COMT) dominate the metabolism of flavonoids in vivo. UGTs are the most major contributors, followed by SULTs and COMT. By contrast, phase I metabolism pathway mediated by CYPs only plays a minor role. Third, the coupling of transporters (such as BCRP and MRPs) and phase II enzymes (UGTs and SULTs) plays an important role in the disposition of flavonoids, especially in the enteroenteric and enterohepatic circulations. Thus, all the above factors should be taken into consideration when studying pharmacokinetics of flavonoids. Here we describe a comprehensive metabolism profile of flavonoids, which will enhance our understanding of the mechanisms underlying the disposition and pharmacological effects of flavonoids in vivo.

Post-column mobile phase adjustment: a strategy to eliminate the contradiction between liquid chromatography and mass spectrometry in the determination of flavonoids in rat plasma.

Flavonoids are a group of important naturally occurring polyphenolic compounds with a wide range of biological effects. In this study, a sensitive liquid chromatography tandem mass spectrometry method was developed to simultaneously determine multiple active flavonoids, including quercetin (Que), kaempferol (Kae), apigenin (Api), isorhamnetin (Iso), luteolin (Lut), and naringenin (Nar), in rat plasma. To achieve a satisfied peak shape and LC separation, formic acid with the concentration between 0.05 and 0.2%, or in some case 5%, was generally used to acidify the LC mobile phase in reported studies. Here we found that even 0.05% formic acid could lead to strong mass signal suppression, and the absence of formic acid could reverse the signal suppression but cause serious peak tailing. There is an irreconcilable contradiction between liquid chromatography (LC) and mass spectrometry (MS). In order to simultaneously satisfy LC and MS, LC mobile phase with 0.00075% formic acid and post column mobile phase adjustment with 0.0677% ammonium solution in isopropanol were applied. Compared with the conventional method with mobile phase containing 0.05% formic acid, the mass signal response of Que, Kae, Api, Iso, Lut, Nar, and Oka increased 26.2, 18.6, 13.6, 23.5, 17.5, 15.6 and 15.4 fold, respectively. In addition, the post column mobile phase addition exhibited the better peak shape for the reduction of analytes longitudinal diffusion. The method has been fully validated according to FDA guidelines within the linear range between 0.328 ng mL⁻¹ and 168 ng mL⁻¹, and successfully applied to a pilot pharmacokinetic study of rats after administering 5.43 g kg⁻¹ Pollen of Brassica campestris.

Pharmacokinetic Interaction of astragaloside IV with atractylenolide I and prim-O-glucosylcimifugin in male Sprague Dawley rats.

Astragaloside IV, atractylenolide I, and prim-O-glucosylcimifugin are main medicinal components of the traditional Chinese medicine prescription Yu-ping-feng which is composed of three herbs: Astragalus membranaceus, Atractylodes macrocephala, and Saposhnikovia divaricata. This study is aimed to assess the influence of atractylenolide I and prim-O-glucosylcimifugin on the pharmacokinetic profile of astragaloside IV so as to investigate the pharmacokinetic mechanisms of the Yu-ping-feng prescription. Fifteen Sprague Dawley rats were randomized to three groups; astragaloside IV, astragaloside IV plus atractylenolide I, and a combination of astragaloside IV, atractylenolide I, and prim-O-glucosylcimifugin were respectively administered to rats of these three groups via intragastric gavage. Serum samples were collected at different times after drug administration, and serum concentrations of astragaloside IV and atractylenolide I were simultaneously detected using HPLC-electrospray ionization-MS. Compared with administration of astragaloside IV alone, concentrations of astragaloside IV in the serum were significantly increased when it was given in combination with atractylenolide I or atractylenolide I+prim-O-glucosylcimifugin, with higher values for Cmax (p = 0.019 and p = 0.033 compared with astragaloside IV + atractylenolide I and astragaloside IV + atractylenolide I + prim-O-glucosylcimifugin groups, respectively) and AUC (p = 0.0052 and p = 0.0047 compared with astragaloside IV + atractylenolide I and astragaloside IV + atractylenolide I + prim-O-glucosylcimifugin groups, respectively). Improvement in mean oral Cmax and mean systemic serum exposure because of the pharmacokinetic interaction between astragaloside IV and atractylenolide I might explain the rationale for the use of multiple herbs in Yu-ping-feng and of combinations of A.membranaceus and A. macrocephala.

Determination of three triterpene alcohols in rat plasma after oral administration of pollen of Brassica campestris based on the utilization of fetal bovine serum as surrogate matrix.

24-Dehydropollinstanol (DEH), 24-methylene cholesterol (MET) and 31-norcycloartenol (NOR) are the functional triterpene alcohols of pollen of Brassica campestris. To study the pharmacokinetics of the above components of pollen of B. campestris in rats, a liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed. To avoid the interference of endogenous MET in rat plasma, fetal bovine serum (FBS) was selected as surrogate matrix and validated. Rat plasma was liquid-liquid extracted, then the chromatographic separation was conducted on a poroshell 120 SB C18 column (2.7µm, 2.1mm×50mm) at 38°C within 5.6min utilizing a gradient elution with a mobile phase consisting of (A) 0.1% formic acid in water and (B) 0.1% formic acid in methanol. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode using positive atmospheric pressure chemical ionization (APCI). The method was validated over the concentration of 9.8-1560ng/ml; the inter-and-intra-day precisions (RSD %) were ≤7.8%, and the accuracies (RE %) were -5.3% to 12.2%, the extraction recovery ranged from 73.5% to 106.9% for all of these analytes, and no obvious matrix effect was observed. The developed method was applied successfully to study the pharmacokinetics of DEH, MET and NOR in rats after oral administration of pollen of B. campestris.

Pharmacokinetic-pharmacodynamic evaluation of the major component astragaloside IV on the immunomodulatory effects of Yu-ping-feng prescription.

Yu-ping-feng decoction (YPF), a traditional Chinese medicine (TCM) prescription, is widely used to treat some respiratory tract diseases. This study was aimed to set up a pharmacokinetic-pharmacodynamic (PK-PD) model to assess dose-effect relationships for immunomodulatory effects of YPF in rats and to clarify compatibility mechanisms for TCM prescription system. Serum samples taken after YPF administration were tested on spleen cells of rats in vitro and proliferation ratio of spleen cells was used as end points to evaluate pharmacodynamic properties of immunoregulatory effects of YPF prescription. And with a HPLC-MS method, concentrations of Astragaloside IV (AS), a main component of YPF, were determined to achieve pharmacokinetic parameters after administration of a simplified prescription which is composed with AS, Atractylenolide I and Prim-O-glucosylcimifugin which are representative components of YPF. A plot of serum AS concentrations versus time and effects showed that there was a correlated relationship between AS concentrations and effects of YPF, and the concentration-response curve which was based on an E max model showed a counterclockwise hysteresis manner. A PK-PD model with Sheiner's method was used to describe time course of AS concentration in blood compartment and effect compartment, and main parameters with the PK-PD model were calculated. These results showed that there is a symmetry relationship between serum AS concentrations and responses of serum containing medicine of YPF prescription, which means that AS plays an important role in immunoregulatory effects of YPF. And the investigation on dose-effect relationships has displayed a feasible method to clarify mechanisms of combination for TCM prescriptions.

Anti-inflammatory and immunoregulatory effects of Yupingfeng powder on chronic bronchitis rats.

OBJECTIVE: To investigate the anti-inflammatory and immunoregulatory effects of Yupingfeng (, YPF) Powder and its components in rats. METHODS: A rat chronic bronchitis (CB) model was developed using lipopolysaccharide (LPS) combined with bacillus Calmette Guerin (BCG). YPF, simple recipe Astragalus membranaceus (Fisch.) Bge (AM) and Astragalus membranaceus (Fisch.) Bge plus rhizome of Atractylodes macrocephala Koidz (AM+RA) decoction were administered (intragastric administration, once a day for 21 days) to rats, to prevent and treat CB. Immunoregulatory and anti-inflammatory effects of YPF, AM and AM+RA were tested by serum pharmacology in vitro on splenic lymphocytes of normal rats and alveolar macrophages of CB rats. RESULTS: Inflammation in the pulmonary tissue and the bronchus of CB rats was significantly reduced in the YPF-treatment groups, AM and AM+RA groups demonstrating the efficacy of YPF. Serum samples collected at different times from rats after administration of YPF, AM and AM+RA demonstrated increased proliferation of splenic lymphocytes with area under the effect curve (AUE) of 552.6%, 336.3% and 452.0%, respectively. Treatment of alveolar macrophages with serum samples in YPF, AM or AM+RA group inhibited interleukin-8 (IL-8) in the cell culture media, and the effect was much better in the YPF group compared with AM or AM+RA group, with a higher maximal effect (Emax, P<0.05) and larger AUE (P <0.01 and P<0.05). Moreover, serum from rats treated with AM or AM+RA had similar efficacy, while the efficiency was lower than that treated with YPF. CONCLUSION: YPF demonstrated anti-inflammatory and immunoregulatory effects in a rat model of CB, and timedependent relationships were demonstrated in vitro.