[Synergistic effect and compatibility structure of active anti-inflammatory ingredients from Lamiophlomis rotata based on network pharmacology and component structure theory].

The synergistic effect and compatibility structure of active anti-inflammatory ingredients(iridoid glycosides: shanzhiside methylester and 8-O-acetylshanzhiside methyl ester, flavonoid glycoside: luteoloside, and phenylethanoid glycoside: forsythoside B) from Lamiophlomis rotata were explored based on network pharmacology and component structure theory. In network pharmacology, CTD, SwisseTargetPrediction, and PharmMapper databases were used to collect and screen the targets of all active ingredients. The inflammation-related targets were obtained from CTD and GeneCards databases. The core targets were obtained by Venny 2.1.0, STRING, and Cytoscape 3.9.1. Core targets were annotated by the GO function and enriched by the KEGG pathway based on the DAVID database. In terms of component structure, based on a uniform design method and xylene-induced ear swelling model in mice, tumor necrosis factor-α and interleukin-6 were taken as the dependent variables, and the compatibility relationship among anti-inflammatory ingredients from L. rotata was explored through the quadratic polynomial stepwise regression. In addition, in vivo pharmacological experiments were conducted to verify the results. A network pharmacology study showed that compared with a single ingredient, the combined action of the three ingredients can synergistically exert anti-inflammatory effects through more biological processes, pathways, and targets. Component structure study showed that the optimal structural ratio of shanzhiside methylester and 8-O-acetylshanzhiside methyl ester in the iridoid glycoside ingredient was 1.21∶1. The optimal structural ratio among the three types of ingredients(iridoid glycosides∶phenylethanol glycoside∶flavonoid glycoside) was 4.8∶1.6∶1. In conclusion, each anti-inflammatory ingredient from L. rotata can work synergistically, and there is an optimal compatibility ratio relationship among these ingredients. This work provides a new experimental basis for the intrinsic quality control of L. rotata.

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.

Characterization of anti-CD79b/CD3 bispecific antibody, a potential therapy for B cell malignancies.

High rates of relapse and poor prognosis confer an urgent need for novel therapeutic agents for B cell non-Hodgkin lymphomas (B-NHLs). Herein, we describe a human IgG-like anti-CD79b/CD3 bispecific antibody (IBI38D9-L) that selectively depletes antigen-positive malignant B cells as an alternative treatment option for relapsed or refractory NHL patients. The antitumor activity and mechanism of action of IBI38D9-L were investigated in vitro using B-NHL cell lines and human primary effector cells and in vivo using xenograft models reconstituted with human PBMCs (peripheral blood mononuclear cells). Pharmacokinetic (PK) properties and preclinical toxicology were evaluated in cynomolgus monkeys and HSC-NPG mice. IBI38D9-L exerted potent B cell killing as well as T cell activation and proliferation in a tumor cell-dependent manner in vitro and was active against B-NHL cell lines with various CD79b expression levels. Subcutaneous xenograft tumors in NOG mice engrafted with human PBMCs were eradicated by IBI38D9-L treatment. Moreover, IBI38D9-L-treated mice showed a strong infiltration of activated T cells. In HSC-NPG mice, IBI38D9-L resulted in potent B cell depletion in peripheral blood and induced only slight body weight loss and cytokine release syndrome without significant toxicological findings. In cynomolgus monkeys, IBI38D9-L was well tolerated with good pharmacokinetic profiles. Collectively, these preclinical efficacy and safety data provide strong scientific rationales for using anti-CD79b/CD3 bispecific antibody as a promising therapeutic agent for B cell malignancies.

Lasting antibody and T cell responses to SARS-CoV-2 in COVID-19 patients three months after infection.

The dynamics, duration, and nature of immunity produced during SARS-CoV-2 infection are still unclear. Here, we longitudinally measured virus-neutralising antibody, specific antibodies against the spike (S) protein, receptor-binding domain (RBD), and the nucleoprotein (N) of SARS-CoV-2, as well as T cell responses, in 25 SARS-CoV-2-infected patients up to 121 days post-symptom onset (PSO). All patients seroconvert for IgG against N, S, or RBD, as well as IgM against RBD, and produce neutralising antibodies (NAb) by 14 days PSO, with the peak levels attained by 15-30 days PSO. Anti-SARS-CoV-2 IgG and NAb remain detectable and relatively stable 3-4 months PSO, whereas IgM antibody rapidly decay. Approximately 65% of patients have detectable SARS-CoV-2-specific CD4+ or CD8+ T cell responses 3-4 months PSO. Our results thus provide critical evidence that IgG, NAb, and T cell responses persist in the majority of patients for at least 3-4 months after infection.

Multiomics Profiling Reveals Protective Function of Schisandra Lignans against Acetaminophen-Induced Hepatotoxicity.

The action principles of traditional Chinese medicines (TCMs) feature multiactive components, multitarget sites, and weak combination with action targets. In the present study, we performed an integrated analysis of metabonomics, proteomics, and lipidomics to establish a scientific research system on the underlying mechanism of TCMs, and Schisandra lignan extract (SLE) was selected as a model TCM. In metabonomics, several metabolic pathways were found to mediate the liver injury induced by acetaminophen (APAP), and SLE could regulate the disorder of lipid metabolism. The proteomic study further proved that the hepatoprotective effect of SLE was closely related to the regulation of lipid metabolism. Indeed, the results of lipidomics demonstrated that SLE dosing has an obvious callback effect on APAP-induced lipidic profile shift. The contents of 25 diglycerides (DAGs) and 21 triglycerides (TAGs) were enhanced significantly by APAP-induced liver injury, which could further induce liver injury and inflammatory response by upregulating protein kinase C (PKCß, PKCγ, PKCδ, and PKCθ). The upregulated lipids and PKCs could be reversed to the normal level by SLE dosing. More importantly, phosphatidic acid phosphatase, fatty acid transport protein 5, and diacylglycerol acyltransferase 2 were proved to be positively associated with the regulation of DAGs and TAGs. SIGNIFICANCE STATEMENT: Integrated multiomics was first used to reveal the mechanism of APAP-induced acute liver failure (ALF) and the hepatoprotective role of SLE. The results showed that the ALF caused by APAP was closely related to lipid regulation and that SLE dosing could exert a hepatoprotective role by reducing intrahepatic diglyceride and triglyceride levels. Our research can not only promote the application of multicomponent technology in the study of the mechanism of traditional Chinese medicines but also provide an effective approach for the prevention and treatment of ALF.

Nosocomial cross-infection of hypervirulent Listeria monocytogenes sequence type 87 in China.

BACKGROUND: To investigate the epidemiological and phenotypic characteristics and molecular relatedness of L. monocytogenes, which were cultured from the blood and cerebrospinal fluid (CSF) samples isolated from two neonates. METHODS: In the present case study, two infected neonates were interviewed and epidemiological investigation performed. The phenotypic characteristics and molecular relatedness of L. monocytogenes was characterized by serotyping, pulsed-field gel electrophoresis and whole-genome sequencing (WGS). RESULTS: The field investigation found that the two neonates were born in the same hospital (Hospital B) and admitted to the neonatal department through different channels within half an hour by different nurses, where they were weighed and placed in different but adjacent incubators. Then they were cared for by the same group of nurses that evening. It is worth noting that there was no record of sanitation of the neonatal incubator of neonate-1. The serotype of the two isolated L. monocytogenes were 1/2b, with an indistinguishable pulsotypes and were sequence type (ST) 87. WGS showed that there were no core SNP differences identified. In order to explore the genomic traits associated with L. monocytogenes virulence genes, we identified the Listeria pathogenicity island 4 and found that the genome was devoid of any stress islands. There are no positive results from the environmental samples. Considering the genomic data together with epidemiological evidence and clinical symptoms, insufficient surface cleaning along with the nursing staff caring for these neonates was considered as cross-infection factors. CONCLUSIONS: To our knowledge, this is the first report of a nosocomial cross-infection of L. monocytogenes ST87 between two neonates, which carries the recently identified gene cluster expressing the cellobiose-family phosphotransferase system (PTS-LIPI-4) between two neonates. The test results of environmental samples in the hospital indicate that strict sterilization and patient isolation measures cannot be emphasized enough in neonatal nursing.

Transmission Potential of Asymptomatic and Paucisymptomatic Severe Acute Respiratory Syndrome Coronavirus 2 Infections: A 3-Family Cluster Study in China.

Data concerning the transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in asymptomatic and paucisymptomatic patients are lacking. We report a 3-family cluster of infections involving asymptomatic and paucisymptomatic transmission. Eight of 15 (53%) members from 3 families were confirmed with SARS-CoV-2 infection. Of 8 patients, 3 were asymptomatic and 1 was paucisymptomatic. An asymptomatic mother transmitted the virus to her son, and a paucisymptomatic father transmitted the virus to his 3-month-old daughter. SARS-CoV-2 was detected in the environment of 1 household. The complete genomes of SARS-CoV-2 from the patients were > 99.9% identical and were clustered with other SARS-CoV-2 sequences reported from China and other countries.

Ginsenoside Rb1 exerts neuroprotective effects through regulation of Lactobacillus helveticus abundance and GABAA receptor expression.

BACKGROUND: Ginsenoside Rb1 (Rb1), one of the most abundant protopanaxadiol-type ginsenosides, exerts excellent neuroprotective effects even though it has low intracephalic exposure. PURPOSE: The present study aimed to elucidate the apparent contradiction between the pharmacokinetics and pharmacodynamics of Rb1 by studying the mechanisms underlying neuroprotective effects of Rb1 based on regulation of microflora. METHODS: A pseudo germ-free (PGF) rat model was established, and neuroprotective effects of Rb1 were compared between conventional and PGF rats. The relative abundances of common probiotics were quantified to reveal the authentic probiotics that dominate in the neuroprotection of Rb1. The expressions of the gamma-aminobutyric acid (GABA) receptors, including GABAA receptors (α2, ß2, and γ2) and GABAB receptors (1b and 2), in the normal, ischemia/reperfusion (I/R), and I/R+Rb1 rat hippocampus and striatum were assessed to reveal the neuroprotective mechanism of Rb1. RESULTS: The results showed that microbiota plays a key role in neuroprotection of Rb1. The relative abundance of Lactobacillus helveticus (Lac.H) increased 15.26 fold after pretreatment with Rb1. I/R surgery induced effects on infarct size, neurological deficit score, and proinflammatory cytokines (IL-1ß, IL-6, and TNF-α) were prevented by colonizing the rat gastrointestinal tract with Lac.H (1 × 109 CFU) by gavage 15 d before I/R surgery. Both Rb1 and Lac.H upregulated expression of GABA receptors in I/R rats. Coadministration of a GABAA receptor antagonist significantly attenuated neuroprotective effects of Rb1 and Lac.H. CONCLUSION: In sum, Rb1 exerts neuroprotective effects by regulating Lac.H and GABA receptors rather than through direct distribution to the target sites.

Comparative analysis of constitutes and metabolites for traditional Chinese medicine using IDA and SWATH data acquisition modes on LC-Q-TOF MS.

Identification of components and metabolites of traditional Chinese medicines (TCMs) employing liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF MS) techniques with information-dependent acquisition (IDA) approaches is increasingly frequent. A current drawback of IDA-MS is that the complexity of a sample might prevent important compounds from being triggered in IDA settings. Sequential window acquisition of all theoretical fragment-ion spectra (SWATH) is a data-independent acquisition (DIA) method where the instrument deterministically fragments all precursor ions within the predefined m/z range in a systematic and unbiased fashion. Herein, the superiority of SWATH on the detection of TCMs' components was firstly investigated by comparing the detection efficiency of SWATH-MS and IDA-MS data acquisition modes, and sanguisorbin extract was used as a mode TCM. After optimizing the setting parameters of SWATH, rolling collision energy (CE) and variable Q1 isolation windows were found to be more efficient for sanguisorbin identification than the fixed CE and fixed Q1 isolation window. More importantly, the qualitative efficiency of SWATH-MS on sanguisorbins was found significantly higher than that of IDA-MS data acquisition. In IDA mode, 18 kinds of sanguisorbins were detected in sanguisorbin extract. A total of 47 sanguisorbins were detected when SWATH-MS was used under rolling CE and flexible Q1 isolation window modes. Besides, 26 metabolites of sanguisorbins were identified in rat plasma, and their metabolic pathways could be deduced as decarbonylation, oxidization, reduction, methylation, and glucuronidation according to their fragmental ions acquired in SWATH-MS mode. Thus, SWATH-MS data acquisition could provide more comprehensive information for the component and metabolite identification for TCMs than IDA-MS.

The improved performance of MALDI-TOF MS on the analysis of herbal saponins by using DHB-GO composite matrix.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an excellent analytical technique for rapid analysis of a variety of molecules with straightforward sample pretreatment. The performance of MALDI-TOF MS is largely dependent on matrix type, and the development of novel MALDI matrices has aroused wide interest. Herein, we devoted to seek more robust MALDI matrix for herbal saponins than previous reported, and ginsenoside Rb1, Re, and notoginsenoside R1 were used as model saponins. At the beginning of the present study, 2,5-dihydroxybenzoic acid (DHB) was found to provide the highest intensity for saponins in four conventional MALDI matrices, yet the heterogeneous cocrystallization of DHB with analytes made signal acquisition somewhat "hit and miss." Then, graphene oxide (GO) was proposed as an auxiliary matrix to improve the uniformity of DHB crystallization due to its monolayer structure and good dispersion, which could result in much better shot-to-shot and spot-to-spot reproducibility of saponin analysis. The satisfactory precision further demonstrated that minute quantities of GO (0.1 µg/spot) could greatly reduce the risk of instrument contamination caused by GO detachment from the MALDI target plate under vacuum. More importantly, the sensitivity and linearity of the standard curve for saponins were improved markedly by DHB-GO composite matrix. Finally, the application of detecting the Rb1 in complex biological sample was exploited in rat plasma and proved it applicable for pharmacokinetic study quickly. This work not only opens a new field for applications of DHB-GO in herbal saponin analysis but also offers new ideas for the development of composite matrices to improve MALDI MS performance.

Identification of subgroups of patients with type 2 diabetes with differences in renal function preservation, comparing patients receiving sodium-glucose co-transporter-2 inhibitors with those receiving dipeptidyl peptidase-4 inhibitors, using a supervised machine-learning algorithm (PROFILE study): A retrospective analysis of a Japanese commercial medical database.

AIMS: To investigate the effects of sodium-glucose co-transporter-2 (SGLT2) inhibitors vs. dipeptidyl peptidase-4 (DPP-4) inhibitors on renal function preservation (RFP) using real-world data of patients with type 2 diabetes in Japan, and to identify which subgroups of patients obtained greater RFP benefits with SGLT2 inhibitors vs. DPP-4 inhibitors. METHODS: We retrospectively analysed claims data recorded in the Medical Data Vision database in Japan of patients with type 2 diabetes (aged ≥18 years) prescribed any SGLT2 inhibitor or any DPP-4 inhibitor between May 2014 and September 2016 (identification period), in whom estimated glomerular filtration rate (eGFR) was measured at least twice (baseline, up to 6 months before the index date; follow-up, 9 to 15 months after the index date) with continuous treatment until the follow-up eGFR. The endpoint was the percentage of patients with RFP, defined as no change or an increase in eGFR from baseline to follow-up. A proprietary supervised learning algorithm (Q-Finder; Quinten, Paris, France) was used to identify the profiles of patients with an additional RFP benefit of SGLT2 inhibitors vs. DPP-4 inhibitors. RESULTS: Data were available for 990 patients prescribed SGLT2 inhibitors and 4257 prescribed DPP-4 inhibitors. The proportion of patients with RFP was significantly greater in the SGLT2 inhibitor group (odds ratio 1.27; P = 0.01). The Q-Finder algorithm identified four clinically relevant subgroups showing superior RFP with SGLT2 inhibitors (P < 0.1): no hyperlipidaemia and eGFR ≥79 mL/min/1.73 m2 ; eGFR ≥79 mL/min/1.73 m2 and diabetes duration ≤1.2 years; eGFR ≥75 mL/min/1.73 m2 and use of antithrombotic agents; and haemoglobin ≤13.4 g/dL and LDL cholesterol ≥95.1 mg/dL. In each profile, glycaemic control was similar in the two groups. CONCLUSION: SGLT2 inhibitors were associated with more favourable RFP vs. DPP-4 inhibitors in patients with certain profiles in real-world settings in Japan.

An auxiliary matrix for routine analysis of small molecules and biological macromolecules using matrix-assisted laser desorption ionization mass spectrometry.

The great hurdles related with matrix-assisted laser desorption/ionization (MALDI) analysis are inhomogeneous crystallization, poor reproducibility, and low sensitivity. To effectively improve the performance of MALDI mass spectrometry (MS), graphene oxide (GO) was first utilized as an auxiliary matrix of the conventional matrices, including 2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydoxycyanocinnamic acid (CHCA), 2,4,6-trihydroxyacetophenone (THAP), and 3,5-dimethoxy-4-hydroxycinnamic acid (SA), for the analysis of small molecules and biological macromolecules on different MALDI MS systems. The results revealed that the DHB-GO composite matrix could provide much superior crystal homogenization, better reproducibility, higher sensitivity, and more excellent linearity for the statins' tissue imaging on iMScope than the single-use DHB matrix. Moreover, the DHB-GO dramatically improved the spot-to-spot and shot-to-shot reproducibility, crystal homogenization, sensitivity, and linearity of MALDI-TOF MS for statins' analysis in dried droplet. The capability of THAP on the analysis of lipids, similarly, could be greatly enhanced by the combined use of GO. THAP-GO composite matrix was expected to be widely used in the MALDI MS-based liposome studies. It was also found that CHCA-GO could provide superior analytical performance for peptides. The sensitivity and reproducibility of intact proteins could be greatly improved by SA-GO composite matrix. More importantly, the better reproducibility produced by the composite matrices sufficiently indicated that low concentration (0.1 mg mL-1) of GO almost did not cause contamination to MALDI MS system. Thus, GO was proved to be a versatile auxiliary matrix for the MALDI MS-based routine analysis of small molecules and biological macromolecules. Graphical abstract ᅟ.

Systematically identifying the hepatoprotective ingredients of schisandra lignan extract from pharmacokinetic and pharmacodynamic perspectives.

BACKGROUND: Herbal medicines (HMs) have been proven to be productive sources of leads for the development of drugs. To date approximately 150 lignans have been identified from Schisandra sphenanthera. Hepatoprotective activity is a well-known characteristic of schisandra lignans, yet the authentic types of active lignans are still not well known. PURPOSE: The present study aimed to develop a reliable and efficient strategy for identifying the hepatoprotective ingredients of schisandra lignan extract (SLE). METHODS: SLEs were prepared by extracting Schisandra sphenanthera powder using 10%, 50% and 90% ethanol (w/w 1:10) combining 5-fold volume of ethyl acetate. The schisandra lignans in SLEs were qualitatively analyzed based on liquid chromatography hybrid ion trap time-of-flight mass spectrometry (LCMS-IT-TOF). Preparative liquid chromatography (PLC) was used to collect ingredient fractions. The hepatoprotective activity of schisandra lignans was systematically investigated on in vivo and in vitro models. RESULTS: The SLE extracted by 50% ethanol and 5-fold volume of ethyl acetate (50%SLE) had the highest lignan content and exhibited significantly stronger hepatoprotective activity than other SLEs (P <  0.01). The hepatoprotective effect of 50%SLE mainly attributed to the SLE segment which collected from 12 to 22 min by PLC. Schisantherin A (Sth A) was confirmed as the most promising hepatoprotective drug in Schisandra sphenanthera due to high content in crude materials, high exposure level in vivo and high efficiency on APAP-induced hepatotoxicity. CONCLUSION: The hepatoprotective ingredients of SLEs were systematically investigated based on the presently developed approach, and Sth A was identified as the optimum hepatoprotective candidate in Schisandra sphenanthera.

Is the combinational administration of doxorubicin and glutathione a reasonable proposal?

The combinational administration of antioxidants and chemotherapeutic agents during conventional cancer treatment is among one of the most controversial areas in oncology. Although the data on the combinational usage of doxorubicin (DOX) and glutathione (GSH) agents have been explored for over 20 years, the duration, administration route, and authentic rationality have not yet been fully understood yet. In the current study, we systematically investigated the pharmacokinetics (PK) and pharmacodynamics (PD) with both in vivo and in vitro models to elucidate the influence of GSH on the toxicity and efficacy of DOX. We first studied the cardioprotective and hepatoprotective effects of GSH in Balb/c mice, H9c2, and HL7702 cells. We showed that coadministration of exogenous GSH (5, 50, and 500 mg/kg per day, intragastric) significantly attenuated DOX-induced cardiotoxicity and hepatotoxicity by increasing intracellular GSH levels, whereas the elevated GSH concentrations did not affect the exposure of DOX in mouse heart and liver. From PK and PD perspectives, then the influences of GSH on the chemotherapeutic efficacy of DOX were investigated in xenografted nude mice and cancer cell models, including MCF-7, HepG2, and Caco-2 cells, which revealed that administration of exogenous GSH dose-dependently attenuated the anticancer efficacy of DOX in vivo and in vitro, although the elevated GSH levels neither influenced the concentration of DOX in tumors in vivo, nor the uptake of DOX in MCF-7 tumor cells in vitro. Based on the results we suggest that the combined administration of GSH and DOX should be contraindicated during chemotherapy unless DOX has caused serious hepatotoxicity and cardiotoxicity.

Activated charcoal significantly improved the reliability of methods for quantitative analysis of endogenous substances in biological specimens: Glutathione and cysteine as cases.

When developing a quantitative assay for exogenous or endogenous compounds, guidelines for method validation normally recommend that the biological specimens should be prepared in corresponding authentic matrices, yet "analyte-free authentic matrices" is in general not available. It is generally known that GSH and CYS are endogenous compounds and present in both prokaryotes and eukaryotes. Herein, an efficient approach for the quantitative analysis of endogenous substances in biological specimens was developed, and glutathione (GSH) & cysteine (CYS) were chosen as model endogenous substances. Activated carbon (AC), a common adsorbent for the adsorption of environmental pollutants, was used to remove the endogenous GSH and CYS and prepare "GSH&CYS-free biological matrix". The endogenous GSH and CYS in mouse plasma, blood and liver homogenate were found can be almost removed via incubating with 100 mg of AC for 2 h. After optimizing the derivatization reagents, internal standard and analytical parameters, a reliable quantitative assay of GSH and CYS in mouse plasma, blood and liver homogenate was developed and validated on LC-ESI-MS/MS using corresponding AC-adsorbed mouse biological matrices. The validation results indicated that the developed method provided suitable accuracy, sensitivity, specificity and high throughput for the analysis of GSH and CYS. Finally, the developed LC-ESI-MS/MS assay was successfully applied to measure the concentrations of GSH and CYS in liver injury mice. The presently developed methodology could be widely applied in the quantitative analysis of endogenous compounds in various complex mixtures such as biological, herbal and environmental samples.

Pharmacokinetic and pharmacodynamic evidence for developing an oral formulation of octreotide against gastric mucosal injury.

Among the somatostatin analogues, octreotide (OCT) is the most commonly used in clinic via intravenous or subcutaneous injection to treat various diseases caused by increased secretion of growth hormone, gastrin or insulin. In order to assesse the feasibility of developing oral formulations of OCT, we conducted systematical pharmacokinetic and pharmacodynamic analyses of OCT in several animal models. The pharmacokinetic studies in rats showed that intragastric administration of OCT had extremely low bioavailability (<0.5%), but it could specifically distribute to the gastric mucosa due to the high expression of somatostatin receptor 2 (SSTR2) in the rat stomach. The pharmacodynamic studies revealed that intragastric administration of OCT dose-dependently protected against gastric mucosal injury (GMI) in mice with WIRS-induced mouse gastric ulcers, which were comparable to those achieved by intravenous injection of OCT, and this effect was markedly attenuated by co-administration of CYN-154806, an antagonist of SSTR2. In pyloric ligation-induced ulcer mice, we further demonstrated that OCT significantly reduced the secretion of gastric acid via down-regulating the level of gastrin, which was responsible for the protective effect of OCT against GMI. Overall, we have provided pharmacokinetic and pharmacodynamic evidence for the feasibility of developing an oral formulation of OCT. Most importantly, the influence of SSTR2 on the pharmacokinetics and pharmacodynamics of OCT suggested that an oral formulation of OCT might be applicable for other clinical indications, including neuroendocrine neoplasms and pituitary adenoma due to the overexpression of SSTR2 on these tumor cells.

Low Cerebral Exposure Cannot Hinder the Neuroprotective Effects of Panax Notoginsenosides.

A bidirectional route of communication between the gastrointestinal tract and the central nervous system, termed the "gut-brain axis," is becoming increasingly relevant to treatment of cerebral damage. Panax Notoginsenoside extract (PNE) is popular for prevention and treatment of cardio-cerebrovascular ischemic diseases although plasma and cerebral exposure levels are extremely low. To date, the mechanisms underlying the neuroprotective effects of PNE remain largely unknown. In the present study, the neuroprotective effects of PNE were systematically studied via investigation of the regulation by PNE of the gastrointestinal microbial community and γ aminobutyric acid (GABA) receptors. The results demonstrated that pretreatment with PNE exerted a remarkable neuroprotective effect on focal cerebral ischemia/reperfusion (I/R) injury in rats, and the efficiency was attenuated in germ-free rats. Pretreatment with PNE could significantly prevent downregulation of Bifidobacterium longum (B.L) caused by I/R surgery, and colonization by B.L could also exert neuroprotective effects. More importantly, both PNE and B.L could upregulate the expression of GABA receptors in the hippocampus of I/R rats, and coadministration of a GABA-B receptor antagonist could significantly attenuate the neuroprotective effects of PNE and B.L. The study above suggests that the neuroprotective effects of PNE may be largely attributable to its regulation of intestinal flora, and oral treatment with B.L was also useful in therapy of ischemia/reperfusion injury (I/R) by upregulating GABA-B receptors.

An integrated strategy for the quantitative analysis of endogenous proteins: A case of gender-dependent expression of P450 enzymes in rat liver microsome.

Liquid chromatography mass spectrometry based methods provide powerful tools for protein analysis. Cytochromes P450 (CYPs), the most important drug metabolic enzymes, always exhibit sex-dependent expression patterns and metabolic activities. To date, analysis of CYPs based on mass spectrometry is still facing critical technical challenges due to the complexity and diversity of CYP isoforms besides lack of corresponding standards. The aim of present work consisted in developing a label-free qualitative and quantitative strategy for endogenous proteins, and then applying to the gender-difference study for CYPs in rat liver microsomes (RLMs). Initially, trypsin digested RLM specimens were analyzed by the nanoLC-LTQ-Orbitrap MS/MS. Skyline, an open source and freely available software for targeted proteomics research, was then used to screen the main CYP isoforms in RLMs under a series of criteria automatically, and a total of 40 and 39 CYP isoforms were identified in male and female RLMs, respectively. More importantly, a robust quantitative method in a tandem mass spectrometry-multiple reaction mode (MS/MS-MRM) was built and optimized under the help of Skyline, and successfully applied into the CYP gender difference study in RLMs. In this process, a simple and accurate approach named 'Standard Curve Slope" (SCS) was established based on the difference of standard curve slopes of CYPs between female and male RLMs in order to assess the gender difference of CYPs in RLMs. This presently developed methodology and approach could be widely used in the protein regulation study during drug pharmacological mechanism research.

Optimization and evaluation of MALDI TOF mass spectrometric imaging for quantification of orally dosed octreotide in mouse tissues.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-TOF-MSI) has received considerable attention in recent years since it allows molecular mapping of diverse bimolecular in animal/plant tissue sections, although some barriers still exist in absolute pixel-to-pixel quantification. Octreotide, a synthetic somatostatin analogue, has been widely used to prevent gastrointestine bleeding in the clinic. The aim of the present study is to develop a MALDI-TOF-MSI method for quantitatively visualizing spatial distribution of octreotide in mouse tissues. In this process, a structurally similar internal standard was spotted onto tissue section together with matrix solution to minimize signal variation and give excellent quantitative results. The 2,5-dihydroxybenzoic acid was chosen as the most suitable matrix via comparing the signal/noise generated by MALDI-TOF-MSI after cocrystallization of octreotide with different matrix candidates. The reliability of MALDI-TOF-MSI, with respect to linearity, sensitivity and precision, was tested via measuring octreotide in fresh tissue slices at different concentrations. The validated method was then successfully applied to visualize the distribution of octreotide in mouse tissues after oral administration of octreotide at 20mg/kg. The results demonstrated that MALDI-TOF-MSI could not only clearly visualize the spatial distribution of octreotide, but also make the calculation of the key pharmacokinetic parameters (Tmax and t1/2) possible. More importantly, the tissue concentration-time curves of octreotide determined by MALDI-TOF-MSI agreed well with those measured based on LC-MS/MS.These findings illustrate the potential of MALDI-TOF-MSI in pharmacokinetic profiling during drug development.

Bioanalytical assay development and validation for simultaneous quantification of five schisandra lignans in rat primary hepatocytes based on LC-MS/MS: application to a real-time uptake study for Schisandra Lignan Extract.

Schisandra lignans, mainly including schizandrol A, schizandrol B, schisantherin A, schizandrin A, schizandrin B, etc., are the major active ingredients of Schisandra chinensis. In the present study, a robust liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the simultaneous quantification of schisandra lignans in rat primary hepatocytes. Lovastatin was used as an internal standard, and chromatographic separation was achieved on a Shimadzu C18 column with a gradient elution at the flow rate of 0.2 mL/min. All of the analytes were detected in multiple reaction monitoring mode with positive electrospray ionization since the sodium adduct ion [M + Na]+ was observed as the most intensive peak in the MS spectrum. For schizandrol A, schisantherin A and schizandrin A, the dynamic range was within 2-1000 ng/mg protein, and the linear range of schizandrol B and schizandrin B was from 5 to 1000 ng/mg protein. The intra- and inter-day precision was <15% and the accuracy (relative error) ranged from -15 to 15%. No significant variation was observed in the stability tests. The validated method was then successfully applied to the time-dependent uptake study for the Schisandra Lignan Extract in rat primary hepatocytes.