Because of the tumor barrier and the microenvironment of tumor tissue, the treatment of tumor immunotherapy is limited and increased adverse reactions. In recent years, nano-preparation has made many new advances in the process of tumor immunotherapy for regulating immune deficiency, remodeling tumor tissue microenvironment, and enhancing the effect of immunotherapy. This article mainly describes the microenvironmental factors of affecting the efficacy of tumor immunotherapy, and reviews the strategies of nano-preparation for remodeling the tumor microenvironment.

OBJECTIVE: To investigate the stability of baicalein-7-O-diglucoside. METHODS: The chromatographic purity of baicalein-7-O-diglucoside and its solution in different condition was analyzed by HPLC-DAD. The degradation products of baicalein-7-O-diglucoside in its solvent were identified by LC-MS. Three degradation products in the solvent of baicalein-7-O-diglucoside were identified with applying electrospray ionization(ESI) source in the positive ion mode.RESULTS: ① Baicalein-7-O-diglucoside was stable with light and in 40, 80 and 105 ℃ within the inspection time in solid state. ②Baicalein-7-O-diglucoside in water was stable in room temperature and degrades quickly in 50%methanol and 50% ethanol. ③The degradation products of baicalein-7-O-diglucoside in solvent were 7-methoxy baicalein, 7-ethoxy baicalein and baicalein. The above research provides that baicalein-7-O-diglucoside is stable in solid state. Baicalein-7-O-diglucoside degrades in 50% methanol and 50% ethanol. CONCLUSION: Water should be used as the solvent for solution of baicalein-7-O-diglucoside. The solution of baicalein-7-O-diglucoside should be made up before use or stored in the refrigerator immediately after preparation.

OBJECTIVE: To conduct a single dose toxicology study of humanized anti-HER2 antibody drug conjugate for injection (HS630) and small molecular maytansine (DM1) in Sprague-Dawley rats. METHODS: Rats were divided randomly into nine groups including HS630 blank control group (0 mg•kg-1), DM1 vehicle control group (0 mg•kg-1), positive control group (Kadcyla®, 60 mg•kg-1), HS630 low-, middle-, high-dose groups (6, 20, 60 mg•kg-1) and DM1 low-, middle-, high-dose groups (0.10, 0.20, 0.40 mg•kg-1). Each group had twenty rats with female and male in half. Each rat was administered intravenously once. Animals were observed clinical symptoms consisting of behavior, fur and feces daily as well as body weight and food consumption twice or three times per week. Dissection was executed at D2 and D21 to examine gross anatomy with histopathological changes and weight main tissues. RESULTS: Rats given 60 mg•kg-1 HS630 appeared some adverse effects that 1/20 animal was dead. It was found that maximal tolerance dose of HS630 was 20 mg•kg-1 equivalent to 0.34 mg•kg-1 DM1. High dose could lead body weight and food consumption reduced and organ weights changed including liver, kidney, spleen and lung increased and testicle and epididymis decreased. Histopathological changes were observed in liver, spleen, lung, thymus, pancreas, kidney, mesenteric glands, intestinum, seminal vesicle, prostate, testicle, epididymis, adrenal gland, thyroid gland, pituitary body, eye, tongue, sternum (marrow), bone, skin, injection site. Rats given Kadcyla® showed the similar side effects with HS630. Rats given 20 mg•kg-1 HS630, abnormality weren′t observed in clinical symptom, body weight and food consumption. It could lead kidney and lung weighted. Histopathological changes were found in liver, spleen, thymus, pancreas, lung, kidney, mesenteric glands, duodenum, adrenal gland, pituitary body, skin. Rats given DM1 exhibited worse adverse effects that 2/20 animals were dead at 0.40 mg•kg-1 level. The maximal tolerance dose of DM1 was 0.20 mg•kg-1. High dose could lead body weight and food consumption reduced and organ weights changed including liver, spleen and adrenal gland increased and thymus decreased. Histopathological changes were found in liver, spleen, thymus, kidney, mesenteric glands, duodenum, intestinum, seminal vesicle, adrenal gland, pituitary body, eye, tongue, sternum (marrow), bone, skin. CONCLUSION: Rats given single dose of humanized anti-HER2 antibody-drug conjugate for injection (HS630) and chemical maytansine (DM1) respectively, the RESULTS: show that HS630, a kind of ADC products, have similar toxicological profile with Kadcyla® and exhibit better tolerability and wider safety margin when given the comparable dosage with DM1 based on the RESULTS: of tolerance, clinical symptoms, organ weight and histopathological findings.

OBJECTIVE: To investigate intermedin protects against at rat renal tubular epithelial cells(NRK-52E) hypoxia-reoxygenation injury through suppresses endoplasmic reticulum stress(ERS)-related apoptosis. METHODS: The NRK-52E cells were divided into 4 groups. One of them was control group; the other three model groups were exposed to H/R condition, following the intervention of single H/R, primitive vector and highly expressed was used IMD vector, respectively. The cell survival rate was detected by MTT; annexin V-FITC/PI double-stained cell apoptosis detection kit for detection of apoptosis; real-time PCR and Western-blot were used to detect expression of ERS molecules such as GRP78, CHOP and Caspase 12. ER specific resident fluorochrome dapoxyl was used to observe ER morphological changes. RESULTS: MTT RESULTS: showed that the survival rate of NRK-52E cells in H/R group was significantly decreased compared with the control group, and the cell survival rate was increased significantly in IMD group. The apoptosis rate of H/R increased significantly, and the mRNA and protein expressions of GRP78, CHOP and Caspase 12 in H/R group were all up-regulated significantly, IMD gene transfer markedly decreased the apoptosis index compared with that of H/R. The mRNA and protein expressions of GRP78, CHOP and Caspase 12 in IMD group were all down-regulated significantly. Dapoxyl staining demonstrated that the structure of the ER was severely destroyed in H/R, the fluorescence density showed nonuniform distribution, accompanied by notable vacuoles in the ER; Over-express IMD decreased the injury of the ER. CONCLUSION: During rat renal tubular epithelial cells NRK-52E H/R injury may occur activation of ERS apoptotic pathway, IMD inhibites ERS-related apoptosis, reduces renal tubular epithelial cell apoptosis and reduces renal IRI.

OBJECTIVE: To find disease biomarkers of cerebral ischemia by establishing cerebral ischemia-reperfusion injury model of rats and comparing with healthy rats. METHODS: Cerebral ischemia-reperfusion model of rats was established by modified Longa′s intraluminal suture method. After 24 h of modeling, plasma and brain tissue from the model group and the control group were collected for plasma biochemical indicators and metabolites analysis. Brain slices and plasma biochemical indicators were used to demonstrate the successful modeling. Gas chromatography-mass spectrometry was adopted to conduct targeted metabolomics studies on free fatty acids in rat plasma, tridecanoic acid (C13∶0) and 2,6-di-tert-butyl-4-methylphenol (BHT) were added as internal standard and antioxidant during sample preparation. Methylation was carried out using concentrated sulfuric acid/CH3OH as a derivatization reagent, and a non-polar stationary phase column was selected for chromatographic separation. RESULTS: Comparing the model group with the blank group, four differential metabolites were obtained, namely palmitic acid, stearic acid, oleic acid and linoleic acid, which were up-regulated throughout the metabolic pathway. The RESULTS of the relevant metabolic pathway analysis showed that the linoleic acid metabolic pathway had significant changes in the disease process. CONCLUSION: In the fatty acid anabolic pathway, the linoleic acid metabolic pathway changes significantly during the onset and treatment of cerebral ischemia-reperfusion. The effect of preventing and treating cerebral ischemia-reperfusion injury can be achieved by controlling the level of linoleic acid. This study evaluates the prognosis of cerebral ischemia reperfusion injury by measuring the plasma of model rats, explores its cause and pathogenesis, and laid a foundation for the study of disease pathology and the mechanism of drug action.

OBJECTIVE To design N dodecanol modified docetaxel(DTX) prodrug, prepare nanostructured lipid carrier(NLC) and investigate in vitro antitumor activity and in vivo pharmacodynamic. METHODS Nanostructured lipid carrier (DNLC) encapsulating n-dodecanol-modified DTX prodrug was prepared by ultrasonic method. The formulation was optimized by single-factor experiment and response surface optimization. The accumulated rates of DTX degraded from DNLC in different media was evaluated by high performance liquid chromatography (HPLC). The morphology of DNLC was observed by transmission electron microscopy (TEM). The particle size and PDI of DNLC were determined by Malvern particle size analyzer. The long-term stability of the preparations was investigated. In vitro cytotoxicity of DNLC was measured by MTT method. In vivo pharmacodynamics of DNLC were performed in 4T1 tumor xenograft balb/c mice using saline and DTX-Sol as control. RESULTS n-Dodecanol-modified DTX prodrug was synthesized and used to prepare DNLC. The optimal formulation was as following: mass ratio of emulsifier to co-emulsifier (Km) of 1∶3, solid-liquid lipid ratio of 1.43∶1, drug-lipid ratio of 1∶10, the emulsifier concentration of 60 mg•mL-1, the temperature of 70 ℃ and the stirring speed of 800 r•min-1. DNLC had a round appearance and a uniform spherical shape. And the particle size and PDI remained substantially stable within 30 d. The accumulated rates of DTX degraded from DNLC in PBS (pH 7.4), PBS (pH 7.4) containing 10 mmol•L-1 DTT and 10 mmol•L-1 H2O2 was (9.07±0.01)%, (21.52±0.35)% and (96.72±4.12)% at 24 h, respectively. After incubation of DTX-Sol and DNLC with 4T1 cells for 72 h, IC50 of DTX-Sol and DNLC were (1.2±0.2) and (13.2±4.3)nmol•L-1, respectively. The cytotoxicity of DTX-Sol group was stronger than that of DNLC group. At the end of the pharmacodynamics, the tumor volumes of the mice in saline, DTX-Sol and DNLC groups were (1 930.39±215.20), (1 013.64±138.65), and (765.16±177.43)mm3, respectively. And the change percentage of body weight in saline, DTX-Sol and DNLC groups were (-19.69±4.44)%, (-14.85±3.61)% and (-2.61±1.70)%. There were significant differences in tumor volume and body weight between the DNLC and DTX-Sol group (P<0.05). CONCLUSION The prepared DNLC shows good stability, redox sensitivity, obvious anti-tumor effect and lower toxicity. These RESULTS could provide a new experimental basis for the development of DTX prodrug loaded nano-drug delivery system.

OBJECTIVE: To evaluate the pharmacokinetics and bioequivalence of levomilnacipran hydrochloride sustained release capsules in Beagle dogs. METHODS: An open, randomized,two-periods trial design was used. HPLC method was established to determine levomilnacipran hydrochloride in plasma samples of beagle dogs. Pharmacokinetic parameters and bioequivalence were evaluated. RESULTS: The pharmacokinetic parameters of levomilnacipran hydrochloride after oral adminstration of test or reference preparations were as follows: ρmax were (394.06±18.22), (384.88±25.65) ng•mL-1; AUC0-72 h were (5 903.86±107.51), (5 396.63±62.63) ng•h•mL-1; AUC0-∞ were (6 325.90±158.88), (6 091.14±121.35) ng•h•mL-1, respectively. The relative bioavailability of F0-72 h(%) and F0-∞(%) of the test/reference formulation were 109.40%, 103.85%; the 90% CIs for the test/reference ratio of ρmax, AUC0-72 h, and AUC0-∞ were 75.11% to 129.61%, 99.35% to 119.44%, and 89.90% to 117.78%, respectively. CONCLUSION: The RP-HPLC method is simple, efficient and accurate which can be used for the determination of levomilnacipran hydrochloride plasma concentration. The test capsules are bioequivalent to the reference capsules.

OBJECTIVE: To establish a high performance liquid chromatography combined with pulsed amperometric detection(HPLC-PAD)method for determination of potency of neomycin sulfate. METHODS: An improved HPLC-PAD method from EP method for determination of the content and related substances of neomycin sulfate was established and validated. The study of impurity profile of neomycin sulfate was completed by LC-IT-TOF method with the help of on-line desalination using a suppressor; and the main components in neomycin sulfate were clarified combining the RESULTS of impurity profile and minimum inhibitory concentrations of the main components and impurities. The semi-preparative liquid chromatography-evaporative light scattering detector(ELSD) was self-assembled, highly purified neomycin B and neomycin C were prepared and their structural confirmation was also conducted. The contents of highly purified neomycin B and neomycin C were determined by means of mass balance method. The potencies of highly purified neomycin B and neomycin C were determined by three-dose antibiotic microbial assay and the conversion factors between contents of neomycin B and neomycin C and their potencies were calculated separately and then a formula for the calculation of potency of neomycin sulfate from the content of main components of neomycin B and neomycin C was obtained.At last, a verification experiment for the accuracy of the conversion factor and the formula were designed and a serial of tests were carried out to investigate the interaction and the verification for the actual sample. RESULTS: The improved HPLC-PAD method was superior to the European Pharmacopoeia method in the separation ability and stability, and was suitable for accurate quantification of various components of neomycin sulfate and related substance inspection. The successful removal of trifluoroacetic acid in the mobile phase by the technology of desalination on-line using a suppressor broke a new way for the study of impurity profile of aminoglycoside such as neomycin sulfate. Combining the impurity profile with the RESULTS of MIC it was clarified that the main activity components of neomycin sulfate were neomycin B and neomycin C. Highly purified neomycin B and neomycin C were successfully prepared. A conversion factor for the transition from potency to purity of neomycin sulfate was obtained through experiments and calculations and was verified successfully. CONCLUSION: It is feasible to replace the microbial assay by HPLC-PAD method for determining the potency of neomycin sulfate.

OBJECTIVE: To study the response stability of free radical center-induced α-H cleavage on linear ion trap mass spectrometry (LTQ-MS), and discuss the interaction mechanism in ion trap and the effects of hydrogen and deuterium hybrid loss on quantitation precision. METHODS: The special cleavage mode of D6-ornithine lactam was used to distinguish different α-H cleavage, and the factors that might affect the abundance stability were inferred by mechanism discussion and falsification. RESULTS: The quantitation precision of ion pairs with only hydrogen loss(115.1→97.1;121.1→103.1) and threonine instead of D6-ornithine lactam (115.1→70.1;120.1→74.0)was significantly better than that of ion pairs involving hydrogen-deuterium hybrid loss(115.1→70.1;121.1→75.1). When 115.1→70.1;121.1→75.1 were used, the quantitation precision of tandem quadrupole mass spectrometry was significantly better than that of LTQ-MS. CONCLUSION: The instability of ion abundance, the effect of quality discrimination and the effect of ion concentration saturation are not significant factors affecting the stability of LTQ detection signal. Combined with the fragmentation characteristics of D6-ornithine lactam and Matthew′s equation, it is proposed that the space charge in the trap is the main reason for the instability of the detection signal.

OBJECTIVE: To establish a method for detecting the impurity components in human serum albumin by electrospray ionization mass spectrometry (ESI-Q-TOF MS) and investigate the impurity components in 28 batches of post-marketing albumin products from 27 companies. METHODS: According to Chinese Pharmacopoeia′s General Principles 4121, gel filtration column (Hiprep16/60 SepharylS-200HR) was used to separate the protein components. The dimer and multimer components were collected, then desalted, concentrated and digested by trypsin. The peptide was eluted by BEH C18(2.1 mm×100 mm,130) column with mobile phase consisting of water (0.1% formic acid)-acetonitrile in gradient elution mode. Chromatography-mass spectrometry was used to determine the secondary sequences of the peptides and the searching software (PLGS3.0) was used to identify the impurity protein components in albumin. RESULTS: A total of 23 impurity proteins were detected, among which apolipoprotein A-II, haptoglobin, and hempoexin, α-1B-glycoprotein and α-2-HS-glycoprotein were the impurity proteins common to all enterprise products; α-albumin (VE binding protein), N-acetyl-L-alanine amidase, haptoglobin-related proteins, hemoglobin (β subunit, α subunit), transthyretin and other proteins were found in the products of most companies. The number of heterologous proteins in each enterprise was between 8-17, and the numbers of impurity proteins in 27 companies were quite different. CONCLUSION: This method can be used for the investigation of unknown components in human serum albumin, which provides a guidance for the optimization of human albumin production process.