Upregulation of glutamate transporter 1 by mTOR/Akt pathway in astrocyte culture during oxygen-glucose deprivation and reoxygenation.

Astrocyte-specific glutamate transporter subtype 1 (GLT-1) plays an important role in influencing glutamate excitatory toxicity and preventing the death of excitatory toxic neurons. Although the mammalian target of rapamycin (mTOR)/protein kinase B(Akt)/nuclear factor kappa B signaling cascade is involved in the upregulation of astrocytic GLT-1 in oxygen-glucose deprivation (OGD), it is unclear whether the mTOR/Akt pathway is involved in astrocytic GLT-1 upregulation in OGD and reoxygenation (OGD/R). In this study, we found that the treatment of cultured astrocytes with rapamycin and triciribine led to the decreased astrocytes' protrusions, smaller nuclei, and an increased apoptotic rate. The inhibitors of mTOR complex 1 significantly increased the expression levels of phosphorylated Akt-Ser473 (p-Akt), phosphorylated Akt-Thr308(p-Akt), and GLT-1, while Akt-specific inhibitors blocked GLT-1 expression, suggesting that the mTOR/Akt pathway is involved in GLT-1 upregulation. We further demonstrated that astrocytes under OGD/R adapted to environmental changes through the mTOR/Akt pathway, mainly by altering cell morphology and apoptosis and upregulating the expression levels of p-Akt and GLT-1. Our results suggested that astrocytes may adapt to short-term ischemic-reperfusion injury by regulating cell morphology, apoptosis and GLT-1 upregulation.

Hydra-Elastin-like Polypeptides Increase Rapamycin Potency When Targeting Cell Surface GRP78.

Rapalogues are powerful therapeutic modalities for breast cancer; however, they suffer from low solubility and dose-limiting side effects. To overcome these challenges, we developed a long-circulating multiheaded drug carrier called 5FA, which contains rapamycin-binding domains linked with elastin-like polypeptides (ELPs). To target these "Hydra-ELPs" toward breast cancer, we here linked 5FA with four distinct peptides which are reported to engage the cell surface form of the 78 kDa glucose-regulated protein (csGRP78). To determine if these peptides affected the carrier solubility, this library was characterized by light scattering and mass spectrometry. To guide in vitro selection of the most potent functional carrier for rapamycin, its uptake and inhibition of mTORC1 were monitored in a ductal breast cancer model (BT474). Using flow cytometry to track cellular association, it was found that only the targeted carriers enhanced cellular uptake and were susceptible to proteolysis by SubA, which specifically targets csGRP78. The functional inhibition of mTOR was monitored by Western blot for pS6K, whereby the best carrier L-5FA reduced mTOR activity by 3-fold compared to 5FA or free rapamycin. L-5FA was further visualized using super-resolution confocal laser scanning microscopy, which revealed that targeting increased exposure to the carrier by ∼8-fold. This study demonstrates how peptide ligands for GRP78, such as the L peptide (RLLDTNRPLLPY), may be incorporated into protein-based drug carriers to enhance targeting.

The bioequivalence of a single dose of a generic solifenacin succinate tablet or Vesicare in fasting or postprandial healthy Chinese adults.

OBJECTIVE: The present study evaluated the bioequivalence of the test preparation (5 mg generic solifenacin succinate tablet, produced by Jiangsu Deyuan Pharmaceutical Co., Ltd.) or the reference preparation (5 mg solifenacin succinate tablets with the trade name Vesicare, produced by Astellas Pharma Europe B.V.) in either fasting or postprandial states in healthy Chinese subjects. MATERIALS AND METHODS: The present study was designed as an open-label, randomized, single-center, single-dose, dual-cycle, dual-crossover, fasting/postprandial study. 56 healthy Chinese subjects (28 each in the fasting and postprandial groups) were enrolled. WinNolin software (version 7.0 or above) was used to calculate the pharmacokinetic parameters. RESULTS: In the fasting and postprandial group, pharmacokinetic analysis and bioequivalence analysis were carried out based on the blood concentration-time data. The 90% confidence interval of the geometric mean ratio of Cmax, AUC0-t, and AUC0-∞ of the test preparation and the reference preparation were both within the acceptance range of 80.00 - 125.00%. CONCLUSION: A single administration of a 5-mg tablet of either the test preparation, generic solifenacin succinate, or the reference preparation, solifenacin succinate with the brand-name Vesicare, was safe and bioequivalent in healthy Chinese subjects in either fasting or postprandial states according to the criteria of bioequivalence of the State Drug Administration of China.

Bioequivalence of daclatasvir hydrochloride tablets in healthy Chinese subjects.

OBJECTIVE: The aim of the present study was to evaluate the bioequivalence and safety of two types of daclatasvir hydrochloride tablets administered to healthy Chinese subjects under fasting and postprandial conditions. MATERIALS AND METHODS: A total of 72 healthy Chinese subjects were randomly divided into two groups: the fasting group (n = 36) and the postprandial group (n = 36). A dose of 60 mg of both the test and reference preparations of the daclatasvir hydrochloride tablets was taken orally under fasting and postprandial conditions. RESULTS: The main plasma pharmacokinetic parameters of the test and reference preparations in the fasting group were as follows: T1/2 was 9.82 ± 1.00 and 9.67 ± 0.99 hours, respectively; tmax was 1.00 hour in both; Cmax was 1,528.25 ± 428.80 and 1,504.25 ± 414.50 ng/mL-1, respectively; AUC0-t was 14,553.04 ± 4,013.26 and 14,391.97 ± 4,078.18 h/ng/mL-1, respectively; the AUC0-∞ was 14,660.80 ± 4,018.37 and 14,494.85 ± 4,095.57 ng/mL-1, respectively. Meanwhile, the main plasma pharmacokinetic parameters of the test and reference preparations in the postprandial group were as follows: T1/2 was 10.18 ± 1.38 and 10.18 ± 1.69 hours, respectively; tmax was 2.00 and 1.75 hours, respectively; Cmax was 974.92 ± 248.50 and 981.44 ± 237.11 ng/mL-1, respectively; AUC0-t was 9,597.00 ± 3,094.28 and 9,982.83 ± 3,512.07 h/ng/mL-1, respectively; AUC0-∞ was 9,712.92 ± 3,130.43 and 10,113.97 ± 3,593.47 ng/mL-1, respectively. CONCLUSION: Both types of daclatasvir hydrochloride tablets demonstrated good safety levels in healthy Chinese subjects under both fasting and postprandial conditions. Moreover, the two preparations were bioequivalent.

[Safety and Tolerance of Healthy People to Injection of Astragalosides-a New Drug for Coronary Heart Disease].

OBJECTIVES: To assess the safety and tolerance of healthy volunteers to as tragalosides injection (AGI), and to determine a safe dose range for phase II clinical trial. METHODS: A total of 62 healthy volunteers participated in this study, with 26 being given a single AGI of 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, or 600 mL and 36 subjects being given 500 mL, 400 mL, 200 mL or 300 mL of AGI once a day for 7 d. Discomfortsymptoms, vital signs and safety problems were recorded 3 d and 7 d after the administration of AGI. The results were analyzed. RESULTS: Of the 62 participants, 40 adverse events (AEs) were reported by 31 participants, which included 23 mild adverse reactions (ADRs) and 4 moderate ADRs. Nine AEs were reported by 9 participants with single AGI, including 7 ADRs. Fourteen AEs were reported by 10 participants with 500 mL and 400 mL multiple AGI, including 12 ADRs occurred in 9 participants.Seventeen AEs were reported by 12 participants with 300 mL and 300 mL multiple AGI, including 3 mild ADRs. The main ADRs included abnormal liver function [slightly elevated glutamic pyruvic transaminase (ALT), glutamic oxaloacetic transaminase (AST),and serum total bilirubin (TBil)], low blood potassium, increased urine red blood cell count, rash, and phlebitis. CONCLUSIONS: The maximum tolerance is 600 mL for single-dose treatment, and 400 mL for multiple-dose (7 d). The dose guidance given in this study should be examined its effects and safety in patients with coronary heart disease in phase II clinical trial.

Off-target autophagy inhibition by SHP2 allosteric inhibitors contributes to their anti-tumor activity in RAS-driven cancers.

Aberrant activation of RAS-MAPK signaling is common in cancer, and efforts to inhibit pathway components have yielded drugs with promising clinical activities. Unfortunately, treatment-provoked adaptive resistance mechanisms inevitably develop, limiting their therapeutic potential. As a central node essential for receptor tyrosine kinase mediated RAS activation, SHP2 has emerged as an attractive cancer target. Consequently, many SHP2 allosteric inhibitors are now in clinical testing. Here we discovered a previously unrecognized off-target effect associated with SHP2 allosteric inhibitors. We found that these inhibitors accumulate in the lysosome and block autophagic flux in a SHP2-independent manner. We showed that off-target autophagy inhibition by SHP2 allosteric inhibitors contributes to their anti-tumor activity. We also demonstrated that SHP2 allosteric inhibitors harboring this off-target activity not only suppress oncogenic RAS signaling but also overcome drug resistance such as MAPK rebound and protective autophagy in response to RAS-MAPK pathway blockage. Finally, we exemplified a therapeutic framework that harnesses both the on- and off-target activities of SHP2 allosteric inhibitors for improved treatment of mutant RAS driven and drug resistant malignancies such as pancreatic and colorectal cancers. Brief Summary: SHP2 allosteric inhibitors elicit off-target autophagy blockade that can be exploited for improved treatment of RAS-driven and drug-resistant cancers.