The Novel Curcumin Derivative 1g Induces Mitochondrial and ER-Stress-Dependent Apoptosis in Colon Cancer Cells by Induction of ROS Production.

Reactive oxygen species (ROS) play an important role in cellular metabolism. Many chemotherapeutic drugs are known to promote apoptosis through the production of ROS. In the present study, the novel curcumin derivative, 1g, was found to inhibit tumor growth in colon cancer cells both in vitro and in vivo. Bioinformatics was used to analyze the differentially expressed mRNAs. The mechanism of this effect was a change in mitochondrial membrane potential caused by 1g that increased its pro-apoptotic activity. In addition, 1g produced ROS, induced G1 checkpoint blockade, and enhanced endoplasmic reticulum (ER)-stress in colon cancer cells. Conversely, pretreatment with the ROS scavenging agent N-acetyl-l-cysteine (NAC) inhibited the mitochondrial dysfunction caused by 1g and reversed ER-stress, cell cycle stagnation, and apoptosis. Additionally, pretreatment with the p-PERK inhibitor GSK2606414 significantly reduced ER-stress and reversed the apoptosis induced by colon cancer cells. In summary, the production of ROS plays an important role in the destruction of colon cancer cells by 1g and demonstrates that targeted strategies based on ROS represent a promising approach to inhibit colon cancer proliferation. These findings reveal that the novel curcumin derivative 1g represents a potential candidate therapeutics for the treatment of colon cancer cells, via apoptosis caused by mitochondrial dysfunction and endoplasmic reticulum stress.

Pharmacokinetics and safety of pegylated recombinant human granulocyte colony-stimulating factor in children with acute leukaemia.

AIMS: This open-label, phase I study evaluated the pharmacokinetics and safety of pegylated recombinant human granulocyte colony-stimulating factor (PEG-rhG-CSF) for the treatment of chemotherapy-induced neutropenia in children with acute leukaemia. METHODS: PEG-rhG-CSF was administered as a single 100 mcg/kg (3 mg maximum dose) subcutaneous injection at the end of each chemotherapy period when neutropenia occurred. Blood samples were obtained from patients treated with PEG-rhG-CSF. PEG-rhG-CSF serum concentrations were determined by an enzyme-linked immunosorbent assay. Population pharmacokinetic (PPK) analysis was implemented using the nonlinear mixed-effects model. Short-term safety was evaluated through adverse events collection (registered at clinicaltrials.gov identifier: 03844360). RESULTS: A total of 16 acute leukaemia patients (1.8-13.6 years) were included, of whom two (12.5%) had grade 3 neutropenia, six (37.5%) had grade 4 neutropenia, and eight (50.0%) had severe neutropenia. For PPK modelling, 64 PEG-rhG-CSF serum concentrations were obtainable. A one-compartment model with first-order elimination was used for pharmacokinetic data modelling. The current weight was a significant covariate. The median (range) of clearance (CL) and area under the serum concentration-time curve (AUC) were 5.65 (1.49-14.45) mL/h/kg and 16514.75 (6632.45-54423.30) ng·h/mL, respectively. Bone pain, pyrexia, anaphylaxis and nephrotoxicity were not observed. One patient died 13 days after administration, and the objective assessment of causality was that an association with PEG-rhG-CSF was "possible". CONCLUSIONS: The AUC of PEG-rhG-CSF (100 mcg/kg, 3 mg maximum dose) in paediatric patients with acute leukaemia were similar to those of PEG-rhG-CSF (100 mcg/kg) in children with sarcoma. PEG-rhG-CSF is safe, representing an important therapeutic option for chemotherapy-induced neutropenia in paediatric patients with acute leukaemia.

Prediction of Unbound Ceftriaxone Concentration in Children: Simple Bioanalysis Method and Basic Mathematical Equation.

The pharmacological activity of ceftriaxone depends on the unbound concentration. However, direct measurement of unbound concentrations is obstructive, and high individual variability of the unbound fraction of ceftriaxone was shown in children. We aim to evaluate and validate a method to predict unbound ceftriaxone concentrations in pediatric patients. Ninety-five pairs of concentrations (total and unbound) from 92 patients were measured by the bioanalysis method that we developed. The predictive performance of the three equations (empirical in vivo equation, disease-adapted equation, and multiple linear regression equation) was assessed by the mean absolute prediction error (MAPE), the mean prediction error (MPE), the proportions of the prediction error within ±30% (P30) and ±50% (P50), and linear regression of predicted versus actual unbound levels (R2). The average total and unbound ceftriaxone concentrations were 126.18 ± 81.46 µg/ml and 18.82 ± 21.75 µg/ml, and the unbound fraction varied greatly from 4.75% to 39.97%. The MPE, MAPE, P30, P50, and R2 of the empirical in vivo equation, disease equation, and multiple linear equation were 0.17 versus 0.00 versus 0.06, 0.24 versus 0.15 versus 0.27, 63.2% versus 89.5% versus 74.7%, 96.8% versus 97.9% versus 86.3%, and 0.8730 versus 0.9342 versus 0.9315, respectively. The disease-adapted equation showed the best predictive performance. We have developed and validated a bioanalysis method with one-step extraction pretreatment for the determination of total ceftriaxone concentrations, and a prediction equation of the unbound concentration is recommended. The proposed method can facilitate clinical practice and research on unbound ceftriaxone in children. (This study has been registered at ClinicalTrials.gov under identifier NCT03113344.).

Ursolic acid exhibits anti-inflammatory effects through blocking TLR4-MyD88 pathway mediated by autophagy.

There is an urgent need for effective treatments to reduce the large and growing burden of acute kidney injury (AKI) and its consequences. Inflammation is believed to play a vital role in the pathophysiology of AKI. Macrophage autophagy is considered protective against inflammation. Previous study discovered that ursolic acid (UA), a natural pentacyclic triterpene carboxylic acid found in many plants as apples, bilberries, cranberries and so on, promoted cancer cell autophagy. In the present study, we aimed to explore the effect of UA on ameliorating AKI and the role of macrophage autophagy in the context of inflammation. The data from in vivo experiments showed that pretreatment of mice with UA significantly suppressed xylene-induced ear oedema as well as protected against LPS-induced AKI. Related mechanisms were further studied through in vitro experiment. As expected, UA decreased inflammatory factors TNF-α, IL-6 and IL-1ß secretion in macrophages in response to lipopolysaccharide (LPS) stimulation. Furthermore, UA blocked LPS-induced TLR4/MyD88 pathway. More importantly, enhanced autophagy of macrophages by UA through increasing the expression of both LC3B and Beclin-1 led to alter macrophage function. What is more, similar to UA, autophagy inhibitor 3-MA obviously decreased inflammation factors releases hinting the vital role of autophagy in regulating inflammation. In all, above study suggested that UA is a potential anti-inflammatory natural compound for treating AKI by inducing autophagy.

Identifying risk factors for high-dose methotrexate-induced toxicities in children with acute lymphoblastic leukemia.

BACKGROUND: Whether monitoring of the methotrexate (MTX) concentrations after high-dose MTX (HD-MTX) infusion can predict toxicities is still controversial, especially when HD-MTX therapy is used in the treatment of children with acute lymphoblastic leukemia (ALL), which is different than the previous schedules. The relationship between patient characteristics and severe adverse events (AEs) has yet to be determined. OBJECTIVE: To analyze the relationship between the MTX concentration and toxicities and to identify the risk predictors from patient characteristics for severe AEs during HD-MTX therapy in children with ALL. METHODS: We conducted a retrospective study on children with ALL who were treated with 388 HD-MTX infusions. The chi-square test and univariate and logistic regression analyses were used to analyze the relationship between the MTX concentrations and toxicities and to identify predictors for severe AEs. RESULTS: Febrile neutropenia (P=0.000) and vomiting (P=0.034) were more likely to occur if the infusion had an MTX level ≥1 µmol/L at 44 h, but other toxicities had no correlations with MTX concentration. Predictive factors for toxicities were as follows: higher risk stratification and higher values of albumin (ALB) for leucopenia, higher values of white blood cell count (WBC) for anemia, higher values of ALB and creatinine (Cr) for neutropenia, higher risk stratification and higher 44-h MTX concentration for febrile neutropenia, higher values of alanine transferase (ALT) for elevated ALT, higher values of ALT for elevated aspartate transferase (AST), and higher values of total bilirubin (TBil) for vomiting. CONCLUSION: Routine monitoring of 44-h MTX concentrations is essential to identify patients at high risk of developing febrile neutropenia and vomiting. This study may provide a reference for clinicians to distinguish patients with a relatively high risk of severe AEs based on certain characteristics before HD-MTX infusion.

Determination and pharmacokinetic study of isothiouronium-modified pyrimidine-substituted curcumin analog (1G), a novel antitumor agent, in rat plasma by liquid chromatography-tandem mass spectrometry.

1G, a novel derivative of curcumin, exhibits promising anticancer activities in various cancer cell lines. To support its further pharmacological studies, a liquid chromatography-tandem mass spectrometry method was developed and validated in accordance with FDA's Guidance. After extraction by protein precipitation, analytes were separated by a 4.5 min gradient elution (water/0.1% formic acid and methanol) on a reverse-phase C18 column at 40 °C. The multiple reaction monitoring mode was used for quantification on a triple quadrupole mass spectrometer with positive ionization. The assay was linear over the concentration range of 5-1000 ng/mL with a correlation coefficient (r) greater than 0.99. Values of intra- and inter-day precision and accuracy were satisfactory, i.e. <10.1% for precision and within ± 14.5% for accuracy. No obvious matrix effect was observed. Recovery of the analyte was higher than 95.3%. 1G was stable during the whole analytic process. The validated method was successfully applied to the pharmacokinetic study of 1G after intravenous and intraperitoneal administration in rats. Favorable pharmacokinetic profiles were demonstrated, including good abdominal absorption (F = 62.58%), moderate clearance and high extravascular distribution. Results indicated that as a novel antitumor agent, 1G exhibited acceptable pharmacokinetic properties for further in vivo pharmacologic evaluation.

Some pharmacokinetic parameters of salvianolic acid A following single-dose oral administration to rats.

CONTEXT: Salvianolic acid A (Sal A) is a hydrophilic bioactive compound isolated from Salvia miltiorrhiza Bunge (Lamiaceae). It exerts beneficial effects after oral administration on diabetic complications. OBJECTIVE: To systematically study the absorption, distribution and excretion of Sal A after single-dose oral administration. MATERIALS AND METHODS: Animal experiments were conducted in Sprague-Dawley rats. Plasma was sampled at designated times after oral doses of 5, 10 and 20 mg/kg, and an intravenous dose of 50 µg/kg. Tissues were harvested at 10, 60 and 120 min postdosing. Bile, urine and feces were collected at specified intervals before and after dosing. Absorption and distribution characteristics were analyzed by LC-MS, and excretion characteristics were analyzed by UPLC-MS/MS. The Caco-2 cell model was applied to investigate potential mechanisms. RESULTS: The Cmax (5 mg/kg: 31.53 µg/L; 10 mg/kg: 57.39 µg/L; 20 mg/kg: 111.91 µg/L) of Sal A increased linearly with doses (r> 0.99). The calculated absolute bioavailability was 0.39-0.52%. Transport experiment showed poor permeability and the ratio of PB-A to PA-B was 3.13-3.97. The highest concentration of Sal A was achieved in stomach followed by small intestine and liver, and it could also be detected in brain homogenate. Approximately 0.775% of its administered dose was excreted via feces, followed by bile (0.00373%) and urine (0.00252%). DISCUSSION AND CONCLUSIONS: These results support the future development of Sal A as an oral drug for the treatment of diabetic complications. Future research should be conducted to investigate the reason for its poor bioavailability and improve this situation.

Perfluorooctylbromide nanoparticles for ultrasound imaging and drug delivery.

Perfluorooctylbromide nanoparticles (PFOB NPs) are a type of multifunctional nanotechnology that has been studied for various medical applications. Commercial ultrasound contrast agents (UCAs) suffer from the following limitations: short half-lives in vivo, high background signal and restricted distribution in the vascular circulation due to their micrometer dimensions. PFOB NPs are new potential UCAs that persist for long periods in the circulatory system, possess a relatively stable echogenic response without increasing the background signal and exhibit lower acoustic attenuation than commercial UCAs. Furthermore, PFOB NPs may also serve as drug delivery vehicles in which drugs are dissolved in the outer lipid or polymer layer for subsequent delivery to target sites in site-targeted therapy. The use of PFOB NPs as carriers has the potential advantage of selectively delivering payloads to the target site while improving visualization of the site using ultrasound (US) imaging. Unfortunately, the application of PFOB NPs to the field of ultrasonography has been limited because of the low intensity of US reflection. Numerous researchers have realized the potential use of PFOB NPs as UCAs and thus have developed alternative approaches to apply PFOB NPs in ultrasonography. In this article, we review the latest approaches for using PFOB NPs to enhance US imaging in vivo. In addition, this article emphasizes the application of PFOB NPs as promising drug delivery carriers for cancer and atherosclerosis treatments, as PFOB NPs can transport different drug payloads for various applications with good efficacy. We also note the challenges and future study directions for the application of PFOB NPs as both a delivery system for therapeutic agents and a diagnostic agent for ultrasonography.

Docetaxel prodrug self-assembled nanosystem: Synthesis, formulation and cytotoxicity.

Conventional drug delivery systems of docetaxel (DTX) are challenged with low drug loading efficiency and potential carriers-induced toxicity. In this work, a docetaxel prodrug self-assembled nanosystem was designed and synthesized by conjugating docetaxel with oleic acid (OA) exploring a thioether as the linker, which is redox-sensitive to the redox environment within tumor cells. Notably, the carrier-free nanomedicine which does not need any carrier has obviously high drug loading that reaches 58%. Moreover, the cytotoxicity of DTX-S-OA maintains an equal level with DTX. The novel prodrug conjugate therefore has a promising perspective as carrier-free nanomedicine for cancer therapy due to its high drug loading property, redox-sensitive release and long circulation mechanism.

Ubenimex suppresses Pim-3 kinase expression by targeting CD13 to reverse MDR in HCC cells.

Hepatocellular carcinoma (HCC) is one of the most serious cancers, with rapid progression and high mortality. However, chemotherapy of HCC is hindered by multi-drug resistance (MDR). It is urgent, therefore, to explore new approaches for overcoming MDR of HCC cells. Ubenimex, an inhibitor of CD13, has been used as an immuno-enhancer for treating hematological neoplasms and other solid tumors. Here, we demonstrate that Ubenimex can also reverse MDR in the HCC cell lines HepG2/5-FU and Bel7402/5-FU. Ubenimex inhibits the expression of the proto-oncogene, Pim-3, which is accompanied by decreased expression of BCL-2 and BCL-XL, decreased phosphorylation of Bad, and increased tumor apoptosis. Moreover, Ubenimex decreases expression of the MDR-associated proteins P-gp, MRP3 and MRP2 to enhance intracellular accumulation of Cisplatin, for which down-regulation of Pim-3 is essential. Our results reveal a previously uncharacterized function of Ubenimex in mediating drug resistance in HCC, which suggests that Ubenimex may provide a new strategy to reverse MDR and improve HCC sensitivity to chemotherapeutic drugs via its effects on Pim-3.

Effects of L-carnitine on high glucose-induced oxidative stress in retinal ganglion cells.

BACKGROUND: Oxidative stress plays a role in diabetic retinopathy. L-Carnitine is an endogenous mitochondrial membrane compound. OBJECTIVE: To elucidate the protective effects of L-carnitine on high glucose-induced oxidative stress in retinal ganglion cells (RGCs). METHODS: Hoechst 33258 staining was used to estimate cell loss. Mitochondrial function was predicted by mitochondrial membrane potential (ΔΨm) measurement. The expression of apoptosis-related protein was measured by Western blotting. Assays for reactive oxygen species (ROS) accumulation, lipid peroxidation, total antioxidative capacity (T-AOC) and antioxidant defense enzymes were completed to explain the antioxidative capacity of L-carnitine. RESULTS: L-Carnitine (12 h) inhibited high glucose-mediated cell loss and restored mitochondrial function including a reversion of ΔΨm loss and cytochrome c release. Cell apoptosis triggered by high glucose was also inhibited by L-carnitine, characterized by the downregulation of caspase-9, caspase-3 and Bax/Bcl-2. Furthermore, L-carnitine inhibited high glucose-induced ROS production and lipid peroxidation and promoted endogenous antioxidant defense components including superoxide dismutase, glutathione peroxidase, catalase and T-AOC in a concentration-dependent manner. CONCLUSIONS: L-Carnitine may protect RGCs from high glucose-induced injury through the inhibition of oxidative damage, mitochondrial dysfunction and, ultimately, cell apoptosis.

Transferrin- and folate-modified, double-targeted nanocarriers for gene delivery.

CONTEXT: Surface modification of nanocarriers with specific ligands defines a new biological identity, which assist in targeting and internalization of the nanocarriers to specific cell populations, such as cancers and disease organs. OBJECTIVE: This study aimed to develop systemically administrable dual ligands modified nanocarriers, which could target the cells through receptor-mediated pathways to increase the nuclear uptake of genetic materials. MATERIALS AND METHODS: In the present work, transferrin (Tf) and folate (Fa) were linked onto polyethylene glycol-phosphatidylethanolamine (PEG-PE) separately to get transferrin-PEG-PE (T-PEG-PE) and folate-PEG-PE (F-PEG-PE) ligands for the surface modification of carriers. The in vivo transfection efficiency of the novel dual ligands modified (D-modified) vectors were evaluated in tumor-bearing animal models. RESULTS: D-Modified solid lipid nanoparticles/enhanced green fluorescence protein plasmid (D-SLN/pEGFP) has a particle size of 226 nm and a gene-loading quantity of 90%. D-SLN/pEGFP displayed over 30% higher transfection efficiency than unmodified SLN/pEGFP and single ligand modified particles in HepG2 cells. CONCLUSION: It could be concluded that Tf and Fa could function as excellent active targeting ligands to improve the cell-targeting ability of the carriers and the resulting dual ligands modified vectors could be applied as a promising active targeting gene delivery system.

Dual ligands modified double targeted nano-system for liver targeted gene delivery.

CONTEXT: It is now well established that the surface of nanocarriers with specific ligands defines a new biological identity, which assist in targeting and internalization of the nanocarriers to specific cell populations, such as cancers and disease organs. OBJECTIVE: The aim of this study is to develop systemically administrable dual ligands modified nano-system which could both target cancer cells and macrophages in the liver. METHODS: Transferrin (Tf) and mannan (M) were linked onto polyethylene glycol-phosphatidylethanolamine (PEG-PE) and PE separately to get transferrin-PEG-PE (T-PEG-PE) and mannan-PE (M-PE) ligands for the surface modification of carriers. The in vivo transfection efficiency of the novel dual ligands modified (D-modified) vectors were evaluated in tumor bearing animal models. RESULTS: D-modified solid lipid nanoparticles/enhanced green fluorescence protein plasmid (D-SLN/pEGFP) has a particle size of 198 nm and a gene loading quantity of 89%. D-SLN/pEGFP displayed over 25% higher transfection efficiency than M-PE modified SLN/pEGFP (M-SLN/pEGFP) in HepG2 cells and T-PEG-PE modified SLN/pEGFP (T-SLN/pEGFP) in Kupffer cells (KCs) isolated from mice. CONCLUSION: It could be concluded that T-PEG-PE and M-PE could function as excellent active targeting ligands to improve the cell targeting ability of the carriers and the dual ligands modified vectors could be applied as a promising active targeting gene delivery system.

Two potent cytochrome P450 2D6 inhibitors found in Rhodiola rosea.

OBJECTIVES: Throughout the world, in particular in Russia, Northern Europe and China, Rhodiola species are used as herb supplements. Previously, we found that the extract of Rhodiola rosea, one of the most widely used Rhodiola species, had an inhibitory effect on the catalytic activity of cytochrome P450 2D6. Here, its inhibitory components were identified. METHODS: A human liver microsomal in vitro system was used with dextromethorphan as substrate. The production rate of destrorphan, a metabolite of dextromethorphan, was used to measure enzyme activity. The concentration of destrorphan in the samples was measured using LC-MS/MS. Inhibitory activity of eight main components from Rhodiola rosea was evaluated. RESULTS: Rhodiosin and rhodionin showed inhibitory activity with IC50 values of 0.761 microM and 0.420 microM, respectively. The other components showed no obvious inhibition (with a residual enzyme activity of more than 90%). Both rhodiosin and rhodionin were determined to be non-competitive inhibitors with Ki values of 0.769 microM and 0.535 microM. CONCLUSION: Two of the main Rhodiola rosea compounds, rhodiosin and rhodionin, can inhibit cytochrome P450 2D6 non-competitively with high specificity which could have implications for interactions with co-administered drugs.

LYP3, a new bestatin derivative for aminopeptidase N inhibition.

Aminopeptidase N (APN) is a ubiquitous enzyme overexpressed on tumor cells and plays an important role in angiogenesis and metastasis of tumor. Bestatin as an effective inhibitor of aminopeptidase N is used for complementary treatment of cancer with other drugs. In this work, we reformed the structure of bestatin to a new derivative LYP3 to improve the water solubility and effectiveness. The inhibitory activity of LYP3 against APN was evaluated in vitro.