The pharmacokinetic study on the interaction between nobiletin and anemarsaponin BII in vivo and in vitro

ContextThe interaction between nobiletin and anemarsaponin BII could affect the pharmacological activity of these two drugs during their combination.ObjectiveThe co-administration of nobiletin and anemarsaponin BII was investigated to explore the interaction and the potential mechanism.Materials and methodsMale Sprague-Dawley rats were only orally administrated with 50 mg/kg nobiletin as the control and another six rats were pre-treated with 100 mg/kg anemarsaponin BII for 7 d followed by the administration of nobiletin. The transport and metabolic stability of nobiletin were evaluated in vitro, and the effect of anemarsaponin BII on the activity of CYP3A4 was also assessed to explore the potential mechanism underlying the interaction.ResultsThe increasing C_max (2309.67 ± 68.06 μg/L vs. 1767.67 ± 68.86 μg/L), AUC (28.84 ± 1.34 mg/L × h vs. 19.57 ± 2.76 mg/L × h), prolonged t_1/2 (9.80 ± 2.33 h vs. 6.24 ± 1.53 h), and decreased clearance rate (1.46 ± 0.26 vs. 2.42 ± 0.40) of nobilein was observed in rats. Anemarsaponin BII significantly enhanced the metabolic stability of nobiletin in rat liver microsomes (half-life increased from 31.56 min to 39.44 min) and suppressed the transport of nobiletin in Caco-2 cells (efflux rate decreased from 1.57 ± 0.04 to 1.30 ± 0.03). The inhibitory effect of anemarsaponin BII on CYP3A4 was also found with an IC₅₀ value of 10.23 μM.Discussion and conclusionsThe interaction between anemarsaponin BII and nobiletin was induced by the inhibition of CYP3A4, which should draw special attention in their clinical co-administration.     

Pharmacokinetic study on the interaction between succinic acid and irbesartan in rats and its potential mechanism

ContextSuccinic acid and irbesartan are commonly used drugs in cardiovascular disease treatment. The interaction might occur during their co-administration, which was still unclear.ObjectiveTo reveal the effect of succinic acid on the metabolism of irbesartan and its potential mechanism.Materials and methodsThe Sprague-Dawley rats (n = 6) were treated with a single dose of 30 mg/kg irbesartan (control) or the co-administration with the pre-treatment of 200 mg/kg succinic acid for 7 d. The effect of succinic acid on the metabolic stability and the activity of CYP2C9 was evaluated in rat liver microsomes.ResultsSuccinic acid increased the AUC (5328.71 ± 959.31 μg/L × h vs. 3340.23 ± 737.75 μg/L × h) and prolonged the half-life of irbesartan (from 12.79 ± 0.73 h to 20.59 ± 6.35 h). The T_max (2.83 ± 0.75 h vs. 3.83 ± 1.10 h) and clearance rate (3.46 ± 1.13 L/h/kg vs. 6.91 ± 1.65 L/h/kg) of irbesartan was reduced by succinic acid. Consistently, succinic acid improved the metabolic stability (half-life from 23.32 ± 3.46 to 27.35 ± 2.15 min, intrinsic clearance rate from 59.43 ± 6.12 to 50.68 ± 5.64 μL/min/mg protein). Succinic acid was also found to inhibit the activity of CYP2C9 with the IC₅₀ value of 13.87 μM.Discussion and conclusionsSuccinic acid increased the system exposure of irbesartan via inhibiting CYP2C9. The experiment design of this study also provides a reference for the further validation of this interaction in humans.     

Baicalein inhibits the pharmacokinetics of simvastatin in rats via regulating the activity of CYP3A4

ContextBaicalein and simvastatin possess similar pharmacological activities and indications. The risk of their co-administration was unclear.ObjectiveThe interaction between baicalein and simvastatin was investigated to provide reference and guidance for the clinical application of the combination of these two drugs.Materials and methodsThe pharmacokinetics of simvastatin was investigated in Sprague–Dawley rats (n = 6). The rats were pre-treated with 20 mg/kg baicalein for 10 days and then administrated with 40 mg/kg simvastatin. The single administration of simvastatin was set as the control group. The rat liver microsomes were employed to assess the metabolic stability and the effect of baicalein on the activity of CYP3A4.ResultsBaicalein significantly increased the AUC_(0–t) (2018.58 ± 483.11 vs. 653.05 ± 160.10 μg/L × h) and C_max (173.69 ± 35.49 vs. 85.63 ± 13.28 μg/L) of simvastatin. The t_1/2 of simvastatin was prolonged by baicalein in vivo and in vitro. The metabolic stability of simvastatin was also improved by the co-administration of baicalein. Baicalein showed an inhibitory effect on the activity of CYP3A4 with the IC₅₀ value of 12.03 μM, which is responsible for the metabolism of simvastatin.Discussion and conclusionThe co-administration of baicalein and simvastatin may induce drug-drug interaction through inhibiting CYP3A4. The dose of baicalein and simvastatin should be adjusted when they are co-administrated.     

Pharmacokinetic study on the interaction between pachymic acid and bavachin and its potential mechanism

ContextPachymic acid and bavachin are commonly used drugs in the therapy of lung cancer.ObjectiveThe co-administration of pachymic acid and bavachin was investigated to evaluate their potential drug-drug interaction.Materials and methodsThe pharmacokinetics of bavachin (10 mg/kg) was studied in male Sprague-Dawley (SD) rats in the presence of pachymic acid (5 mg/kg) (n = 6). The rats without pre-treatment of pachymic acid were set as the control and the pre-treatment of pachymic acid was conducted for 7 days before the administration of bavachin. The effect of pachymic acid on the activity of CYP2C9 was also estimated in rat liver microsomes with corresponding probe substrates.ResultsPachymic acid influenced the pharmacokinetic profile of bavachin with the increased AUC (32.82 ± 4.61 vs. 19.43 ± 3.26 μg/L/h), the prolonged t_1/2 (3.21 ± 0.65 vs. 2.32 ± 0.28 h), and the decreased CLz/F (307.25 ± 44.35 vs. 523.81 ± 88.67 L/h/kg) in vivo. The metabolic stability of bavachin was enhanced by pachymic acid and the transport of bavachin was inhibited by pachymic acid. Pachymic acid was found to inhibit the activity of CYP2C9 with the IC₅₀ of 21.25 µM as well as the activity of P-gp.Discussion and conclusionThe interaction between pachymic acid and bavachin results from the inhibition of CYP2C9 and P-gp. The dose of bavachin should be adjusted when combining with pachymic acid. The study design can be generalized to a broader study population with adjustment in the dose.     

Effects of ginkgo leaf tablet on the pharmacokinetics of rosiglitazone in rats and its potential mechanism

ContextGinkgo leaf tablet (GLT), a traditional Chinese herbal formula, is often combined with rosiglitazone (ROS) for type 2 diabetes mellitus treatment. However, the drug-drug interaction between GLT and ROS remains unknown.ObjectiveTo investigate the effects of GLT on the pharmacokinetics of ROS and its potential mechanism.Materials and methodsThe pharmacokinetics of 10 mg/kg ROS with 100/200 mg/kg GLT as single-dose and 10-day multiple-dose administration were investigated in Sprague-Dawley rats. In vitro, the effects of GLT on the activity of CYP2C8 and CYP2C9 were determined in recombinant human yeast microsomes and rat liver microsomes with probe substrates.ResultsThe t_1/2 of ROS increased from 2.14 ± 0.38 (control) to 2.79 ± 0.37 (100 mg/kg) and 3.26 ± 1.08 h (200 mg/kg) in the single-dose GLT administration. The AUC_0-t (139.69 ± 45.46 vs. 84.58 ± 39.87 vs. 66.60 ± 15.90 h·μg/mL) and t_1/2 (2.75 ± 0.70 vs. 1.99 ± 0.44 vs. 1.68 ± 0.35 h) decreased significantly after multiple-dose GLT treatment. The IC₅₀ values of quercetin, kaempferol, and isorhamnetin, GLT main constituents, were 9.32, 7.67, and 11.90 μmol/L for CYP2C8, and 27.31, 7.57, and 4.59 μmol/L for CYP2C9. The multiple-dose GLT increased rat CYP2C8 activity by 44% and 88%, respectively.Discussion and conclusionsThe metabolism of ROS is attenuated in the single dose of GLT by inhibiting CYP2C8 and CYP2C9 activity, and accelerated after the multiple-dose GLT treatment via inducing CYP2C8 activity in rats, indicating that the clinical dose of ROS should be adjusted when co-administrated with GLT.     

In vitro study on the effect of peucedanol on the activity of cytochrome P450 enzymes

ContextPeucedanol is a major extract of Peucedanum japonicum Thunb. (Apiaceae) roots, which is a commonly used herb in paediatrics. Its interaction with cytochrome P450 enzymes (CYP450s) would lead to adverse effects or even failure of therapy.ObjectiveThe interaction between peucedanol and CYP450s was investigated.Materials and methodsPeucedanol (0, 2.5, 5, 10, 25, 50, and 100 μM) was incubated with eight human liver CYP isoforms (CYP1A2, 2A6, 3A4, 2C8, 2C9, 2C19, 2D6, and 2E1), in pooled human liver microsomes (HLMs) for 30 min with specific inhibitors as positive controls and untreated HLMs as negative controls. The enzyme kinetics and time-dependent study (0, 5, 10, 15, and 30 min) were performed to obtain corresponding parameters in vitro.ResultsPeucedanol significantly inhibited the activity of CYP1A2, 2D6, and 3A4 in a dose-dependent manner with IC₅₀ values of 6.03, 13.57, and 7.58 μM, respectively. Peucedanol served as a non-competitive inhibitor of CYP3A4 with a K_i value of 4.07 μM and a competitive inhibitor of CYP1A2 and 2D6 with a K_i values of 3.39 and 6.77 μM, respectively. Moreover, the inhibition of CYP3A4 was time-dependent with the K_i/K_inact value of 5.44/0.046 min/μM.Discussion and conclusionsIn vitro inhibitory effect of peucedanol on the activity of CYP1A2, 2A6, and 3A4 was reported in this study. As these CYPs are involved in the metabolism of various drugs, these results implied potential drug-drug interactions between peucedanol and drugs metabolized by CYP1A2, 2D6, and 3A4, which needs further in vivo validation.     

In vitro differences in toddalolactone metabolism in various species and its effect on cytochrome P450 expression

ContextToddalolactone, the main component of Toddalia asiatica (L.) Lam. (Rutaceae), has anticancer, antihypertension, anti-inflammatory, and antifungal activities.ObjectiveThis study investigated the metabolic characteristics of toddalolactone.Materials and methodsToddalolactone metabolic stabilities were investigated by incubating toddalolactone (20 μM) with liver microsomes from humans, rabbits, mice, rats, dogs, minipigs, and monkeys for 0, 30, 60, and 90 min. The CYP isoforms involved in toddalolactone metabolism were characterized based on chemical inhibition studies and screening assays. The effects of toddalolactone (0, 10, and 50 µM) on CYP1A1 and CYP3A5 protein expression were investigated by immunoblotting. After injecting toddalolactone (10 mg/kg), in vivo pharmacokinetic profiles using six Sprague–Dawley rats were investigated by taking 9-time points, including 0, 0.25, 0.5, 0.75, 1, 2, 4, 6 and 8 h.ResultsMonkeys showed the greatest metabolic capacity in CYP-mediated and UGT-mediated reaction systems with short half-lives (T_1/2) of 245 and 66 min, respectively, while T_1/2 of humans in two reaction systems were 673 and 83 min, respectively. CYP1A1 and CYP3A5 were the major CYP isoforms involved in toddalolactone biotransformation. Induction of CYP1A1 protein expression by 50 μM toddalolactone was approximately 50% greater than that of the control (0 μM). Peak plasma concentration (C_max) for toddalolactone was 0.42 μg/mL, and T_max occurred at 0.25 h post-dosing. The elimination t_1/2 was 1.05 h, and the AUC_0–t was 0.46 μg/mL/h.ConclusionsThese findings demonstrated the significant species differences of toddalolactone metabolic profiles, which will promote appropriate species selection in further toddalolactone studies.     

Alpinetin suppresses CYP3A4, 2C9, and 2E1 activity in vitro

ContextAlpinetin, the major active constitutes of Alpinia katsumata Hayata (Zingiberaceae), has been demonstrated to possess the activity of anti-breast cancer. Cytochrome P450 enzymes (CYP450s) plays vital roles in the biotransformation of various drugs.ObjectiveTo assess the effect of alpinetin on the activity of CYP450s and estimate the inhibition characteristics.Materials and methodsThe activity of CYP450s was evaluated in pooled human liver microsomes with corresponding substrates and marker reactions. The effect of alpinetin was compared with blank control (negative control) and corresponding inhibitors (positive control). The dose-dependent and time-dependent experiments were conducted in the presence of 0, 2.5, 5, 10, 25, 50, and 100 μM alpinetin and incubated for 0, 5, 10, 15, and 30 min.ResultsAlpinetin suppressed CYP3A4, 2C9, and 2E1 activity. All the inhibitions were significantly influenced by alpinetin contration with the IC₅₀ values of 8.23 μM (CYP3A4), 12.64 μM (CYP2C9), and 10.97 μM (CYP2E1), respectively. The inhibition of CYP3A4 was fitted with the non-competitive model with a Ki value of 4.09 μM and was time-dependent with KI and Kinact values of 4.67 min and 0.041 μM⁻¹, respectively. While CYP2C9 and 2E1 were inhibited by alpinetin competitively with Ki values of 6.42 (CYP2C9) and 5.40 μM (CYP2E1), respectively, in a time-independent manner.Discussion and conclusionThe in vitro inhibitory effect of alpineticn on CYP3A, 2C9, and 2E1 implied the potential interaction of alpinetin or its origin herbs with the drugs metabolised by those CYP450s, which needs further in vivo validation.     

Effects of tumour necrosis factor-α on the coronary circulation of the rat isolated perfused heart: a potential role for thromboxane A2 and sphingosine

1. The actions of tumour necrosis factor-α (TNF-α) on the coronary circulation were investigated in the rat isolated heart, perfused under constant flow, recirculating conditions.
2. An early increase in coronary perfusion pressure (CPP) was observed upon treatment with TNF-α (increase in CPP 10 min after TNF-α treatment: 45±12 mmHg vs control: 15±4 mmHg, P<0.05). The role of sphingosine, prostanoids and endothelins, in this coronary constrictor action, was investigated with the use of pharmacological inhibitors and antagonists.
3. The TNF-α induced increase in coronary tone was blocked by indomethacin, 10 μM (increase in CPP after 10 min: 13±4 mmHg vs TNF-α alone, P<0.05).
4. The thromboxane receptor antagonist GR32191, 10 μM, attenuated the TNF-α induced coronary constriction (12±2 mmHg vs TNF-α alone, P<0.05), as did the joint thromboxane A₂ synthesis inhibitor and receptor antagonist ZD1542, 10 μM (8±1 mmHg vs TNF-α alone, P<0.05).
5. The ceramidase inhibitor N-oleoylethanolamine (NOE), 1 μM, also blocked the TNF-α induced response (8±4 mmHg vs TNF-α alone, P<0.05).
6. In contrast, the coronary constrictor action of TNF-α was not inhibited by the endothelin_A/B receptor antagonist bosentan, 3 μM (38±9 mmHg vs TNF-α, P=NS).
7. These data indicated that the early coronary vasoconstriction induced by TNF-α was mediated by both thromboxane A₂ and sphingosine, suggesting an interaction between both the sphingomyelinase and phospholipase A₂ metabolic pathways.

     

Aaptamine – a dual acetyl – and butyrylcholinesterase inhibitor as potential anti-Alzheimer’s disease agent

ContextAlzheimer’s disease (AD) is a neurodegenerative disorder that affects millions of people worldwide. Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are promising therapeutic targets for AD.ObjectiveTo evaluate the inhibitory effects of aaptamine on two cholinesterases and investigate the in vivo therapeutic effect on AD in a zebrafish model.Materials and methodsAaptamine was isolated from the sponge Aaptos suberitoides Brøndsted (Suberitidae). Enzyme inhibition, kinetic analysis, surface plasmon resonance (SPR) and molecular docking assays were used to determine its inhibitory effect on AChE and BuChE in vitro. Zebrafish were divided into six groups: control, model, 8 μM donepezil, 5 , 10  and 20 μM aaptamine. After three days of drug treatment, the behaviour assay was performed.ResultsThe IC₅₀ values of aaptamine towards AChE and BuChE were 16.0 and 4.6 μM. And aaptamine directly inhibited the two cholinesterases in the mixed inhibition type, with K_i values of 6.96 ± 0.04 and 6.35 ± 0.02 μM, with K_d values of 87.6 and 10.7 μM. Besides, aaptamine interacts with the crucial anionic sites of AChE and BuChE. In vivo studies indicated that the dyskinesia recovery rates of 5 , 10  and 20 μM aaptamine group were 34.8, 58.8 and 60.0%, respectively, and that of donepezil was 63.7%.Discussion and conclusionsAaptamine showed great potential to exert its anti-AD effects by directly inhibiting the activities of AChE and BuChE. Therefore, this study identified a novel medicinal application of aaptamine and provided a new structural scaffold for the development of anti-AD drugs.     

Pogostone inhibits the activity of CYP3A4, 2C9, and 2E1 in vitro

ContextPogostone possesses various pharmacological activities, which makes it widely used in the clinic. Its effect on the activity of cytochrome P450 enzymes (CYP450s) could guide its clinical combination.ObjectiveTo investigate the effect of pogostone on the activity of human CYP450s.Materials and methodsThe effect of pogostone on the activity of CYP450s was evaluated in human liver microsomes (HLMs) compared with blank HLMs (negative control) and specific inhibitors (positive control). The corresponding parameters were obtained with 0–100 μM pogostone and various concentrations of substrates.ResultsPogostone was found to inhibit the activity of CYP3A4, 2C9, and 2E1 with the IC₅₀ values of 11.41, 12.11, and 14.90 μM, respectively. The inhibition of CYP3A4 by pogostone was revealed to be performed in a non-competitive and time-dependent manner with the K_i value of 5.69 μM and the KI/K_inact value of 5.86/0.056/(μM/min). For the inhibition of CYP2C9 and 2E1, pogostone acted as a competitive inhibitor with the K_i value of 6.46 and 7.67 μM and was not affected by the incubation time.Discussion and conclusionsThe inhibitory effect of pogostone on the activity of CYP3A4, 2C9, and 2E1 has been disclosed in this study, implying the potential risk during the co-administration of pogostone and drugs metabolized by these CYP450s. The study design provides a reference for further in vivo investigations to validate the potential interaction.     

Evodiamine decreased the systemic exposure of pravastatin in non-alcoholic steatohepatitis rats due to the up-regulation of hepatic OATPs

ContextPatients with non-alcoholic steatohepatitis (NASH) may have a simultaneous intake of pravastatin and evodiamine-containing herbs.ObjectiveThe effect of evodiamine on the pharmacokinetics of pravastatin and its potential mechanisms were investigated in NASH rats.Materials and methodsThe NASH model was conducted with feeding a methionine choline-deficient (MCD) diet for 8 weeks. Sprague-Dawley rats were randomised equally (n = 6) into NASH group, evodiamine group (10 mg/kg), pravastatin group (10 mg/kg), and evodiamine (10 mg/kg) + pravastatin (10 mg/kg) group. Normal control rats were fed a standard diet. Effects of evodiamine on the pharmacokinetics, distribution, and uptake of pravastatin were investigated.ResultsEvodiamine decreased C_max (159.43 ± 26.63 vs. 125.61 ± 22.17 μg/L), AUC_0-t (18.17 ± 2.52 vs. 14.91 ± 2.03 mg/min/L) and AUC_0-∞ (22.99 ± 2.62 vs. 19.50 ± 2.31 mg/min/L) of orally administered pravastatin in NASH rats, but had no significant effect in normal rats. Evodiamine enhanced the uptake (from 154.85 ± 23.17 to 198.48 ± 26.31 pmol/mg protein) and distribution (from 736.61 ± 108.07 to 911.89 ± 124.64 ng/g tissue) of pravastatin in NASH rat liver. The expression of Oatp1a1, Oatp1a4, and Oatp1b2 was up-regulated 1.48-, 1.38-, and 1.51-fold by evodiamine. Evodiamine decreased the levels of IL-1β, IL-6, and TNF-α by 27.82%, 24.76%, and 29.72% in NASH rats, respectively.Discussion and conclusionsEvodiamine decreased the systemic exposure of pravastatin by up-regulating the expression of OATPs. These results provide a reference for further validation of this interaction in humans.     

Effects of β2-agonist- and dexamethasone-treatment on relaxation and regulation of β-adrenoceptors in human bronchi and lung tissue

1. Long-term treatment with β₂-adrenoceptor agonists can lead to a decreased therapeutic efficacy of bronchodilatation in patients with obstructive pulmonary disease. In order to examine whether or not this is due to β-adrenoceptor desensitization, human bronchial muscle relaxation was studied in isolated bronchial rings after pretreatment with β₂-adrenoceptor agonists. Additionally, the influence of pretreatment with dexamethasone on desensitization was studied.
2. The effect of β₂-agonist incubation alone and after coincubation with dexamethasone on density and affinity of β-adrenoceptors was investigated by radioligand binding experiments.
3. In human isolated bronchi, isoprenaline induces a time- and concentration-dependent β-adrenoceptor desensitization as judged from maximal reduction in potency by a factor of 7 and reduction of 73±4% in efficacy of isoprenaline to relax human bronchial smooth muscle.
4. After an incubation period of 60 min with 100 μmol l⁻¹ terbutaline, a significant decline in its relaxing efficacy (81±8%) and potency (by a factor 5.5) occurred.
5. Incubation with 30 μmol l⁻¹ isoprenaline for 60 min did not impair the maximal effect of a subsequent aminophylline response but led to an increase in potency (factor 4.4).
6. Coincubation of dexamethasone with isoprenaline (120 min; 30 μmol l⁻¹) preserved the effect of isoprenaline on relaxation (129±15%).
7. In radioligand binding experiments, pretreatment of lung tissue for 60 min with isoprenaline (30 μmol l⁻¹) resulted in a decrease in β-adrenoceptor binding sites (B_max) to 64±1.6% (P<0.05), while the antagonist affinity (K_D) for [³H]-CGP-12177 remained unchanged.
8. In contrast, radioligand binding studies on lung tissue pretreated with either dexamethasone (30 μmol l⁻¹) or isoprenaline (30 μmol l⁻¹) plus dexamethasone (30 μmol l⁻¹) for 120 min did not lead to a significant change of B_max (160±22.1% vs 142.3±28.7%) or K_D (5.0 nmol l⁻¹ vs 3.5 nmol l⁻¹) compared to the controls.
9. In conclusion, pretreatment of human bronchi with β-adrenoceptor agonists leads to functional desensitization and, in lung tissue, to down-regulation of β-adrenoceptors. This effect can be counteracted by additional administration of dexamethasone. Our model of desensitization has proved useful for the identification of mechanisms of β-adrenoceptor desensitization and could be relevant for the evaluation of therapeutic strategies to counteract undesirable effects of long-term β-adrenoceptor stimulation.

     

Gambogic amide inhibits angiogenesis by suppressing VEGF/VEGFR2 in endothelial cells in a TrkA-independent manner

ContextGambogic amide (GA-amide) is a non-peptide molecule that has high affinity for tropomyosin receptor kinase A (TrkA) and possesses robust neurotrophic activity, but its effect on angiogenesis is unclear.ObjectiveThe study investigates the antiangiogenic effect of GA-amide on endothelial cells (ECs).Materials and methodsThe viability of endothelial cells (ECs) treated with 0.1, 0.15, 0.2, 0.3, 0.4, and 0.5 μM GA-amide for 48 h was detected by MTS assay. Wound healing and angiogenesis assays were performed on cells treated with 0.2 μM GA-amide. Chicken eggs at day 7 post-fertilization were divided into the dimethyl sulfoxide (DMSO), bevacizumab (40 μg), and GA-amide (18.8 and 62.8 ng) groups to assess the antiangiogenic effect for 3 days. mRNA and protein expression in cells treated with 0.1, 0.2, 0.4, 0.8, and 1.2 μM GA-amide for 6 h was detected by qRT-PCR and Western blots, respectively.ResultsGA-amide inhibited HUVEC (IC₅₀ = 0.1269 μM) and NhEC (IC₅₀ = 0.1740 μM) proliferation, induced cell apoptosis, and inhibited the migration and angiogenesis at a relatively safe dose (0.2 μM) in vitro. GA-amide reduced the number of capillaries from 56 ± 14.67 (DMSO) to 20.3 ± 5.12 (62.8 ng) in chick chorioallantoic membrane (CAM) assay. However, inactivation of TrkA couldn’t reverse the antiangiogenic effect of GA-amide. Moreover, GA-amide suppressed the expression of VEGF and VEGFR2, and decreased activation of the AKT/mTOR and PLCγ/Erk1/2 pathways.ConclusionsConsidering the antiangiogenic effect of GA-amide, it might be developed as a useful agent for use in clinical combination therapies.     

Salidroside protects against ventilation-induced lung injury by inhibiting the expression of matrix metalloproteinase-9

ContextSalidroside, a compound extracted from Rhodiola rosea L. (Crassulaceae), possesses many beneficial pathological effects.ObjectiveTo explore the effect of salidroside on ventilator-induced lung endothelial dysfunction in vivo and in vitro.Materials and methodsIn vivo, male ICR mice were divided into sham, ventilation, salidroside, and ventilation plus salidroside groups. The mice were ventilated for 4 h, salidroside (50 mg/kg) was administrated intraperitoneally before ventilation, dexamethasone (Dex) (5 mg/kg) was used as a positive control. In vitro, mouse lung vascular endothelial cells (MLVECs) were treated with salidroside, MMP-9 siRNA, and BAY11-7082 (10 μM), and then exposed to cyclic stretch for 4 h. Afterward, lung tissues and MLVECs were collected for further analysis.ResultsSalidroside pre-treatment significantly reversed the expression of vascular endothelial cadherin (VE-cadherin) and zonula occluden-1 (ZO-1) proteins in cyclic stretch-treated MLVECs (0.46 ± 0.09 vs. 0.80 ± 0.14, 0.49 ± 0.05 vs. 0.88 ± 0.08) and ventilated lung tissues (0.56 ± 0.06 vs. 0.83 ± 0.46, 0.49 ± 0.08 vs. 0.80 ± 0.12). The results further indicated that salidroside inhibited the expression of matrix metalloproteinase-9 (MMP-9), whereas knockdown of its expression restored the expression levels of VE-cadherin (0.37 ± 0.08 vs. 0.85 ± 0.74) and ZO-1 (0.48 ± 0.08 vs. 0.81 ± 0.11) in stretched MLVECs. Meanwhile, salidroside inhibited the NF-κB signalling pathway and alleviated lung injury.ConclusionsSalidroside protected against stretch-induced endothelial barrier function, improving lung injury after ventilation. Thus, salidroside may be a promising therapeutic agent for patients with MV-induced lung injury.     

β-Hydroxyisovalerylshikonin regulates macrophage polarization via the AMPK/Nrf2 pathway and ameliorates sepsis in mice

ContextThe potential anti-inflammatory bioactivities of β-hydroxyisovalerylshikonin (β-HIVS) remain largely unknown.ObjectiveThis study investigated the anti-inflammatory effects and underlying mechanisms of β-HIVS.Materials and methodsRAW 264.7 cells stimulated with LPS (100 ng/mL) for 24 h were treated with the non-cytotoxic doses of β-HIVS (0.5 or 1 μM, determined by MTT and Trypan blue staining), qRT-PCR and FCM assay were used to examine macrophage polarization transitions. Western blotting was used to evaluate the activation of the AMPK/Nrf2 pathway. In vivo, C57BL/6 mice were randomly divided into vehicle control, LPS (10 mg/kg), and β-HIVS (2.5 mg/kg) combined with LPS (10 mg/kg) groups, blood samples, BALF, and lung tissues of mice were subjected to ELISA, qRT-PCR, FCM, and H&E staining.Resultsβ-HIVS (1 μM) inhibited LPS-induced expression of M1 macrophage markers (TNF-α: 0.29-fold, IL-1β: 0.32-fold), promoted the expression of M2 macrophage markers (CD206: 3.14-fold, Arginase-1: 3.98-fold) in RAW 264.7 cells; mechanistic studies showed that β-HIVS increased the expression of nuclear Nrf2 (2.04-fold) and p-AMPK (3.65-fold) compared with LPS group (p < 0.05). In vivo, β-HIVS decreased the levels of pro-inflammatory cytokines (TNF-α: 1130.41 vs. 334.88 pg/mL, IL-1β: 601.89 vs. 258.21 pg/mL in serum; TNF-α: 893.07 vs. 418.21 pg/mL, IL-1β: 475.22 vs. 298.54 pg/mL in BALF), decreased the proportion of M1 macrophages (77.83 vs. 68.53%) and increased the proportion of M2 macrophages (13.55 vs. 19.56%) in BALF, and reduced lung tissue damage and septic mice survival (p < 0.05).ConclusionsThese results indicate that β-HIVS may be a new potential anti-inflammatory agent.     

Salidroside orchestrates metabolic reprogramming by regulating the Hif-1α signalling pathway in acute mountain sickness

ContextRhodiola crenulata (Hook. f. et Thoms.) H. Ohba (Crassulaceae) is used to prevent and treat acute mountain sickness. However, the mechanisms underlying its effects on the central nervous system remain unclear.ObjectiveTo investigate the effect of Rhodiola crenulata on cellular metabolism in the central nervous system.Materials and methodsThe viability and Hif-1α levels of microglia and neurons at 5% O₂ for 1, 3, 5 and 24 h were examined. We performed the binding of salidroside (Sal), rhodiosin, tyrosol and p-hydroxybenzyl alcohol to Hif-1α, Hif-1α, lactate, oxidative phosphorylation and glycolysis assays. Forty male C57BL/6J mice were divided into control and Sal (25, 50 and 100 mg/kg) groups to measure the levels of Hif-1α and lactate.ResultsMicroglia sensed low oxygen levels earlier than neurons, accompanied by elevated expression of Hif-1α protein. Salidroside, rhodiosin, tyrosol, and p-hydroxybenzyl alcohol decreased BV-2 (IC₅₀=1.93 ± 0.34 mM, 959.74 ± 10.24 μM, 7.47 ± 1.03 and 8.42 ± 1.63 mM) and PC-12 (IC₅₀=6.89 ± 0.57 mM, 159.28 ± 8.89 μM, 8.65 ± 1.20 and 8.64 ± 1.42 mM) viability. They (10 μM) reduced Hif-1α degradation in BV-2 (3.7-, 2.5-, 2.9- and 2.5-fold) and PC-12 cells (2.8-, 2.8-, 2.3- and 2.0-fold) under normoxia. Salidroside increased glycolytic capacity but attenuated oxidative phosphorylation. Salidroside (50 and 100 mg/kg) treatment increased the protein expression of Hif-1α and the release of lactate in the brain tissue of mice.ConclusionsThese results suggest that Sal induces metabolic reprogramming by regulating the Hif-1α signalling pathway to activate compensatory responses, which may be the core mechanism underlying the effect of Rhodiola crenulata on the central nervous system.     

Modelling of the pharmacodynamic interaction between phenytoin and sodium valproate

1. Treatment of epilepsy with a combination of antiepileptic drugs remains the therapeutic choice when monotherapy fails. In this study, we apply pharmacokinetic-pharmacodynamic modelling to characterize the interaction between phenytoin (PHT) and sodium valproate (VPA).
2. Male Wistar rats received a 40 mg kg⁻¹ intravenous dose of PHT over 5 min either alone or in combination with an infusion of VPA resulting in a steady-state concentration of 115.5±4.9 μg ml⁻¹. A control group received only the infusion of VPA. The increase in the threshold for generalized seizure activity (ΔTGS) was used as measure of the anticonvulsant effect.
3. PHT pharmacokinetics was described by a pharmacokinetic model with Michaelis-Menten elimination. The concentration-time course and plasma protein binding of PHT were not altered by VPA. The pharmacokinetic parameters V_max and K_m were, respectively, 294±63 μg min⁻¹ and 7.8±2.4 μg ml⁻¹ in the absence of VPA and 562±40 μg min⁻¹ and 15.6±0.9 μg ml⁻¹ upon administration in combination with VPA.
4. A delay of the onset of the effect relative to plasma concentrations of PHT was observed. The assessment of PHT concentrations at the effect site was based on the effect-compartment model, yielding mean k_e0 values of 0.128 and 0.107 min⁻¹ in the presence and absence of VPA, respectively.
5. A nonlinear relationship between effect-site concentration and the increase in the TGS was observed. The concentration that causes an increase of 50% in the baseline TGS (EC_50%TGS) was used to compare drug potency. A shift of EC_50%TGS from 13.27±3.55 to 4.32±0.52 μg ml⁻¹ was observed upon combination with VPA (P<0.01).
6. It is concluded that there is a synergistic pharmacodynamic interaction between PHT and VPA in vivo.

     

α-Amylase and dipeptidyl peptidase-4 (DPP-4) inhibitory effects of Melicope latifolia bark extracts and identification of bioactive constituents using in vitro and in silico approaches

ContextMelicope latifolia (DC.) T. G. Hartley (Rutaceae) was reported to contain various phytochemicals including coumarins, flavonoids, and acetophenones.ObjectiveThis study investigates the antidiabetic and antioxidant effects of M. latifolia bark extracts, fractions, and isolated constituents.Materials and methodsMelicope latifolia extracts (hexane, chloroform, and methanol), fractions, and isolated constituents with varying concentrations (0.078–10 mg/mL) were subjected to in vitro α-amylase and dipeptidyl peptidase-4 (DPP-4) inhibitory assay. Molecular docking was performed to study the binding mechanism of active compounds towards α-amylase and DPP-4 enzymes. The antioxidant activity of M. latifolia fractions and compounds were determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging and β-carotene bleaching assays.ResultsMelicope latifolia chloroform extract showed the highest antidiabetic activity (α-amylase IC₅₀: 1464.32 μg/mL; DPP-4 IC₅₀: 221.58 μg/mL). Fractionation of chloroform extract yielded four major fractions (CF₁–CF₄) whereby CF₃ showed the highest antidiabetic activity (α-amylase IC₅₀: 397.68 μg/mL; DPP-4 IC₅₀: 37.16 μg/mL) and resulted in β-sitosterol (1), halfordin (2), methyl p-coumarate (3), and protocatechuic acid (4). Isolation of compounds 2–4 from the species and their DPP-4 inhibitory were reported for the first time. Compound 2 showed the highest α-amylase (IC₅₀: 197.53 μM) and β-carotene (88.48%) inhibition, and formed the highest number of molecular interactions with critical amino acid residues of α-amylase. The highest DPP-4 inhibition was exhibited by compound 3 (IC₅₀: 911.44 μM).Discussion and conclusionsThe in vitro and in silico analyses indicated the potential of M. latifolia as an alternative source of α-amylase and DPP-4 inhibitors. Further pharmacological studies on the compounds are recommended.     

Interactions between loreclezole,chlormethiazole and pentobarbitone at GABAA receptors: functional and binding studies

1. Interations were investigated between loreclezole, chlormethiazole and pentobarbitone as potentiators of depolarization responses mediated by γ-aminobutyric acid_A (GABA_A) receptors on afferent nerve terminals in the rat cuneate nucleus in vitro. These drugs were also compared as modulators of [³H]-flunitrazepam (FNZ) binding to synaptic membranes prepared from rat whole brain homogenate.
2. In rat cuneate nucleus slices, the drugs shifted muscimol log dose–response lines to the left in an approximately parallel fashion with the result that 200 μM chlormethiazole potentiated muscimol responses by 0.567±0.037 log unit (mean±s.e.mean, n=4) while loreclezole gave a maximal potentiation at 10 μM of only 0.121±0.037 (n=6) log unit and 0.071±0.039 (n=22) at 50 μM.
3. While 50 μM chlormethiazole and 30 μM pentobarbitone showed no significant interactions between each other when potentiating muscimol responses in combination, 50 μM loreclezole in combination with either chlormethiazole or pentobarbitone attenuated their potentiating effects, possibly by inducing desensitization of GABA_A receptors.
4. In the [³H]-FNZ binding studies on well-washed membranes, loreclezole enhanced binding to a maximum of 47.3±2.83% of control (mean±s.e.mean, n=3) at 300 μM. Scatchard analysis revealed no change in B_max but a decrease in K_D for [³H]-FNZ from 3.9±0.29 nM to 2.7±0.10 nM (mean±s.e.mean, n=4) in the presence of 100 μM loreclezole. In contrast, 100 μM chlormethiazole caused no potentiation. A small component of the enhancement by loreclezole could be blocked by 100 μM bicuculline and could also be blocked by 100 μM chlormethiazole. It seems likely that the effects on [³H]-FNZ binding are due predominantly to direct actions of the drugs on the GABA_A receptor and are separate from the GABA-potentiating effects.
5. The results indicate distinctly different profiles of action for loreclezole, chlormethiazole and pentobarbitone on GABA_A receptors.