Surplus fatty acid synthesis increases oxidative stress in adipocytes and lnduces lipodystrophy.

Adipocytes are the primary sites for fatty acid storage, but the synthesis rate of fatty acids is very low. The physiological significance of this phenomenon remains unclear. Here, we show that surplus fatty acid synthesis in adipocytes induces necroptosis and lipodystrophy. Transcriptional activation of FASN elevates fatty acid synthesis, but decreases NADPH level and increases ROS production, which ultimately leads to adipocyte necroptosis. We identify MED20, a subunit of the Mediator complex, as a negative regulator of FASN transcription. Adipocyte-specific male Med20 knockout mice progressively develop lipodystrophy, which is reversed by scavenging ROS. Further, in a murine model of HIV-associated lipodystrophy and a human patient with acquired lipodystrophy, ROS neutralization significantly improves metabolic disorders, indicating a causal role of ROS in disease onset. Our study well explains the low fatty acid synthesis rate in adipocytes, and sheds light on the management of acquired lipodystrophy.

Predictors of vessel quantitative flow ratio loss in patients with severely calcified lesions after rotational atherectomy.

BACKGROUND: Previous studies have established that moderately to severely calcified lesions (MSCL) are associated with high rates of major adverse cardiovascular events, even when drug-eluting stents are implanted after rotational atherectomy (RA). Yet, the changes in coronary function indexes during follow-ups have never been investigated. The quantitative flow ratio (QFR), a novel coronary function index, has been increasingly adopted in daily practice in recent years. METHODS: A total of 111 MSCL patients were retrospectively enrolled in this study. The vessel QFR (QFRv) loss was defined as post-percutaneous coronary intervention QFRv minus follow-up QFRv. The study subjects were divided into high QFRv loss (n = 51) and low QFRv loss (n = 60) groups according to the binary method. The obtained predictors of QFRv loss were then analyzed. RESULTS: The results showed that the final burr-to-vessel ratio (B to V ratio) in the high QFRv loss group decreased significantly compared to the low QFRv loss group (p < 0.01). The univariate and multivariate regression analyses indicated that the final B to V ratio was an excellent predictor of QFRv loss. The cut-off value of the final B to V ratio for QFRv loss prediction was 0.50 (sensitivity: 50.98%, specificity: 68.33%, and area under the curve: 0.627 [95% confidence interval: 0.530-0.717], p < 0.05). Additionally, the target vessel failure incidence in the high QFRv loss group was higher than in the low QFRv loss group (p < 0.01). CONCLUSIONS: An increased burr-to-vessel ratio can prevent QFRv loss in patients with MSCLs after RA, an effect that might be closely associated with a low target vessel failure incidence.

Prognostic value of infarct-related-lead Tpeak-Tend/QT ratio in patients with ST-segment elevation myocardial infarction.

Tpeak-Tend/QT ratio (Tp-e/QT) in patients with ST-segment elevation myocardial infarction (STEMI) is reportedly associated with major adverse cardiac events (MACEs). However, Tp-e/QT measurement methods are controversial, and few studies have clarified the effect of different Tp-e/QT measures on prognosis. Our study is the first to investigate the advantages of the Tp-e/QT measured by infarct-related-lead method in predicting MACEs during hospitalization and long-term mortality in patients with STEMI. A total of 427 STEMI patients undergoing primary percutaneous coronary intervention (PCI) were included in this study. The Tp-e/QT before PCI was measured by traditional 12-lead method and infarct-related-lead method. Outcomes were tested using comparative statistics, logistic regression, receiver operating characteristic (ROC) curve and Kaplan-Meier survival analysis. There were 62 (14.5%) patients who had MACEs in-hospital. Logistic regression showed that the Tp-e/QT in infarct-related-lead was an independent predictor (p < 0.001). The area under the ROC curve (AUC) of the Tp-e/QT in infarct-related-lead was larger than that in the Tp-e/QT in traditional 12-lead (0.889 vs 0.741), and the optimal cutoff value was 0.32. The three-year survival rate of patients in the infarct-related-lead Tp-e/QT < 0.32 group was better than Tp-e/QT ≥ 0.32 group in Kaplan-Meier survival analysis (93.9 vs 87.0%). When stratified according to infarct-related arteries, the results showed that the common odds ratio of patients in Tp-e/QT ≥ 0.32 group occurred MACEs was 1.562, P = 0.038. The infarct-related-lead Tp-e/QT performed better than the traditional 12-lead Tp-e/QT in predicting poor prognosis.

Significant association between the endothelial lipase gene 584C/T polymorphism and coronary artery disease risk.

Several studies have investigated a potential association between the endothelial lipase gene (LIPG) 584C/T polymorphism and susceptibility to coronary artery disease (CAD), but a uniform conclusion is yet to be reached. To better evaluate the true relationship between the LIPG 584C/T polymorphism and the risk of CAD, a meta-analysis of 14 case-control studies with 9731 subjects was performed. Relevant articles published through August 2020 were searched in the CNKI, PubMed, Embase and Web of Science databases. Thirteen articles, including 14 eligible case-control studies with 4025 cases and 5706 controls, were enrolled in the present meta-analysis. The Newcastle-Ottawa Scale (NOS) scores of the case-control studies ranged from 6 to 8. The pooled results indicated that there is a significant association between the LIPG 584C/T polymorphism and CAD in the homozygote comparison model and the allelic comparison model. Subgroup analyses revealed that the LIPG 584C/T mutation significantly decreased the risk of CAD in the subgroups of African, CAD, hospital-based (HB), and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) populations in some genetic models. No publication bias was found in our meta-analysis, which certifies the robustness of the current meta-analysis. Trial sequential analysis (TSA) also confirmed the stability of our results. The results of our meta-analysis indicate that the LIPG 584C/T polymorphism plays a protective role in the incidence of CAD. More high-quality case-control studies on various ethnicities are needed to confirm our results.

Melatonin inhibits macrophage infiltration and promotes plaque stabilization by upregulating anti-inflammatory HGF/c-Met system in the atherosclerotic rabbit: USPIO-enhanced MRI assessment.

Macrophage plays critical roles in the pathogenesis of atherosclerosis (AS), and is an attractive target for detecting and treating vulnerable plaque. Our previous study showed that melatonin (MLT) ameliorated AS by suppressing the pro-inflammatory Toll-like receptor 4/nuclear factor kappa B system in high-fat-fed rabbit. However, it is unknown whether the anti-atherosclerotic properties of MLT are associated with the upregulation of anti-inflammatory hepatocyte growth factor (HGF)/mesenchymal-epithelial transition factor (c-Met) system. In present study, we examined whether MLT could inhibit macrophage infiltration and promote plaque stabilization by upregulating HGF/c-Met system with ultrasmall superparamagnetic iron oxide (USPIO)-enhanced magnetic resonance imaging (MRI) assessment in AS rabbit. Rabbits in this study were randomly divided into three groups and treated with a standard diet, high-fat diet, and high-fat diet plus 10 mg/kg/day MLT for 12 weeks, respectively. MLT treatment significantly reversed spotty signal void in 3D-TOF MRI, standard signal intensity reduction in T2WI MRI and aortic luminal area reduction in 2D-TOF MRI of the atherosclerotic abdominal aorta 72 h after USPIO injection. It also decreased serum interleukin-6 (IL-6), intima/media thickness ratio of the abdominal aorta, CD68 and iron-positive areas in the aortic intima, and increased serum IL-10, HGF and c-Met protein expression and the accumulation of vascular smooth muscle cell and collagen fiber in the aortic intima of AS rabbit. Our data demonstrated that MLT significantly decreased plaque macrophage infiltration and promoted plaque stability in AS rabbit assessed by USPIO-enhanced MRI. Remarkably, it was very first revealed that upregulation of anti-inflammatory HGF/c-Met system might contribute to the atheroprotective mechanisms of MLT.

Telmisartan prevents angiotensin II-induced endothelial dysfunction in rabbit aorta via activating HGF/Met system and PPARγ pathway.

Telmisartan with partial activation of peroxisome proliferator-activated receptor γ (PPARγ) powerfully reduces blood pressure, improves endothelial function and lipid metabolism. Hepatocyte growth factor/mesenchymal-epithelial transition factor (HGF/Met) system in the local vasculature plays a pivotal role in maintaining normal endothelial function. This study is aimed to evaluate whether telmisartan directly prevents angiotensin II (Ang II)-induced endothelial dysfunction (ED) via activating HGF/Met system and/or PPARγ pathway. The isolated aortic rings of rabbits were incubated with Ang II (0.01-1 µM), telmisartan (0.1-10 µM), SU11274 (5 µM) as a specific Met inhibitor, GW9662 (10 µM) as a PPARγ antagonist alone or a combination for 6 h. Ang II obviously inhibited the mRNA and protein expression of HGF, Met and PPARγ, and the accumulative concentration-relaxation of the aortic rings to acetylcholine, among which the inhibitory effect of 1 µM Ang II was most significant. By contrast, telmisartan significantly increased the mRNA and protein expression of HGF, Met, and PPARγ, thus preventing Ang II-induced ED in a dose-dependent pattern. However, SU11274, GW9662 or a combination of both partially abolished the protective effects derived from telmisartan, with the effect of SU11274 exceeding that of GW9662. These results demonstrate that Ang II-induced ED in rabbit aortic rings in vitro can be prevented by telmisartan through selective PPARγ-modulating pathway. Moreover, this study indicates for the first time that activating HGF/Met system in the local vasculature is involved in the protective mechanism of telmisartan.

Melatonin ameliorates vascular endothelial dysfunction, inflammation, and atherosclerosis by suppressing the TLR4/NF-κB system in high-fat-fed rabbits.

Vascular endothelial dysfunction (VED) and inflammation contribute to the initiation and progression of atherosclerosis. Melatonin (MLT) normalizes lipid profile, improves endothelial function, and possesses anti-inflammatory properties. However, the precise mechanisms are still unclear. This study investigated whether MLT could ameliorate VED, inflammation, and atherosclerosis by suppressing the Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) system in high-fat-fed rabbits. Rabbits were randomly divided into three groups that received a standard diet (control group), high-cholesterol diet (atherosclerosis group), or high-cholesterol diet plus 10 mg/kg/day MLT (MLT group) for 12 wk. After treatment, high-fat diet significantly increased serum lipid and inflammatory markers in rabbits in atherosclerosis group compared with that in control group. In addition, high-fat diet also induced VED and typical atherosclerotic plaque formation and increased intima/media thickness ratio, which were significantly improved by MLT therapy as demonstrated in MLT group. Histological and immunoblot analysis further showed that high-fat diet enhanced the expressions of TLR4, myeloid differentiation primary response protein (MyD88), and NF-κB p65, but decreased inhibitor of NF-κB (IκB) expression. By contrast, MLT therapy decreased the expressions of TLR4, MyD88, and NF-κB p65 and increased IκB expression. This study has demonstrated that MLT ameliorates lipid metabolism, VED, and inflammation and inhibits the progression of atherosclerosis in high-fat-fed rabbits. Moreover, our study indicates for the first time that suppression of the TLR4/NF-κB system in local vasculature with atherosclerotic damage is important for the protective effects of MLT.

Effects of recombinant adenovirus hepatocyte growth factor gene on myocardial remodeling in spontaneously hypertensive rats.

BACKGROUND AND OBJECTIVES: Myocardial hypertrophy and fibrosis are important determinants of congestive heart failure. Previous work has shown that hepatocyte growth factor (HGF) can reduce acute myocardial injury and tissue fibrosis. This study was designed to examine the effects of HGF on myocardial remodeling following sustained hypertension. METHODS AND RESULTS: There were 4 experimental groups (n = 6) that included spontaneously hypertensive rats (SHRs) injected with 0.1 mL of adenovirus (Ad)-null into the left ventricular (LV) free wall, SHR injected with 0.1 mL of Ad-HGF gene (5 × 10(9) pfu/mL), and SHR injected with 0.1 mL of normal saline, and Wistar Kyoto rats injected with 0.1 mL of Ad-null served as control. At 4 weeks after injection, rats were sacrificed, and HGF expression, myocardial fibrosis, and LV function were determined. We observed that HGF protein expression was reduced in the hearts of SHR (P < .05 vs normal control) and it was markedly increased in SHR injected with Ad-HGF (P < .01 vs SHR injected with Ad-null). Myocardial fibrosis, collagen I, LV mass index (LVMI), and LV end-diastolic pressure (LVEDP) were increased and -dP/dtmax was decreased in SHR injected with Ad-null or normal saline (P < .01 vs normal control). Upregulation of myocardial HGF expression in SHR significantly suppressed myocardial fibrosis, collagen I content, LVMI, LVEDP, and increased -dP/dtmax (all P < .05 vs SHR-Ad-null, n = 6). CONCLUSIONS: These findings indicate that HGF expression is attenuated in hypertrophic and fibrotic myocardium of SHR. The forced increase in HGF exerts a salutary effect on myocardial fibrosis, collagen I expression, and hemodynamic parameters.

Metabonomic profiling of TASTPM transgenic Alzheimer's disease mouse model.

Identification of molecular mechanisms underlying early stage Alzheimer's disease (AD) is important for the development of new therapies against and diagnosis of AD. In this study, nontargeted metabonomics of TASTPM transgenic AD mice was performed. The metabolic profiles of both brain and plasma of TASTPM mice were characterized using gas chromatography-mass spectrometry and compared to those of wild-type C57BL/6J mice. TASTPM mice were metabolically distinct compared to wild-type mice (Q2Y=0.587 and 0.766 for PLS-DA models derived from brain and plasma, respectively). A number of metabolites were found to be perturbed in TASTPM mice in both brain (D-fructose, L-valine, L-serine, L-threonine, zymosterol) and plasma (D-glucose, D-galactose, linoleic acid, arachidonic acid, palmitic acid and D-gluconic acid). In addition, enzyme immunoassay confirmed that selected endogenous steroids were significantly perturbed in brain (androstenedione and 17-OH-progesterone) and plasma (cortisol and testosterone) of TASTPM mice. Ingenuity pathway analysis revealed that perturbations related to amino acid metabolism (brain), steroid biosynthesis (brain), linoleic acid metabolism (plasma) and energy metabolism (plasma) accounted for the differentiation of TASTPM and wild-type mice. Our results provided insights on the pathogenesis of APP-induced AD and reinforced the role of TASTPM in drug and biomarker development.

Metabolomic response of human skin tissue to low dose ionizing radiation.

Understanding how human organs respond to ionizing radiation (IR) at a systems biology level and identifying biomarkers for IR exposure at low doses can help provide a scientific basis for establishing radiation protection standards. Little is known regarding the physiological responses to low dose IR at the metabolite level, which represents the end-point of biochemical processes inside cells. Using a full thickness human skin tissue model and GC-MS-based metabolomic analysis, we examined the metabolic perturbations at three time points (3, 24 and 48 h) after exposure to 3, 10 and 200 cGy of X-rays. PLS-DA score plots revealed dose- and time-dependent clustering between sham and irradiated groups. Importantly, delayed metabolic responses were observed at low dose IR. When compared with the high dose at 200 cGy, a comparable number of significantly changed metabolites were detected 48 h after exposure to low doses (3 and 10 cGy) of irradiation. Biochemical pathway analysis showed perturbations to DNA/RNA damage and repair, lipid and energy metabolisms, even at low doses of IR.

Impaired neuronal insulin signaling precedes Aß42 accumulation in female AßPPsw/PS1ΔE9 mice.

Reduced glucose utilization is likely to precede the onset of cognitive deficits in Alzheimer's disease (AD). Similar aberrant glucose metabolism can also be detected in the brain of several AD mouse models. Although the cause of this metabolic defect is not well understood, it could be related to impaired insulin signaling that is increasingly being reported in AD brain. However, the temporal relationship between insulin impairment and amyloid-ß (Aß) biogenesis is unclear. In this study using female AßPPsw/PS1ΔE9 mice, we found that the level of Aß40 was fairly constant in 6- to 15-month-old brains, whereas Aß42 was only significantly increased in the 15-month-old brain. In contrast, increased levels of IRß, IGF-1R, IRS1, and IRS-2, along with reduced glucose and insulin content, were detected earlier in the 12-month-old brains of AßPPsw/PS1ΔE9 mice. The reduction in brain glucose content was accompanied by increased GLUT3 and GLUT4 levels. Importantly, these changes precede the significant upregulation of Aß42 level in the 15-month-old brain. Interestingly, reduction in the p85 subunit of PI3K was only apparent in the 15-month-old AßPPsw/PS1ΔE9 mouse brain. Furthermore, the expression profile of IRß, IRS-2, and p85/PI3K in AßPPsw/PS1ΔE9 was distinct in wild-type mice of a similar age. Although the exact mechanisms underlining this connection remain unclear, our results suggest a possible early role for insulin signaling impairment leading to amyloid accumulation in AßPPsw/PS1ΔE9 mice.

Fixed-dose telmisartan/hydrochlorothiazide in comparison with losartan/hydrochlorothiazide in decreasing serum hepatocyte growth factor and improving endothelial dysfunction in hypertensive patients.

Combined treatment with an angiotensin II receptor blocker and hydrochlorothiazide (HCT) is advocated to control hypertension (HT). Hepatocyte growth factor (HGF) may be a new marker to evaluate endothelial dysfunction (ED), which is a potential target in treating HT. The aim of the present study was to compare the effects of Telmisartan/HCT with Losartan/HCT on serum HGF and ED in hypertensive patients. Hypertensive patients were randomly divided into a Telmisartan/HCT (group T) or Losartan/HCT group (group L) and received one tablet of either drug per day for 8 weeks. Serum HGF, nitric oxide (NO), plasma von Willebrand factor (vWF), and endothelin (ET) were measured before treatment and after 8 weeks of treatment. Twenty healthy subjects were selected as controls (control group). HGF, vWF, ET, and the ET/NO ratio were higher, and NO was lower in hypertensive patients than those in the control group (all P < 0.01). After treatment for 8 weeks, HGF, vWF and ET decreased, and NO increased significantly in both groups (all P < 0.01). The reductions in BP and HGF and increase in NO (DeltaNO) were not significantly different between the two groups (all P > 0.05), but the reductions in vWF and ET (DeltaET) and DeltaET/DeltaNO ratio were more obvious in group T than in group L (all P < 0.01). There was no significant correlation between the changes in most of the measured parameters and the extent of BP reduction in either group. Both Telmisartan/HCT and Losartan/HCT could decrease serum HGF and improve ED, which was independent of the antihypertensive effects. However, the improvement in ED may be superior with Telmisartan/HCT than Losartan/HCT when the BP-lowering effects are the same.

Simultaneous determination of irinotecan (CPT-11) and SN-38 in tissue culture media and cancer cells by high performance liquid chromatography: application to cellular metabolism and accumulation studies.

A simple and sensitive HPLC method was developed to simultaneously determine CPT-11 and its major metabolite SN-38 in culture media and cell lysates. Camptothecin (CPT) was used as internal standard (I.S.). Compounds were eluted with acetonitrile-50 mM disodium hydrogen phosphate buffer containing 10 mM sodium 1-heptane-sulfonate, with the pH adjusted to 3.0 using 85% (w/v) orthophosphoric acid (27/73, v/v) by a Hyperclon ODS (C18) column (200 mm x 4.6 mm i.d.), with detection at excitation and emission wavelengths of 380 and 540 nm, respectively. The average extraction efficiencies were 96.9-108.3% for CPT-11 in culture media and 94.3-107.2% for CPT-11 in cell lysates; and 87.7-106.8% for SN-38 in culture media and 90.1-105.6% for SN-38 in cell lysates. Within- and between-day precision and accuracy varied from 0.1 to 10.3%. The limit of quantitation (precision and accuracy <20%) was 5.0 and 2.0 ng/ml for CPT-11 and 1.0 and 0.5 ng/ml for SN-38 in culture media and cell lysates, respectively. This method was successfully applied to quantitate the cellular accumulation and metabolism of CPT-11 and SN-38 in H4-II-E, a rat hepatoma cell line.

A mechanistic study on altered pharmacokinetics of irinotecan by St. John's wort.

Irinotecan (CPT-11) is an important anticancer drug in management of advanced colon cancer. A marked protective effect on CPT-11-induced blood and gastrointestinal toxicity is obtained by combination of St. John's wort (SJW) in recent clinical and rat studies. However, the mechanism is unclear. This study aimed to explore the effects of SJW on the pharmacokinetics of CPT-11 and its major metabolites (SN-38 and SN-38 glucuronide) in rats and the underlying mechanisms using several in vitro models. Short-term (3 days) and long-term (14 days) pretreatment with SJW were conducted in rats to examine the effects of co-administered SJW on the plasma pharmacokinetics of CPT-11, SN-38 and SN-38 glucuronide. Rat liver microsomes and a rat hepatoma cell line, H4-II-E cells, were utilized to study the effects of aqueous and ethanolic extracts (AE and EE) and major active components (hyperforin, hypericin and quercetin) of SJW on CPT-11 and SN-38 metabolism and intracellular accumulation. Co-administered SJW for consecutive 14 days significantly decreased the initial plasma concentration (C0) of CPT-11, the area under the concentration-time curve (AUC(0-10hr)) and maximum plasma concentration (Cmax) of SN-38. The ethanolic extracts (EE) of SJW at 5 microg/ml significantly decreased SN-38 glucuronidation by 45% (P < 0.05) in rat hepatic microsomes. Pre-incubation of aqueous SJW extracts (AE) at 10 microg/ml, SJW EE at 5 microg/ml, and quercetin at 10 microM significantly increased the glucuronidation of SN-38 in H4-II-E cells. A 2-hr pre-incubation of quercetin (100 microM) significantly increased the intracellular accumulation of CPT-11 (P < 0.05). However, pre-incubation of hypericin (20 nM and 200 nM) and hyperforin (1 microM) significantly decreased the intracellular accumulation of CPT-11. In addition, pre-incubation of hypericin, SJW EE and quercetin significantly increased the intracellular accumulation of SN-38. Aqueous and ethanolic SJW extracts and its major active components did not alter the plasma protein binding of CPT-11 and SN-38. These results indicated that the aqueous and ethanolic extracts of SJW and its major active components could markedly alter glucuronidation of SN-38 and intracellular accumulation of CPT-11 and SN-38, which probably provides partial explanation for the altered plasma pharmacokinetics of CPT-11 and SN-38 and the antagonizing effects on the toxicities of CPT-11. Further studies are needed to explore the role of both pharmacokinetic and pharmacodynamic components in the protective effect of SJW against the toxicities of CPT-11.

Drug-herb interactions: eliminating toxicity with hard drug design.

By searching the literatures, it was found that a total of 32 drugs interacting with herbal medicines in humans. These drugs mainly include anticoagulants (warfarin, aspirin and phenprocoumon), sedatives and antidepressants (midazolam, alprazolam and amitriptyline), oral contraceptives, anti-HIV agents (indinavir, ritonavir and saquinavir), cardiovascular drug (digoxin), immunosuppressants (cyclosporine and tacrolimus) and anticancer drugs (imatinib and irinotecan). Most of them are substrates for cytochrome P450s (CYPs) and/or P-glycoprotein (PgP) and many of which have narrow therapeutic indices. However, several drugs including acetaminophen, carbamazepine, mycophenolic acid, and pravastatin did not interact with herbs. Both pharmacokinetic (e.g. induction of hepatic CYPs and intestinal PgP) and/or pharmacodynamic mechanisms (e.g. synergistic or antagonistic interaction on the same drug target) may be involved in drug-herb interactions, leading of altered drug clearance, response and toxicity. Toxicity arising from drug-herb interactions may be minor, moderate, or even fatal, depending on a number of factors associated with the patients, herbs and drugs. Predicting drug-herb interactions, timely identification of drugs that interact with herbs, and therapeutic drug monitoring may minimize toxic drug-herb interactions. It is likely to predict pharmacokinetic herb-drug interactions by following the pharmacokinetic principles and using proper models that are used for predicting drug-drug interactions. Identification of drugs that interact with herbs can be incorporated into the early stages of drug development. A fourth approach for circumventing toxicity arising from drug-herb interactions is proper design of drugs with minimal potential for herbal interaction. So-called "hard drugs" that are not metabolized by CYPs and not transported by PgP are believed not to interact with herbs due to their unique pharmacokinetic properties. More studies are needed and new approached are required to minimize toxicity arising from drug-herb interactions.

St. John's wort attenuates irinotecan-induced diarrhea via down-regulation of intestinal pro-inflammatory cytokines and inhibition of intestinal epithelial apoptosis.

Diarrhea is a common dose-limiting toxicity associated with cancer chemotherapy, in particular for drugs such as irinotecan (CPT-11), 5-fluouracil, oxaliplatin, capecitabine and raltitrexed. St. John's wort (Hypericum perforatum, SJW) has anti-inflammatory activity, and our preliminary study in the rat and a pilot study in cancer patients found that treatment of SJW alleviated irinotecan-induced diarrhea. In the present study, we investigated whether SJW modulated various pro-inflammatory cytokines including interleukins (IL-1beta, IL-2, IL-6), interferon (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) and intestinal epithelium apoptosis in rats. The rats were treated with irinotecan at 60 mg/kg for 4 days in combination with oral SJW or SJW-free control vehicle at 400 mg/kg for 8 days. Diarrhea, tissue damage, body weight loss, various cytokines including IL-1beta, IL-2, IL-6, IFN-gamma and TNF-alpha and intestinal epithelial apoptosis were monitored over 11 days. Our studies demonstrated that combined SJW markedly reduced CPT-11-induced diarrhea and intestinal lesions. The production of pro-inflammatory cytokines such as IL-1beta, IFN-gamma and TNF-alpha was significantly up-regulated in intestine. In the mean time, combined SJW significantly suppressed the intestinal epithelial apoptosis induced by CPT-11 over days 5-11. In particular, combination of SJW significantly inhibited the expression of TNF-alpha mRNA in the intestine over days 5-11. In conclusion, inhibition of pro-inflammatory cytokines and intestinal epithelium apoptosis partly explained the protective effect of SJW against the intestinal toxicities induced by irinotecan. Further studies are warranted to explore the potential for STW as an agent in combination with chemotherapeutic drugs to lower their dose-limiting toxicities.

A mechanistic study on reduced toxicity of irinotecan by coadministered thalidomide, a tumor necrosis factor-alpha inhibitor.

Dose-limiting diarrhea and myelosuppression compromise the success of irinotecan (7-ethyl-10-[4-[1-piperidino]-1-piperidino]carbonyloxycamptothecin) (CPT-11)-based chemotherapy. A recent pilot study indicates that thalidomide attenuates the toxicity of CPT-11 in cancer patients. This study aimed to investigate whether coadministered thalidomide modulated the toxicities of CPT-11 and the underlying mechanisms using several in vivo and in vitro models. Diarrhea, intestinal lesions, cytokine expression, and intestinal epithelial apoptosis were monitored. Coadministered thalidomide (100 mg/kg i.p. for 8 days) significantly attenuated body weight loss, myelosuppression, diarrhea, and intestinal histological lesions caused by CPT-11 (60 mg/kg i.v. for 4 days). This was accompanied by inhibition of tumor necrosis factor-alpha, interleukins 1 and 6 and interferon-gamma, and intestinal epithelial apoptosis. Coadministered thalidomide also significantly increased the systemic exposure of CPT-11 but decreased that of SN-38 (7-ethyl-10-hydroxycampothecin). It significantly reduced the biliary excretion and cecal exposure of CPT-11, SN-38, and SN-38 glucuronide. Thalidomide hydrolytic products inhibited hydrolysis of CPT-11 in rat liver microsomes but not in primary rat hepatocytes. In addition, thalidomide and its major hydrolytic products, such as phthaloyl glutamic acid (PGA), increased the intracellular accumulation of CPT-11 and SN-38 in primary rat hepatocytes. They also significantly decreased the transport of CPT-11 and SN-38 in Caco-2 and parental MDCKII cells. Thalidomide and PGA also significantly inhibited P-glycoprotein (PgP/MDR1), multidrug resistance-associated protein (MRP1)- and MRP2-mediated CPT-11 and SN-38 transport in MDCKII cells. These results provide insights into the pharmacodynamic and pharmacokinetic mechanisms for the protective effects of thalidomide against CPT-11-induced intestinal toxicity.

Small interfering RNA-mediated silencing of cytochrome P450 3A4 gene.

RNA interference (RNAi) is a specific and powerful tool used to manipulate gene expression and study gene function. The cytochrome P450 3A4 (CYP3A4) can metabolize more than 50% of drugs. In the present study, we investigated whether vector-expressed small interfering RNAs (siRNAs) altered the CYP3A4 expression and function using the Chinese hamster cell line (V79) overexpressing CYP3A4 (CHL-3A4). Three different siRNA oligonucleotides (3A4I, 3A4II, and 3A4III) were designed and tested for their ability to interfere with CYP3A4 gene expression. Our study demonstrated that transient transfection of CHL-3A4 cells with the 3A4III siRNAs, but not 3A4I and II, significantly reduced CYP3A4 mRNA levels by 65% and protein expression levels by 75%. All these siRNAs did not affect the expression of CYP3A5 at both mRNA and protein levels in V79 cells overexpressing CYP3A5. Transfection of CHL-3A4 cells with 3A4III siRNAs significantly diminished the cytotoxicity of two CYP3A4 substrate drugs, cyclophosphamide and ifosfamide, in CHL-3A4 cells, with the IC50 increased from 55 to 210 microM to >1000 microM. Nifedipine at 5.78, 14.44, and 28.88 microM was significantly (P < 0.01) depleted by approximately 100, 40, and 22%, respectively, in S9 fractions from CHL-3A4 cells compared with parental CHL-pIC19h cells. In addition, transfection of the CHL-3A4 cells with vectors expressing the 3A4III siRNAs almost completely inhibited CYP3A4-mediated nifedipine metabolism. This study demonstrated, for the first time, the specific suppression of CYP3A4 expression and function using vector-based RNAi technique. The use of RNAi is a promising tool for the study of cytochrome P450 family function.

Pharmacokinetic mechanisms for reduced toxicity of irinotecan by coadministered thalidomide.

The clinical use of irinotecan (CPT-11) is hindered by dose-limiting diarrhea and myelosuppression. Recent clinical studies indicate that thalidomide, a known tumor necrosis factor-alpha inhibitor, ameliorated the toxicities induced by CPT-11. However, the mechanisms for this are unknown. This study aimed to investigate whether combination of thalidomide modulated the toxicities of CPT-11 using a rat model and the possible role of the altered pharmacokinetic component in the toxicity modulation using in vitro models. The toxicity model was constructed by treatment of healthy rats with CPT-11 at 60 mg/kg per day by intravenous (i.v.) injection. Body weight, acute and delayed-onset diarrhea, blood cell counts, and macroscopic and microscopic intestinal damages were monitored in rats treated with CPT-11 alone or combined therapy with thalidomide at 100 mg/kg administered by intraperitoneal (i.p.) injection. Single dose and 5-day multiple-dose studies were conducted in rats to examine the effects of concomitant thalidomide on the plasma pharmacokinetics of CPT-11 and its major metabolites SN-38 and SN-38 glucuronide (SN-38G). The effect of CPT-11 on thalidomide's pharmacokinetics was also checked. Rat liver microsomes and a rat hepatoma cell line, H4-II-E cells, were used to study the in vitro metabolic interactions between these two drugs. H4-II-E cells were also used to investigate the effect of thalidomide and its hydrolytic products on the transport of CPT-11 and SN-38. In addition, the effect of thalidomide and its hydrolytic products on rat plasma protein binding of CPT-11 and SN-38 was examined. Administration of CPT-11 by i.v. for 4 consecutive days to rats induced significant body weight loss, decrease in neutrophil and lymphocyte counts, severe acute- and delayed-onset diarrhea, and intestinal damages. These toxicities were alleviated when CPT-11 was combined with thalidomide. In both single-dose and 5-day multiple-dose pharmacokinetic study, coadministered thalidomide significantly increased the area under the plasma concentration-time curve (AUC) of CPT-11, but the AUC and elimination half-life (t(1/2)) of SN-38 were significantly decreased. However, CPT-11 did not significantly alter the pharmacokinetics of thalidomide. Thalidomide at 25 and 250 microM and its hydrolytic products at a total concentration of 10 microM had no significant effect on the plasma protein binding of CPT-11 and SN-38, except for that thalidomide at 250 microM caused a significant increase in the unbound fraction (f(u)) of CPT-11 by 6.7% (P < 0.05). The hydrolytic products of thalidomide (total concentration of 10 microM), but not thalidomide, significantly decreased CPT-11 hydrolysis by 16% in rat liver microsomes (P < 0.01). The formation of both SN-38 and SN-38G from CPT-11, SN-38 glucuronidation, or intracellular accumulation of both CPT-11 and SN-38 in H4-II-E cells followed Michaelis-Menten kinetics with the one-binding site model being the best fit for the kinetic data. Coincubation or 2-hr preincubation of thalidomide at 25 microM and 250 microM and its hydrolytic products at 10 microM did not show any significant effects on CPT-11 hydrolysis and SN-38 glucuronidation. However, preincubation of H4-II-E cells with thalidomide (250 microM), its hydrolytic products (total concentration of 10 microM), or phthaloyl glutamic acid (one major thalidomide hydrolytic product, 10 microM) significantly increased the intracellular accumulation of SN-38, but not CPT-11 (P < 0.01). The dose-limiting toxicities of CPT-11 were alleviated by combination with thalidomide in rats and the pharmacokinetic modulation by thalidomide may partially explain its antagonizing effects on the toxicities of CPT-11. The hydrolytic products of thalidomide, instead of the parental drug, modulated the hepatic hydrolysis of CPT-11 and intracellular accumulation of SN-38, probably contributing to the altered plasma pharmacokinetics of CPT-11 and SN-38. Further studies are needed to explore the role of both pharmacokinetics and pharmacodynamic components in the protective effect of thalidomide against the toxicities of CPT-11.