Cerebrospinal fluid metabolic profiling reveals divergent modulation of pentose phosphate pathway by midazolam, propofol and dexmedetomidine in patients with subarachnoid hemorrhage: a cohort study.

BACKGROUND: Agitation is common in subarachnoid hemorrhage (SAH), and sedation with midazolam, propofol and dexmedetomidine is essential in agitation management. Previous research shows the tendency of dexmedetomidine and propofol in improving long-term outcome of SAH patients, whereas midazolam might be detrimental. Brain metabolism derangement after SAH might be interfered by sedatives. However, how sedatives work and whether the drugs interfere with patient outcome by altering cerebral metabolism is unclear, and the comprehensive view of how sedatives regulate brain metabolism remains to be elucidated. METHODS: For cerebrospinal fluid (CSF) and extracellular space of the brain exchange instantly, we performed a cohort study, applying CSF of SAH patients utilizing different sedatives or no sedation to metabolomics. Baseline CSF metabolome was corrected by selecting patients of the same SAH and agitation severity. CSF components were analyzed to identify the most affected metabolic pathways and sensitive biomarkers of each sedative. Markers might represent the outcome of the patients were also investigated. RESULTS: Pentose phosphate pathway was the most significantly interfered (upregulated) pathway in midazolam (p = 0.0000107, impact = 0.35348) and propofol (p = 0.00000000000746, impact = 0.41604) groups. On the contrary, dexmedetomidine decreased levels of sedoheptulose 7-phosphate (p = 0.002) and NADP (p = 0.024), and NADP is the key metabolite and regulator in pentose phosphate pathway. Midazolam additionally augmented purine synthesis (p = 0.00175, impact = 0.13481) and propofol enhanced pyrimidine synthesis (p = 0.000203, impact = 0.20046), whereas dexmedetomidine weakened pyrimidine synthesis (p = 0.000000000594, impact = 0.24922). Reduced guanosine diphosphate (AUC of ROC 0.857, 95%CI 0.617-1, p = 0.00506) was the significant CSF biomarker for midazolam, and uridine diphosphate glucose (AUC of ROC 0.877, 95%CI 0.631-1, p = 0.00980) for propofol, and succinyl-CoA (AUC of ROC 0.923, 95%CI 0.785-1, p = 0.000810) plus adenosine triphosphate (AUC of ROC 0.908, 95%CI 0.6921, p = 0.00315) for dexmedetomidine. Down-regulated CSF succinyl-CoA was also associated with favorable outcome (AUC of ROC 0.708, 95% CI: 0.524-0.865, p = 0.029333). CONCLUSION: Pentose phosphate pathway was a crucial target for sedatives which alter brain metabolism. Midazolam and propofol enhanced the pentose phosphate pathway and nucleotide synthesis in poor-grade SAH patients, as presented in the CSF. The situation of dexmedetomidine was the opposite. The divergent modulation of cerebral metabolism might further explain sedative pharmacology and how sedatives affect the outcome of SAH patients.

Cystine supplementation rebalances the redox homeostasis of microenvironment in non-small cell lung cancer cells and reverses their resistance to docetaxel.

Continuous docetaxel (DTX) treatment of non-small cell lung cancer induces development of drug resistance, but the mechanism is poorly understood. In this study we performed metabolomics analysis to characterize the metabolic patterns of sensitive and resistant A549 non-small cell lung cancer cells (A549/DTX cells). We showed that the sensitive and resistant A549 cells exhibited distinct metabolic phenotypes: the resistant cells were characterized by an altered microenvironment of redox homeostasis with reduced glutathione and elevated reactive oxygen species (ROS). DTX induction reprogrammed the metabolic phenotype of the sensitive cells, which acquired a phenotype similar to that of the resistant cells: it reduced cystine influx, inhibited glutathione biosynthesis, increased ROS and decreased glutathione/glutathione disulfide (GSH/GSSG); the genes involved in glutathione biosynthesis were dramatically depressed. Addition of the ROS-inducing agent Rosup (25, 50 µg/mL) significantly increased P-glycoprotein expression and reduced intracellular DTX in the sensitive A549 cells, which ultimately acquired a phenotype similar to that of the resistant cells. Supplementation of cystine (1.0 mM) significantly increased GSH synthesis, rebalanced the redox homeostasis of A549/DTX cells, and reversed DTX-induced upregulation of P-glycoprotein, and it markedly improved the effects of DTX and inhibited the growth of A549/DTX in vitro and in vivo. These results suggest that microenvironmental redox homeostasis plays a key role in the acquired resistance of A549 cancer cells to DTX. The enhancement of GSH synthesis by supplementary cystine is a promising strategy to reverse the resistance of tumor cells and has potential for translation in the clinic.

Rebalancing of the gut flora and microbial metabolism is responsible for the anti-arthritis effect of kaempferol.

Kaempferol is a natural flavonol that possesses various pharmacological activities, including anti-arthritis effects, yet the underlying mechanisms remain controversial. To evaluate the anti-arthritis efficacy and the underlying mechanisms of kaempferol, collagen-induced arthritis (CIA) mice were treated with kaempferol intragastrically (200 mg · kg-1 · d-1) and intraperitoneally (20 mg · kg-1 · d-1). Pharmacodynamic and pharmacokinetic studies showed that the oral administration of kaempferol produced distinct anti-arthritis effects in model mice with arthritis in terms of the spleen index, arthritis index, paw thickness, and inflammatory factors; the bioavailability (1.5%, relative to that of the intraperitoneal injection) and circulatory exposure of kaempferol (Cmax = 0.23 ± 0.06 ng/mL) and its primary metabolite kaempferol-3-O-glucuronide (Cmax = 233.29 ± 89.64 ng/mL) were rather low. In contrast, the intraperitoneal injection of kaempferol caused marginal anti-arthritis effects, although it achieved a much higher in vivo exposure. The much higher kaempferol content in the gut implicated a potential mechanism involved in the gut. Analysis of 16S ribosomal RNA revealed that CIA caused imbalance of 14 types of bacteria at the family level, whereas kaempferol largely rebalanced the intestinal microbiota in CIA mice. A metabolomics study showed that kaempferol treatment significantly reversed the perturbation of metabolites involved in energy production and the tryptophan, fatty acid and secondary bile acid metabolisms in the gut contents of the CIA mice. In conclusion, we demonstrate for the first time that the high level of kaempferol in the gut regulates the intestinal flora and microbiotic metabolism, which are potentially responsible for the anti-arthritis activities of kaempferol.

Compound danshen dripping pills normalize a reprogrammed metabolism of myocardial ischemia rats to interpret its time-dependent efficacy in clinic trials: a metabolomic study.

INTRODUCTION: Clinical trials of Compound danshen dripping pills (CDDP) indicated distinct improvement in patients with chronic stable angina. Daily fluctuation of therapeutic effect agreed with a peak-valley PK profile during a 4-week CDDP regimen, but stabilized after 8-week treatment. OBJECTIVES: This article aims to explore the underlying mechanism for the time-dependent drug efficacy of the up-down fluctuation or stabilization in clinic trials. METHODS: A rat model of myocardial ischemia was established via isoproterenol induction. Metabolomics was employed to analyze the energy-related substances both in circulatory system and myocardium in the myocardial ischemia model. RESULTS: CDDP treatment ameliorated myocardial ischemia, reversed the reprogramming of the metabolism induced by ISO and normalized the level of most myocardial substrates and the genes/enzymes associated with those metabolic changes. After 1- or 2-week treatment, CDDP regulated plasma and myocardial metabolome in an analogous, time-dependent way, and modulated metabolic patterns of ischemic rats that perfectly matched with the fluctuated or stabilized effects observed in clinical trials with 4 or 8-week treatment, respectively. CONCLUSION: Metabolic modulation by CDDP contributes to the fluctuated or stabilized therapeutic outcome, and is a potential therapeutic approach for myocardial ischemia diseases.

Paeoniflorin inhibits Th1 and Th17 cells in gut-associated lymphoid tissues to produce anti-arthritis activities.

Paeoniflorin shows distinct anti-arthritis and immunoregulatory activities, but its rather low bioavailability via oral administration greatly challenges its known mechanism of in vivo activity. Our data showed that oral administration, instead of intraperitoneal injection, of paeoniflorin significantly reduced the polyarthritis index by 44.4%, reduced paw swelling by 18.4% and delayed the onset of arthritis in collagen-induced arthritis (CIA) mice. Oral paeoniflorin treatment also downregulated the systemic pro-inflammatory cytokines IL-6 (by 52.2%), TNF-α (by 57.7%) and IL-1ß (by 34.1%). A pharmacokinetic study revealed that the maximal plasma concentration of paeoniflorin after oral administration was 4.8 ± 1.9 µM in the CIA mice, much lower than the effective concentration in vitro (30 µM). In contrast, paeoniflorin was highly concentrated in the gut content, intestine and Peyer's patches. T cell analysis showed that paeoniflorin markedly reduced transcription factors of Th1 and Th17, inhibited Th1 by 22.2% and 23.1% and Th17 by 43.2% and 25.4% (p < 0.05) in the mesenteric lymph node and Peyer's patches, respectively. Paeoniflorin did not have a significant impact on Th1 and Th17 in the spleen. For the first time, these data suggest that paeoniflorin accumulates in the intestine and primarily modulates Th1 and Th17 responses in the mesenteric lymph nodes and Peyer's patches, rather than in the spleen, to exert anti-arthritis effects.

Aneurysmal Subarachnoid Hemorrhage Onset Alters Pyruvate Metabolism in Poor-Grade Patients and Clinical Outcome Depends on More: A Cerebrospinal Fluid Metabolomic Study.

Cerebral metabolism alterations influence cerebrospinal fluid (CSF) composition and are sensitive to brain injury. In subarachnoid hemorrhage (SAH) patients, Fisher scale, Hunt-Hess scale, and World Federation of Neurological Societies (WFNS) grading scale evaluating SAH severity are inadequate to predict long-term outcome; therefore, in an effort to determine metabolite pattern disparity and discover corresponding biomarkers, we designed an untargeted CSF metabolomic study covering a broad range of metabolites of SAH patients with different severity and outcome. The present study demonstrated the SAH altered the cerebrospinal fluid metabolome involving carbohydrate, lipid, and amino acid metabolism. Pyruvate metabolism was enhanced in SAH patients with Hunt-Hess scale above III, and the CSF pyruvate level was significantly associated with WFNS grading scale above III. There is no significant variation among CSF metabolome in SAH patients with merely different amounts and distribution of bleeding. SAH patients with unfavorable outcome present upregulated CSF amino acids level and enhanced lipid biosynthesis. The present study provides a novel possibility of early identification of patients who might possess unfavorable outcome and further clarification of the underlying pathophysiology.

The aldose reductase inhibitor epalrestat exerts nephritic protection on diabetic nephropathy in db/db mice through metabolic modulation.

Epalrestat is an inhibitor of aldose reductase in the polyol pathway and is used for the management of diabetic neuropathy clinically. Our pilot experiments and accumulated evidences showed that epalrestat inhibited polyol pathway and reduced sorbitol production, and suggested the potential renal protection effects of epalrestat on diabetic nephropathy (DN). To evaluate the protective effect of epalrestat, the db/db mice were used and exposed to epalrestat for 8 weeks, both the physiopathological condition and function of kidney were examined. For the first time, we showed that epalrestat markedly reduced albuminuria and alleviated the podocyte foot process fusion and interstitial fibrosis of db/db mice. Metabolomics was employed, and metabolites in the plasma, renal cortex, and urine were profiled using a gas chromatography-mass spectrometry (GC/MS)-based metabolomic platform. We observed an elevation of sorbitol and fructose, and a decrease of myo-inositol in the renal cortex of db/db mice. Epalrestat reversed the renal accumulation of the polyol pathway metabolites of sorbitol and fructose, and increased myo-inositol level. Moreover, the upregulation of aldose reductase, fibronectin, collagen III, and TGF-ß1 in renal cortex of db/db mice was downregulated by epalrestat. The data suggested that epalrestat has protective effects on DN, and the inhibition of aldose reductase and the modulation of polyol pathway in nephritic cells be a potentially therapeutic strategy for DN.

Combined treatment with apatinib and docetaxel in A549 xenograft mice and its cellular pharmacokinetic basis.

Apatinib, a small-molecule inhibitor of VEGFR-2, has attracted much attention due to its encouraging anticancer activity in third-line clinical treatment for many malignancies, including non-small cell lung cancer (NSCLC). Its usage in second-line therapy with chemotherapeutic drugs is still under exploration. In this study we investigated the antitumor effect of apatinib combined with docetaxel against NSCLC and its cellular pharmacokinetic basis. A549 xenograft nude mice were treated with apatinib (100 mg/kg every day for 20 days) combined with docetaxel (8 mg/kg, ip, every four days for 5 times). Apatinib significantly enhanced the antitumor effect of docetaxel and alleviated docetaxel-induced liver damage as well as decreased serum transaminases (ALT and AST). LC-MS/MS analysis revealed that apatinib treatment significantly increased the docetaxel concentration in tumors (up to 1.77 times) without enhancing the docetaxel concentration in the serum, heart, liver, lung and kidney. Furthermore, apatinib decreased docetaxel-induced upregulation of P-glycoprotein in tumors. The effects of apatinib on the uptake, efflux and subcellular distribution of docetaxel were investigated in A549 and A549/DTX (docetaxel-resistant) cells in vitro. A cellular pharmacokinetic study revealed that apatinib significantly increased cellular/subcellular accumulation (especially in the cytosol) and decreased the efflux of docetaxel in A549/DTX cells through P-gp, while apatinib exerted no significant effect on the cellular pharmacokinetics of docetaxel in A549 cells. Consequently, the IC50 value of docetaxel in A549/DTX cells was more significantly decreased by apatinib than that in A549 cells. These results demonstrate that apatinib has potential for application in second-line therapy combined with docetaxel for NSCLC patients, especially for docetaxel-resistant or multidrug-resistant patients.

Pharmacokinetic and metabolomic analyses of the neuroprotective effects of salvianolic acid A in a rat ischemic stroke model.

Salvianolic acid A (SAA), a water-soluble phenolic acid isolated from the root of Dan Shen, displays distinct antioxidant activity and effectiveness in protection against cerebral ischemia/reperfusion (I/R) damage. However, whether SAA can enter the central nervous system and exert its protective effects by directly targeting brain tissue remains unclear. In this study, we evaluated the cerebral protection of SAA in rats subjected to transient middle cerebral artery occlusion (tMCAO) followed by reperfusion. The rats were treated with SAA (5, 10 mg/kg, iv) when the reperfusion was performed. SAA administration significantly decreased cerebral infarct area and the brain water content, attenuated the neurological deficit and pathology, and enhanced the anti-inflammatory and antioxidant capacity in tMCAO rats. The concentration of SAA in the plasma and brain was detected using LC-MS/MS. A pharmacokinetic study revealed that the circulatory system exposure to SAA was equivalent in the sham controls and I/R rats, but the brain exposure to SAA was significantly higher in the I/R rats than in the sham controls (fold change of 9.17), suggesting that the enhanced exposure to SAA contributed to its cerebral protective effect. Using a GC/MS-based metabolomic platform, metabolites in the serum and brain tissue were extracted and profiled. According to the metabolomic pattern of the tissue data, SAA administration significantly modulated the I/R-caused perturbation of metabolism in the brain to a greater extent than that in the serum, demonstrating that SAA worked at the brain tissue level rather than the whole circulation system. In conclusion, a larger amount of SAA enters the central nervous system in ischemia/reperfusion rats to facilitate its protective and regulatory effects on the perturbed metabolism.

In vitro assessment of the glucose-lowering effects of berberrubine-9-O-ß-D-glucuronide, an active metabolite of berberrubine.

Berberrubine (BRB) is the primary metabolite of berberine (BBR) that has shown a stronger glucose-lowering effect than BBR in vivo. On the other hand, BRB is quickly and extensively metabolized into berberrubine-9-O-ß-D-glucuronide (BRBG) in rats after oral administration. In this study we compared the pharmacokinetic properties of BRB and BRBG in rats, and explored the mechanisms underlying their glucose-lowering activities. C57BL/6 mice with HFD-induced hyperglycemia were administered BRB (50 mg·kg-1·d-1, ig) for 6 weeks, which caused greater reduction in the plasma glucose levels than those caused by BBR (120 mg·kg-1·d-1) or BRB (25 mg·kg-1·d-1). In addition, BRB dose-dependently decreased the activity of α-glucosidase in gut of the mice. After oral administration of BRB in rats, the exposures of BRBG in plasma at 3 different dosages (10, 40, 80 mg/kg) and in urine at different time intervals (0-4, 4-10, 10-24 h) were dramatically greater than those of BRB. In order to determine the effectiveness of BRBG in reducing glucose levels, we prepared BRBG from the urine pool of rats, and identified and confirmed it through LC-MS-IT-TOF and NMR spectra. In human normal liver cell line L-O2 in vitro, treatment with BRB or BRBG (5, 20, 50 µmol/L) increased glucose consumption, enhanced glycogenesis, stimulated the uptake of the glucose analog 2-NBDG, and modulated the mRNA levels of glucose-6-phosphatase and hexokinase. However, both BBR and BRB improved 2-NBDG uptake in insulin-resistant L-O2 cells, while BRBG has no effect. In conclusion, BRB exerts a stronger glucose-lowering effect than BBR in HFD-induced hyperglycemia mice. Although BRB significantly stimulated the insulin sensitivity and glycolysis in vitro, BRBG may have a greater contribution to the glucose-lowering effect because it has much greater system exposure than BRB after oral administration of BRB. The results suggest that BRBG is a potential agent for reducing glucose levels.

Isoflavones and phytosterols contained in Xuezhikang capsules modulate cholesterol homeostasis in high-fat diet mice.

AIM: Xuezhikang (XZK), an extract of red yeast rice, has been widely used in traditional Chinese medicine to treat cardiovascular disease. Three fractions F1, F2 and F3 (primarily containing isoflavones, monacolins or phytosterols, respectively) are extracted from Xuezhikang capsules. In this study we evaluated the lipid-lowering effects of these fractions and explored the potential mechanisms of actions. METHODS: Mice treated with a high-fat diet (HFD) were orally administered lovastatin (10 mg·kg(-1)·d(-1)), XZK (1200 mg·kg(-1)·d(-1)), F1 (27.5 mg·kg(-1)·d(-1)), F2 (11.3 mg·kg(-1)·d(-1)) or F3 (35 mg·kg(-1)·d(-1)) for 10 weeks. Lipids were measured using commercial enzymatic kits, and the mRNA and protein levels of genes involved in cholesterol and bile acid homeostasis were evaluated using qRT-PCR and Western blot analysis, respectively. RESULTS: XZK increased the fecal excretion of lipids and bile acids, reduced serum TC, TG and LDL-C levels by 40%, 55% and 46%, respectively, and increased serum HDL-C by 31%. Administration of F1 repressed serum TC and TG by 24% and 52%, respectively, and elevated hepatic synthesis of CYP7A1. It also increased hepatic elimination of bile acids in the fecal excretions by 79% through upregulating BSEP and downregulating NTCP. Administration of F3 decreased serum TC, TG and LDL-C levels by 33%, 29% and 39%, respectively, and increased serum HDL-C by 28%, significantly reduced intestinal absorption of cholesterol by inhibiting the transcription of NPC1L1, and elevated excretion of TC, FC and CE by 96%, 72% and 101%, respectively. Administration of F2 showed pharmacological effects similar to those of lovastatin. CONCLUSION: Isoflavones and phytosterols in XZK exert cholesterol-lowering effects in HFD mice through mechanisms that differ from those of lovastatin. Isoflavones and phytosterols act in a complimentary manner: through enhancing the elimination of bile acids and reducing intestinal cholesterol absorption, respectively.

[Metabolomic approach to evaluating the effect of the mixed decoction of kelp and licorice on system metabolism of SD rats].

The aim of the study is to evaluate the effects of the single and mixed decoction of Thallus laminariae (kelp) and Glycyrrhiza glabra (licorice) on the metabolism and their difference. The mixed decoction of kelp and licorice and the single decoction were made and intragastrically administered to the SD rats. The effect on system metabolism, the toxicity of liver and kidney were assessed by GC-MS profiling of the endogenous molecules in serum, routine biochemical assays and histographic inspection of tissues from SD rats, separately. The mixed decoction of kelp and licorice induced more obvious pathological abnormalities in SD rats than a single decoction of kelp, while the extracts of licorice did not show any pathological change. Neither the mixed, nor the single decoction showed abnormal histopathology. After intragastric administration of extracts for 5 days, the mixed decoction induced a decrease of ALT (no significant change in the groups of single decoction) and an increase of BUN (so did the single decoction of kelp). Metabolomic profile of the molecules in serum revealed that the metabolic patterns were all obviously affected for the three groups, i.e., the mixed and single decoction of kelp and licorice. The rats given with the single decoction of kelp showed a similar pattern to that of the mixed decoction, indicating that the kelp primarily contributed the perturbation of metabolism for the mixed decoction. All three groups induced a decrease of branched chain amino acids, TCA cycle intermediates and glycolysis intermediates (e.g., pyruvic acid and lactic acid) and an increase of 3-hydroxybutyric acid. Kelp decoction showed stronger potential in reducing TCA cycle intermediates and glycolysis intermediates than the other two groups, while the levels of branched chain amino acids were the lowest after licorice extracts were given. These results suggested that the effect of the mixed decoction on metabolism was closely associated with both kelp and licorice. The continuous administration of single decoction of kelp and the mixed decoction of licorice and kelp resulted in pathological abnormalities in kidney of SD rats. The mixed decoction of kelp and licorice distinctly perturbed sera molecules and hence system metabolism, which showed associated with those of kelp and licorice. Although the metabolic effect was associated with both kelp and licorice, the results suggested kelp contributed to it primarily.

Modulation of the pentose phosphate pathway alters phase I metabolism of testosterone and dextromethorphan in HepG2 cells.

AIM: The pentose phosphate pathway (PPP) is involved in the activity of glucose-6-phosphate dehydrogenase (G6PD) and generation of NADPH, which plays a key role in drug metabolism. The aim of this study was to investigate the effects of modulation of the PPP on drug metabolism capacity in vitro. METHODS: A pair of hepatic cell lines, ie, the cancerous HepG2 cells and normal L02 cells, was used. The expression of CYP450 enzymes, p53 and G6PD in the cells were analyzed. The metabolism of testosterone (TEST, 10 µmol/L) and dextromethorphan (DEM, 1 µmol/L), the two typical substrates for CYP3A4 and CYP2D6, in the cells was examined in the presence of different agents. RESULTS: Both the expression and metabolic activities of CYP3A4 and CYP2D6 were considerably higher in HepG2 cells than in L02 cells. The metabolism of TEST and DEM in HepG2 cells was dose-dependently inhibited by the specific CYP3A4 inhibitor ketoconazole and CYP2D6 inhibitor quinidine. Addition of the p53 inhibitor cyclic PFT-α (5, 25 µmol/L) in HepG2 cells dose-dependently enhanced the metabolism of DEM and TEST, whereas addition of the p53 activator NSC 66811 (3, 10, 25 µmol/L) dose-dependently inhibited the metabolism. Furthermore, addition of the G6PD inhibitor 6-aminonicotinamide (5, 15 µmol/L) in HepG2 cells dose-dependently inhibited the metabolism of DEM and TEST, whereas addition of the PPP activity stimulator menadione (1, 5, 15 µmol/L) dose-dependently enhanced the metabolism. CONCLUSION: Modulation of p53 and the PPP alters the metabolism of DEM and TEST, suggesting that the metabolic flux pattern of PPP may be closely involved in drug metabolism and the individual variance.

Pharmacokinetic interactions between 20(S)-ginsenoside Rh2 and the HIV protease inhibitor ritonavir in vitro and in vivo.

AIM: 20(S)-Ginsenoside Rh2 (Rh2) has shown potent inhibition on P-glycoprotein (P-gp), while most HIV protease inhibitors are both substrates and inhibitors of P-gp and CYP3A4. The aim of this study was to investigate the potential pharmacokinetic interactions between Rh2 and the HIV protease inhibitor ritonavir. METHODS: The effects of Rh2 on the cellular accumulation and transepithelial transport of ritonavir were studied in Caco-2 and MDCK-MDR1 cells. Male rats were administered Rh2 (25 or 60 mg/kg, po) or Rh2 (5 mg/kg, iv), followed by ritonavir (25 mg/kg, po). The P-gp inhibitors verapamil (20 mg/kg, po) or GF120918 (5 mg/kg, po) were used as positive controls. The concentrations of ritonavir in plasma, bile, urine, feces and tissue homogenates were analyzed using LC-MS. RESULTS: Rh2 (10 µmol/L) significantly increased the accumulation and inhibited the efflux of ritonavir in Caco-2 and MDCK-MDR1 cells, as verapamil did. But Rh2 did not significantly alter ritonavir accumulation or transport in MDCK-WT cells. Intravenous Rh2 significantly increased the plasma exposure of ritonavir while reducing its excretion in the bile, and oral verapamil or GF120918 also increased plasma exposure of ritonavir but without changing its excretion in the bile. Interestingly, oral Rh2 at both doses did not significantly change the plasma profile of ritonavir. Moreover, oral Rh2 (25 mg/kg) significantly elevated the ritonavir concentration in the hepatic portal vein, and markedly increased its urinary excretion and tissue distribution, which might counteract the elevated absorption of ritonavir. CONCLUSION: Rh2 inhibits the efflux of ritonavir through P-gp in vitro. The effects of Rh2 on ritonavir exposure in vivo depend on the administration route of Rh2: intravenous, but not oral, administration of Rh2 significantly increased the plasma exposure of ritonavir.