Patient experiences of implantable cardiac monitoring in hypertrophic cardiomyopathy: an exploratory study.

AIMS: Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease. Insertable cardiac monitors (ICMs) are increasingly used in this population to provide closer monitoring, with the potential for notification systems. However, little is known regarding the psychological impact this information may have on patients. The Abbott Confirm Rx™ ICM has the capability of connecting to the patient's smartphone to enable active participation in their care, as well as two-way communication between the patient and their care providers. This study aimed to explore individuals' experiences of having a smartphone-enabled ICM to monitor for arrhythmias in HCM. METHODS AND RESULTS: Semi-structured interviews were conducted with 10 participants. Utilizing a grounded theory approach, the interview guide was modified based on emerging themes throughout the study. Reflexive thematic analysis was applied to categorize interview data into codes and overacting themes, with each interview independently coded by two study members. Analysis revealed three key themes: (i) psychological impact, (ii) educational needs, and (iii) technology expectations. Participants reported that receiving feedback from ICM transmissions resulted in improved symptom clarity, providing reassurance, and aiding implantable cardioverter defibrillator decision-making. Some participants reported uncertainty regarding when to send manual transmissions. Lastly, participants reported the app interface did not meet expectations with regard to the amount of data available for patients. CONCLUSION: Overall, utilizing a smartphone app to facilitate two-way communication of ICM transmissions was well accepted. Future directions include addressing gaps in educational needs and improvements in the patient interface with increased access to data.

Bluetooth-Enabled Implantable Cardiac Monitors and Two-Way Smartphone Communication for Patients With Hypertrophic Cardiomyopathy.

Background: Sudden cardiac death (SCD) risk stratification in hypertrophic cardiomyopathy (HCM) currently relies on arrhythmic burden quantification by 24 or 48-hour Holter monitoring. Whether this approach adequately captures arrhythmic burden, compared with longer-term continuous monitoring, is unclear. We sought to assess the long-term incidence of nonsustained ventricular tachycardia (NSVT) in HCM patients at low or moderate SCD risk, using implantable cardiac monitors (ICMs) paired with a novel Bluetooth-enabled 2-way communication platform. Methods: This prospective, single-arm, observational study enrolled 33 HCM patients. Patients were implanted with an Abbott (Chicago, IL) Confirm Rx ICM and monitored using a protocolized care pathway. Results: A total of 20 patients (60.6%) had ≥ 1 episode of NSVT recorded on the ICM, the majority of whom had previous Holter monitors that did not identify NSVT (60%, n = 12). A total of 71 episodes of NSVT were detected. Median time to first NSVT detection was 76.5 days (range: 0-553 days). A total of 19 patients underwent primary prevention implantable cardioverter defibrillator implantation during an average follow-up of 544 days (range: 42-925 days). A total of 172,112 automatic transmissions were received, and 65 (0.04%) required clinical follow-up. A total of 325 manual transmissions were received and managed. A total of 14 manual transmissions (4.3%) required follow-up, whereas 311 (95.7%) were managed solely with a text message. Conclusions: Surveillance and reporting systems utilizing 2-way communication enabled by novel ICMs are feasible and allow remote management of patients with HCM. Prolonged monitoring with ICMs identified more patients with nonsustained arrythmias than did standard Holter monitoring. In many cases, this information impacted both SCD risk stratification and patient management.

Contexte: La stratification du risque de mort cardiaque subite (MCS) dans la cardiomyopathie hypertrophique (CMH) dépend actuellement de la quantification de la charge arythmique par une surveillance Holter de 24 ou 48 heures. Il n'est pas clair si cette approche permet d'évaluer adéquatement la charge arythmique, comparativement à une surveillance continue à plus long terme. Nous avons cherché à évaluer la fréquence à long terme de la tachycardie ventriculaire non soutenue (TVNS) chez des patients atteints de CMH à risque faible ou modéré de MCS, au moyen de moniteurs cardiaques implantables (MCI) couplés à une nouvelle plate-forme de communication bidirectionnelle utilisable avec Bluetooth. Méthodologie: Cette étude par observation prospective comportant un seul groupe a été menée auprès de 33 patients atteints de CMH. Les patients ont reçu un MCI Confirm Rx d'Abbott (Chicago, États-Unis) et ont été surveillés dans le cadre d'un parcours de soins reposant sur un protocole. Résultats: Au total, 20 patients (60,6 %) ont eu au moins un épisode de TVNS enregistré par le MCI. La majorité de ces patients portaient déjà un moniteur Holter qui n'a pas décelé de TVNS (60 %, n = 12). Au total, 71 épisodes de TVNS ont été détectés. Le temps médian écoulé avant la première détection de TVNS était de 76,5 jours (fourchette : 0-553 jours). Au total, 19 patients se sont fait poser un défibrillateur cardioverteur implantable en prévention primaire pendant un suivi moyen de 544 jours (fourchette : 42-925 jours). En tout, 172 112 transmissions automatiques ont été reçues, et 65 (0,04 %) ont nécessité un suivi clinique. Par ailleurs, 325 transmissions manuelles ont été reçues et traitées. De ce nombre, 14 transmissions (4,3 %) ont nécessité un suivi, tandis que 311 (95,7 %) ont été traitées uniquement au moyen d'un message texte. Conclusions: Les systèmes de surveillance et de signalement utilisant une communication bidirectionnelle rendue possible grâce aux nouveaux MCI sont réalisables et permettent une prise en charge à distance des patients atteints d'un CMH. La surveillance prolongée par un MCI a permis de déceler plus d'arythmies non soutenues que la surveillance Holter type. Dans de nombreux cas, ces renseignements ont eu un effet positif tant sur la stratification du risque de MCS que sur la prise en charge des patients.

Management of traumatic haemorrhage in the Berger's space of a 4-year-old child.

We are reporting an unusual case of haemorrhage in the Berger's space after an episode of blunt ocular trauma in an eye of a 4-year-old boy, who enjoyed premorbid normal vision. A secondary posterior subcapsular cataract developed as a complication of the haemorrhage after 6 months of observation. Surgery comprised cataract extraction, removal of a residual retrolenticular haematoma via opening of posterior continuous curvilinear capsulorhexis (CCC), anterior vitrectomy and placement of an intraocular lens. This yielded a reasonable visual outcome. Complete spontaneous resolution of haemorrhage in the Berger's space is unlikely and may cause secondary cataracts in children. We suggest early intervention in such conditions in order to prevent the development of amblyopia.

Successful Treatment of Lasiodiplodia theobromae Keratitis - Assessing the Role of Voriconazole.

PURPOSE: The aim of this paper is to report the successful management of the first case of Lasiodiplodia theobromae keratitis in Hong Kong. METHODS: We conducted a case report. RESULTS: A 43-year-old Chinese male with a history of diabetes developed left eye keratitis after a trauma during tree felling. Fungal keratitis was diagnosed using a confocal microscope on day 1, and L. theobromae was confirmed from the culture. He was given oral voriconazole, topical natamycin, and topical and intracameral amphotericin B. The patient's condition improved after the initial treatment. However, there was a slow progression to descemetocele formation and impending perforation due to corneal melting. Penetrating keratoplasty was performed at 8 weeks after presentation. Final visual recovery was good with no recurrence of infection. The cornea remained clear. CONCLUSIONS: We report the first case of L. theobromae keratitis in Hong Kong, and it is the only case so far that involved the use of oral voriconazole in the combination therapy. Early recognition with the aid of confocal microscopy allowed the early start of treatment. The use of newer antifungal voriconazole topically and orally combined with topical amphotericin B appeared to be useful in the eradication of the fungus and prevention of recurrence. Intracameral antifungals might have improved the clinical management.

Chemical and pharmacological evaluations on the extract of Scutellaria baicalensis Georgi (Huang-Qin) prepared by various extraction methods.

BACKGROUND: This study reported a comprehensive approach (comparing the extraction yields, chemical profiles, antioxidant properties and CYP450-inhibitory effects) to evaluated the effectiveness of various extraction methods [microwave-assisted extraction using water (MAE-W), heat reflux extraction using water (HRE-W), ultrasonic extraction using 70 % ethanol and ultrasonic extraction using ethanol (UE-E)] for Huang-Qin (HQ), the dried root of Scutellaria baicalensis Georgi. RESULTS: The HQ extraction efficiency by MAE-W was the best. The chemical profiles of extracts obtained using HRE-W and MAE-W were similar; whereas more flavones but less flavone glycosides were detected in the UE-E extract. There was no difference in the antioxidant properties among different extracts. In vitro human liver microsome assays illustrated that all extracts possessed herb-drug interaction potentials but the UE-E extract are shown with a potent interaction with CYP3A4-metabolized drugs. CONCLUSION: MAE-W is a favorable method for the preparation of HQ extracts based on extraction yield, pharmacological properties and safety.

Interaction effects on cytochrome P450 both in vitro and in vivo studies by two major bioactive xanthones from Halenia elliptica D. Don.

The major components, 1-hydroxy-2,3,5-trimethoxy-xanthone (HM-1) and 1,5-dihydroxy-2,3-dimethoxy-xanthone (HM-5) isolated from Halenia elliptica D. Don (Gentianaceae), could cause vasodilatation in rat coronary artery with different mechanisms. In this work, high-performance liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LCMS-IT-TOF) was used to clarify the metabolic pathways, and CYP450 isoform involvement of HM-1 and HM-5 were also studied in rat. At the same time, in vivo inhibition effects of HM-1 and ethyl acetate extracts from origin herb were studied. Three metabolites of HM-5 were found in rat liver microsomes (RLMs); demethylation and hydroxylation were the major phase I metabolic reactions for HM-5. Multiple CYP450s were involved in metabolism of HM-1 and HM-5. The inhibition study showed that HM-5 inhibited Cyp1a2, 2c6 and 2d2 in RLMs. HM-1 inhibited activities of Cyp1a2, Cyp2c6 and Cyp3a2. In vivo experiment demonstrated that both HM-1 and ethyl acetate extracts could inhibit Cyp3a2 in rats. In conclusion, the metabolism of xanthones from the origin herb involved multiple CYP450 isoforms; in vitro, metabolism of HM-5 was similar to that of its parent drug HM-1, but their inhibition effects upon CYP450s were different; in vivo, Cyp3a2 could be inhibited by HM-1 and ethyl acetate extracts.

Miltirone Is a Dual Inhibitor of P-Glycoprotein and Cell Growth in Doxorubicin-Resistant HepG2 Cells.

Miltirone (1), an abietane-type diterpene quinone isolated from Salvia miltiorrhiza, possesses anticancer activity in p-glycoprotein (P-gp)-overexpressing human cancer cells. Results of the current study suggest a dual effect of miltirone on P-gp inhibition and apoptotic induction in a human hepatoma HepG2 cell line and its P-gp-overexpressing doxorubicin-resistant counterpart (R-HepG2). Miltirone (1) elicited a concentration-dependent cytotoxicity, with a similar potency (EC50 ≈ 7-12 µM), in HepG2 and R-HepG2 cells. Miltirone (1) (1.56-6.25 µM) produced synergistic effects on doxorubicin (DOX)-induced growth inhibition of R-HepG2 (synergism: 0.3 < combination index < 0.5). Molecular docking studies illustrated that miltirone (1) interacted with the active site of P-gp with a higher binding affinity than DOX, suggesting that it was a P-gp inhibitor. Flow cytometric analysis confirmed miltirone (1) as a competitive inhibitor of P-gp. At non-necrotic concentrations (1.56-25 µM), miltirone (1) activated caspase-dependent apoptotic pathways and triggered the generation of reactive oxygen species (ROS) and ROS-mediated mitogen-activated protein kinase (MAPK) signaling pathways (e.g., p38 MAPK, stress-activated protein kinase/c-Jun N-terminal kinase, and extracellular regulated kinase 1/2) in both HepG2 and R-HepG2 cells. Thus, we conclude that miltirone (1) is a dual inhibitor of P-gp and cell growth in human drug-resistant hepatoma cells.

Danshen (Salvia miltiorrhiza) water extract inhibits paracetamol-induced toxicity in primary rat hepatocytes via reducing CYP2E1 activity and oxidative stress.

OBJECTIVES: This study aimed to investigate the protective effects of Danshen (Salvia miltiorrhiza) water extract (DSE) and its major phenolic acid components against CYP2E1-mediated paracetamol (APAP)-induced hepatic toxicity. METHODS: The protection and underlying mechanisms were detected in CYP2E1 overexpression primary rat hepatocytes by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, alamar blue assay, CYP2E1 inhibition assay and glutathione assay. KEY FINDINGS: After APAP treatment, DSE (0.06-1 mg/ml) significantly increased cell viability in MTT assay. Two major components danshensu (8.2-130.5 µm) and salvianolic acid B (Sal B; 3.3-53.5 µm) mainly contributed to this protection, but rosmarinic acid, protocatechuic aldehyde and Sal A did not. Alamar blue assay showed that DSE, danshensu and Sal B maintained mitochondrial metabolic activity. DSE inhibited CYP2E1 (Ki = 1.46 mg/ml) in a mixed mode in rat liver microsomes in vitro; DSE decreased APAP-induced total glutathione depletion and preserved redox status (GSH/GSSG ratio) in hepatocytes. Danshensu and Sal B did not inhibit CYP2E1 or decrease total glutathione depletion, but preserved redox status. CONCLUSIONS: DSE protected hepatocytes against APAP-induced injury via maintenance of mitochondrial metabolic activity, CYP2E1 inhibition, reduction of total glutathione depletion and preservation of redox status. Danshensu and Sal B were mainly responsible for this protection.

Effects of tanshinones from Salvia miltiorrhiza on CYP2C19 activity in human liver microsomes: enzyme kinetic and molecular docking studies.

OBJECTIVES: This study aimed to investigate the effects of five tanshinones, the lipophilic components from Danshen (Salvia miltiorrhiza), on CYP2C19 activity in pooled human liver microsomes (HLMs). METHODS: The effects of tanshinones on CYP2C19 activity were compared by enzyme inhibition study using omeprazole 5-hydroxylation in pooled HLMs. The inhibition constant (Ki) values and inhibition modes of effective tanshinones were evaluated by enzyme kinetic study. Molecular docking analysis was used to simulate the binding conformations of tanshinones to the active cavity of human CYP2C19. RESULTS: Dihydrotanshinone and miltirone showed potent inhibitory effects on CYP2C19 activity in a concentration-dependent manner. Tanshinone I showed weaker inhibitory effect, whereas tanshinone IIA and cryptotanshinone had no inhibitory effect. Further enzyme kinetic study showed that the inhibition by dihydrotanshinone and miltirone was a mixed type. The effects of tanshinones were also confirmed by a molecular docking study. Besides, the ethanol extract of Danshen also showed a mixed type of inhibition, whereas the water extract had no inhibitory effect. CONCLUSIONS: The current findings demonstrate the inhibition of CYP2C19 activity by the ethanol extract of Danshen and its components tanshinones, implicating the potential herb-drug interactions between Danshen and therapeutic agents metabolized by CYP2C19 in clinical practice.

Reversal of P-glycoprotein (P-gp) mediated multidrug resistance in colon cancer cells by cryptotanshinone and dihydrotanshinone of Salvia miltiorrhiza.

OBJECTIVE: Multidrug resistance (MDR) of cancer cells to a broad spectrum of anticancer drugs is an obstacle to successful chemotherapy. Overexpression of P-glycoprotein (P-gp), an ATP-binding cassette (ABC) membrane transporter, can mediate the efflux of cytotoxic drugs out of cancer cells, leading to MDR and chemotherapy failure. Thus, development of safe and effective P-gp inhibitors plays an important role in circumvention of MDR. This study investigated the reversal of P-gp mediated multidrug resistance in colon cancer cells by five tanshinones including tanshinone I, tanshinone IIA, cryptotanshinone, dihydrotanshinone and miltirone isolated from Salvia miltiorrhiza (Danshen), known to be safe in traditional Chinese medicine. METHODS: The inhibitory effects of tanshinones on P-gp function were compared using digoxin bi-directional transport assay in Caco-2 cells. The potentiation of cytotoxicity of anticancer drugs by effective tanshinones were evaluated by MTT assay. Doxorubicin efflux assay by flow cytometry, P-gp protein expression by western blot analysis, immunofluorescence for P-gp by confocal microscopy, quantitative real-time PCR and P-gp ATPase activity assay were used to study the possible underlying mechanisms of action of effective tanshinones. RESULTS: Bi-directional transport assay showed that only cryptotanshinone and dihydrotanshinone decreased digoxin efflux ratio in a concentration-dependent manner, indicating their inhibitory effects on P-gp function; whereas, tanshinone I, tanshinone IIA and miltirone had no inhibitory effects. Moreover, both cryptotanshinone and dihydrotanshinone could potentiate the cytotoxicity of doxorubicin and irinotecan in P-gp overexpressing SW620 Ad300 colon cancer cells. Results from mechanistic studies revealed that these two tanshinones increased intracellular accumulation of the P-gp substrate anticancer drugs, presumably by down-regulating P-gp mRNA and protein levels, and inhibiting P-gp ATPase activity. CONCLUSIONS: Taken together, these findings suggest that cryptotanshinone and dihydrotanshinone could be further developed for sensitizing resistant cancer cells and used as an adjuvant therapy together with anticancer drugs to improve their therapeutic efficacies for colon cancer.

Metabolite profiling analysis of FR429, an ellagitannin purified from Polygonum capitatum, in rat and human liver microsomes, cytosol and rat primary hepatocytes in vitro.

FR429, an ellagitannin (a type of polyphenol), is isolated and purified from Polygonum capitatum Buch.-Ham.ex D. Don which is the original herbal medicine of the "Re-Lin-Qing" formula used clinically to treat urinary tract infection in China. FR429 has been investigated for its antitumor potential in tumor-bearing nude mice in vivo, but its in vitro anti-tumor effect in hepatoma cell lines was low. Thus, it was of our interest to investigate its metabolism pathways for supporting its in vivo antitumor potential. The metabolic profiles of FR429 were studied in vitro by liquid chromatography coupled to ion trap time-of-flight mass spectrometry. Total eight metabolites were identified in rat and human liver microsomes, cytosol, and rat primary hepatocytes in vitro. Ellagic acid, a reported anti-angiogenic agent, was one of the main metabolites in these biological matrices. Methylated metabolites catalyzed by catechol-O-methyl transferase (COMT) were observed mainly in the in vitro incubation with rat liver cytosol, which was verified by using a COMT specific inhibitor entacapone and supported by molecular docking analysis. Methylated and sulfated metabolites were also found in rat primary hepatocytes in a time-dependent manner. In conclusion, the in vitro metabolism pathways of FR429 were hydrolysis, methylation and sulfation. The anti-tumor effects of its major metabolites should be further studied.

Evaluation and SAR analysis of the cytotoxicity of tanshinones in colon cancer cells.

AIM: This study was designed to evaluate the anti-cancer actions of tanshinone I and tanshinone IIA, and six derivatives of tanshinone IIA on normal and cancerous colon cells. Structure activity relationship (SAR) analysis was conducted to delineate the significance of the structural modifications of tanshinones for improved anti-cancer action. METHOD: Tanshinone derivatives were designed and synthesized according to the literature. The cytotoxicity of different compounds on colon cancer cells was determined by the MTT assay. Apoptotic activity of the tanshinones was measured by flow cytometry (FCM). RESULTS: Tanshinone I and tanshinone IIA both exhibited significant cytotoxicity on colon cancer cells. They are more effective in p53(+/+) colon cancer cell line. It was also noted that the anti-cancer activity of tanshinone I was more potent and selective. Two of the derivatives of tanshinone IIA (N1 and N2) also exhibited cytotoxicity on colon cancer cells. CONCLUSION: The anti-colon cancer activity of tanshinone I was more potent and selective than tanshinone IIA, and is p53 dependent. The derivatives obtained by structural modifications of tanshinone IIA exhibited lower cytotoxicity on both normal and colon cancer cells. From steric and electronic characteristics point of view, it was concluded that structural modifications of ring A and furan or dihydrofuran ring D on the basic structure of tanshinones influences the activity. An increase of the delocalization of the A and B rings could enhance the cytotoxicity of such compounds, while a non-planar and small sized D ring region would provide improved anti-cancer activity.

In vitro identification of cytochrome P450 isoforms responsible for the metabolism of 1-hydroxyl-2,3,5-trimethoxy-xanthone purified from Halenia elliptica D. Don.

1-Hydroxyl-2,3,5-trimethoxyxanthone (HM-1) is one of the main constituents extracted from Halenia elliptica D. Don, which is a traditionally used Tibetan medicinal plant. The aim of this study was to illustrate the proposed metabolic pathways of HM-1 and identify which cytochrome P450 (CYP450) isoforms involved in its metabolism by using pooled human liver microsomes (HLMs) and recombinant CYP450 isoforms with selective chemical inhibitors. Metabolites were identified by high performance liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LCMS(n)-ESI-IT-TOF) and nuclear magnetic resonance spectroscopy (hydrogen-1 NMR and carbon-13 NMR). Three metabolites (M1-M3) were identified, which demonstrated that demethylation and hydroxylation were the major Phase I metabolic reactions for HM-1 in HLMs. The structure of another metabolite (M4) was still unclear. The enzymatic kinetics of M1 (K(m)=23.19±14.20 µM) and M2 (Km=32.06±17.09 µM) exhibited substrate inhibition; whereas, the formation of M3 (K(m)=5.73±0.70 µM) and M4 (K(m)=16.43±5.12 µM) displayed Michaelis-Menten kinetics. The intrinsic clearance (V(max)/K(m)) of M3 was highest among these metabolites, suggesting that M3 was the major metabolite of HM-1. Moreover, CYP3A4 and CYP2C8 were the primary CYP450 isoform responsible for the metabolism of HM-1. CYP1A2, CYP2A6, CYP2B6, CYP2C9 and CYP2C19 were also involved in HM-1 metabolism, especially in the formation of M3. This study finally provides evidence of substrate inhibition and metabolism-based drug-drug interaction for the medicinal preparations containing HM-1 used in clinic.

Acute simvastatin inhibits K ATP channels of porcine coronary artery myocytes.

BACKGROUND: Statins (3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors) consumption provides beneficial effects on cardiovascular systems. However, effects of statins on vascular KATP channel gatings are unknown. METHODS: Pig left anterior descending coronary artery and human left internal mammary artery were isolated and endothelium-denuded for tension measurements and Western immunoblots. Enzymatically-dissociated/cultured arterial myocytes were used for patch-clamp electrophysiological studies and for [Ca(2+)]i, [ATP]i and [glucose]o uptake measurements. RESULTS: The cromakalim (10 nM to 10 µM)- and pinacidil (10 nM to 10 µM)-induced concentration-dependent relaxation of porcine coronary artery was inhibited by simvastatin (3 and 10 µM). Simvastatin (1, 3 and 10 µM) suppressed (in okadaic acid (10 nM)-sensitive manner) cromakalim (10 µM)- and pinacidil (10 µM)-mediated opening of whole-cell KATP channels of arterial myocytes. Simvastatin (10 µM) and AICAR (1 mM) elicited a time-dependent, compound C (1 µM)-sensitive [(3)H]-2-deoxy-glucose uptake and an increase in [ATP]i levels. A time (2-30 min)- and concentration (0.1-10 µM)-dependent increase by simvastatin of p-AMPKα-Thr(172) and p-PP2A-Tyr(307) expression was observed. The enhanced p-AMPKα-Thr(172) expression was inhibited by compound C, ryanodine (100 µM) and KN93 (10 µM). Simvastatin-induced p-PP2A-Tyr(307) expression was suppressed by okadaic acid, compound C, ryanodine, KN93, phloridzin (1 mM), ouabain (10 µM), and in [glucose]o-free or [Na(+)]o-free conditions. CONCLUSIONS: Simvastatin causes ryanodine-sensitive Ca(2+) release which is important for AMPKα-Thr(172) phosphorylation via Ca(2+)/CaMK II. AMPKα-Thr(172) phosphorylation causes [glucose]o uptake (and an [ATP]i increase), closure of KATP channels, and phosphorylation of AMPKα-Thr(172) and PP2A-Tyr(307) resulted. Phosphorylation of PP2A-Tyr(307) occurs at a site downstream of AMPKα-Thr(172) phosphorylation.

Enzyme kinetic and molecular docking studies for the inhibitions of miltirone on major human cytochrome P450 isozymes.

Previous studies have shown that major tanshinones isolated from Danshen (Salvia miltiorrhiza) inhibited human and rat CYP450 enzymes-mediated metabolism of model probe substrates, with potential in causing herb-drug interactions. Miltirone, another abietane type-diterpene quinone isolated from Danshen, has been reported for its anti-oxidative, anxiolytic and anti-cancer effects. The aim of this study was to study the effect of miltirone on the metabolism of model probe substrates of CYP1A2, 2C9, 2D6 and 3A4 in pooled human liver microsomes. Miltirone showed moderate inhibition on CYP1A2 (IC(50)=1.73 µM) and CYP2C9 (IC(50)=8.61 µM), and weak inhibition on CYP2D6 (IC(50)=30.20 µM) and CYP3A4 (IC(50)=33.88 µM). Enzyme kinetic studies showed that miltirone competitively inhibited CYP2C9 (K(i)=1.48 µM), and displayed mixed type inhibitions on CYP1A2, CYP2D6 and CYP3A4 with K(i) values of 3.17 µM, 24.25 µM and 35.09 µM, respectively. Molecular docking study further confirmed the ligand-binding conformations of miltirone in the active sites of these human CYP450 isoforms, and provided some information on structure-activity relationships for the CYPs inhibition by tanshinones. Taken together, CYPs inhibitions of miltirone were weaker than dihydrotanshinone, but stronger than cryptotanshinone, tanshinone I and tanshinone IIA.

Increased glutathione and mitogen-activated protein kinase phosphorylation are involved in the induction of doxorubicin resistance in hepatocellular carcinoma cells.

AIM: The human hepatocellular carcinoma (HCC) cell line HepG2 can easily acquire resistance to doxorubicin. However, the mechanism of action is unclear. METHODS: In the present study, we used confocal microscopy, flow cytometry and other methods to reveal the mechanisms by which HepG2 cells acquire doxorubicin resistance. RESULTS: Our results showed that R-HepG2 cells, a doxorubicin-resistant sub-line of HepG2, exhibited decreased intracellular accumulation of doxorubicin and increased expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 when compared with HepG2 cells. R-HepG2 cells also harbored higher levels of glutathione and increased expression of glutathione peroxidase. Furthermore, we demonstrated that the phosphorylation of mitogen-activated protein kinases (p38 and c-jun-N-terminal kinases), IkBα and CREB were increased in R-HepG2 cells. Specific p38 inhibitor SB203580 decreased P-gp expression. The multi-kinase inhibitor sorafenib tosylate also significantly suppressed the phosphorylation of these proteins and inhibited the expression of P-gp. CONCLUSION: These findings reveal that the drug resistance could be acquired through mitogen-activated protein kinase-dependent upregulation of P-gp. This mechanism protects R-HepG2 cells from the anticancer action of doxorubicin.

Metabolic studies of 1-testosterone in horses.

1-Testosterone (17ß-hydroxy-5α-androst-1-en-3-one), a synthetic anabolic steroid, has been described as one of the most effective muscle-building supplements currently on the market. It has an anabolic potency of 200 as compared to 26 for testosterone. Apart from its abuse in human sports, it can also be a doping agent in racehorses. Metabolic studies on 1-testosterone have only been reported for human in the early seventies, whereas little is known about its metabolic fate in horses. This paper describes the studies of in vitro and in vivo metabolism of 1-testosterone in horses, with the aim of identifying the most appropriate target metabolites to be monitored for controlling the misuse or abuse of 1-testosterone in racehorses. Six in vitro metabolites, namely 5α-androst-1-ene-3α,17ß-diol (T1a), 5α-androstane-3ß,17ß-diol (T2), epiandrosterone (T3), 16,17-dihydroxy-5α-androst-1-ene-3-one (T4 & T5), and 5α-androst-1-ene-3,17-dione (T6), were identified. For the in vivo studies, two thoroughbred geldings were each administered orally with 800 mg of 1-testosterone by stomach tubing. The results revealed that the parent drug and eight metabolites were detected in urine. Besides the four in vitro metabolites (T1a, T2, T3, and T5), four other urinary metabolites, namely 5α-androst-1-ene-3ß,17α-diol (T1b), 5α-androst-1-ene-3ß,17ß-diol (T1c), 5α-androstane-3α,17α-diol (T7) and 5α-androstane-3ß,17α-diol (T8) were identified. This study shows that the detection of 1-testosterone administration is best achieved by monitoring the parent drug, which could be detected for up to 30 h post-administration.

Danshensu is the major marker for the antioxidant and vasorelaxation effects of Danshen (Salvia miltiorrhiza) water-extracts produced by different heat water-extractions.

Some of the major components of Danshen (Salvia miltiorrhiza), a widely used Chinese herbal medicine rich in phenolic acids, are thermosensitive and may degrade to other phenolic acids during extractions with heating. The chemical profiles of Danshen water-extract may vary with different heat water extraction at different temperatures, affecting the composition and bioactivity of the extracts. In this study, six water-extracts of Danshen obtained from heat reflux water extraction and microwave-assisted extraction with water (MAE-W) at different temperatures were tested for their composition and pharmacological effects. Among these extracts, the third-round MAE-W (100°C) extract had the highest phenolic acids and tanshinones contents, with the strongest antioxidant activity in 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) assay and ferric reducing/antioxidant potential (FRAP) assay. This extract also showed the strongest inhibitory effects on 2,2'-azobis-2-amidinopropane (AAPH)-induced hemolysis in human red blood cells, hydrogen peroxide-induced apoptosis in rat heart H9c2 cells and the highest relaxation effects on rat basilar artery. The antioxidant effects of Danshen water-extracts linearly correlated to their relaxation effects (r=0.895-0.977). Through multiple linear regression analysis, danshensu was found to be the most significant marker in the antioxidant and vasodilation effects of Danshen water-extract, while tanshinone IIA as the marker on hydrogen peroxide-induced apoptosis in rat heart H9c2 cells. Danshensu is, therefore, a useful marker for the quality control of Danshen water-extracts in antioxidant and vasodilation, while tanshinone IIA for anti-apoptotic potential of different extracts.

Enzyme kinetic and molecular docking studies on the metabolic interactions of 1-hydroxy-2,3,5-trimethoxy-xanthone, isolated from Halenia elliptica D. Don, with model probe substrates of human cytochrome P450 enzymes.

Halenia elliptica D. Don is a Tibetan herb and medicinal preparations containing Halenia elliptica have been commonly used for the treatment of hepatitis B virus infection in China. The metabolism of 1-hydroxy-2,3,5-trimethoxy-xanthone (HM-1) to its metabolites is mediated through cytochrome P450 enzymes. This study aimed to investigate the herb-drug interaction potential of HM-1 by studying its effects on the metabolism of model probe substrates of five major CYP450 isoforms in human liver microsomes. HM-1 showed moderate inhibitory effects on CYP1A2 (IC50 = 1.06 µM) and CYP2C9 (IC50 = 3.89 µM), minimal inhibition on CYP3A4 (IC20 = 11.94 µM), but no inhibition on model CYP2D6 (dextromethorphan) and CYP2E1 (chlorzoxazone) probe substrates. Inhibition kinetic studies showed that the K(i) values of HM-1 on CYP1A2, CYP2C9 and CYP3A4 were 5.12 µM, 2.00 µM and 95.03 µM, respectively. HM-1 competitively inhibited testosterone 6ß-hydroxylation (CYP3A4) but displayed mixed type inhibitions for phenacetin O-deethylation (CYP1A2) and tolbutamide 4-hydroxylation (CYP2C9). Molecular docking study confirmed the inhibition modes of HM-1 on these human CYP isoforms.

Herb-drug interactions with Danshen (Salvia miltiorrhiza): a review on the role of cytochrome P450 enzymes.

Danshen, the dried root and rhizome of Salvia miltiorrhiza Bunge, is a widely used medicinal plant for the treatment of cardiovascular diseases in China and a complementary medicine in the West. Danshen is indexed in the 2010 Chinese Pharmacopoeia, with more than 35 formulations and concoctions containing Danshen water-extracts, ethanolic extracts or their combination, which are rich in phenolic acids and different levels of tanshinones. There are rare reports on the adverse effects of Danshen preparations. It is, however, well-known that Danshen leads the anticoagulation failure of warfarin. The Danshen-warfarin interaction may be mediated via both pharmacodynamic and pharmacokinetic mechanisms. This review does not summarize recent progress, but the effects of Danshen and its active ingredients on the interactions of cytochrome P450 (CYP450) and drug transporters, as well as the analysis of ingredients, and the metabolism and pharmacokinetics that are related to these interactions. Tanshinones play significant roles in the inhibition and induction of several CYP450 isozymes. It can be concluded that precautions should be taken when using Danshen preparations rich in tanshinones for CYP-related herb-drug interactions.