Bufalin-Loaded Multifunctional Photothermal Nanoparticles Inhibit the Anaerobic Glycolysis by Targeting SRC-3/HIF-1α Pathway for Improved Mild Photothermal Therapy in CRC.

Purpose: Compared with traditional photothermal therapy (PTT, >50°C), mild PTT (≤45°C) is a promising strategy for tumor therapy with fewer adverse effects. Unfortunately, its anti-tumor efficacy is hampered by thermoresistance induced by overexpression of heat shock proteins (HSPs). In our previous study, we found bufalin (BU) is a glycolysis inhibitor that depletes HSPs, which is expected to overcome thermotolerance of tumor cells. In this study, BU-loaded multifunctional nanoparticles (NPs) were developed for enhancing the mild PTT of colorectal cancer (CRC). Methods: Fe3O4 NPs coated with the polydopamine (PDA) shell modified with polyethylene glycol (PEG) and cyclic arginine-glycyl-aspartic peptide (cRGD) for loading BU (Fe3O4@PDA-PEG-cRGD/BU NPs) were developed. The thermal variations in Fe3O4@PDA-PEG-cRGD/BU NPs solution under different conditions were measured. Glycolysis inhibition was evaluated by measuring the glucose uptake, extracellular lactate, and intracellular adenosine triphosphate (ATP) levels. The cellular cytotoxicity of Fe3O4@PDA-PEG-cRGD/BU NPs was analyzed using a cell counting kit-8 assay, Calcein-AM/PI double staining, and flow cytometry in HCT116 cells. The magnetic resonance imaging (MRI) performance and anti-tumor therapeutic efficacy of Fe3O4@PDA-PEG-cRGD/BU NPs were evaluated in HCT116-tumor bearing mice. Results: Fe3O4@PDA-PEG-cRGD/BU NPs had an average diameter of 260.4±3.5 nm, the zeta potential of -23.8±1.6 mV, the drug loading rate of 1.1%, which had good thermal stability, photothermal conversion efficiencies and MRI performance. In addition, the released BU not only killed tumor cells but also interfered with glycolysis by targeting the steroid receptor coactivator 3 (SRC-3)/HIF-1α pathway, preventing intracellular ATP synthesis, and combating HSP-dependent tumor thermoresistance, ultimately strengthening the thermal sensitivity toward mild PTT both in vitro and in vivo. Conclusion: This study provides a highly effective strategy for enhancing the therapeutic effects of mild PTT toward tumors.

Comparative study on pharmacokinetics of extract from Bufonis venenum and its liposomes.

Bufadienolides and indolealkylamines, the primary active components in Bufonis venenum, have rapid clearance from the body with a short half-life, leading to low bioavailability. Moreover, Bufadienolides and indolealkylamines are associated with serious adverse effects. In order to reduce the toxicities, minimize the adverse effects and simultaneously load lipophilic bufadienolides and hydrophilic indolealkylamines with satisfactory drug loading and encapsulation rate, we prepared Bufonis venenum extract-liposomes (BVE-LP). To compare the pharmacokinetic differences between Bufonis venenum extract (BVE) and its liposomes, ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay was established to simultaneously detect the 12 chemical components in rat plasma. Results of pharmacokinetic study in SD rats showed that the highest exposure based on the area under the plasma concentration-time curve (AUC0-t) was obtained by 5-hydroxytryptamine with a mean value of 267.097 µg/L*h in BVE-LP group, which was 72.36 % higher as compared to that obtained in Bufonis venenum extract (BVE) group with a mean value of AUC0-t of 154.966 µg/L*h. The exposure (AUC0-t) of desacetylcinobufotalin, desacetylcinobufagin, arenobufagin and telocinobufagin in BVE-LP group was 111.89 %, 94.13 %, 134.08 %, and 92.94 % higher when compared to that in BVE group. With the employment of liposomes, there was an obvious decrease in the clearance of bufotenidine, desacetylcinobufotalin, gamabufotalin, arenobufagin, and telocinobufagin. The terminal half life (t1/2) of desacetylcinobufotalin, gamabufotalin, arenobufagin, and telocinobufagin in BVE-LP group was increased by 185.96 %, 292.64 %, 196.78 % and 131.29 % when compared to that in BVE group. All the results above indicated that a slower elimination rate and more exposure of some bufadienolides and indolealkylamines was obtained by BVE-LP when compared to BVE group, which verified BVE-LP could provide a therapeutic option for effective delivery of Bufonis venenum in clinical. DATA AVAILABILITY: The data will be shared on reasonable request to the corresponding author.

Advances on Delivery System of Active Ingredients of Dried Toad Skin and Toad Venom.

Dried toad skin (TS) and toad venom (TV) are the dried skin of the Bufo bufo gargarizans Cantor and the Bufo melanostictus Schneider, which remove the internal organs and the white secretions of the skin and retroauricular glands. Since 2005, cinobufacini preparations have been approved by the State Food and Drug Administration for use as adjuvant therapies in the treatment of various advanced cancers. Meanwhile, bufalenolides has been identified as the main component of TS/TV, exhibiting antitumor activity, inducing apoptosis of cancer cells and inhibiting cancer cell proliferation or metastasis through a variety of signaling pathways. However, clinical agents frequently face limitations such as inherent toxicity at high concentrations and insufficient tumor targeting. Additionally, the development and utilization of these active ingredients are hindered by poor water solubility, low bioavailability, and rapid clearance from the bloodstream. To address these challenges, the design of a targeted drug delivery system (TDDS) aims to enhance drug bioavailability, improve targeting within the body, increase drug efficacy, and reduce adverse reactions. This article reviews the TDDS for TS/TV, and their active components, including passive, active, and stimuli-responsive TDDS, to provide a reference for advancing their clinical development and use.

Novel Strategies for Solubility and Bioavailability Enhancement of Bufadienolides.

Toad venom contains a large number of bufadienolides, which have a variety of pharmacological activities, including antitumor, cardiovascular, anti-inflammatory, analgesic and immunomodulatory effects. The strong antitumor effect of bufadienolides has attracted considerable attention in recent years, but the clinical application of bufadienolides is limited due to their low solubility and poor bioavailability. In order to overcome these shortcomings, many strategies have been explored, such as structural modification, solid dispersion, cyclodextrin inclusion, microemulsion and nanodrug delivery systems, etc. In this review, we have tried to summarize the pharmacological activities and structure-activity relationship of bufadienolides. Furthermore, the strategies for solubility and bioavailability enhancement of bufadienolides also are discussed. This review can provide a basis for further study on bufadienolides.

Simultaneous determination of resibufogenin and its eight metabolites in rat plasma by LC-MS/MS for metabolic profiles and pharmacokinetic study.

BACKGROUND: Resibufogenin is one of the main active compounds of Venenum Bufonis and exhibits diverse pharmacological activities. It is brought into focus for its potency in heart failure and cancer therapy. PURPOSE: The purpose of this study was to establish a convenient and effective method which was used to simultaneously determine the resibufogenin and its metabolites in rat plasma for further understanding the metabolic profiles of resibufogenin in vivo and pharmacokinetic study by LC-MS/MS. METHODS: The analytes were separated on a BEH C18 column with a mobile phase of water containing 0.05% formic acid and acetonitrile under gradient elution at a flow rate of 0.4 ml/min. Resibufogenin and its eight metabolites were quantified in positive electrospray ionization and MRM mode with transitions of m/z 385.5→349.2 for resibufogenin; m/z 513.7→145.3 for IS (internal standard); m/z 401.23→365.21, m/z 417.23→285.21 and m/z 385.24→349.21 for three main metabolites (hydroxylated-resibufogenin; dihydroxylated-resibufogenin and 3-epi-resibufogenin, respectively). RESULTS: This method was successfully validated with a good linearity over the concentration ranges of 1-200 ng/ml for resibufogenin and the correlation coefficients was more than 0.990. The lower limit of quantification was 1 ng/ml and the precision and accuracy values were less than 15%. The method was applied to study the metabolic profiles of resibufogenin in rat plasma after oral administration of 20 mg/kg. The results indicated that the metabolic reactions of resibufogenin were mainly hydroxylation, dihydroxylation, dehydrogenation and isomerization. Totally eleven metabolites were identified, among which eight were successfully quantified. CONCLUSION: The results could provide further research foundation for the mechanisms study of activity and toxicity in vivo and facilitate the appropriate clinical application of resibufogenin.

Simultaneous Determination of Bufalin and Its Nine Metabolites in Rat Plasma for Characterization of Metabolic Profiles and Pharmacokinetic Study by LC⁻MS/MS.

Characterization and determination of metabolites to monitor metabolic pathways play a paramount role in evaluating the efficacy and safety of medicines. However, the separation and quantification of metabolites are rather difficult due to their limited contents in vivo, especially in the case of Chinese medicine, due to its complexity. In this study, an effective and convenient method was developed to simultaneously quantify bufalin and its nine metabolites (semi-quantitation) in rat plasma after an oral administration of 10 mg/kg to rats. The prototype and metabolites that were identified were subsequently quantified using positive electrospray ionization in multiple reaction monitoring (MRM) mode with transitions of m/z 387.4→369.6 and 387.4→351.3 for bufalin, m/z 513.7→145.3 for IS, and 387.4→369.6, 419.2→365.2, and 403.2→349.2 for the main metabolites (3-epi-bufalin, dihydroxylated bufalin, and hydroxylated bufalin, respectively). The method was validated over the calibration curve range of 1.00-100 ng/mL with a limit of quantitation (LOQ) of 1 ng/mL for bufalin. No obvious matrix effect was observed, and the intra- and inter-day precisions, as well as accuracy, were all within the acceptable criteria in this method. Then, this method was successfully applied in metabolic profiling and a pharmacokinetic study of bufalin after an oral administration of 10 mg/kg to rats. The method of simultaneous determination of bufalin and its nine metabolites in rat plasma could be useful for pharmacokinetic-pharmacodynamic relationship research of bufalin, providing experimental evidence for explaining the occurrence of some adverse effects of Venenum Bufonis and its related preparations.

In situ phase transition of microemulsions for parenteral injection yielding lyotropic liquid crystalline carriers of the antitumor drug bufalin.

In this work, we used the small angle X-ray scattering (SAXS) method for controlled preparation of in situ forming sustained-release carriers for the antitumor drug bufalin (BUF), which has very poor solubility and a considerable cardiotoxicity in a non-encapsulated state. To that aim, we exploited the pseudo-ternary phase diagram of an oil(O)/surfactant(S)/water(W) system containing medium chain capric/caprylic triglycerides (MCT) and a co-surfactant blend of Macrogol (15)-hydroxystearate (Solutol HS 15) and sorbitan monooleate (Span 80). Two compositions with different oil contents (sample B and C) were selected from the microemulsion region of the phase diagram in order to study the effect of the aqueous environment on their structural behavior. A phase transition from a microemulsion (ME) to a liquid crystalline phase (LC) was established by SAXS upon progressive dilution. The drug bufalin (BUF) was encapsulated in the microemulsions with low viscosity, whereas the release of the drug occurred from the in situ generated lamellar liquid crystalline structures. The formulations were characterized by SAXS, dynamic light scattering (DLS), cryogenic transmission electron microscopy (Cryo-TEM), rheology, drug loading and encapsulation efficiency, and in vitro release profiles. A correlation was suggested between the structures of the in situ phase-transition formed LCME formulations, the differences in their viscosities and drug release profiles. The performed cytotoxicity, cell apoptosis and pharmacokinetic experiments showed an enhanced bioavailability of BUF after encapsulation. These results suggest potential clinical applications for the obtained safe in situ phase-transition sustained-release formulations of BUF.

Systemic delivery of the anticancer agent arenobufagin using polymeric nanomicelles.

Arenobufagin (ABG) is a major active component of toad venom, a traditional Chinese medicine used for cancer therapy. However, poor aqueous solubility limits its pharmacological studies in vivo due to administration difficulties. In this study, we aimed to develop a polymeric nanomicelle (PN) system to enhance the solubility of ABG for effective intravenous delivery. ABG-loaded PNs (ABG-PNs) were prepared with methoxy poly (ethylene glycol)-block-poly (d,l-lactic-co-glycolic acid) (mPEG-PLGA) using the solvent-diffusion technique. The obtained ABG-PNs were 105 nm in size with a small polydispersity index of 0.08. The entrapment efficiency and drug loading were 71.9% and 4.58%, respectively. Cellular uptake of ABG-PNs was controlled by specific clathrin-mediated endocytosis. In addition, ABG-PNs showed improved drug pharmacokinetics with an increased area under the curve value (a 1.73-fold increase) and a decreased elimination clearance (37.8% decrease). The nanomicelles showed increased drug concentrations in the liver and lung. In contrast, drug concentrations in both heart and brain were decreased. Moreover, the nanomicelles enhanced the anticancer effect of the pure drug probably via increased cellular uptake of drug molecules. In conclusion, the mPEG-PLGA-based nanomicelle system is a satisfactory carrier for the systemic delivery of ABG.

Degradation of Mcl-1 through GSK-3ß Activation Regulates Apoptosis Induced by Bufalin in Non-Small Cell Lung Cancer H1975 Cells.

BACKGROUND/AIMS: Mcl-1, an anti-apoptotic Bcl-2 family member, is often overexpressed in non-small cell lung cancer (NSCLC). Bufalin has been reported to induce apoptosis in various tumor cells. However, there is no report showing that bufalin could downregulate Mcl-1 expression in NSCLC. METHODS: Cell proliferation was analyzed by cell counting kit-8 (CCK-8) assay in H1975 cells. Cell apoptosis was detected by flow cytometry. Mcl-1 mRNA was detected by RT-PCR. The expression of apoptosis-associated proteins in H1975 cells was detected by western blotting. The levels of Mcl-1 ubiquitination and NOXA were analyzed by Immunoprecipitation assay. RESULTS: Cell growth was inhibited by bufalin in a time and dose-dependent manner. Bufalin induced apoptosis in NSCLC cells by activating caspase cascades and downregulating Mcl-1 expression. However, overexpression of Mcl-1 diminished bufalin-induced apoptosis. Furthermore, bufalin did not reduce Mcl-1 mRNA expression in H1975 cells, but strongly promoted Mcl-1 protein degradation. Proteasome inhibitor MG132 markedly prevented the degradation of Mcl-1 and blocked bufalin-induced Mcl-1 reduction. Bufalin did not significantly affect NOXA protein levels, but downregulated the expression of p-GSK-3ß. GSK-3 inhibitor and GSK-3ß siRNA resulted in increased levels of Mcl-1 and reversed the bufalin-induced Mcl-1 degradation. CONCLUSION: Bufalin induced cell apoptosis in H1975 cells may be through downregulation of Mcl-1. Proteasomal degradation of Mcl-1 via GSK-3ß activation was involved in bufalin-induced apoptosis.

TXNIP/TRX/NF-κB and MAPK/NF-κB pathways involved in the cardiotoxicity induced by Venenum Bufonis in rats.

Venenum Bufonis (VB) is a widely used traditional medicine with serious cardiotoxic effects. The inflammatory response has been studied to clarify the mechanism of the cardiotoxicity induced by VB for the first time. In the present study, Sprague Dawley (SD) rats, were administered VB (100, 200, and 400 mg/kg) intragastrically, experienced disturbed ECGs (lowered heart rate and elevated ST-segment), increased levels of serum indicators (creatine kinase (CK), creatine kinase isoenzyme-MB (CK-MB), alanine aminotransferase (ALT), aspartate aminotransferase (AST)) and serum interleukin (IL-6, IL-1ß, TNF-α) at 2 h, 4 h, 6 h, 8 h, 24 h, and 48 h, which reflected that an inflammatory response, together with cardiotoxicity, were involved in VB-treated rats. In addition, the elevated serum level of MDA and the down-regulated SOD, CAT, GSH, and GPx levels indicated the appearance of oxidative stress in the VB-treated group. Furthermore, based on the enhanced expression levels of TXNIP, p-NF-κBp65, p-IκBα, p-IKKα, p-IKKß, p-ERK, p-JNK, and p-P38 and the obvious myocardial degeneration, it is proposed that VB-induced cardiotoxicity may promote an inflammatory response through the TXNIP/TRX/NF-κB and MAPK/NF-κB pathways. The observed inflammatory mechanism induced by VB may provide a theoretical reference for the toxic effects and clinical application of VB.

Interspecies variation in phase I metabolism of bufalin in hepatic microsomes from mouse, rat, dog, minipig, monkey, and human.

1. Bufalin (BF), one of the major bioactive compounds in traditional Chinese medicine (TCM) Chansu, has been found with various pharmacological and toxicological effects. This study aims to investigate the species differences in phase I metabolism of BF in hepatic microsomes from human and five common experimental animals. 2. Metabolite profiling demonstrated that two major metabolites were formed in liver microsomes from human and animal species in NADPH-generating system. Two major metabolites were identified as 5ß-hydroxyl-bufalin and 3-keto-bufalin, with the help of authentic standards. CYP3A was assigned as the main isoform involved in both 5ß-hydroxylation and 3-oxidation in all studied liver microsomes. The apparent kinetic parameters including substrate affinity and catalytic efficiency for 5ß-hydroxylation and 3-oxidation of BF were also determined. 3. In summary, CYP3A mediated 5ß-hydroxylation and 3-oxidation were two major metabolic pathways of BF in hepatic microsomes from human and five studied animals, but kinetic analysis demonstrated that the intrinsic clearances of these two metabolic pathways were much different among various species. The qualitative and quantitative interspecies study indicated that minipig exhibited the similar metabolic profile, kinetic behaviors and intrinsic metabolic clearances of BF phase I biotransformation in comparison with that of human.

Marinobufagenin in essential hypertension and primary aldosteronism: a cardiotonic steroid with clinical and diagnostic implications.

BACKGROUND: The cardiotonic steroid marinobufagenin (MBG) is increasingly suggested to be responsible for some of the cardiovascular injury that has been previously attributed to aldosterone. We examined the clinical correlates of circulating MBG concentrations in hypertensive patients and tested the hypothesis that MBG serves as a reliable diagnostic tool for detecting primary aldosteronism (PA). METHODS: Plasma MBG concentrations (mean: 0.51±0.25 nmol/l) were measured in the morning fasting samples in 20 patients with PA and 20 essential hypertensive (EH) controls matched for age, sex, body mass index, renal function, urinary sodium and intake of antihypertensive medication (mean age: 51.6 years; 52.2% women). RESULTS: Overall, plasma MBG was directly correlated with plasma aldosterone, aldosterone to active renin ratio (AARR), diastolic blood pressure, mean carotid intima-media thickness, serum sodium, urinary protein to creatinine ratio and inversely with serum potassium levels. Plasma MBG levels were significantly higher in patients with PA compared to EH (mean: 0.68±0.12 versus 0.35±0.24 nmol/l; p<0.001). ROC analysis yielded a greater AUC for plasma MBG compared to the AARR, PAC and serum potassium levels for detecting PA. Youden's Index analyses yielded the optimal plasma MBG cut-off score for diagnosing PA at >0.49 nmol/l with specificity and sensitivity values of 0.85 and 0.95, respectively, which were higher than those at the optimum AARR cut-off at >3.32 ng/dl/µU/ml. CONCLUSIONS: In a well-characterized cohort, values of plasma MBG were significantly related to clinical correlates of cardiovascular and renal disease. Plasma MBG emerged as a valuable alternative to the AARR for screening of PA.

Pharmacokinetics and tissue distribution of five bufadienolides from the Shexiang Baoxin Pill following oral administration to mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Shexiang Baoxin Pill (SBP) is a well-known composite formula of traditional Chinese medicine (TCM), widely used to treat cardiovascular diseases such as angina pectoris and myocardial infarction. Bufadienolides are major active compounds of Venenum Bufonis, which is one of the seven materiamedicas that comprise the Shexiang Baoxin Pill. Previous pharmacokinetics studies of bufadienolides have typically used a single medicinal material delivered to rats. In this study, we have chosen the mouse, a more proper animal model than the rat, to investigate the in vivo pharmacokinetics and tissue distribution of bufadienolides from the Shexiang Baoxin Pill. MATERIALS AND METHODS: The concentrations of bufadienolides in plasma and tissues were identified using high performance liquid chromatography-tandem mass spectrometry (HPLC-ESI-MS/MS). The samples were prepared by liquid-liquid extraction with ethyl acetate, and the separation of bufadienolides was achieved using an ACQUITY HSS T3 column by gradient elution using water (containing 0.1% formic acid) and acetonitrile as the mobile phase at a flow rate of 0.3 mL/min. The pharmacokinetic parameters were determined using non-compartmental analysis. RESULTS: The results showed that the five bufadienolides were rapidly absorbed and distributed into the body. The pharmacokinetic curve showed double peaks after oral administration. The major tissue depots for resibufogenin, bufalin, and bufotalin in mice were the intestines, lung and kidney, whereas the major tissue depots of gamabufotalin and arenobufagin were the intestines, liver and kidney. CONCLUSION: The information gained from this research provides a meaningful insight for the clinical applications of the Shexiang Baoxin Pill.

Immunoliposome co-delivery of bufalin and anti-CD40 antibody adjuvant induces synergetic therapeutic efficacy against melanoma.

Liposomes constitute one of the most popular nanocarriers for improving the delivery and efficacy of agents in cancer patients. The purpose of this study was to design and evaluate immunoliposome co-delivery of bufalin and anti-CD40 to induce synergetic therapeutic efficacy while eliminating systemic side effects. Bufalin liposomes (BFL) conjugated with anti-CD40 antibody (anti-CD40-BFL) showed enhanced cytotoxicity compared with bufalin alone. In a mouse B16 melanoma model, intravenous injection of anti-CD40-BFL achieved smaller tumor volume than did treatment with BFL (average: 117 mm(3) versus 270 mm(3), respectively); the enhanced therapeutic efficacy through a caspase-dependent pathway induced apoptosis, which was confirmed using terminal deoxynucleotidyl transferase-mediated dUTP-Fluorescein nick end labeling and Western blot assay. Meanwhile, anti-CD40-BFL elicited unapparent body-weight changes and a significant reduction in serum levels of tumor necrosis factor-α, interleukin-1ß, interleukin-6, interferon-γ, and hepatic enzyme alanine transaminase, suggesting minimized systemic side effects. This may be attributed to the mechanism by which liposomes are retained within the tumor site for an extended period of time, which is supported by the following biodistribution and flow cytometric analyses. Taken together, the results demonstrated a highly promising strategy for liposomal vehicle transport of anti-CD40 plus bufalin that can be used to enhance antitumor effects via synergetic systemic immunity while blocking systemic toxicity.

Preparation of bufalin-loaded pluronic polyetherimide nanoparticles, cellular uptake, distribution, and effect on colorectal cancer.

A large number of studies have shown that bufalin can have a significant antitumor effect in a variety of tumors. However, because of toxicity, insolubility in water, fast metabolism, short half-life, and other shortcomings, its application is limited in cancer therapy. In this study, we explored the anti-metastatic role of bufalin-loaded pluronic polyetherimide nanoparticles on HCT116 colon cancer-bearing mice. Nanoparticle size, shape, drug loading, encapsulation efficiency, and in vitro drug release were studied. Also, cellular uptake of nanoparticles, in vivo tumor targeting, and tumor metastasis were studied. The nanoparticles had a particle size of about 60 nm and an encapsulation efficiency of 75.71%, by weight. The in vitro release data showed that free bufalin was released faster than bufalin-loaded pluronic polyetherimide nanoparticles, and almost 80% of free bufalin was released after 32 hours. Nanoparticles had an even size distribution, were stable, and had a slow release and a tumor-targeting effect. Bufalin-loaded pluronic polyetherimide nanoparticles can significantly inhibit the growth and metastasis of colorectal cancer.

Simultaneous determination of seven bufadienolides in rat plasma after oral administration of Shexiang Baoxin Pill by liquid chromatography-electrospray ionization-tandem mass spectrometry: application to a pharmacokinetic study.

A liquid chromatography-electrospray ionization-tandem mass spectrometry method was described for the simultaneous determination of resibufogenin, bufalin, gamabufotalin, telibufagin, arenobufagin, cinobufagin and bufotalin in rat plasma. Plasma samples were pretreated by liquid-liquid extraction with ethyl acetate. Chromatographic separation was carried out on an ACQUITY HSS T3 column with gradient elution using mobile phase consisting of acetonitrile-0.1% formic acid in water at a flow rate of 0.3 mL/min. All analytes showed good linearity over a wide concentration range (r>0.99). The lower limit of quantification was in the range of 0.5-10 ng/mL for seven bufadienolides. The mean recovery of the analytes ranged from 94.36 to 104.18%. The intra- and inter-day precisions were in the range of 1.74-13.78% and the accuracies were between 89.37 and 101.38%. The validated method was successfully applied to a pharmacokinetic (PK) study of the seven bufadienolides in rat plasma after oral administration of Shexiang Baoxin Pill (SBP). The selected PK marker compounds with typical efficacy/toxicity may provide a practical solution for marker compound selection and dosage design for the therapeutic drug monitoring and PK study of SBP in its clinical applications.

Quantitative determination of arenobufagin in rat plasma by ultra fast liquid chromatography-tandem mass spectrometry and its application in a pharmacokinetic study.

A rapid, sensitive, and selective ultra fast liquid chromatography-tandem mass spectrometry method was developed for quantitative determination of arenobufagin in rat plasma. Sample pretreatment involved a one-step protein precipitation with methanol using 0.1mL rat plasma. The separation was carried out on a Shim-pack XR-ODS II (75mm×2.0mm, i.d. 2.1µm) column with gradient elution at a flow rate of 0.30mLmin(-1). The mobile phase was acetonitrile and 0.1% formic acid in water. A post-column switching valve was applied to reduce the matrix effect. The detection was performed on a triple-quadruple tandem mass spectrometer in the multiple reaction monitoring mode after electrospray ionization. Linear calibration curves for arenobufagin were obtained over the concentration range 1.056-1056ngmL(-1), with a lower limit of quantification of 1.056ngmL(-1). The intra-day and inter-day precision values were lower than 15% and the accuracy ranged from 5.4% to 9.8% at all quality control levels. The method was successfully applied to the determination and pharmacokinetic study of arenobufagin in rat plasma following intraperitoneal administration.

Enhancement of skin permeation of bufalin by limonene via reservoir type transdermal patch: formulation design and biopharmaceutical evaluation.

A reservoir-type transdermal delivery system (TDS) of bufalin was designed and evaluated for various formulation variables like different penetration enhancers, formulation matrix, rate controlling membranes as well as biopharmaceutical characteristics. Hairless mouse skin was used in permeation experiments with Franz diffusion cells. In vitro skin permeation study showed that terpenes, especially d-limonene was the most effective enhancer when ethanol and PG were used as the vehicle with a synergistic effect. Among different rate controlling membranes, ethylene vinyl acetate (EVA) membrane containing 19% vinyl acetate demonstrated a more suitable release rate for bufalin than the other membranes. In vivo pharmacokinetic study of the bufalin patch in rat showed steady-state of bufalin from 3h to 12 h. In vivo release rate and cumulative amount analyzed by deconvolution method demonstrated the sustained release of bufalin as long as the patch remained on the animal for at least 12 h. The MRT increased from 1h of IV administration to 9h of transdermal administration. In vitro permeation across mouse skin was found to have biphasic correlation with plasma AUC in the in vivo pharmacokinetic study. Current in vitro-in vivo correlation (IVIVC) enabled the prediction of pharmacokinetic profile of bufalin from in vitro permeation results. In conclusion, current reservoir transdermal patch containing 10% D-limonene as a permeation enhancer, 40% ethanol, 30% PG and 15% carbopol-water gel complex provided an improved sustained release of bufalin through transdermal administration. The bufalin patch was successfully applied to biopharmaceutical study in rats and demonstrated the feasibility of this transdermal formulation for future development and clinical trials.

Bufalin-loaded mPEG-PLGA-PLL-cRGD nanoparticles: preparation, cellular uptake, tissue distribution, and anticancer activity.

BACKGROUND: Recent studies have shown that bufalin has a good antitumor effect but has high toxicity, poor water solubility, a short half-life, a narrow therapeutic window, and a toxic dose that is close to the therapeutic dose, which all limit its clinical application. This study aimed to determine the targeting efficacy of nanoparticles (NPs) made of methoxy polyethylene glycol (mPEG), polylactic-co-glycolic acid (PLGA), poly-L-lysine (PLL), and cyclic arginine-glycine-aspartic acid (cRGD) loaded with bufalin, ie, bufalin-loaded mPEG-PLGA-PLL-cRGD nanoparticles (BNPs), in SW620 colon cancer-bearing mice. METHODS: BNPs showed uniform size. The size, shape, zeta potential, drug loading, encapsulation efficiency, and release of these nanoparticles were studied in vitro. The tumor targeting, cellular uptake, and growth-inhibitory effect of BNPs in vivo were tested. RESULTS: BNPs were of uniform size with an average particle size of 164 ± 84 nm and zeta potential of 2.77 mV. The encapsulation efficiency was 81.7% ± 0.89%, and the drug load was 3.92% ± 0.16%. The results of in vitro cytotoxicity studies showed that although the blank NPs were nontoxic, they enhanced the cytotoxicity of bufalin in BNPs. Drug release experiments showed that the release of the drug was prolonged and sustained. The results of confocal laser scanning microscopy indicated that BNPs could effectively bind to human umbilical vein endothelial cells. In the SW620 xenograft mice model, the BNPs could effectively target the tumor in vivo. The BNPs were significantly more effective than other NPs in preventing tumor growth. CONCLUSION: BNPs had even size distribution, were stable, and had a slow-releasing and tumor-targeting effect. BNPs significantly inhibited colon cancer growth in vitro and in vivo. As a novel drug carrier system, BNPs are a potentially promising targeting treatment for colon cancer.

Marinobufagenin predicts and resibufogenin prevents preeclampsia: a review of the evidence.

OBJECTIVE: The purpose of this review is to provide information detailing the existing evidence with regard to the hypothesis that marinobufagenin (MBG) is an important etiologic and predictive factor in preeclampsia (PE). In addition, evidence describing the role of the antagonist to MBG, resibufogenin (RBG), in the prevention and/or treatment of this disorder is provided. STUDY DESIGN: The studies outlined were performed in an animal model of PE, in in vitro experiments, and in human studies. RESULTS: Data have been obtained that strongly support the hypothesis that ~60 to 70% of PE patients demonstrate elevations in urinary and serum MBG levels. In the animal model, the entire syndrome can be prevented by the administration of RBG beginning early in pregnancy. CONCLUSION: Expanded human trials of MBG as a predictor of the later development of PE are warranted as are studies of the efficacy and safety of RBG as a preventative/therapy.