Comparing the pharmacokinetics of 13α,21-dihydroeurycomanone and eurycomanone exclusively enriched in Eurycoma longifolia extracts and their spermatogenesis enhancement in andrographolide-induced oligospermia in rats.

OBJECTIVES: The quassinoids eurycomanone (EN) and 13α,21-dihydroeurycomanone (DHY) of Eurycoma longifolia Jack are reported to enhance spermatogenesis. This study aims to profile the pharmacokinetics of DHY, a minor and hitherto unstudied constituent, evaluate its spermatogenesis enhancement property and compare these attributes with that of the predominant EN. METHODS: Crude Eurycoma longifolia extract was chromatographed into a DHY-enriched extract (DHY-F) and an EN-enriched extract (EN-F). Male Sprague-Dawley rats were administered intravenously and orally with both extracts and their plasma levels of both quassinoids were determined. The extracts were then tested for their spermatogenesis augmentation ability in normal rats and an andrographolide-induced oligospermia model. KEY FINDINGS: Chromatographic enrichment resulted in a 28-fold increase of DHY in DHY-F and a 5-fold increase of EN in EN-F compared with non-chromatographed crude extracts. DHY showed better oral bioavailability (1.04 ± 0.58%) than EN (0.31 ± 0.19%). At 5 mg/kg, EN exhibited higher efficacy in spermatogenesis enhancement in normal rats and restoration of oligospermia to normal sperm profile versus DHY. CONCLUSIONS: Despite the better pharmacokinetic profile of DHY, EN remains the main chemical contributor to plant bioactivity. DHY-F and EN-F represent improvements in developing Eurycoma longifolia as a potential phytomedicine for male infertility particularly oligospermia.

Pharmacokinetic study on bruceoside A revealed the potential role of quassinoid glycosides for the anticancer properties of Fructus Bruceae.

Bruceoside A, an abundant quassinoid glycoside in Fructus Bruceae, was chosen for the pharmacokinetic study. It is the first case report on the pharmacokinetic study of quassinoid glycosides so far. A sensitive, accurate, and repeatable UHPLC-MS/MS method was developed for the determination of bruceoside A and its major metabolite. The results showed bruceoside A could be transformed into the potent anticancer component brusatol in vivo, rather than its direct deglycosylated metabolite bruceosin. And the intestinal bacteria were proposed to take a potential role during such transformation. Based on the present study, it could be concluded that the quassinoid glycosides possessing weak activities in vitro could do contribution to the anticancer properties of Fructus Bruceae in vivo via transforming into more active metabolites.

Self-nanoemulsifying drug delivery system of bruceine D: a new approach for anti-ulcerative colitis.

BACKGROUND: Bruceine D (BD) is a major bioactive component isolated from the traditional Chinese medicinal plant Brucea javanica which has been widely utilized to treat dysentery (also known as ulcerative colitis [UC]). METHODS: To improve the water solubility and absolute bioavailability of BD, we developed a self-nanoemulsifying drug delivery system (SNEDDS) composing of MCT (oil), Solutol HS-15 (surfactant), propylene glycol (co-surfactant) and BD. The physicochemical properties and pharmacokinetics of BD-SNEDDS were characterized, and its anti-UC activity and potential mechanism were evaluated in TNBS-induced UC rat model. RESULTS: The prepared nanoemulsion has multiple beneficial aspects including small mean droplet size, low polydispersity index (PDI), high zeta potential (ZP) and excellent stability. Transmission electron microscopy showed that nanoemulsion droplets contained uniform shape and size of globules. Pharmacokinetic studies demonstrated that BD-SNEDDS exhibited enhanced pharmacokinetic parameters as compared with BD-suspension. Moreover, BD-SNEDDS significantly restored the colon length and body weight, reduced disease activity index (DAI) and colon pathology, decreased histological scores, diminished oxidative stress, and suppressed TLR4, MyD88, TRAF6, NF-κB p65 protein expressions in TNBS-induced UC rat model. CONCLUSION: These results demonstrated that BD-SNEDDS exhibited highly improved oral bioavailability and advanced anti-UC efficacy. In conclusion, our current results provided a foundation for further research of BD-SNEDDS as a potential complementary therapeutic agent for UC treatment.

Pharmacokinetic, metabolic profiling and elimination of brusatol in rats.

Brusatol, a quassinoid isolated from the traditional Chinese medicine Brucea javanica, has been reported to be an inhibitor of Nrf2 pathway and has great potential to be developed into a novel chemotherapeutic adjuvant. However, the in vivo process of brusatol has not been comprehensively explained yet. Therefore, this paper focused on the pharmacokinetic metabolism and excretion of brusatol in rats using a simple and reproducible LC-MS/MS method. The results indicated that the plasma concentration of brusatol decreased rapidly; the average cumulative excretion rate in urine was 5.82% during 24 h, and 0.71% in bile during 12 h. High-resolution mass spectrometry was applied for the identification of metabolites; as a result, four metabolites were detected and the structure was tentatively deduced on the base of the MS2 data, Compound Discoverer 2.0 and Mass Frontier 7.0 software. Hydroxylation, hydrolysis and glucuronidation were suggested as major metabolic pathways in vivo. The in vivo process and detection of metabolites of brusatol might improve the understanding of the mechanism of its anticancer effect and provide valuable information for its safety estimation, which will be essential to the new drug development.

Redox-sensitive micelles composed of disulfide-linked Pluronic-linoleic acid for enhanced anticancer efficiency of brusatol.

Brusatol (Bru) exhibits promising anticancer effects, with both proliferation inhibition and chemoresistance amelioration activity. However, the poor solubility and insufficient intracellular delivery of Bru greatly restrict its application. Herein, to simultaneously utilize the advantages of Pluronics as drug carriers and tumor microenvironment-responsive drug release profiles, a flexible amphiphilic copolymer with a polymer skeleton, that is, Pluronic® F68 grafting with linoleic acid moieties by redox-reducible disulfide bonds (F68-SS-LA), was synthesized. After characterization by 1H-nuclear magnetic resonance and Fourier transform infrared spectroscopy, the redox-sensitive F68-SS-LA micelles were self-assembled in a much lower critical micelle concentration than that of the unmodified F68 copolymer. Bru was loaded in micelles (Bru/SS-M) with high loading efficiency, narrow size distribution, and excellent storage stability. The redox-sensitive Bru/SS-M exhibited rapid particle dissociation and drug release in response to a redox environment. Based on the enhanced cellular internalization, Bru/SS-M achieved higher cytotoxicity in both Bel-7402 and MCF-7 cells compared with free Bru and nonreducible micelles. The improved anticancer effect was attributed to the remarkably decreased mitochondrial membrane potential and increased reactive oxygen species level as well as apoptotic rate. These results demonstrated that F68-SS-LA micelles possess great potential as an efficient delivery vehicle for Bru to promote its anticancer efficiency via an oxidation pathway.

A Rapid and Sensitive UPLC-MS/MS Method for Quantification of Bruceantinol in Rat Plasma and Its Application to a Pharmacokinetic Study.

Bruceantinol (BOL), a quassinoid compound isolated from the fruits of Brucea javanica, has been reported to have cytotoxic and antibacterial effects. In this study, a rapid, sensitive, and specific ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) method was developed for the quantitative determination of BOL in rat plasma. The samples were treated by simple liquid-liquid extraction with ethyl acetate and separated on an UPLC BEH C18 column (2.1 mm × 50 mm) using a 3-min gradient elution scheme, which consists of water (0.1% v/v, formic acid) and methanol (0.1%, v/v, formic acid) to achieve the separation of BOL and sinomenine (IS) with high selectivity. The electrospray ionization source was used in positive ion mode; the multiple reaction monitoring quantified the target fragment ions m/z 629.6 → 569.5 for BOL and m/z 330.5 → 207.3 for IS. This work was evaluated with regards to the specificity, extraction recovery, matrix effect, linearity, accuracy, precision, stability, and dilution integrity. This approach was used to examine the pharmacokinetics of BOL in rats after oral (0.3 mg/kg) and intravenous (0.15 mg/kg) administration. BOL presented fast excretion and very low oral bioavailability.

HPLC-MS/MS method for bioavailability study of bruceines D & E in rat plasma.

Bruceines D and E are quassinoids from seeds of Brucea javanica (L.) Merr. exhibiting hypoglycemia effect. The crude drug is used as a traditional medicine by diabetes patients. The aim of this study is to understand the bioavailability and pharmacokinetics of both the bruceines D & E. A rapid and sensitive HPLC-MS/MS method was developed and validated for the quantification of both quassinoids, bruceines D & E in rat plasma. Both the bruceines D & E were separated with the Zorbax SBC-18 column with gradient elution and mobile phase system of acetonitrile and deionized water with 0.1% formic acid at a flow rate of 0.5mL/min. Analytes were detected in multiple reaction monitoring (MRM) mode with electrospray positive ionization. The quassinoids, namely bruceines D & E were detected with transitions of m/z 411.2→393.2 and m/z 395.2→377.2, respectively. Another quassinoid, eurycomanone was used as the internal standard with transition of m/z 409.2→391.2. The method was validated and conformed to the regulatory requirements. The validated method was applied to pharmacokinetic and bioavailability studies in rats. The pharmacokinetic study indicated both bruceine D and E were rapidly absorbed into the circulation system and reached its peak concentration at 0.54±0.34h and 0.66±0.30h, respectively. Bruceine E was eliminated slower than Bruceine D with t1/2 value almost increased two-fold compared to Bruceine D. In conclusion, a rapid, selective and sensitive HPLC-MS/MS method was developed for the simultaneous determination of both the bruceines D and E in rat plasma. Both bruceines D and E displayed poor oral bioavailability.

Determination of brusatol in plasma and tissues by LC-MS method and its application to a pharmacokinetic and distribution study in mice.

OBJECTIVES: The quassinoid brusatol, which can be isolated from Brucea javanica (L.) Merr., becomes popularly studied because of its anti-tumor activity. In order to further investigate brusatol and extend its applications, a sensitive analytical method for determination of brusatol in biological samples is essential. However, few methods had been reported until now. In this study, a highly sensitive and reproducible LC-MS method for simultaneous quantification of brusatol in mouse plasma and tissues was developed and validated. METHOD: Plasma samples and tissue homogenate were extracted with diethyl ether after addition of the internal standard solution(IS). The supernatant was blown to dryness with nitrogen and residual was reconstituted with 100µl of methanol. The separation was performed on an Intersil ODS-3 column and gradient elution was conducted with the mobile phase of water and methanol (0-5min 47:53, 5-5.5min 47:53-10:90, 5.5-9min 10:90, posttime 4min 47:53) at a flow rate of 0.8mL/min. Quantification was performed in the selected ion monitoring (SIM) mode at m/z 543.2 for brusatol and 220.0 for IS (ornidazole). The method was validated by analyzing quality control plasma and tissue homogenate samples, and was applied to analyze samples obtained from mice after injections of brusatol via the tail vein. RESULTS: With ornidazole as the internal standard, calibration curve of the method ranged from 10 to 320ng/ml for plasma and 10-240ng/ml for tissues. Recovery rate of brusatol from plasma and tissues were between 71.09%-94.91%. Relative standard deviation (RSD) for inter- and intra-day precision was less than 15%, and the accuracy was between 96.1%-111.8%. The pharmacokinetics and distribution study of brusatol in mice after three single doses via the tail vein were carried out based on this method. The concentration of brusatol in plasma decreased rapidly and a more than 10 fold concentration of brusatol was found as compared to that in other tissues. CONCLUSIONS: This is the first reported LC-MS method for detecting brusatol in tissues and can accurately determine the concentrations of these compounds in plasma and different tissues. Further research on the metabolism of brusatol in vivo is still needed.

Determination of eurycomanone in rat plasma using hydrophilic interaction liquid chromatography-tandem mass spectrometry for pharmacokinetic study.

In this study, a rapid, sensitive, and reliable hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for the determination of eurycomanone in rat plasma was developed and validated. Plasma samples were pretreated with a protein precipitation method and quercitrin was used as an internal standard (IS). A HILIC silica column (2.1 × 100 mm, 3 µm) was used for hydrophilic-based chromatographic separation, using the mobile phase of 0.1% formic acid with acetonitrile in gradient elution at a flow rate of 0.25 mL/min. Precursor-product ion pairs for multiple-reaction monitoring were m/z 409.1 → 391.0 for eurycomanone and m/z 449.1 → 303.0 for IS. The linear range was 2-120 ng/mL. The intra- and inter-day accuracies were between 95.5 and 103.4% with a precision of <4.2%. The developed method was successfully applied to the pharmacokinetic analysis of eurycomanone in rat plasma after oral dosing with pure compound and E. longifolia extract. The Cmax and AUC0-t , respectively, were 40.43 ± 16.08 ng/mL and 161.09 ± 37.63 ng h/mL for 10 mg/kg eurycomanone, and 9.90 ± 3.97 ng/mL and 37.15 ± 6.80 ng h/mL for E. longifolia extract (2 mg/kg as eurycomanone). The pharmacokinetic results were comparable with each other, based on the dose as eurycomanone.

Brusatol overcomes chemoresistance through inhibition of protein translation.

The NRF2 pathway activates a cell survival response when cells are exposed to xenobiotics or are under oxidative stress. Therapeutic activation of NRF2 can also be used prior to insult as a means of disease prevention. However, prolonged expression of NRF2 has been shown to protect cancer cells by inducing the metabolism and efflux of chemotherapeutics, leading to both intrinsic and acquired chemoresistance to cancer drugs. This effect has been termed the "dark side" of NRF2. In an effort to combat this chemoresistance, our group discovered the first NRF2 inhibitor, the natural product brusatol, however the mechanism of inhibition was previously unknown. In this report, we show that brusatol's mode of action is not through direct inhibition of the NRF2 pathway, but through the inhibition of both cap-dependent and cap-independent protein translation, which has an impact on many short-lived proteins, including NRF2. Therefore, there is still a need to develop a new generation of specific NRF2 inhibitors with limited toxicity and off-target effects that could be used as adjuvant therapies to sensitize cancers with high expression of NRF2.

Ailanthone targets p23 to overcome MDV3100 resistance in castration-resistant prostate cancer.

Androgen receptor (AR) antagonist MDV3100 is the first therapeutic approach in treating castration-resistant prostate cancer (CRPC), but tumours frequently become drug resistant via multiple mechanisms including AR amplification and mutation. Here we identify the small molecule Ailanthone (AIL) as a potent inhibitor of both full-length AR (AR-FL) and constitutively active truncated AR splice variants (AR-Vs). AIL binds to the co-chaperone protein p23 and prevents AR's interaction with HSP90, thus resulting in the disruption of the AR-chaperone complex followed by ubiquitin/proteasome-mediated degradation of AR as well as other p23 clients including AKT and Cdk4, and downregulates AR and its target genes in PCa cell lines and orthotopic animal tumours. In addition, AIL blocks tumour growth and metastasis of CRPC. Finally, AIL possesses favourable drug-like properties such as good bioavailability, high solubility, lack of CYP inhibition and low hepatotoxicity. In general, AIL is a potential candidate for the treatment of CRPC.

Determination of a potential antitumor quassinoid in rat plasma by UPLC-MS/MS and its application in a pharmacokinetic study.

A sensitive UPLC-MS/MS method was developed and validated for the determination of brusatol in rat plasma. Chromatographic separation was carried out on a C18 column using methanol and 10mM ammonium acetate containing 0.1% (v/v) formic acid (55:45, v/v). The lower limit of quantification (LLOQ) was 1.0 ng/mL for brusatol in plasma. The intra- and inter-day precision for the analyte ranged from 3.2% to 9.2% and 1.3% to 7.8%, and the accuracy was between 97.3% and 108.5%. The method was successfully applied in a pharmacokinetic study of brusatol following intravenous injection (0.5, 1.0, and 2.0mg/kg) of brusatol.

Development of a validated LC-MS/MS method for the determination of ailanthone in rat plasma with application to pharmacokinetic study.

Ailanthone, a natural compound isolated from Chinese herb Ailanthus altissima, has drawn a lot of attention for its antitumor activity. In this study, a simple and sensitive method for determination of ailanthone in rat plasma was developed for the first time, using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Brusatol was used as an internal standard. Separation was achieved on an Agilent Zorbax Eclipse Plus C18 column with gradient elution using water-methanol as mobile phase at a flow rate of 0.2 mL/min. A triple quadrupole mass spectrometer operating in the negative electrospray ionization mode with multiple reaction monitoring (MRM) was used to detect ailanthone and IS transitions of 375.2 → 301.1 and 519.1 → 437.4, respectively. The lower limit of quantification was 5 ng/mL with a linear range of 5-2000 ng/mL. The intra- and inter-day accuracy (RE) ranged from -3.6 to 1.5% and -0.7 to 4.7% and the intra- and inter-day precision (RSD) was between 2.8-6.7% and 3.1-8.0%. The validated method has been successfully applied to a pharmacokinetic study of ailanthone in rats. The elimination half-lives (t1/2) were 105.5 ± 13.6, 113.3 ± 39.6, and 95.8 ± 23.9 min after single intravenous administration of 0.5, 1.0, and 1.5mg/kg ailanthone, respectively. The area under the plasma concentration versus time curve (AUC0-6h) and initial plasma concentration (C0) were linearly related to dose.

Physico-chemical effects of the major quassinoids in a standardized Eurycoma longifolia extract (Fr 2) on the bioavailability and pharmacokinetic properties, and their implications for oral antimalarial activity.

A simple validated LC-UV method for the phytochemical analysis of four bioactive quassinoids, 13alpha(21)-epoxyeurycomanone (EP), eurycomanone (EN), 13alpha,21-dihydroeurycomanone (ED) and eurycomanol (EL) in rat plasma following oral (200 mg/kg) and intravenous administration (10 mg/kg) of a standardized extract Fr 2 of Eurycoma longifolia Jack was developed for pharmacokinetic and bioavailability studies. The extract Fr 2 contained 4.0%, 18.5%, 0.7% and 9.5% of EP, EN, ED and EL, respectively. Following intravenous administration, EP displayed a relatively longer biological half-life (t1/2 = 0.75 +/- 0.25 h) due primarily to its lower elimination rate constant (k(e)) of 0.84 +/- 0.26 h(-1)) when compared with the t1/2 of 0.35 +/- 0.04 h and k(e) of 2.14 +/- 0.27 h(-1), respectively of EN. Following oral administration, EP showed a higher C(max) of 1.61 +/- 0.41 microg/mL over that of EN (C(max) = 0.53 +/- 0.10 microg/mL). The absolute bioavailability of EP was 9.5-fold higher than that of EN, not because of chemical degradation since both quassinoids were stable at the simulated gastric pH of 1. Instead, the higher log K(ow) value of EP (-0.43) contributed to greater membrane permeability over that of EN (log K(ow) = -1.46) at pH 1. In contrast, EL, being in higher concentration in the extract than EP, was not detected in the plasma after oral administration because of substantial degradation by the gastric juices after 2 h. Similarly, ED, being unstable at the acidic pH and together with its low concentration in Fr 2, was not detectable in the rat plasma. In conclusion, upon oral administration of the bioactive standardized extract Fr 2, EP and EN may be the only quassinoids contributing to the overall antimalarial activity; this is worthy of further investigation.

Bioavailability and pharmacokinetic studies of eurycomanone from Eurycoma longifolia.

A validated HPLC analysis of eurycomanone (1), a bioactive quassinoid, in rat plasma following oral and intravenous administration of Eurycoma longifolia Jack extract was developed for pharmacokinetic and bioavailability studies. Relatively high plasma eurycomanone concentrations were detected after an intravenous injection of 10 mg/kg extract F2 containing 1.96 mg/kg of the quassinoid. However, it declined rapidly to zero after 8 h. Its mean elimination rate constant (k(e)), biological half-life (t(1/2)), volume of distribution (V(d)) and clearance (CL) were 0.88 +/- 0.19 h (-1), 1.00 +/- 0.26 h, 0.68 +/- 0.30 L/kg and 0.39 +/- 0.08 L/h/kg, respectively. Following oral administration of eurycomanone, its Cmax and Tmax values were detected as 0.33 +/- 0.03 microg/mL and 4.40 +/- 0.98 h, respectively. The plasma concentration of the quassinoid after oral administration was much lower than after intravenous application in spite of the oral dose being 5 times higher. The results indicate that eurycomanone is poorly bioavailable when given orally. A comparison of the AUC (0-->infinity) obtained orally to that obtained after an intravenous administration (normalized for dose differences) revealed that the absolute bioavailability of the compound was low with 10.5 %. Furthermore, the compound appeared to be well distributed in the extravascular fluids because of its relatively high V(d) value. The poor oral bioavailability was not attributed to instability problems because eurycomanone has been shown to be stable under different pH conditions. Thus, its poor oral bioavailability may be due to poor membrane permeability in view of its low P value and/or high first-pass metabolism.