Betulinic acid and its spray dried microparticle formulation: In vitro PDT effect against ovarian carcinoma cell line and in vivo plasma and tumor disposition.

The race against ovarian cancer continue to motivate the research worldwide. It is known that many antitumor drugs have limited penetration into solid tumor tissues due to its microenvironment, thus contributing to their low efficacy. Therapeutic modalities have been exploited to elicit antitumor effects based on microenvironment of tumor, including Photodynamic therapy (PDT). Prospection of natural small molecules and nanotechnology are important tools in the development of new ways of obtaining photoactive compounds that are biocompatible. The Betulinic acid (BA) has shown potential biological effect as bioactive drug, but it has low water solubility. Thus, in the present study, owing to the poor solubility of the BA, its free form (BAF) was compared to a spray dried microparticle betulinic acid/HP-ß-CD formulation (BAC) aiming to assess the BAF and BAC efficacy as a photosensitizer in PDT for application in ovarian cancer. BAF and BAC were submitted to assays in the presence of LED (λ = 420 nm) under different conditions (2.75 J/cm2, 5.5 J/cm2, and 11 J/cm2) and in absence of irradiation, after 5 min or 4 h of contact with ovarian carcinoma cells (A2780) or fibroblast murine cells (3T3). Furthermore, HPLC-MS/MS and MALDI-MSI methods were developed and validated in plasma and tumor of mice proving suitable for in vivo studies. The results found a greater photoinduced cytotoxic effect for the BAC at low concentration for A2780 when irradiated with LED with similar results for fluorescence microscopy. The results motivate us to continue the studies with the BA as a potential antitumor bioactive compound.

An LC-ESI/MS/MS method for the determination of lupeol via precolumn derivatization and its application to pharmacokinetic studies in rat plasma.

Lupeol, a phytosterol and triterpene, is widely found in edible fruits and vegetables, and has been reported to exhibit a spectrum of pharmacological activities against various disease conditions. In the present study, a derivative generated by the reaction of lupeol with p-toluenesulfonyl isocyanate was ionizable and fragmentable in the negative mode by electrospray ionization/tandem mass spectrometry. Based on this simple chemical derivatization, a liquid chromatography-electrospray ionization/tandem mass spectrometry method was developed and validated for the quantification of lupeol in rat plasma. The calibration curves were linear (r2 > 0.999) over concentrations from 2.5 to 250 ng/ml for lupeol. The method had an accuracy of 96.0-109.4%, and the intra- and inter-day precisions (RSD) were within ± 15%. The stability data showed that no significant degradation occurred under the experimental conditions. The mean recoveries at three quality control levels were within 88.7-95.7%. No significant matrix effects (105.3-109.8%) were observed in rat plasma. This method was successfully applied to the pharmacokinetic study of lupeol in rat plasma after oral administration.

A Phase 1 Study to Assess the Relative Bioavailability, Food Effect, and Safety of a Tablet Formulation of GSK2838232, a Novel HIV Maturation Inhibitor in Healthy Participants After Single and Repeated Doses.

GSK2838232 is a novel, potent HIV-1 maturation inhibitor for use in regimen-based combination antiretroviral therapy from a once-daily oral dose boosted with a pharmacoenhancer (ritonavir or cobicistat). This phase 1 study in healthy participants was conducted in 2 parts. Part 1 (n = 14) assessed the relative bioavailability of single doses of a 200-mg GSK2838232 tablet and capsule formulation boosted with 100 mg ritonavir in fed and fasted (tablet-only) subjects. Part 2 (n = 10) assessed the pharmacokinetics of repeated 500-mg once-daily doses of GSK2838232 without a pharmacoenhancing boosting agent. In part 1, GSK2838232 demonstrated comparable bioavailability following a single dose of 200 mg GSK2838232 as capsule and tablet formulations in combination with ritonavir (RTV) under fed conditions, with lower intrasubject variability observed for the tablet formulation. In part 2, following administration of 500 mg GSK2838232 once daily for 11 days under fed conditions, Cmax , AUC0-τ , and Cτ showed a small degree of accumulation (1.2- to 1.3-fold) of GSK2838232. The median tmax was approximately 4 hours on both day 1 and day 11 when given with food. The mean t½ was approximately 23 hours on day 11. Steady-state concentrations were achieved by day 3 with a geometric mean steady-state Cτ on day 11 of 28 ng/mL. The tablet formulation was generally well tolerated as a single 200-mg dose with RTV under fed and fasted conditions and following administration of multiple daily doses (11 days) of 500 mg unboosted.

Development and validation of a liquid chromatography-tandem mass spectrometry method for quantification of Lupeol in plasma and its application to pharmacokinetic study in rats.

Lupeol, a phytosterol and triterpene, possesses numerous medicinal properties against cancer, inflammation, arthritis, diabetes, heart diseases, etc. A novel, sensitive, specific and reproducible method for quantification of Lupeol in rat plasma using liquid chromatography combined with atmospheric pressure chemical ionization (APCI) tandem mass spectrometry (LC-MS/MS) was developed and validated as per regulatory guidelines. Sample preparation was simple and fast which consisted of one-step protein precipitation using acetonitrile. Testosterone was used as an internal standard. HyPurity Advance column was used to develop the chromatography method using 0.1% formic acid in water and acetonitrile as mobile phases by gradient elution. APCI positive ion mode was used for mass spectrometric detection. Multiple reaction monitoring (MRM) transitions of m/z 409.5 [M + H - H2O]+→137.3 for Lupeol and m/z 289.1 [M + H]+→97.1 for Testosterone were used for quantification. The method was validated over a linear concentration range of 5-5000 ng/mL with a correlation coefficient (r2) of ≥ 0.99. This method showed acceptable accuracy (89.52-97.10%), precision (%CV ≤ 10.75%) and recovery with a negligible matrix effect. Lupeol was found to be stable in the stock solution, autosampler condition and also in plasma for four freeze-thaw cycles, 6 h at ambient temperature and 30 days at -20°C. This method was successfully applied to measurement of Lupeol in plasma samples from pharmacokinetic study in rats and can be easily extended to human pharmacokinetic studies.

A sensitive liquid chromatography-mass spectrometry method for the simultaneous determination in plasma of pentacyclic triterpenes of Olea europaea L.

Table olives are especially rich in pentacyclic triterpenic compounds, which exert several biological activities. A crucial step in order to know if these compounds could contribute to the beneficial and healthy properties of this food is their measurement in blood. Therefore, the present study describes a simple and accurate liquid-liquid extraction followed by LC-QqQ-MS analysis for the simultaneous determination of the main pentacyclic triterpenes from Olea europaea L. in rat plasma. The method was validated by the analysis of blank plasma samples spiked with pure compounds, obtaining a linear correlation, adequate sensitivity with a limit of quantification ranging from 1nM for maslinic acid to 10nM for uvaol. Precision and accuracy were lower than 10% in all cases and recoveries were between 95 and 104%. The oral administration of olives to rats and its determination in plasma verified that the established methodology is appropriate for bioavailability studies.

Development of a UPLC-TQ/MS Approach for the Determination of Eleven Bioactive Components in Haizao Yuhu Decoction Plus-Minus Haizao and Gancao Drug Combination after Oral Administration in a Rat Model of Hypothyroidism.

Haizao Yuhu Decoction (HYD) has been used for approximately 500 years and is well-known in Traditional Chinese Medicine for its efficacy in the treatment of thyroid-related diseases. In this study, a rapid liquid chromatography-tandem mass spectrometry method was developed for the determination of liquiritin, naringin, hesperidin, peimine, liquiritigenin, glycyrrhizic acid, bergapten, nobiletin, osthole, and glycyrrhetinic acid in rat plasma to investigate the pharmacokinetic profile of different HYD prescriptions in a rat model of hypothyroidism. The differences in pharmacokinetic parameters among the groups were compared by Student's t-test. The pharmacokinetic profile of liquiritin, naringin, hesperidin, peimine, liquiritigenin, glycyrrhizic acid, bergapten, nobiletin, osthole, and glycyrrhetinic acid showed significant differences between Haizao and Gancao anti-drug combination and other herbs in HYD. These results may contribute to the rational clinical use of HYD and reveal the compatibility profile of the Haizao and Gancao anti-drug combination.

Biopharmaceutical and pharmacokinetic characterization of asiatic acid in Centella asiatica as determined by a sensitive and robust HPLC-MS method.

ETHNOPHARMACOLOGICAL RELEVANCE: Asiatic acid is one of the main components in the herb Centella asiatica, which is a well-known herbal medicine for its excellent pharmacological effects. To enhance the development potentials of asiatic acid as a chemopreventative agent, there is a great need to further understand its biopharmaceutical and pharmacokinetic properties. The aim of this research is to clarify the mechanisms of absorption and metabolism of asiatic acid, and explore its biopharmaceutical and pharmacokinetic properties in rats by using a sensitive and robust HPLC-MS method. MATERIALS AND METHODS: Male Sprague-Dawley rats were randomly assigned to 2 groups and administered with asiatic acid by oral and intravenous administration. Plasma concentrations of asiatic acid were determined at designated points and main pharmacokinetic parameters were estimated. The absorption of asiatic acid was investigated by using Caco-2 cell line absorption model in vitro and rat intestinal perfusion model in situ. The metabolic rate of asiatic acid was investigated by incubating it in rat liver microsome system in vitro. In addition, the solubility of asiatic acid in aqueous solution was also determined by using HPLC-MS method. RESULTS: The absolute oral bioavailability of asiatic acid is 16.25%. It was found that the permeability of asiatic acid is more than 10(-5) in the Caco-2 cell monolayer and rat intestinal perfusion model, and its main absorption region is the jejunum in rats. The metabolic rate of asiatic acid in rat liver microsomes, t1/2, is 9.493min, which shows that asiatic acid can be metabolized rapidly. The solubility of aisiatic acid was 0.1583mgmL(-1), and its poor solubility will result in low bioavailability. CONCLUSIONS: The asiatic acid in a variety of matrixes was analyzed by using a sensitive and specific HPLC-MS method, and its absolute oral bioavailability in rats was very low. Asiatic acid can be metabolized rapidly in rat liver microsomes, and has good permeability across Caco-2 monolayer cell and rat intestine perfusion. It can be deduced that the low bioavailability of asiatic acid results from poor solubility and rapid metabolism.

[LC-MS quantification and pharmacokinetics of the multi-constituents of Huangqin Tang in rat plasma after different single oral doses].

The current study aims to investigate the pharmacokinetic properties of Huangqin Tang on different oral doses. An LC-MS method for simultaneous determination of flavonoids and terpenoids in rat plasma was developed and validated. Plasma samples were treated with hydrochloric acid (containing 1% ascorbic acid), precipitated with acetonitrile, separated on a Zorbax SB-C18 column, detected by single quadruple mass spectrometry with an electrospray ionization interface, and quantified using selected ion monitoring mode. All pharmacokinetic parameters were processed by non-compartmental analysis using WinNonlin software. The results of specificity, linearity, intra-day and inter-day precisions, accuracy, and stability for LC-MS assay were suitable for the quantification of paeoniflorin, baicalin, wogonoside, baicalein, wogonin, oroxylin A, glycyrrhizic acid and glycyrrhetinic acid in rat plasma. The concentration-time profiles of baicalin, wogonoside, baicalein, wogonin, oroxylin A and glycyrrhizic acid showed double-peak phenomenon after Huangqin Tang was orally administered at 40 g x kg(-1) dose; all eight constituents in rat plasma showed good dose-exposure relationship within the dosage of 10-40 g x kg(-1); although plasma concentrations were different, the flavonoids with the same backbone showed the similar fate in the body with the corresponding dosage. In conclusion, the LC-MS assay was successfully applied for the pharmacokinetic study of multi-constituents of Huangqin Tang with different doses. Additionally, these constituents demonstrated good pharmacokinetic properties in the body.

Analytical strategy to reveal the in vivo process of multi-component herbal medicine: a pharmacokinetic study of licorice using liquid chromatography coupled with triple quadrupole mass spectrometry.

Although various techniques have been employed to analyze drug metabolites, the metabolism of multi-component herbal medicine has seldom been fully addressed. In contrast to chemical drugs, a number of compounds in herbal medicine could get into circulation and then be metabolized. Moreover, these compounds may have metabolic interactions which make their pharmacokinetics (PK) even more complicated. The present work aims to elucidate the multi-component pharmacokinetics of a herbal medicine, and to demonstrate how PK behaviors were altered by co-existing constituents. Licorice (Glycyrrhiza uralensis Fisch.), a most commonly used herbal medicine, was chosen as a model. A strategy was proposed to compare the PK profiles of licorice extract with those of nine single compounds. These compounds were major bioactive constituents of licorice, and represented various structural types (flavanone, chalcone, isoflavone, saponin, and coumarin). We established a segmented selected reaction monitoring LC/MS/MS method to simultaneously monitor 63 licorice metabolites in rat plasma, and obtained the PK profiles of 55 metabolites. The results indicated that interactions among licorice compounds altered their PK behaviors in 4 aspects: improvement in bioavailability for aglycones (133- and 109-fold increase for liquiritigenin and isoliquiritigenin, respectively), prolongation in system circulation for glycosides (0.3h delay in T(max) for liquiritin apioside and isoliquiritin apioside), decrease of potential toxicity for saponins such as glycyrrhizic acid, and shift in plasma distribution for phase II metabolites. This is the first attempt to systematically reveal the in vivo process of licorice. Moreover, the study indicates noticeable interactions to alter pharmacokinetics among licorice compounds, which may be characteristic for herbal medicines.

A liquid chromatography/electrospray ionization tandem mass spectrometric method for quantification of asiatic acid from plasma: application to pharmacokinetic study in rats.

RATIONALE: Asiatic acid (AA), a pentacyclic triterpene from Centella asiatica (L.) Urban, has shown numerous therapeutic activities. However, none of the published works to date has used high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) for determination of AA from biological fluids. Therefore, the present paper describes a sensitive HPLC/electrospray ionization (ESI)-MS/MS method for quantification of AA in rat plasma. METHODS: Ammonium adduct formation of AA was essential in the development of a sensitive method with the rat plasma samples being pre-treated by a simple solid-phase extraction method. The separation was achieved on a Cosmosil C(18) column using a gradient mobile phase flow. Detection was performed using an Applied Biosystems API Q-Trap 2000 mass spectrometer equipped with an ESI source operated in positive mode with colchicine used as internal standard. RESULTS: An eight-point calibration curve over the concentration range of 1.02-407.88 ng/mL for AA from rat plasma provided an optimum linear detector response (with r(2) >0.9983). The mean percentage recovery (n = 3) for the low, middle and high quality control samples was 91.23 ± 1.88%, 90.36 ± 0.55% and 89.71 ± 0.21%, respectively. The intra-day and inter-day precision and accuracy of the quality control samples were within ≤5% and ±7% correspondingly. CONCLUSIONS: The developed method was validated as per US FDA guidelines and applicability demonstrated by successful measurement of AA from plasma following oral administration of C. asiatica extracts to Wistar rats. The results suggest that the method could be applied to therapeutic monitoring of AA and pharmacokinetic studies in human volunteers.