Enhancement of cytotoxicity and induction of apoptosis by cationic nano-liposome formulation of n-butylidenephthalide in breast cancer cells.

Breast cancer is the second most common malignancy in women. Current clinical therapy for breast cancer has many disadvantages, including metastasis, recurrence, and poor quality of life. Furthermore, it is necessary to find a new therapeutic drug for breast cancer patients to meet clinical demand. n-Butylidenephthalide (BP) is a natural and hydrophobic compound that can inhibit several tumors. However, BP is unstable in aqueous or protein-rich environments, which reduces the activity of BP. Therefore, we used an LPPC (Lipo-PEG-PEI complex) that can encapsulate both hydrophobic and hydrophilic compounds to improve the limitation of BP. The purpose of this study is to investigate the anti-tumor mechanisms of BP and BP/LPPC and further test the efficacy of BP encapsulated by LPPC on SK-BR-3 cells. BP inhibited breast cancer cell growth, and LPPC encapsulation (BP/LPPC complex) enhanced the cytotoxicity on breast cancer by stabilizing the BP activity and offering endocytic pathways. Additionally, BP and LPPC-encapsulated BP induced cell cycle arrest at the G0/G1 phase and might trigger both extrinsic as well as intrinsic cell apoptosis pathway, resulting in cell death. Moreover, the BP/LPPC complex had a synergistic effect with doxorubicin of enhancing the inhibitory effect on breast cancer cells. Consequently, LPPC-encapsulated BP could improve the anti-cancer effects on breast cancer in vitro. In conclusion, BP exhibited an anti-cancer effect on breast cancer cells, and LPPC encapsulation efficiently improved the cytotoxicity of BP via an acceleration of entrapment efficiency to induce cell cycle block and apoptosis. Furthermore, BP/LPPC exhibited a synergistic effect in combination with doxorubicin.

Liposome Consolidated with Cyclodextrin Provides Prolonged Drug Retention Resulting in Increased Drug Bioavailability in Brain.

Although butylidenephthalide (BP) is an efficient anticancer drug, its poor bioavailability renders it ineffective for treating drug-resistant brain tumors. However, this problem is overcome through the use of noninvasive delivery systems, including intranasal administration. Herein, the bioavailability, drug stability, and encapsulation efficiency (EE, up to 95%) of BP were improved by using cyclodextrin-encapsulated BP in liposomal formulations (CDD1). The physical properties and EE of the CDD1 system were investigated via dynamic light scattering, transmission electron microscopy, UV-Vis spectroscopy, and nuclear magnetic resonance spectroscopy. The cytotoxicity was examined via MTT assay, and the cellular uptake was observed using fluorescence microscopy. The CDD1 system persisted for over 8 h in tumor cells, which was a considerable improvement in the retention of the BP-containing cyclodextrin or the BP-containing liposomes, thereby indicating a higher BP content in CDD1. Nanoscale CDD1 formulations were administered intranasally to nude mice that had been intracranially implanted with temozolomide-resistant glioblastoma multiforme cells, resulting in increased median survival time. Liquid chromatography-mass spectrometry revealed that drug biodistribution via intranasal delivery increased the accumulation of BP 10-fold compared to oral delivery methods. Therefore, BP/cyclodextrin/liposomal formulations have potential clinical applications for treating drug-resistant brain tumors.

Pharmacokinetic Profiling of Butylidenephthalide and Alisol B in Danggui-Shaoyao-San in Rats.

BACKGROUND AND OBJECTIVE: Danggui-Shaoyao-San (DSS), a famous Chinese formula, has been widely used to treat gynecological disorders since ancient times and has recently showed efficacy in treating Alzheimer's disease. Butylidenephthalide (BDPH) and alisol B (ALI) are recognized as the primary active ingredients of DSS. The objectives of the present study were to evaluate the pharmacokinetic comparative study of BDPH and ALI in herbal extracts and their purified forms. METHOD: A sensitive and specific high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) methodology was developed to determine the concentration level of BDPH and ALI in rat plasma. This approach enables a real-time pharmacokinetics profiling of BDPH and ALI in DSS extracts as well as their purified forms in rats after oral administration. RESULTS: The validated method showed an evident linearity over a wide range of dosages (r > 0.99) with sensitivity down to 1.0 ng/mL for each analyte. The extraction recovery of the analyte ranged from 80.8 to 99.1%. The pharmacokinetic parameters were significantly different in herbal extracts and their purified forms. The results showed that the absorption of both BDPH and ALI from DSS extracts was significantly greater compared with their purified forms. CONCLUSIONS: A highly specific, sensitive and rapid HPLC-MS/MS method was developed and applied for the determination of BDPH and ALI in rat plasma. It was found that BDPH and ALI had higher bioavailability in the DSS extract compared with their purified forms.

Liquid chromatography-tandem mass spectrometry evaluation of the pharmacokinetics of a diacid metabolite of norcantharidin loaded in folic acid-targeted liposomes in mice.

A previous study has reported diacid metabolite (DM) as the stable form of norcantharidin (NCTD), which is almost 100% metabolized to DM-NCTD. However, the unreliable pharmacokinetic characteristics of DM-NCTD could result in low bioavailability, hindering the clinical use of DM-NCTD in the treatment of diseases. A liposomal drug delivery system could overcome the shortcomings of DM-NCTD by improving the relative bioavailability (Fr), reducing drug toxicity, and increasing the therapeutic efficacy. However, there are no data concerning the pharmacokinetics of a DM-NCTD-loaded liposomal drug delivery system in animals, which is required for assessing its safety profile. Therefore, a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of DM-NCTD in mouse plasma. Standard curves were linear (r=0.9966) over the range 10.0-1.00×10(4)ng/ml in mouse plasma with a lower limit of quantification (LLOQ) of 10ng/ml. This study successfully investigated the pharmacokinetics of DM-NCTD and DM-NCTD encapsulated in polyethylene glycol (PEG)-Liposomes (DM-NCTD/PEG-Liposome) or folic acid (FA)-PEG-Liposomes (DM-NCTD/FA-PEG-Liposome) in Kunming mice after a single intravenous dose of 2mg/kg. The plasma profile data of the three groups adhered to a two-compartment model. Compared with the DM-NCTD group, the Liposome groups had longer circulation times following intravenous administration in mice, and the Fr of DM-NCTD increased significantly (P<0.05). Furthermore, the area under the concentration-time curve (AUC) declined with an increase in the volume of distribution (Vd) from the PEG-Liposome to the FA-PEG-Liposome groups, which indicates a more efficient removal of the drug from the plasma of the FA-PEG-Liposome group. This result suggests a possible increased risk of DM-NCTD intoxication in normal tissues with FA-PEG-Liposomes. Based on this study, further investigation of the biodistribution of DM-NCTD/FA-PEG-Liposomes in healthy animals is warranted. In addition, the plausibility of formulating a safe DM-NCTD-loaded system without increasing toxicity against normal tissues needs to be determined.

Metabolic conversion from co-existing ingredient leading to significant systemic exposure of Z-butylidenephthalide, a minor ingredient in Chuanxiong Rhizoma in rats.

Pharmacokinetic (PK) study of medicinal herbs is a great challenge, because which component(s) is(are) the bioactive ingredients is largely unknown. Most of the reported PK studies of herbs focused on the major ingredients regardless of their in vivo bioactivities, while PK of components with low content in herbs is often ignored. The present study demonstrates how PK study can reveal potential importance of a low content ingredient to the herbal bioactivities using Z-butylidenephthalide (BuPh), a bioactive phthalide present in a significantly low quantity in medicinal herb Chuanxiong Rhizoma, as an example. PK of BuPh was investigated in rats using Chuanxiong extract, fraction containing BuPh and ligustilide, and pure BuPh, respectively. The results demonstrated that remarkable blood concentrations of BuPh were observed after administration of the herbal extract and its systemic exposure was significantly different between BuPh given in pure and mixed forms. More interestingly, AUC of BuPh via intake of fraction (9.3-fold) and extract (4.5-fold) was significantly greater than that obtained from pure BuPh, which was further evidenced to be mainly due to metabolic conversion from ligustilide, a major component in Chuanxiong. Our findings revealed that although it naturally occurred in low amount, BuPh reached significant systemic concentrations via metabolic conversion from ligustilide. Moreover, our results demonstrated that PK study is one of crucial and inevitable steps for revealing in vivo bioactive ingredients of herbal medicines, and such studies should be more appropriate to focus on in vivo profile of the ingredients co-existing in herbs rather than only studying them individually.

Local interstitial delivery of z-butylidenephthalide by polymer wafers against malignant human gliomas.

We have shown that the natural compound z-butylidenephthalide (Bdph), isolated from the chloroform extract of Angelica sinensis, has antitumor effects. Because of the limitation of the blood-brain barrier, the Bdph dosage required for treatment of glioma is relatively high. To solve this problem, we developed a local-release system with Bdph incorporated into a biodegradable polyanhydride material, p(CPP-SA; Bdph-Wafer), and investigated its antitumor effects. On the basis of in vitro release kinetics, we demonstrated that the Bdph-Wafer released 50% of the available Bdph by the sixth day, and the release reached a plateau phase (90% of Bdph) by the 30th day. To investigate the in situ antitumor effects of the Bdph-Wafer on glioblastoma multiforme (GBM), we used 2 xenograft animal models-F344 rats (for rat GBM) and nude mice (for human GBM)-which were injected with RG2 and DBTRG-05MG cells, respectively, for tumor formation and subsequently treated subcutaneously with Bdph-Wafers. We observed a significant inhibitory effect on tumor growth, with no significant adverse effects on the rodents. Moreover, we demonstrated that the antitumor effect of Bdph on RG2 cells was via the PKC pathway, which upregulated Nurr77 and promoted its translocation from the nucleus to the cytoplasm. Finally, to study the effect of the interstitial administration of Bdph in cranial brain tumor, Bdph-Wafers were surgically placed in FGF-SV40 transgenic mice. Our Bdph-Wafer significantly reduced tumor size in a dose-dependent manner. In summary, our study showed that p(CPP-SA) containing Bdph delivered a sufficient concentration of Bdph to the tumor site and effectively inhibited the tumor growth in the glioma.

A biomimetic pH-responsive polymer directs endosomal release and intracellular delivery of an endocytosed antibody complex.

Poly(propylacrylic acid) (PPAAc) is a synthetic pH-responsive polymer that has been shown to disrupt cell membranes at low pH values typical of the endosome, but not at physiological pH, suggesting its use as an endosomal-releasing agent [Murthy et al. J. Controlled Release 61, 137-43]. We have constructed an antibody-targeted biotherapeutic model to investigate whether PPAAc can enhance intracellular trafficking of proteins to the cytoplasm. A ternary complex composed of a biotinylated anti-CD3 antibody, streptavidin, and biotinylated PPAAc was fluorescently labeled, and its intracellular fate was analyzed by confocal microscopy, flow cytometry, and quantitative western blotting of cell fractionates. The 64.1 anti-CD3 antibody was previously shown to direct receptor-mediated endocytosis in the Jurkat T-cell lymphoma cell line and was rapidly trafficked from the endosome to the lysosomal compartment. The antibody-streptavidin complex was also rapidly internalized to the endosomal/lysosomal compartment and retained there, as evidenced by punctate regions of fluorescence observed by confocal fluorescence microscopy. In samples containing the ternary complex of antibody, streptavidin, and PPAAc-biotin, diffuse fluorescence in the cytoplasm was observed, indicating that PPAAc enhanced translocation to the cytoplasm. This was confirmed by western blotting analysis of the isolated cytoplasm. Flow cytometry results demonstrated that neither streptavidin nor PPAAc caused nonspecific uptake of the complex, nor did they inhibit antibody-mediated endocytosis. The striking enhancement of protein delivery to the cytoplasm by complexed PPAAc suggests that this polymer could provide a new delivery agent for therapeutic, vaccine, and diagnostics development.

Distribution, metabolism and excretion of butylidenephthalide of Ligustici chuanxiong rhizoma in hairless mouse after dermal application.

The absorption, distribution and excretion of butylidenephthalide after dermal application to hairless mouse have been examined with [8-14C]butylidenephthalide. By the investigation of the whole body autoradiogram and liquid scintillation analysis, it was indicated that the transdermally applied butylidenephthalide quickly permeate into peripheral circulation system without accumulation in the skin and then distribute into lung, liver, bile and kidney. The total radioactivity, however, was decreased due to excretion into urine, and in the case of i.v.-administration, 80% of the administered butylidenephthalide was excreted into urine within 24 h, while only 5% was excreted into feces within 24 h. Then, the metabolite in urine was determined to be a cysteine conjugate by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method. Thus, it has been concluded that after dermal application butylidenephthalide quickly permeates through skin into peripheral circulation system; distributes to lung, liver, bile and kidney; and then excreted into urine as a cysteine adduct.

Determination of Z-butylidenephthalide in plasma by high-performance liquid chromatography.

An HPLC assay is described for the determination of Z-butylidenephthalide (Z-Bdph) in plasma. Plasma samples were cleaned up by extraction with 2% chloroform in n-hexane. Z-Bdph was separated on a normal-phase silica column with a mobile phase of chloroform-n-hexane (1:1). The limit of quantitation with UV detection at 254 nm for Z-Bdph in plasma was 0.01 microgram/ml. The recovery of Z-Bdph by organic solvent extraction of plasma was 99.5%. The intra-day and inter-day coefficients of variation and relative errors for Z-Bdph determination in plasma were both less than 10%. The present method was applied to pharmacokinetic studies of Z-Bdph in plasma after intravenous administration to rabbits.

Generation of hexahydrophthalic anhydride atmospheres in a controlled human-use test chamber.

A method for generating controlled atmospheres of hexahydrophthalic anhydride (HHPA) in an 8 m3 exposure chamber was developed. The permeation principle was used for gaseous HHPA generation. HHPA concentration was monitored by sampling on XAD-2 tubes and by a Fourier-transform infrared (FTIR) spectrometer using the partial least-square quantitative method. The repeatability of the FTIR was 5%, the reproducibility 12%, and the limit of detection 10 micrograms/m3. A bubbler method determined the sum of HHPA and HHP acid by using gas chromatography/mass spectrometry detection after derivatization with methanol/boron trifluoride. The precision of the work-up procedure was 3% and the recovery was 94% at 300 ng sampled amount of HHPA. The limit of detection was 10 ng HHPA. The variation in the permeation rate was 3% over 3 days. Different concentrations in the exposure chamber were generated by changing the temperature of the permeation tubes. The generated HHPA concentration range, at human exposure, was 3-90 micrograms/m3. The concentration at one temperature was reproducible even after major changes in the temperature. The coefficient of variation (CV) of six samples from different places in the breathing zone was 3%. The variation in the concentration, during an 8-hour human exposure at 10 micrograms/m3, was 3%. Time-weighted averages (8 hour) for human exposures of 10 micrograms/m3 (CV = 15%; n = 6); 37 micrograms/m3 (CV = 5%; n = 5); and 81 micrograms/m3 (CV = 6%; n = 9) were obtained at intended concentrations of 10 micrograms/m3, 40 micrograms/m3, and 80 micrograms/m3. The loss of HHPA in the exposure chamber was 54% (CV = 17%).(ABSTRACT TRUNCATED AT 250 WORDS)

Toxicokinetics and biological monitoring in experimental exposure of humans to gaseous hexahydrophthalic anhydride.

Six healthy volunteers were exposed to gaseous hexahydrophthalic anhydride (HHPA) concentrations of 10, 40, or 80 micrograms.m-3 (65, 260 or 520 nmol.m-3, respectively) for 8 h. The respiratory uptake of the inhaled HHPA was almost complete. Rapid increases in plasma and urinary levels of hexahydrophthalic acid (HHP acid) were seen. During the first 4 h after the end of exposure, the half-time of HHP acid in plasma was about 2 h. A corresponding decay was seen in urine. The correlations (r > 0.90) between the air concentrations of HHPA and the levels of HHP acid in plasma and urine were close. They were even closer (r > 0.96) when the total respiratory uptake of HHPA was used. Urinary pH adjustment by intake of ammonium chloride or sodium hydrogen carbonate did not significantly alter the excretion of HHP acid. The results show that the analysis of HHP acid in plasma or urine is useful as a biological monitor for exposure to HHPA.

Induction of mast cell sensitization by chemical allergens: a comparative study.

In previous investigations we have shown that chemical allergens of different classes induce in mice qualitatively divergent immune responses. Respiratory allergens provoke substantial increases in total serum concentration of IgE. In contrast, contact allergens which are known or suspected not to cause respiratory sensitization or which at most have only a very limited potential to do so, have little or no effect on total serum IgE. Such differences, we propose, provide a novel approach for the prospective identification of chemicals with potential to cause respiratory allergy. In the absence of a robust method for the direct measurement of respiratory hypersensitivity reactions in mice we have sought in the present study to determine whether the IgE responses induced in mice by respiratory allergens are specific and of sufficient magnitude to cause the active sensitization of mast cells in vivo, a prerequisite for immediate hypersensitivity, including acute-onset respiratory hypersensitivity. Topical exposure of BALB/c mice to concentrations of > or = 10% of the human respiratory allergen trimellitic anhydride (TMA) caused the specific sensitization of peritoneal mast cells in situ as measured by the conjugate-induced release of [3H]-5-hydroxytryptamine in vitro. Experiments were performed also with 2,4-dinitrochlorobenzene (DNCB), a contact allergen that fails to induce respiratory hypersensitivity. Treatment of mice with concentrations of DNCB of comparable immunogenicity failed to cause mast cell sensitization. These data demonstrate that a known human chemical respiratory allergen induces in mice specific mast-cell-sensitizing IgE antibody and reinforce the value of the mouse as a model for the evaluation of respiratory sensitization potential.

Occupational exposure to hexahydrophthalic anhydride: air analysis, percutaneous absorption, and biological monitoring.

Urinary hexahydrophthalic acid (HHP acid) levels were determined in 20 workers occupationally exposed to hexahydrophthalic anhydride (HHPA) air levels of 11-220 micrograms/m3. The levels of HHP acid in urine increased rapidly during exposure and the decreases were also rapid after the end of exposure. The elimination half-time of HHP acid was 5 h, which was significantly longer than in experimentally exposed volunteers, possibly indicating distribution to more than one compartment. There was a close correlation between time-weighted average levels of HHPA in air and creatinine-adjusted levels of HHP acid in urine collected during the last 4 h of exposure (r = 0.90), indicating that determination of urinary HHP acid levels is suitable as a method for biological monitoring of HHPA exposure. An air level of 100 micrograms/m3 corresponded to a postshift urinary HHP acid level of ca. 900 nmol/mmol creatinine in subjects performing light work for 8 h. Percutaneous absorption of HHPA was studied by application of HHPA in petrolatum to the back skin of three volunteers. The excreted amounts of HHP acid in urine, as a fraction of the totally applied amount of HHPA, were within intervals of 1.4%-4.5%, 0.2%-1.3%, and 0%-0.4% respectively, indicating that the contribution from percutaneous absorption is of minor importance in a method for biological monitoring.

Controlled gastric emptying. II. In vitro erosion and gastric residence times of an erodible device in beagle dogs.

An erodible gastric retention device fabricated from various polymeric blends was examined in vitro for its dissolution properties and in vivo in fasting dogs for assessment of its gastric retention potential. Dissolution studies were conducted with extruded rods of polymer blends to assess their potential as candidates for the erodible component of a gastrically retained device. Based on results from dissolution studies, rods of poly(ortho ester)/polyethylene blends (POE/PE) (45% erosion at pH 1.5 and 24 hr) were used to fabricate arms for tetrahedron-shaped devices. Corners for the tetrahedral device were fabricated from Silastic 382 loaded with 15% barium sulfate for X-ray visualization. Beagle dogs were dosed with tetrahedron-shaped test devices administered in gelatin capsules and gastric retention monitored by X ray over a 24-hr period. A comparison of in vitro erosion rates and in vivo performance of various polymer blends indicated a definite trend for increased gastric retention of devices made from the more slowly eroding blends. The results indicate that the blending of erodible and nonerodible polymers is a valid approach for obtaining materials that will provide the necessary structural properties to achieve gastric retention yet lose integrity within a desired time.