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Oral hepatic cancer therapy by gold nanoparticle based sirna complex with bile acid transporter
Abstracts / Drug Metabolism and Pharmacokinetics 32 (2017) S27eS107
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were compared, and their impacts on DOX drug detection by mass spectrometry was evaluated. The results showed that mixing bile matrix with blank plasma at a ratio of 1:3 (v/v), in combination with an optimized protein precipitation, resulted in a “cleaner” sample preparation than LLE or the protein precipitation approach alone. Fifty mL of the bile sample was mixed with 150 mL of blank rat plasma, and then 600 mL of acetonitrile was added. Following centrifugation, the resulting supernatant was diluted at a 1:4 ratio (v/v) with deionized water containing 30% acetonitrile and 0.1% formic acid. Five mL of the diluted sample was injected onto Phenomenex Kinetex 2.6 um Biphenyl 100A, 2.1 x 50.0 mm column using Acquity UPLC, followed by detection using a Xevo TQS LCMS system (Waters Corporation). Successive MRM experiments were conducted and the data were processed using quantification tools in MassLynx (Waters Corporation). We demonstrated that a measurement of 1.0-1000 ng/mL (r2 ¼ 0.995127, LLOQ ¼ 1.0 ng/mL) DOX in rat bile was achieved. This was 10 times more sensitive than the detection achieved using an acetonitrile precipitation technique for sample processing (LLOQ 10.0 ng/mL). In addition, the reproducibility of the measurement was significantly improved, the precision of both intra- and inter-run were below 15% CV. The bile concentration vs. time profile of DOX following a single intravenous administration at 3 mg/kg was obtained in rats, and the biliary excretion was calculated. The results are comparable with published data (Ballet F, Vrignaud P, Robert J, Rey C, and Poupon R; Hepatic extraction, metabolism and biliary excretion of doxorubicin in the isolated perfused rat liver. Cancer Chemother Pharmacol 19: 240- 245, 1987). In conclusion, combining the addition of blank plasma into bile with protein precipitation can be used successfully for drug concentration analysis in bile to support biliary excretion evaluation in drug development.
target. CA102N is a Nimesulide derivative (NiNH2) and sodium hyaluronate (HA) conjugate synthesized covalently through amidation reaction susceptible to lysosomal hydrolysis. In order to characterize CA102N with respect to identity, purity and stability, in this study a comprehensive analysis was performed using a variety of analytical techniques including NMR, FTIR, UPLC and LC-MS/MS. The presence of a newly formed covalent bond between the drug and HA was confirmed by 1 H-NMR and 13C-NMR. FTIR spectrum showed broad peaks at 1614, 1410, 1045 cm-1 indicative of C-O-C, C-O, C¼O stretching of HA and peaks at 1487~1612 cm-1 (aromatic C-C stretching) and 1319, 1213, 1153 cm-1 (C-N, S¼O stretching) unique to NiNH2. The drug content (% of DS) in the polymer conjugate was determined by 1 H-NMR and UPLC studies at 245 nm, specific spectrum of NiNH2. Free NiNH2 and rearrangement products were also quantified and shown to be <1% of total HA content, no other impurities were detected in CA102N samples. Further, enzymatic cleavage using chondroitinase AC, was employed for characterization and quantitative measurement of conjugated active moietie by LC-MS and LC-MS/MS. Several saccharideNiNH2 conjugates were identified as potential active metabolites of CA102N. CA102N was also demonstrated to be stable in plasma samples with <1% of free NINH2 release, crucial for the delivery of intact CA102N to target tumors.
P63 DRUG ACTIVITY SCREENING BASED ON MICROSOMES-HYDROGEL SYSTEM IN PREDICTING METABOLISM INDUCED ANTITUMOR ACTIVITY OF OROXYLIN A
Colorectal cancers have been known to easily metastasize to the liver and their hepatic metastases are responsible for the cause of death in half of those patients. Furthermore, only less than 10% of patients with hepatic metastasis can be salvaged by the liver resection, so currently, 10-year overall survival rate has been reported to be only 26%. In this research, we focus the oral siRNA delivery for the treatment of hepatic metastases colon cancer. Herein the gold nanoparticles (AuNPs) were used to link with siRNA by gold-thiol interaction. And the taurocholic acid (TCA), which is a kind of bile acids, was conjugated with glycol chitosan (GC) to assign the property for oral administration and delivery of the siRNA to hepatic site to gold nanoparticles. Animal model of hepatic metastases colon cancer was used to elucidate the therapeutic ability of fabricated AR-GT100 nanoparticle (GC-TCA coated-AuNP based siRNA complex). Ultimately, herein oral delivery of fabricated AR-GT100 nanoparticles suggested a possibility to treat various hepatic diseases, particularly metastatic colorectal cancer, via enterohepatic circulation.
Huiying Yang, Jianfeng Li, Yuanting Zheng, Lu Zhou, Shanshan Tong, Bei Zhao, Weimin Cai. Fudan University, Shanghai, China A novel microsomes-hydrogel added cell culture system (MHCCS) was employed in the antitumor activity screening of natural compounds, aiming to achieve drug screening with better in vivo correlation, higher initiative to explore the potential active metabolites, and investigation of the antitumor mechanism from the perspective of metabolism. MTT assay and cell apoptosis detection showed that test drug oroxylin A (OA) had enhanced cytotoxicity and W with reduced cytotoxicity on MCF-7 cell line upon MHCCS incubation. In vivo antitumor evaluations including tumor volume change and tumor apoptosis also demonstrated that OA induced higher tumor inhibition than W at the same dosage (40 or 60 mg/kg). UPLC-Q-TOF and semi-preparative HPLC were utilized for OA metabolites separation and characterization. Nine major metabolites were collected and their antitumor activities were screened using MTT assay. Metabolites M318 exhibited higher cytotoxicity than OA and other metabolites. 1H NMR spectrums, HPLC and TOF MS/MS results revealed that OA was catalyzed into its active metabolite M318 via a ring-opening reaction. M318 was unstable and continued to lose a molecular of water into the inactive metabolite M300. The antitumor mechanism of M318 was further explored by cell apoptosis, cell cycle arrest, and gene chip assays. M318 induced significant cell apoptosis and S-phase arrest through affecting tumor survival related genes. While, OA affected genes more related to tumor invasion and metastasis. In conclusion, our MHCCS could be a useful tool for drug activity screening from a perspective of metabolism. P64 CHARACTERIZATION OF CA102N, A HYALURONIC ACID CONJUGATE FOR THE TREATMENT OF COLORECTAL CANCER Eskouhie Tchaparian, David Healthcare, Taipei, Taiwan
Chu.
Preclinical
Research,
DRUG
HolyStone
Hyaluronic Acid conjugated drugs (HACD), are drug delivery systems designed for preferential transport of active pharmacological agent to their
P65 ORAL HEPATIC CANCER THERAPY BY GOLD NANOPARTICLE BASED SIRNA COMPLEX WITH BILE ACID TRANSPORTER Sung-Hun Kang 1, Eun Ju Hyun 1, Yong-kyu Lee 1, Kwang Jae Cho 2. 1 Korea National University of Transportation, Chungju, South Korea; 2 Uijeongbu St. Mary’s Hospital, Uijeongbu, South Korea
P66 PHARMACOKINETIC CONTROL OF CYCLOSPORINE A EMPLOYING NOVEL NANO-MATRIX PARTICLES FOR INHALATION Hideyuki Sato 1, Hiroki Suzuki 1, Keisuke Yakushiji 1, Jennifer Wong 2, Yoshiki Seto 1, Robert Prud'homme 3, Hak-Kim Chan 2, Satomi Onoue 1. 1 University of Shizuoka, Shizuoka, Japan; 2 The University of Sydney, New South Wales, Australia; 3 Princeton University, Princeton, NJ, USA This study was undertaken to evaluate the biopharmaceutical properties of cyclosporine A (CsA)-loaded nano-matrix particles for inhalation. Nanomatrix particles of CsA with mannitol (nCsAm) were prepared by a flash nano-precipitation technique employing a multi-inlet vortex mixer and evaluated in terms of physicochemical properties, anti-inflammatory effect in the rat model of airway inflammation, pharmacokinetic behavior, and distributions of CsA to side-effect-related organs after intratracheal administration. In nCsAm, spherical nano-particles of CsA were covered with mannitol and the mean particle size was 1.3 mm. The in vitro Next Generation Impactor analysis demonstrated very good inhalation performance with a fine particle fraction value of 65.8%. Intratracheal administration of nCsAm (100 mg-CsA/rat) significantly attenuated the recruitment of inflammatory cells into the airway in the rat model of airway inflammation, followed by suppression of the inflammatory biomarkers. After
S45
were compared, and their impacts on DOX drug detection by mass spectrometry was evaluated. The results showed that mixing bile matrix with blank plasma at a ratio of 1:3 (v/v), in combination with an optimized protein precipitation, resulted in a “cleaner” sample preparation than LLE or the protein precipitation approach alone. Fifty mL of the bile sample was mixed with 150 mL of blank rat plasma, and then 600 mL of acetonitrile was added. Following centrifugation, the resulting supernatant was diluted at a 1:4 ratio (v/v) with deionized water containing 30% acetonitrile and 0.1% formic acid. Five mL of the diluted sample was injected onto Phenomenex Kinetex 2.6 um Biphenyl 100A, 2.1 x 50.0 mm column using Acquity UPLC, followed by detection using a Xevo TQS LCMS system (Waters Corporation). Successive MRM experiments were conducted and the data were processed using quantification tools in MassLynx (Waters Corporation). We demonstrated that a measurement of 1.0-1000 ng/mL (r2 ¼ 0.995127, LLOQ ¼ 1.0 ng/mL) DOX in rat bile was achieved. This was 10 times more sensitive than the detection achieved using an acetonitrile precipitation technique for sample processing (LLOQ 10.0 ng/mL). In addition, the reproducibility of the measurement was significantly improved, the precision of both intra- and inter-run were below 15% CV. The bile concentration vs. time profile of DOX following a single intravenous administration at 3 mg/kg was obtained in rats, and the biliary excretion was calculated. The results are comparable with published data (Ballet F, Vrignaud P, Robert J, Rey C, and Poupon R; Hepatic extraction, metabolism and biliary excretion of doxorubicin in the isolated perfused rat liver. Cancer Chemother Pharmacol 19: 240- 245, 1987). In conclusion, combining the addition of blank plasma into bile with protein precipitation can be used successfully for drug concentration analysis in bile to support biliary excretion evaluation in drug development.
target. CA102N is a Nimesulide derivative (NiNH2) and sodium hyaluronate (HA) conjugate synthesized covalently through amidation reaction susceptible to lysosomal hydrolysis. In order to characterize CA102N with respect to identity, purity and stability, in this study a comprehensive analysis was performed using a variety of analytical techniques including NMR, FTIR, UPLC and LC-MS/MS. The presence of a newly formed covalent bond between the drug and HA was confirmed by 1 H-NMR and 13C-NMR. FTIR spectrum showed broad peaks at 1614, 1410, 1045 cm-1 indicative of C-O-C, C-O, C¼O stretching of HA and peaks at 1487~1612 cm-1 (aromatic C-C stretching) and 1319, 1213, 1153 cm-1 (C-N, S¼O stretching) unique to NiNH2. The drug content (% of DS) in the polymer conjugate was determined by 1 H-NMR and UPLC studies at 245 nm, specific spectrum of NiNH2. Free NiNH2 and rearrangement products were also quantified and shown to be <1% of total HA content, no other impurities were detected in CA102N samples. Further, enzymatic cleavage using chondroitinase AC, was employed for characterization and quantitative measurement of conjugated active moietie by LC-MS and LC-MS/MS. Several saccharideNiNH2 conjugates were identified as potential active metabolites of CA102N. CA102N was also demonstrated to be stable in plasma samples with <1% of free NINH2 release, crucial for the delivery of intact CA102N to target tumors.
P63 DRUG ACTIVITY SCREENING BASED ON MICROSOMES-HYDROGEL SYSTEM IN PREDICTING METABOLISM INDUCED ANTITUMOR ACTIVITY OF OROXYLIN A
Colorectal cancers have been known to easily metastasize to the liver and their hepatic metastases are responsible for the cause of death in half of those patients. Furthermore, only less than 10% of patients with hepatic metastasis can be salvaged by the liver resection, so currently, 10-year overall survival rate has been reported to be only 26%. In this research, we focus the oral siRNA delivery for the treatment of hepatic metastases colon cancer. Herein the gold nanoparticles (AuNPs) were used to link with siRNA by gold-thiol interaction. And the taurocholic acid (TCA), which is a kind of bile acids, was conjugated with glycol chitosan (GC) to assign the property for oral administration and delivery of the siRNA to hepatic site to gold nanoparticles. Animal model of hepatic metastases colon cancer was used to elucidate the therapeutic ability of fabricated AR-GT100 nanoparticle (GC-TCA coated-AuNP based siRNA complex). Ultimately, herein oral delivery of fabricated AR-GT100 nanoparticles suggested a possibility to treat various hepatic diseases, particularly metastatic colorectal cancer, via enterohepatic circulation.
Huiying Yang, Jianfeng Li, Yuanting Zheng, Lu Zhou, Shanshan Tong, Bei Zhao, Weimin Cai. Fudan University, Shanghai, China A novel microsomes-hydrogel added cell culture system (MHCCS) was employed in the antitumor activity screening of natural compounds, aiming to achieve drug screening with better in vivo correlation, higher initiative to explore the potential active metabolites, and investigation of the antitumor mechanism from the perspective of metabolism. MTT assay and cell apoptosis detection showed that test drug oroxylin A (OA) had enhanced cytotoxicity and W with reduced cytotoxicity on MCF-7 cell line upon MHCCS incubation. In vivo antitumor evaluations including tumor volume change and tumor apoptosis also demonstrated that OA induced higher tumor inhibition than W at the same dosage (40 or 60 mg/kg). UPLC-Q-TOF and semi-preparative HPLC were utilized for OA metabolites separation and characterization. Nine major metabolites were collected and their antitumor activities were screened using MTT assay. Metabolites M318 exhibited higher cytotoxicity than OA and other metabolites. 1H NMR spectrums, HPLC and TOF MS/MS results revealed that OA was catalyzed into its active metabolite M318 via a ring-opening reaction. M318 was unstable and continued to lose a molecular of water into the inactive metabolite M300. The antitumor mechanism of M318 was further explored by cell apoptosis, cell cycle arrest, and gene chip assays. M318 induced significant cell apoptosis and S-phase arrest through affecting tumor survival related genes. While, OA affected genes more related to tumor invasion and metastasis. In conclusion, our MHCCS could be a useful tool for drug activity screening from a perspective of metabolism. P64 CHARACTERIZATION OF CA102N, A HYALURONIC ACID CONJUGATE FOR THE TREATMENT OF COLORECTAL CANCER Eskouhie Tchaparian, David Healthcare, Taipei, Taiwan
Chu.
Preclinical
Research,
DRUG
HolyStone
Hyaluronic Acid conjugated drugs (HACD), are drug delivery systems designed for preferential transport of active pharmacological agent to their
P65 ORAL HEPATIC CANCER THERAPY BY GOLD NANOPARTICLE BASED SIRNA COMPLEX WITH BILE ACID TRANSPORTER Sung-Hun Kang 1, Eun Ju Hyun 1, Yong-kyu Lee 1, Kwang Jae Cho 2. 1 Korea National University of Transportation, Chungju, South Korea; 2 Uijeongbu St. Mary’s Hospital, Uijeongbu, South Korea
P66 PHARMACOKINETIC CONTROL OF CYCLOSPORINE A EMPLOYING NOVEL NANO-MATRIX PARTICLES FOR INHALATION Hideyuki Sato 1, Hiroki Suzuki 1, Keisuke Yakushiji 1, Jennifer Wong 2, Yoshiki Seto 1, Robert Prud'homme 3, Hak-Kim Chan 2, Satomi Onoue 1. 1 University of Shizuoka, Shizuoka, Japan; 2 The University of Sydney, New South Wales, Australia; 3 Princeton University, Princeton, NJ, USA This study was undertaken to evaluate the biopharmaceutical properties of cyclosporine A (CsA)-loaded nano-matrix particles for inhalation. Nanomatrix particles of CsA with mannitol (nCsAm) were prepared by a flash nano-precipitation technique employing a multi-inlet vortex mixer and evaluated in terms of physicochemical properties, anti-inflammatory effect in the rat model of airway inflammation, pharmacokinetic behavior, and distributions of CsA to side-effect-related organs after intratracheal administration. In nCsAm, spherical nano-particles of CsA were covered with mannitol and the mean particle size was 1.3 mm. The in vitro Next Generation Impactor analysis demonstrated very good inhalation performance with a fine particle fraction value of 65.8%. Intratracheal administration of nCsAm (100 mg-CsA/rat) significantly attenuated the recruitment of inflammatory cells into the airway in the rat model of airway inflammation, followed by suppression of the inflammatory biomarkers. After