Development and multicenter validation of an LC-MS-based bioanalytical method for antisense therapeutics.

Background: Many bioanalytical methods for antisense oligonucleotides (ASOs) using LC-MS have been reported. However, no data have been available on the reproducibility and robustness of a single bioanalytical method for ASOs. As such, in the current study, we evaluated the reproducibility and robustness of LC-MS-based bioanalytical methods for ASOs in multiple laboratories. Methods/Results: Seven independent laboratories were included in this study. Mipomersen was measured by ion-pairing LC-MS (IP-LC-MS) as a model ASO using different LC-MS. The validation results of calibration curve, accuracy, precision and selectivity met the criteria of conventional bioanalytical method validation guidelines using LC/GC-MS for drugs in all laboratories. Meanwhile, carryover (>20%) was detected in three laboratories. Conclusion: We first demonstrated the multicenter-validated IP-LC-MS bioanalytical method for ASOs. Our data showed that the method was sensitive, robust and reproducible. However, the occurrence of carryover should be carefully monitored in its future application.

Development of a bioanalytical method for an antisense therapeutic using high-resolution mass spectrometry.

Background: Ion-pairing reverse-phase LC coupled with high-resolution mass spectrometry (IP-LC/HRMS) has gained attention in oligonucleotide therapeutic bioanalyses owing to its high sensitivity and selectivity. However, optimization and validation of IP-LC/HRMS-based methods are rare. The objective of this study is the development of a sensitive and reproducible IP-LC/HRMS-based bioanalytical method using clinically approved mipomersen as a model for antisense oligonucleotides. Materials & methods/results: Mipomersen was extracted from rat plasma using Clarity OTX SPE and quantified by IP-LC/HRMS. The calibration range was 0.5-250.0 ng/ml. The developed method met the general regulatory criteria for accuracy, precision, carry-over, selectivity, matrix effect and dilution integrity. Conclusion: A highly sensitive and reliable method for mipomersen measurement with potential antisense oligonucleotide bioanalysis applications has been developed.

Lipid profiling of pre-treatment plasma reveals biomarker candidates associated with response rates and hand-foot skin reactions in sorafenib-treated patients.

Sorafenib is a multi-kinase inhibitor for treatment of advanced hepatocellular carcinoma (HCC). Beyond its clinical benefit against advanced HCC, the efficacy and safety of sorafenib chemotherapy are critical concerns. In this study, we addressed the lipid profiles associated with the efficacy and safety of sorafenib chemotherapy. Plasma samples from HCC patients before sorafenib chemotherapy (N = 44) were collected and subjected to lipidomic analysis. We measured the levels of 176 lipids belonging to 8 classes of phosphoglycerolipids, 2 classes of sphingolipids, 3 classes of neutral lipids, and 4 other classes of lipids. To characterize lipids associated with efficacy, we compared the responder group (N = 21; partial response and stable disease) with non-responder group (N = 22; progressive disease). To characterize lipids associated with hand-foot skin reaction (HFSR), we compared the susceptible group (N = 12; grade 2 and 3) with non-susceptible group (N = 32; grade 0 and 1). The levels of 8 lipids, including phosphatidylcholine (PC)[34:2], PC[34:3]a, PC[35:2], PC[36:4]a, PC[34:3e], acylcarnitine (Car)[18:0], cholesterol ester[20:2], and diacylglycerol (DG)[34:2], were significantly lower in the responder group, and 6 out of 8 these lipids contained FA(18:2). In addition, the levels of 7 lipids (Car[12:0], Car[18:0], Car[18:1], Car[20:1] and fatty acid amides (FAA[16:0], FAA[18:0], and FAA[18:1]b)) were significantly lower in the group susceptible to HFSR. Our comprehensive lipidomics study using samples from sorafenib-treated patients with HCC revealed that significant differences in the lipid profiles of pre-treatment plasma were associated with sorafenib efficacy and sorafenib-induced HFSR. Validation using another set of patient plasma samples and elucidating the molecular basis of these changes will lead to better treatment with sorafenib chemotherapy.

Ether-phosphatidylcholine characterized by consolidated plasma and liver lipidomics is a predictive biomarker for valproic acid-induced hepatic steatosis.

Valproic acid (VPA) is known to induce hepatic steatosis due to mitochondrial toxicity in rodents and humans. In the present study, we administered VPA to SD rats for 3 or 14 days at 250 and 500 mg/kg and then performed lipidomics analysis to reveal VPA-induced alteration of the hepatic lipid profile and its association with the plasma lipid profile. VPA induced hepatic steatosis at the high dose level without any degenerative changes in the liver on day 4 (after 3 days dosing) and at the low dose level on day 15 (after 14 days dosing). We compared the plasma and hepatic lipid profiles obtained on day 4 between the VPA-treated and control rats using a multivariate analysis to determine differences between the two groups. In total, 36 species of plasma lipids and 24 species of hepatic lipids were identified as altered in the VPA-treated group. Of these lipid species, ether-phosphatidylcholines (ePCs), including PC(16:0e/22:4) and PC(16:0e/22:6), were decreased in both the plasma and liver from the low dose level on day 4, however, neither an increase in hepatic TG level nor histopathological hepatic steatosis was observed at either dose level on day 4. Hepatic mRNA levels of glycerone-phosphate O-acyltransferase (Gnpat), which is a key enzyme for biosynthesis of ePC, was also decreased by treatment with VPA along with the decrease in ePCs. In conclusion, the changes in ePCs, (PC[16:0e/22:4] and PC[16:0e/22:6]), have potential utility as predictive biomarkers for VPA-induced hepatic steatosis.

Arachidonic acid-containing phosphatidylcholine characterized by consolidated plasma and liver lipidomics as an early onset marker for tamoxifen-induced hepatic phospholipidosis.

Lipid profiling has emerged as an effective approach to not only screen disease and drug toxicity biomarkers but also understand their underlying mechanisms of action. Tamoxifen, a widely used antiestrogenic agent for adjuvant therapy against estrogen-positive breast cancer, possesses side effects such as hepatic steatosis and phospholipidosis (PLD). In the present study, we administered tamoxifen to Sprague-Dawley rats and used lipidomics to reveal tamoxifen-induced alteration of the hepatic lipid profile and its association with the plasma lipid profile. Treatment with tamoxifen for 28 days caused hepatic PLD in rats. We compared the plasma and liver lipid profiles in treated vs. untreated rats using a multivariate analysis to determine differences between the two groups. In total, 25 plasma and 45 liver lipids were identified and altered in the tamoxifen-treated group. Of these lipids, arachidonic acid (AA)-containing phosphatidylcholines (PCs), such as PC (17:0/20:4) and PC (18:1/20:4), were commonly reduced in both plasma and liver. Conversely, tamoxifen increased other phosphoglycerolipids in the liver, such as phosphatidylethanolamine (18:1/18:1) and phosphatidylinositol (18:0/18:2). We also examined alteration of AA-containing PCs and some phosphoglycerolipids in the pre-PLD stage and found that these lipid alterations were initiated before pathological alteration in the liver. In addition, changes in plasma and liver levels of AA-containing PCs were linearly associated. Moreover, levels of free AA and mRNA levels of AA-synthesizing enzymes, such as fatty acid desaturase 1 and 2, were decreased by tamoxifen treatment. Therefore, our study demonstrated that AA-containing PCs might have potential utility as novel and predictive biomarkers for tamoxifen-induced PLD. Copyright © 2017 John Wiley & Sons, Ltd.

Development of a novel gene silencer pyrrole-imidazole polyamide targeting human connective tissue growth factor.

Pyrrole-imidazole (PI) polyamide can bind to specific sequences in the minor groove of double-helical DNA and inhibit transcription of the genes. We designed and synthesized a PI polyamide to target the human connective tissue growth factor (hCTGF) promoter region adjacent to the Smads binding site. Among coupling activators that yield PI polyamides, 1-[bis(dimethylamino)methylene]-5-chloro-1H-benzotriazolium 3-oxide hexafluorophosphate (HCTU) was most effective in total yields of PI polyamides. A gel shift assay showed that a PI polyamide designed specifically for hCTGF (PI polyamide to hCTGF) bound the appropriate double-stranded oligonucleotide. A fluorescein isothiocyanate (FITC)-conjugated PI polyamide to CTGF permeated cell membranes and accumulated in the nuclei of cultured human mesangial cells (HMCs) and remained there for 48 h. The PI polyamide to hCTGF significantly decreased phorbol 12-myristate acetate (PMA)- or transforming growth factor-ß1 (TGF-ß1)-stimulated luciferase activity of the hCTGF promoter in cultured HMCs. The PI polyamide to hCTGF significantly decreased PMA- or TGF-ß1-stimulated expression of hCTGF mRNA in a dose-dependent manner. The PI polyamide to hCTGF significantly decreased PMA- or TGF-ß1-stimulated levels of hCTGF protein in HMCs. These results indicate that the developed synthetic PI polyamide to hCTGF could be a novel gene silencer for fibrotic diseases.