Therefore, the aim of this study was to investigate the effect of HA on PDL cell compatibility, proliferation, and differentiation in vitro.Hyaluronic acid (HA) has been reported to have a positive effect on periodontal wound healing following nonsurgical and surgical therapy. However, to date, a few basic Either non-cross-linked (HA_ncl) or cross-linked (HA_cl) HA was investigated. Human PDL cells were seeded in 7 conditions as follows (1) Control tissue culture plastic (TCP) (2) dilution of HA_ncl (1:100), (3) dilution of HA_ncl (1:10), 4) HA_ncl directly coated onto TCP, (5) dilution of HA_cl (1:100), 6) dilution of HA_cl (1:10) and (7) HA_cl directly coated onto TCP. Samples were then investigated for cell viability using a live/dead assay, an inflammatory reaction using real-time PCR and ELISA for MMP2, IL-1 and cell proliferation via an MTS assay. Furthermore, the osteogenic potential of PDL cells was assessed by alkaline phosphatase(ALP) activity, collagen1(COL1) and osteocalcin(OCN) immunostaining, alizarin red staining, and real-time PCR for genes encoding Runx2, COL1, ALP, and OCN.Both HA_ncl and HA_cl showed high PDL cell viability (greater than 90%) irrespective of the culturing conditions. Furthermore, no significant difference in both mRNA and protein levels of proinflammatory cytokines, including MMP2 and IL-1 expression was observed. Both diluted HA_ncl and HA_cl significantly increased cell numbers compared to the controlled TCP samples at 3 and 5 days. HA_ncl and HA_cl in standard cell growth media significantly decreased ALP staining, COL1 immunostaining and down-regulated early osteogenic differentiation, including Runx2, COL1, and OCN mRNA levels when compared to control samples. When osteogenic differentiation medium (ODM) was added, interestingly, the expression of early osteogenic markers increased by demonstrating higher levels of COL1 and ALP expression; especially in HA 1:10 diluted condition. Late stage osteogenic markers remained inhibited.Both non-cross-linked and cross-linked HA maintained high PDL cell viability, increased proliferation, and early osteogenic differentiation. However, HA was consistently associated with a significant decrease in late osteogenic differentiation of primary human PDL cells. Future in vitro and animal research is necessary to further characterize the effect of HA on periodontal regeneration. Hyaluronic acid  is an anionic, nonsulfated glycosaminoglycan and considered an optimal biomaterial for tissue engineering, given its broad expression in a connective tissue as well as the significant role it plays during organogenesis, cell migration and development in general –4. Non-cDue to the growing use of HA in dentistry, HA has also been hypothesized to have influences on periodontal regeneration . The manFurthermore, other advantages of HA include its anti-inflammatory activity promoting soft and hard tissue healing response, which may be of significant interest during periodontal regeneration . Based oTherefore, the aim of the present study was to investigate the effects of HA_ncl and HA_cl on PDL cells by stimulating cells under 3 different conditions of either diluted samples with HA (co-existing at 2 concentrations of 1:10 and 1:100 dilutions) or directly by precoating HA onto tissue culture plastic. The PDL cells cultured with either HA_ncl or HA_cl were assessed for cell viability at 24 h, inflammatory cytokines expression at 1 day, cell proliferation at 1, 3 and 5 days and osteogenic differentiation marker expression at 7 days and 14 days.Streptococcus [HA was kindly provided by Regedent  utilizing 2 compositions of HA including non-cross-linked native HA  as well as a cross-linked HA . HyaDENT (HA_ncl) contains a formulation of 14.0 mg/mL of sodium hyaluronate  control TCP (2) dilution of HA_ncl (1:100), (3) dilution of HA_ncl (1:10), (4) HA_ncl directly coated, (5) dilution of HA_cl (1:100), (6) dilution of HA_cl (1:10) and (7) HA_cl directly coated, on chamber slides . At 1 day post cell seeding, cells were evaluated using a live-dead staining assay according to the manufacturer’s protocol  as previously described [Primary human PDL cells were seeded in at a density of 12,500 cells / cmescribed . FluoresPDL cells were seeded in 24-well plates at a density of 10,000 cells per well in a 24 well culture plate with the same conditions, (1) control TCP (2) dilution of HA_ncl (1:100), (3) dilution of HA_ncl (1:10), (4) HA_ncl directly coated onto TCP, (5) dilution of HA_cl (1:100), (6) dilution of HA_cl (1:10) and (7) HA_cl directly coated onto TCP. Cells were quantified using a fluorescent MTS assay  at 1, 3 and 5 days for cell proliferation as previously described . At desiPDL cells were first cultured for 1 day with HA in order to investigate its inflammatory marker expressions, including matrix metalloproteinase-2 (MMP2) and interleukin-1 (IL-1). Moreover, in order to investigate the effects of HA on the osteogenic differentiation, the cells were stimulated for 7 days within each concentration of HA with and without osteogenic differentiation medium (ODM), which consisted of DMEM supplemented with 10% FBS, 1% antibiotics, 50 μg/mL ascorbic acid (Sigma) and 10 mM β-glycerophosphate (Sigma) to promote osteogenic differentiation as previously described . The RNAThe supernatant culture media were collected at 1 and 3 days post cell seeding. MMP2  and IL-1β/IL-F2  were quantified using an ELISA assays  according to manufacturer’s protocol as previously described , 32. BriPDL cells were stimulated within each concentration of HA with and without ODM. At 7 days, cells were quantified for alkaline phosphatase expression utilizing a cell imaging system. Alkaline phosphatase activity was monitored using a Leukocyte alkaline phosphatase kit  as previously described . PDL celAt 14 days post PDL cell seeding, the cells were fixed with 4% formaldehyde for 10 min, followed by permeabilized within PBS containing 0.2% Triton X-100 and blocked in PBS containing 1% bovine serum albumin  for 1 h. Subsequently, cells incubated overnight at 4 °C either with a polyclonal rabbit to collagen type I antibody  or a polyclonal rabbit to osteocalcin antibody  at a dilution of 1:75 in PBS containing 1% BSA. After washing with PBS, cells were incubated for 1 h at 37 °C with TR-conjugated-goat-anti-rabbit antibodies  (1:100) diluted in PBS containing 1% BSA. Prior to viewing, samples were mounted with Vectashield containing DAPI nuclear staining . Images were captured from each surface with an OLYMPUS BX51 fluorescence microscope. The optical density (OD) of the fluorescent staining was quantified from 3 independent experiments using Image J software.Alizarin red staining was performed to determine the presence of extracellular matrix mineralization. PDL cells were stimulated for 14 days within each concentration of HA in ODM. After 14 days, cells were fixed in 96% ethanol for 15 min and stained with 0.2% alizarin red solution  in water (pH 6.4) at room temperature for 1 h as previously described . All imap values < 0.05 was considered significant) by GraphPad Prism 6.0 software .All experiments were performed in triplicate with three independent experiments for each condition. Mean and standard error (SE) were analyzed for statistical significance using a one-way analysis of variance with Tukey post hoc test  in the presence of HA at various concentrations Fig. . MoreoveThereafter, the primary human PDL cells were investigated for their ability to differentiate when cultured with HA_ncl and HA_cl Figs. , 6 and 7Moreover, it was generally found that HA_ncl significantly decreased ALP staining. HA_cl treatment demonstrated no significant changes in the ALP staining at 7 days likely as a result of the cross-linking HA Fig. . InteresCells were further investigated by OCN immunostaining and alizarin red staining when induced by HA_ncl and HA_cl treatment Figs. , and 7. In the present study, the effect of high molecular weight (HMW) HA_ncl and HA_cl were evaluated on in vitro periodontal ligament cell behavior. It was first found that both HA_ncl and HA_cl did not elucidate PDL cell apoptosis even at high concentrations Fig. . Both coWe then sought to investigate the inflammatory response of HA on PDL cells. It has previously been shown that low molecular weight  HA, but not the native HMW HA molecules , stimulated inflammatory cells . NakatanThereafter, the effects of HA demonstrated an increase in PDL cell proliferation Fig. . During The effect of HMW HA on cell differentiation has however had controversial findings. To date, most studies reported that cell differentiation was increased by low-MW HA , 41–43. In the present study, the expression of osteogenic markers stimulated by HA_ncl and HA_cl was investigated with and without ODM. Both HA_ncl and HA_cl down-regulated Runx2, COL1a2 and OCN mRNA levels, suggesting that HA treatment of PDL cells inhibits osteogenesis in a regular cell culture medium Fig. . MoreoveFor periodontal regeneration, HMW HA has previously been investigated in a dog intrabony defect model and neither significant periodontal tissue nor bone tissue regeneration was observed . InteresBoth HA_ncl and HA_cl were shown to be extremely biocompatible and at both concentrations were associated with a significant increase in PDL cell numbers. The early osteogenic differentiation markers such as Runx2, COL1 and ALP were significantly downregulated in standard cell growth media. Interestingly, with the addition of ODM, the expression of early osteogenic markers was shown to significantly increase COL1 and ALP levels, especially at higher concentration (1:10) utilizing both HA_ncl and HA_cl diluted condition. Nevertheless, the late stage osteogenic marker such as OCN as well as calcium formation was inhibited regardless of the ODM addition. Future in vitro models including in inflammatory conditions, as well as animal research, are necessary to further characterize the optimal uses as well as delivery systems of HA for improved clinical use.


The present study aims to investigate the protective effects of ω-3 polyunsaturated fatty acids (ω-3PUFAs) against high-fat diet induced male mouse reproductive dysfunction and to explore circadian regulation mechanisms. Male C57BL/6 mice were randomly divided into three groups and fed a normal chow diet , a high-fat diet (HFD group) or a HFD supplemented with fish oil (FO group) for 12 weeks. After 12 weeks of feeding, the body weight and the ratio of perinephric and epididymal fat weight to body weight were significantly higher in the HFD group compared with the CON group. The supplement of fish oil rich in ω-3PUFAs only slightly reduced the HFD-induced obesity but remarkably ameliorated HFD-induced dyslipidemia, sexual hormones disorder, testicle lesions and germ cell apoptosis. Fish oil supplementation restored the expression of steroid synthesis associated genes in HFD fed mouse and flattened the HFD-induced oscillations in circadian genes’ expression. Fish oil supplementation prevented HFD-induced male mouse reproductive dysfunction and modified the rhythmic expression of testosterone synthesis related genes. P450scc) is the key rate-limited enzyme in testosterone synthesis, catalyzes the oxidative cleavage of the side-chain of cholesterol to form pregnenolone [3β-Hsd) and reduced by 17β-hydroxysteroid dehydrogenase (17β-Hsd) to yield testosterone [Star and P450scc, leading to reproductive dysfunction [Star and P450scc and to improve testosterone synthesis, thereby modifying reproduction [Lipid metabolism is closely associated with reproduction. Epidemiological data have revealed that chronic high fat intake is accompanied by a rise of the incidence of metabolic diseases, such as obesity and non-alcoholic fatty liver disease, as well as the morbidity of reproductive dysfunction . The relnenolone ,9,10. Thosterone . Some stoduction . Our preoduction .Bmal1, Clock, Per1/2, Cry1/2, Rev-erbα, Pparα, Sirt1 and Rorα, construct a transcriptional/translational feedback loop to control the circadian clock [Most mammalian activities, including the sleep–wake cycle, hormone secretion, energy metabolism and immune response, present typical circadian rhythms and are regulated by the body’s internal clock ,15. The an clock . The rhyan clock .The quantity and components of daily food play crucial roles in reproductive rhythms . In normIn the current study, we aimed to investigate whether fish oil ameliorates high-fat-diet-induced male sexual hormones metabolic chaos and reproductive disorder in a rhythm-dependent way and to explore the effects of fish oil on the circadian expression of genes involved in testosterone synthesis and testicle function in high-fat diet fed male mice.After only 4 weeks’ feeding, the body weight of mice in both the high-fat diet (HFD) and fish oil (FO) groups increased significantly compared with the control diet (CON) group a. After The ratio of testes weight/body weight decreased in the HFD and FO groups compared with the CON group a. The epp < 0.001) and which was restored in the FO group. The apoptosis index was found to be higher in the HFD group compared with the control group; after fish oil was added, the apoptosis index went down markedly and no significant difference was seen between the FO group and the CON group  and slightly reduced with the intake of fish oil  (p < 0.05) and the expression of Bmal1, Clock genes was rescued by fish oil feeding. However, no differences were found at ZT12 between the three groups. The rhythmic expression of clock genes between ZT0 and ZT12 were analyzed. The testicle mRNA levels of Bmal1, Clock, Cry1, Cry2, Rorα and Pparα genes at ZT0 and ZT12 showed no remarkable differences in the CON group (p > 0.05). In the HFD group, all the examined clock genes presented oscillated expression (p < 0.05) and the oscillations of Bmal1, Clock, Per1, Cry1, Rorα and Pparα genes were abated in the FO group  and Sf-1 pathways. To our knowledge, this is the first finding that high-fat feeding disturbed normal testicular rhythms, disrupted the circadian expression of clock core genes and steroidogenesis-related gene expression in testicles, which may lead to reproductive dysfunction. Furthermore, the protective effects of fish oil against a high-fat diet and the induced disorder of testosterone synthesis, may be partly attributed to the rescue of testicular rhythms.In conclusion, fish oil rich in ω-3PUFAs can ameliorate the chaos of sexual hormones synthesis in high-fat induced obese male mice by increasing the number of Leydig cells and inhibiting apoptosis in reproductive cells and then stimulating the expression of w/w) (HFD group), a fish-oil-rich diet with 6.5% fish oil (w/w) and a total of 42 kcal% fat with 0.5% cholesterol (w/w) (FO group). Detailed information on these diets is presented in This animal study was conducted according to the Guidelines for the Care and Use of Experimental Animals, and the protocol was approved by Laboratory Animal Ethics Committee of Wuhan Polytechnic University (ID Number: 20160709003) on 9 July 2016. Male C57BL/6 mice  were purchased from the Center for Disease Control of Hubei province  and were housed in a 12 h day/night cycle with a room temperature of 22 °C ± 1 °C. Mice were randomly allocated to three groups  and each group was fed one of the following three diets for 12 weeks: a normal chow diet with 10 kcal% fat (CON group), a high fat diet with 42 kcal% fat and 0.5% cholesterol (2 suffocation. Blood was distributed into a heparinized tube (1.5 mL) and centrifuged at 1000× g for 15 min at 4 °C and plasma was collected and stored at −80 °C until analysis. Concurrently, the testes, perinephric fat and epididymal fat were rapidly removed, rinsed with 0.9% sodium chloride solution and weighed. Isolated testes were kept at −80 °C or fixed in 10% neutral buffered formalin.At the end of 12th week of feeding, mice were sacrificed by way of COPlasma TC , TG , estradiol  and testosterone  were determined using respective kits according to the manufacturer’s instructions.Fixed testes were processed for HE staining according to a previously described method . In eachTotal RNAs from mouse testes were extracted using the TRIzol reagent . cDNA was synthesized using the primeScript™ RT Reagent Kit . A 20μL amplification reaction consisted of iTaq Universal SYBR Green Supermix  with 300 nM of both reverse and forward primers. All reactions were performed on the RT-qPCR system . The thermal cycling conditions were for 2 min at 50 °C and 3 min at 95 °C, followed by 40 repeats at 95 °C for 15 s, 60 °C for 20 s and 72 °C for 30 s. Primer sets used for q-PCR are listed in 2 (pH 7.4), 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1mM phenylmethylsulfonyl fluoride (PMSF), 10 mM NaF and 1 mM Na3VO3 and centrifuged at 15,000× g for 15 min. Protein concentrations were measured using the bicinchoninic acid (BCA) assay . Equal quantities of protein (20–40 µg) were separated with denaturing sodium dodecyl sulphate 10% polyacrylamide gels under reducing and denaturing conditions and were subsequently transferred onto a polyvinylidene difluoride (PVDF) membrane . Membranes were blocked with 5% (w/v) bovine serum albumin (BSA) and 0.1% Tween in Tris-buffered saline (pH 7.4) at 37 °C for 2 h, followed by hybridization with an anti-LHR , anti-STAR , anti-P450SCC , anti-β-actin  primary antibody (1:1000 dilution) at 4 °C overnight. After washing three time in Tris-buffered saline containing 0.1% Tween 20, membranes were hybridized with a horseradish peroxidase-conjugated rabbit immunoglobulin G secondary antibody for 1 h at room temperature. Specific protein expression levels were normalized to β-actin for total protein analyses. The blot was detected with the ChemiDoc™ MP System detection system . The membranes were scanned and the sum optical density was quantitatively analyzed by Image Lab software .Testicular samples were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA-Nap < 0.05.Data are presented as mean ± SD. A one-way ANOVA and Tukey’s post hoc analysis were conducted to establish significant differences. All analyses were performed with Graphpad Prism  statistical software. Differences were considered statistically significant at


Strain J5 is phylogenetically far from well-known representatives within the Bradyrhizobium japonicum linage. The complete genome showed the largest single chromosomal (10.1 Mb) and symbiosis island (998 kb) among complete genomes of soybean bradyrhizobia.Soybean bradyrhizobia form root nodules on soybean plants and symbiotically fix N Bradyrhizobium diazoefficiens and B. japonicum, in temperate areas  approach  with default parameters. The final draft assembly produced two contigs totaling 10.3 Mb in size and with an input read coverage of 28×. Row reads generated from the Illumina MiSeq sequencing procedure were trimmed using CLC Genomics Workbench version 8.5.1  with the following parameters: ambiguous limit, 2; quality limit, 0.05; number of 5′ terminal nucleotides, 20; number of 3′ terminal nucleotides, 5; and minimum number of nucleotides in reads, 70. Trimmed whole-genome shotgun reads were mapped to contigs to update them, with the following CLC Genomics Workbench parameters: mismatch cost, 2; insertion cost, 3; deletion cost, 3; length fraction, 0.5; similarity fraction, 0.8; and no masking. As a result, a single circular contig was assembled. The sequence was annotated using the NCBI Prokaryotic Genome Automatic Annotation Pipeline (http://www.migap.org) and GenomeMatcher  from the nodule of a soybean plant grown in Shibetsu City in Hokkaido, Japan, in 1998. The genome was sequenced using the PacBio RSII  and Illumina MiSeq  platforms. Genomic DNA libraries were prepared for PacBio sequencing using the SMRTbell template prep kit version 1.0, while Illumina libraries were prepared using the Nextera DNA sample preparation kit (Illumina). The resulting reads, generated from the PacBio RSII sequencing procedure, were assembled http://ggdc.dsmz.de/distcalc2.php) (B. japonicum strain J5 and strains USDA6 and E109 (0.0441 to 0.0439) was apparently larger than that between USDA6 and E109 (0.0003), indicating that J5 was far from the well-known representatives within the B. japonicum linage in terms of genomics as well. As for denitrification genes relevant to global warming  and 9,067 coding sequences (CDSs). The genome size and CDS numbers were the highest among complete genomes of soybean bradyrhizobia. The length of symbiosis island A ( warming , the J5 B. japonicum strain J5 has been deposited at the GenBank under the accession number CP017637.The genome sequence of


Escherichia coli, Salmonella enterica, Salmonella enteritidis, and Salmonella choleraesuis and spasmolytic effect on contraction on rat ileum trips were determinate. Eight organic extracts (hexanic and methanolic) of albedo (mesocarp) and flavedo (pericarp) of two varieties  of Citrus sinensis (L.) Osbeck of Yucatán, México, were studied. Additionally, chromatographic fingerprints were obtained and correlated with their pharmacological effects. MAN, MAV, and HFN extract caused inhibition against S. choleraesuis (MIC: 1000 µg/mL) and S. enteritidis (MIC: 1000 µg/mL). Regarding the spasmolytic effect, the Valencian extracts variety was more efficient on spontaneous contraction, HAV (Emax = 51.98 ± 1.98%), MAV (Emax = 35.98 ± 1.42%), HFV (Emax = 68.91 ± 4.14%), and MFV (Emax = 51.28 ± 2.59%), versus National variety, HAN (Emax = 43.80 ± 6.32%), MAN (Emax = 14.62 ± 1.69%), HFN (Emax = 64.87 ± 3.04%), and MFN (Emax = 31.01 ± 3.92%). Chromatographic fingerprints of HFV and HFN were found to have some similar signals that belong to monoterpenes, whereas for HAN and HAV similar signals were found belonging to fatty acids and triterpenoids. Methanolic extracts showed signals of (1) furfural, (2) furfural acetone (3) furfuraldehyde and (4) β–sitosterol compounds. Flavedo portion of C. sinensis possessed spasmolytic effect on rat ileum strips and antibacterial activity against Salmonella strains. This species is source for obtaining bioactive compounds with therapeutic potential in the treatment of infectious diarrhea.Antibacterial activity on ATCC strains of Citrus sinensis (L.) Osbeck or sweet orange is consumed all over the world for being an excellent source of vitamin C and for its powerful antioxidant properties that build up the immune system. The orange fruit is composed of three sections, an external layer (peel), named flavedo, epicarp, or exocarp, a white portion below the exocarp, named albedo or mesocarp, and the innermost portion, the endocarp, that contains vesicles with juice and seeds [ C. sinensis cultivated in Yucatán.Diarrhea is the passage of three or more loose or liquid stools per day, or more frequently than normal for the individual. It is usually a symptom of gastrointestinal infection, which can be caused by a variety of bacterial, viral, and parasitic organisms . Severe nd seeds , 6. Orannd seeds . These bnd seeds –11. RepoPapaverine HCl, dimethyl sulfoxide (DMSO), and amikacin were purchased from Sigma-Aldrich Co. . Ethylic ether, n-hexane, and methanol were purchased from High-Purity Co.  ACS grade. Stock solutions of extracts were made using distilled water and freshly prepared the same day of experimentation. Citrus sinensis (L.) Osbeck National and Valencian varieties were collected in a crop local in Akil, Yucatán, México (20°14.9′N and 98°20.1′W) in December 2009. Plant material was authenticated by Salvador Flores Guido, PhD, from the Botany Department of Facultad de Veterinaria y Zootecnia of Universidad Autónoma de Yucatán (UADY). Voucher herbarium specimens were obtained and a specimen plant was deposited at UADY's herbarium “Alfredo Barrera Marín.”Fruits of Citrus sinensis National and Valencian varieties were successively extracted in a soxhlet apparatus first with n-hexane and then with methanol; the solutions were concentrated to dryness in a rotary evaporator  at 45°C. Extraction yields  were for hexanic extract of albedo National variety , methanolic extract of albedo National variety , hexanic extract of flavedo National variety , methanolic extract of flavedo National variety , hexanic extract of albedo Valencian variety , methanolic extract of albedo Valencian variety , hexanic extract of flavedo Valencian variety , and methanolic extract of flavedo Valencian variety . To carry out the experiments, all extracts were dissolved in a mixture of water: DMSO. Final concentration of DMSO inside the organ chamber never exceeded 0.1%.Firstly, mature fruits were washed with distilled water and, then, flavedo portion (peel) was separated mechanically, to get the albedo portion (white portion beneath the peel). The juice was extracted and the vegetal material was dried in a herbal desiccator at 50°C for three days to be grounded. Dried albedo (210 g) and flavedo (425 g) of Escherichia coli (ATCC 128), Salmonella enterica (ATCC 14028), Salmonella enteritidis (ATCC 22177), and Salmonella choleraesuis (ATCC 10708). The bacterial stock cultures were incubated for 24 h at 37°C on nutrient agar. Stocks cultures were retained at −20°C to use.The microorganisms used in the present investigation included reference strains from American Type Culture Collection (ATCC), Citrus sinensis (L.) Osbeck National and Valencian varieties were evaluated for antimicrobial activity against Escherichia coli, Salmonella enterica, Salmonella enteritidis, and Salmonella choleraesuis using the microdilution minimum inhibitory concentration (MIC) and minimum bacterial concentration (MBC) assays [The eight organic extracts  of) assays , 13. Theμg/mL with DMSO as diluent. The microtiter plates were prepared adding 100 μL of sterile nutrient broth into each well. Thereafter, the organic extracts and the positive control (Amikacin 12.5 μg/mL) were added at a volume of 100 μL. The organic extracts were serially diluted to reach concentrations of 1000, 500, 250, 125, 62.5, 31.2, 15.6, 7.8, 3.9, 1.9, 0.97, and 0.48 μg/mL. Negative control was also included (nutrient broth with DMSO).The eight organic extracts were diluted to a concentration of 1000 μL of inoculum (at 0.5 McFarland) was added to each well and the plates were incubated for 24 h at 37 ± 1°C. After incubation, 100 μL of INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride) at 0.02% was added to the reaction mixture, and then it was incubated at 37 ± 1°C in orbital shaking for 30 minutes. The interaction of the microorganisms (when viable) with INT gives rise to a color change from colorless to a reddish-pink color. The wells with the lowest dilution changes observed were considered as the MIC for these tested samples.Finally, 100 Healthy male Wistar rats were used and maintained under standard laboratory conditions with free access to food and water. All animal procedures were conducted in accordance with our Federal Regulations for Animal Experimentation and Care  and appr2PO4, 1.2; MgSO4, 1.2; CaCl2, 1.5; NaHCO3, 20; and glucose 11.4; pH, 7.4) constantly bubbled with an O2 : CO2 (95 : 5) mixture. Changes in tension were recorded by force transducers Grass-FT03  connected to analyzer MP150  as described previously by Estrada-Soto et al., 2010 [Rats (250–300 g body weight) were killed and abdominal dissection was carried out to extract the ileum. It was cleaned from excrement and adjacent and connective tissue and then cut into strips 2 cm long. Then, the tissue sections were assembled using stainless steel hooks under an optimal tension in chambers at 37°C containing Krebs-Henseleit solution . The effect of organic extracts and positive control on spontaneous contraction of ileum rings was determined by comparing the mean of the muscular tone and frequency inscribed by tissue contractions before and after addition of the test materials. Muscular tone was calculated from the tracings using Acknowledge Software (BIOPAC 4.1).Tissue segments (≈2 cm) were placed in organ baths containing 14 mL of KHS. All tissues were assembled with stainless steel hooks under an optimal tension of 1 g in organ baths with KH solution. After equilibration (15 min), a 10 min control period was recorded. The eight organic extracts , positive control , and vehicle (DMSO 1%) were added to the bath in a volume of 100  post hoc test. p < 0.05 was considered to imply significance of the pharmacological effects in the experiments.The experimental results are expressed as mean of five experiments ± standard error of mean (SEM). Concentration responses curves (CRC) were plotted, and experimental data in the CRC were adjusted using the fit-sigmoidal (Hill equation) in the program Microcal™ Origin 8.6 . Statistical analysis was conducted using one-way ANOVA, followed by Tukey'sμL of the hexane solution (5 mg/mL) containing the extract was injected in split mode (50 : 1 ratio by 1 min) in a gas chromatograph  equipped with a mass selective detector  and a fused silica capillary column  of 30 m × 0.25 mm × 0.25 μm coated with cross-linked 5% phenyl-95% methyl polysiloxane. High purity (>99.999%) helium was used as carrier gas, at 0.8 mL/min with constant pressure. The oven temperature was programmed, 45°C for 3 min, and then increased 4°C/min to 250°C and stood by for 5 min; then a last increase of 20°C/min to 325°C took place, with a total time of 80 min for the analysis of each extract.All the extracts were derivatized with boron trifluoride-methanol (BTF/MeOH) for gas chromatography analyses. 5 mg of each extract was added to 10 mL of BTF/MeOH and the mixture was heated under reflux for 20 min, and then the solutions were partitioned with 5 mL of n-hexane. 1 Citrus sinensis (L.) O., commonly known as “sweet orange,” has been used for hundreds of years for its medicinal and nutritional properties [ C. sinensis fruits are a main source of important phytochemical nutrients and for a long time they have been valued for their wholesome nutritious and antioxidant properties. Bactericide activity of flavedo portion has been investigated previously but albedo (mesocarp) is less investigated.operties . C. sine C. sinensis L. (Osbeck) are known. Their albedo and flavedo were subject to extraction process exhaustively and subsequently were evaluated for bactericidal activity and ex vivo spasmolytic effect. Additionally, chromatographic fingerprints of each extract were obtained and helped to correlate the pharmacological activity with the presence of secondary metabolites.In Yucatán, México, two varieties of Citrus sinensis (L. Osbeck) National and Valencian varieties were studied in different concentrations  against four bacterial strains . The results of the antibacterial activities are presented in  Citrus sinensis (L. Osbeck) National variety presented significant activity. The HFN had antimicrobial activity against S. enteritidis presenting MIC values of 1000 μg/mL and MAN had activity against S. choleraesuis also with a MIC of 1000 μg/mL. Regarding the extracts of Citrus sinensis (L. Osbeck) Valencian variety, only MAV had activity against S. enteritidis with a MIC of 1000 μg/mL. The MBC was not determined for any of the strains tested as the required concentrations of the extracts were above the concentrations examined.The antibacterial activities of the eight organic extracts  of Citrus sinensis National variety extracts . The CRC of hexanic extract of flavedo (HFN) is significantly shifted to the left when compared to HAN, MAN, and MFN. HFN was more efficient (Emax = 64.87 ± 3.04%) and potent (EC50 = 300 μg/mL) than HAN , MAN , and MFN . On the other hand,  C. sinensis Valencian variety extracts . Again, the hexanic extract of flavedo (HFV) was more efficient (Emax = 68.91 ± 4.14%) and potent (EC50 = 287.46 μg/mL) compared with HAV , MAV , and MFV  and it was also more powerful than HFN . All extracts of Valencian variety were more efficient that National variety as shown in Emax = 95%).Many compounds eluted from capillary gas chromatography in each extract, so the chromatograms were divided into three sections. The first one includes chemical compounds with the lowest molecular weights (MW) as monoterpenes and sesquiterpenes; the second part consists of compounds with middle MW, mainly fatty acids between C14 to C18, and the last section consists of compounds with the highest MW containing triterpenoidic molecules. According to the spasmolytic effect and antibacterial activity of hexanic flavedo extracts from both varieties were significantly more efficient and potent on spasmolytic effect than other extracts. We compared fingerprints of hexanic extracts. Chromatographic fingerprint analysis of HFV and HFN suggests the presence of monoterpenes , fatty acids , and triterpenic  compounds, respectively. The comparison of chromatographic fingerprints of Valencian (HFV) and National (HFN) hexanic flavedo extracts showed similar signals in the chromatogram .β-sitosterol acetate, (10) β-sitosterol, (11) stigmastan-3-5-diene, and the last one (12) β-tocopherol.The monoterpenes (1) 1,3,8-p-methatriene, (2) D-limonene, (3) dehydrocymene, and (4) thujol were eluted in the first section (low MW) around minutes 10 to 25. In the second section  four main fatty acids were found: (5) palmitic, (6) linoleic, (7) oleic and (8), stearic. In the last section  four signals were identified as triterpenoids: (9) β-Sitosterol and Stigmastan-3-5-diene, only representing 2% of the total area of the chromatogram. The albedo extracts fingerprints (HAN and HAV) were significantly different from flavedo extracts, especially in the first section, which corresponded to small molecules with monoterpenic structure. The fingerprints of HAV and HAN  that were less efficient on the ex vivo assay revealed monoterpenes , fatty acids , and triterpenoids .The fingerprint analysis from hexanic albedo extracts (HAN and HAV) only showed a few signals in the second section of the chromatogram . These sβ-sitosterol.On the other hand, albedo (MAN and MAV) and flavedo (MFN and MFV) methanolic extracts  showed sThe methanolic extracts of both varieties showed higher yields in the extraction process compared to hexanic extracts. It has been described that metabolic content is greater in medicinal plants extracts obtained with solvents of medium to high polarity such as methanol . Waxes a Salmonella sp. and E. coli. Similar data is reported by Ou et al., 2015, where antibacterial activities of cold-pressed and distilled essential oils of C. paradise and C. grandis (L.) Osbeck resulted in a MIC 20 mg/mL for Salmonella sp. and E. coli [μg/mL for HFN, MAN, and MAV on S. enteritidis and S. choleraesuis. On the other hand, hexanic flavedos of both varieties showed significant spasmolytic effect (>60%) on ileum strips isolated from rat when compared to hexanic albedos and methanolic extracts.Chanthaphon et al., 2008, evaluated the antimicrobial activity of essential oils of kaffir lime, lime, pomelo, acidless orange, Chugun orange, and Nech kumquat , obtaini E. coli . Compare2+ influx [Chromatographic fingerprint analysis of HFV and HFN suggests the presence of monoterpenes, fatty acids, and triterpenic compounds in higher proportion with respect to HAV and HAN  and it is related to pharmacological activity. Flavedo's extract of national and Valencian varieties showed an efficient spasmolytic effect, possibly related to triterpenic or monoterpene compounds, since they are in greater quantity with respect to hexanic extracts of albedo (HAV and HAN). The antibacterial activity is only related to the amount of monoterpenes since the flavedo hexanic extract of national variety (38.2% of monoterpenes) was more active than flavedo hexanic extract of Valencian variety (8.1% of monoterpenes). Antimicrobial properties, anti-inflammatory activity, antiulcer activity, and antifungal activity has been reported in triterpenic compounds . Also Pr+ influx . Monoter+ influx . Several+ influx –25. De S+ influx . Besides+ influx .Citrus sinensis (L.) Osbeck National variety had significative spasmolytic ex vivo effect and antibacterial activity due to presence of monoterpenic and triterpenic compounds in flavedo portion. This species represents an important source for obtaining bioactive compounds with therapeutic potential in the treatment of infectious diarrhea.


More importantly, β1 integrin expression was significantly correlated with patient outcome. In summary, our study indicated that β1 integrin could regulate TNBC cells migration, invasion, drug sensitivity, and be a potential prognostic biomarker in TNBC patient survival.Triple negative breast cancer (TNBC) displays higher risk of recurrence and distant metastasis. Due to absence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), TNBC lacks clinically established targeted therapies. Therefore, understanding of the mechanism underlying the aggressive behaviors of TNBC is required for the design of individualized strategies and the elongation of overall survival duration. Here, we supported a positive correlation between β1 integrin and malignant behaviors such as cell migration, invasion, and drug resistance. We found that silencing of β1 integrin inhibited cell migration, invasion, and increased the sensitivity to anti-cancer drug. In contrast, activation of β1 integrin increased cell migration, invasion, and decreased the sensitivity to anti-cancer drug. Furthermore, we found that silencing of β1 integrin abolished Focal adhesion kinese (FAK) mediated cell survival. Overexpression of FAK could restore cisplatin-induced apoptosis in β1 integrin-depleted cells. Consistent to in vitro data, β1 integrin expression was also positively correlated with FAK ( Triple negative breast cancer (TNBC) is a specific breast cancer subtype that is immunohistochemically negative for expressions of prognostic markers including estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) . TNBC acIntegrins, which are transmembrane receptors in a large family of 18α and 8β heterodimeric transmembrane proteins, were well known as adhesion molecules in mediating cell-extracellular matrix (ECM) interaction . IntegriRecent attention has been focused on the role of β1 integrin in malignant phenotype . PreviouTo analyze the role of β1 integrin on TNBC cells, we first compared the basal expression of β1 integrin in normal human mammary epithelial cell (M10) and three TNBC cells . The effects of β1 integrin on TNBC cells motility were investigated by using a standard wound-healing assay. As shown in To further verify the role of β1 integrin in contributing cell mobility, we next determined the role of β1 integrin in the process of EMT. As performed in In order to further clarify the role of β1 integrin in TNBC cells, we next examined if β1 integrin plays a regulatory role in the drug sensitivity of TNBC cells. To further characterize the details of the mechanism in β1 integrin-mediating drug sensitivity, we next sought to identify the downstream signal pathway involved in TNBC. As shown in p = 0.039, 0.038, and 0.004 respectively).High expression of β1 integrin has been reported in many cancer types but is still unclear in TNBC. In order to confirm our in vitro data, immunohistochemical staining was used to investigate the relationships between β1 integrin and other clinicopathological parameters in biopsy specimens from 67 TNBC patients. The patients had an average age of 51 ± 10 years (range 32–81 years) and an average tumor size of 2.5 ± 1.5 cm (range 0.13–7.5 cm). p = 0.002). The univariate analysis (p = 0.0004), nodal stage (p < 0.0001), metastatic stage (p = 0.0019), and tumor recurrence (p = 0.0436). Multivariate Cox regression analysis of β1 integrin expression, age, tumor grade, nodal stage, metastatic stage, and tumor recurrence showed that β1 integrin expression was a significant independent predictor of overall survival (p = 0.0476). Taken together, the experimental data indicate that β1 integrin has potential use as biomarker of TNBC survival and as a biomarker of TNBC cell migration, invasion, and drug resistance.We further investigated whether β1 integrin expression was associated with patient survival. The Kaplan-Meier test was used to compare the survival time between patients with high and low β1 integrin expression. analysis  revealedHigh level of β1 integrin has been associated with poor outcomes in many types of tumors including colon cancer, pancreatic cancer, lung cancer, ovarian cancer and breast cancer . PreviouBreast cancer is a heterogeneous disease. Seeking and developing a personalized therapy is an important issue in improving outcomes of clinical patients. Pre-clinical and clinical data have suggested that TNBC could be more sensitive to platinum-based chemotherapy in patients with defective homologous recombination repair. However, several randomized trials in evaluating platinum efficacy indicated conflicting results for long-term outcomes . TherefoThe dissemination of cancer cells from the primary tumor, and their invasion in and out of blood vessels has been linked to EMT-related processes . TherebyBesides, β1 integrin is associated with drug resistance in several cancer types ,45,46. FIn summary, we reported that high β1 integrin expression is correlated with low survival rates and advanced metastatic status in TNBC patients. β1 integrin also plays a regulatory role in TNBC cell migration, invasion, and EMT. Additionally, β1 integrin could regulate drug sensitivity in TNBC cells. Taken together, our study clearly indicates that β1 integrin could not only be a potential prognostic biomarker in TNBC patients, but targeting β1 integrin-FAK-AKT axis could be an effective therapeutic strategy in β1 integrin overexpressing TNBC cells as well. It has been showed that the level of β1 integrin is strictly regulated by miRNA ,49. Here2 atmosphere. TNBC cancer cells   and H184B5F5/M10 human mammary epithelial cell  were used in this study. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) and DMEM/F12 medium with 10% fetal bovine serum (FBS)  and antibiotics at 37 °C in a 5% COAntibodies against β1 integrin , FAK , phospho-FAK(Y397) , Akt , phospho-Akt (S473) , Caspase 3 , cleaved PARP , p44/42 MAPK (ERK1/2) , phospho-p44/42 MAPK  , Vimentin , N-Cadherin , β-Catenin , GAPDH  and E-Cadherin  were purchased from Cell Signaling Technology . Mouse IgG1 negative control  was from Biolegend . Anti-β1 integrin activating antibody (MAB1951Z (P4G11), 10 μg/mL) was obtained from Millipore Corporation .TM mammalian protein extraction buffer, Thermo Scientific, Rockford, IL, USA) and cellular debris was removed by centrifugation. Proteins were loaded on an SDS-polyacrylamide gel and transferred to nitrocellulose, and immunoblotting was performed by using the indicated antibodies.Proteins extraction and immunoblotting were performed as previously described . In brieShort interfering RNA (siRNA) for human β1 integrin  and negative control  were purchased from Dharmacon . MDA-MB-231 and HS578T cells were transfected with 100 nM nontargeting and specific siRNA using Lipofectamine 2000 and Opti-MEM medium according to the standard protocol provided by Invitrogen . Constitutively active EGFP-FAK expression constructs were a gift obtained from Chen Hong-Chen’s lab . β1 integrin silencing cells were transfected with the appropriate amount of expression construct and control empty vector using Lipofectamine 2000 and Opti-MEM medium as mention earlier.4 cells were plated in each well of a 24-well plate. After treated with different doses of cisplatin, cells were stained with MTT solution  and incubated for 1–2 h. Finally, the formazan crystals were solubilized in 200μL DMSO  and measured at 560 nm.1 × 102 incubator for 24 h. Images were captured at 0 and 24 h. The experiments were repeated in triplicate.Cells of each group were cultured in 6-well plates. The confluent cell monolayer was made a wound area by a 200 μL pipette tip. Cells were then incubated in culture medium at 37 °C in a 5% COCell invasion assay were performed as previously described . In brieFormalin-fixed, paraffin-embedded blocks of tissues from 67 TNBC patients were selected from the Department of Pathology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan. Institutional Review Board approval for using these human tissues in this study was given by the Research Ethics Committee of the Kaohsiung Medical Hospital (IRB: KMUHIRB-E(II)-20150086) on 22 June 2015.Blocks of tissue samples embedded in paraffin were sectioned into thicknesses of 4 μm. The samples were de-paraffinized, rehydrated, and then autoclaved at 121 °C for 9 min in pH 6.0 DAKO target retrieval solution  to induce antigen retrieval. In each section, endogenous peroxidase was blocked by incubation in 3% hydrogen peroxide  for 10 min. Sections were incubated with β1 integrin and FAK  primary antibodies at room temperature for 1 h. The DAKO REAL Envision Detection kit  was then applied for 1 h. Finally, sections were incubated in 3’3-diaminobenzidine for 5 min, counterstained with Mayer’s Hematoxylin, and mounted. Negative controls were prepared by replacing the primary antibodies with non-immune serum. β1 integrin expression in different patients was scored according to Yao et al.  in whicht test was used to compare the difference of groups. P value less than 0.05 was considered statistically significant. All statistic analyses were performed using SPSS 19.0 software .Expression of β1 integrin in TNBC tissues as determined by immunohistochemical staining was compared and assessed by Chi-square test. To evaluate the use of β1 integrin for TNBC prognosis, survival curves were performed by Kaplan-Meier method. A Cox proportional hazards model was used to evaluate univariate comparisons of overall survival with clinicopathologic variables. Two-tailed Student’s


N-acetyl galactosamine  is a high-affinity ligand for hepatocyte-specific asialoglycoprotein receptors. Conjugation with GalNAc3 via a trishexylamino (THA)-C6 cluster significantly enhances antisense oligonucleotide (ASO) potency. Herein, the biotransformation, disposition, and elimination of the THA cluster of ION-681257, a GalNAc3-conjugated ASO currently in clinical development, are investigated in rats and monkey. Rats were administered a single subcutaneous dose of 3H-radiolabeled (3H placed in THA) or nonradiolabeled ION-681257. Mass balance included radiometric profiling and metabolite fractionation with characterization by mass spectrometry. GalNAc3-conjugated ASOs were extensively distributed into liver. The THA-C6 triantenerrary GalNAc3 conjugate at the 5′-end of the ASO was rapidly metabolized and excreted with 25.67 ± 1.635% and 71.66 ± 4.17% of radioactivity recovered in urine and feces within 48 hours postdose. Unchanged drug, short-mer ASOs, and linker metabolites were detected in urine. Collectively, 14 novel linker associated metabolites were discovered including oxidation at each branching arm, initially by monooxidation at the β-position followed by dioxidation at the α-arm, and lastly, tri and tetra oxidations on the two remaining β-arms. Metabolites in bile and feces were identical to urine except for oxidized linear and cyclic linker metabolites. Enzymatic reaction phenotyping confirmed involvement of N-acetyl-β-glucosaminidase, deoxyribonuclease II, alkaline phosphatase, and alcohol + aldehyde dehydrogenases on the complex metabolism pathway for THA supplementing in vivo findings. Lastly, excreta from monkeys treated with ION-681257 revealed the identical series as observed in rat. In summary, our findings provide an improved understanding of GalNAc3-conjugated-ASO metabolism pathways which facilitate similar development programs.Triantennary  Mechanisms of action of ASOs are comprehensive and rely on the oligonucleotide chemistry, and other aspects, including the selected target mRNA binding site and may include RNase H degradation, 5′capping inhibition, and/or translation arrest.N-acetyl galactosamine  is a targeting ligand for ASOs which displays pronounced affinity and selectivity to the asialoglycoprotein receptor, a major cell surface receptor in hepatocytes. The design of GalNAc3 as a major targeting ligand is such that the oligonucleotide is functionalized with a tri-carbohydrate cluster in a tri-antennary format of established dimensions to allow recognition and enhanced binding affinity to facilitate hepatocyte uptake. GalNAc3-ASO conjugates  are administered subcutaneously with a rapid rate of uptake and eventual endocytotic clearance by the asialoglycoprotein receptor. A variety of GalNAc3-oligonucleotide conjugates with different spatial geometries have shown great promise effectively widening the therapeutic window for treatment of several genetic diseases.4 Targeted delivery using GalNAc3-conjugated ASOs has been shown to enhance hepatocyte potency greater than 10-fold in animals and likewise, numerous other investigations for RNA therapeutics exploit the GalNAc delivery platform to achieve enhanced receptor binding.8Triantennary 9 Hence, the distribution, metabolism, and fate of unconjugated ASOs are well established, yet the metabolic pathway of the THA cluster containing GalNAc3 as a targeting moiety has never been reported and is important to help understand the efficacy and safety of the conjugated ASO.In light of these observations, and based on internal data, previous animal studies revealed that GalNAc sugars and the trishexylamino (THA) cluster were rapidly cleaved within minutes to hours from GalNAc-conjugated ASOs following subcutaneous (SC) administrations, releasing unconjugated ASOs in liver to exhibit their pharmacological effects. It is well understood that unconjugated ASOs are slowly metabolized to chain-shortened oligonucleotide metabolites by nucleases and eliminated in urine.11 Such studies with radiolabeled drug for mass balance determinations remain a strong desire before new chemical entities expand into larger phase-3 clinical trials.12 As such, metabolic profiling offers early risk estimations and guides drug synthesis to improve stability, identify soft-spots of lead compounds, reduce in vivo degradation potential, and better ensure nonreactive intermediates, nonbioactivation, and reduce adverse drug reactions. Drug metabolism remains a critical issue for drug development and concerns regarding drug metabolism safety studies are warranted.14 Preclinical radiolabel mass balance studies remain invaluable and indispensable allowing a comprehensive and cohesive understanding of the in vivo behavior and action of drugs prior to exposure in large clinical studies.16 Clearly understanding the metabolic fate of 5′THA-GalNAc3-ASOs offers important insights into the extent of linker absorption, disposition, rate and routes of clearance mechanisms. Better assessments of the necessity for safety and/or relevant drug–drug interaction (DDI) studies are identified which lead development programs into a more complete path to overall product safety and successful clinical translation.Metabolite proportionality and cross-species evaluations are essential for early drug development and assist preclinical safety studies that expedite clinical development.in vivo models and in vitro phenotyping to describe the role of disposition, metabolism and excretion and definite major elimination pathways of GalNac-conjugated ASOs are provided. Our report aims to provide a detailed account into (i) describing absorption, disposition, rate and route of elimination by mass balance, (ii) radiochromatographic profiling and characterization of metabolites by mass spectrometry, (iii) comprehensive identification of metabolizing enzymes, (iv) describing the overall biotransformation pathway, and lastly, (v) a cross species determination of common metabolic fate in rats and monkey. Herein, this study attempts to describe a comprehensive investigation into the biotransformation of the THA cluster on ION 681257, a 2′-O-(2-methoxyethyl) modified mixed PS/PO backbone ASO conjugated with a THA-C6-triantennary GalNAc3 at the 5′-end, targeting human lipoprotein (a) (Apo(a)) in rats and monkeys .In the current work, for the first time, mechanistic investigations using both 3H-ION 681257 and tissue distribution are listed in Figure 2, (n = 2 rats). By total mass balance and compared with the percentage of normalized dose excreted in urine and feces it was found that 25.67 ± 1.635% and 71.66 ± 4.17% of the radioactive dose were accounted for by renal and biliary excretion, respectively. Radioactivites in both whole blood and plasma including both a 24- and 168-hour timepoint made up less than 0.1% of the cumulative radioactivity. Rapid disposition was evident with nearly complete excretion by 24 hours. As observed in Figure 2b, <3% of dose remained in major tissues (liver and kidney) at both 24 and 168 hours (n = 2 rats). Sample excreta including urine and feces were extracted and diluted to100,000 disintegrations per minute per 20 µl injection and assayed by radio-high-performance liquid chromatography (HPLC) detection using both basic and acidic mobile phase conditions. Metabolites observed contained ionizible functional groups evident at varying pH conditions. Chromatographic separation and near baseline resolution for most of the major metabolites were obtained using a shallow reversed phase gradient with most all species eluted prior to the introduction of (35%) acetonitrile in the gradient . The intact dosing solution profile was not altered by pH; however, mostly for all of the metabolites pH-dependent effects evident by a shift in retention time across the chromatogram were observed .Mass balance (percentages) of cumulative radioactivity following a single SC dose of The radiochromatographic profile for urine was distinguishable from bile and feces with a few similar shared peaks toward the end of the gradient, however, bile and feces were very near identical. Two significant peaks for bile and feces were obtained under acidic conditions . However, using a basic buffer system , metabolites were substantially more polar and resolved well. Fraction collection of all of the major metabolite species was then performed for both the basic and acidic buffer system to evaluate the total distribution of radioactivity for each purified species. The pH-dependent separation determined by radiochromatography was also confirmed by liquid scintillation counting (LSC) of the fractionated metabolites. Bile duct cannulation was also examined to evaluate bile excreta from 0 to 2 hours, and 6 to 8 hours to determine the relative rate of metabolite formation.− with an averaged full mass corresponding to m/z 8,635. An abundance of nuclease cleaved species into short-mer oligonucleotides was also observed and corresponded to major ([M−H])− mass to charge values of m/z 2,867, 3,187, and 3,532. The remaining material was accounted for by an early eluting species; although unidentified, we suspect a small and highly polar fragment of the THA linker.A separate experiment using male Sprague Dawley rats was investigated with rats divided into four dosing groups for single-dose SC administration of saline, 7.5, 30, or 100 mg/kg of nonradiolabeled ION 681257. Urine and feces were collected between 0 and 6 and 6–24 hours postdose. Following fractionation of all major species using the same chromatographic separation as described previously, each separate metabolite fraction was assayed by liquid chromatography mass spectrometry (LC–MS). As revealed in the mass spectra of urine extracts, the major species was determined to be intact ASO-conjugate, identical to that in the dosing solution. The full mass spectra charge envelope for the intact parent conjugated oligonucleotide was observed at various charge states ([M−H])+ representing the major species  were extracted and combined into a single chromatogram  with a clear pattern of major mass shifts of m/z ±14 and m/z ±48 present.Following fraction collection of bile and fecal samples of the high dose group, metabolites were assayed by LC–MS using a shortened gradient. High abundance masses were determined with similarities compared across each fraction of both bile and feces. Unique masses not present in control samples were tabulated. Major ions ([M+H])Figure 4a–h). In several cases, a distinctive similarity in the product ion spectra depicting common fragmentation ions was detected. A major nonoxidized cyclized linker (M5) relating to the THA cluster was observed to fragment to [M+H]+ in ESI (+) at m/z 721.4, corresponding to a loss of 189 Da while monooxidized cyclic linker (M6) is observed to have two large fragment ions with losses of both (189 and 203 Da) corresponding to both the loss of a single nonoxidized and monooxidized branching arm (positions A and either b1–b3). Major metabolite (M7), a dioxidized linker, was shown to have a major loss of 203 Da alone  corresponding to fully oxidized branching arms as terminal oxidations to carboxylic acids. Product ion spectra of the proposed cyclic metabolites were seemingly much more resistant to ion bombardment even at higher collision energies. Product ion spectra patterns are comparable for the noncyclized metabolite series and focused on the cleavage of various branching arm substituents from the major cluster . Noncyclic metabolite series from (M8) are feasible due to the presence or absence of distinctive fragmentation ions including losses of  in the series. Of note, when each substituent arm is fully oxidized the loss of 189 Da is absent . Product ion spectra reveal the initial oxidation on either of the b1–b3 branching arms, while the second oxidation occurs at position a1 while the third and fourth oxidations occur on either of the b1–b3 positions.The major metabolite series was evaluated using MS/MS fragmentation to obtain individual product ion spectra and fragmentation patterns , −2 (M2), and −3 (M3) sugars from the cluster . The intact drug substance in absence of β-Nag revealed a fully conjugated and intact GalNAc3–ASO complex with [M−H]− in ESI (−) revealed a major ion  corresponding to the −5 charge state observed in the full mass spectra. However, upon addition of β-Nag in the rapid incubation, [M−H]− masses corresponding to −1 sugar (M1) , −2 sugars (M2) , and −3 sugars (M3)  were observed, each as a distinctive loss of a sugar from the intact parent conjugate (P) .Enzymatic incubation of the intact parent GalNAc3-ASO (−0 sugar), in addition to −1 and −3 sugar standards corresponding to the conjugated-ASO absent two or all GalNAc moieties, respectively. Neat standards were incubated in the presence of DNase II for 0, 4, 8, and 24 hours to evaluate cleavage of the oligonucleotide from the cluster. From the total ion chromatogram depicted in  for the −0 sugar standard, following incubation with DNase, the intact conjugate is observed with the presence of numerous metabolites. The mass spectra reveal the cleavage of the oligonucleotide from the GalNAc3 cluster. High abundance full mass [M−H]− in ESI (−) revealed a (−5) m/z ion, 1,422.4, corresponding to the free ASO. The cleaved GalNAc3 cluster was observed  in addition to numerous sugar deletions of the linker cluster including the GalNAc3 cluster with −1 sugar deletion , and −2 sugar deletions . Moreover two high abundance peaks also observed in excreta extracts corresponded to two distinctive metabolites observed as m/z 926.7 corresponding to the linear (M8) with PO4 (M4), and m/z 908.6 as the hydrolyzed and stable cyclic linker (M5). Following incubation with DNase II for the −1 and −3 sugar standards, the chromatographic profile was quite different. The major products observed included the intact parent (−4) m/z, 2,005.1, free ASO (−5) m/z 1,422.3, and both metabolites identified from the previous set (M4) m/z 926.7, and (M5) m/z 908.6, this time in much greater abundance. Following peak abundance assignment for the various species in  it was found that the −0, −1, and −3 sugar standards formed esterified cyclic species 4.7-, 4.3-, and 6.0-fold greater than linear linker containing PO4 .Neat standards consisted of both intact-conjugated parent GalNAcFigure 6a with major peaks of interest near 14.2 minutes. Mass spectra of the −3 sugar standard treated with DNase II alone is observed in  depicting the major formation of both M4 and M5. DNase II treatment followed by alkaline phosphatase treatment results are observed in  and observation of [M+H]+ in ESI (+) reveals both the formation of m/z 848.6 (M8) linker in high abundance and the presence of cyclized linker m/z 910.5 (M5), both of which were also observed in urine, bile, and feces. These high abundance masses were likewise not observed in the corresponding control .Following incubation with DNase II using a neat −3 sugar standard, samples were protein precipitated and dried down. Substrates were then reconstituted in 1 mol/l diethanolamine buffer 0.40 mmol/l MgCl pH 9.8 and incubated with 10 U/ml of alkaline phosphatase for 2 hours at 37 °C. The total ion chromatogram of samples including time 0 controls is depicted in Figure 6e,f, a [M+H]+ in ESI (+) mass shift of m/z 2 was revealed, representing the oxidation of (M8) standard substrate (m/z 848.5) into the subsequent aldehyde (m/z 846.5). A separate reaction was run in parallel with the products from the initial ADH reaction reconstituted directly into 50 mmol/l 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) containing 1 mmol NAD+ and 1.4 U/ml of ALDH. Following a brief 2-hour incubation, a significant reduction of (m/z 846.5) and formation of both mono (m/z 862.5) and dioxidations (m/z 876.4) representing M9 and M10, respectively, were observed in the corresponding mass spectra . It is important to note that the oxidations occur on any of the b1–3 positions of M8.Metabolite (M8) standard was investigated as a potential substrate of alcohol dehydrogenase for oxidation into an aldehyde, which is further oxidized by ALDH. The initial reaction included an incubation of metabolite standard (M8), 1 mmol nicotinamide adenine dinucleotide (NAD+), with 76.4 U of ADH incubated for 2 hours at 37 °C. From the total ion chromatogram in 3-conjugated ASO, ION 681257 and feces were collected from 12 to 24 hours. The chromatographic profile revealed an initial series of oxidations (M5–7) corresponding to 909.5, 923.5, and 937.5 Da, respectively . In addition the similar ion pattern was observed in ESI (+) revealing the identical m/z ± 48 mass shift at m/z ± 14 intervals corresponding to metabolites (M8–12), 847.5, 861.5, 875.5, 889.5, and 903.5 Da. No accumulation of any of the metabolite series was observed in plasma following the multiple dosing regimens.A cross species comparison of monkey was evaluated following a 12 mg/kg SC administration of GalNAc3-conjugated-2′-MOE (methoxyethyl) ASO in rats, and monkey. To the best of our knowledge, to date this is the first report for GalNAc3 biotransformation . A rapid clearance of metabolites within the first 24 hours allowed effective in vivo and in vitro characterizations facilitating the identification of 14 novel GalNAc-ASO linker associated metabolites by radiometric and/or spectroscopic methods. Based on the fragmentation of metabolites, a common biotransformation pathway involving multiple oxidations was discovered. Apart from providing convincing evidence that helps us understand the elimination and disposition mechanisms and identify key metabolizing enzymes, and initial insight into the in vivo metabolic fate provides knowledge of potential pharmacological activity, cross species metabolite susceptibility, and overall metabolite safety.In this study, we have characterized the disposition and metabolism of ION-681257, a GalNAcn = 4) samples obtained from (n = 2) rats, was discovered in addition to the presence of short-mer oligonucleotides (15.0 ± 3.38%). As expected, short-mer formation from the ASO occurred rapidly as a result of 5′ and 3′-exo and endonuclease mediated metabolism seemingly more prevalent due to excess unbound free drug.20 Additionally, a pair of nonoxidized linker metabolites (M5 and M8),  and a single unidentified species (28.6 ± 2.35% of urine radioactivity) were discovered. Despite several attempts including varying column chemistries and chromatographic conditions, the single unidentified species in urine remained unaccounted for yet composed 7.35 ± 0.60 of total radioactivity by dose excreted. We might suspect the role of esterase activity on an amide bond of the conjugate forming a very polar and low molecular weight species, yet this is unconfirmed. We revealed the cyclized linker formation (M5) by an internal esterification and loss of water from linear intermediate (M4) from the intact parent, we also determine alkaline phosphatase responsible for generation of (M8). As a major accumulating and elimination organ, and in similar s.c. dosing studies in rats using3H-radiolabeled ASOs, the liver has been shown to contain the greatest proportion of dose administered.21 Thus, we suspected a large role of hepatic metabolism for THA linker and discovered major bile and feces metabolites including (M5) and (M8)  and a novel and unique series of oxidized metabolites as (M6–M7) and (M9–M12). A pattern with both a m/z ± 48, and m/z ± 14 mass shift in the spectra represented both the light- and heavy-oxidized series. The major mass spectral differences for the oxidized metabolite series were accounted for by the number of oxidations on each branching arm of the cluster with the total possibility of major ions representing losses of 189, 203, 213, and 227 Da.Our initial assessment revealed the presence of intact parent ASO-linker-conjugate in urine with minor linker related metabolites, along with significant metabolites and no parent conjugated or free ASO in bile and feces. We next sought to collect bile and evaluate metabolite excretion in bile to identify and distinguish from those formed in the gastrointestinal tract and excreted in feces. For this approach, bile duct cannulated rats were used to reveal the rate of metabolite formation and order of appearance. Seemingly the rate of metabolite formation in bile of (M5–7) from 0 to 2 hours was maximal compared with (M9–11). Numerous less abundant peaks were observed in the bile and feces radiochromatograms, likely as nonfully metabolized aldehyde intermediates related to (M5 and M8). In urine, a large abundance of intact, nonmetabolized, parent GalNAc-conjugated-ASO corresponding to (47.1 ± 3.76%) of urine radioactivity), , −2 (M2) and −3 (M3) sugars. DNase II revealed cleavage of the intact linker cluster from the oligonucleotide. DNase II incubation of the intact and minus sugar standards also revealed the formation of a reactive linear intermediate (M4) which we propose may undergo internal esterification and loss of H2O forming a cyclized linker (M5), also observed in urine, bile, and feces. The cyclized linker formation for the −0, −1, and −3 sugar standards had ~4.7, 4.3, and 6.0 fold higher abundances for the formation of esterified species (M5) compared with the linear intermediate (M4), (m/z +18) over 24 hours, respectively. We revealed that DNase II cleaves the oligonucleotide selectively leaving the −PO4 moiety on the linker cluster liberating free ASO.Following Reaction phenotyping using DNase II in an initial reaction followed by alkaline phosphatase revealed the formation of (M8) from linear linker (M4). Following DNase II incubation alone the presence of (M8) was negligible due to absence of alkaline phosphatase to hydrolyze (M4) into (M5) for (M8) formation. The profile revealed the prehydrolyzed linear linker (M4) as responsible for (M8) catalyzed by alkaline phosphatase. Following elimination of the GalNAc moiety from the linker as well as clevage of the linker cluster from the oligonucleotide, we demonstrated that alkaline phosphatase may generate major metabolite (M8) also seen in the urine, bile, and feces.m/z 2 units due to the conversion of (M8) to an aldehyde using alcohol dehydrogenase. Using this newly formed substrate in subsequent reactions with ALDH formed both mono and dioxidized metabolites (M9 and M10). We suspect the in vitro formation of tri and tetra-oxidized species to be much less abundant and more difficult to observe. Although not confirmed in vitro, we presume that (M5) can be further oxidized into metabolites (M6 and M7) as observed in vivo. Based on our findings, the conversion to an aldehyde by ADH and further oxidation by ALDH may generate the metabolites (M9–M12) and (M6–7) observed in the bile and feces of rats and monkey feces.Our final phenotyping study confirmed that alcohol dehydrogenase metabolized (M8) into an aldehyde prior to oxidation by ALDH. Metabolite (M8) endured a loss of 22 This observation is also in line with similar animal and human model evaluations for unconjugated 2′-O-(2-methoxyethyl) modified ASOs which likewise demonstrate strong consistency of PK/PD relationships across species.24 Comparisons of metabolite exposures in both rat and monkey evaluating proportionality allow better confidence for safety coverage in preclinical species.Lastly, we were able to confirm that the major metabolites formed in rat are also present in monkey feces, suggesting a similar primary pathway of metabolism.3-ASO-associated metabolites, with no detectable accumulation in plasma, we anticipate a much reduced potential for pharmacological activity and relative toxicity, with improved safety. Our drug metabolism research brings us strategically closer to complying with regulatory guidances regarding metabolite safety assessments set by the US Food and Drug Administration, in addition to the European Medicines Agency.26Ultimately multiple dosing studies provide essential information into accumulation properties and elucidate the necessity for drug–drug interaction studies. However, based on the short retention and rapid elimination of the 5′THA-GalNAc3-conjugated MOE ASO was well absorbed, rapidly distributed into liver tissues and exhibited extensive metabolism of the parent compound and linker conjugate. Based on the cumulative excretion of tritiated linker-related material in urine and feces, animals revealed at least 91.5% of dose was absorbed within the first 24 hours of dosing. An insignificant amount of radioactivity (<1%), and no major metabolites were recovered in circulation. Sugar deletions, oligonucleotide cleavage, and the formation of numerous oxidized species from the cluster were observed prior to biliary excretion (~71%) and renal excretion (~25%) following SC administration. Urine extracts contained the same metabolites except for those most extensively oxidized. Several GalNAc–ASO-conjugate metabolizing enzymes were identified in vitro including N-acetyl-β-glucosaminidase, DNase II, alkaline phosphatase, and alcohol and ALDH, each with seemingly similar metabolites formed from our in vivo findings. A total of four novel metabolites  were identified in the urine, and eight in the bile and feces . The primary clearance mechanism of linker in rats was found to be attributed to phase-1 oxidations on the terminal position of each branching arm of the linker cluster. The current study is the first account of 14 different metabolites detected both in vitro and/or in vivo with a similar metabolic profile obtained for two animal species.Taken together, this study revealed that ION-681257, a GalNAcMaterials. Potassium phosphate was purchased from Corning Life Sciences . HPLC-grade water, acetonitrile, methanol, formic acid, ammonium acetate, ammonium bicarbonate, dimethyl sulfoxide, ethylenediaminetetraacetic acid (EDTA), phenol:chloroform:isoamyl alcohol (25:24:1), triethylamine, hexafluoroisopropanol, triethylammonium bicarbonate, N-acetyl-β-glucosaminidase from jack beans, deoxyribonuclease II from porcine spleen (Lot 029K7360V), alkaline phosphatase (Lot SLBL7932V) from bovine intestinal mucosa, acid phosphatase (Lot 053M4028V) from potato, alcohol dehydrogenase (Lot SLBH7396V) from saccharomyces cerevisiae, and ALDH (Lot BCBK5982V) from bakers yeast were purchased from Sigma-Aldrich . NAD+ (Lot 01) was purchased from Selleck Chemicals LLC . Chlorox bleach was obtained from VWR Scientific . Luna 3 μm C18 (2), 2.0 × 150 mm, and 96-well Strata X polymeric reversed phase SPE cartridges (33 µm) were purchased from Phenomenex . Xbridge column  was purchased from Waters . Ultima Gold XR scintillation cocktail was purchased from PerkinElmer Life Science . Ultra free-MC 0.22 µm GV durapore centrifugal filter units were purchased from Merck Millipore . Gimble mount swivel holder, 12-inch tether, dual luer swivel, quick connect dual harness, extension line , and injection caps were purchased from SAI Infusion Technologies .Drug substance synthesis and radiolabeling. GalNAc3-conjugated 20mer ION 681257 targeting human lipoprotein (a) (Apo (a)) containing a 2′-O-(2-methoxyethyl) modified mixed phosphorothioate, phosphodiester (PS/PO) backbone was internally radiolabeled with [3H] incorporated into the glutaric acid moiety of the stable THA-C6 cluster and conjugated at the 5′ end. [3H]-ION 681257  was synthesized in-house at Ionis Pharmaceuticals  with a specific activity of 35.91 μCi/mg and a radiochemical purity of 90% determined by radio-HPLC. To minimize the potential for3H2O exchange, drug substance was formulated and mass balance studies were initiated within a week following synthesis. Chemical structure of the compound and tritium radiolabel position on the conjugate is provided in Figure 1. The comprehensive structure–activity relationship of the nonradiolabeled N-acetylgalactosamine-conjugated ASO has been described.27 Authentic standards of nonradiolabeled material including intermediate metabolite (M8), -conjugated ION 681257 in addition to intact GalNAc3-conjugated 681257 were all synthesized in-house as materials used for chromatographic and mass spectrometric assessment during the study, structures provided in Table 1.In vivo mass balance and radiometric assessment of 3H-GalNAc3-ASO (ION 681257). All animals and procedures conducted in the study were in compliance and approved by the Ionis Pharmaceuticals Institutional Animal Care and Use Committee (protocol: P-0226-100028). Male Sprague Dawley rats aged between 8 and 9 weeks (250–300 g) were obtained from Charles River Laboratories . The rats were maintained on a 12-hour light dark cycle housed at 23 °C with 70% humidity with access to food and water ad libitum. Rats were weighed the day of dosing and administered a single SC dose of 3H-ION (681257) at 4.3 mg/kg at a specific activity of 35.9 µCi/mg and total radioactivity of 38.2 µCi per rat using a dosing volume of 1 ml/kg at the nape of the neck. Three male rats were used in study with two dosed equally as described including one as a saline control. Rats were housed separately into assigned metabolic cages and urine, feces, and cage washes were collected separately including a predose, 0–6, 6–24, and every 24 hours up to 1 week for radiometric measurements. Whole blood was collected at 24 hours and 1 week at the time of necropsy in addition to major and minor tissues. Rats were anesthetized using isoflurane followed by euthanization by cardiac puncture and cervical dislocation. Whole blood was collected into K2 EDTA tubes and immediately centrifuged at 2,000g for 10 minutes to collect plasma. Weights of urine, feces, cage rinses, plasma, and tissues were recorded and a 100 µL or 100 mg aliquot of urine, cage wash, plasma, or tissue homogenate was added directly into liquid scintillation vials with the addition of 5 ml Ultima Gold XR scintillation cocktail for LSC. Whole blood was processed by weighing 100–150 mg into sctinillation vials with 2 ml of soluene 350 and 2 ml of isopropyl alcohol with incubation at 55 °C for 5 hours and cooled overnight. Then 550 µl of 30% H2O2 was added and incubated at 55 °C for 30 minutes, after cooling 100 µl was added into 10-mL cocktail buffer and light adapted for 4 hours. Feces were diluted 4× (w/v) with 200 mg in filter sterilized purified water and homogenized into a uniform paste. Samples were then decolorized, solubilized with 2 ml of Clorox bleach and incubated at 55 °C for 5 hours. After solubilization, a 100-µl aliquot was added directly to liquid scintillation vials with the addition of 10-ml Ultima Gold XR scintillation cocktail and were maintained in the dark overnight prior to LSC. Sample aliquots were further evaluated by HPLC with radiometric detection for bioanalytical method development to profile metabolites.In vivo non-radiometric evaluation, dosing with GalNAc3-ASO (ION 681257). Male Sprague Dawley rats (n = 4) between 250 and 300 g were divided into four dosing groups with one saline dosing vehicle control and one for each dose group at a 7.5, 30, and 100 mg/kg dose, respectively. Rats were dosed using a single SC administration using a dosing volume of 1 ml/kg and immediately housed into individual metabolic cages as described previously with access to food and water ad libitum. Urine and feces from each rat were collected between 0–6 and 6–24 hours postdose. At the end of the study, rats were sacrificed as previously described.Bile duct cannulated rat study. Male Sprague Dawley rats between 250 and 300 g with bile duct and duodenal catheterizations were obtained from Charles River Laboratories with dual portharnesses. Five dosing groups were used including a saline dosing vehicle control, a 7.5, 30, and 100 mg/kg dose of ION-681257, respectively, in addition to 3H-ION (681257) dosed at 20 mg/kg with 60 µCi of total administered radioactivity. Predose bile was collected from each rat and all compounds were administrated as a single SC dose to the nape of the neck. For each rat (n = 1), a tether was attached to the bile port with the duodenal port capped for bile flow through an extension line held by a tether. The line was then passed through a swivel so that the outlet collection line outlet maintained below each rat with sampling tubes maintained on ice. Bile was collected from each rat in 2-hour intervals between 0–2, 2–4, 4–6, and 6–8 hours, respectively, with a collection rate of ~2.0 ml per hour. A 2 µl aliquot of each sample from the3H-ION (681257) dosing group was added in combination with 5.0 ml of scintillation cocktail for LSC to determine total radioactivity and percent dose excreted. At the end of the study rats were euthanized. See Supplementary Materials and Methods for additional details.Sample preparation and isolation of metabolites for LC–MS/radiochromatography. Initial method development was performed using a Waters 2690 Alliance HPLC Separations Module, Waters  coupled with a v2 radiometric detector, LabLogic . During radiometric profiling, all extracts including  were first assayed by liquid scintillation individually to obtain respective total radioactive counts. Samples were then diluted accordingly to standardize and normalize the radioactivity of each sample to 100,000 disintegrations per minute per 20 µl injection, or 5,000 disintegrations per minute/µl. A 100-µl aliquot of urine collected between 0 and 6 hours from both 4.3 mg/kg 3H-ION (681257) dosed rats containing roughly 500,000 disintegrations per minute was added to separate Ultra free-MC 0.22 µm GV durapore centrifugal filter units and centrifuged for 4 minutes at 10,000g at room temperature.Bile obtained from3H-ION (681257) dosed rats at 20 mg/kg with 60 µCi of total administered radioactivity was also processed in the same manner. All filtrates were removed and dried down under ambient temperature using a Speed-Vap evaporator, Horizon Technology . An aliquot of feces from rats dosed with 4.3 mg/kg 3H-ION (681257) was weighed to 120 mg and 1.5 ml of extraction buffer containing (methanol and H2O), 80:20 volume by volume (v/v) was added.28 Approximately 100 mg of ceramic beads was added to each tube and samples were pulverized for 2 minutes. Tubes were then centrifuged at 2,000g for 5 minutes. The supernatant was removed, with all samples added to centrifugal units, and recentrifuged at 10,000g for 4 minutes, followed by filtrate evaporation. All samples  were then reconstituted with 100 µL of mobile phase at a 95:5 v/v solution of (A1 or A2) with B1, respectively, which consisted of either (A1), , or (A2), , with (B) acetonitrile, for both, respectively. A Luna 3μm C18 (2) 2.0 × 150-mm column maintained at 25 °C using an injection volume of 20 µl at an initial flow rate of 0.3 ml/minute was used. For chromatographic separation of metabolites, a gradient elution was setup programming 5% B from T0–2, 8% B at T8, 11% B at T14, 14% B at T20, 17% B at T26, 20% B at T32, 23% B at T38, 26% B at T44, 29% B at T50, 95% B at T50.01 with a 4-minute hold at 95% and a 10-minute equilibration at initial conditions. Fraction collection every 2-minute intervals using the same gradient was performed to further isolate and purify metabolites with the entire eluent from each fraction added to 5 ml of Ultima Gold XR scintillation cocktail for LSC to evaluate radioactivity.Profiling, structure elucidation, and characterization of metabolites by LC–MS. Structure elucidations were performed in several ways including radio-chromatography, liquid chromatography mass spectrometry (LC–MS), and tandem mass spectrometry (MS–MS). Structure elucidation of metabolites was initially evaluated by scanning for a range of biotransformation possibilities.29 Individual fraction collected extracts, each consisting of 2-minute collected intervals were evaluated by LC–MS using an Agilent 1200 series binary pump liquid chromatography system interfaced with an Agilent 6520 Quadrupole Time-of-Flight Mass Spectrometer (QTof-MS), .g with all samples injected and evaluated separately. The initial mobile phase for negative polarity ionization consisted of 25 mmol/l ammonium bicarbonate solution at pH 7.0 and acetonitrile, 95:5 v/v, and the flow rate was held at 0.25 ml/minute. A linear gradient at initial conditions was held for 0–2 minutes, followed by 10% B from 2 to 5min, 35% B from 5 to 15min, and 95% B from 15 to 16 min with a 2-minute hold, followed by a 6-minute reequilibration at 5% B with a total run time of 24 minutes. The same gradient was used for positive polarity ionization except that mobile phase (A) consisted of 0.1% aqueous formic acid. Electrospray ionization was used in both negative and positive ion mode, and source conditions were set as follows: drying gas (N2) temperature at 350 °C, and flow rate of 11 l/minute, nebulizer set at 45 psi, and capillary voltage set to 4,000 V. Initial analysis was carried out in full scan mode using a mass to charge (m/z) range of 350–2,500 with a scan rate of 2.0 spectra per second using a fragmentor voltage of 120 V with reference lock mass enabled at 5 psi.Fractionated samples of bile, feces, and urine collected from both basic and acidic gradient conditions were dried down and reconstituted in initial mobile phase conditions followed by filtration using a 0.22-μm pore size centrifugal filter unit and centrifuged for 4 minutes at 10,0002O with 50-μm EDTA as the reconstitution buffer. For ion pairing, chromatographic separation was achieved using an Xbridge column  at a flow rate of 0.25 ml/minute using a gradient of (A) 15 mm TEA, 400 mm HFIP buffer pH 7.8 with (B) methanol, held at 15% B initially and ramped to 50% B to 9 minute, followed a second ramp for 1 minute to 70% B and maintained for 1 minute before returning to initial conditions with a 5-minute postrun using the same source conditions as described. Oligonucleotide related peaks and metabolites were evaluated in the total ion chromatogram and averaged for respective m/z values for high abundance ions. Multiple charged ion envelope for oligonucleotide related metabolites was observed with a common range of charged species up to a (−12) charge state.Additionally an ion-pairing method was also evaluated for both dosing solution and urine fractionated extracts and included H31 Targeted tandem mass spectrometry (MS/MS) was then applied to sort five maximal high abundance precursor ions of each fractionated extract per cycle using a mass range of m/z 500–1500 at a scan rate of 1.5 spectra per second to obtain product ion spectra. An automated ramping collision energy table was used with m/z 400 with Z1 at 35, and Z2 at 30V, respectively, m/z 1,000 with Z1 at 60, and 55 V respectively, and m/z 2,000 with Z1 at 70, and Z2 65 V, respectively.MassHunter workstation software version B.06.00 of build 6.0.633.10,  with molecular feature extraction was used to identify unique mass traces of metabolites common in each species compared with each fractionated matrix control sample. Potential compounds were background subtracted and initially filtered to restrict the number and ratio of elements and isotopic patterns in addition to peak abundance.4 containing and non-PO4 containing metabolites a mass shift difference of m/z ± 48 was investigated in the spectra, presumably by internal esterification, loss of water, and cyclization.Radiochromatographic profiles were directly compared with the mass spectra of fractionated extracts of bile, feces, and urine, and of the controls, respectively. Metabolite masses in each fraction were compared with corresponding theoretical masses of biotransformed reference compounds. For most cases, a correlation of fragment masses of metabolites with proposed theoretical structures was compared with the MS/MS fragmentation patterns of available reference material . Proposed structures and MS/MS fragmentation patterns were compared with key fragments of the (M8) reference standard. To differentiate between both PONomenclature and assignment of metabolites. Initially metabolite (M8) was characterized as an intermediate metabolite of the GalNAc3 cluster and used to assign other metabolites. Metabolites clearly identified by radiometric detection, chromatographic retention time, and mass spectral features were named in order of suspected formation (M1–M14). Metabolite (M8) contains four branching arms  with structural uniformity for the b1–3 series. Nonmetabolized parent compound is designated as (P) and pronounced but uncharacterized peaks are labeled as (X). Proposed structures and naming of all metabolites is described in Table 1.In vitro GalNAc3 cleavage by β-N-acetylglucosamindase. GalNAc3-conjugated ION 681257 was evaluated at 100 µmol/l and combined with a 100× dilution of β-N-acetylglucosamindase (β-Nag) at (122 U/ml) and incubated at for 30 minutes at 37 °C on a shaker set to 100 rpm. Reactions were terminated by freezing at −80 °C and samples were basified with NH4OH and extracted with phenol/chloroform/isoamyl alcohol (25:24:1) and centrifuged at 13000g for 4 minutes to separate aqueous and organic layers. Aqueous supernatants were removed and an additional back-extraction was performed. Following the liquid liquid extraction, solid phase extraction was performed. Solid phase extraction plates were equilibrated with 2 ml of acetonitrile followed by 2 ml of 1 mol/l triethylammonium bicarbonate. Samples were loaded onto the cartridges followed by a rinse with 2 ml of 0.1 mol/l TEAB, 2 ml of H2O, 2ml of 2% NH4OH , and 2ml of 10% acetonitrile in 0.1 mol/l TEAB. Samples were eluted with 1.5 ml of 70% acetonitrile in 0.1 mol/l TEAB and run through a protein precipitation plate prior to LC–MS analysis.DNase II digestion of GalNAc conjugated and −3GalNAc-conjugated ASO. GalNAc3-conjugated ION 681257 and −3GalNAc-conjugated ASO were diluted in water to 0.2 mg/ml, deoxyribonuclease II (DNase II) containing 757 U/ml was prepared at 0.4 mg/ml in a reaction buffer containing 20 mmol/l sodium acetate, pH 4.6, with 5 mmol/l MgCl2 as a cofactor. The digest was initiated by addition of 100 µl of DNase II to 100 µl of substrate. Samples were incubated for 0, 4, 8, and 24 hours at 37 °C. Reactions were terminated by the addition of 0.4 ml of ice-cold methanol with brief vortexing and centrifuged at 10,000g for 3 minutes followed by evaporation using a speedvac under ambient temperature. Samples were reconstituted in 100 µl of mobile phase and transferred to an autosampler vial for LC–MS under negative polarity electrospray ionization using a scan range of m/z 800–2,200.In vitro hydrolysis study using alkaline phosphatase. Lyophilized −3 sugar standard was reconstituted to form a 0.2 mg/ml solution in 20-mmol/l sodium acetate, pH 4.6, with 5 mmol/l MgCl2. To initiate the reaction, 75 µL of DNase II (0.4 mg/ml dissolved in the same buffer system) was added with 75 µl of the standards briefly mixed and incubated at 37 °C for 18 hours. Following digestion, 0.3 ml of ice-cold methanol was added, vortexed, and centrifuged at 10,000g for 3 minutes, the supernatant was removed and dried down. Stock alkaline phosphatase was diluted to 10 U/ml using 1 mol/l diethanolamine containing 0.50 mmol/l MgCl, pH 9.8, and 200 µl was added to the previous substrate directly. Samples were vortexed and incubated at 37 °C for 2 hours followed by the addition of 0.4 ml of methanol. Samples were mixed and centrifuged at 10,000g for 3 minutes. The supernatant was dried down and reconstituted in 100 µl of mobile phase . Positive polarity electrospray ionization using a −mass scan range of m/z 700–1000 was used.In vitro generation of oxidized metabolites: enzymatic studies using ADH and ALDH. As a potential substrate for alcohol dehydrogenase (ADH), M8 corresponding to the (THA linker) was prepared in 50 mmol/l HEPES pH 8.0 at 5.23 mmol. ADH (382 U/ml) 1.0 mg/ml and NAD+ at 1.0 mg/ml were both dissolved in the same buffer. The final reaction was set up using a total volume of 0.2 ml and included 123.5 µl of 50 mmol HEPES, 10 µl of 5.23 mmol M8 substrate, 26.5 µl of 1 mmol NAD+, and initiated with the addition of 40 µl ADH enzyme (76.4 U). Samples were incubated for 2 hours at 37 °C. Reactions were terminated with the addition of 2× volume of ice cold methanol, and vortexing. Samples were centrifuged for 3 minutes at 10,000g and the supernatant was dried down and reconstituted in mobile phase for LC–MS. Positive polarity electrospray ionization using a mass-scan range of m/z 500–1,000 was used Additional samples were processed in the same method but reconstituted in 50 mmol/l HEPES pH 8.0 and not mobile phase to be used as the substrate for ALDH. Potential metabolites M9–12 were carried over from the initial ADH incubation and used as the substrate with the addition of 26.5 µl of 1.0 mg/ml NAD+ and 173.5 µl of ALDH (1.4 U/ml). Samples were incubated for 2 hours at 37 °C, stopped, and assayed as described for ADH.Figure S1. Metabolites observed in rat bile (n = 2) as individually fraction collected and combined into a single chromatogram.Figure S2. Representative GalNAc3-associated metabolites confirmed in monkey feces by liquid chromatography mass spectrometry using positive polarity electrospray ionization following administration of 12 mg/kg ION 681257.Table S1. Mass balance with excretion route and percentage of total dose excreted.Supplementary Materials and Methods