FDA-Approved Excipient N,N-Dimethylacetamide Attenuates In-Vitro and In-Vivo In ammatory Bowel Disease

Jagadish Koya Saint John's University: St John's University Tong Shen Regor Therapeutics Group Geming Lu Icahn School of Medicine at Mount Sinai Alex Gauthier Saint John's University: St John's University Lin Mantell Saint John's University: St John's University Charles Ashby Saint John's University: St John's University Sandra Reznik (  rezniks@stjohns.edu ) Saint John's University: St John's University https://orcid.org/0000-0002-6272-7198

. One driver of these pro-in ammatory responses is high mobility group box 1 (HMGB1), a ubiquitous nuclear protein that activates innate immune responses when released from cells (Yang 2020). HMGB1 has been recently recognized as a therapeutic target in various in ammatory diseases (Yang et al. 2010, Andersson et al. 2011, Kang et al. 2014; Andersson et al. 2018), including IBD (Hu et al. 2015).
We have previously reported that N,N-dimethylacetamide (DMA), a widely used drug excipient, formerly believed to be inert, attenuates in ammation-induced preterm birth in mice by preventing nuclear factor kappa B (NF-kB) activation (Sundaram et al. 2013.) We have also shown that DMA suppresses cytokine secretion in lipopolysaccharide (LPS)-challenged RAW 264.7 cells, tumor necrosis factor alpha (TNFa)induced human placental JEG-3 cells and LPS-stimulated human placental explants (Pekson et al. 2016) speci cally by acting on the NF-kB pathway. Ghayor et al. have shown that DMA prevents osteoporosis in rats via the inhibition of osteoclast mediated bone resorption (Ghayor et al. 2017) and enhances bone regeneration impaired by excess in ammation (Siegenthaler et al.2020). In other re-purposing studies, DMA has been shown to prevent high-fat diet induced weight gain (Bhattacharya et al. 2019) and has been investigated as a potential reversible contraceptive (Khera et al. 2020

Methods
Cell culture and reagents.
The human monocyte THP-1 (ATCC TIB-202) cell line; the human colon epithelial cell lines HT-29, HCT-116 and SW 620; and the mouse macrophage RAW 264.7 cell line were purchased from the American Type Culture Collection (Manassas, VA, USA). HT-29, HCT-116, SW 620 and RAW 264.7 cells were cultured in complete growth media (CGM) using Dulbecco's Modi ed Eagle's Medium (DMEM) with 4.5 g/L glucose, L-glutamine and sodium pyruvate (Cellgro, Corning, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Atlanta Biologics, Lawrenceville, GA) and 1% penicillin-streptomycin (Cellgro, Corning, NY). THP-1 cells were grown in Roswell Park Memorial Institute (RPMI) medium with Lglutamine (Cellgro, Corning, NY) supplemented with 10% heat-inactivated FBS and 1% penicillinstreptomycin. T84 cells were grown in complete growth medium, which is a 1:1 Dulbecco's Modi ed Eagle's Medium (DMEM) and Ham's F12 medium with 2.5 mM L-glutamine and supplemented with 5% heat-inactivated fetal bovine serum (FBS). All cells were maintained in an incubator set at 37°C and 5% CO 2 and allowed to grow to 80-90% con uency before being sub-cultured or used in experiments. Purity of DMA was con rmed by gas chromatography-mass spectroscopy. All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) or VWR (Bridgeport, NJ). A solution of 3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) was prepared in phosphate buffered saline (PBS) at a concentration of 5mg/ml. HCT-116, SW620, and HT-29 cells were seeded at 18,000 cells/well whereas THP-1 cells were seeded at 30,000 cells/well in 96-well plates and incubated at 37°C and 5% CO 2 overnight. The next day, cells were washed with phosphate buffered saline At the end of 24 h of treatment, 20 µl of MTT solution (5 mg/ml) (Alfa Aesar, Ward Hill, MA) was added to each well at a nal concentration of 0.5 mg/ml. After incubation for 2 h at 37°C and 5% CO 2 , media was aspirated and dimethyl sulfoxide (DMSO) (100 µl/well) (BDH, Randor, PA) was added to dissolve formed purple formazan crystals. The plates were shaken on an orbital microplate shaker for 10 min to ensure complete solubilization of the crystals and the absorbance of the resulting purple solution was measured at 570 nm using an Opsys MR microplate reader (Dynex Technologies, Chantilly, VA).
Sandwich ELISA was used to determine the levels of various cytokines and chemokines such as TNF-α, Interleukin (IL)-6, IL-1β, IL-8, monocyte chemoattractant protein (MCP), granulocyte-macrophage colonystimulating factor (GM-CSF)-1 and IL-10 and high mobility group box 1 protein (HMGB1) in cell culture supernatants. Ready-SET-Go! sandwich ELISA kits (eBioscience, San Diego, CA) for various human cytokines and chemokines were used as per manufacturers' protocols. First, the optimal sample dilution for each target cytokine was determined to ensure that the sample absorbance readings fall within the range of their respective standard curves. Standard curves were produced by serially diluting lyophilized or recombinant standards, provided with the kits, according to the manufacturers' instructions.
As per manufacturers' protocols, clear at-bottom Maxisorp 96-well plates (Thermo Fisher Scienti c, Waltham, MA) were coated and incubated overnight at 4°C with capture antibody diluted in coating buffer for each analyte. The following day, each well was washed with washing buffer (1X PBS with 0.05% Tween 20) three times and then blocked for non-speci c binding sites with assay buffer for one h.
Samples were added to the wells in duplicate undiluted or diluted with assay buffer and incubated at for 2 h at room temperature. After the incubation period, each well was washed three times and the detection antibody diluted in assay buffer for the respective analytes was added and samples were incubated for one more h. Following the detection antibody incubation each well was washed thrice and avidin-HRP labeled secondary antibody diluted in assay buffer was added and incubated for another 30 min. Before proceeding to the last step, wells were washed ve times and tetramethylbenzidine (TMB) substrate was added and the samples were incubated for 15 min, which resulted in a blue-colored solution. After 15 min., the reaction was concluded by adding a stop solution (1M H 3 PO 4 ), turning the blue solution to yellow and the sample absorbance was measured at 450 nm using an Opsys MR microplate reader. The concentration of each analyte was interpolated using a second order polynomial equation created from the standard curve, using GraphPad Prism 6 software, and then the value was multiplied by the dilution factor used in the assay to calculate the concentration of the analyte in the original undiluted sample.
To study the mechanism of DMA's effect on cytokine secretion, its effect on nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) expression in LPS stimulated THP-1 cells was examined. To perform the experiment, THP-1 cells were seeded at a density of 3 x 10 6 per T25 cm 2 tissue culture ask and incubated overnight at 37°C and 5% CO 2 using respective CGM. The next day, cells were washed with sterile PBS and pre-treated with different concentrations of DMA (0.1, 1 or 10 mM) or plain CGM for the untreated group, for 2 h. Then, LPS was added to all cells, except for the untreated group, at a nal concentration of 1 µg/ml and cells were incubated for another 30 min. Whole cell lysates were prepared at the end of the respective treatment periods as described below.
Preparation of whole cell lysates.
Whole cell lysates were prepared using a modi ed protocol as described by Abcam. Cells were scraped off using a cell scraper (Greiner Bio-one, Monroe, NC) and collected in a pre-chilled 5 ml snap cap centrifuge tube and then centrifuged at 14,000 x g for 2 min. The old medium was aspirated from the 5 ml tubes and cells were washed twice with ice-cold PBS. The washed pellets were again collected into 1.5 ml pre-chilled centrifuge tubes and were then centrifuged at 1000 x g for 5 min. The supernatants were removed without disturbing the cell pellets, which were washed with ice-cold PBS once again and centrifuged at the same settings as mentioned above. Again, the supernatants were removed without disturbing the cell pellets and the pellets were resuspended in 65 µl of radio immunoprecipitation assay (RIPA) lysis and extraction buffer (G-Biosciences, St. Louis, MO). The RIPA lysis buffer was freshly supplemented with EDTA-free protease inhibitor cocktail set III (Calbiochem, San Diego, CA) (1:200 dilution) and with phenylmethylsulfonyl uoride (PMSF) (Calbiochem, San Diego, CA) at a concentration of 100 mM in ethanol (1:100 dilution). The cell pellets were thoroughly mixed with the prepared lysis buffer and were kept on ice with vortexing for 5 sec every 10 min., for a total of 30 min. The lysed cells were centrifuged at 14,000 x g for 20 min. at 4°C (Eppendorf 5424R, Hauppauge, NY). The whole cell lysates were collected in labelled 0.65 ml microcentrifuge tubes and stored at -80°C until further analysis.
Automated capillary western blot analysis (WES Simple Western).
WES, an automated capillary-based electrophoresis system (ProteinSimple, San Jose, CA) was used for performing the protein expression analysis. The total protein concentration of each experimental sample was calculated using Pierce bicinchoninic acid (BCA) protein assay (Thermo Fisher Scienti c, Waltham, MA). The volumes of the lysates used for WES analysis were determined based on total protein concentrations, using a bovine serum albumin (BSA) (Thermo Fisher Scienti c, Waltham, MA) standard curve. The nal concentration of protein loaded into the WES plate is optimized to be 1µg/ml. All the reagents required for WES were prepared as per the manufacturer's instructions. The provided 10X sample buffer was diluted using ultrapure water to 0.1X, which was used to dilute lysates. First, all standard pack reagents (DTT, 5X Fluorescent master mix and biotinylated ladder) were prepared. Then the lysates were mixed with prepared 5X uorescent master mix in a 4:1 ratio to have the nal concentrations of the lysates at 1 µg/µl. Finally, the lysates were heated at 95°C for 5 min.
The prepared lysates, blocking reagent, primary antibodies, HRP-conjugated secondary antibodies, chemiluminescent substrate mix (Luminol:Peroxide mixture in 1:1 ratio) and wash buffer were dispensed into designated wells in an assay plate provided by the manufacturer and as per the manufacturer's instructions. The assay plate, along with the respective capillary cartridge, was placed into the WES instrument (ProteinSimple, San Jose, CA), which carries out all further assay steps automatically using default settings. Anti-I Bα antibody(Cell Signaling technologies, Danvers, MA), diluted 1/50, was used as the primary antibody and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (Cell Signaling Technologies, Danvers, MA), diluted 1/10,000, was used for the gel loading control. All primary antibodies were diluted in an antibody diluent provided by the manufacturer and the optimal dilution for each primary antibody was determined using Simple Western antibody database as a reference. In approximately 3 h, molecular weight and quantitative signals for target proteins were automatically reported by the Compass software (ProteinSimple, San Jose, CA). Protein expression was analyzed using ImageJ software (NIH, Bethesda, MD) and normalized to that of GAPDH.

Animals.
Nine-week-old male C57Bl/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME). All In vivo studies on the effect of DMA on dextran sodium sulphate (DSS)-induced colitis.
Colitis was induced with DSS. A total of ten male C57Bl/6 mice weighing between 20 and 28 g were given drinking water containing 2.5% DSS. Mice were then randomly assigned to two groups. Group I mice (n = 5) were negative controls and were injected intraperitoneally (ip) with 0.2 mL of PBS once a day for nine days. Group II mice (n = 5) were injected ip with 0.2 mL of 33% DMA (2.1 g/kg) once a day for four days. One sham mouse was allowed to drink plain drinking water and injected with 0.2 mL PBS once a day for nine days. Mice were euthanized on the ninth day of the experiment and necropsied. The intestines were removed in their entirety for histologic examination.
Histological evaluation and grading.
Several sections of the distal portion of the colon from each mouse were xed in formalin, para n embedded, sectioned at 4 µM and stained with hematoxylin and eosin. Sections were examined by a practicing anatomic pathologist, who was blinded to the experimental conditions (S.E.R.), and scored, following the method described by Laroui et al. (2012). Each section was scored for severity of in ammation (0 = rare in ammatory cells in the lamina propria, 1 = increased in ammatory cells in the lamina propria, 2 = con uence of in ammatory cells extending into the submucosa, 3 = transmural in ammation); crypt injury (0 = intact crypts, 1 = loss of the basal one third of crypts, 2 = loss of the basal two thirds of crypts, 3 = loss of entire length of crypts, 4 = focal erosion of epithelial surface, 5 = con uent areas of erosion of the epithelium); and ulceration (0 = absence of ulceration, 1 = 1 or 2 foci of ulceration, 2 = 3 or 4 foci of ulceration, 3 = con uent ulceration). Scores were added to yield maximum histologic grade of 11. Sections were examined with a Nikon Eclipse 80i light microscope and images were captured with a Nikon Digital Sight camera (Nikon, Melville, NY).

Statistical analysis.
All the data are represented as the mean +/-SEM of at least four independent experiments. The data were analyzed using GraphPad Prism 6 software (San Diego, CA, USA). The statistical signi cance among and between groups was tested using a one-way analysis of variance (ANOVA), followed by Tukey's multiple comparison post hoc test for the cell viability, cytokine secretion, and immunoblotting protein analyses. The Kruskal Wallis test was used to analyze differences in histologic scores. The a priori signi cance value was P < .05.

Cell viability.
The effect of various concentrations of DMA on the viability of HT-29, HCT-116, SW620 and THP-1 cells was determined in order to identify a non-toxic range of concentrations that could be used in further experiments. DMA was found to produce no signi cant change in cell viability at concentrations up to at least 10 mM in all four cell lines in unstimulated cells ( Supplementary Fig. 1), in all four cell lines with stimulation by 1 µg mL − 1 LPS (Supplementary Fig. 2) and in the three colonic epithelial cell lines with stimulation by TNFα (Supplementary Fig. 3). Therefore, in vitro assays were carried out with no more than Page 8/20 DMA attenuates IL-8 secretion from HT-29, HCT-116, SW620 cells.
While colonic epithelial cells are likely a relatively minor source of in ammatory cytokines in vivo, monocytes and macrophages contribute signi cantly to the cytokine response in IBD. Therefore, we tested the ability of DMA to suppress cytokine secretion in THP-1 cells, a human monocyte line. In THP-1 cells stimulated with 1 µg mL − 1 LPS, DMA signi cantly reduced IL-1β secretion at 10 mM (Fig. 3A), IL-6 secretion at 1 and 10 mM (Fig. 3B), IL-8 secretion at 10 mM (Fig. 3C), MCP-1 secretion at 10 mM (Fig. 3D) and IL-10 secretion at 10 mM (Fig. 3E). BAY 11-7082, an NF-kB inhibitor (is used at 5 µM as a positive control.

DMA prevents IκBα degradation in THP-1 cells.
To test whether DMA affects levels of IκBα in THP-1 cells, the time course of IκBα degradation in THP-1 cells stimulated with 1 µg mL − 1 LPS was examined, using immunoblotting. Lowest levels of IκBα were found in cell lysates at 30 min (Supplemental Fig. 4). Therefore, DMA's effect on IκBα degradation was evaluated in THP-1 cells stimulated with LPS for 30 min. DMA protected IκBα from LPS-induced degradation at 0.1, 1 and 10 mM (Fig. 4).
DMA attenuates HMGB1 secretion from RAW 264.7 cells at micromolar concentrations.
HMGB1 secretion re ects oxidative stress, a key component of the pathogenesis of IBD. In addition, HMGB-1 activates the NF-kB pathway. Interestingly, DMA prevented HMGB1 secretion from LPS stimulated RAW 264.7 cells, a well characterized mouse macrophage cell line known to secrete HMGB1, at all concentrations tested (Fig. 5).
Control DSS challenged mice (n = 5) all developed bloody diarrhea by the eighth day of the experiment, whereas all the DMA rescued DSS challenged mic (n = 5) developed bloody diarrhea on the ninth day of the study. In addition, histologic analysis of intestinal sections from the DSS control mice showed in ammation, crypt injury and ulceration, but these effects were attenuated by DMA with signi cant reduction in crypt injury (P < .05) and total histologic scores (P < .05, Fig. 6). Mean histologic scores for in ammation in DSS challenged mice with and without DMA rescue were 2.4 ± 0.9 and 3.0, respectively. Mean scores for crypt injury for the two groups were 3.8 ± 0.8 and 5.0, respectively (P < .05). Mean scores for ulceration for these two groups were 2.2 ± 0.8 and 3, respectively. Finally, mean total histologic scores were 8.4 ± 2.1 and 11.0, respectively (P < .05). The negative control mouse raw histologic scoring data are summarized in Supplementary Table 1.

Discussion
While the precise etiology of IBD remains elusive, it is clear that NF-kB driven pro-in ammatory responses play a central role. Current pharmacotherapeutic approaches to IBD. such as sulfasalazine and biologics, work by preventing activation of the NF-kB pathway either directly or indirectly (Khanna and Marshall 2017, Hindryckx and D'Haens Geert 2017). However, sulfasalazine, while it attenuates IBD symptoms and is readily accessible, does not show e cacy in some patients and, like all sulfonamides, is associated with hepatotoxicity (European Association for the Study of the Liver 2019). On the other hand, biologics, such as monoclonal antibodies, may have greater e cacy, but remain extremely expensive (Chen et al. 2018). DMA is an inexpensive, non-toxic widely used drug excipient, which has been previously shown by us and others to inhibit NF-kB (Sundaram et al. 2013, Ghayor et al. 2017) and which has been proven to be non-toxic in pediatric cancer patients found to have plasma concentrations in the mM range (Hempel et al. 2007). Therefore, we hypothesized that DMA would attenuate DSS-induced colitis.
After establishing the highest non-toxic concentration of DMA in cell viability assays, we proceeded to investigate the effect of DMA on in ammatory mediators (cytokines and chemokines) in human IECs and in human monocytes. Initially, we screened for the presence of cytokines and chemokines including TNFα, IL-6, IL-1β, IL-8, IL-10, MCP-1, and GM-CSF in the three IEC cell lines and the monocytes. The stimulated IECs, which are not immune in origin, only upregulated the production of IL-8. Chemokine IL-8 recruits neutrophils to sites of tissue injury (Wéra et al., 2016) and triggers the formation of crypt abscesses, a predominant feature in UC. Moreover, levels of neutrophils in colonic biopsies from UC patients are proportional to levels of disease severity (Mitsuyama et al., 1994;Daig et al., 1996). DMA, at 10 mM, attenuated IL-8 secretion from the IECs stimulated with either LPS or TNFa, which is consistent with our previous nding that DMA decreases neutrophil counts in placentas harvested from LPS-stimulated pregnant C57Bl/6 mice (Sundaram et al., 2013).
In addition to investigating DMA's effect on IL-8 secretion from IECs, we tested DMA's effect on the secretion of various cytokines from LPS-stimulated THP-1 monocytes. Monocytes reside under the lamina propria in the intestine, express toll-like receptor (TLR)-4 on their surface and play an active part in producing chronic gut in ammation (Rugtveit et al. 1994, Matricon et al., 2010). Cytokines IL-6, IL-1β and IL-10 and chemokines IL-8 and MCP-1 were signi cantly reduced with treatment of DMA at 10 mM in THP-1 cells. Consistent with these results, levels of IL-6 and IL-1β are positively correlated with disease severity in both Crohn's disease and UC (Reinisch et al., 1999;Ligumsky et al., 1990). While IL-10 is classically considered an anti-in ammatory cytokine, it has also been reported to show immune stimulatory effects by upregulating major histocompatibility class II expression in B-lymphocytes, inducing cytotoxic T-cell differentiation (Li and He, 2004). Moreover, IL-10 de cient mice develop colitis (Keubler et al. 2015). It is likely that IL-10 is acting as a pro-in ammatory cytokine in our cultured THP-1 cells.
Importantly, we show here that DMA inhibits degradation of the NF-kB inhibitory molecule IkBa in THP-1 cells. This nding is consistent with our previously reported result that DMA prevents IkBa degradation in RAW 264.7 cells (Pekson et al. 2016) and suggests one mechanism whereby DMA prevents NF-kB driven up-regulation of cytokines and chemokines. In addition, we demonstrate that DMA decreases HMGB1 secretion from RAW 264.7 cells. HMGB1, when secreted, acts as a pro-in ammatory mediator. DMA's ability to prevent HMGB1 release from cells, therefore, reinforces its effect on NF-kB signaling. Interestingly, DMA prevents HMGB1 secretion at 0.1 mM, whereas much higher concentrations of DMA are required to produce its effects on cytokine levels. This suggests that while decreased HMGB1 release may contribute to DMA's anti-in ammatory effects, reduced HMGB1 secretion alone does not fully account for DMA's mechanism of action. Ghayor et al. have also shown that DMA is a bromodomain ligand (Ghayor et al. 2017), a result that we have con rmed in our laboratory. Taken together, the data suggest that DMA, a small, highly soluble molecule, may act via multiple mechanisms.
Finally, we show here, for the rst time, that DMA attenuates DSS-induced colitis in a murine model. In particular, DMA prevented the formation of crypt abscesses, a hallmark feature of UC in human patients.
Further preclinical and clinical studies should be done to explore DMA's potential role as drug therapy for IBD.

Conclusion
DMA attenuates in ammatory responses in in-vitro models of IBD, including cytokine-and chemokinesecreting human IECs and monocytes; prevents HMGB1 release from RAW 264.7 cells; and decreases the severity of DSS-induced colitis and crypt abscesses in particular. It prevents NF-kB transcriptional activity by inhibiting IkBa degradation and may act by multiple mechanisms. DMA should be further investigated as potential drug therapy for IBD.

Declarations
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Supplementary Files
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