5-Aminosalicylic Acid Azo-Linked to Procainamide Acts as an

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5-Aminosalicylic acid azo-linked to procainamide acts as an anticolitic mutual prodrug via additive inhibition of nuclear factor kappaB. Wooseong Kim, Joon Nam, Sunyoung Lee, Seongkeun Jeong, and Yunjin Jung Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00294 • Publication Date (Web): 26 Apr 2016 Downloaded from http://pubs.acs.org on April 28, 2016

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Molecular Pharmaceutics

5-Aminosalicylic acid azo-linked to procainamide acts as an anti-colitic mutual prodrug via additive inhibition of nuclear factor kappaB.

Wooseong Kim, Joon Nam, Sunyoung Lee, Seongkeun Jeong and Yunjin Jung

College of Pharmacy, Pusan National University, Busan, Republic of Korea 609-735

Running title: 5-Aminosalicylic acid azo-linked to procainamide

*To whom correspondence should be addressed: Yuniin Jung College of Pharmacy, Pusan National University, Pusan 609-735, Republic of Korea Tel. 051-510-2527, Fax. 051-513-6754, [email protected]

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Abstract To improve the anti-colitic efficacy of 5-aminosalicylic acid (5-ASA), a colon-specific mutual prodrug of 5-ASA was designed. 5-ASA was coupled to procainamide (PA), a local

anesthetic,

via

an

azo

bond

to

prepare

5-(4-{[2-

(diethylamino)ethyl]carbamoyl}phenylazo)salicylic acid (5-ASA-azo-PA). 5-ASA-azoPA was cleaved to 5-ASA and PA up to about 76% at 10 h in the cecal contents while remaining stable in the small intestinal contents. Oral gavage of 5-ASA-azo-PA and sulfasalazine, a colon-specific prodrug currently used in clinic, to rats showed similar efficiency in delivery of 5-ASA to the large intestine and PA was not detectable in the blood after 5-ASA-azo-PA administration. Oral gavage of 5-ASA-azo-PA alleviated 2,4,6-trinitrobenzene sulfonic acid-induced rat colitis. Moreover, combined intracolonic treatment with 5-ASA and PA elicited an additive ameliorative effect. Furthermore, combined treatment with 5-ASA and PA additively inhibited nuclear factor-kappaB (NFκB) activity in human colon carcinoma cells and inflamed colonic tissues. Finally, 5-ASA-azo-PA administered orally was able to reduce inflammatory mediators, NFκB target gene products, in the inflamed colon. 5-ASA-azo-PA may be a colon-specific mutual prodrug acting against colitis, and the mutual anti-colitic effects occurred at least partly through the cooperative inhibition of NFκB activity.

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Keywords 5-Aminosalicylic acid, Procainamide, Colon-specific prodrug, Colitis, Nuclear factor kappaB

Abbreviations

IBD, inflammatory bowel disease; 5-ASA, 5-aminosalicylic acid; PA, procainamide; 5ASA-azo-PA, 5-aminosalicylic acid azo-linked to procainamide; TNF, tumor necrosis factor; COX-2, cyclooxygenese-2; iNOS, inducible nitric oxide synthase; CINC, cytokine-induced neutrophil chemoattractant; TNBS, 2,4,6-trinitrobenzene sulfonic acid; MPO, myeloperoxidase; CDS, colonic damage score

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Introduction Inflammatory bowel disease (IBD), usually referring to ulcerative colitis (UC) or Crohn’s disease (CD), is a chronic inflammation in the gut. Although IBD’s etiology is still elusive, the exaggerated immune reaction of the host with antigens derived from food and/or microbes is largely responsible for the disease 1-3. In addition, the imbalance in autonomic nerves innervated in the gut has also been suggested as an etiological factor 4. Adrenergic preponderance may induce intestinal mucosal injury probably via vasoconstriction and accelerated epithelial turnover.

5,

6

. In line with this

pathophysiology, rectal treatment with local anesthetics, likely soothing hyperactive autonomic nerves and the unregulated immune response, alleviates colonic inflammation in animal colitis models7-9. 5-Aminosalicylic acid (5-ASA) is most widely used for the treatment of mild-tomoderate active UC and CD

10, 11

. Rapid and extensive absorption of 5-ASA in the

upper intestine prevents the therapeutic use of 5-ASA itself as an anti-IBD drug

10

. In

addition to the low potency of 5-ASA for IBD treatment, adverse effects from systemically absorbed 5-ASA, such as nephrotic syndrome

12, 13

, necessitate the anti-

IBD drug to be specifically delivered to the large intestine where inflammation occurs mainly, being achieved by pharmaceutical formulation or prodrug approach. 14, 15.

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To improve the therapeutic activity of 5-ASA against colitis, we designed a colonspecific mutual 5-ASA prodrug. Based on the beneficial effects of local anesthetics on colitis, procainamide (PA), pharmacologically classified as an anti-arrhythmic and a local anesthetic 16, was linked to 5-aminosalicylic acid via an azo bond to produce PAconjugated 5-ASA (5-ASA-azo-PA). 5-ASA-azo-PA would release 5-ASA and PA in the large intestine after microbial azo-reduction to amines

17

, presumably cooperating to

ameliorate inflammation. PA, considered as a colon-specific carrier as well as a therapeutic agent against colitis, was selected as the local anesthetic to conjugate with 5-ASA because it is less lipophilic than other local anesthetics and has the potential to interfere with the activity of NFκB, an anti-colitic drug target

18, 19

. A colon-specific carrier should be hydrophilic

to restrict systemic absorption of the colon-specific prodrug and the carrier 17. In this study, 5-ASA azo-linked to PA (5-ASA-azo-PA) was synthesized and evaluated as a colon-specific mutual 5-ASA prodrug acting against colitis along with therapeutic comparison with sulfasalazine, a colon-specific 5-ASA prodrug used currently in clinic. In addition, molecular pharmacology of the anti-colitic mutual action was suggested based on the in vitro/vivo results.

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Materials and methods Materials Salicylic acid, sodium nitrite (NaNO2), sulfamic acid, and 5-ASA were purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan). Procainamide (PA) HCl, sulfasalazine, and TNBS were purchased from Sigma Chemical Co. Inc. (St. Louis, MO, USA). All ELISA analysis kits were obtained from R & D Systems (Minneapolis, MN, USA). All other chemicals were reagent-grade, commercially available products. IR spectra and 1

H-NMR spectra were recorded on a Varian FT-IR spectrophotometer (Varian, Palo Alto,

CA, USA) and a Varian AS 500 spectrometer. The chemical shifts in NMR spectra are in ppm downfield from tetramethylsilane. Electrospray ionization mass spectrometry (ESI-MS) spectra were obtained by using an Agilent 65360 Q-TOF (Agilent, Santa Clara, CA, USA). Animals Six-week-old male Sprague-Dawley (SD) rats were purchased from Samtako Bio Korea (Kyeong-gi-do, South Korea) and housed in the animal care facility at Pusan National University, Busan, South Korea. Rats were grouped as follows: Normal, TNBS control, and TNBS/drug-treated groups. Each group consisted of five rats. The SD rats were housed in the university animal facility with controlled temperature, humidity, and

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dark/light cycle. The animal protocol used in this study was reviewed and approved by the Pusan National University–Institutional Animal Care and Use Committee (PNU– IACUC) for ethical procedures and scientific care. Synthesis of 5-(4-[2-(diethylamino)ethyl]carbamoylphenylazo)salicylic acid (5ASA-azo-PA) To procainamide HCl (2 mmol) dissolved in 3 ml of pre-chilled 18% hydrochloric acid was added NaNO2 (4 mmol), followed by stirring for 1 h at 4 °C before the addition of sulfamic acid (2 mmol). The resulting solution was added to salicylic acid (6 mmol) dissolved in 0.1 M NaOH (3 ml) and then stirred at 4 °C for 1 h, followed by reaction at room temperature for 6 h. pH was adjusted to 9–10 during the reaction. The precipitate was filtered and washed three times with ethanol/acetone (1:1) followed by drying in a vacuum oven. Yield: 75 %; mp: 258–260 °C; IR (nujol mull), νmax (cm−1): 1628 (C=O, carboxylic), 1654 (C=O, amide); 1H-NMR (DMSO-d6): δ = 1.21 (t, 6H, J = 15.5 Hz), 3.27 (m, 6H), 3.63 (q, 2H, J = 11 Hz), 6.78 (d, 2H, J = 18 Hz), 7.79 (dd, 1H, J1 = 18 Hz, J2 = 5 Hz), 7.83 (d, 2H, J = 16.5 Hz), 8.0 (d, 2H, J = 16.5 Hz), 8.21 (d, 1H, J = 5 Hz), 8.8 (t, 1H, J = 11 Hz). ESI-MS m/z 385.1873 [M+H]+, 407.1694 [M+Na]+.

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Apparent partition coefficient and pH stability To a solution of 5-ASA-azo-PA (10 ml, 25 µM) in pH 7.4 isotonic phosphate buffer presaturated with 1-octanol was added 1-octanol pre-saturated with the pH 7.4 isotonic phosphate buffer (10 ml). The mixture was shaken for 12 h and then left to stand for 3 h for phase separation at room temperature. The concentration of 5-ASA-azo-PA in the aqueous phase was determined by using a UV spectrophotometer. The apparent partition coefficients were calculated as described previously 20. 5-ASA-azo-PA, placed in pH 1.2 hydrochloric acid buffer or in pH 6.8 isotonic phosphate buffer (500 µM, USP XXIII), was incubated at 37 °C for 10 h. At a predetermined time interval, a 20-µl portion of each solution was removed and the concentrations of drugs were analyzed by using HPLC (Gilson, Middleton, WI, USA). High-performance liquid chromatography analysis HPLC analysis using a reversed-phase C18 column was performed as in a previous paper 21

. A mobile phase comprising methanol and 10 mM pH 7.4 phosphate buffer (1:9, v/v)

containing 0.5 mM tetrabutylammonium chloride was used and the eluate was monitored at 323 nm (for 5-ASA) and 277 nm (for PA). The retention times of 5-ASA and PA were 7.1 and 15.5 min, respectively.

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Incubation of drugs with the contents of the gastrointestinal tract Male SD rats (250–260 g) were sacrificed by CO2 followed by a midline incision. The contents of each segment of intestinal tract, the proximal small intestine (PSI), distal small intestine (DSI), and cecum, was collected to prepare a 20% (w/v) suspension in pH 6.8 isotonic phosphate buffer as described previously

22 23

, . To the 20% suspension

(0.5 ml), either 5-ASA-azo-PA or sulfasalazine (as a control) in pH 6.8 isotonic phosphate buffer (0.5 ml, 1 mM) was added and incubated at 37 °C under nitrogen (for the cecal contents). The samples were centrifuged at 10,000 × g for 5 min. To the 0.1 ml portion of the supernatants, 0.9 ml of methanol was added, followed by vortexing and then centrifuging at 20,000 × g at 4 °C for 10 min. The concentrations of 5-ASA and PA in the supernatants were determined by using HPLC. Oral and rectal administration of drugs After starvation for 24 h except for water, male SD rats (250-260 g) were anesthetized with isoflurane. For oral administration, sulfasalazine (30 mg/kg) or 5-ASA-azo-PA (28.94 mg/kg, equivalent to the amount of 5-ASA in 30 mg of sulfasalazine) suspended in pH 7.4 phosphate buffer saline (PBS, 1 ml) was administered to rats once a day by oral gavage (Jungdo-BNP, Seoul, South Korea) 72 h after induction of colitis. For rectal administration, 5-ASA (20 mM, 500 µl) and/or PA (20 mM, 500 µl) in PBS were

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instilled into the colon using a rubber cannula (OD, 2 mm) as in oral gavage. The rats were sacrificed for evaluation of the anti-colitic activity 7 days after medication was received. Plasma concentration of drugs After starvation for 24 h except for water, male SD rats were anesthetized with isoflurane and cannulated with polyethylene tubing SP 45 (OD, 0.96 mm; Natsum, Japan) through the femoral artery. PA (17.7 mg/kg) or 5-ASA-azo-PA (17.7 mg equivalent of PA/kg) in PBS (1.0 ml) was administered by oral gavage. Blood samples were collected from the femoral artery with a heparinized syringe at appropriate time intervals and centrifuged at 4,000 × g for 10 min. PA in the plasma was analyzed by HPLC following precipitation of plasma proteins by addition of 9-fold volume of methanol. Induction and evaluation of inflammation TNBS-induced rat colitis was generated by the method reported previously 24, 25. Drugs were administered via oral or rectal route once a day and the rats were sacrificed 7 days after medication was received. Colonic injury was assessed by calculating colonic damage score (CDS) according to the scoring system set previously

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24, 25

. Four

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independent observers blinded to the treatment assessed the CDS. Myeloperoxidase (MPO) activity was measured in the distal colon (4 cm) as described previously 25. Cell culture Human colon carcinoma cell lines HCT116 and murine macrophage RAW264.7 cells (ATCC, Manassas, VA, USA) were grown in Dulbecco’s Modified Eagle’s medium supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) and penicillin/streptomycin. Recombinant human TNF-α was obtained from R & D systems (Minneapolis, MN, USA). LPS was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Luciferase assay and Western blot Cells, plated in 6-well plates to be 50–60% confluent, were 24 h-transfected with an NFκB-dependent luciferase plasmid (a gift from Dr. M. Birrer, NCI) and CMV Renilla luciferase plasmid (Promega, Madison, WI, USA) using Fugene (Roche, CA, USA) as a transfection reagent and luciferase activities were measured 8 h later as described in a previous paper 26 Tissue lysates of the inflamed distal colon were prepared by disruption and homogenization of the tissues (1 g) in 3 ml of ice cold RIPA buffer [50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.7% Na deoxycholate, 1% NP-40, 150 mM NaCl, 0.3 µM

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aprotinin, 1 µM pepstatin and 1 mM PMSF], which was incubated on ice for 30 min followed by centrifugation at 10,000 × g at 4 °C for 10 min. Tissue extracts were subjected to Western blot analysis after electrophoretic separation using 7.5 or 10% SDS-PAGE gels. COX-2 and iNOS proteins in the tissue homogenates were detected using a monoclonal anti-COX-2 antibody and an anti-iNOS (NOS-2) antibody (Santa Cruz Biotechnology). For cellular iNOS and COX-2 proteins, the proteins were detected in whole cell lysates obtained by using RIPA buffer. Supersignal chemiluminescence substrate (Pierce, Rockford, IL, USA) was used to visualize signals. Experiments were performed in duplicate and normalized with antibodies to α-tubulin (Santa Cruz Biotechnology). IL-6, IL-8 and CINC-3 immunoassay To measure cytokine-induced neutrophil chemoattractant-3 (CINC-3) and interleukin-6 (IL-6) in the inflamed tissues and IL-8 (neutrophil chemotactic factor) in cell culture supernatants, ELISA for each cytokine was performed according to the manufacturer’s instructions (R & D systems).

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Data analysis The results are expressed as mean ± standard error of the mean (SEM). One-way ANOVA followed by Tukey’s HSD test or the Mann Whitney U test (for CDS) were used to test the difference between the data. Differences with α or P < 0.05 were considered significant.

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Results Synthesis of 5-ASA-azo-PA 5-ASA-azo-PA was synthesized by a simple synthetic process, as shown in Fig. 1. The final product (5-ASA-azo-PA) was precipitated in pH 9.0 buffer. The mass spectrum showed that approximately 95% of the final product was detected as [MH]+ at m/z 385.1873 and the rest was likely its sodium salt detected at m/z 407.1694. The carbonyl stretching bands in the IR spectra were found at 1654 cm-1 and 1628 cm-1. The carbonyl band ascribed to the benzamide group in PA was shifted from 1635 cm-1 to 1654 cm-1 while the carbonyl band in the carboxylic group in 5-ASA was shifted from 1651 cm-1 to 1628 cm-1. These shifts were owing to the formation of the azo-bond between 5-ASA and PA, which enabled a zwitterion to be formed. In line with this, the protons adjacent to the nitrogen in the alkyl amine moiety in PA (in 5-ASA-azo-PA) were detected at chemical shifts similar to those in PA hydrochloride while the protons in PA were detected at lower chemical shifts. 1H-NMR data for PA hydrochloride and PA are shown in Supporting information 1. Colon targetability of 5-ASA-azo-PA We investigated whether 5-ASA-azo-PA was colon-specific both in vitro and in vivo. The partition coefficient was measured as 0.326 in an n-octanol/pH 6.8 buffer system,

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indicating that the prodrug is relatively hydrophilic. When the prodrug was incubated in the small intestinal contents, and pH 1.2 and pH 6.8 buffers, either 5-ASA or PA was not detected in the media along with no change in the level of the prodrug. On the contrary, 5-ASA-azo-PA was cleaved to 5-ASA and PA in the cecal contents, and the percent cleavage of the prodrug was about 67% at 5 h and 76% at 10 h (Fig. 2A). To verify whether the colonic activation of 5-ASA-azo-PA was performed by microbial enzymes, the cecal contents were autoclaved as reported

27

followed by the addition of 5-ASA-

azo-PA. Neither 5-ASA nor PA was generated up to 24 h. To further examine colon targetability, 5-ASA-azo-PA was administered orally to rats and 5-ASA was detected in the cecum. To compare delivery efficiency with a current colon-specific prodrug of 5ASA, sulfasalazine was subjected to the same experiment. As shown in Fig. 2B, 5-ASA and PA began to be detected in the cecum at 2 h and reached the maximal concentration 4 h after oral gavage of the prodrug. Compared with sulfasalazine, a slightly greater amount of 5-ASA was detected at time points later than 4 h. Colonic delivery of 5-ASA reduces the side effects by decreasing its systemic absorption 17. However, it was not clear whether absorption of PA delivered to the large intestine was limited, thereby avoiding the possible cardiovascular effects of PA. To examine this, PA was monitored in the blood after oral gavage of 5-ASA-azo-PA. This

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experiment was repeated with PA. As shown in Fig. 2C, PA was not detectable with oral gavage of 5-ASA-azo-PA while detected up to about 31 µM after oral gavage of PA, implying that the systemic absorption of PA may be limited by colonic delivery of PA, thus probably avoiding the systemic effect. 5-ASA-azo-PA ameliorated TNBS-induced rat colitis by combined action of 5-ASA and PA We examined whether 5-ASA-azo-PA ameliorated TNBS-induced colitis rats. The colonic damage score (CDS) and the myeloperoxidase (MPO) activity were determined 7 days after oral gavage of the prodrug once a day. As shown in Fig 3A exhibiting severity of colonic injury by TNBS-induced inflammation, the TNBS control colon was massively covered with scab caused by the hemorrhagic necrosis of the mucosa and suffered from severe stricture and extensive serosal adhesion to the adjacent organs while no damage was observed in the normal colon. Oral gavage of 5-ASA-azo-PA significantly healed the damaged colon. Photos of the colons are shown in Supporting information 2. In parallel, Fig. 3B showed that MPO activity in colitic rats treated with 5-ASA-azo-PA via oral gavage was lowered to about 47% of the TNBS control. When designing 5-ASA-azo-PA, PA was expected to potentiate anti-colitic activity of 5-ASA. Therefore, we assessed whether PA did so. A 500-µl aliquot of 5-ASA (20 mM) and/or

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PA (20 mM) in PBS was instilled into the inflamed colons through the rectal route once a day. CDS and MPO activities were determined 7 days later. The dose of 5-ASA and PA was decided based on a previous paper 28. As shown in Fig. 3C and D, both 5-ASA and PA alleviated the colonic damage and reduced MPO activities in the inflamed colon, and combined treatment with 5-ASA and PA showed an additive improvement in the inflammatory indices. Photos of the colons are shown in Supporting information 3. Anti-colitic effects of 5-ASA-azo-PA are attributable to suppression of inflammatory mediators in the inflamed colon Our data demonstrate that the therapeutic effects of 5-ASA-azo-PA likely resulted from an additive anti-colitic effect of 5-ASA and PA. It was investigated how the carrier PA exerted such an effect. Since, in addition to its local anesthetic activity, PA derivatives as well as 5-ASA inhibit the activity of NFκB

18, 29

, a drug target for the treatment of

colitis, we examined whether PA and 5-ASA could exert NFκB inhibition in an additive manner. Human colon carcinoma HCT116 cells, transfected with an NFκB-dependent luciferase plasmid, were stimulated with TNF-α in the presence of 5-ASA and/or PA. As shown in Fig. 4A, TNF-α increased the luciferase activities by up to 9.6-fold. TNFα induction of luciferase was attenuated by either PA or 5-ASA, and combined PA and 5-ASA

treatment elicited additive inhibition. To confirm this,

17


TNF-α or


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lipopolysaccharide (LPS)-mediated expression of NFκB target gene products was monitored in the presence of 5-ASA and/or PA. Consistently, as shown in Fig. 4B and C, combined PA and 5-ASA treatment additively reduced TNF-α secretion of IL-8, a neutrophil chemoattractant, (in HCT116 cells) and LPS induction of COX-2 and iNOS (in murine macrophage RAW 246.7). We examined whether the results of the cell-based experiments could be reproduced in vivo. To do this, NFκB-dependent inflammatory mediators, IL-6, CINC-3, COX-2, and iNOS, were analyzed in the inflamed colon after rectal administration of 5-ASA and/or PA. As shown in Fig. 4D, E, and F, the elevated levels of IL-6 (Fig. 4D) and CINC-3 (Fig. 4E) were detected in the inflamed colon. Combined treatment with PA and 5-ASA lowered the inflammatory mediators at a greater level than single treatment with either PA or 5-ASA. For COX-2 and iNOS (Fig. 4F), we could not assess an additive effect from the combined treatment because 5-ASA alone almost completely suppressed the induction of COX-2 and iNOS in our experimental conditions. 5-ASA-azo-PA is as effective at improving inflammatory indices as sulfasalazine. Our data suggest that 5-ASA-azo-PA would elicit greater anti-colitic activity than sulfasalazine (5-ASA azo-linked to sulfapyridine) where 5-ASA but not sulfapyridine has anti-colitic effects. To examine this, the anti-colitic activity of sulfasalazine was

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compared with that of 5-ASA-azo-PA after oral gavage of sulfasalazine to colitic rats. As shown in Fig 5A and B, sulfasalazine reduced CDS and MPO, which was comparable to 5-ASA-azo-PA (Fig 5A and B). In parallel, no significant difference in anti-inflammatory activities of the two prodrugs was exhibited as the inflammatory mediators were determined in the inflamed colon (Fig 5C, D and E). This observation is not consistent with our results showing that 5-ASA-azo-PA acted as a mutual prodrug of 5-ASA. To provide a plausible explanation, we examined whether sulfapyridine, known as the inactive carrier of sulfasalazine

30

, affected NFκB activity. As shown in

Supporting information 4, in line with no anti-colitic activity of the sulfa drug, no NFκB inhibitory effect was detected when sulfapyridine and/or 5-ASA was subjected to experiments to access NFκB activity. Since a colon-specific prodrug and its parent drug may coexist until complete conversion of the prodrug in the large intestine 17. Thus, we examined whether sulfasalazine cooperated with 5-ASA to inhibit NFκB activity. Cells stimulated with TNF-α were treated with sulfasalazine and/or 5-ASA and NFκB activities were determined. Sulfasalazine concentration lower than that of 5-ASA was used to simulate the therapeutic concentrations in the large intestine following sulfasalazine administration

31, 32

. As shown in Fig 6A and B, sulfasalazine inhibited

NFκB activity, consistent with previous papers

33 34

, and sulfasalazine potentiated the

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ability of 5-ASA to inhibit NFκB activity, suggesting that anti-colitic effects of sulfasalazine may be exerted partly through mutual action.

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Discussion In this study, we prepared and evaluated 5-ASA azo-linked to procainamide (5-ASAazo-PA) as a colon-specific mutual prodrug. 5-ASA-azo-PA delivered 5-ASA and PA to the large intestine and exhibited anti-colitic activity in a TNBS-induced rat colitis model, which were comparable to sulfasalazine, a colon-specific 5-ASA prodrug currently being used in clinic. Rectal administration of combined 5-ASA and PA elicited additive anti-colitic effects compared with those of either 5-ASA or PA. Moreover, the molecular data suggest that the anti-colitic effects of 5-ASA-azo-PA are partly attributable to 5ASA and PA cooperating to inhibit NFκB. Our in vitro and in vivo data suggest that 5-ASA-azo-PA can deliver 5-ASA and PA to the large intestine. The delivery efficiency of 5-ASA-azo-PA seems to be comparable to a commercially available colon-specific 5-ASA prodrug, sulfasalazine, as similar cecal accumulation of 5-ASA was observed after oral gavage of the two 5-ASA prodrugs. Given a previous paper demonstrating that the colonic amount of 5-ASA is at least 5 times greater with oral gavage of sulfasalazine than that of 5-ASA31, it is very likely that 5-ASA-azo-5-ASA efficiently delivers 5-ASA and PA to the large intestine. These results also support large intestinal conversion of 5-ASA-azo-PA to 5-ASA and PA, which is consistent with the in vitro observation that 5-ASA-azo-PA released 5-

21



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ASA and PA in the cecal contents while remaining stable in the small intestinal contents. The prodrug activation is likely to occur by microbial enzymes such as azoreductases as the conversion of the prodrug to 5-ASA and PA did not take place in the autoclaved cecal contents where microbial enzymes are inactivated 27. Our data showing that oral gavage of 5-ASA-azo-PA did not afford PA detectable in the blood while a substantial amount was detected after PA administration suggest that colonic delivery of PA can limit its systemic absorption. Since PA is an anti-arrhythmic agent and may cause arrhythmia in the normal heart

16

, restriction of absorption is an

important issue in the therapeutic switching of PA to an anti-colitic agent. In addition, given that a large percentage of IBD patients under long-term sulfasalazine therapy experience moderate to serious side effects, such as agranulocytosis and hypospermia, owing to systemic absorption of sulfapyridine generated from sulfasalazine in the large intestine 35, 5-ASA-azo-PA may have toxicological advantages over sulfasalazine. Our data show that combined intracolonic treatment with 5-ASA and PA exerted better anti-colitic effects than did treatment with 5-ASA. These data suggest that PA contributed to the anti-colitic effects of 5-ASA-azo-PA. Actually, it is not surprising that local anesthetics have beneficial effects in colitis

8, 9

. Therefore, PA likely acts a colon-

specific carrier as well as a therapeutic agent, thus satisfying the conditions to be a

22


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mutual prodrug 17. Moreover, we suggest that the combined effect occurs at least partly via cooperative inhibition of an anti-inflammatory target, NFκB

19

. This argument is

based on the data demonstrating that PA as well as 5-ASA inhibited NFκB activities, and additive NFκB inhibition was elicited by combined treatment with PA and 5-ASA in cells. In parallel, combined intracolonic treatment with PA and 5-ASA reduced the levels of NFκB target gene products in the inflamed colon, which was more effective than single treatment with either 5-ASA or PA. Although 5-ASA-azo-PA is very likely a colon-specific mutual prodrug acting against colitis, significant therapeutic superiority to sulfasalazine was not observed in our experimental conditions. Our data showed that 5-ASA-azo-PA was not more effective than the current colon-specific 5-ASA prodrug in improving inflammatory indices such as CDS, MPO, and inflammatory mediators. This result is not what was expected since sulfapyridine, the carrier of sulfasalazine, does not have any anti-colitic activity

30

. We suggest that the discrepant result may be ascribed to the anti-colitic

activity of sulfasalazine itself. A number of papers demonstrate that sulfasalazine, likely coexisting with 5-ASA in the inflamed large intestine until complete conversion to 5ASA and sulfapyridine, has anti-NFκB activity 34, 36. This hypothesis is supported by our data showing that sulfasalazine and 5-ASA elicited an additive inhibitory effect on

23



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NFκB, which was not observed with treatment with 5-ASA-azo-PA and 5-ASA. However, considering that local anesthetics exhibits anti-colitic activity likely via their effects on enteric nerves

8, 9

, the NFκB-related hypothesis does not fully explain the

therapeutic discrepancy. Thus, it could also be owing to limitations of the animal colitis model to differentiate such therapeutic differences. Animal experiments with more elaborate experimental conditions, such as optimizing the TNBS amount for colitis induction and treatment period, may be required to conclusively determine the therapeutic advantage of 5-ASA-azo-PA over sulfasalazine. Despite no significant therapeutic superiority to sulfasalazine in our experiments, 5ASA-azo-PA may have advantage over sulfasalazine. Procainamide, a local anesthetic, likely lessens colitis symptoms such as abdominal pain

37

and improves the large

intestinal functions by down-regulating overactive sympathetic nerves 38, 39. In addition, 5-ASA-azo-PA may have improved toxicological property considering the harmful side effects of sulfasalazine result from the carrier, sulfapyridine, absorbed systemically 40. Collectively, 5-ASA-azo-PA is a potential colon-specific mutual prodrug acting against colitis, and cooperative NFκB inhibition may be a molecular mechanism for the mutual anti-colitic action of the prodrug.

24


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Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2009-0083538) and NRF2014H1A2A10221270.

Declaration of interest: The authors report no conflicts of interest.

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Figure legends Fig. 1. Synthesis of 5-(4-{[2-(diethylamino)ethyl]carbamoyl}phenylazo)salicylic acid (5-aminosalicylic acid azo-linked to procainamide, 5-ASA-azo-PA). Fig. 2. 5-ASA-azo-PA is a colon-specific prodrug converted to 5-ASA and PA. (A) 5-ASA-azo-PA (10 mM) was incubated in the cecal contents and the mixture of the small intestinal contents and mucosa suspended in pH 6.8 PBS (10%). At appropriate time intervals, the levels of 5-ASA and PA in the samples were determined by HPLC. Drugs (%) represents percent of drugs (5-ASA and PA) generated from 5-ASA-azo-PA. (B) Male SD rats (250–260 g) were starved for 24 h except for water. Sulfasalazine (30 mg/kg) or 5-ASA-azo-PA (28.9 mg/kg, equivalent to 5-ASA amount in 30 mg of sulfasalazine) suspended in pH 7.4 phosphate buffer saline (PBS, 1 ml) was administered orally to rats by gavage. The rats were sacrificed and a midline incision was made to obtain the cecal contents. Drugs in the cecal contents were analyzed by HPLC. (C) PA (17.7 mg/kg, equivalent to 28.9 mg of 5-ASA-azo-PA) or 5-ASA-azo-PA (28.9 mg/kg) was administered orally to rats and the blood was collected at predetermined intervals from a cannulated femoral artery. PA was analyzed in the blood by HPLC. The data in A, B and C represent mean ± SEM (n = 5).

33



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Fig. 3. 5-ASA-azo-PA ameliorates experimental colitis by combined action of 5ASA and PA. (A and B) 5-ASA-azo-PA (28.9 mg/kg) was administered orally to TNBS-induced colitis rats once a day and the rats were sacrificed after treatment with each drug for 7 days. (A) CDS were assigned for each group.

*:

α < 0.05 vs. TNBS control (B) Using

the distal colon (4 cm), myeloperoxidase activities were measured. *: P < 0.05 vs. TNBS control (C and D) 5-ASA (20 mM) or/and PA (20 mM) in pH 6.8 PBS buffer (300 µl) was administered rectally to TNBS-induced colitis rats once a day and the rats were sacrificed after treatment with each drug for 7 days. (C) CDS were assigned for each group. *: α < 0.05 vs. TNBS control, #: < 0.05. (D) Using the distal colon (4 cm), myeloperoxidase activities were measured. *: P < 0.05, **: P < 0.01 vs. TNBS control; #: P < 0.05. The data in A, B, C and D represent mean ± SEM (n = 5). Fig. 4. PA potentiates the ability of 5-ASA to inhibit NFκB activity and expression of inflammatory mediators. (A) Colon carcinoma HCT116 cells were cotransfected with NFκB-dependent luciferase plasmid and CMV Renilla luciferase plasmid and subsequently treated for 6 h with TNF-α (10 ng/ml) in the presence of 5-ASA (10 mM) and/or PA (10 mM). Reporter activities were measured and normalized to CMV Renilla luciferase activity. *: P < 0.05;

34


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**:

P < 0.01 vs. group treated with TNF-α alone; #: P < 0.05. (B) HCT116 cells were

stimulated with TNF-α for 6 h in the presence of 5-ASA (10 mM) and/or PA (10 mM). Levels of IL-8 were monitored in the supernatants. *: P < 0.05 vs. group treated with TNF-α alone; **: P < 0.01 vs. group treated with TNF-α alone; #: P < 0.05. (C) RAW 264.7 cells were stimulated with LPS for 4 h in the presence of 5-ASA (10 mM) and/or PA (10 mM). Levels of iNOS and COX-2 protein were monitored in the whole cell lysates. (D, E, and F) 5-ASA (20 mM) and/or PA (20 mM) in pH 6.8 PBS buffer (300 µl) were administered rectally to TNBS-induced colitis rats once a day and the rats were sacrificed after treatment with each drug for 7 days. The levels of IL-6 (D), CINC-3 (E), and COX-2 and iNOS (F) were determined in the inflamed colons. *: P < 0.05; **: P < 0.01 vs. TNBS control; #: P < 0.05. The data in A, B, D, and E represent mean ± SEM (n = 5). Fig. 5. 5-ASA-azo-PA is as effective at improving inflammatory indices as sulfasalazine. (A and B) Sulfasalazine (30 mg/kg) or 5-ASA-azo-PA (28.9 mg/kg, equivalent to the amount of 5-ASA in 30 mg of sulfasalazine) was administered orally to TNBS-induced colitis rats once a day and the rats were sacrificed 7 day after treatment with each drug. (A) CDS were assigned for each group.

*:

α < 0.05 vs. TNBS control (B) Using the

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distal colon (4 cm), myeloperoxidase activities were measured. *: P < 0.05 vs. TNBS control. Inflammatory mediators, IL-6 (C), CINC-3 (D) and COX-2 and iNOS (E) were determined in the inflamed colons. The data in A, B, C and D represent mean ± SEM (n = 5). *: P < 0.05; **: P < 0.01 vs. TNBS control. Fig. 6. Sulfasalazine potentiates the ability of 5-ASA to inhibit NFκB activity. (A) Human colon carcinoma HCT116 cells were cotransfected with NFκB-dependent luciferase plasmid and CMV Renilla luciferase plasmid for 1 day, followed by 6 htreatment with TNF-α (10 ng/ml) in the presence of 5-ASA and/or sulfasalazine. Reporter activities were measured and normalized to CMV Renilla luciferase activity. *: P < 0.05;

**:

P < 0.01 vs. group treated with TNF-α alone; #: P < 0.05. (B) HCT116

cells were stimulated with TNF-α for 6 h in the presence of 5-ASA and/or sulfasalazine. Levels of IL-8 were monitored in the supernatants. *: P < 0.05; **: P < 0.01 vs. group treated with TNF-α alone; #: P < 0.05.

36


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Supporting information Supporting information 1. 1H-NMR data of procainamide HCl and procainamide Supporting information 2. Photos of the distal colons of rats after oral gavage of drugs Supporting information 3. Photos of the distal colon of rats after rectal administration of drugs Supporting information 4. Effect of sulfapyridine and/or 5-ASA on NFκB activity

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Fig 1. 190x142mm (300 x 300 DPI)


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Fig 2. 190x142mm (300 x 300 DPI)



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Fig 3. 190x142mm (300 x 300 DPI)


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Fig 4ABC. 190x142mm (300 x 300 DPI)



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Fig 4DEF. 190x142mm (300 x 300 DPI)


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Fig 5AB. 190x142mm (300 x 300 DPI)



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Fig 5CDE. 190x142mm (300 x 300 DPI)


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Fig 6. 190x142mm (300 x 300 DPI)



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Table of Contents Graphic 254x190mm (300 x 300 DPI)


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5-Aminosalicylic Acid Azo-Linked to Procainamide Acts as an

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