Gastroprotective [6]-Gingerol Aspirinate as a Novel Chemopreventive Prodrug of Aspirin for Colon Cancer

A growing body of research suggests daily low-dose aspirin (ASA) reduces heart diseases and colorectal cancers. However, the major limitation to the use of aspirin is its side effect to cause ulceration and bleeding in the gastrointestinal tract. Preclinical studies have shown that ginger constituents ameliorate ASA-induced gastric ulceration. We here report the design and synthesis of a novel prodrug of aspirin, [6]-gingerol aspirinate (GAS). Our data show that GAS exerts enhanced anti-cancer properties in vitro and superior gastroprotective effects in mice. GAS was also able to survive stomach acid and decomposed in intestinal linings or after absorption to simultaneously release ASA and [6]-gingerol. We further present that GAS inactivates both COX-1 and COX-2 equally. Our results demonstrate the enhanced anticancer properties along with gastroprotective effects of GAS, suggesting that GAS can be a therapeutic equivalent for ASA in inflammatory and proliferative diseases without the deleterious effects on stomach mucosa.

Scientific RepoRts | 7:40119 | DOI: 10.1038/srep40119 found an increased risk of developing cardiovascular condition with the use of selective COX-2 inhibitors [19][20][21][22] . Therefore, rather than selective COX-2 inhibitors, the advancement of new aspirin-like molecules with gastroprotective effects would be a welcome development.
The potential long-term therapy of aspirin-like molecules without deleterious side effects in clinical trials prompted our current investigations. The development of ginger ingredient 6G and aspirin in one molecule has never been reported before. We herein report the design and synthesis of [6]-gingerol aspirinate (GAS). The molecular basis of pharmacological properties of GAS was further probed as well.

Results
Synthesis and structural elucidation of GAS and AMS. GAS was prepared by acylation of 6G with O-acetylsalicyloyl chloride in the presence of Et 3 N at 0 °C (Fig. 1a). The structure of GAS was confirmed by NMR experiments (1D-and 2D-) and mass spectra ( Fig. 1b and c). In detail, GAS had a molecular ion at m/z 457 [M + H] + (295 + 162), indicating a molecular formula of C 26 H 32 O 7 , suggesting that GAS is a [6]-gingerol aspirinate. Aspirin residue and 6G moiety in the structure of GAS were identified by HMBC correlations (Fig. 1b). The attachment of ASA residue at OH-4 in the aromatic ring of 6G moiety through an ester bond was established by a weak HMBC crosspeak between H-5 (δ H 7.02) and C-7″ (δ C 162.6, C= O) (Fig. 1b). This linkage was further supported by NOESY correlations observed between OAc-2″ (δ H 2.29, CH 3 CO) and OMe-3 (δ H 3.79) as well as H-5 (δ H 7.02), and between OMe-3 (δ H 3.79) and H-6″ (δ H 8.23) (Fig. 1b). Major MS/MS fragments at m/z 439 (loss of water), m/z 415 (loss of acetyl group), and m/z 397 (loss of both acetyl group and water) were also in agreement with the above interpretation ( Fig. 1c and Table 1). Thus, the structure of GAS was identified as 4-(5-hydroxy-3-oxodecyl)-2-methoxyphenyl 2-acetoxybenzoate, which is a novel compound.
Acetylation of methyl salicylate (MS) by Ac 2 O in pyridine gave methyl aspirinate (AMS) (Fig. 1a). The structure of AMS was confirmed by interpretation of its 1 H-and 13 C-NMR spectra 23 .
GAS has enhanced anticancer activities in human colon cancer cells than the combination of 6G and ASA, as well as AMS. Subsequently, individual drugs 6G and ASA, an equimolar mixture of 6G and ASA (6G + ASA), and GAS were evaluated for their anticancer activities against human colon cancer cells HCT-116 and HT-29 by MTT assays. As seen, GAS was more active (IC 50 s: 75.97 μ M in HCT-116, and 84.49 μ M in HT-29) than individual drugs 6G (IC 50 > 100 μ M in both cells) and ASA (IC 50 > 100 μ M in both cells), as well as 6G + ASA (IC 50 > 100 μ M in both cells) ( Fig. 2a and b). Likewise, colony formation assays showed that GAS had greater antiproliferative activity than 6G + ASA and individual drugs 6G and ASA, and could inhibit colony formations in both cancer cells in a dose-dependent manner (Fig. 3). In particular, GAS could visually suppress the growth of colonies in cancer cells HCT-116 and HT-29 at concentrations of 20 μ M and 40 μ M, respectively, compared to control ( Fig. 3a and c). At a concentration of 60 μ M, GAS nearly completely suppressed the colony formation in HCT-116 by 100% and in HT-29 by 97% ( Fig. 3b and d). Whereas, at this concentration, both 6G + ASA and 6G were less active than GAS in both cell lines, and only inhibited the growth of colonies in HCT-116 by 76% and 66%, and in HT-29 by 74% and 68%, respectively ( Fig. 3b and d). To decide if gingerol ester in GAS is specifically constitutive of inhibitory effects of this compound on the growth of cancer cells, a simple methyl ester of aspirin (AMS) was synthesized and used along with MS as controls. We found that both MS and AMS exerted similar cytotoxicities to aspirin (IC 50 > 100 μ M in both cells for each), and were less active than GAS (Fig 2c and d). These observations demonstrated that gingerol aspirinate has anticancer potential rather than methyl aspirinate and non-covalent complex 6G + ASA. Our findings identified that GAS has enhanced anticancer properties, suggesting a lower-dose use of GAS in long-term therapy than ASA in clinical trials.

GAS alleviates gastric ulceration in mice.
To investigate if GAS effectively reduces the common side effects of ASA, we determined acute gastric lesions and bleeding following intragastric gavage of ASA, ASA + 6G, and GAS in mice. Photographs of the stomach were taken and digitized. As seen in Fig. 4a, the macroscopic images showed that the hemorrhagic gastric lesions coated with coagulated blood in the ASA administered group were more apparent than the 6G + ASA or GAS treated group, verifying that both GAS treatment and the co-administration of 6G with ASA effectively attenuated lesion formation caused by ASA. The measured areas of gastric lesions further demonstrated that GAS significantly inhibited the ASA-induced stomach injury in mice by 75% (p < 0.05), and has better protective effects on stomach mucosa than the combination of 6G and ASA (6G + ASA) (20% inhibition) (Fig. 4b). Our results demonstrate the remarkable gastroprotective properties of GAS. The greater potency of GAS also suggests different pharmacokinetics and biodistribution parameters when administration of 6G and ASA as a single molecule (GAS).
Metabolism and distribution of GAS in mice. LC-tandem mass spectroscopic data determined the metabolism and biodistribution of GAS in mice. 6G, salicyluric acid (SUA), and salicylic acid (SA) were identified as the major metabolites of GAS in mouse gastrointestinal (GIT) contents, GIT tissues, and biofluids by direct comparison of their respective tandem mass spectra and retention times with authentic standards (Table 1 and  (Table 1) 24,25 . Under positive ESI mode, GAS was found to be almost intact in the stomach, partly hydrolyzed in the proximal intestine and then fully decomposed in the distal intestine to release 6G and ASA in 2 h after oral administration ( Fig. 5a-f). 6G was further transformed into gingerdiols M1 and M2 in the distal intestine and colon ( Fig. 5e-h), and the remaining 6G was transited out of body through feces (Fig. 5o). Likewise, 6G was extensively distributed in the tissues of stomach and colon ( Fig. 5i-l), and predominantly presented in plasma in 2 h after treatment ( Fig. 5m and n). On the other hand, SA, a major metabolite of ASA 25 , was abundant in the tissues of stomach and colon, plasma, feces, and urine under a negative ESI mode ( Fig. 5i′-p′ ). Meantime, we found SUA, another metabolite of ASA 25 , in the feces and urine samples ( Fig. 5o′-p′ ). Our results demonstrate that GAS can survive stomach acid and is decomposed to simultaneously release ASA and 6G in intestinal linings. These findings suggest that GAS has resistance to disintegration in the stomach, thereby causing less upper-gastrointestinal bleeding (UGIB) than plain ASA. Also, the departed 6G may simultaneously attenuate the gastrointestinal complications caused by ASA due to its anti-ulcerative effects 3,6 .
GAS inactivates COX-1 and COX-2. GAS has been found to enhance anti-cancer properties of ASA while reducing the risk of gastric toxicity in a fashion similar to those selective COX-2 inhibitors 19,20 . To determine the selectivity of GAS toward COX enzymes, a Western blotting assay was performed. We found that, unlike selective COX-2 inhibitors, GAS significantly inhibits the expression of both COX-1 and COX-2 in a dose-dependent manner in human colon cancer cells HT-29, compared to the control (DMSO) (p < 0.05 for all) ( Fig. 6a and b). Particularly, GAS exerted similar inhibitory effects against both COX-1 and COX-2 at tested concentrations (30% vs 24% at 40 μ M; 40% vs 38% at 80 μ M; and 71% vs 62% at 100 μ M) ( Fig. 6a and b). Furthermore, Western blotting assays also demonstrated that GAS exerted significantly inhibitory effects towards COX-1 and COX-2 at a concentration of 80 μ M, when compared to individuals ASA and 6G, and the mixture of 6G and ASA (6G + ASA) at a same concentration, respectively (p < 0.05 for all) ( Fig. 6c and d). Our results demonstrate that GAS is a nonselective NSAID. The sustained inhibition of COX-1 in nonselective NSAIDs provides an antiplatelet effect, which may partly reduce the cardiovascular risk associated with COX-2 inhibition, such as Naproxen 18 , implicating the advantages of "balanced" inhibitions of COX-1 and COX-2 in nonselective NSAIDs.

Discussion
To our knowledge, this is the first study to incorporate 6G, the pungent principal of fresh ginger with gastroprotective effects 6,10 , into ASA moiety to form one molecule. We found joint use of non-covalent complex of 6G and ASA (6G + ASA) could significantly suppress the acute injury of stomach induced by ASA in mice, which is in agreement with the important role of 6G in stomachic medications in previous studies 6,10 . We further demonstrated that the covalent complex of 6G and ASA (GAS) exerted significantly improved gastroprotective effects than 6G + ASA in mice. The enhanced gastroprotection of GAS could be a result of several contributions. Firstly, GAS survives mouse stomach acids, thereby decreasing the risk of UGIB. Secondly, GAS was decomposed to simultaneously release 6G and ASA in the mouse intestine or after absorption, and concomitant 6G in mouse organs, tissues, and plasma would immediately restore or prevent the local injury caused by ASA. In addition, small amounts of GAS in the tissue of mouse stomach and colon may, at least, exert its own gastroprotection. Our findings also showed the improved anticancer activities of GAS compared to ASA, 6G, and 6G + ASA, in human colon cancer cells. This observation could be due to the structural skeleton composed of 6G and ASA with higher anticancer properties than those of individuals, or 6G + ASA, similar to our previous findings on the combination of resveratrol and ASA 25 . AMS and MS failed to strengthen the anticancer activity of aspirin, suggesting that methyl ester makes little contribution to aspirin while gingerol ester is a specific building block of GAS for various beneficial physiological effects of aspirin. It is quite understandable that, without gingerol ester in the structure, methyl aspirinate has no ability to reduce the risk of gastric toxicity caused by aspirin, unlike gingerol aspirinate (GAS). In addition, when 6G and ASA were co-administrated (6G + ASA), multiple aspects, including drug interactions, the definitive exposure to the desired targets, and individual pharmacokinetics and biodistribution, should be concerned. These factors may also affect the pharmacological properties of 6G + ASA as a joint use. In contrast, the use of GAS could apparently overcome these difficulties. Our findings suggested that use of an even lower dose of GAS may achieve the equal efficacy of ASA, thereby attenuating the risk of gastrointestinal complications caused by ASA. However, the molecular basis and mechanism of actions of GAS underlying the improved anticancer properties need to be further investigated in the future.
In order to reduce gastrointestinal damage, enteric-coated aspirin and buffered formulations have also been developed to delay the release of ASA beyond the stomach or increase gastrointestinal solubility of ASA in clinical trials 26,27 . Results from endoscopic studies carried out in health volunteers indicated that enteric-coated or buffered formulations caused less gastric erosion and microbleeding than plain ASA [28][29][30] . However, use of low   doses of these formulations in patients carries no clear beneficial effects on gastrointestinal symptoms compared to regular aspirin 26,27,31 . Our findings featured the enhanced anticancer activities of GAS together with significant gastroprotective effects compared to plain ASA. GAS can be an attractive alternative in patients who are at risk of ASA-induced ulceration, as a means of reducing the cardiovascular risk and gastrointestinal complications associated with ASA. Long-term studies estimating clinical GI events are warranted to confirm the clinical GI safety profile of GAS in the future.

Materials and Methods
Chemistry. General Methods. Reactions were monitored by analytical thin-layer chromatography (TLC) on 250 μm silica gel plates (GF254) (Merck) and visualized under UV light. The products were isolated and purified by column chromatography (CC) using silica gel (Sorbent Technologies). 1 H and 13 C NMR spectra were recorded on a Bruker AVANCE 600 MHz spectrometer (Brucker, Inc., Silberstreifen, Rheinstetten, Germany) using TMS as an internal standard. Chemical shifts (δ) are expressed in ppm. Coupling constants (J) are expressed in Hz, and multiplicities are indicated by s (singlet), d (doublet), t (triplet), q (quartet), and br (broad). The 13 C NMR spectra are proton decoupled. Aspirin was purchased from MP Biomedicals (Solon, OH). Methyl salicylate was procured from Sigma-Aldrich (St. Louis, MO). 6G was purified from ginger extracts in our lab 32 . Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) or VWR BDH Chemicals (Radnor, PA), and were used without further purification. All synthesized compounds were> 95% pure.

LC-ESI/MS analysis. LC-ESI/MS was performed with a Thermo-Finnigan Spectra System consisting of an
Ultimate 3000 degasser, an Ultimate 3000 RS pump, an Ultimate 3000 RS autosampler, an Ultimate 3000 RS column compartment, and an LTQ Velos Pro ion trap mass spectrometer (Thermo Electrom) equipped with an electrospray ionization (ESI) interface. A Gemini C18 column (5 μ m, 3.0 mm i.d. × 150 mm, Phenomenex, Torrance, CA) was employed for the chromatographic separation. The mobile phase consisted of 5% aqueous methanol with 0.1% formic acid (mobile phase A) and 95% aqueous methanol with 0.1% formic acid (mobile phase B). The gradient elution was performed for 45 min at a flow rate of 0.3 mL/min. The gradient was initiated at 0% B and held constant for 3 min, followed by a linear increase to 55% from 5 to 15 min; to 100% from 15 to 30 min, and then held constant for 10 min. The column was then re-equilibrated with 0% B for 5 min. The injection volume of each sample was 10 μ L. The LC eluent was introduced into the ESI interface. The positive ion polarity mode was set for the ESI source with the ion spray voltage at approximately 3.5 kV. Nitrogen gas was used as the sheath gas at a flow rate of 34 arb and the aux gas at a flow rate of 10 arb. Optimized parameters, including temperature (300 °C), voltage of the capillary (45 V), and voltage of the tube lens offset (120 V), were tuned using authentic 6G. Selected-ion monitoring (SIM) mode was used to search GAS and its metabolites. Data dependent scanning was performed to obtain the MS/MS spectra. The collision-induced dissociation (CID) was conducted with an isolation width of 1.2 Da and normalized collision energy of 35 for both MS 2 and MS 3 . The mass range was measured from 50 to 800 m/z. Data acquisition and analysis were performed using Xcalibur 2.0 (Thermo Electron, San Jose, CA).

Synthesis of GAS.
To a solution of 6G (294 mg, 1.0 mmol, 1.0 eq.) and Et 3 N (0.42 mL, 3.0 mmol, 3.0 eq.) in DCM (5 mL) at 0 °C was added a solution of O-acetylsalicyloyl chloride (398 mg, 2.0 mmol, 2.0 eq.) in DCM (2 mL) dropwise. After addition was completed, the mixture was stirred at 0 °C for 2 h, and quenched with water (1 mL) followed by adding 10 mL of ethyl acetate (EA). The organic phase was washed with water (10 mL × 2) and brine (10 mL × 1), dried over Na 2 SO 4 , and filtered. The filtrate was evaporated in vacuo to give a residue, which was purified by CC (H/E = 4:1, 3:1, and 2:1) to get the final product, GAS, 450 mg (yield: 99%), as a yellow oil. 1   Dose-dependent inhibition of GAS on cyclooxygenases, COX-1 (a) and COX-2 (b) in human colon cancer cells HT-29. Cell extracts after treatment for 48 h with respective 0, 40, 80, and 100 μ M of GAS were performed to Western blot analysis. And inhibitory effects of ASA, 6G, 6G + ASA, and GAS on COX-1 (c) and COX-2 (d) in human colon cancer cells HT-29, respectively. Cell extracts after treatment for 48 h with respective DMSO, ASA, 6G, 6G + ASA, and GAS at a concentration of 80 μ M were harvested for Western blot analysis. Similar results were obtained in three independent experiments. Results are mean ± SD (n = 3). Bar, standard error. *p < 0.05. All statistical tests are unpaired Student's t tests, two-tailed, compared to control (DMSO) or corresponding compounds). ASA, aspirin; 6G + ASA, an equimolar mixture of 6G and ASA; GAS, [6]-gingerol aspirinate.
Scientific RepoRts | 7:40119 | DOI: 10.1038/srep40119 Synthesis of AMS. To a solution of methyl salicylate (608 mg, 4.0 mmol, 1.0 eq.) in pyridine (4.0 mL) was added acetic anhydride (816 mg, 8.0 mmol, 2.0 eq.) dropwise. After incubation at 37 °C overnight, pyridine was removed under reduced pressure. The residue was subjected to CC (H/E = 30:1, 20:1, and 10:1) to give AMS (776 mg, yield: 100%), as a white solid. 1  After the cells were cultured for 48 h, the medium was aspirated and cells were treated with 200 μ L fresh medium containing 2.41 mmol/L 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumomide (MTT). After incubation for 3 h at 37 °C, the medium containing MTT was aspirated, 100 μ L of DMSO was added to solubilize the formazan precipitate, and plates were shaken gently for 1 h at room temperature. Absorbance values were derived from the plate reading at 550 nm on a Biotek microtiter plate reader (Winooski, VT). The reading reflected the number of viable cells and was expressed as a percentage of viable cells in the control. Both HCT-116 and HT-29 cells were cultured in McCoy's 5 A medium. The media used above was supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% glutamine, and the cells were kept in a 37 °C incubator with 95% humidity and 5% CO 2 . The IC 50 values were conducted by using GraphPad Prism software (version 5).
Colony formation assay. Human colon cancer cells HCT-116 (1000 cells per well) were seeded in 6-well culture plates for 24 h, and HT-29 (2000 cells per dish) were seeded in 60 mm cell culture dish for 24 h. The medium were then incubated with the compounds in DMSO in a 37 °C incubator with 5% CO 2 . After 2 weeks, colonies were washed with phosphate-buffered saline (PBS), then stained with a mixture of 6.0% glutaradehyde and 0.5% crystal violet for 30 min at room temperature, rinsed in water, air-dried, and then photographed. The numbers of the colony were counted by using ImageJ software (version 1.48 V).
Animal study. Experiments with mice were carried out according to protocols approved by the Institutional Review Board for the Animal Care and Facilities Committee at North Carolina Research Campus (No: 13-014). The methods were carried out in accordance with the approved guidelines. Six-week-old female CF-1 mice were purchased from Charles River Laboratories Inc (Durham, NC) and allowed to acclimate for at least 1 week prior to the start of the experiment. The mice were housed 5 per cage and maintained in air-conditioned quarters with a room temperature of 20 ± 2 °C, relative humidity of 50 ± 10%, and an alternating 12-h light/dark cycle. Mice were fed AIN-93G diet from Research Diet (New Brunswick, NJ) and water, and were allowed to eat and drink ad libitum.
Metabolism of GAS. GAS in DMSO was administered to mice by oral gavage (200 mg/kg). Stomach contents and tissue, and intestine contents and tissue were collected at 1 h and 2 h, respectively, after administration of vehicle (control group, n = 5), and GAS (treated group, n = 5). Urine and fecal samples were collected in metabolism cages (5 mice per cage) in 24 h after administration. Blood samples were collected by cardiac puncture and plasma was obtained after centrifugation at 8,000× g for 5 min at 4 °C. The samples were stored at -80 °C until analysis.
Sample Preparation. For the metabolic profile study, ~100 mg of each fecal sample (control and treated) was weighted and put into individual tubes. Samples were homogenized with 1.0 mL of MeOH/H 2 O (1:1) with 0.2% AA for 5 min using an Omni Bead Ruptor Homogenizer (Kennesaw, GA) and then centrifuged at 13,200 × g for 10 min at 4 °C. The supernatant (250 μ L) was collected and diluted 5 times for analysis. Enzymatic deconjugation of the urine and plasma samples were performed as described previously with slight modifications 33 . In brief, 50 μ L of urine or plasma sample from each group (control group and GAS-treated group) were treated with β -glucuronidase (250 U) and sulfatase (3 U) for 24 h at 37 °C. The reaction was stopped by adding an equal volume of MeOH with 1% AA and centrifuged at 13,200 × g for 10 min at 4 °C, then the supernatant was transferred into vials for LC/MS analysis. For the preparation of mouse tissue samples (stomach, small intestine, and colon), 50 mg of thawed tissue was homogenized with 300 μ L of sodium acetate buffer (pH 5.4) and then centrifuged at 13,200 × g for 10 min at 4 °C, an accurately measured portion of the supernatant was collected and treated with a mixture of β-glucuronidase and sulfatase as described above, the hydrolyzed tissue sample was subjected to LC/MS for analysis.
Gastric ulceration. The proper amount of ASA, 6G + ASA, or GAS was dissolved in DMSO and ethanol (2% of final volume, respectively) and then suspended in 10% Tween-80 to 2 mL (166.72 mM as final concentration for each tested compound).
Animal Treatments. The mice were fasted for 24 h (with as libitum access to water) in metabolic cages and then were administered aspirin (200 mg/kg body weight), 6G + ASA (6G was given to mice in the same molar dose of aspirin), and GAS (GAS was given to mice in the same molar dose of aspirin) suspensions by oral gavage. At 4 h after the administration of test compounds, the mice were sacrificed by euthanization with isofluorane and then blood was collected from the heart. After sacrifice, mouse stomachs were removed and cut open along the greater curvature. The gastric contents were emptied, and the stomachs were then washed with PBS. Gastric mucosal lesions were observed under microscope at 10 × magnification and the size of the all lesions were measured and estimated manually. The inner surface was photographed by Nikon stereoscopic zoom microscope (SMZ1500) with DXM 1200 C digital camera system (0.7 × Relay lens) under Nikon Element Software (Nikon Corporation, Kanagawa, Japan). Next the gastric mucosal tissues were removed, frozen in liquid nitrogen and stored at -80 °C.
Statistical Analysis. For simple comparisons between two groups, two-tailed Student's t-test was used. A p-value of less than 0.05 was considered statistically significant in all the tests.