ABSTRACT

In this work, a curcumin-diglutaric acid (CurDG) prodrug was synthesized by conjugation of curcumin with glutaric acid via an ester linkage. The water solubility, partition coefficient, release characteristics, and antinociceptive activity of CurDG were compared to those of curcumin. The aqueous solubility of CurDG (7.48 μg/mL) is significantly greater than that of curcumin (0.068 μg/mL). A study in human plasma showed that the CurDG completely releases curcumin within 2 h, suggesting the ability of CurDG to serve as a prodrug of curcumin. A hot plate test in mice showed the highest antinociceptive effect dose of curcumin at 200 mg/kg p.o., whereas CurDG showed the same effect at an effective dose of 100 mg/kg p.o., indicating that CurDG significantly enhanced the antinociceptive effect compared to curcumin. The enhanced antinociceptive effect of CurDG may be due to improved water solubility and increased oral bioavailability compared to curcumin.

Curcuma longa L. (turmeric) is a member of the ginger or Zingiberaceae family. The main bioactive compounds in the turmeric rhizome are curcuminoids, a mixture of three diarylheptanoids compounds named curcumin (Figure 1), demethoxycurcumin and bisdemethoxycurcumin, with curcumin as the main constituent [1]. The antioxidant, anti-inflammatory, anti-cancer and potential chemotherapeutic properties of curcumin have been widely studied [24] and long-term administration of curcumin has been shown to be safe [5]. Curcumin and its derivatives have also been investigated as antibiotic, antifungal and antimalarial agents [6,7]. Curcumin also inhibits cell proliferation and angiogenesis, and induces apoptosis and cell cycle arrest in tumor cell lines [8]. However, the development of curcumin as a therapeutic agent has been hindered by its low water solubility, poor absorption, and rapid metabolism and elimination, all of which lead to poor oral bioavailability. Therefore, structural modification of curcumin has been used to achieve higher water solubility and metabolic stability, with the goal of improving bioavailability [9].

Figure 1.

Synthesis scheme of curcumin-diglutaric acid (CurDG).

Approaches to chemical modification of curcumin fall into three broad categories based on mimics, derivatives and prodrugs. The goal of the prodrug approach is to sustain or enhance pharmacological properties while improving physicochemical properties and delivery. Diacids such as maleic acid, succinic acid and glutaric acid are often chosen as prodrug carriers for enhancement of solubility due to their safety profiles and long-term historical use [1014]. In addition to solubility and safety aspects, the conjugated prodrugs with dicarboxylic acids can also contribute the salt formation, which is of benefit for developing formulations suitable for different administration routes such as parenteral dosage forms for intravenous administration. Currently, a co-crystal technology has been utilized as a new drug delivery platform for insoluble and/or unstable drugs. Prodrugs with dicarboxylic acids provide the potential of forming co-crystal with bases, and therefore can further enhance the drug’s solubility and stability. In an aspect of the prodrug design, introducing certain dicarboxylic acids such as glutaric acid to drug molecules has generated intramolecularly-activated prodrugs, which have an advantage on avoiding the interspecies and interindividual variability of prodrugs via an enzymatic bioactivation [15]. The mechanism and rate for cyclization-activated hydrolysis of prodrugs are based on the length of aliphatic hydrocarbon and the reactivity of nucleophilic attack on a carbonyl carbon of an ester by a terminal carboxyl group, resulting in the release of active moiety from the prodrug molecule.

Curcumin diethyl disuccinate (CDD), a succinic acid ester prodrug of curcumin synthesized in our laboratory [14], has better aqueous stability in phosphate buffer at pH 7.4, and has prominent cytotoxic effects in human epithelial colorectal adenocarcinoma cells and an antinociceptive effect in mice [14,16]. However, this curcumin prodrug is less soluble than curcumin due to the addition of ethyl succinyl ester at phenolic functional groups via esterification.

Recently, curcumin monoglucuronide (CMG), an aqueous soluble prodrug of curcumin, was synthesized and evaluated on its pharmacokinetics in rats and anti-cancer activity in tumor-bearing mice with the HCT116 human colon cancer cell line [17]. CMG failed to improve the plasma level of curcumin after oral administration, however, a high level of free curcumin was achieved after intravenous administration of CMG. In addition, intravenous administration of CMG demonstrated a higher tumor reduction in mice in comparison with intravenous administration of curcumin. Therefore, a highly water-soluble prodrug of curcumin would be an alternative approach to improve pharmacological effects of curcumin.

In this study, a glutaric acid conjugate of curcumin, curcumin-diglutaric acid (CurDG, Figure 1) was synthesized to increase the water solubility of curcumin. The release of parent curcumin from CurDG in human plasma was determined to ensure that CurDG can serve as a prodrug of curcumin. We show that CurDG increased water solubility and enhanced antinociceptive effect after oral administration in mice compared to curcumin.

Materials and methods

All reagents and solvents were purchased from commercial sources and used without further purification. 1H- and 13C-NMR spectra were recorded on a Varian Inova 500 MHz spectrometer operated with VnmrJ software. IR spectra were recorded with a Perkin Elmer 1760X. High resolution mass spectra (HRMS) were obtained on a Reflex IV Bruker time-of-flight high-resolution mass spectrometer. Melting points were determined using a differential scanning calorimeter (DSC823e, Mettler Toledo). Amounts of curcumin and CurDG were determined using an Agilent 1200 HPLC system (Agilent Technologies, USA) consisting of a G1312A binary pump equipped with a G1379B degasser and G1367B thermostat autosampler. Analytical separation was carried out on an Alltech Altima C18 column (150 × 4.6 mm id., 5 μm, Grace, IL, USA) with UV detection at 400 nm.

Syntheses of curcumin and CurDG

Curcumin was synthesized using a previously reported method with some modifications [13,14]. In brief, acetylacetone (1.03 mL, 10 mmol), tributyl borate (10.8 mL, 40 mmol) and boric anhydride (0.35 g, 5.0 mmol) were added to ethyl acetate (30.0 mL) and stirred at 50 °C for 15 min to give an acetylacetone-boric oxide complex. Vanillin (3.04 g, 20 mmol) was added to the complex and further stirred at 50 °C for 5 min. n-Butylamine (0.4 mL, 4.1 mmol) was then added dropwise over 40 min at 50 °C. The reaction mixture was refluxed for 4 h, cooled, combined with 1 N HCl (30 mL), and further stirred for 30 min. The organic layer was separated, extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporator. The crude product was purified by crystallization in methanol to give curcumin (2.92 g, 81.0%) as orange crystals; melting point 187–188 °C [18]; IR (KBr) 3,500 (phenolic hydroxyl, OH), 1,626 (enolic ketone, C=O), 1,601 (conjugated diene, C=C), 1,504 (aromatic double bond, C=C), 1,428 (aromatic double bond, C=C), 1,027 (methoxyl, O–CH3) cm−1; 1H-NMR (CDCl3) δ 3.95 (s, 6H), 5.80 (s, 1H), 6.48 (d, J = 15.7 Hz, 2H), 6.94 (d, J = 8.1 Hz, 2H), 7.05 (d, J = 1.8 Hz, 2H), 7.13 (dd, J = 8.1 and 1.8 Hz, 1H), 7.59 (d, J = 15.7 Hz, 2H); 13C-NMR (CDCl3) 183.3, 147.8, 146.8, 140.5, 127.6, 122.8, 121.8, 114.8, 109.6, 101.2, 56.0; HRMS calculated for C21H21O6 [M + H+]: 369.1338; found 369.1335.

In synthesis of CurDG (Figure 1), curcumin (1.47 g, 4 mmol) and triethylamine (1.26 mL, 9 mmol) were dissolved in dichloromethane, followed by gradual addition of glutaric anhydride (1.03 g, 9 mmol) in dichloromethane at 40 °C under a nitrogen atmosphere. The reaction mixture was refluxed under nitrogen for 2 h. After the reaction was complete based on monitoring with TLC, 0.1 N HCl and water were used to remove triethylamine hydrochloride and excess hydrochloric acid, respectively. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The crude product was purified by crystallization from methanol to give CurDG (2.03 g, 85%) as a yellow solid; melting point 158–161 °C, IR (KBr) 3,000–3,500 (broad, hydroxyl group of carboxylic acid), 1,757 (carboxyl group, COOH), 1,700 (carbonyl of phenolate ester), 1,632 (enolic ketone, C=O), 1,599 (conjugated diene, C=C), 1,509 (aromatic double bond, C=C), 1,411 (aromatic double bond, C=C), 1,029 (methoxyl, O-CH3) cm−1; 1H-NMR (acetone-d6) δ 2.05(m, 4H), 2.49 (t, J = 7.3 Hz, 4H), 2.69 (t, J = 7.3 Hz, 4H), 3.91 (s, 6H), 6.09 (s, 1H), 6.88 (d, J = 15.9 Hz, 2H), 7.14 (d, J = 8.1 Hz, 2H), 7.29 (dd, J = 1.7 and 8.1 Hz, 2H), 7.46 (d, J = 1.7 Hz, 2H), 7.67 (d, J = 15.9 Hz, 2H); 13C-NMR (CDCl3) 184.38, 174.12, 171.29, 152.60, 142.53, 140.60, 134.86, 125.28, 124.15, 121.99, 112.56, 102.37, 56.34, 33.41, 32.92, 21.01; HRMS calculated for C31H32O12 [M + Na+]: 619.1786; found 619.1725. The peak assignment in NMR spectra of curcumin and CurDG are shown in Figure S14 (see supplemental information).

Solubility of CurDG

The specific aim of a water solubility study is to investigate the impact of glutaric acid on the water solubility in comparison with curcumin. Solubility of CurDG was measured and compared to curcumin using the standard shake flask method as per an OECD guideline [19]. Curcumin and CurDG (10 mg) were dissolved in hexane (1 mL) and evaporated until dry. Then, 1 mL of water was added to the dried sample and capped. The samples were continuously shaken at 100 rpm for 24 h at 25 °C. After shaking, the samples were centrifuged at 10,000 rpm for 10 min and supernatants were collected. The concentrations of curcumin and CurDG were determined from calibration curves, using HPLC-UV. Experiments were performed in triplicate. In addition to water solubility study, drug solubility in biological buffers at the body temperature (37.0 ± 0.5 °C) i.e. 0.1 M HCl, acetate buffer pH 4.5, and phosphate buffer pH 6.8 was investigated to observe the effect of glutaric acid on buffer solubility in the same manner of water solubility. The calibration curves for determining the solubility of curcumin and CurDG in water were linear over the concentration range of 0.03 μg/mL to 2.60 μg/mL (r  2 > 0.99) and 0.61 μg/mL to 6.06 μg/mL (r  2 > 0.99), respectively. The calibration curves for the solubility in buffers of curcumin and CurDG were 0.028 μg/mL to 3.330 μg/mL for curcumin (r  2 > 0.999) and 0.026 μg/mL to 1.641 μg/mL for CurDG (r  2 > 0.999).

Partition Coefficient of CurDG

The partition coefficient (log Po/w) of CurDG was determined by the shake flask method (distribution between n-octanol and pH 1.2 buffer) using OECD guidelines for testing of chemicals [20]. n-Octanol and pH 1.2 buffer were both saturated at 25 ± 1 °C. To prepare the saturated solvents, two large stock bottles, one containing n-octanol with a sufficient quantity of water and the other containing pH 1.2 buffer with a sufficient quantity of n-octanol, were shaken for 24 h on a mechanical shaker, and left standing until complete separation. The saturated layers were used in the experiment. CurDG was dissolved in three duplicate runs with different solvent ratios at a concentration of 0.1 M HCl (pH 1.2 buffer: n-octanol ratios of 1:1, 2:1 and 1:2). The samples in the test vessel were placed in a mechanical shaker and shaken through 180° about the transverse axis approximately 100 times in 5 min at 25 ± 1 °C. Separation of the two phases was achieved by centrifugation at 4000 rpm at 25 ± 1 °C for 10 min before analysis. The concentration of CurDG in each phase was determined from a calibration curve. The Po/w value was calculated from these data. Six values were obtained from three duplicate runs with different solvent ratios. The six log Po/w values should fall within ± 0.3 units. Log Po/w was calculated from the mean of the six values.

Stability of CurDG

Stock solutions of curcumin and CurDG at 40 μM were prepared in methanol, and then aliquoted in 0.1 M HCl (pH 1.2), 0.1 M acetate buffer (pH 4.5), and phosphate buffer (pH 7.4) at a final concentration of 2 μM. Each solution was left to stand at 37.0 ± 0.5 °C for 24 h. The amounts of curcumin and CurDG were determined against calibration curves at appropriate time intervals using HPLC-UV. Each experiment was performed in triplicate. Kinetic parameters including observed rate constant (k  obs) and degradation half-life (t  1/2) of curcumin and CurDG in buffer solutions were determined by a semi-logarithmic plot of a concentration versus time, and calculated using linear least-squares regression analysis.

Kinetic release of curcumin from CurDG in human plasma

Human blank plasma (Nation Blood Center, Thai Red Cross Society, Bangkok, Thailand) was spiked with a stock solution of CurDG to obtain a final concentration of 2 μM. The spiked plasma sample was incubated at 37.0 ± 0.5 °C for 12 h. The release profile of curcumin was determined at appropriate time intervals by protein precipitation with acetonitrile and analysis by HPLC. The experiment was performed in triplicate. Kinetic parameters (k  obs and t  1/2) were determined by a semi-logarithmic plot of concentration against time and calculated by linear least-squares regression analysis.

Chromatographic conditions

A published reverse-phase HPLC-UV method was used with some modifications [14]. Chromatography was carried out using a gradient system with an autosampler temperature of 4 °C, column temperature of 33 °C, flow rate of 2.0 mL/min, UV detection at a wavelength of 400 nm, and gradient elution with eluents A (2% v/v acetic acid and B (acetonitrile): 0–4 min, isocratic elution A-B (70:30, v/v); 4–5 min, linear gradient A-B (50:50, v/v); 5–10 min, isocratic elution A-B (50:50, v/v); 10–12 min, linear gradient A-B (70:30, v/v); 12–13 min, linear gradient A-B (70:30, v/v); 13–14 min, isocratic elution A-B (70:30, v/v). The injection volume was 20 μL.

Animals

Male ICR mice (18–25 g) were obtained from the National Laboratory Animal Center, Mahidol University, Salaya, Nakhonpathom, Thailand. The mice were housed in the laboratory animal facility at the Faculty of Pharmaceutical Sciences, Chulalongkorn University under standard conditions at 25 ± 2°C and 50–60% relative humidity under a 12 h light/dark cycle. The mice were kept under laboratory conditions for one week prior to the start of the experiment and allowed food and water ad libitum.The experiment protocol was approved by the Institutional Animal Care and Use Committee of the Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand.

Hot-plate test

The hot-plate test was performed as previously described by Boonyarikpunchai et al. [21]. Briefly, male ICR mice weighing 18–25 g (n = 10 per group) were placed on a hot-plate (Harvard Apparatus 28 × 28 cm). The hot-plate was set at 55 ± 0.5 °C and surrounded by a clear Plexiglas® wall cylinder, 20 cm in diameter and 30 cm in height to confine the mice to the heated surface during testing. Only mice with a pretreatment hot-plate latency time less than 45 s were used. Mice were orally administered 0.5% carboxymethyl cellulose (CMC) or various doses of curcumin or CurDG at 25, 50, 100 and 200 mg/kg. The latency to licking of a hind paw or jumping from the surface of the hot-plate was measured. If this behavior was not observed within 45 s, the animal was removed from the hot-plate to avoid tissue damage. The post-drug latency was measured in 7 trials at 15, 30, 45, 60, 90, 120 and 240 min after drug administration. The hot-plate latency was expressed as the mean percent maximum possible effect (%MPE) calculated as below.

Statistical analysis

The results in the in vivo study are represented as mean ± S.E.M. Data were analyzed by one-way analysis of variance (ANOVA) and a post hoc Tukey test for multiple comparisons. The minimum level for significance was set at p < 0.05.

Results

Solubility, partition coefficient and stability of CurDG

The water solubility of CurDG was found to be approximately 100 times higher than that of curcumin. At 25 °C, curcumin was found to have a maximum concentration of 0.068 μg/mL in water, while that of CurDG was 7.48 μg/mL.The solubility as a function of pH study showed that the maximum concentrations of CurDG in 0.1 M HCl and acetate buffer pH 4.5 were less than 0.025 μg/mL whereas the concentration in phosphate buffer pH 6.8 was 1.43 μg/mL after 1.5 h (Table 1).

Table 1.

Solubility of curcumin and CurDG in buffers at 37 °C.

Buffers Curcumin (μg/mL) CurDG (μg/mL)
1.5 h 24 h 1.5 h 24 h
0.1 M HCl (pH 1.2) < 0.025 < 0.025 < 0.025 < 0.025
Acetate buffer (pH 4.5) < 0.025 < 0.025 < 0.025 < 0.025
Phosphate buffer (pH 6.8) < 0.025 < 0.025 1.429 0.026
Buffers Curcumin (μg/mL) CurDG (μg/mL)
1.5 h 24 h 1.5 h 24 h
0.1 M HCl (pH 1.2) < 0.025 < 0.025 < 0.025 < 0.025
Acetate buffer (pH 4.5) < 0.025 < 0.025 < 0.025 < 0.025
Phosphate buffer (pH 6.8) < 0.025 < 0.025 1.429 0.026
Table 1.

Solubility of curcumin and CurDG in buffers at 37 °C.

Buffers Curcumin (μg/mL) CurDG (μg/mL)
1.5 h 24 h 1.5 h 24 h
0.1 M HCl (pH 1.2) < 0.025 < 0.025 < 0.025 < 0.025
Acetate buffer (pH 4.5) < 0.025 < 0.025 < 0.025 < 0.025
Phosphate buffer (pH 6.8) < 0.025 < 0.025 1.429 0.026
Buffers Curcumin (μg/mL) CurDG (μg/mL)
1.5 h 24 h 1.5 h 24 h
0.1 M HCl (pH 1.2) < 0.025 < 0.025 < 0.025 < 0.025
Acetate buffer (pH 4.5) < 0.025 < 0.025 < 0.025 < 0.025
Phosphate buffer (pH 6.8) < 0.025 < 0.025 1.429 0.026

In addition, the partition coefficient was reported as log Po/w. In this study, the log Po/w of CurDG was 1.79. In the stability in buffers study, the amounts of curcumin and CurDG were measured after incubation in 0.1 N hydrochloric acid (pH 1.2), acetate buffer (pH 4.5), and phosphate buffer (pH 7.4) at 37 °C. Semi-logarithmic plots of the concentration of curcumin and CurDG versus time were linear (Figure 2), indicating that the degradation kinetics are pseudo first-order. The degradation rate constants (k  obs) and half-lives (t  1/2) of curcumin and CurDG in the various buffers are shown in Table 2.

Figure 2.

Kinetic plots of (a) curcumin and (b) curcumin-diglutaric acid in buffer solutions.

Table 2.

Kinetic parameters for the stability of curcumin and CurDG in buffers (pH 1.2, 4.5 and 7.4) at 37 °C.

Compounds Kinetic parameters
pH 1.2 pH 4.5 pH 7.4
k  obs (h−1) t  1/2 (h) k  obs (h−1) t  1/2 (h) k  obs (h−1) t  1/2 (h)
Curcumin 0.011 59.74 0.013 50.22 1.24 0.56
CurDG 0.048 14.58 0.033 17.96 2.62 0.26
Compounds Kinetic parameters
pH 1.2 pH 4.5 pH 7.4
k  obs (h−1) t  1/2 (h) k  obs (h−1) t  1/2 (h) k  obs (h−1) t  1/2 (h)
Curcumin 0.011 59.74 0.013 50.22 1.24 0.56
CurDG 0.048 14.58 0.033 17.96 2.62 0.26
Table 2.

Kinetic parameters for the stability of curcumin and CurDG in buffers (pH 1.2, 4.5 and 7.4) at 37 °C.

Compounds Kinetic parameters
pH 1.2 pH 4.5 pH 7.4
k  obs (h−1) t  1/2 (h) k  obs (h−1) t  1/2 (h) k  obs (h−1) t  1/2 (h)
Curcumin 0.011 59.74 0.013 50.22 1.24 0.56
CurDG 0.048 14.58 0.033 17.96 2.62 0.26
Compounds Kinetic parameters
pH 1.2 pH 4.5 pH 7.4
k  obs (h−1) t  1/2 (h) k  obs (h−1) t  1/2 (h) k  obs (h−1) t  1/2 (h)
Curcumin 0.011 59.74 0.013 50.22 1.24 0.56
CurDG 0.048 14.58 0.033 17.96 2.62 0.26

Kinetic release of curcumin from CurDG in human plasma

Kinetic parameters of curcumin release from the CurDG in human plasma were calculated by measuring the decrease in concentration of the conjugate by HPLC. Release of curcumin from CurDG was plotted against time (Figure 3). Degradation of the conjugate in human plasma had pseudo-first order characteristics (Figure 4). The values for k  obs and t  1/2 for CurDG hydrolysis in human plasma were 5.83 h−1 and 0.12 h, respectively.

Figure 3.

The percentage of curcumin release from curcumin-diglutaric acid in human plasma over 12 h incubation at 37 °C.

Figure 4.

Pseudo-first order kinetic plot of curcumin-diglutaric acid hydrolysis in human plasma at 37 °C.

Effect on the hot plate test

The mouse hot-pate test was utilized to examine the efficacy of curcumin and CurDG in producing analgesia. All doses of orally administered curcumin and CurDG (25–200 mg/kg) significantly (p < 0.05) increased the hot-plate latencies when compared to the vehicle group (Figure 5). Curcumin at 200 mg/kg significantly increased the hot-plate latencies (p < 0.05) when compared to other doses of curcumin while CurDG at 100 mg/kg significantly (p < 0.05) increased the hot-plate latencies in comparison with other doses of CurDG. Curcumin at 200 mg/kg and CurDG at 100 mg/kg appeared to be the most effective doses. However, the antinociceptive activity of curcumin at 200 mg/kg is comparable to CurDG at 100 mg/kg.

Figure 5.

Area of analgesia (%MPE-min) of curcumin and curcumin-diglutaric acid. Data are expressed as mean values ± S.E.M. (n = 10). Columns with different superscript letters are significantly different (p < 0.05).

Discussion

Although curcumin has demonstrated its vast biological activities and safety profile, the usage of curcumin as a medicinal agent is limited by its low aqeous solubility and stability. Prodrug approach is a strategy that has been employed to modulate biopharmaceutical and physicochemical properties of curcumin resulting in the improvement of biological activities. Here, we report the physicochemical and pharmacological properties of CurDG, which could be essential for further development of this curcumin prodrug.

During the course of curcumin prodrug development in our lab, CurDG was prepared by another research group and patented as an intermediate for further conjugation with sugar moieties using glutaric acid as a linker [22]. The synthesized CurDG intermediate appeared in the patent has not been yet evelauted for its biological activities while the curcumin-glutaric acid-sugar conjugates demonstrated lower antioxidant activity and the amyloid beta (Aβ) dissolution ability compared to curcumin-clicked-monogalactose conjugate.

In this study, we attempted to investigate the biological activity of CurDG as an analgesic agent. The antinociceptive effects of curcumin and CurDG were evaluated utilizing the standard mouse hot-plate test which is a well-validated model for detection of centrally-acting analgesic agents. The principle of this method is based on measurement of two behavioral responses including hind paw licking and jumping. Results from the hot-plate test indicated that all doses of curcumin and CurDG had significant effects on pain latency compared to controls. Curcumin had the highest antinociceptive effect at 200 mg/kg while CurDG had the highest antinociceptive effect at 100 mg/kg, indicating that CurDG has higher antinociceptive effect than curcumin. However, curcumin and CurDG did not show the dose-dependency in the same dose range used in this study. Similar findings were found in CDD [16], Dysphania graveolens extract [23] and pregabalin [24]. It is possible that the maximum absorption of CurDG was reached at dose 100 mg/kg, hence, the highest dose of CurDG (200 mg/kg) did not show the highest antinociceptive effect. Though curcumin showed the highest antinociceptive effect at 200 mg/kg and the effect seemed to increase with doses higher than 200 mg/kg as shown in Figure 5, our preliminary study demonstrated the downward trend after reaching the maximum pain latency at the dose of 200 mg/kg as well (data not shown). Therefore, further studies should be done to clarify the potency of CurDG in relative to curcumin.

For a drug intended for oral administration, it should have a sufficient water solubility to ensure the dissolution in the gastrointestinal (GI) fluid. Theoretically, for passive transcellular absorption, a drug must dissolve in the GI medium and be able to partition across the lipid membrane of the GI epithelia. Our research demonstrates that glutarylation at both phenolic OH of curcumin greatly enhances the water solubility of curcumin. The superior water solubility of CurDG is derived from the presence of dicarboxylic groups. We evaluated the dissolution of CurDG under the pH conditions similar to those of the GI fluids. The time for solubility as a function of pH study (1.5 h) was set to mimic the usual transit time for a drug along the GI tract under fasting condition [25]. At all tested pH values, curcumin sparingly dissolved into the solutions as suggested by the detected curcumin concentrations were lower than the lowest concentration of our calibration curves. Similarly, at pH 1.2, the solubility of CurDG could not be determined because the amount of soluble CurDG was quite low. We hypothesized that the ionization of the carboxylic groups were suppressed by a high abundant of hydronium ions. Interestingly, the solubility of CurDG at pH 4.5 is not quite different from that of pH 1.2 although it is expected to become ionized and more soluble. At pH 6.8, the solubility of CurDG increases substantially as it is almost fully ionized. Therefore, the better antinociceptive activity of CurDG in comparison with curcumin might be contributed by the higher aqueous solubility along the GI tract. Please be noted that CurDG in phosphate buffer (pH 6.8) is apparently less soluble than in water because the hydrolysis rate is predominant. At 24 h, the solubility of CurDG in phosphate buffer (pH 6.8) is approximately 0.026 μg/mL.

The equilibrium partitioning between n-octanol and an aqueous layer is commonly used to determine the lipophilicity of a compound and is expressed as log Po/w. In this study, the log Po/w of CurDG was found to be lower than the reported values of curcumin, which is ranging from 2.56 to 3.29 [26,27]. This indicated that the lipophilicity of curcumin decreased after glutarylation at both phenolic groups. The reader should note that this log Po/w values was determined from the partition between n-octanol and 0.1 M HCl (pH 1.2). There are two reasons for the selection of pH 1.2 in partition coefficient study. At basic pH, CurDG completely ionizes to yield the dianionic species and cannot partition to the octanol layer. By definition, partition coefficient is referred to the partitioning of unionized species between the two layers. It is therefore suitable to design the study with the environment that the compound of interest is fully unionized. Therefore, pH 1.2 was chosen in this study. In addition, CurDG is rapidly hydrolyzed in basic environment, and hence, the basic pH was not chosen in the partition coefficient study for CurDG.

Stability under physiological pHs is commonly assessed the possibility of a drug substance to survive in biological fluids and reach its target sites. Our research showed the higher k  obs and t  1/2 values in buffers of CurDG compared to those of curcumin, indicating that CurDG is less stable in aqueous environments than curcumin. As expected, the k  obs of CurDG hydrolysis at physiological pH (pH 7.4) is far higher than those at the lower pH because the cyclization-elimination is predominant at pH 7.4. The terminal carboxylic acid groups in CurDG undergo intramolecular nucleophilic attack at the carbonyl carbon of the phenolate ester, leading to acceleration of the hydrolysis rate. These findings are in agreement with other phenol prodrugs with a terminally free carboxylic acid [15,28]. Diglutaric acid conjugation might extend the degradation time of the released curcumin, which allows the molecule to be gradually absorbed through a cell membrane before curcumin release, and hence, provide better the antinociceptive activity.

Prodrugs are pharmacologically inactive molecules that are designed to improve biopharmaceutical and physicochemical properties of the active drugs. They are supposed to release their active parent drug, either chemically or enzymatically, under certain physiological conditions. Blood or plasma is one of matrices responsible to the bioconversion of pharmaceuticals especially ester-containing compounds and it is used to investigate the ability of prodrugs to liberate the active moieties. CurDG, an ester prodrug of curcumin, was totally converted to curcumin in human plasma within 2 h (Figure 3). The hydrolysis of CurDG in human plasma is proposed to be accelerated by plasma esterases, as suggested by the 2 times higher k  obs in plasma in comparison with that of in phosphate buffer (pH 7.4). These results are in consonance with the reported glutaric acid ester of phenol [29]. The hydrolysis of CurDG in human plasma could be a combination of intramolecular cyclization and enzymatic hydrolysis. It is well-known that plasma contains several esterases such as butyrylcholinesterase (BChE), acetylcholinesterase (AChE) and paraoxonase and these esterases can play crucial roles in hydrolysis of ester prodrugs [9,30]. Therefore, the specific type of esterase involving in the hydrolysis of CurDG should be further investigated.

Conclusion

We were able to synthesize CurDG with high purity and in good yield. The solubility of CurDG was markedly improved in comparison with that of curcumin, and the antinociceptive activity in mice was greater for CurDG. The release of curcumin in plasma from CurDG could be derived from the combination between cyclization activation and enzymatic bioactivation. The prodrug approach using glutaric acid conjugated with curcumin significantly improved water solubility, and the greater antinociceptive activity may be due to an increase in oral bioavailability. Hence, CurDG is a promising candidate that may serve as a curcumin-based therapeutic agent.

Author contribution

C. Muangnoi and P. Jithavech performed animal experiments and assisted in manuscript preparation. P. Ratnatilaka Na Bhuket, W. Supasena and W. Wichitnithad performed chemistry-related experiments, collected data and assisted in the preparation of the manuscript. P. Towiwat involved in the design and data interpretation of the animal study. N. Niwattisaiwong and I. S. Haworth contributed to the experimental design and gave comments on the manuscript. P. Rojsitthisak is in charge of the project including the overall experimental design and discussion as well as critical review and revision of the manuscript. All authors reviewed and approved the final version of this manuscript.

Disclosure statement

The authors declare no conflict of interest.

Funding

This work was supported by the Ratchadaphiseksomphot Endowment Fund of Chulalongkorn University (grant number CU-GR_60_24_33_07, P. Towiwat) and the Annual Research Fund of Faculty of Pharmaceutical Sciences, Chulalongkorn University (P. Rojsitthisak).

Supplemental data

Supplemental data for this article can be accessed https://doi.org/10.1080/09168451.2018.1462694

Acknowledgements

We would like to thank the Pharmaceutical Research Instrument Center for instruments access in this study.

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Supplementary data