Physicochemical investigation of a novel curcumin diethyl γ-aminobutyrate, a carbamate ester prodrug of curcumin with enhanced anti-neuroinflammatory activity

Abstract

Curcumin is a polyphenol compound that alleviates several neuroinflammation-related diseases including Alzheimer's disease, Parkinson's disease, multiple sclerosis, epilepsy and cerebral injury. However, the therapeutic efficacy of curcumin is limited by its poor physicochemical properties. The present study aimed to develop a new carrier-linked curcumin prodrug, curcumin diethyl γ-aminobutyrate (CUR-2GE), with improved physicochemical and anti-neuroinflammatory properties. CUR-2GE was designed and synthesized by conjugating curcumin with gamma-aminobutyric acid ethyl ester (GE) via a carbamate linkage. The carbamate linkage was selected to increase stability at acidic pH while GE served as a promoiety for lipophilic enhancement. The synthesized CUR-2GE was investigated for solubility, partition coefficient, stability, and bioconversion. The solubility of CUR-2GE was less than 0.05 μg/mL similar to that of curcumin, while the lipophilicity with log P of 3.57 was significantly increased. CUR-2GE was resistant to chemical hydrolysis at acidic pH (pH 1.2 and 4.5) as anticipated but rapidly hydrolyzed at pH 6.8 and 7.4. The incomplete hydrolysis of CUR-2GE was observed in simulated gastrointestinal fluids which liberated the intermediate curcumin monoethyl γ-aminobutyric acid (CUR-1GE) and the parent curcumin. In plasma, CUR-2GE was sequentially converted to CUR-1GE and curcumin within 1 h. In lipopolysaccharide (LPS)-stimulated BV-2 microglial cells, CUR-2GE effectively attenuated the pro-inflammatory mediators by decreasing the secretion of nitric oxide and cytokines (TNF-α and IL-6) to a greater extent than curcumin due to an increase in cellular uptake. Altogether, the newly developed acid-stable CUR-2GE prodrug is a potential pre-clinical and clinical candidate for further evaluation on neuroprotective and anti-neuroinflammatory effects.

Fig 1. Synthesis of CUR-2GE and CUR-1GE.

Reagents and condition (a) DMAP/ACN 50°C 3 h (b) DMAP/ACN 50°C 24 h with GE-1: curcumin at molar ratio 4.5: 1 (c) DMAP/ACN 50°C 24 h with GE-1: curcumin at molar ratio 1: 2.5.

Fig 2. Representative chromatograms of CUR-2GE incubated at 37°C in buffers at pH 1.2, 4.5, pH 6.8, pH 7.4, and human plasma for 24 h, 24 h, 7 min, 7 min, and 30 min, respectively.

The retention times of curcumin, CUR-1GE and CUR-2GE were 1.6, 2.3 and 2.8 min, respectively, and the retention times of major unknown compounds 1 and 2 were 0.9 and 2.0 min, respectively.

Fig 3. Chemical stability of CUR-2GE in various buffers and human plasma.

Kinetic profiles of CUR-2GE and its hydrolytic products in buffers and human plasma: (A) pH 1.2, (B) 4.5, (C) 6.8, (D) 7.4 and (E) human plasma. Data of CUR-2GE and its hydrolytic products are expressed as the percentage of the initial peak area of CUR-2GE (%Total). (F) Pseudo-first-order kinetic plots of CUR-2GE hydrolysis in buffers and human plasma.

Fig 4. Proposed acid-catalyzed hydrolysis mechanisms of CUR-2GE in buffers at pH 1.2 and 4.5.

Fig 5. Proposed hydrolysis mechanisms of CUR-2GE in buffers at pH 6.8 and 7.4.

Fig 6. UPLC-MS/MS chromatograms of CUR-2GE, curcumin, CUR-1GE.

(A) CUR-2GE, curcumin and CUR-1GE standards at 10 μg/mL, CUR-2GE incubated at 37°C in buffers at (B) pH 1.2 for 24 h, (C) pH 4.5 for 24 h, (D) pH 6.8 for 7 min, (E) pH 7.4 for 7 min and (F) human plasma for 1 h. The peak of CUR-2GE, curcumin and CUR-1GE was shown at the retention times about 2.87, 1.98 and 2.47, respectively. The peak with retention times of 2.18 and 1.69 min represented tautomers of CUR-2GE and CUR-1GE, respectively, as they exhibited the same transition ions. Other peaks were generated from an insource fragmentation.

Fig 7

Proposed mass fragmentations of (A) curcumin, (B) CUR-1GE and (C) CUR-2GE.

Fig 8. In vitro cellular uptake of curcumin and CUR-2GE.

(A) Representative images of curcumin and CUR-2GE uptake in BV-2 microglial cells. The scale bars correspond to 502 μm (center) and 100 μm (right). The fluorescence intensity was further analyzed using ImageJ software. (B) Bar graph showing the fluorescent intensity of a single cell along the dotted line and (C) total average fluorescence intensity of 50 cells. The data in Fig 8C are expressed as mean ± SD (n = 50). ***p <0.001, control vs other treatments, ###p <0.001, Curcumin vs CUR-2GE.

Fig 9. Effects of curcumin and Cur-2GE on cytotoxicity and secretion of pro-inflammatory mediators in LPS-stimulated BV-2 microglial cells.

(A-B) Cytotoxicity, (C) NO, (D) TNF-α, (E) IL-6 and (F) Cell viability. Data are expressed as mean ± SD of three independent experiments. $ $p < 0.01, $ $ $p < 0.001 compared to the control group. ***p < 0.001 compared to the LPS group. ###p < 0.001 significant difference between curcumin and CUR-2GE groups. The differences were analyzed by one-way ANOVA followed by the Bonferroni post hoc test.

Fig 10. Proposed mechanism of CUR-2GE in LPS-stimulated BV-2 microglial cells.

CUR-2GE with improved stability and lipophilicity profiles enhanced cellular uptake of curcumin, leading to greater anti-inflammatory effects in LPS-stimulated BV-2 cells. CUR, curcumin; GE, gamma-aminobutyric acid ethyl ester; IL-6, interleukin 6; LPS, lipopolysaccharide; NO, nitric oxide; TNF-α, tumor necrosis factor α.