Novel Gastroprotective and Thermostable Cocrystal of Dimethyl Fumarate: Its Preparation, Characterization, and In Vitro and In Vivo Evaluation

Abstract

Crystallization has revolutionized the field of solid-state formulations by modulating the physiochemical and release profile of active pharmaceutical ingredients (APIs). Dimethyl fumarate (DF), an FDA-approved first-line drug for relapsing-remitting multiple sclerosis, has a sublimation problem, leading to loss of the drug during its processing. To tackle this problem, DF cocrystal has been prepared by using solvent evaporation technique using nicotinamide as a coformer, which has been chosen based on in silico predictions and their ability to participate in hydrogen bonding. Fourier transform infrared (FT-IR), powder X-ray diffraction (PXRD), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and sublimation analysis have characterized the cocrystal and its thermostability. Comparative analysis of the release profile has been done by the dissolution and pharmacokinetic study of DF and its cocrystal. Formulated cocrystal is noncytotoxic, antioxidant and inhibits interleukin-6 and tissue necrosis factor-α in peripheral blood mononuclear cells induced by lipopolysaccharide. We have obtained a thermostable cocrystal of DF with a similar physicochemical and release profile to that of DF. The formulated cocrystal also provides a gastroprotective effect which helps counterbalance the adverse effects of DF by reducing lipid peroxidation and total nitrite levels.

Figure 1

(a) Molecular structure of DF, (b) molecular structure of MMF, (c) molecular structure of NIC, (d) the 3D interaction between DF and NIC, (e) RMSD of the system, (f) RMSF of the atoms of DF and NIC, and (g) the number of hydrogen bonds between DF and NIC w.r.t. time.

Figure 2

FTIR spectra of DF, NIC, DF–NIC physical mixture, and DF–NIC cocrystal.

Figure 3

TGA thermogram of DF, NIC, DF–NIC PM, and DF–NIC cocrystal.

Figure 4

DSC thermogram of DF, NIC, their physical mixture, and DF–NIC cocrystal.

Figure 5

PXRD pattern of the DF–NIC cocrystal.

Figure 6

Sublimation of DF, its physical mixture, and its cocrystal over 20 days. Data are presented as the mean ± standard error mean (SEM), and error bars represent the SEM (n = 3). *p < 0.05 compared to DF at day 10 and **p < 0.05 compared to DF at day 20 [two-way ANOVA, followed by the Bonferroni post-hoc test].

Figure 7

Percentage cumulative drug release with time for DF and its cocrystal. Data are presented as the mean ± SEM, and error bars represent the SEM (n = 3) *p < 0.05 compared to DF % cumulative release at 120 min [two-way ANOVA, followed by the Bonferroni post-hoc test].

Figure 8

Plasma concentration of MMF over 24 h for DF and DF–NIC cocrystal. Data are presented as the mean ± SEM, and error bars represent the SEM (n = 3) [two-way ANOVA, followed by the Bonferroni post-hoc test].

Figure 9

PBMC viability in terms of % control. Data are presented as the mean ± SEM, and error bars represent the SEM (n = 3) [unpaired t-test].

Figure 10

ROS activity on treatment with DF and DF–NIC cocrystal after LPS induction in PBMC. Data are presented as the mean ± SEM, and error bars represent the SEM (n = 4). ^ap < 0.05 compared to PBMC + vehicle, ^bp < 0.05 compared to PBMC + LPS, ^cp < 0.05 compared to PBMC + LPS + DF [one-way ANOVA, followed by the Newman–Keuls test].

Figure 11

IL-6 activity on treatment with DF and DF–NIC after LPS induction in PBMC. Data are presented as the mean ± SEM, and error bars represent the SEM (n = 3) ^ap < 0.05 compared to PBMC + vehicle, ^bp < 0.05 compared to PBMC + LPS, ^cp < 0.05 compared to PBMC + LPS + DF [one-way ANOVA, followed by the Newman–Keuls test].

Figure 12

TNF-α activity on treatment with DF and DF–NIC cocrystal after LPS induction in PBMC. Data are presented as the mean ± SEM, and error bars represent the SEM (n = 3) ^ap < 0.05 compared to PBMC + vehicle, ^bp < 0.05 compared to PBMC + LPS [one-way ANOVA, followed by the Newman–Keuls test].

Figure 13

Ulcer area (cm²): (a) vehicle control group, (b) treatment with DF, (c) DF–NIC cocrystal, and (d) ulcer area statistics. Data are presented as the mean ± SEM, and error bars represent the SEM (n = 3) ^ap < 0.05 compared to the vehicle control, ^bp < 0.05 compared to DF [one-way ANOVA followed by the Newman–Keuls test].

Figure 14

Ulcer index: (a) control group, (b) acetic acid + vehicle group, (c) acetic acid + cimetidine group, (d) acetic acid + DF, (e) acetic acid + DF–NIC cocrystal, and (f) ulcer index statistics. Data are presented as the mean ± SEM, and error bars represent the SEM (n = 5). ^ap < 0.05 compared to the control, ^bp < 0.05 compared to the acetic acid + vehicle control, ^cp < 0.05 compared to acetic acid + cimetidine, ^dp < 0.05 compared to acetic acid + DF [one-way ANOVA, followed by the Newman–Keuls test].

Figure 15

Lipid peroxidation in the gastric tissue on treatment with DF and DF–NIC after acetic acid-induced ulcers. Data are presented as the mean ± SEM, and error bars represent the SEM (n = 5). ^ap < 0.05 compared to the control, ^bp < 0.05 compared to the acetic acid + vehicle control, ^cp < 0.05 compared to acetic acid + cimetidine, ^dp < 0.05 compared to acetic acid + DF [one-way ANOVA, followed by the Newman–Keuls test].

Figure 16

Total nitrite in the gastric tissue on treatment with DF and DF–NIC after acetic acid-induced ulcers. Data are presented as the mean ± SEM, and error bars represent the SEM (n = 5) ^ap < 0.05 compared to the control, ^bp < 0.05 compared to the acetic acid + vehicle control, ^cp < 0.05 compared to acetic acid + cimetidine, ^dp < 0.05 compared to acetic acid + DF [one-way ANOVA, followed by the Newman–Keuls test].