Dual-targeting for brain-specific drug delivery: synthesis and biological evaluation

Figures & data

Figure 1. The structure of ibuprofen prodrug Glu-Vc-Ibu.

Scheme 1. Synthesis of prodrug Glu-Vc-Ibu. Reagents and conditions: (a) NaH, BnBr, DMF, r.t.; (b) Ac₂O-AcOH, ZnCl₂, r.t.; (c) CH₃ONa, CH₃OH, r.t.; (d) succinic anhydride, DMAP, CH₂Cl₂, r.t.; (e) acetone, acetyl chloride, r.t.; (f) BnBr, K₂CO₃, acetone, reflux; (g) HCl, CH₃CN, 30 °C; (h) 8, DCC, DMAP, CH₂Cl₂, r.t.; (i) ibuprofen, DCC, DMAP, CH₂Cl₂, r.t.; (j) Pd/C, H₂, CH₃OH, r.t.

Table 1. Chemical stability of prodrugs at 37 °C.

		Kinetic constants
Prodrug	pH value	Kdisapp (h^−1)	t1/2 (h)
Glu-Vc-Ibu	2.5	3.84 × 10^−2	18.05
	5.0	1.39 × 10^−2	49.87
	7.4	5.40 × 10^−2	12.84
	8.0	6.25 × 10^−2	11.09
Glu-Ibu	2.5	4.03 × 10^−2	17.20
	5.0	1.94 × 10^−2	35.73
	7.4	6.66 × 10^−2	10.41
	8.0	7.59 × 10^−2	9.13
Vc-Ibu	2.5	3.13 × 10^−2	22.15
	5.0	2.19 × 10^−2	31.65
	7.4	7.69 × 10^−2	9.01
	8.0	8.63 × 10^−2	8.03

Table 2. Metabolic stability of prodrugs in mice plasma extracts and brain homogenate at 37 °C.

		Kinetic constants
Prodrug	Biological matrix	Kdisapp (min^−1)	t1/2 (min)
Glu-Vc-Ibu	Plasma	1.12 × 10^−2	61.89
	Brain	3.25 × 10^−2	21.33
Glu-Ibu	Plasma	0.95 × 10^−2	72.96
	Brain	2.62 × 10^−2	26.46
Vc-Ibu	Plasma	1.35 × 10^−2	51.34
	Brain	4.16 × 10^−2	16.66

Figure 2. Cell viability upon addition of prodrugs in PC12 cells. Cell viability was expressed as a percentage of the control cell culture. Values are represented as mean ± SD (n = 3).

Figure 3. The neuroprotective effect of ibuprofen and the prodrugs. (A) Cell viability, (B) ROS level of ibuprofen and the prodrugs groups after H₂O₂ treatment in vitro, (C) SOD activity of ibuprofen and the prodrugs groups after H₂O₂ treatment in vitro. Cell morphology (D–I) in PC12 cells under hydrogen peroxide condition, (D) untreated PC12 cell, (E) PC12 cell exposed to H₂O₂ for 2 h, (F) PC12 cells pro-incubated with ibuprofen for 4 h and exposed to H₂O₂ for 2 h, (G) PC12 cells pro-incubated with Glu-Ibu for 4 h and exposed to H₂O₂ for 2 h, (H) PC12 cells pro-incubated with Vc-Ibu for 4 h and exposed to H₂O₂ for 2 h, (F) PC12 cells pro-incubated with Glu-Vc-Ibu for 4 h and exposed to H₂O₂ for 2 h. ***p < .001, a indicates significance at p < .05 versus H₂O₂ group, b indicates significance at p < .05 versus ibuprofen group, c indicates significance at p < .05 between Glu-Ibu or Vc-Ibu group and Glu-Vc-Ibu group. Data represent mean ± SD (n = 3).

Figure 4. The neuroprotective effect on brain ischemia model. (A) TTC staining of the brain sections, (B) percentage of TTC staining rate compared with the sham group, (C) SOD activity, (D) MDA level, (E) GSH level. **p < .01, ***p < 0.001, a indicates significance at p < .05 versus saline group, b indicates significance at p < .05 versus ibuprofen group, c indicates significance at p < .05 between Glu-Ibu or Vc-Ibu group and Glu-Vc-Ibu group. Data are presented as mean ± SD (n = 5).

Figure 5. Concentration curves in plasma versus time after administration of Ibu, Glu-Ibu, Vc-Ibu and Glu-Vc-Ibu (hydrolysis, n = 3).

Table 3. Pharmacokinetic parameters in plasma (hydrolysis, n = 3).

Parameters	Ibuprofen	Ibu from Glu-Ibu	Ibu from Vc-Ibu	Ibu from Glu-Vc-Ibu
AUC(0–t) (µg/ml min)	8250.62 ± 412.86	11607.51 ± 354.13	10,495.56 ± 438.25	10,483.82 ± 591.91
MRT (min)	65.47 ± 3.12	92.18 ± 3.74	88.33 ± 5.38	99.46 ± 2.55
Tmax (min)	5	5	5	5
Cmax (µg/ml)	80.03 ± 12.41	103.24 ± 13.45	100.15 ± 8.34	90.17 ± 7.69

Figure 6. Concentration curves in brain homogenate versus time after administration of Ibu, Glu-Ibu, Vc-Ibu and Glu-Vc-Ibu (hydrolysis, n = 3).

Table 4. Pharmacokinetic parameters of ibuprofen in brain homogenate (hydrolysis, n = 3).

Parameters	Ibuprofen	Ibu from Glu-Ibu	Ibu from Vc-Ibu	Ibu from Glu-Vc-Ibu
AUC(0–t) (µg/g min)	2782.42 ± 176.55	5859.63 ± 337.28	6629.24 ± 467.29	7308.11 ± 447.86
MRT (min)	190.28 ± 15.11	164.08 ± 20.52	158.37 ± 9.33	156.77 ± 13.09
Tmax (min)	15	45	45	50
Cmax (µg/g)	11.54 ± 1.49	47.69 ± 5.77	48.54 ± 6.34	60.43 ± 2.37
RE	–	2.10	2.38	2.63
CE	–	4.13	4.20	5.24