Systemic delivery of alpha-asarone with Kolliphor HS 15 improves its safety and therapeutic effect on asthma

Figures & data

Figure 1. Chemical structure of (a) alpha-asarone and (b) Kolliphor HS 15.

Figure 2. The spraying device of ultrasonic atomization in therapeutic effect study.

Figure 3. Transmission electron microscopy (TEM).

Table 1. The stability of the (a) Kolliphor HS 15-ARE at room temperature (25 °C) and (b) Kolliphor HS 15-ARE and CA-ARE after dilution.

Time		0 month	3 months
Panel a
Mean size (nm)		13.73 ± 0.02	14.08 ± 0.02
PDI		0.19 ± 0.01	0.22 ± 0.01
Solubility (mg/ml)		4.31 ± 0.16	4.18 ± 0.16
Drug loading (%)		5.56 ± 0.02	5.39 ± 0.02
		HS15-ARE	CA-ARE
Panel b
Before dilution	Size (nm)	13.7	10.5
	Zeta potential (mV)	−1.0	0.9
After dilution (50 times)	Size (nm)	13.2	10.2 + 245.7 (double park)
	Zeta potential (mV)	−1.2	−9.3

Figure 4. Plot of the fluorescence of pyrene I₁/I₃ intensity ratio versus concentration of Kolliphor HS 15 in normal saline at 25 °C (n = 3).

Table 2. Panel a: Design of the hemolysis assay of the Kolliphor HS 15-ARE and Panel b: the hemolysis assay of the Kolliphor HS 15-ARE.

Panel a
V (ml)	Negative control tube	Positive control tube	HS15-ARE tube
2% Erythrocyte suspension	2.5	2.5	2.5
Normal saline	2.5	/	2.2
Purified water	/	2.5	/
Kolliphor HS 15-ARE	/	/	0.3
Panel b
Time (min)	Negative control	Positive control	HS15-ARE
15 min	−	+	−
30 min	−	++	−
45 min	−	++	−
60 min	−	++	−
120 min	−	++	−
180 min	−	++	−

Evaluation criteria and results: ++ represents all hemolysis; +represents part of hemolysis. −represents no hemolysis.

Figure 5. Mean plasma concentration–time curves of ARE in plasma after intravenous injections of HS 15-ARE and CA-ARE. Each value represents the mean±standard deviation (n = 5).

Table 3. The main pharmacokinetic parameters of HS 15-ARE and CA-ARE in rats (n = 5).

Parameters	HS 15-ARE	CA-ARE
AUC(0–t) (mg/l min)	152.72 ± 25.21	160.49 ± 6.84
AUC(0–∞) (mg/l min)	175.45 ± 29.39	225.18 ± 64.47
MRT(0–t) (min)	53.07 ± 8.88	50.32 ± 8.56
MRT(0–∞) (min)	98.48 ± 30.43	198.97 ± 154.72
t1/2z (min)	102.91 ± 37.67	208.30 ± 133.59
CLz + L (min/kg)	0.12 ± 0.020	0.09 ± 0.02
Cmax (mg/l)	4.97 ± 2.95	5.67 ± 0.89

Each value represents the mean ± SD (n = 5).

Abbreviations: AUC, area under the concentration-time curve; MRT, mean retention time; t_1/2z, elimination half life; CLz, clearance; C_max, maximum plasma concentration.

Figure 6. (a) AUC (mg/l min) for HS 15-ARE and CA-ARE in tissue distribution studies (n = 5). *p < 0.05; **p < 0.01; ***p < 0.001 (compared with CA-ARE). (b) The ARE concentration of Kolliphor HS 15-ARE and CA-AREs in tissues at times (10, 30, 60 and 120 min) post-injection (n = 5).

Figure 7. The histamine levels of different groups of guinea pigs in anaphylaxis study. ***p < 0.001 (compared with CA-ARE).

Table 4. The results of allergy symptoms of four groups of guinea pigs in anaphylaxis study.

Group	Sensitization	Challenge	Allergy symptoms after challenge	Symptom score
10% egg albumen	i.p. on day 1, 3 5	An injection into the vein at the lateral of crus curvilineum on day 15	Five animals died within 5 min	20
Normal saline			Normal	0
HS15-ARE			Normal	0
CA-ARE			Syncope, shortness of breath and gait disorder, but no death	14

Abbreviations: 20 (++++), death: extremely positive of allergic reaction; 14 (+++), gait disorder: strong positive of allergic reaction; 0 (−), normal: no allergic reaction.

Figure 8. (a) The histamine level released into blood in Wistar rats. *p < 0.05 (compared with CA-ARE). (b) The IL-5 content in the serum of each group in Wistar rats. *p < 0.05 (compared with CA-ARE). (c) The IL-5 content in alveolar lavage fluid in Wistar rats. *p < 0.05 (compared with CA-ARE).