Innovative pulmonary targeting of terbutaline sulfate-laded novasomes for non-invasive tackling of asthma: statistical optimization and comparative in vitro/in vivo evaluation

Figures & data

Table 1. Box-Behnken design for optimization of the TBN-NVS.

Factor (independent variables)	Level
	−1	0	+1
X1 : Cholesterol (mg)	20	40	60
X2 : Span 60 (mg)	50	100	150
X3 : Stearic acid (mg)	15	25	50
Responses (dependent variables)	Constraints
Y1 : Encapsulation Efficiency (%)	Maximize
Y2 : Particle size (nm)	Minimize
Y3 : Drug release Q8h (%)	Maximize

TBN: terbutaline sulfate; NVS: novasomes.

Table 2. Composition of TBN-NVS Formulations based on the Box-Behnken design and the observed response variables.

	Independent variables			Dependent variables
Formulation	X1 Cholesterol (mg)	X2 Span 60 (mg)	X3 Stearic acid (mg)	Y1 EE (%)	Y2 PS (nm)	Y3 Q8h (%)	PDI
F1^a	40	100	25	68.93 ± 6.04	233.19 ± 19.46	68.18 ± 1.95	.41
F2	20	150	25	67.28 ± 2.83	311.26 ± 13.63	62.35 ± 2.79	.59
F3	40	50	15	60.31 ± 2.46	205.94 ± 6.82	78.70 ± 1.34	.51
F4	20	50	25	45.72 ± 3.23	300.83 ± 14.02	74.14 ± 0.91	.31
F5^a	40	100	25	68.11 ± 7.21	235.10 ± 21.06	69.05 ± 2.08	.39
F6	40	150	50	69.15 ± 4.41	400.21 ± 32.86	60.47 ± 2.17	.37
F7	60	50	25	34.87 ± 3.23	314.18 ± 12.04	63.56 ± 1.19	.32
F8	20	100	15	63.07 ± 4.65	225.74 ± 17.64	76.93 ± 3.37	.59
F9^a	40	100	25	67.92 ± 8.56	234.37 ± 25.05	67.07 ± 2.21	.38
F10	40	50	50	65.18 ± 4.66	354.83 ± 48.81	65.64 ± 1.44	.51
F11	60	100	50	61.07 ± 4.53	440.24 ± 34.73	60.87 ± 1.95	.39
F12	40	150	15	65.94 ± 5.66	329.25 ± 28.78	70.59 ± 2.53	.49
F13	60	100	15	38.27 ± 2.01	391.11 ± 13.81	68.69 ± 3.69	.52
F14	20	100	50	68.35 ± 7.97	421.21 ± 28.18	70.65 ± 4.29	.49
F15	60	150	25	40.29 ± 5.28	466.36 ± 29.59	55.18 ± 2.61	.56

TBN: terbutaline sulfate; NVS: novasomes; EE%: entrapment efficiency percent; PS: particle size; Q_8h: accumulative % release after 8 h; PDI: polydispersity index.

Drug concentration is constant (10 mg).

Data are mean values (n = 3) ± SD.

a Indicates the center point of the design.

Figure 1. Model diagnostic plots of the three independent variables (a) linear correlation plots between actual and predicted values for various responses of TBN-NVS, (b) normal quantile-quantile plots of residual errors and (c) plot of residual error vs. model predicted responses.

Figure 2. (a) Response 3D plots and (b) cube plot for the effect of cholesterol (X₁), Span 60 (X₂) and stearic acid (X₃) concentrations on the entrapment efficiency percent (Y₁).

Figure 3. (a) Response 3D plots and (b) cube plot for the effect of cholesterol (X₁), Span 60 (X₂) and stearic acid (X₃) concentrations on the particle size (Y₂).

Figure 4. (a) Response 3D plots and (b) cube plot for the effect of cholesterol (X₁), Span 60 (X₂) and stearic acid (X₃) concentrations on the accumulative % drug release after 8 h (Y₃).

Table 3. Results of regression analysis for responses Y₁ (EE%), Y₂ (PS) and Y₃ (Q_8h%).

Response	Model	Adequate precision	R^2	Adjusted R^2	Predicted R^2	SD	CV%	P value
Y1 : EE%	Quadratic	10.39	.9627	.8955	.8859	4.06	6.87	.0046
Y2 : PS	Quadratic	23.58	.9905	.9733	.8494	14.08	4.35	.0002
Y3 : Q8h%	Quadratic	16.58	.9656	.9316	.9193	1.67	2.48	.0016

EE%: entrapment efficiency percent; PS: particle size; Q_8h: accumulative % release after 8 h; R²: coefficient of determination; SD: standard deviation; CV: coefficient of variation.

Table 4. TBN-NVS optimal formulation composition with the laboratory measured and model predicted characteristics.

Factor	Optimal value	Response	Measured value	Predicted value	% Prediction error^a
X1 : Cholesterol (mg)	30.77	Y1 : EE (%)	67.48 ± 5.23	71.00	−5.21
X2 : Span 60 (mg)	90.72	Y2 : PS (nm)	223.89 ± 22.41	209.23	6.55
X3 : Stearic acid (mg)	15.00	Y3 : Q8h (%)	80.11 ± 7.25	78.33	2.22

TBN: terbutaline sulfate; NVS: novasomes.

a % Prediction error = (Measured − Predicted/Measured*100).

Figure 5. Transmission electron micrograph of the optimized TBN-NVS formulation.

Figure 6. Effect of storage on the EE%, particle size and ζ potential of the optimized TBN-NVS formulation.

Table 5. Cascade impaction results of TBN-NVS.

Aerodynamic parameter	Value
TED (µg)	144.64 ± 12.81
TED as percentage of nominal dose (%)	72.32 ± 5.54
FPD (µg)	125.03 ± 6.71
FPF (%)	86.44 ± 6.32
MMAD (µg)	3.30 ± .06

TBN: terbutaline sulfate; NVS: novasomes; TED: total emitted dose; FPD: fine particle dose; FPF: fine particle fraction; MMAD: mass median aerodynamic diameter.

Results are means ± SD (n = 6).

Figure 7. Light photomicrographs showing histopathological sections of (a) control untreated rat lung and (b) rat lung received an intratracheal suspension of TBN-NVS formulation (200X H & E).

Figure 8. TBN plasma concentration time profiles after administration of oral TBN solution, i.t TBN solution and i.t TBN-NVS suspension.

Table 6. The mean pharmacokinetic parameters of TBN in rat plasma following administration of oral TBN solution, i.t TBN solution and i.t TBN-NVS suspension.

Pharmacokinetic parameter	Formulation
	Oral TBN solution	i.t TBN solution	i.t TBN-NVS suspension
Cmax (ng/ml)	77.41 ± 5.33	104.27 ± 10.99^a	65.22 ± 4.21^a ^,b
Tmax (h)	1.00 ± .25	.50 ± .00^a	4.00 ± .00^a ^,b
Ke (h^−1)	.21 ± .03	.13 ± .03^a	.10 ± .004^b
t1/2 (h)	3.30 ± .62	5.33 ± .37^a	6.93 ± .10^a ^,b
AUC0–24 (ng h/ml)	167.99 ± 7.20	218.93 ± 15.87^a	588.84 ± 24.86^a ^,b
AUC0–∞ (ng h/ml)	168.47 ± 11.11	223.41 ± 16.17^a	655.24 ± 27.95^a ^,b
Frel (%)	–	132.61	388.93^b

TBN: terbutaline sulfate; NVS: novasomes.

Data presented are mean ± SD, n = 4.

Using one-way ANOVA followed by Tukey’s post-hoc test.

a p < .05 vs. oral TBN solution.

b p < .05 vs. i.t TBN solution.

Data availability statement

All processed data in this work are incorporated into the article.