Experimental design of ATO-β-CD inclusion complexes
In the context of our experimental design approach, we designated the independent variables as the ATO-β-CD ratio and mixing time, while the dependent variables were identified as the percentage of ATO loading and the cumulative ATO dissolved at the 30-minute mark. The outcomes of these experiments are presented in Table 3. The ATO-β-CD inclusion complexes, prepared in triplicate using the factorial experimental design, were evaluated based on the percentage of ATO loaded and the percentage of cumulative dissolved ATO at the 30-minute interval. The influence of the ATO: β-CD ratio and mixing time, both individually and in combination, was statistically analyzed using two-way ANOVA and Tukey's comparison tests. As a result, when examining the quantity of the active substance (ATO) loaded into the inclusion complexes concerning the ATO: β-CD molar ratio, significant differences were observed between the ratios of 2:3 and 3:7. However, no significant difference was detected between the ratios of 5:5 and 3:7 (p > 0.05). Conversely, no significant disparity was found between the loaded active substance amounts for the ratios of 5:5 and 3:7, 7:3 and 3:7, 3:2 and 2:3, and 7:3 and 5:5. Nonetheless, significant differences were observed between the loaded active substance amounts for the ratios of 2:3 and 3:7, 3:2 and 3:7, 5:5 and 2:3, 7:3 and 2:3, 3:2 and 5:5, and 7:3 and 3:2. Furthermore, when investigating the quantity of loaded active substance and its correlation with mixing time, it becomes evident that mixing time also exerts a significant influence on this ratio. Subsequent statistical analyses employing the same method unveiled the relationship between the cumulative dissolved ATO amount and the ATO: β-CD ratio. Significant differences were discerned between the ratios of 3:2 and 3:7, 3:2 and 2:3, 3:2 and 5:5, and 7:3 and 3:2. However, no significant disparities were found among the other ratios (p > 0.05). Additionally, it can be inferred from the data that inclusion complexes prepared with a mixing time of 20 minutes exhibit statistically higher dissolution rates for ATO when compared to the complexes prepared with a mixing time of 40 minutes.
Table 3
Dependent variable results of experiments with experimental design (n = 3)
Experiment Number
|
ATO:β-CD ratio (molar)
|
Mixing time
(min)
|
ATO quantity (%)
|
Cumulative dissolved amount of ATO ( %)
|
1
|
7:3
|
40
|
88.64
|
82.32
|
2
|
2:3
|
20
|
99.63
|
87.88
|
3
|
7:3
|
40
|
89.27
|
82.14
|
4
|
3:7
|
20
|
58.70
|
82.52
|
5
|
5:5
|
20
|
56.00
|
86.56
|
6
|
7:3
|
20
|
83.14
|
84.08
|
7
|
7:3
|
20
|
84.22
|
84.80
|
8
|
3:2
|
40
|
80.79
|
53.96
|
9
|
5:5
|
20
|
55.68
|
87.32
|
10
|
5:5
|
40
|
42.11
|
74.24
|
11
|
2:3
|
20
|
98.89
|
88.60
|
12
|
3:7
|
40
|
52.84
|
78.64
|
13
|
3:7
|
20
|
59.02
|
82.56
|
14
|
7:3
|
20
|
84.51
|
84.16
|
15
|
2:3
|
40
|
80.01
|
73.92
|
16
|
3:2
|
20
|
84.70
|
43.00
|
17
|
3:2
|
20
|
85.38
|
42.24
|
18
|
3:2
|
40
|
80.48
|
53.68
|
19
|
5:5
|
40
|
44.75
|
75.12
|
20
|
2:3
|
40
|
79.88
|
73.80
|
21
|
3:2
|
40
|
81.06
|
53.84
|
22
|
3:7
|
40
|
53.21
|
80.56
|
23
|
3:7
|
40
|
53.89
|
81.60
|
24
|
5:5
|
40
|
43.06
|
72.20
|
25
|
3:2
|
20
|
85.60
|
42.16
|
26
|
7:3
|
40
|
90.22
|
82.12
|
27
|
2:3
|
20
|
98.52
|
88.80
|
28
|
5:5
|
20
|
56.00
|
86.40
|
29
|
3:7
|
20
|
58,06
|
82.52
|
30
|
2:3
|
40
|
78.60
|
74.80
|
Preparation of ATO-β-CD ODMTs
In our study, we employed two types of superdisintegrants, namely Parteck ODT® and Ac-Di-Sol®, alongside magnesium stearate as a lubricant. Each group of components was utilized in three distinct ratios, specifically 5, 7.5, and 10 for superdisintegrants, and 0.5, 1, and 1.5 for magnesium stearate. The percentage of superdisintegrant and lubricant content was designated as the independent variables, while the friability, hardness, and disintegration time of the orally disintegrating tablets (ODMTs) served as the dependent variables. Our study findings, which were conducted with two replications, underwent rigorous statistical evaluation through ANOVA. The formulation of ODMTs was meticulously designed based on the independent variables within the experimental design, incorporating the most suitable formulation components. These formulations were then prepared using the direct compression method. Furthermore, a series of comprehensive physicochemical assessments were carried out on all 18 ODMT formulations that were prepared as part of this study.
Physicochemical control results of ODMTs
Orally disintegrating tablets (ODMTs) were meticulously prepared according to a 32 factorial experimental design, which was conducted twice to rigorously assess the influence of independent variables on the dependent variables. This experimental design was employed with the purpose of exploring the intricate relationship between the independent variables and the dependent variables. The physicochemical properties of all 18 ODMT formulations are meticulously presented in Table 4. The hardness values of the ODMTs in all formulations were found to surpass the requisite criteria, consistently exceeding 30 N. Furthermore, the diameter-thickness and weight deviation values of the tablets fell comfortably within the predetermined specifications. However, it's noteworthy that in formulations 1 and 5, the percentage friability values exceeded 1%, whereas the remaining formulations exhibited values below this threshold, aligning with our expectations. With the exception of F11 and F16, all ODMTs displayed impressive disintegration times, consistently under 1 minute. For this comprehensive study, a total of 18 formulations were meticulously prepared through the repetition of the preparation process for nine distinct tablet formulations. The dependent variables, specifically hardness, friability, and disintegration time, underwent rigorous statistical analysis, facilitated by the Design Expert 12® software program.
Table 4
Results of physicochemical controls of independent variables examined with DoE
|
Super disintegrant ratio
(%)
|
Lubricant ratio
(%)
|
Disintegration time (min)
|
Friability (%)
|
Hardness (N)
|
Weight variation (mg)
|
Thickness (mm)
|
Diameter (mm)
|
F1
|
7.5
|
1.0
|
50 ± 1.4
|
1.20
|
50.00 ± 2.83
|
120.5 ± 0.7
|
3.21 ± 0.02
|
6.52 ± 0.01
|
F2
|
5.0
|
1.5
|
39 ± 2.1
|
0.35
|
41.50 ± 1.96
|
120.4 ± 0.6
|
3.24 ± 0.01
|
6.53 ± 0.01
|
F3
|
5.0
|
1.0
|
36 ± 3.1
|
0.62
|
40.95 ± 1.70
|
120.1 ± 0.6
|
3.21 ± 0.01
|
6.55 ± 0.01
|
F4
|
10.0
|
0.5
|
21 ± 1.6
|
0.86
|
41.60 ± 1.40
|
120.5 ± 1.0
|
3.35 ± 0.02
|
6.51 ± 0.01
|
F5
|
7.5
|
1.0
|
54 ± 2.2
|
1.80
|
48.90 ± 1.10
|
120.8 ± 0.5
|
3.33 ± 0.01
|
6.53 ± 0.01
|
F6
|
5.0
|
1.0
|
32 ± 2.3
|
0.56
|
45.64 ± 1.42
|
119.6 ± 0.1
|
3.22 ± 0.02
|
6.54 ± 0.01
|
F7
|
5.0
|
0.5
|
14 ± 0.9
|
0.42
|
50.35 ± 1.92
|
120.5 ± 0.8
|
3.24 ± 0.02
|
6.50 ± 0.01
|
F8
|
5.0
|
1.5
|
43 ± 1.1
|
0.46
|
43.70 ± 0.87
|
120.6 ± 0.2
|
3.28 ± 0.03
|
6.54 ± 0.02
|
F9
|
10.0
|
1.0
|
33 ± 2.0
|
0.45
|
46.15 ± 0.35
|
120.4 ± 0.5
|
3.27 ± 0.01
|
6.54 ± 0.01
|
F10
|
7.5
|
0.5
|
49 ± 0.8
|
0.56
|
39.75 ± 1.62
|
120.1 ± 0.7
|
3.21 ± 0.02
|
6.52 ± 0.01
|
F11
|
7.5
|
1.5
|
64 ± 0.5
|
0.22
|
50.20 ± 3.16
|
120.8 ± 0.6
|
3.25 ± 0.02
|
6.53 ± 0.01
|
F12
|
10.0
|
1.0
|
37 ± 1.8
|
0.38
|
46.26 ± 1.90
|
120.6 ± 0.6
|
3.25 ± 0.01
|
6.54 ± 0.01
|
F13
|
10.0
|
1.5
|
38 ± 4.2
|
0.31
|
44.70 ± 1.53
|
120.6 ± 0.8
|
3.32 ± 0.01
|
6.54 ± 0.01
|
F14
|
7.5
|
0.5
|
55 ± 0.6
|
0.62
|
42.55 ± 1.26
|
120.8 ± 0.4
|
3.30 ± 0.03
|
6.52 ± 0.02
|
F15
|
10.0
|
0.5
|
19 ± 1.3
|
0.79
|
45.40 ± 2.61
|
120.1 ± 0.6
|
3.32 ± 0.05
|
6.52 ± 0.01
|
F16
|
7.5
|
1.5
|
62 ± 1.1
|
0.25
|
48.30 ± 2.74
|
120.6 ± 0.4
|
3.26 ± 0.03
|
6.53 ± 0.02
|
F17
|
10.0
|
1.5
|
41 ± 3.3
|
0.28
|
49.20 ± 0.95
|
120.4 ± 0.5
|
3.34 ± 0.01
|
6.53 ± 0.02
|
F18
|
5.0
|
0.5
|
12 ± 1.3
|
0.32
|
50.02 ± 1.84
|
120.3 ± 0.9
|
3.31 ± 0.01
|
6.48 ± 0.03
|
Optimization on ODMTs
Upon conducting ANOVA analysis using the Design Expert 12® software for the independent variables in the ODMTs, we observed that the hardness values of the ODMT formulations remained statistically unaffected by variations in the superdisintegrant and lubricant ratios, well within an acceptable range. However, it became evident that the disintegration time and friability values were significantly impacted by the percentage values of the lubricant and superdisintegrant. Consequently, we generated three-dimensional surface and contour graphics for each independent variable, from which we derived desirable limit values for optimizing the ODMT formulations. The analysis of the dependent variables led us to identify the most suitable formulations: F1 (comprising 10% Parteck ODT® and 0.5% lubricant), F2 (consisting of 10% Parteck ODT® and 1% lubricant), and F10 (containing 10% Ac-Di-Sol® and 1% lubricant). These formulations were chosen based on their adherence to acceptable limit values for the determined dependent variables. Subsequently, we prepared ODMTs utilizing optimized ATO-β-CD inclusion complexes and subjected them to evaluation in tablet form, considering taste, dissolution rate, and physicochemical control results. Among the formulations, F2 exhibited the most desirable characteristics. Subsequently, we conducted in vivo animal experiments specifically with this formulation to further investigate its efficacy and safety.
In vivo ADHD model and treatments
The number and body weights of the pubs from both control and nicotine-exposed mothers were similar. The body weight of the male pubs, both at the time of allocation to the experimental groups and throughout the protocol, remained comparable. Caregivers noted that the offspring of female rats administered nicotine (200 µg/mL) displayed higher activity levels and restlessness compared to the offspring of control rats, an observation that was further corroborated by behavioral tests.
The results of the Open Field Test (OFT) indicated increased mobility in nicotine-exposed animals. Parameters such as time spent in the central zone, entries into the central zone, and rearing numbers were all higher than those observed in the negative control group, where neither mothers nor pubs were exposed to nicotine, before drug treatments were initiated. This difference persisted between the negative and positive control groups, which received a placebo (PLA) but no drug, at the end of the protocol. However, statistical significance was achieved only in rearing and the number of entries into the central zone between the ADHD-modeled and control animals (Table 5, p < 0.05). The administration of ATO in both conventional capsule and developed ODMT formulations improved OFT parameters, significantly reducing grooming times in treated animals compared to pre-treatment values. However, the significant effect of treatment on rearing numbers was observed only in the ODMT-ATO group. Nevertheless, all parameters in the ATO-treated groups were comparable to those in the negative control group (Table 5).
Table 5
The open field test parameters of the experimental groups before and 21 days of treatment
|
Total time in the central zone (seconds)
|
Entries to the central zone
(number/5 minutes)
|
Rearing
(number/5 minutes)
|
Total Grooming Time
(seconds)
|
|
Before
|
After
|
Before
|
After
|
Before
|
After
|
Before
|
After
|
Negative Control
(n = 6)
|
2.0 ± 1.5
|
3.0 ± 2.0
|
1.9 ± 1.6
|
3.1 ± 1.0
|
14.1 ± 4.5
|
17.0 ± 3.9
|
41.4 ± 11.2
|
28.6 ± 2.6
|
Positive Control
(n = 6)
|
3.9 ± 1.2
|
3.3 ± 0.8
|
9.4 ± 3.2**
|
8.7 ± 2.3**
|
22.6 ± 2.9**
|
21.4 ± 1.1**
|
38.7 ± 6.9
|
43.0 ± 10.8**
|
ODMT-Capsule
(n = 6)
|
5.6 ± 2.6
|
4.0 ± 2.0
|
5.0 ± 1.2**
|
3.4 ± 0.9
|
24.6 ± 3.2**
|
17.0 ± 5.0*
|
37.9 ± 5.4
|
25.9 ± 8.3*
|
ODMT-ATO
(n = 6)
|
2.7 ± 0.9
|
3.7 ± 1.2
|
5.1 ± 1.8**
|
6.0 ± 2.8
|
22.3 ± 4.1**
|
22.7 ± 5.3
|
41.7 ± 12.8
|
26.1 ± 3.1*
|
The data was presented as the mean ± S.E.M. *p < 0.05 versus before the treatment, **p < 0.05 versus negative control group |
The results of the Barnes Maze Test (BMT) demonstrated impaired learning and memory functions in nicotine-exposed animals, as evidenced by significantly increased total errors and errors until the animal found the target for the first time in ADHD-modeled animals (Fig. 1A and B). Other parameters measured in BMT, such as latency and time spent in the target quadrant, supported our model, although statistical significance was not reached.
When evaluating the effect of ATO in BMT, it was observed that ATO-treated animals, whether in the form of ODMT or capsule, performed better. Errors, total errors, and time spent in the target quadrant of the maze were significantly improved in ATO-receiving groups and differed from the positive control animals that received no drug (Fig. 1A, B, D). The latency parameter of the BMT improved in all groups; however, the significance between pre- and post-treatment values was valid only for the ODMT-ATO group (Fig. 1C).
The Novel Object Recognition Test (NORT) assessed attention primarily, and the parameters evaluated were discrimination and preference indices, as well as the time spent around the new object and the number of times the animal touched the new object. Pretreatment values indicated decreased attention in nicotine-exposed animals compared to control animals, as evidenced by lower discrimination and preference indices (Fig. 2A and B). Both indices improved in the second trials; however, the groups were similar.
Serum ATO levels were measured using the HPLC method from blood samples collected at the end of the protocol in both ATO-treated and PLA-administered animals. No significant difference was observed between the capsule form, representing the commercially available ATO preparation, and the developed ODMT form (p > 0.005). However, significantly higher serum drug levels were measured in ATO-treated animals (Fig. 3).