- *Corresponding Author:
- Paravastu Venkata Kamala Kumari

Vignan Institute of Pharmaceutical Technology, Beside VSEZ, Duvvada, Visakhapatnam, Andhra Pradesh 530049, India

**E-mail:**[email protected]

Date of Received | 07 June 2020 |

Date of Revision | 01 January 2021 |

Date of Acceptance | 12 June 2021 |

Indian J Pharm Sci 2021;83(3):575-583 |

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms

## Abstract

Current research has been done to develop statistically and optimized immediate release tablets for eslicarbazepine. Nineteen formulations of eslicarbazepine acetate immediate release tablets are prepared by wet granulation. The three-dimensional Box-Behnken design at three levels (33) was developed to study how selected independent variables affect dependent responses. After the initial test, three independent items were selected as follows; polyvinylpyrrolidone (A), tapioca starch (B) and galbanum gum (C). Responses to measure were the time of disintegration (Y1) and in vitro drug release at 45 min (Y2). In accordance with the independent variables, three different levels were established as the lowest, highest and middle variables tested. The predicted formula formulated with the Box-Behnken statistical design consisted of polyvinylpyrrolidone, tapioca starch and galbanum gum at the optimum levels of 2.49, 2.77 and 3 respectively.

## Keywords

Eslicarbazepine, polyvinylpyrrolidone, galbanum, Box-Behnken, anti-epilepsy

Organizing, conducting and interpreting test results by performing a small number of trials are achieved through experimental design. Independent variables if by chance variated, scientists use experimental design in the production of their product/process to demonstrate their knowledge of it. The design space is done using design concepts like Design of Experiments (DoE) through the introduction of easy-to-use software tools and the regulatory guidelines are encouraged. If many independent factors vary in a random way, then it is impossible to determine what influenced the outcome. The DoE anticipates easy-to-use techniques even though the features vary at a reasonable level[1]. There are many purposes for a DoE application: studies to identify the most influential factors affecting the product/process being studied; full or fractional designs for the measurement of factorial results; indepth response studies that are particularly helpful in optimization, mixture designs etc. Optimization is used to obtain a combination of factors related to the response and robustness is used as a final test before product release, to ensure that it stays within the specification. The primary goal of any experimental study is to find a relationship between independent variables (factors) and dependent variables (results, outcomes) within the experimental framework[2]. Factors such as ingredients level, material properties, independent variable processing parameters as well and dependent variables are product properties or parameters that reflect the dependable process performance.

The test (experimental) design, in general, is used to simultaneously study the effects of multiple independent variables (factors) on response variability; therefore, it is a multivariate analysis method[3]. A large number of objects can be learned by performing a small number of tests in a simple test design where the important object can be easily identified using a full factorial design. Response design varies in atleast three levels and a wide range of factors of different levels (using experimental designs) is used.

Quality by design (QbD) is a proposal for a pharmaceutical product development program that focuses on identifying product performance, optimizing and improving process efficiency under critical quality specifications[4]. Multivariate analysis with a small number of experiments can be easily done with Box-Behnken design (BBD) face response surface methodology (RSM) and D-optimal design. The model-equation is produced by BBD to understand the relationship between variable (independent) and quality (dependent) responses. BBD is an independent quadratic design in which the treatment combination is centered at the edges of the process area and in the center. BBD designs are rotatable (or close to rotating) and require three levels of each factor[5].

Eslicarbazepine ((S)-licarbazepine, (S)-10-hydroxycarbazepine),
a compound with anti-epileptic
properties is found in eslicarbazepine acetate and is
also one of the most effective active oxcarbazepine
metabolites after oral administration[6]. High plasma
concentrations (t_{max}) are detected in 1-4 h. Plasma
protein binding is low (<40 %). Eslicarbazepine acetate
has a half-life of 13-20 h. Intestinal absorption of
carbamazepine is slow and variable, possibly due to its
slow dissolution in intestinal fluid. The concentration of
peak serum is usually obtained between 4 and 8 h after
oral administration of the tablets and the bioavailability
of immediate release tablets is found to be in the order
of 75-85 %.

Typical oral drug products, such as tablets and capsules, are designed to dispense active medication immediately after oral administration. In the development of generic drug products, no deliberate effort was made to change the rate of drug release rate. Immediate release products lead to faster drug absorption and the onset of associated pharmacodynamic effects[7]. In the case of prodrugs, the pharmacodynamic activity may slow down due to active drug conversion by hepatic or intestinal metabolism or by chemical hydrolysis. Alternatively, in typical oral products containing lipophilic drugs, drug absorption may be delayed due to a slow dissolution or selective absorption throughout the gastrointestinal (GI) tract, which also led to the delayed onset in time.

The pattern of drug release from modified-release (MR) dosage forms is deliberately altered from the conventional (immediate release) formulation of the dose to achieve the desired therapeutic goal or better patient compliance. The term MR drug product is used to describe products that change the duration and/ or rate of drug substance. Immediate release of drugs is appropriate for drugs with a long shelf life, high bioavailability, low clearance and a low elimination half-life. But the main requirement for immediate release of the drug is poor solubility and the action of drug[8].

## Materials and Methods

**Materials:**

Eslicarbazepine acetate was received as a gift sample from Ami life sciences Pvt. Ltd. Croscarmellose sodium; polyvinylpyrrolidone (PVP) K30 was obtained from Yarrow Chem. Products, Mumbai, tapioca starch and maize starch were obtained from Surya min chem., magnesium stearate, talc was obtained from Molychem. Galbanum was obtained from a local retailer.

**Methods:**

**Preparation of immediate release tablets:** Nineteen
different formulations using natural binders of
eslicarbazepine acetate tablets were prepared using
the ingredients by wet granulation technique as given
in **Table 1**[9]. Croscarmellose sodium was used as a
superdisintegrant, tapioca starch, maize starch and
galbanum were used as natural additives and PVP was
used as a synthetic binder. Magnesium stearate was
used as a lubricant and talc as a glidant[10]. Various
concentrations of 1 %, 2 % and 3 % were prepared by
wet granulation technique using drug and excipients.
The granules were passed through a number ≠ sieve
no. 10 and then dried in hot air oven at 60°. The dried
granules were passed through ≠ sieve no. 22 and were
lubricated with magnesium stearate and talc. Eventually
the dried granules were compressed as tablets with a
MiniPress.

Independent variables | Symbols | Levels | ||
---|---|---|---|---|

Lowest | Central | Highest | ||

PVP | A | 1 | 2 | 3 |

Tapioca starch | B | 1 | 2 | 3 |

Galbanum gum | C | 1 | 2 | 3 |

Constraints | ||||

Dependent variables | Y1 | Minimize | ||

Y2 | Minimize |

**Table 1: **Summarizes the Ranges and Constraints of Independent and Dependent Variables, Respectively

**Drug content analysis:**

A total of ten tablets were weighed and powdered. The powder equivalent to eslicarbazepine acetate was then dissolved in small amounts of methanol and made up to the required volume with distilled water[10]. The sample aliquot was then analyzed spectrophotometrically (Elite UV-150 double beam spectrophotometer) at 222 nm after serial dilution.

**Disintegration time:**

In the disintegration time study, the tablets were taken and placed in each tube of the disintegration apparatus in a 1 l beaker containing 900 ml of distilled water and the disintegration time was recorded at 37±2º.

*In vitro* dissolution studies:

United States Pharmacopeia (USP) dissolution test
equipment (Model-DS 8000) type 2 (paddle) was
used to carry the *in vitro* dissolution study. 900 ml of
dissolution medium (0.1 N hydrochloric acid (HCl))
was taken in a basket and -0.75 % sodium lauryl sulfate
(SLS) was added to the basket[11]. The temperature was
maintained at 37±0.5°. The paddle speed was set at
50 rpm. The sample (5 ml) was filtered and diluted
with 0.1 N HCl before being analyzed by UV
Spectrophotometer (Elite UV-150 double beam
spectrophotometer) at 222 nm.

**Experimental design:**

BBD at three levels (33) was developed to study
how the selected independent variables influence
dependent responses[12]. BBD was developed using the
experimental version of Design-Expert^{®} software 12.
The design consisted of replicated center points and
a group of points located in the center of each edge
of a multidimensional cube that lowered the area of
interest. The polynomial equation generated by this
experimental design is given as follows:

Y_{0}=b_{0}+b_{1}X_{1}+b_{2}X_{2}+b_{3}X_{3}+b_{4}X_{1}X_{2}+b_{5}X_{2}X_{3}+b_{6}X_{1}X_{3} +
b_{7}X_{12}+b_{8}X_{22}+b_{9}X_{32}

Where, Y_{0}=dependent variable; X_{1}, X_{2} and X_{3}=
independent variable coded levels; b_{0}=intercept; b_{1} to
b_{9}=regression coefficients.

After preliminary experiments, three independent
variables were chosen as follows; PVP (A), tapioca
starch (B) and galbanum gum (C). The responses
to be measured were disintegration time (Y1) and
*in vitro* drug release at 45 min (Y2). Corresponding
to the independent variables, three different levels
were established as the lowest, the highest and central
values of the tested variables (**Table 1**). The matrix
of 19 experimental formulations was constructed as
represented in **Table 2**.

Factor 1 | Factor 2 | Factor 3 | Response 1 | Response 2 | |
---|---|---|---|---|---|

Run | A: PVP (mg) | B: Tapioca starch (mg) | C: Galbanum gum (mg) | Disintegration time (s) | In vitro drug release at 45 min (%) |

1 | 2 | 2 | 2 | 18 | 88.98 |

2 | 2 | 3 | 1 | 25 | 86.36 |

3 | 1 | 2 | 3 | 20 | 87.45 |

4 | 2 | 2 | 2 | 18 | 88.98 |

5 | 3 | 1 | 2 | 30 | 79.89 |

6 | 2 | 1 | 3 | 25 | 86.36 |

7 | 2 | 2 | 2 | 18 | 89.98 |

8 | 2 | 2 | 2 | 18 | 88.96 |

9 | 2 | 2 | 2 | 18 | 88.96 |

10 | 2 | 2 | 2 | 18 | 89.96 |

11 | 1 | 2 | 1 | 20 | 89.96 |

12 | 1 | 1 | 2 | 20 | 87.54 |

13 | 2 | 1 | 1 | 25 | 86.36 |

14 | 3 | 2 | 1 | 30 | 79.89 |

15 | 1 | 3 | 2 | 20 | 87.56 |

16 | 2 | 2 | 2 | 25 | 86.65 |

17 | 3 | 3 | 2 | 30 | 79.89 |

18 | 3 | 2 | 3 | 30 | 79.99 |

19 | 2 | 3 | 3 | 25 | 87.45 |

**Table 2: **Modeling of the Response in the Experimental Design

According to the suggested experimental design,
19 formulations (including 3 center points) were
prepared experimentally in triplicate and characterized
(**Table 2**). The obtained data were fitted to the appropriate
models (linear, 2-FI and quadratic) and analyzed by the
one-way analysis of variance (ANOVA). The models
were explained by polynomial equations and their
related three-dimensional (3D) response surface plots
were created by design-expert^{®} software[13]. For the
purpose of model reduction and better predictability,
the step-wise method was applied for the elimination of
non-significant parameters. The main, interacting and
quadratic effects of independent variables are (A, B and
C), (AB, AC and BC) and (A^{2}, B^{2} and C^{2}) respectively.

Dependent variables | p-value | Best fitted model | Lack of fit | Adequate precision | Predicted R^{2} |
Adjusted R^{2} |
R^{2} |
---|---|---|---|---|---|---|---|

Y1 (Disintegration time) | 0.0072 | Quadratic | Insignificant (p>0.05) | 5.7427 | 0.8027 | 0.7101 | 0.8550 |

Y2 (in vitro drug release at 45 min) |
<0.0001 | Quadratic | Insignificant (p>0.05) | 14.1521 | 0.8377 | 0.9323 | 0.9661 |

**Table 3:** Model Characteristics

The best significant fitted model was analyzed
by ANOVA for the prediction of particle size as summarized in **Table 3**. This table demonstrates that the
model is significant (p<0.05) whereas lack of fit is nonsignificant
(p>0.05), which implies that the proposed
model is adequate for prediction of the response[14,15]. The
characteristics of the best-fitted model are summarized
in **Table 3**. It could be observed from the table that the
proposed quadratic model was significant (p<0.05)
while lack of fit was non-significant (p>0.05), which
connotes that the proposed model was appropriate for
prediction of the response.

**Optimization of formulation components:**

Optimization process was carried out by relying on desirability measurement to get the levels of tested variables that could agree with the desirable responses[16,17]. Based on the required criteria, a suggestion was displayed with a desirability range from 0 to 1 where the desirability value towards 1 indicated the preference of response to its ideal value. Furthermore, the optimized formulation was determined and subjected to comparison of predicted and experimental values.

## Results and Discussion

Statistical analysis of data was done. The validity
of the utilized design was examined by standard
error graph shown in **fig. 1**. This graph indicated the
values of standard error of prediction for areas in the
design space[18]. It was satisfactory to obtain relatively
minimum values of standard error close to1or lower
across the area of interest. The results revealed that the
standard error was ranged between 0.378 and 0.866,
hence implying the efficient potential of prediction of
the design.

In the present study, ANOVA was applied at 95 %
confidence level to evaluate the model significance. The
model p-values observed for Y1 and Y2 responses were
0.0072 and <0.0001 respectively. This declared that the
independent variables manifested significant effects on
the tested responses away from experimental errors or
chances. Besides, this illustration would be confirmed
by greater values of F-ratio where their low values
elucidated more error in the model. The rank order of the model predicting the capability of responses was
determined as follow; Y2>Y1 which was based on
small p-values and high values of F-ratios. In addition,
lack of fit values could be used to inspect the efficiency
of model taking into consideration of their p-values
where non-significant values of lack of fit were good
and fitted the satisfactory model[19]. The values of
lack of fit for the observed dependent responses were
1.00 and 0.6314 with p-values of 0.0072 and <0.0001
for Y1 and Y2 respectively (**Table 3**). This concluded
that lack of fit values was not significant and the chance
for these large values due to noise was 0.72 % and
0.1 % respectively. Total 19 formulations were prepared
for optimization of the 3 independent variables (A, B
and C) and then characterized to analyze the influence
exerted on the observed dependent responses (Y1 and
Y2).

Effect of independent variables on disintegration
time (Y1) was measured. Results mentioned in
**Table 3** showed the significance of the model because
of the high F-ratio (5.90) with p-value of 0.0072. This
revealed that the chance for this large F-ratio to occur
due to noise is only 0.72 %. In our study, A, B, C, BC,
A^{2} were significant terms owing to their significant
p-values[20], otherwise, insignificant p-values greater
than 0.1 were indicative for insignificant model terms.
The predicted regression analysis (R^{2}) of 0.8027 was
in feasible agreement with the adjusted R^{2} of 0.7101
where the difference between them was less than 0.2.
Also, adequate precision quantified the ratio of signal to noise. The desirable adequate precision of 5.7427
(greater than 4) indicated an adequate signal and the
model could navigate the design space as shown in **fig. 2**.

The Model F-value of 5.90 implies the model is
significant. There is only a 0.72 % chance that an
F-value, this large could occur due to noise, p-values
less than 0.0500 indicate model terms are significant.
In this case A, B^{2}, C^{2} are significant model terms.
Values greater than 0.1000 indicate the model terms are
not significant. If there are many insignificant model
terms, model reduction may improve your model.
The lack of fit F-value of 0.00 implies the lack of fit
is not significant relative to the pure error. There is a
100.00 % chance that a lack of fit F-value this large
could occur due to noise. Non-significant lack of fit is
good.

The polynomial equation attained for this model was:

Y1=0.0533-0.0083*A-0.0050*A^{2}-0.0067*B^{2}-
0.0067*C^{2}

According to the regression equation, the positive
sign in the equation indicates synergistic effects and the negative sign means antagonistic effects on the
studied response. This equation stated that the variables
have lesser impact on disintegration time[21,22]. The
relationship between independent and dependent
variables on disintegration time was studied by plotting
the 3D response surface graphs (**fig. 3, fig. 4** and **fig. 5**).

Effect of independent variables on *in vitro* drug
release at 45 min (Y2) was measured. As presented by
**Table 3**, the high F-ratio of 28.53 with p-value of
<0.0001 implied that the model was significant and
there was only a 0.01 % chance that this F-ratio occurred
due to noise. In this model, A, B, C, A^{2}, B^{2}, C^{2} were
significant model terms because of their significant
p-values, while other terms were not significant. Also,
the predicted R^{2} (0.8377) was in reasonable agreement
with the adjusted R^{2} (0.9323). The desirable adequate
precision of 14.1521 pointed out that the model could
express the design space. The effect of independent
variables on in vitro drug release of immediate release
tablets along with linear correlation between predicted
and actual response was shown in **fig. 6**.

The polynomial equation was determined as follow:
Y^{2}=0.0112+0.0006*A-0.0001*AC+0.0005*A^{2}+0.000
2*B^{2}+0.0001C^{2}

The relationship between independent and dependent
variables on *in vitro* drug release at 45 min was studied
by plotting the 3D response surface graphs (**fig. 7,
fig. 8** and **fig. 9**).

The optimized formulation with maximum desirability
were prepared and evaluated for the validation of
model. The result of predicted and observed response
for the optimized formulation of immediate release
tablets with maximum desirability are shown in
**Table 4**.

Response | Predicted mean | Predicted median* | Observed |
---|---|---|---|

Disintegration time | 27.1005 | 26.6637 | 23.00 |

in vitro drug release |
84.646 | 84.6353 | 86.37 |

**Table 4: **Predicted and Observed Responses of Formulation Composition with Maximum Desirability Suggested Box-Behnken Statistical Design

**Table 4** predicted and observed responses of formulation
composition with maximum desirability suggested
Box-Behnken statistical design.

The values were in goodness of agreement. This is
providing confirmation for the productive design
validity and optimization. The desirability values of the
numerical optimization process are shown in **fig. 10** and
**fig. 11**.

The optimized formula predicted for the optimized formulation of eslicarbazepine immediate release tablets through Box-Behnken statistical design consisted of PVP, tapioca starch and galbanum gum at optimum level of 2.49, 2.77 and 3 respectively.

With the help of BBD, three factors at three levels (33)
were selected to study how the independent variables
influence the dependent responses after preparation
of immediate release tablets of eslicarbazepine. After
preliminary experiments, three independent variables were chosen as follows; PVP (A), tapioca starch (B)
and galbanum gum (C). The responses to be measured
were disintegration time (Y1) and *in vitro* drug release
at 45 min (Y2). Corresponding to the independent
variables, three different levels were established
as the lowest, the highest and central values of the
tested variables. The matrix of nineteen experimental
formulations was constructed. Standard error graph
was satisfactory to obtain relatively minimum values
of standard error close to1or lower across the area
of interest. The optimized formula predicted for the
optimized formulation of eslicarbazepine immediate
release tablets through Box-Behnken statistical design
consisted of PVP, tapioca starch and galbanum gum at
optimum level of 2.49, 2.77 and 3 respectively.

**Funding:**

No financial funding.

**Conflicts of interest:**

The authors declared no conflict of interest.

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