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^{1}Pontifícia Universidade Católica–PUCPR, Escola de Saúde e Biociências, Av. da União, 500, 85902–532 Toledo, PR, Brazil

^{2}Divisão de Desenvolvimento Farmacotécnico e Biotecnológico, Fundação Ezequiel Dias, Rua Conde Pereira Carneiro, 80 – Gameleira, 30510–010, Belo Horizonte, Minas Gerais, Brazil

^{3}Universidade Federal do Rio Grande do Sul, Faculdade de Farmácia, Av. Ipiranga 2752, 90610–000, Porto Alegre, RS, Brazil.

- Corresponding Author:
- Sausen Tiago R

Tiago Rafael Sausen, Pontifícia

Universidade Católica – PUCPR

Escola de Saúde e Biociências, Av. da União

500, 85902–532 Toledo, PR, Brazil

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

**Received Date:** 06-06-2013; **Accepted Date:** 08-07-2013

**Citation: **Sausen Tiago R, Fialho Sílvia L, Mayorga Paulo. “Design, Optimization and Evaluation of Clozapine tablets by response Surface Analysis”. Int. J. Drug Dev. & Res., July-September 2013, 5 (3): 333-340

**Copyright:** © 2013 Tiago Rafael Sausen et al, publisher and licensee IYPF. This is an Open Access article which permits unrestricted noncommercial use, provided the original work is properly cited.

The purpose of this work was, applying experimental design methodology on tablet formulation development by direct compression, to evaluate the influences of magnesium stearate and sodium croscarmelose quantities upon clozapine tablets, by a Central Composite Design. The results were fitted to non-linear regression and a second order equation was used to plot response surface graphics. The results showed that hardness and friability were influenced by magnesium stearate quantities, decreasing the mechanical resistance of tablets, and the sodium croscarmelose quantities caused a linear decreased on disintegration time and a increased on dissolution efficiency of tablets, on the studied experimental field.

Central composite design, Direct compression, Experimental statistical design

Direct compression is a straightforward, easiest to control, and least expensive method to produce tablets because of its advantage that possess fewer processing stages, which can increase the productivity and consequently reduce the final cost of the product [1,2]. Besides, with the elimination of heat and moisture effects, it becomes the most appropriate process for hygroscopic and thermo-sensitive drugs. However, as few drugs have the mechanical and physical properties that allow direct compression, successful tablet productions by this process is mainly dependent on the excipients that make the pharmaceutical blend [3,4]. So, the choice of excipients is extremely critical in formulating direct compression tablets.

It is well known that traditional experimentation involves a good deal of effort and time, especially when complexes processes are evaluated. Most of the experiment on tablet formulation development is still performed in an unsystematic way, by changing the levels of each variable, or factor, at a time, and keeping all the others variables constant in order to study the effects of that specific variable on the selected response. Statistical experimental design is a well-established concept for planning and execution of informative experiments. In this approach, process variables are first “screened” to determine which variable is important to the outcome, and then follow the “optimization”, when the best settings for the important variables are determined [5].

In this way, response surface methodologies have been successfully applied in drug development. The use of experimental statistical design such as Central Composite Design allows to evaluate, in an effective and systematic way, the differences among the batches and makes possible to plot surface response graphics, which allows to evaluate the influence of each variable, which can be ranked according to its effect on the whole response [5,6,7,8,9].

The aim of this work was, by using an experimental statistic design, to evaluate the influences of magnesium stearate and sodium croscarmelose quantities on the clozapine tablets hardness, friability, disintegration time and dissolution efficiency, by applying factorial design and response surface methodologies. Clozapine is a dibenzodiazepinic compound used in psychoses treatment to control schizophrenia [10].

**Materials**

The following raw materials were used:
clozapine (Medapi Farmacêutica Ltda, Brazil),
sodium croscarmellose (Explocel^{®}, Blanver, Brazil),
magnesium stearate (Henrifarma Produtos
Químicos e Farmacêuticos Ltda, Brazil), colloidal
silicon dioxide (Aerosil^{®} 200, Blanver, Brazil),
microcrystalline cellulose (Microcel^{®} 101, Blanver,
Brazil), and spray-dried lactose (New Zealand).

**Clozapine tablets preparation**

Thirteen formulations were prepared using
a Central Composite Design, whose experimental
matrix is showed on **Table 1**. The proportions of
magnesium stearate (STE) and sodium croscarmellose (SCC) were established
empirically, according the usual concentration
described on literature, ranging from 0.5 to 2.0 %
to STE and 2.0 to 4.0 % to SCC. To all the
formulations, colloidal silicon dioxide 0.5 % (w/w)
and a mixture of microcrystalline cellulose and
spray-dried lactose, in a proportion of 70:30 (w/w)
were added.

Run | STE (coded) | SCC (coded) | STE (%) | SCC (%) |
---|---|---|---|---|

1 | – 1.00 | – 1.00 | 0.50 | 2.00 |

2 | 1.00 | – 1.00 | 2.00 | 2.00 |

3 | – 1.00 | 1.00 | 0.50 | 4.00 |

4 | 1.00 | 1.00 | 2.00 | 4.00 |

5 | 0.00 | – 1.41 | 1.25 | 1.59 |

6 | 0.00 | 1.41 | 1.25 | 4.41 |

7 | – 1.41 | 0.00 | 0.19 | 3.00 |

8 | 1.41 | 0.00 | 2.31 | 3.00 |

9 | 0.00 | 0.00 | 1.25 | 3.00 |

10 | 0.00 | 0.00 | 1.25 | 3.00 |

11 | 0.00 | 0.00 | 1.25 | 3.00 |

12 | 0.00 | 0.00 | 1.25 | 3.00 |

13 | 0.00 | 0.00 | 1.25 | 3.00 |

where : STE – magnesium stearate and SCC – sodium croscarmelose.

**Table 1:** Central Composite Design matrix used to produce clozapine tablets.

The powders were thoroughly mixed using
an ERWEKA AR 400 mixing device (Erweka
Apparatebau, Heusenstamm, Germany) at 20
rpm. The tablets were produced in a rotative
tablet press (Picolla D3 – 8, Riva^{®}), with a
compressional force of 15 kN. Biconcave tablets
with a diameter of 7 mm were obtained.

**Hardness**

The hardness of tablets (n = 10) was measured using an Erweka TBH TAG FTCQ 003 model hardness tester.

**Friability**

Tablet friability was calculated as the percentage weight loss of 20 tablets after 100 rotations per minute in a Roche. J. Engelsmann (Ludwigshafen, Germany) friability apparatus.

**Disintegration time**

The disintegration time was measured in purified water at 37 ± 1 ºC and the results represent a calculated average of six determinations.

**Dissolution test**

Dissolution of clozapine tablets was performed according to the United States Pharmacopoeia [11] proposed method: apparatus I (basket) at 100 rpm, in 900 mL of pH 4.0 buffer acetate at 37 ± 1 ºC as dissolution medium, using a Pharma Test, PTW S III type dissolution tester (Hamburg, Germany). Sink conditions were maintained during dissolution. Samples (n=6) were collected and then analyzed on a Hewlett–Packard 8452 A spectrophotometer (Hewlett-Packard, USA) at 290 nm for the drug content, using the Dissolution Test Software vs. 03.01. The dissolution efficiency of clozapine tablets, calculated by the area under curve, was obtained using the equation [1]

[1]

where t_{i} is the i^{th} time point, y_{i} is the percentage of
dissolved product at time t_{i}.

**Experimental statistical design and statistical
analysis**

In this study, the factors selected were amount of
magnesium stearate (STE) and sodium
croscarmellose (SCC). The response criteria
evaluated were hardness, friability, disintegration
time and dissolution efficiency of tablets. A
preliminary evaluation of the factor levels was
performed using a 2^{2} factorial design without
replication in order to define the experimental
field. After that, the experimental design was
transformed into a Central Composite Design,
according **Table 1**. The data were adjusted to a
polynomial second order equation by the leastsquare
method and the respective response surfaces were modeled using the results from
Composite Central Design (StatGraphics^{®} Plus
version 5.1, Statistical Graphics Corp., USA). The
model was validated statistically by ANOVA by
means of calculation and evaluation of the
multiple-correlation coefficients and estimation of
the lack-of-fit, using Equation [2]:

[2]

where Y = response (hardness, friability, disintegration time, dissolution efficiency), x1 and x2 = equation coefficients (STE and SCC amount) and β0.... β22 = regression coefficients.

Hardness, friability, disintegration time and
dissolution efficiency of the formulation produced
according to the central composite design are
shown in **Table 2**.

Run | H | F | DT | DE |
---|---|---|---|---|

1 | 89.20 N | 0.12 % | 3.95 min | 98.99 % |

2 | 64.30 N | 0.31 % | 3.32 min | 99.57 % |

3 | 91.10 N | 0.16 % | 2.30 min | 101.05 % |

4 | 66.50 N | 0.39 % | 2.05 min | 101,93 % |

5 | 78.40 N | 0.07 % | 4.58 min | 95.05 % |

6 | 70.70 N | 0.31 % | 2.02 min | 101.71 % |

7 | 87.50 N | 0.06 % | 3.02 min | 100.89 % |

8 | 62.30 N | 0.26 % | 2.71 min | 101.37 % |

9 | 64.40 N | 0.10 % | 2.75 min | 98.91 % |

10 | 59.10 N | 0.10 % | 2.75 min | 98.91 % |

11 | 67.00 N | 0.25 % | 2.7 min | 100.16 % |

12 | 60.60 N | 0.11 % | 3.12 min | 100.15 % |

13 | 65.30 N | 0.20 % | 3.07 min | 99.57 % |

**Table 2:** Results for tablets Hardness (H), Friability (F), Disintegration Time (DT) and Dissolution Efficiency (DE).

The results from **Table 2** were used to fit an
appropriated second order model from each
dependent variable and the general equation
was adjusted by a non-linear regression to the STE
and SCC factors, allowing the determination of
constant, linear, quadratic and interaction terms.

The mathematical model that describes the hardness was:

H = 159.831 - 43.603 × STE - 39.414 × SCC + 0.0984 × STE SCC + 11.645 × STE^{2} + 6.407 × SCC^{2} [3]

where H = hardness; STE = STE factor; SCC = SCC factor; STESCC = factor interaction

The results from the multiple regression
coefficient calculated from equation 3 indicated
that about 93 % of the experimental variance
could be explained (r^{2} = 0.927). Once the lack-offit
test was not significant (F_{(P 0.95; FG 3.4)} = 6.59 >
2.18), the experimental variation could be
ascribed to a randomized error that was not
related to the experimental model. Thus, the
regression model expressed by equation 3
appears to be satisfactory to describe the tablet
hardness behavior.

Considering the ANOVA and the t-test
results (**Table 3**), with the exception of the
interaction between the STE and the SCC factors,
the linear and quadratic coefficients had a
significant effect on the estimated response. The
STE quadratic term had the higher effect on the
tablet hardness, followed by the SCC quadratic
term, and both showed a positive effect (hardness
increase). The STE and SCC linear terms were also
significant, showing a negative effect on the
tablet hardness.

Term | Coefficient | Standard Error | t calculated |
---|---|---|---|

1 | 159.831 | 17.665 | 9.048 |

STE | – 43.603 | 10.675 | 4.085* |

SCC | – 39.414 | 9.870 | 3.993* |

Interaction | 0.0984 | 2.677 | 0.0367 |

STE2 | 11.645 | 2.709 | 4.299* |

SCC2 | 6.407 | 1.529 | 4.190* |

* significant for T = 0.95

**Table 3:** Results of the t-test for equation 3 coefficients

Term | Coefficient | Standard Error | t calculated |
---|---|---|---|

1 | 0.274 | 0.321 | 0.854 |

STE | – 0.0185 | 0.194 | 0.0952 |

SCC | – 0.180 | 0.179 | 1.005 |

Interaction | 0.0133 | 0.0486 | 0.275 |

STE2 | 0.0382 | 0.0492 | 0.778 |

SCC2 | 0.0368 | 0.0278 | 1.325 |

**Table 4:** Results of the t-test for equation 4 coefficients

The response surface (**Figure 1a**) and the
corresponding contour-plot (**Figure 1b**) graphs showed that tablet hardness was almost
independent of the SCC concentration, but an
increase in the STE concentration caused a
decreased in tablet hardness. When the SCC
concentration were lower than 2.5 % and higher
than 3.5 %, a slight increase in tablet hardness,
was observed on the experimental field.

To the friability parameter F, the equation [4] was obtained:

F = 0.274 - 0.0185 × STE- 0.180 × SCC 0.0133 × STE × SCC + 0.0382 × STE^{2} + 0.0368 SCC^{2} [4]

where F = friability; STE = STE factor; SCC = SCC factor; STESCC = factor interaction

The results from the ANOVA test were not
significant (P > 0.05), so the equation 4 was not
satisfactory to describe the tablet friability
behavior. The lower multiple regression coefficient
value (r^{2} = 0.728) showed that the equation is
adequate, but the mathematical model proposed was not able to differ the obtained
response (friability) and the graphic background
noise. As the results from the t-test showed that the
proposed model was not satisfactory either,
(coefficients not significant – P > 0.05), the
response surface and the contour-plot graphics
could not be plotted.

It can be observed that the hardness and friability were more susceptible to the STE concentration, and that this excipient caused a decreased in the tablet mechanical resistance. The formulations with lower STE concentration produced tablets with higher hardness and lower friability. As the lubricant particles cover the formulation components surface, they act as a mechanical barrier, interfering on the mixture binding properties and consequently producing tablets mechanically weaker [12,13].

The following mathematical model was estimated to the disintegration time, according the central composite design, resulting the equation [5]:

DT = 7.013-0.716 × STE-1.714 × SCC + 0.127 × STE × SCC + 0.169 × STE^{2} + 0.123 × SCC^{2} [5]

where DT = disintegration time; STE = STE factor; SCC = SCC factor; STESCC = factor interaction

The equation 5 shows that about 95 % of
experimental variance could be explained by the
multiple regression coefficient calculated (r^{2} =
0.953). As the lack-of-fit test was not significant (F _{(P0.95; FG 3.4)} = 6.59 > 1.43), a possible experimental
variation could be ascribed to a randomized error
that was not related to the experimental model,
and the regression model expressed by equation
5 seems to be satisfactory to describe the
disintegration time behavior of these tablets.

According to **Table 5**, it was possible to assume
that the mathematical model proposed to explain
the tablet disintegration time behavior was
adequate, and the experimental variance could
be attributed to a randomized error that was not
related to the experimental model.

Term | Coefficient | Standard Error | t calculated |
---|---|---|---|

1 | 7.120 | 0.937 | 7.599 |

STE | – 0.135 | 0.529 | 0.256 |

SCC | – 1.824 | 0.559 | 3.264* |

Interaction | 0.119 | 0.117 | 1.019 |

STE2 | – 0.178 | 0.139 | 1.239 |

SCC2 | 0.146 | 0.0847 | 1.592 |

* significant for T = 0.95

**Table 5:** Results of the t-test for equation 5 coefficients

The results from the t-test (**Table 5**) showed that
the disintegration time was only influenced by
SCC concentration, where the SCC lineal term
was the main responsible for the decrease in the
tablets disintegration time. All the others equations
components had no statistical significance on the
response. The response surface (**Figure 2a**) and
the contour-plot graphics (**Figure 2b**) showed that
the disintegration time decreased when higher
SCC concentrations are combined with higher STE
concentrations. It can be observed that the lower
disintegration time was obtained when the SCC
concentration was higher than 3.5 % and the STE
concentration was higher than 2.0 %.

Tablets containing higher SCC concentrations showed lower disintegration time. The decrease in the disintegration time observed when the SCC concentration increase was also verified in early works [14,15,16], which proves the high efficiency of the disintegrant used in the production of tablets by direct compression. As the STE is a hygroscopic excipient, one expected that its presence would result in an increase in the disintegration time of tablets. However, in the studied experimental field, STE presented an unexpected effect, therefore reducing the disintegration time when its concentration was increased, demonstrating an anomalous behavior of the STE considering the disintegration time.

Equation [6] was fitted to the dissolution efficiency parameter, DE:

DE = 93.080 - 4.171 × STE + 4.168 × SCC + 0.998 × STE × SCC + 1.691 × STE^{2} + 0.427 × SCC^{2} [6]

where: DE = dissolution efficiency; STE = STE factor; SCC = SCC factor; STESCC = factor interaction.

The analysis of the regression indicates that
equation 6 was valid to describe the tablets
dissolution efficiency (r^{2} = 0.856) and the
experimental variance can be attributed to the
pure experimental error and does not depend on
the adjustment model to the experimental data (F_{(P 0.95; FG 3.4)} = 6.59 > 3.70). The results of the t-test for
the equation coefficients (**Table 6**) demonstrate
that the dissolution efficiency were influenced by
the quadratic component of the STE
concentration, followed by the linear component
of the SCC concentration. All the others
coefficients had no statistical significance on the
dissolution efficiency.

Term | Coefficient | Standard Error | t calculated |
---|---|---|---|

1 | 93.080 | 3.912 | 23.792 |

STE | – 4.171 | 2.364 | 1.764 |

SCC | 4.618 | 2.186 | 1.907* |

Interaction | 0.0998 | 0.593 | 0.168 |

STE2 | 1.691 | 0.600 | 2.819* |

SCC2 | – 0.427 | 0.339 | 1.260 |

* significant for T = 0.95

**Table 6:** Results of the t-test for equation 6 coefficients

The response surface (**Figure 3a**) and the contourplot
graphic (**Figure 3b**) showed that the
dissolution efficiency was mainly affected by SCC
concentration, where an increase in the SCC
concentration caused a faster disintegration and
higher tablet dissolution efficiency. The increase in
the STE concentration did not change the
dissolution efficiency, however, when the STE
concentration was higher than 2 %, an increase in
the tablet dissolution efficiency was observed. The
higher the SCC proportion, the faster the tablet
disintegration and thereof the clozapine release.

The STE effect was best observed on the
dissolution efficiency than on the disintegration
time, strengthening the STE anomalous behavior
upon the disintegration time. According to the
surface response (**Figure 3**), an increase in STE
concentration cause a decrease in the
disintegration time, however, this increase does
not reduce the dissolution efficiency values in the
same extent.

As the development of pharmaceutical products involves effort and time, a very efficient way to enhance the value of research, to minimize the process development time and obtain information concerning the influence of the different excipients is through designed experiments. Using experimental design, such as Central Composite Design, where a given number of experiments are selected out of many possible ones, is a good way in order to obtain a statistically optimized design.

According the Central Composite Design proposed for this work, the STE concentration influenced the tablet mechanical resistance, affecting the hardness in a negative way, decreasing the obtained values, and the friability in a positive way, increasing the obtained values. The SCC proportion determined a linear decrease in the disintegration time, causing a faster clozapine release from the tablets, thereby increasing tablets dissolution efficiency. It was possible to verify a STE anomalous behavior, because the increase on its amount results in a decrease in disintegration time. However, this increase does not cause an increase on the tablet dissolution efficiency, on the experimental field.

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