3.2.2. Regression Equations

Based on the experiment data, the coefficients and their *p*-values of the fitted full quadratic equations calculated by Expert-Design 8.0.6 (Stat-Ease Inc., Minneapolis, MN, USA) are listed in Table 4. The final equations consisted of only statistically significant coefficients. It is clear that the citric acid concentration (*X*1) and ADEC coating weight gain (*X*3) showed significant effects on the drug release rate throughout the dissolution period, while the weight gain of the dissolution-rate controlling layer (*X*2) showed only a weak effect during the dissolution period. The impact of *X*1 on the drug release rate verified the effectiveness of the first-step control on the drug release rate, which could be explained by its impact on the pHM, as extensively reported in the literature [36,37]. The effect of *X*3 on the drug release rate could be attributed to its control on the drug diffusion rate, which was considered as the second-step control [38]. Besides, an interaction effect of *X*1 and *X*3 was observed on the response of *Y*1 and *Y*3, which might be explained by a speculation that *X*3 also showed an effect on the release rate of citric acid. Additionally, it seemed that *X*3 played a more dominant role on the final drug release, as high coefficients of the main, interaction, and quadratic effects of *X*3 were observed in *Y*3. From the statistic results in Table 4, we could conclude that both the dissolution-rate and diffusion-rate controlling steps have significant effects on the drug release rate.



\* *p*-value < 0.05.

## 3.2.3. Response Surface Plots

The relationship between the dependent and independent variables was further elucidated using a 3D response surface plot, which is useful to see the effect of two factors on the response at one time while the third factor is kept at a constant level. The effects and interactions between concentration of citric acid (*X*1), the sub coating weight gain (*X*2), and ADEC coating weight gain (*X*3) on the finial drug release (*Y*3) are given in Figure 5. The similar impacts of the three factors on the other responses (*Y*1 and *Y*2) can be seen in Supplementary Materials Figures S2 and S3. As illustrated in Figure 5a,b, it was clear to see that *X*2 showed little effect on *Y*3 irrespective of the levels of other two factors. This was attributed to the fact that the dissolution-rate controlling layer is made up of aqueous polymer, which was dissolved before 12 h.

**Figure 5.** Contour plots showing the effects of (**a**) *X*1 and *X*2, (**b**) *X*2 and *X*3, and (**c**) *X*1 and *X*3 on the response *Y*3.

The effects of citric acid concentration (*X*1) and the ADEC coating weight gain (*X*3) on *Y*3 are depicted in Figure 5c. While *X*3 was kept at low level, the increase of *X*1 from 1% to 3% showed no effect on *Y*3, which kept nearly constant at above 90%. While at a high level of *X*3, the increase of *X*1 from 1% to 3% resulted in a significant decrease of *Y*3 from 85% to 60%. The result indicated that an interaction effect of the two factors existed on the drug release rate, as mentioned in Section 3.2.2. At a high level of *X*3, an effective diffusion barrier was formed on the surface of the pellets [38], which significantly reduced the diffusion rate of loxoprofen. However, the release rate of loxoprofen was not solely controlled by the diffusion-controlling layer. For example, a nearly complete release of loxoprofen (>85%) was observed at a high level of *X*3 (Figure 5c), when *X*1 was kept at a low level of 1%. Therefore, the release rate of loxoprofen was a combined result of the two controlling steps. At a low level of *X*3, the citric acid was soon released regardless of its concentrations, which resulted in a quick increase of the pHM and a fast drug release rate. When the concentration of citric acid and ADEC coating weight gain were kept at high levels, both the drug dissolution and diffusion rate were reduced, which resulted in a prominent decrease of the drug release rate.

#### 3.2.4. Design Space and Formulation Parameters Optimization

Design space was defined by the ICH Q8 as the relationship between the process inputs (material attributes and process parameters) and the critical quality attributes that have been demonstrated to provide assurance of quality [39]. The wider the design space is, the more robust and flexible the process is to resist variations [40]. As the response surface models of the output parameters as a function of selected variables were given, design space of *X*1, *X*2, and *X*3 was determined by applying constraints on *Y*1 (<30%), *Y*2 (50–70%), and *Y*3 (>90%). The yellow overlap region of ranges for the three responses in Figure 6a–c show the proposed design space of the citric acid concentration *X*1 and the ADEC coating weight gain *X*3 at three di fferent levels of the sub-coating weight gain *X*2. As shown in Figure 6a, there was no design space of *X*1 and *X*3 at the low level of *X*2. Additionally, Figure 6c depicted a narrow design space of *X*1 and *X*3 at high level of X2, which would increase the di fficulty of the operation process since an accurate coating load of ADEC must be achieved during the manufacturing process. While at the medium level of *X*2 (Figure 6b), the design space was expanded, which showed a less strict field of ADEC coating level. As the design space depicted the ranges of the formulation parameters for achieving the desired quality of product, the levels of the three factors for the optimal formulation must be set within the design space. Considering the robustness and flexibility, parameters of the optimal formulation were set at the medium level of sub coating weight gain with the CA concentration and coating level of ADEC at 2.5% and 11.0% respectively. The model predicted a release profile of 19.87% at 2 h, 64.48% at 6 h, and 91.71% at 12 h. To verify these values, a new batch of the optimal formulation was prepared. The obtained release data of the optimal formulation were in close agreemen<sup>t</sup> with the predicted values with a maximum percentage error of 11.73% at the initial release (data not showed).

**Figure 6.** Design space of operating variables of the citric acid (CA) concentration and aqueous dispersion of ethyl cellulose (ADEC) coating level (**a**) at the low level of the sub-layer coating weight gain, (**b**) at the medium level of the sub-layer coating weight gain, and (**c**) at the high level of the sub-layer coating weight gain (yellow zone: design space; grey zone: failure space).

#### *3.3. Simultaneous Release of CA and LXP from the Optimal Formulation in Di*ff*erent Dissolution Media*

In order to evaluate the effect of pH on drug release, various media simulating different physiology pH values were applied. As shown in Figure 7, dissolution tests were performed in pH 1.0 HCl, pH 4.5 and pH 6.8 phosphate buffer solutions and water. Furthermore, the release profiles of CA were also investigated in these media. As illustrated in Figure 7, drug release profiles were pH-independent at pH above 4.5, and showed similar release profiles to that of CA.

**Figure 7.** Loxoprofen and citric acid released from sustained release pellets in different dissolution media. (**a**) pH 1.0 HCl (**b**) pH 4.5 phosphate buffer (**c**) pH 6.8 phosphate buffer (**d**) water (means ± SD, *n* = 3).

Although the solubility of LXP was pH-independent in media with pH above 4.5 (Supplementary Materials Figure S1), it seemed that the dissolution media were not the reason for this pH-independent release behavior. As the drug showed a completed release within 3 h without the incorporation of CA inside the pellets (Figure 3, formulation with the CA concentration of 0%), it should exhibit a similar release for the optimal formulation as the dissolution media were also above 4.5. In the contrast, the optimal formulation showed sustained release for almost 12 h. It was the pHM created by CA, which showed similar release profiles at pH above 4.5, that accounted for the pH-independent release profiles of LXP (Figure 7). As mentioned before, the saturated solution pH of CA was 0.4 [36], which was much lower than that of the dissolution media except the pH 1.0 HCl. However, with the release of CA during the dissolution period, the amount of CA left inside the optimal pellets was insufficient to maintain a constant pHM inside the pellets. The pHM was gradually increased, which resulted in a consistent enhancement of the drug solubility. In addition, matching sustained release profiles of LXP and CA were achieved in Figure 7.

While at pH 1.0, it was the dissolution media that showed a major effect on the drug release rate. With the decrease of CA, the pHM would soon be increased up to above 1.0. However, the dissolution media that penetrated into the pellets provided a stable pHM inside the pellets, which in turn resulted in a different release profile to the other three. In conclusion, the dissolution media and the incorporated CA played a combined effect on the drug release rate. In media with high pH values, the CA showed

a greater e ffect on the drug release rate. While in media with low pH values, it was the dissolution media that dominated the drug release rate.

### *3.4. Release Mechanism Studies*

In order to elucidate the transport mechanism of LXP in the optimal formulation, di fferent mathematical models were applied to analyze the kinetics of the release data. As shown in Table 5, the incorporation of CA into the sub-layer resulted in abnormal release kinetics of this ADEC coating system, as the *n* value for Ritger–Peppas was 0.7422, which is between 0.45 and 0.89, indicating a non-Fick di ffusion [41]. Additionally, a general empirical equation of Weibull distribution model with *r2* of 0.9944 was more appropriate to describe the release process of the optimal formulation. In the model, the derived estimate of *b* value was calculated to be 1.38, which represented a sigmoid shape curve (*b* > 1) for the release profile [41]. The initial slow release representing the starting part of the sigmoid curve was a result of several factors. As reported previously, the permeability of water through the EC coating is much faster than the permeability of the compound [38], which might contribute to the initial slow release of LXP. Besides, the decreased dissolution rate created by the initial low pH M inside the pellets also played an important role on the initial slow release. Furthermore, the hydration of the hydroxypropyl methyl cellulose (HPMC) inside the pellets during the initial dissolution period could also inhibit the initial drug release rate. Thereafter, due to the saturation of water inside the pellets and the disruption of the dissolution-rate controlling layer, the drug release rate was dominated by the dissolution- and di ffusion-rate control, which resulted in a sustained release profile.

**Table 5.** Models simulated for the drug release profiles of the optimal formulation.


### *3.5. Scanning Electron Photomicrographs*

Figure 8 shows the scanning electron photomicrographs of the optimal pellets. The surface of pellets were smooth (Figure 8a,b), and no crack could be seen. Besides, layers of the dissolution-rate controlling layer and the di ffusion-rate controlling layer were clearly seen in the cross-section of the coating pellets (Figure 8c,d). These results indicated that a successful procedure had been developed for manufacturing the sustained release pellets.

**Figure 8.** SEM photographs of pellets with double coating layers: (**a**) Surface of sustained release pellets with 70 magnifications, (**b**) surface of sustained release pellets with 450 magnifications, (**c**) cross-section of sustained release pellets with 70 magnifications, (**d**) cross-section of sustained release pellets with 350 magnifications.

## *3.6. Pharmacokinetic Studies*

The pharmacokinetic studies of the optimal pellets and the commercial tablets were investigated on fasted beagles. The profile of mean plasma concentrations of LXP versus time is shown in Figure 9. The main pharmacokinetic parameters are summarized in Table 6. As shown in Figure 9, the plasma concentration of the commercial tablet quickly increased and reached the peak concentration of 5.16 μg/mL at 0.5 h after administration. Then it dropped down and was only 0.2 μg/mL at 6 h. This was attributed to the short half-life (t1/2 = 64.46 min) of LXP [12], which resulted in a quick elimination of the drug in vivo. The optimal formulation reached the maximum plasma concentration of 2.40 μg/mL at 5 h after administration, and the drug concentration fell slowly even at 12 h, when the drug concentration was 0.15 μg/mL, in contrast with the undetectable drug concentration in plasma for the commercial tablet 8 h after administration.

**Figure 9.** Plasma drug concentrations vs. time after oral administration of conventional tablets (60 mg) and sustained release pellets (90 mg).

**Table 6.** The pharmacokinetic parameters of loxoprofen after oral administration of the optimal sustained release pellets and commercial tablets in beagle dogs (*n* = 6).


As a pro-drug, LXP inhibits the activities of cyclooxygenase-1 and -2 (COX-1 and COX-2) by its active metabolite trans-OH LXP, of which the IC50 values for COX-1 and COX-2 were 0.38 and 0.12 μM, respectively [42]. It has been reported that the concentration of trans-OH LXP, which was the major metabolite of LXP, was equal to more than half of the LXP concentration detected in plasma [12]. As the LXP concentration range in plasma of the optimal pellets was 0.6–10.0 μM, we can deduce that the concentration of trans-alcohol LXP after administration of the optimal pellet would be higher than the IC50 of the trans-OH LXP. Therefore, a therapy concentration of trans-OH LXP in plasma after administration of the optimal pellets would be maintained for almost 12 h. As the frequency of dosage of the optimal formulation was reduced to two times a day, the patient's compliance would be better improved. It has been reported that the incidence of gastric lesions after administration of LXP in rats showed a dose-dependent manner. Additionally, the amount of PGE2, which has a strong protective effect on the GI mucosa, also decreased in a concentration-dependent manner after treatment of LXP within the concentration range of 1.0 μM to 1.0 M [14,43]. Therefore, the risk of GI lesions would be significantly decreased, since the initial burst release disappeared in vivo and the Cmax of LXP was significantly decreased from 20 to 10 μM after administration of the optimal formulation. Besides, a less fluctuant drug concentration in plasma was achieved for the optimal pellets in Figure 9. The significant difference (*p* < 0.05) of AUC0–∞/dose between the test formulation and conventional tablet might be caused by the limited GI absorption site of LXP, which would need further investigation. The relative bioavailability of the test formulation was 87.16% compared with the reference, and it would be improved in patients as a prolonged GI transit time has been reported in humans [44].
