2.10.3. Bioavailability Study

Non-compartmental pharmacokinetic analysis was applied to calculate parameters such as Tmax, Cmax, AUC0–t, and AUC0–∞ from the plasma concentration-time curve data using WinNonlin software (version 1.5, Pharsight Corp. Mountain View, CA, USA). The relative bioavailability of the optimal formulation to the commercial tablet (reference) was calculated using the following Equation (2):

$$\text{Relative bioavailability} = \frac{A \text{UC}\_{T0-\text{os}} \times X\_R}{A \text{UC}\_{R0-\text{os}} \times X\_T} \times 100\% \tag{2}$$

where *XR* and *XT* were the administered dose of the reference and test respectively. Results were presented as means ± standard deviation. A one-way ANOVA (SPSS, version 19) with *p* < 0.05 as a level of significance was applied to examine the differences of Cmax and AUC0–∞ between the test and reference.

### **3. Results and Discussions**

#### *3.1. Impact of CA on Drug Release*

Formulations with different concentrations of CA in the sub-layer were developed to evaluate the effect of pH-modifier on the drug release rate, while the dissolution-controlling layer and ADEC coating levels were kept at 8% and 11% respectively. The results in Figure 2 illustrated that formulation without CA showed a fast release of LXP (>80% within 2 h), while the drug release within 2 h was decreased to 40% at a CA concentration of 1%. Additionally, the release rate continued to decrease with the increase of CA concentration, which showed 16.37%, 11.34%, and 7.77% of LXP release within the first 2 h. At the CA concentration of 1%, a completed drug release was finished within 6 h. While at higher CA concentrations, there were still 21.80% (2.5% CA) and 32.43% (4.0% CA) of the initial drug amount released after 6 h.

**Figure 2.** Effect of citric acid concentration on the drug release within different intervals.

As a pH modifier, CA was aimed to modulate the pHM inside the systems. For pH-sensitive compound, its solubility is more appropriate to be described as the solubility in the diffusion layer at the surface of the dissolving particles [25]. Therefore, according to the Noyes–Whitney theory, the dissolution rate of LXP was much more dependent on the solubility in the low pHM beneath the

di ffusion-controlling layer, other than the dissolution media. Theoretically, drug release rate from a coherent film coating system is controlled by both the coating level and the drug concentration gradient across the coating film, which obeyed the Fick's di ffusion law. As the film coating level was kept constant, drug release rate was predominantly controlled by the drug concentration gradient, which was determined by the dissolution rate of LXP inside the pellets. Therefore, as the drug release was significantly decreased with the increase of CA concentrations (Figure 2), the first step of developing a dissolution-rate controlling layer proved to work.

Furthermore, simultaneous release profiles in Figure 3 were constructed to investigate the impact of dynamic release process of CA on the drug release rate. In formulations with lower CA concentrations, with the release of CA during the dissolution period, pH M could be changed from 0.4 (the saturated solution pH of CA) to the approximate equilibrium pH of the dissolution medium [36]. As the solubility of LXP changed approximately 300 times within this pH range (Supplementary Materials Figure S1), the drug dissolution rate was significantly dependent on the amount of CA left inside the pellets. Therefore, matching release profiles of LXP and CA were observed at low concentrations of CA.

**Figure 3.** The simultaneous release profiles of citric acid and loxoprofen from sustained release pellets at di fferent concentrations of citric acid (*n* = 3).

While in formulations with higher CA concentrations, as a su fficient amount of CA remained inside the pellets at the end of the dissolution period, e.g., 30% or 40% of the initial CA amount left at the concentration of 4% or 6% respectively, a constant and e ffective pH M was achieved inside the pellets, which resulted in no further decrease of the drug release rate (Figure 3). A similar phenomenon was observed in a matrix tablet containing dipyridamole, due to a constant and e ffective pH M maintained by fumaric acid inside the dosage form, no further enhancement of the dipyridamole release was observed after increasing the fumaric acid concentration to 40% [37]. Therefore, a discrepancy of the release profiles between CA and LXP was observed in formulations with higher CA concentrations.

### *3.2. Release Experiments and Statistical Evaluation*

### 3.2.1. Testing of Drug Release

Experimental variables and observed responses of all the 15 formulations were listed in Table 2. And their drug dissolution profiles were displayed in Figure 4. At a low level of CA concentration, most of the formulations (Formulation Nos. 6, 9, 11) showed a fast drug release except Formulation No. 7, which had a high coating level of ADEC. The fast release in Formulation Nos. 6, 9, 11 was

attributed to an inefficient pHM inside the pellets and the short diffusion pathway of ADEC coating, which could be identified as the failure of the first- and second-step control. As a prolonged diffusion pathway was developed in Formulation No. 7, the release rate of LXP was significantly decreased. While at a low coating level of ADEC, most of the formulations (Formulation Nos. 1, 11, 14) showed a fast drug release rate, expect Formulation No. 13, which was incorporated with a high concentration of CA. The fast release in Formulation Nos. 1, 11, 14 could be explained by a failure of the second-step control, as a short diffusion pathway created by the low coating level of ADEC was unable to retard the release rate of LXP. However, when 3% of the CA concentration was applied in Formulation No. 13, the effective control of the drug dissolution-rate could compensate for the failure of the diffusion-rate control to achieve a sustained release of LXP.

**Figure 4.** Dissolution profiles of loxoprofen in formulations prepared by the Box-Behnken design experiments (**a**) Formulation Nos. 1–5, (**b**) Formulation Nos. 6–10, (**c**) Formulation Nos. 11–15.
