1. Introduction
As a major global chronic disease, non-alcoholic fatty liver disease (NAFLD) has a disease spectrum that includes simple non-alcoholic fatty liver, non-alcoholic fatty hepatitis, and related liver cirrhosis and liver cell carcinoma [
1]. NAFLD is associated with a bleak prognosis, including cirrhosis, hepatocellular carcinoma, and even death [
1]. Recent trends in the treatment of NAFLD have focused on lifestyle modification [
2], bariatric surgery [
3], and medical therapy [
4,
5]. Given the considerable risk associated with surgery, the exploration of new therapeutic agents against NAFLD is essential but challenging. IMM−H014 (previously known as WS117) is a novel collateral phenyl-structured compound with a new mechanism of action involving a nuclear factor NF−E2-related factor agonist. IMM–H014 has anti-inflammatory activity and can increase insulin sensitivity, which may be useful in the treatment of NAFLD [
6].
In the treatment of chronic diseases, IMM-H014 requires long-term administration, and patients with NAFLD require continuous and effective medical care. IMM-H014 is easily absorbed in the gastrointestinal tract, demonstrating a 96.3% oral bioavailability in rats, a short plasma drug concentration peak time (T
max) (0.5 h), and a short plasma elimination half-life (t
1/2) (rats, 1.8 h; beagle dogs, 3.5 h) [
7]. Drugs with a short half-life are given at shorter intervals and more frequently. To improve patient compliance, it is recommended that IMM-H014 should possess an extended-release (ER) profile after oral administration. ER solid dosage forms for oral administration may be effective for avoiding frequent dosing and may improve long-term therapeutic management with drug molecules that exhibit a narrow therapeutic range and/or are rapidly cleared from the blood [
8].
Inert polymer matrices have been widely used as skeleton materials to adjust the release rate in controlled-release delivery formulates [
9,
10]. The mechanism of action of hydrophilic polymer matrix systems, which are widely used in controlled drug delivery, is based on the gel layer that is formed by hydrating the polymer. The gel layer controls the drug release rate [
11,
12,
13]. The release of water-soluble drugs in vitro controls the diffusion of the gel layer outside, which depends on the gel’s viscosity. In contrast, the release of poorly water-soluble drugs is dependent on the dissolution of the polymer [
14,
15].
Cellulose derivatives have been widely used as hydrogel matrices for controlled drug delivery, among which, hydroxypropyl methylcellulose (HPMC) is the most widely applied given that it is easy to use, widely available to purchase, and has low/no toxicity [
16]. Drug release is controlled by a gel layer formed on the matrix surface due to the hydration of HPMC, through which the loaded drug diffuses [
17].
In matrix tablets prepared with HPMC, a gel layer is produced on the surface of the tablets upon aquation. At a higher use level of HPMC, the greater degree of entanglement of the linear polymer chains results in “virtual crosslinking,” leading to the formation of a more robust gel layer [
18]. HPMC is a versatile polymer for the production of tablets and is widely accepted as a pharmaceutical excipient for oral administration [
19,
20]. HPMC can be used to control the release behavior of hydrophilic and hydrophobic drugs through swelling, diffusion, and erosion processes [
21].
In this study, we explored the feasibility of producing IMM-H014 extended-release tablets using a direct compression method based on hydrophilic polymeric matrices of HPMC. The effect of the polymer concentration on the in vitro and in vivo drug release rate was researched to establish the preferred formulation in terms of modified release. Furthermore, a point-by-point in vitro–in vivo correlation (IVIVC) was developed to relate the percentage of drug dissolved to the percentage of drug absorbed. The changes in drug absorption in the body can be evaluated by IVIVC based on the in vitro dissolution when the formulation is changed slightly [
22].
3. Materials and Methods
3.1. Materials
IMM-H014 was produced in the author’s laboratory; IMM-H014 extended-release tablets (batch Nos. 1, 2, and 3) were produced by Pharmaron Ningbo Co., Ltd. (Ningbo, China); hydroxypropyl methylcellulose (HPMC) was purchased from Shin–Etsu Chemical Co., Ltd. (Dalian, China); lactose was purchased from DFE pharma GmbH & Co. KG. (Shanghai, China); silicon dioxide and magnesium stearate were purchased from Anhui Sunhere Pharmaceutical Excipients Co., Ltd. (Huainan, China); and acetonitrile (HPLC grade) and methanol (HPLC grade) were purchased from Honeywell. The other reagents were analytical grade.
Animal: Beagles (female or male, purchased from Beijing Marshall Biotechnology Co., Ltd. Beijing, China) were used for in vivo pharmacokinetic studies. During the experiment, the beagles were given a standard diet and allowed to drink freely. The study followed ethical guidelines and the protocol was approved by the Laboratory Animal Ethics Committee of the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (animal ethical clearance protocol number: 00005367, 2022. 12.)
3.2. Solubility Test
3.2.1. Preparation Methods for Solubility Media
The saturation solubility of IMM-H014 in pH 2.0, pH 4.5, pH 6.8, and pH 12 media were investigated. These media were prepared as follows:
- (1)
pH 2.0 hydrochloric acid solution: 0.9 mL of hydrochloric acid was diluted with water to 1000 mL.
- (2)
pH 4.5 acetate-buffered solution: 2.99 g of sodium acetate was weighed, to which 1.6 mL glacial acetic acid was added and diluted with water to 1000 mL.
- (3)
pH 6.8 phosphate-buffered solution: 6.8045 g of monopotassium phosphate and 0.896 g of sodium hydroxide were weighed and dissolved in an appropriate amount of water, and the solution was then diluted with water to 1000 mL.
- (4)
pH 12 sodium hydroxide solution: 0.4 g of sodium hydroxide was dissolved with 1000 mL of water.
3.2.2. Determination Method for Solubility Studies
An appropriate amount of the API was added to different pH media at 37.0 ± 0.5 °C, before shaking the flask for 24 h, filtering, and taking the continuous filtrate. The continuous filtrates were then diluted stepwise with the respective media to a suitable concentration within the range of 20–60 μg/mL, and were used as the test solutions. The amount of the dissolved drug was quantified by HPLC, as described in
Section 3.6.4.
3.3. Production of the Immediate-Release (IR) Capsule of IMM-H014
The appropriate amount of the IMM-H014 active pharmaceutical ingredient (API) was loaded into the gelatin capsule shell as the IMM-H014 immediate-release preparation.
3.4. Production of IMM-H014 ER Tablets
IMM-H014 ER tablets were produced by pressing the powder directly. The required quantities of drug and ingredients were passed through an 80-mesh screen (stainless steel). IMM-H014, HPMC, and lactose were mixed completely using the equivalent addition method. Then, after adding silicon dioxide and magnesium, the sample was mixed for a further 10 min. Finally, the tablets were compressed using a flat-faced 8 mm punch. Each tablet contained 35 mg of IMM-H014, with the tablet weight and hardness being maintained at 200 mg and between 100 and 130 N, respectively. The composition of the tablets is shown in
Table 6.
3.5. Evaluation of Granules
3.5.1. Angle of Repose
The granules’ angle of repose was measured using the funnel method. The granules were precisely weighed and located in the funnel, and the height of the funnel was adjusted so that the cusp of the funnel just touched the top of the granule cone. The granules ran freely through the funnel to the surface. The diameter of the particle cone was evaluated and the angle of repose was determined using Equation (1).
where h is the height of the granule cone and r is the radius of the granule cone.
3.5.2. Bulk Density
Approximately 5.0 g of particles, which was weighed without any aggregation, was placed in a 10.0 mL measuring cylinder to acquire the volume. The cylinder was dropped from a height of 2.5 cm on a flat surface every 2 s until there was no further change in volume. The loose bulk density (
LBD) and tapped bulk density (
TBD) data were calculated using Equations (2) and (3).
3.5.3. Compressibility Index
Carr’s Index [
23] was used to determine the compressibility index of granules, and was calculated using Equation (4).
3.6. Evaluation of Tablets
3.6.1. Weight Variation Test
An electronic balance was used to weigh 20 tablets that were taken randomly from each formulation, and the weight values were determined in milligrams (mg) (Mettler Toledo, Oakland, CA, USA).
3.6.2. Hardness Test
Six tablets were taken randomly from each formulation and determined using a hardness tester, with hardness values reported in newtons (N) (Tianda Tianfa Instrument, Tianjin, China).
3.6.3. Friability Test
Approximately 6.5 g of tablets was taken from each formulation and accurately weighed and placed in the friability tester (Xixin Instrument, Tianjin, China), which was set to 100 revolutions for 4 min. After the resulting tablets were dedusted and reweighed, the percentage weight lost was counted as the friability using Equation (5).
3.6.4. Content Determination
Twenty tablets were taken at random from each lot and weighed before being placed in a mortar and being porphyrized with a pestle. A quantity equivalent to 5 mg of IMM-H014 (40 mg of powder) was pulled out with 100 mL of hydrochloric acid solution (0.1 N) and supersonically extracted over 30 min. Then, the resulting 10 mL solution was filtered through a polytetrafluoroethylene filter membrane (PTFE, 0.45 μm pore size, Jinteng, Tianjin, China). A C18 column was used in the stationary phase. The collected samples (20 μL) were analyzed using HPLC (Shimazu LC-20AT, Kyoto, Japan) with a UV detector wavelength of 230 nm. IMM–H014 was separated under a mobile phase consisting of ammonium formate PBS (pH 4.0) to acetonitrile at a 60:40 (v/v) ratio using a C18 column (4.6 mm × 25 cm, 5.0μm). The flow velocity was 0.7 mL/min and the column temperature was 35 °C. The linearity of IMM-H014 ranged from 20.01 μg/mL to 60.02 μg/mL. The validation was conducted following the ICH guidelines.
3.6.5. In Vitro Release Studies
A USP type II dissolution apparatus was used to test the in vitro release of IMM–H014 tablets. The dissolution profiles of five dissolution media were surveyed. The first dissolution medium was 700 mL of 0.01 N HCl (pH 2.0). At 2 h, 200 mL of a 5.25 g/L (m/v) sodium phosphate solution was added to adjust the pH of the media to 7.5. The other dissolution mediums were a 0.01 N HCl solution, pH 4.5 PBS, water, and pH 6.8 PBS, where the volume of the dissolution medium was 900 mL; the speed was 50 rpm, and and the temperature was maintained at 37 ± 0.5 °C throughout all experiments. The drug release was quantified by ultraviolet–visible spectrophotometry at a wavelength of 230 nm. As a result, different dynamic equations were in accordance with the data of the first media, including zero-order, first-order, Higuchi, and Ritger–Peppas models.
The similarity factor (f
2) was calculated to compare the release profiles of the IMM–H014 ER tablets during the production and stability study periods, which were defined by Equation (6) [
24].
where
n is the number of sampling times, and
Rt and
Tt are the dissolution values of the reference and test samples at each timing, respectively [
24].
3.6.6. Stability Studies
The IMM–H014 tablets were packed in aluminum–aluminum blister packaging and placed under the acceleration conditions (40 °C, RH75%) for 6 months. The similarity factor (f2) of the dissolution curve, appearance, and content were used as the index of inspection.
3.7. Drug Release Mechanism
The following mathematical models with different equations were used to analyze the description of in vitro dissolution [
25].
where the drug release amount at time t is M
t, and the final drug release amount is M
∞. The zero-order release rate constant is k
0; the first-order release rate constant is k
1; and k
H is the zero-order release rate constant [
25].
In the Ritger–Peppas model, the diffusion mechanism is expressed by the value of
n, with
n ≤ 0.45 corresponding to a Fickian diffusion mechanism, 0.45 <
n < 0.89 corresponding to a diffusion and erosion skeleton common action mechanism, and
n ≥ 0.89 corresponding to an erosion skeleton mechanism [
26]. For every model considered, the best fit is indicated by the correlation coefficient (r), where an r closer to 1 indicates a better fitting effect.
3.8. In Vivo Pharmacokinetics Study in Beagles
3.8.1. Study Design
The study was conducted to compare the pharmacokinetics of the IMM-H014 ER tablets to those of the IMM-H014 IR tablets, following the administration of single doses equivalent to 75 mg (three tablets per dose) in a two-treatment, two-period (time interval was 7 days) crossover design. Ten healthy beagle dogs of either sex were selected for the experiment and randomly divided into two groups (five dogs in each). Before the study, the dogs were fasted for approximately 12 h, with water provided freely. The sampling time points were different due to the different release rates of the ER and IR preparations. Venous blood samples (1–2 mL) were withdrawn into heparinized tubes 0, 0.08, 0.17, 0.25, 0.33, 0.5, 0.75, 1, 2, 3, 5, 8, 12, 24, 36, and 48 h after administration of the IR preparation, while the sampling times for the ER tablets were 0, 0.25, 0.5, 1, 2, 3, 5, 8, 12, 24, 36, and 48 h after administration. The blood samples were promptly centrifuged at 4 °C and 4000 rpm for 10 min to isolate the plasma, which was stored at −20 °C until analysis. A validated HPLC–MS analytical technique was developed to estimate the drug concentration in plasma samples, and the pharmacokinetic parameters (Cmax, Tmax, MRT, and AUC) were estimated.
3.8.2. Statistical Analysis
The plasma concentration of IMM–H014 was plotted versus time to exhibit the pharmacokinetic profiles. Statistical analysis was executed using DAS2.0 software (version 2.0, Mathematical Pharmacology Professional Committee, Shanghai, China). The key parameters of pharmacokinetics, such as C
max, T
max, MRT, and AUC, were analyzed. Statistical significance was defined as
p < 0.05. The relative bioavailability (F) was calculated using the AUC
0–t of IR and ER tablets [
27].
3.9. IVIVC
The IVIVC was developed to relate the percentage of in vitro drug dissolution to the percentage of in vivo drug absorption and was used for drug development. Based on a good correlation, the in vivo pharmacokinetic profile can be determined using the in vitro dissolution rate alone.
The fraction of the drug absorbed (
Fa) was calculated by the Wagner–Nelson equation [
28], as shown in Equation (7).
where
Fa is the fraction of the drug absorbed, C
t is the concentration of the drug in the plasma at time point
t, k is the elimination rate constant, AUC
0–t is the calculated area under the plasma concentration curve from zero to time t, and AUC
0–∞ is the calculated area under the plasma concentration curve from time zero to infinity [
28]. The percentage of drug absorption (
Fa) at the specified timing was drawn against the percentage of drug dissolved in vitro at the same timepoint. The pertinence between the in vitro release and in vivo absorption was assessed by the linear regression coefficient (R).
4. Conclusions
In this study, IMM-H014 was developed as an ER preparation to solve the problems of a short oral administration half-life, short administration interval, and high-frequency administration of IMM-H014. IMM-H014 ER tablets composed of hydrophilic polymers were produced via a direct powder pressing method. The optimal formulation of IMM-H014 ER tablets was determined to accelerate stability and remained stable over a 6-month period. For the in vitro dissolution studies, the optimal formulation showed an obvious ER compared to the IR preparation. The results from the in vivo pharmacokinetics study in beagle dogs also clearly indicate that the AUC of the IMM-H014 ER tablets was equivalent to that of the IR preparation, the relative bioavailability was 97.9%, the Cmax demonstrated a decrease of around 1/3, and the Tmax and MRT were meaningfully lengthened, with a visible ER impression. Moreover, the IVIVC correlation coefficient (R2) was 0.9509, suggesting that the prepared tablets had a good correlation between the in vitro release and pharmacokinetic effect. Therefore, absorption in vivo can be predicted by the release in vitro.