Solid Self-Microemulsifying Drug Delivery System for Improved Oral Bioavailability of Relugolix: Preparation and Evaluation

Introduction

According to the latest World Health Organization global cancer statistics for 2021, there were over 19.3 million new cancer cases worldwide and over 10 million cancer deaths.¹ In the world, prostate cancer is the most common cancer and the second leading cause of cancer deaths in men. According to statistics, about 10% of women at childbearing age suffer from endometriosis.² Leuprolide is the first-line drug for these diseases, but it has toxic side effects and low efficacy.³ Relugolix (RLGL) is an orally active non-peptide gonadotropin-releasing hormone receptor antagonist used in the treatment of various sex hormone-related disorders.^4,5 Its chemical structure formula is shown in Figure 1A. RLGL has higher biosafety and efficacy than leuprolide, but its poor water solubility leads to low oral bioavailability.^6,7 A marketing application for Relugolix was submitted to the FDA in 2019 and approved in 2020. Since then, Relugolix has been marketed under the trade name Orgovyx. Relugolix may become the most important drug in the treatment of prostate cancer in the future due to its convenience, small side effects. However, its poor water solubility leads to only 12% oral bioavailability, and a drug delivery system is urgently needed to improve its oral bioavailability.

Figure 1 (A) Chemical structure of relugolix. Solubility of RLGL in various oils (B), surfactants (C) and co-surfactants (D). All values in μg/g, data are expressed as means ± SD (n=3).

Self-microemulsifying drug delivery system (SMEDDS) has gained wide attention for its ability to improve the oral bioavailability of poorly water-soluble or lipophilic drugs. It is a homogeneous mixture containing oil, surfactant and co-surfactant that can spontaneously form a clarified system (particle size less than 100 nm) when dispersed in water.^8–10 The possible mechanisms by which self-microemulsion can promote oral absorption of drugs and improve their oral bioavailability are increased solubility and improved drug dissolution, the large amount of surfactants on the surface of self-microemulsions which increase the penetration into intestinal epithelial cells and promote the drug absorption, certain emulsifiers which can inhibit the efflux of drugs caused by P-glycoprotein, the lipid component of the prescription which can be degraded by pancreatic enzymes and biles to form smaller microemulsion droplets and bile salt micelles thereby increasing drug solubility and transmembrane transport efficiency and the lipid component of the oil phase which can also facilitate the absorption of drugs by lymphatically overcoming the first-pass effect of drugs and obstacles to the absorption of large molecules through the epithelial cells of the gastrointestinal tract.^11–15 The main reason that few self-emulsifying formulations are currently on the market is that all commercial liquid-based self-microemulsion products are encapsulated in hard gelatin capsules or soft gels, and the following disadvantages arise: the complexity of the production process, resulting in high production costs, compatibility issues between formulation components and capsule shells and possible capsule leakage after long-term storage.^16–21

The solid self-microemulsifying drug delivery system (S-SMEDDS) is a solid dosage form that combines the advantages of both self-emulsifying and solid formulations and overcomes the disadvantages of SMEDDS.^22,23 S-SMEDDS offer great advantages in terms of controlled drug release, prolonged gastric residence time and improved permeability, in addition to improving the absorption of poorly water-soluble drugs.^24–26 Moreover, S-SMEDDS can improve drug stability compared to SMEDDS.²⁷ As mentioned above S-SMEDDS may be an effective means to improve oral bioavailability of RLGL.

In this article, we report the preparation of a novel solid self-microemulsifying drug delivery system of RLGL (RLGL-S-SMEDDS) that is expected to have better therapeutic efficacy than RLGL-suspensions. To confirm our hypothesis, we prepared a self-microemulsion formulation for oral administration and performed detailed formulation optimization to select the appropriate excipients for oral administration. The morphology, particle size, zeta potential, thermodynamic and kinetic stability were examined. The solid carriers were screened for powder logical characterization. In vitro cellular uptake experiments, we established a Caco-2 cell monolayer model and a Caco-2 cell co-incubation model with lymphocytes to study the cellular uptake and transport of RLGL-S-SMEDDS. In vivo pharmacokinetic studies were carried out for determination of oral bioavailability of the drug. Histopathological analyses were made for preliminary evaluation of formulation safety.

Materials and Methods

Materials

Relugolix was from Takeda Pharmaceutical Industry Co., Ltd. (99%, Tokyo, Japan). Isopropyl myristate, ethanol, ethyl oleate, PEG 400 and Tween 80 were purchased from Aladdin Biochem Tech. Co., Ltd. (Shanghai, China). Transcutol HP (Gattefosse, France), Solutol HS15 and Kolliphor P188 (BASF Ltd., Shanghai, China) were obtained as gift samples. Hydrophilic-200 silica and Sylysiak 320 were purchased from Yien Chemical Technology Co., Ltd. (Shanghai, China). Triton X-100, Dulbecco’s modified Eagle’s medium (DMEM), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and trypsin were purchased from Beijing Solarbio Sci&tech. Co., Ltd. (Beijing, China). Corning Matrigel Matrix were purchased from Corning Life Sciences (Wujiang) Co., Ltd. (Jiangsu, China). All the other chemicals and reagents were of analytical or HPLC grade and obtained from commercial sources.

Formulation and Optimization of RLGL-SMEDDS

Solubility Study

The solubility data of RLGL in various oils, surfactants and co-surfactants were determined by shake flask method. Excess RLGL was added into each tube containing the excipients (2 g), and the tubes were vortexed for 10 min and incubated at 25 °C for 48 h in THZ-82 digital water bath thermostat (Jiangsu Chenyang Electronics Co., Ltd. China) to reach equilibrium. The content of RLGL was measured by HPLC method.

Compatibility Test

To visually evaluate the self-emulsifying ability of a certain oil, surfactant and co-surfactant combination, a compatibility test was conducted according to the literature. Briefly, selected oils, surfactants and co-surfactants were mixed for 5 min in 4:4.5:1.5 (w/w) using a vortex mixer, and the above mixture was diluted by water to 20-fold its volume at 37 °C with gentle agitation.²⁸

Construction of Pseudoternary Phase Diagram

Pseudoternary phase diagrams were constructed to obtain the concentration range of components for microemulsions.²⁹ The weight ratio of surfactant to co-surfactant (Km) was varied as 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1. For each pseudoternary phase diagram at a specific surfactant/co-surfactant weight ratio, the oily mixtures of oil, surfactant and co-surfactant were prepared with the weight ratio of oil to the mixture of surfactant and co-surfactant at 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1, respectively. Water was added drop by drop to each oily mixture under proper magnetic stirring at 37 °C until the mixture became clear at a certain point.³⁰ The concentrations of the components were recorded in order to complete the pseudoternary phase diagrams, and then the contents of oil, surfactant, co-surfactant and water at appropriate weight ratios were selected based on these results.

Experimental Design

The most appropriate ratio of K_m and oil was determined using central composite design (CCD). In this study, 13-run, two-factor, two-level CCD statistical screening design was used to optimize the formulation factors and the effects of Km and oil phase ratio on particle size and drug loading were investigated.^31,32 The statistical analysis was performed using Design-Expert^® software (version 8.0.6.1).

Preparation of RLGL-SMEDDS

Ethyl Oleate (0.26 g), Solutol HS15 (0.49 g), Transcutol HP (0.25 g) and RLGL (4.8 mg) was successively placed in a clean vial, vortex ultrasonically mixed, and stirred in a 70 °C thermostatic water bath until the RLGL were completely dissolved. In the bottle, a clear and transparent RLGL-SMEDDS was obtained.

Characterization of RLGL-SMEDDS

Particle Size, Zeta Potential and Morphology

The samples were diluted with distilled water under gently stirring for 5 min and the particle size, polydispersity index (PDI), and zeta potential were measured by dynamic light scattering using a Brookhaven system (NanoBrook-173Plus, Brookhaven Instrument, USA).

The freshly prepared RLGL-SMEDDS sample was diluted 50-fold with distilled water, and an appropriate amount of the solution was dropped on a microporous copper mesh, dried naturally, stained with 2% phosphotungstic acid, and observed under a transmission electron microscope (TEM, H-600, Hitachi, Japan).

Effect of pH and Robustness to Dilution

In this paper, RLGL-SMEDDS was diluted 50-fold, 100-fold and 250-fold with distilled water, pH 1.2 and pH 6.8 buffers, respectively and left for 24 h. The phase transparency, drug precipitation and phase separation of the system were observed.

Cloud Point

The self-microemulsion was diluted with distilled water and stored in a water bath. The initial temperature of the water bath was 25 °C and was increased 5 °C every 10 min and the corresponding cloud point temperature was read visually at the first sign of turbidity.

Stability Studies

SMEDDS were subjected to different stress conditions including heating/cooling, freeze/thaw and centrifugation stresses. During this study, RLGL-SMEDDS was heated to 45 °C and held for 36 h, then cooled to 4 °C and held for 36 h, the formulation was subjected to three such hot and cold cycles. RLGL-SMEDDS was also kept at −20 °C for 36 h and then at 4 °C for 36 h, and the formulation underwent 3 such freeze/thaw cycles. In addition, centrifugal stress studies were performed with the preparation being under centrifugation at 5000 rpm for 30 min. After each stress study, the preparation was examined for phase separation and drug precipitation.

Formulation and Optimization of RLGL-S-SMEDDS

The S-SMEDDS was prepared by solid adsorption method, harnessing one of the water-soluble carriers (PEG 4000, PEG 6000, mannitol and lactose) or the water-insoluble carriers (Sylysiak 320 and Hydrophilic-200 silica).^33,34 The liquid was added drop by drop from the microemulsion to 1 g of adsorbent, ground well while adding. When the powder was slightly moist and each adsorbent material was saturated with adsorption, the amount of liquid self-microemulsion adsorbed was recorded as the maximum adsorption amount of that material. The particle size, PDI and in vitro drug release of different solid self-microemulsions were determined to screen for suitable solid adsorbent carriers.

Characterization of RLGL-S-SMEDDS

Particle Size, Zeta Potential

The samples were diluted with distilled water by gently stirring for 5 minutes, and centrifuged at 1000 rpm for 10 min. The particle size, polydispersity index, and zeta potential were measured by dynamic light scattering using a Brookhaven system (NanoBrook-173Plus, Brookhaven Instrument, USA).

Scanning Electron Microscopy

The morphologies of RLGL, Hydrophilic-200 silica and RLGL-S-SMEDDS were recorded by scanning electron microscope (SEM, Regulus 8100, Japan). Samples were fixed on a brass specimen club using a double-sided carbon conductive adhesive tape and the SEM images were observed at 15 kV accelerating voltage.

In vitro Release Study

The in vitro dissolution test of RLGL (2 mg) and S-SMEDDS (equivalent to 2 mg of drug) was carried out utilizing ZRS-6G basket tablet dissolution tester (Tianda, China). Experiments were carried out in 100 mL of 1% tween 80 dissolution medium at different pH (1.2, 6.8 and 7.4), 100 rpm and 37± 0.5 °C.1 mL was withdrawn from the dissolution medium at different predetermined time points and filtered through 0.22 μm organic filter membrane. The volume withdrawn was replaced with 1 mL of fresh dissolution solution to maintain a constant volume. The samples were suitably diluted and the drug content was determined by HPLC at 291 nm. All data were obtained in triplicate and expressed as a percentage of cumulative drug release.

Powder X-Ray Diffraction

Powder X-ray diffractograms of RLGL, Hydrophilic-200 silica, physical mixtures of RLGL and Hydrophilic-200 silica, and RLGL-S-SMEDDS were recorded using X-ray diffractometer (Bruker, D8 ADVANCE, Germany).The experiment was conducted at room temperature using monochromatic Cu/K-alfa radiation (1.542 Å) and analyzed between 5 and 60° (2θ). The voltage and current used were 40 kV and 40 mA, respectively and the chart speed was 10 mm/sec (2θ).

Flow Properties

The flowability and compressibility of solid self-microemulsion powders were evaluated by measuring three indices: rest angle (θ), Hausner ratio (HR), and Karl index (CI).

A funnel was taken and fixed on a wire stand so that its tip was fixed at a specific height (h) and the mixed solid self-microemulsion sample was poured into the funnel so that the tip of the formed vertebral powder just touches the tip of the funnel and the radius (r) of the powder pile is recorded at this point. Calculate rest angle (θ), tanθ= h /r.

A certain amount of solid self-microemulsion powder sample was taken and put into a 20 mL measuring cylinder and tapped until the volume no longer changed. The volumes before and after tapping were recorded as V_before and V_after respectively. Calculate the Hausner ratio of the sample, HR=V_before /V_after.

The stack density (ρb) of the sample powder was calculated as ρb= mass of powder/ V_before, and the vibrational density (ρt) was calculated as ρt = mass of powder/ V_after. Based on the experimental measurement data, the Karl index (CI) was calculated as CI (%) = (ρt - ρb)/ ρt × 100%.

Cell Studies

Cell Culture

Caco-2 human colon carcinoma cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), while Raji cells were from Procell Life Science&Technology Co.,Ltd. (Wuhan, China). Cells were respectively cultured in Roswell Park Memorial Institute (RPMI) 1640 or Minimum Essential Medium (MEM) with 10% (v/v) Fetal Bovine Serum (FBS), 1% (v/v) penicillin (100 U/mL) and streptomycin (0.1 mg/mL) in 5% CO₂ atmosphere and 95% relative humidity at 37 °C.

Cytotoxicity

The cytotoxicity of Free RLGL, RLGL-SMEDDS and RLGL-S-SMEDDS on Caco-2 cells were investigated by MTT method.^35,36 Caco-2 cells were inoculated in 96-well plates at a cell density of 1×10⁴ well⁻¹ and incubated for 24 h allowing cells to adhere mutually. The cells were exposed to various preparations and incubated at various concentrations for 24 h, respectively. The absorbance value of each well was measured at a wavelength of 490 nm using a microplate reader.

Cellular Uptake

Coumarin 6 (COU) as a fluorescent marker used in cell experiments.^37,38 4×10⁵ Caco-2 cells were seeded in 6-well plates, incubated at 37 °C, 5% CO₂ for 24 h, then the old medium was discarded and the cells were washed twice with PBS. Serum medium diluted COU solution (Free COU) and COU-S-SMEDDS (COU final concentration of 300 ng mL⁻¹) were both seeded in three wells of the same plate and incubated at 37 °C for 2 h. After incubation, the supernatant was aspirated, and the Caco-2 cells were washed 3 times with pre-cooled PBS, trypsinized, centrifuged at 1500 rpm for 5 min, and washed twice with 1 mL PBS, redispersed into 500 μL PBS, and blowed evenly. 300 mesh cells were sieved into a flow tube, and the fluorescence intensity of COU was measured by flow cytometry, Ex/Em=488/530 nm, and no less than 10⁴ cells were analyzed at each time.

Transporter Pathways in Caco-2 Cells

To investigate intestinal transporter pathways, Caco-2 cells were pretreated with efflux inhibitors, including verapamil (10 µg/mL), probenecid (0.2 mm) and elacridar (10 mm), at 37 °C for 1 h. Then, COU, COU-S-SMEDDS were mixed with efflux inhibitors and incubated with the cells for 3 h, respectively. After incubation, intracellular COU was detected by flow cytometry as described above.

The Effect of Excipients on the Intestinal Transporter

To investigate the effect of excipients on the intestinal transporter, Caco-2 cells were incubated with COU-EO, COU-Solutol HS15 and COU-Transcutol HP, respectively.^39,40 The concentration of the excipient is consistent with the prescription concentration and the final COU concentration was 300 ng/mL.

Transcellular Permeation Study

To establish an in vitro Caco-2 cell monolayer model, Caco-2 cells were placed in 12-well transwell plates with 0.4 µm pore polycarbonate film inserts (Corning, NY, USA).⁴¹ For each transwell insert, 0.5 mL of cell culture medium containing 2×10⁵ cells was added in the upper compartment and 1.5 mL of cell culture medium was added in the lower compartment. The medium of both compartments was changed every other day for the first 14 days and daily for the next 7 days. The integrity of Caco-2 cell monolayers was assessed by measuring transepithelial electrical resistance (TEER) and sodium fluorescein permeability. TEER was measured using a Millicell^® ERS-2 voltameter (Bedford, MA, USA). After 21 days of incubation, cells with TEER values exceeding 500 Ω were selected for subsequent transport studies. After modeling, sodium fluorescein was added to the apical to test the membrane permeability.

Where TEER_T is the measured TEER value of the Transwell chamber with cells (Ω); TEER_C is the measured TEER value of the Transwell chamber without cells (Ω); A is the area of the Transwell chamber (cm²).

To study transepithelial transport, 0.5 mL of free COU, COU-S-SMEDDS, COU-EO, COU-Solutol HS15 and COU-Transcutol HP were added to the apical side (AP) and 1.5 mL of PBS acceptor was added to the basal side (BP), similarly, 1.5 mL of these samples were added to the BP side and 0.5 mL of acceptor was added to the AP side. After 3 h, the COU content of the samples was measured by fluorescence spectrophotometer. The apparent permeability coefficient (P_app) of COU was calculated using the following equation:

Where dQ/dt is the permeability rate (µg/s), C₀ is the initial concentration in the donor chamber (mg/mL), and A is the surface area of the filter (cm²), which was 1.12 cm² in this study.

Transport in Lymphocyte

After normal culture of Caco-2 monolayer cells up to 15 days, Raji B cells at a density of 1×10⁶ cells well⁻¹ were added to the basal side of the transwell, and the culture medium on the apical side was changed daily, and that on the basal side was changed every half day.^15,42 The culture continued until the 21st day. Then 0.5 mL of free COU, COU-S-SMEDDS was added to the apical side and 1.5 mL of PBS acceptor was added to the basal side, similarly, 1.5 mL of these samples were added to the BP side and 0.5 mL of acceptor was added to the AP side. COU concentration was detected by method described above after 3 h of incubation at 37 °C.

Pharmacokinetic Study

To further understand the bioavailability of the formulation in vivo, experiments were conducted using Sprague-Dawley (SD) rats. SD rats were purchased from Hunan Slaughter Jingda Laboratory Animal Co., Ltd. (Hunan, China, No.430727220101126967). Female nude mice were placed in a pathogen-free environment with 5 mice per cage. The animals were acclimatized for 4–7 days before experimentation, fed with standard diet and allowed water ad libitum. All animal experiments were approved by the Ethics Committee of Nanchang University in accordance with the Guidelines for the Ethical Review of Laboratory Animal Welfare (GB/T 35892–2018) and ARRIVE 2.0.

Single oral administration: 12 female SD rats (250 ± 20 g) were randomly divided into 2 groups evenly, fasted for 12 h, free of drinking water, and underwent oral gavage of RLGL-suspension and RLGL-S-SMEDDS at a dose of 10.8 mg/kg, respectively. At 0.5, 1, 2, 3, 4, 6, 8, 12 and 24 h after administration, about 0.5 mL of blood was taken from the eyelids and placed in a heparinized EP tube, centrifuged at 10,000 rpm for 10 min, and the supernatant plasma was obtained. The drug concentration was determined by HPLC. DAS 3.0 pharmacokinetic software analysis.

Toxicological Study

After 7 days of PBS, S-SMEDDS, Solutol HS15 and Transcutol HP administration, the heart, liver, spleen, lung and kidney tissues of rats were dissected and fixed in 4% paraformaldehyde solution. The tissues were routinely taken, dehydrated, paraffin embedded, prepared to 4 μm thickness, HE stained, observed by light microscopy and photographed (microscope: Leica MD 1000, imaging system: Leica Application Suite). The pathology of each tissue was observed, photographed and analyzed.

HPLC Analysis

For the determination of RLGL solubility and in vitro drug release study, concentration of RLGL was determined by using HPLC. A 4.6×150 mm (3.5 μm particle size) Agilent Extend-C₁₈ was used. The column temperature was maintained at 40 °C, while the flow rate was maintained at 0.7 mL/min. The mobile phase used water (0.05 mol/L monopotassium phosphate, pH=3) and acetonitrile (68:32[v/v]). Finally, the samples’ concentrations were measured under ultraviolet detection at 291 nm.

Statistical Analysis

All the data are shown as means ± standard deviation (SD) and statistically evaluated using a one-way analysis of variance (ANOVA). P < 0.05 (*), P < 0.01 (**), P < 0.001 (***) and P < 0.0001 (****) were considered statistically significant.

Results and Discussion

Formulation and Optimization of RLGL-SMEDDS

Solubility Studies

Screening oil, surfactant and co-surfactant with maximum solubility potential is beneficial to optimum drug loading. The solubilities of RLGL in various oils, surfactants, and co-surfactants are shown in Figure 1B–D. Among the tested oils, the highest solubility of RLGL was detected in Isopropyl myristate (IPM), followed by Oleic acid and Ethyl Oleate (EO). Therefore, IPM, Oleic acid and EO were chosen as the oil phases for further studies. The three surfactants with the highest solubility for RLGL, namely, Labrasol, Solutol HS15 and Tween 80, were selected for further study. The co-surfactants were ordered as Transcutol HP, Ethanol and Propylene glycol.

Compatibility Tests

Compatibility testing is a key step in the design of formulations. Through the compatibility test, different oils, surfactants and co-surfactants possessing various emulsifying abilities can be compatible with each other in a mixture, which is beneficial to the optimization of the combination of oil, surfactant and co-surfactants.⁴³ By visual grading, IPM and Oleic acid as the oil phase could not form Grade “A” emulsion, indicating that IPM and Oleic acid were inferior in compatibility with surfactants and co-surfactants. When EO was combined with Tween 80 or Solutol HS15, Grade “A”, “B” or “B~C” emulsion can be formed. The best compatibility was produced when Ethyl Oleate was mixed with Solutol HS15. The emulsifying effects of co-surfactants were all good, but ethanol and propylene glycol have certain toxicity and Transcutol HP has the maximum solubility to RLGL. EO is widely used in the food, cosmetics, and pharmaceutical industries with good safety.^44,45 According to reports (Gurjar et al, 2018), Solutol HS 15 has an inhibitory effect on p-gp, and Transcutol HP has the best effect on improving oral bioavailability of RLGL. The above compatibility results once again confirmed that it is a good choice to use EO as the oil phase, Solutol HS15 as the surfactant and Transcutol HP as the co-surfactant.

Construction of Phase Diagrams

As shown in Figure 2A, when Km was 1:9, 2:8, and 3:7, no clear and transparent microemulsion formed, suggesting that the emulsification effect of Transcutol HP was weaker than that of Solutol HS15. When the Km ratio was 9:1, the microemulsion was turbid. The area of microemulsion was smaller at 8:2, indicating that the single emulsifier was not effective. The area of microemulsion formed at Km 7:3 and 6:4 was the largest and equal. However, this range is still too large. In order to obtain the appropriate Km when the amount of surfactant is as small as possible, CCD Experimental design was used to further determine the ratio of each component.

Figure 2 (A) Pseudoternary phase diagrams of the system composed of oil (Ethyl Oleate), surfactant (Solutol HS15), cosurfactant (Transcutol HP) and water. The black regions represent the regions of microemulsion (n=3). (B) Response surface plots (3D) showing the effect of oil (X1) and Km (X2) additions on particle size and drug loading. (C) Size distribution of RLGL-SMEDDS in water. (D) Transmission electron micrograph of RLGL-SMEDDS 50 nm.

Experimental Design

CCD is the most frequently used method for response surface design. In this study, the oil content (X₁) and Km (X₂) were chosen as the two independent variables. A total of 13 experiments were conducted, and the response data for all experimental runs of CCD. The best-fit model was expressed in terms of coded factors as follows:

ANOVA was used to validate the obtained models. It was shown that the p for both particle size and solubility regression equations were less than 0.01, implying a highly significant difference between the experimental models for this test. F was greater than 0.05, which was not significant, indicating that the quadratic term regression equation was a better fit, and that other factors interfered less with the model and the experimental error was relatively small.

As can be seen in Figure 2B, with the proportion of fixed oil phase in a certain range, the particle size increases slightly with the increase of Km, and the value of fixed Km increases with the increase of the proportion of oil phase. When the ratio of fixed oil phase was in a certain range, the drug loading increases slightly with the increase of Km while the value of fixed Km decreases with the increase of the ratio of oil phase. Overall, considering the side effects of a large number of surfactants and co-surfactants, the optimal formulations were as follows: Ethyl Oleate at 26% (w/w), Solutol HS15 at 49% (w/w), and Transcutol HP at 25% (w/w). The prediction error is less than 5%, indicating that the actual response is very close to the predicted value.

Characterization of RLGL-SMEDDS

In SMEDDS, the efficiency of self-emulsification can be assessed by measuring the emulsification rate and particle size distribution.⁴⁶ The latter is a key factor in determining the rate and extent of drug release and absorption. The particle size of RLGL-SMEDDS is 23.84 ± 0.43 nm (Figure 2C), PDI is less than 0.1, Zeta potential is −8.15 ± 3.63 mV, and self-emulsification time is less than 30s. Figure 2D shows the transmission electron microscope image of RLGL-SMEDDS. The results show that RLGL-SMEDDS is regular spherical or quasi-spherical with a size of about 25 nm, which is consistent with the particle size measurement of uniform distribution and good dispersion.^47–49

After oral administration, SMEDDS is expected to gradually dilute upon contact with gastrointestinal fluids. Therefore, the stability of SMEDDS to dilution was monitored by diluting it 50, 100, and 250 times with distilled water as well as pH 1.2 and pH 6.8 buffers. It was observed that all the emulsions obtained were clarified and transparent without drug precipitation and phase separation, demonstrating their robustness to dilution. This also indicates the possibility of uniform release of the drug in vivo, in which case the formulation is expected to dilute gradually.

The cloud point is an important parameter for evaluating the stability of SMEDDS formulations.⁵⁰ The formulation is cloudy due to phase separation and drug precipitation, which can also hinder drug release. The cloud point temperature of the formulations was determined as in the range of 75–80 °C. This not only indicates a good thermal stability of the formulation but also gives an indication of unimpeded drug release.

Thermodynamic stability refers to the kinetic stability of the formulation, and the self-microemulsion should be able to tolerate various conditions in the gastrointestinal tract. The absence of precipitation and phase separation observed after heating/cooling, freeze/thaw and centrifugation stresses indicates good stability. The stability of RLGL-SMEDDS was good after 7 days at 4 °C. Precipitation was not observed, and particle size and PDI did not change significantly. This means that RLGL-SMEDDS can maintain good stability under these conditions.

Formulation and Optimization of RLGL-S-SMEDDS

Figure 3A shows the adsorption capacity of SMEDDS by different carriers, and that by water-soluble carriers is generally small. Then, the drug release in vitro was investigated. S-SMEDDS prepared by the water-soluble carrier could be completely released in water.

Figure 3 (A) Solid carrier adsorption capacity (n=3). (B) In vitro release of RLGL under different pH conditions (n=3). In vitro release of Hydrophilic-200 silica (C) and sylysiak 320 (D) under different pH conditions (n=3).

As shown in Figure 3B, the solubility of RLGL is pH dependent. 17% can be dissolved in 12 h at pH 1.2, while less than 5% can be dissolved in 12 h at pH 6.8 and pH 7.4. As shown in Figure 3C, the release of RLGL from solid formulations is significantly higher than that of RLGL. As for RLGL, there is a higher release of up to 90% at pH 1.2 for 5 min and 80% at pH 6.8 and 7.4 for 5 min. Solid self-microemulsion in contact with water results in rapid formation of O/W microemulsion. Because the drug is already dissolved in the mixture, the usual rate-limiting dissolution step required for crystallizing drugs is avoided.

As shown in Figure 3D, the two water-insoluble carriers released more than 80% within 5 minutes in different media. Therefore, two water-insoluble carriers with larger adsorption capacity were selected. After the two carriers were redispersed in water, it was discovered that the particle size was similar and the PDI was small. In conclusion, Hydrophilic-200 silica was selected as the solid adsorbent.

Characterization of RLGL-S-SMEDDS

Scanning electron microscopy showed the morphology of the various solid formulations (Figure 4A). The RLGL as an active pharmaceutical ingredient shows a crystalline structure, Hydrophilic-200 silica is a collection of particles with many pores on the surface. After being adsorbed, the pores on the surface of Hydrophilic-200 silica disappeared and no obvious RLGL bulk structure was observed, which indicated that the SMEDDS was adsorbed onto Hydrophilic-200 silica by the adsorption phenomenon.

Figure 4 (A) Scanning electron micrographs of RLGL, Hydrophilic-200 silica and RLGL-S- SMEDDS. (B) Power X-ray diffraction of RLGL, Hydrophilic-200 silica, Physical mixture of RLGL and Hydrophilic-200 silica, and RLGL-S-SMEDDS. (C) Cell viabilities of Caco-2 cells after 24 h treatment with free RLGL, S-SMEDDS, Transcutol HP and Solutol HS15 at equivalent doses of RLGL (n=3).

As shown in Figure 4B, the high crystallinity of RLGL is reflected in the diffraction angles of 7.4°, 8.9°, 9.9°, 12.1°, 16.6° and 17.3°. These characteristic peaks are still observed in physical mixture and absent in S-SMEDDS, indicating that the physical state of RLGL is completely transformed from the crystalline to the amorphous state in the S-SMEDDS formulation.

S-SMEDDS has good flow characteristics, angle of repose (33.7°-34.2°), Hausner ratio (1.15–1.18), and compressibility (13.3–15.5%). From these results, it can be concluded that S-SMEDDS have good flow characteristics and compression properties, which contribute to the production of tablets with good cohesive properties and content uniformity.

Cytotoxic Studies

The Caco-2 cell monolayer model was chosen for this study. As an in vitro model, it is reliable and approved by the FDA and pharmaceutical companies for studying the cytotoxicity and behavior of developed agents drugs in the intestine. MTT results are shown in Figure 4C. RLGL, solid formulation and surfactant Transcutol HP, Solutol HS15 showed a better safety profile after 24 h incubation with Caco-2 cells within 0.1–20μg/mL concentrations (cell survival rate >90%). The excipients used in this prescription are relatively safe. They have been reported to be widely used in the food, cosmetic or pharmaceutical industries, which is consistent with the results of this experiment. Therefore, the present formulation has a good biosafety profile.

Cellular Uptake

The results of the flow cytometry experiment are shown in Figure 5A. The uptake of COU-S-SMEDDS is three times higher than that of free COU. This may be due to the rapid dispersion of the solid formulation in water to form O/W microemulsions with very small particle size, which penetrate the cell membrane more easily, and the surfactant in the prescription inhibits the efflux of P-gp and reduces the pumping of the drug. The exact mechanism needs to be further investigated.

Figure 5 (A) Flow cytometric quantification of intracellular uptake of COU-S-SMEDDS and Free COU by Caco-2 cells after 3 h incubation at 37 °C (n=3). (B) Flow cytometric quantification of intracellular uptake of COU, COU+Elacridar, COU+Verapamil and COU+Probenecid in Caco-2 cells (n=3). (C) Flow cytometric quantification of intracellular uptake of S-SMEDDS, S-SMEDDS +Elacridar, S-SMEDDS+Verapamil and S-SMEDDS+Probenecid in Caco-2 cells (n=3). (D) Flow cytometric quantification of intracellular uptake of COU, COU + Ethyl Oleate, COU +Transcutol HP and COU+Solutol HS15 in Caco-2 cells (n=3). Statistical difference between groups: p > 0.05 ns and p < 0.01**.

Efflux Pathways in Caco-2 Cells

To better understand the mechanism of cellular efflux in drug, the specific efflux protein inhibitors verapamil, probenecid and elacridar were used to inhibit P-glycoprotein, multidrug resistance-associated protein 2 and breast cancer resistance protein, respectively. As shown in Figure 5C, the uptake of free COU pretreated with verapamil and probenecid was significantly higher than that of untreated free COU. The addition of verapamil and probenecid inhibited efflux proteins, confirming that the efflux of Caco-2 cells involves the P-gp and multidrug resistance-associated protein-2 pathways. Elacridar had no effect on cellular uptake.

The Effect of Excipients on the Intestinal Transporter

These were incubated S-SMEDDS with Caco-2 cells after pretreatment with three efflux protein inhibitors. The uptake results are shown in Figure 5B, with no significant difference between with and without efflux protein inhibitors. This result may be explained by that it is the surfactant that acts as an efflux protein inhibitor. We co-incubated the prescribed Ethyl Oleate, Transcutol HP and Solutol HS15 with COU, and the experimental results are shown in Figure 5D that no significant change in uptake was observed when COU was co-incubated with Transcutol HP and Ethyl Oleate, and a significant increase in uptake was observed when COU was co-incubated with Solutol HS 15. This suggests that Solutol HS 15 can effectively inhibit the intestinal transporter to increase drug uptake.

Transport Within Caco-2 Cell Monolayers

The cell monolayer with a resistance above 550 Ω was finally selected for the experiment by measuring the transmembrane resistance. As shown in Figure 6A, S-SMEDDS showed a significant increase in transport from AP-BP compared to free COU, along with a slight increase in transport from BP-AP. As shown in Figure 6B, free COU transport was increased after administration with Solutol HS 15 while no significant change was observed after co-administration with EO and Transcutol HP, which was due to the inhibition of intestinal transporter by Solutol HS15. However, the transport of free COU was less efficient than S-SMEDDS because the solid formulation formed an O/W microemulsion with water, and the small particle size further enhanced the transport. This is consistent with the results of our cellular uptake experiments.

Figure 6 (A) the apparent permeability coefficient (Papp) of COU and S-SMEDDS. (B) the apparent permeability coefficient (Papp) of COU with Solutol HS15, Ethyl Oleate and Transcutol HP (n=3). (C) Transmembrane resistance for Caco-2 cell monolayer and co-culture model with lymphocytes for 21 days (n=3). (D) The apparent permeability coefficient (Papp) of COU and S-SMEDDS in Caco-2 cells monolayer and co-culture model with lymphocytes (n=3). Statistical difference between groups: p > 0.05 ns and p < 0.001***.

Transport in Lymphocyte

The intestinal cells in the intestine are surrounded by the lamina propria and are connected to abundant capillaries and lymphatic vessels. Once the drug crosses the intestinal epithelium, it can be absorbed through the lymphatic vessels. Drugs transported through the intestinal lymphatic system enter the body’s circulation without first passing through the liver which would enhance oral bioavailability. To investigate whether S-SMEDDS can be absorbed by lymphatic vessels, we established a Caco-2 cell monolayer co-cultured model with lymphocytes. As shown in Figure 6C, compared to the normal Caco-2 cell monolayer, the transmembrane resistance of co-culture model decreased after the addition of lymphocytes and eventually to 2/3 of that of the normal cell monolayer. This implies that the tightly connected cell monolayer was opened and the cell model was successfully established. As shown in Figure 6D, the apparent permeability coefficient of S-SMEDDS was significantly increased in the co-culture model, indicating that the preparation was available for uptake by lymphocytes.⁵¹ There was also an increase in uptake from the BP-AP side because the tight cell monolayer was opened.

Pharmacokinetic Study

The main pharmacokinetic parameters of RLGL-suspension and RLGL-S-SMEDDS obtained by non-compartment mode are shown in Table 1 and Figure 7A. At the same RLGL dose, the maximum plasma concentration (Cmax) of the RLGL-S-SMEDDS administration group was 3.2 times higher than that of RLGL suspension, and the area under the concentration-time curve (AUC_0-∞) of the RLGL-S-SMEDDS administration group was 1.9 times higher than that of RLGL suspension, with no significant differences in other parameters. The reasons for the improved oral absorption and bioavailability of RLGL-S-SMEDDS may be the following: improved bioavailability through intestinal lymphatic transport avoiding the overtake effect, improved solubility of RLGL owing to S-SMEDDS, allowing drug remaining dissolved, the absorption-promoting effect of oil and surfactant, Solutol HS15 inhibiting intestinal transporter to improve absorption,58 and the small particle size of O/W-type microemulsion formed by S-SMEDDS avoiding phagocytosis by the reticuloendothelial system to prolong systemic circulation time.^52–59 Therefore, it can be concluded that S-SMEDDS can effectively improve the oral bioavailability of RLGL.

Table 1 Main Parameters of RLGL-Suspensions and RLGL-S-SMEDDS in vivo Pharmacokinetics (Mean ± SD, n=6)

Figure 7 (A) Blood concentration-time profiles after oral administration of RLGL-S-SMEDDS and RLGL-suspensions (n=6). (B) Histological examination of heart, liver, spleen, lung and kidney tissues from animals treated with PBS, Solutol HS15, Transcutol HP and S-SMEDDS by hematoxylin and eosin (H&E) staining, scale bar 200 mm.

Toxicology Studies

The results of pathological examination on organs in each group are shown in Figure 7B. The results of all groups were similar to that of the PBS group, and no significant pathological changes were found in the heart, liver, spleen, lung and kidney. Based on the above findings, it can be confirmed that the surfactant has good biosafety at the administered dose and the solid self-microemulsion is non-toxic.

Conclusion

In the study, RLGL-S-SMEDDS was prepared and evaluated. It has uniform particle size distribution, good thermodynamic and kinetic stability and qualified powderological properties. The in vitro drug release for RLGL-S-SMEDDS was significantly enhanced compared to that for free RLGL. In vitro studies showed that the uptake of RLGL-S-SMEDDS by Caco-2 cells was three times higher than that of free RLGL by Caco-2 cells; in vivo studies indicated that RLGL-S-SMEDDS had a higher area under the drug-time curve than the RLGL-suspensions. The in vivo cytotoxicity assay and in vitro HE staining analysis showed that the various excipients have good biosafety and the solid self-microemulsions are non-toxic. In conclusion, the S-SMEDDS prepared in this study can improve oral bioavailability of RLGL and is biosafe, which is expected to provide a new idea and method for clinical application.