A self-microemulsion enhances oral absorption of docetaxel by inhibiting P-glycoprotein and CYP metabolism

Oral absorption of docetaxel was limited by drug efflux pump p-glycoprotein (P-gp) and cytochrome P450 enzyme (CYP 450). Therefore, co-loading agent that inhibits P-gp and CYP450 in self-nanoemulsifying drug delivery systems (SMEs) is considered a promising strategy for oral delivery of docetaxel. In this study, curcumin was selected as an inhibitor of P-gp and CYP450, and it was co-encapsuled in SMEs to improve the oral bioavailability of docetaxel. SMEs quickly dispersed in water within 20 s, and the droplet size was 32.23 ± 2.21 nm. The release rate of curcumin from DC-SMEs was higher than that of docetaxel in vitro. Compared with free docetaxel, SMEs significantly increased the permeability of docetaxel by 4.6 times. And competitive experiments showed that the increased permeability was the result of inhibition of p-gp. The half-life and oral bioavailabilty of DC-SMEs increased about 1.7 times and 1.6 times than docetaxel SMEs, which indicated that its good pharmacokinetic behavior was related to the restriction of hepatic first-pass metabolism. In conclusion, DC-SME was an ideal platform to facilitate oral delivery of docetaxel through inhibited P-gp and CYP 450.


Introduction
Oral administration has become the first choice in clinics due to its safety and convenience, while chemotherapy drugs are still mainly through intravenous route. The main limitations of oral chemotherapy are poor solubility and permeability, especially for BCS IV anti-tumor drugs [1]. Therefore, how to improve poor oral bioavailability is the key to the development of oral chemotherapeutic agents.
Docetaxel as the first-line anti-tumor agent was currently used for prostate cancer and non-small-cell lung cancer (NSCLC) [2,3]. The recommended dose schedule was intravenously administered docetaxel every 3 weeks, which was restricted by myelosuppression in clinics. Although weekly docetaxel was less toxic [4], it was rarely used in practice because of its inconvenience to patients. To address these challenges, docetaxel oral solid dispersion formulations have been developed and evaluated in clinical trials, including ModraDoc001 capsule and Modra-Doc006 tablet [5,6]. Unfortunately, limited oral bioavailability caused therapeutic failures. In recent years, the development of nanotechnology has greatly improved the solubility and toxic side effects of drugs, such as PLGA nanoparticles [7], solid lipid nanoparticles [8], mesoporous silica nanoparticles (MSNs) [9], mesoporous carbon nanoparticles (MCNs) [10], and self-nanoemulsifying drug delivery systems (SMEs) [11]. Among these nanosystems, SME was considered an ideal oral drug delivery strategy because of its high solubilization potential and thermodynamic stability [12]. SME was a lipid-based oral drug delivery system, which was composed of oil, emulsifier, and co-emulsifier. When exposed to an aqueous environment of the gastrointestinal tract (GI), a water-containing (O/W)-type microemulsion will be automatically formed. Thus, the drug molecules within SMEs not only enhanced solubility and membrane permeability, but also the stability Le Tong and ZeYang Zhou contributed equally. against the GI environment. SMEs have been applied to a series of drugs, including docetaxel and nifedipine [11,13]. However, oral absorption of SMEs loaded with docetaxel was still hampered by P-gp and CYP450 [5]. To address these issues, co-administration of the CYP450 inhibitor ritonavir was a feasible scheme. However, different pharmacokinetic behaviors and dose-limited toxicity (diarrhea) in a phase I clinical trial affected the development of oral docetaxel [14]. Cui et al. [15] reported novel SMEs for the oral co-delivery of docetaxel and cyclosporine A (CsA), which strongly enhanced the oral bioavailability of docetaxel. Although CsA has great potential in improving the oral absorption of docetaxel, its use in clinical applications still faced great challenges due to its acute nephrotoxicity, severe hypertension, and neurotoxicity [16,17]. Curcumin, a natural polyphenol, exhibits several pharmacological effects including anti-oxidant and anti-inflammatory [18]. Several studies have demonstrated that curcumin was a potent inhibitor of P-gp and has been frequently used for reversing P-gp-mediated efflux [19]. Some pieces of evidence suggested that the combination of curcumin can effectively enhance the treatment of docetaxel [20,21]. Curcumin was also reported as an effective inhibitor of CYP450 and the IC50 of CYP3A4 was 11.93±3.49 μM [22]. Thus, the down-regulation of intestinal CYP3A in vivo could significantly improve the oral bioavailability of drugs with strong first pass metabolism. Curcumin was generally considered to be a safe compound for human beings, even if the oral doses of 8 g/day for 3 months [23]. However, instability, strong hydrophobicity and rapid metabolism in the GI tract resulted in a low oral bioavailability of curcumin. Thus, we hypothesized that encapsulation of docetaxel and curcumin into SMEs could improve the oral bioavailability and therapeutic effect.
Herein, we reported unique co-loaded SMEs for efficient oral delivery of docetaxel and curcumin. The prepared SMEs were optimized by simplex lattice method analysis and characterized by the droplet size, zeta potential, morphology, stability, and in vitro drug release. The permeability of SMEs was studied by using Caco-2 cell monolayer. Finally, a pharmacokinetics study was conducted in rats to evaluate the oral bioavailability of co-loaded SMEs. In this study, we demonstrated that co-loaded SMEs significantly increased the oral bioavailability of docetaxel by inhibiting P-gp and CYP450 enzymes.

Solubility of docetaxel (D) and curcumin (C)
The solubilities of docetaxel and curcumin were measured according to previous reports [13]. Briefly, excessive docetaxel and curcumin were added into various oils, surfactants, and co-surfactants, and then the mixtures were shaken at 100 rpm under 37 °C for 72 h. Thereafter, the undissolved drug was separated by centrifugation at 12,000 rpm for 15 min. The contents of docetaxel and curcumin were determined by HPLC.

Construction of pseudo-ternary-phase diagram (PTPD) and preparation of DC-self-microemulsions (DC-SMEs)
In order to optimize the microemulsion region (MA), a pseudo-ternary-phase diagram was constructed by mixing water, oil, surfactant, and co-surfactant [24]. In brief, MCT was employed as the oil phase, and EL40 and DGME were selected as surfactant and co-surfactant. The weight ratio of oil to surfactant was from 9:1 to 5:5. And the weight ratio of surfactant to co-surfactant was from 9:1 to 1:9. The mixtures were titrated dropwise into deionized water at 37 °C with stirring of 300 r/min, and the phase behavior of the pseudo-ternary system (clarity and flowability) was monitored. When the mixture becomes cloudy and phase separation was observed, the titration end point is set. Based on these results, the MA was labelled in the ternary phase diagram using OriginPro 2019C 64-bit software.
Based on PTPD results, simplex lattice method analysis was used to optimize the ratio of the oil phase, surfactant, and co-surfactant. The percentage of oil phase (X 1 ), surfactant (X 2 ), and co-surfactant (X 3 ) were set as independent variables. The particle size (Y 1 ; nm) and drug (docetaxel and curcumin) loading efficiency (Y 2 , Y 3 ; mg/g) were set as response variables. The percentage of independent variables of X 1 was in the range of 10-25%, X 2 was in the range of 45-60%, and X 3 was in the range of 30-45%. The results were analyzed by using the software Design-Expert 8.0.6. The fitted polynomial equations were drawn in 3D response surfaces.

Particle size, zeta potential, and morphology of DC-SMEs
After being diluted 100 times with distilled water, the particle size and zeta potential of DC-SMEs were measured by a dynamic light scattering (DLS) particle size analyzer (NanoZSE, Malvern Instruments Ltd., UK). The morphology of the DC-SMEs formulation was observed by a transmission electron microscope (TEM, Tecnai 12, Philips, Holland).

Encapsulation efficiency (EE) and drug loading (DL)
Ultra-filtration method was used to determine the entrapment efficiency of DC-SMEs. Briefly, an aliquot (1 ml) of DC-SMEs was placed in an ultra-filtering centrifuge tube, and centrifuged at high speed (10,000 rpm) for 10 min to separate the free drug from DC-SMEs. Then, the free drugs were determined by HPLC, and the EE of docetaxel and curcumin were calculated by using the following formula: (where C Free refers to the free of docetaxel or curcumin in DC-SMEs, and C total refers to the total input amount in the preparation. In addition, V represents the volume of DC-SMEs; W represents the amount of ingredients. All samples were performed in triplicates.)

Stability of DC-SMEs
In order to evaluate the validity period of DC-SMEs, stability experiments were carried out. The DC-SMEs were stored at 4 °C for 30 days. After dilution with water, the droplet size and polydispersity index (PDI) of DC-SMEs were measured on day 0, day 5, day 10, day 15, and day 30. In addition, we also studied the stability of self-microemulsions in pH 1.2 and pH 6.8 buffer solutions.

In vitro drug release of DC-SMEs
In vitro release of DC-SMEs was determined by the dialysis method. In brief, free docetaxel and curcumin and DC-SMEs were injected into dialysis bags (8000 ~ 14,000 Da) and placed in a 250 ml pH 1.2 and pH 6.8 phosphate buffer containing 20% DMSO. Then, a series of samples were collected at 0.5, 1, 2, 4, 8, and 12 h. The concentrations of docetaxel and curcumin were analyzed by the HPLC method. All the experiments were carried out in triplicate.

Cytotoxicity study of DC-SMEs
The cytotoxicity of DC-SMEs in Caco-2 cells in vitro was determined by MTT assay. Briefly, cells were seeded in 96-well plates with 1 × 10 5 cells per well in 200 µl of complete medium. After 72 h exposure to the DC-SMEs at 37 °C, cell viability was determined by MTT assay. All experiments were carried out in triplicate.

Caco-2 cell permeability of DC-SMEs and the contribution of P-gp protein
The permeability of DC-SMEs was evaluated by the transcellular transport experiment using Caco-2 monolayer. Briefly, Caco-2 cells were seeded on a 12-well transwell insert at the density of 1.0 × 10 5 cells/well for 21 days. Transepithelial resistance (TEER) increased by more than 250 was used for the permeability experiment. The permeability was determined on both the apical side and basolateral side. After incubation with the drug solutions (docetaxel and curcumin) or D-SMEs and DC-SMEs for 30 min, the samples were collected at 37 °C at 15, 30, 45, 60, 90, and 120 min from the apical side or basolateral side of the plate. All the experiments were conducted in triplicate. The concentration of docetaxel was measured by the UPLC-MS/MS method, and the apparent permeability coefficient (P app ) was calculated by the following formula: where dCr/dt is the steady-state permeability rate (cm/s), Vr is the receiver volume, A is the diffusion area of the monolayer (cm 2 ), and C 0 is the initial concentration of docetaxel. The concentrations were determined by the UPLC-MS/MS method.
In order to evaluate the role of P-gp protein in permeability, a competitive inhibition experiment was performed. Briefly, Caco-2 cells' monolayer was incubated with DC-SMEs or a mixture of curcumin and docetaxel at 37 °C for 45 min. And sterile PBS with or without verapamil as a control. After washing with cold PBS, the Caco-2 cells were homogenized with PBS. The samples were collected and centrifuged at 13,000 rpm for 10 min, and the supernatant was analyzed by UPLC-MS/MS, and P app was calculated by the formula.

In vivo pharmacokinetic studies of DC-SMEs
The experimental plan was in line with institutional guidelines and approved by the Animal Health and Use Committee of the Guang Xi University of Chinese Medicine.
Wister rats were randomly divided into two groups (n = 4), and were given free docetaxel or DC-SME orally (with a docetaxel equivalent dose of 20 mg/kg), respectively. Aliquots of blood samples were collected at 0.5, 1, 2, 3, 4, 6, 8, 12, and 24 h, then transferred to heparinized tubes (10 mg/ml) and centrifuged at 12,000 r for 3 min. In order to calculate the absolute bioavailability, a physical mixture of curcumin and docetaxel was administered to rats by intravenous injection at a dose of 2 mg/kg. Aliquots of blood samples were collected at 2, 5, 10, 20, and 30 min and 1, 2, 4, 8, 12, and 24 h. The supernatant was kept at − 20 °C until it was analyzed. The absolute bioavailability was calculated by the following equation: where F refers to the absolute bioavailability, AUC po refers to the area of oral, D iv refers to the dose of intravenous, AUC iv refers to the area of intravenous, and D po refers to the dose of oral.

Statistical analysis
Statistical analysis was conducted using the Bonferroni t-test after ANOVA for group comparison and the Student t-test for two-group comparison at the p < 0.05 level. Results are expressed as mean ± standard deviation (SD) from at least three individual samples.

Construction of pseudo-ternary phase diagram (PTPD) and preparation of DC-self-microemulsions (DC-SMEs)
SME is a clear and isotropic system composed of oil, surfactant, and co-surfactant.
When exposed to the water environment of the gastrointestinal tract (GI), the ternary phase of SMEs spontaneously emulsified, forming an oil-in-water nanoemulsion with a droplet size of 10-250 nm. Therefore, it is very important to detect the solubility of drugs in three phases. Based on the results of solubility, we found that MCT, cremophor EL40, Tween 80, Tween 20, DGME, and PEG 400 were the most effective in developing SMES because of their high solubility of docetaxel and curcumin (Table S1).
Surfactants and co-surfactants can be freely distributed between the oil and water phase as a modifier to reduce the interfacial tension and increase the stability of the emulsion, which are beneficial to the formation of the SMEs. In order to select appropriate co-emulsifiers and emulsifiers, a PTPD was plotted in the absence of curcumin and docetaxel. When MCT was used as the oil phase, the PTPDs of the different co-emulsifiers and emulsifiers are shown in Fig. 1a-f.
Comparing the microemulsion area of the three-group data, it was found that the microemulsion area with PEG400 as a co-emulsifier was larger than that with DGME as a coemulsifier, so PEG400 was selected as a co-emulsifier. It is well known that the hydrophilic-lipophilic balance (HLB) value is an important index for the selection of surfactants. The high HLB value indicates that the emulsifying ability of the emulsifier is strong. Although cremophor EL40, Tween 80, and Tween 20 could form O/W microemulsions with the same co-emulsifier (PEG400 or DGME), there was quite a difference in their emulsification ability. As shown in Fig. 1, cremophor EL 40 exhibited a higher microemulsion area than Tween 80 and Tween 20, so it was selected as an emulsifier. And the HLB of Tween 20 (HLB16.7) was higher than cremophor EL40 (HLB15) and Tween 80 (HLB15) [25]. These results indicated the emulsification ability of surfactant is closely related to HLB and solubility. In the case of high drug solubility, the larger HLB of the surfactant is beneficial for SMEs.
Based on the results of PTPD, simplex lattice method analysis (SLMA) was used to optimize the ratio of the oil phase, co-emulsifiers, and emulsifiers. Design-Expert 8.0.6 software was used to analyze the interaction between the independent variables and response variables. 3D response surfaces are plotted in Fig. 2. The polynomial regression equation was calculated as follows: Particle size: Drug loading of curcumin: Drug loading of docetaxel: The p values are lower than 0.05, and the model fits well. According to the optimization result, the optimal percentage of oil for DC-SMEs was 10.0% (w/w) and the emulsifiers and co-emulsifiers were 52.5% (w/w) and 37.5% (w/w), respectively. As shown in Table S2, there is no significant difference between the predicated and measured value of particle size and drug loading (the deviation was less than 2%), which indicated that the obtained fitting equation could accurately describe the relationship between the independent variables and response variables [12]. Figure 2 shows that with the increase of cremophor EL 40 and PEG400, particle size decreases. By contrast, the drug loading of curcumin and docetaxel gradually increased with the increase of cremophor EL 40 and PEG 400, and then decreased slightly.

Characterization of DC-SMEs
After being diluted 100 times with deionized water, the droplet size, zeta potential, and TEM image of DC-SMEs were measured and the results are shown in Fig. 3. It was observed that SMEs quickly dispersed in water, pH 6.8 and pH 1.2 buffer within 20 s, and the appearance of microemulsion was light yellow. The DLS results showed that the droplet size and zeta potential of DC-SMEs were 32.23 ± 2.21 nm and − 16.25 ± 3.72 mv, respectively. These results indicated that prepared SMEs had an excellent self-nanoemulsifying ability. TEM images clearly showed that many spherical microemulsion droplets are formed after hydration, and they are spherical with a diameter of 30 nm, which was in good agreement with DLS. Generally speaking, the droplet size of SMEs smaller than 300 nm is considered to be more suitable for endocytosis [26]. Thus, the optimal particle size of 30 nm will show excellent intestinal permeability and oral bioavailability. In addition, the stability results showed that after 30 days of storage at 4 °C and incubated with pH 6.8 and pH 1.2 buffer for 15 h, there was no obvious change in the appearance, particle size, and PDI, which indicated that DC-SMEs had good stability ( Fig. 3 and Fig. S1). The encapsulation rate of curcumin and docetaxel was determined by the ultra-filtration centrifugation method. The encapsulation rate of curcumin and docetaxel is 98.26% ± 0.02% and 97.65 ± 0.04%, respectively. The results showed that curcumin and docetaxel could be successfully incorporated into the self-microemulsion system.
In vitro release experiment of DC-SMEs was performed in pH 6.8 and pH 1.2 phosphate buffer containing 20% DMSO, which was the sink condition of docetaxel. After 12-h incubation in pH 6.8 buffer, the cumulative release rates of docetaxel and curcumin were 69.2 ± 8.9% and 78.0 ± 3.1%, respectively (Fig. 4). In contrast, docetaxel and curcumin showed similar cumulative release behaviors at pH 1.2. The steady release behavior indicated that there is a strong interaction between drugs and SMEs, which may provide protection for drugs in the gastrointestinal tract. Compared to DC-SMEs, D-SMEs showed a similar release pattern of docetaxel, which indicated that co-loading curcumin did not interfere with docetaxel release. Notably, curcumin exhibited a higher release rate than docetaxel in DC-SMEs. The released curcumin may Fig. 1 PTPD of various surfactants, co-surfactants, oil, and water. a MCT/cremophor EL40/DGME system; b MCT/cremophor EL40/ PEG400 system; c MCT/TW80/DGME system; d MCT/TW80/ PEG400 system; e MCT/TW20/DGME system; f MCT/TW20/ PEG400 system. Microemulsion regions of the ternary plot are expressed in dots 1 3 inhibit p-gp, which was beneficial to the absorption of the released docetaxel, thus improving oral bioavailability. Therefore, co-encapsulation of docetaxel and curcumin in SMEs was a potential platform for promoting drug dissolution, release, and absorption.

Cytotoxicity study of DC-SMEs
In order to evaluate the cytotoxicity of DC-SMEs, Caco-2 cells were used to carry out an MTT assay. As shown in Fig. 5, no obvious cytotoxicity was observed in the presence of docetaxel, curcumin, and DC-SMEs ranging from 1 to 20 μM, which was used in the following permeability evaluation.

Caco-2 cell permeability of DC-SMEs and the contribution of P-gp protein
The membrane permeability was determined by calculating P app using a Caco-2 monolayer, and the results are Fig. 3 a, b Appearance of DC-SMEs; c morphology of DC-SMEs; d, e droplet size and zeta potential distribution of DC-SMEs; f the particle size and zeta potential of DC-SMEs storage at 4 °C for 30 days (n = 3) Fig. 4 The cumulative release profile of DC-SMEs and D-SMEs in pH 6.8 (a) and pH 1.2 (b) phosphate buffer containing 20% DMSO (n = 3) shown in Fig. 6 In the free docetaxel group, the P app was (0.3 ± 0.05) × 10 −6 cm/s, which suggested a poor permeability of docetaxel. By contrast, loading docetaxel in D-SMEs increased the permeability of docetaxel by 1.8 times, which indicated the larger surface area of nanoparticles promoting drug release and transmembrane. Although D-SMEs exhibited better permeability than free docetaxel, the P app of D-SMEs was significantly higher in the B-A direction than in the A-B direction ((1.1 ± 0.08) × 10 −6 cm/s vs (2.4 ± 0.13) × 10 −6 cm/s) [27]. These results indicated that D-SMEs can not overcome the efflux effect of the P-gp. In order to reduce the efflux effect of P-gp, a typical inhibitor verapami [28] (or curcumin [19]) was co-incubated with docetaxel. The results suggested the P app of docetaxel was increased by 4.6 and 3.8 times in the presence of verapami and curcumin. By contrast, DC-SMEs showed a similar P app, which indicated that the improvement in permeability of DC-SMEs was due to the inhibition of P-gp by curcumin. Therefore, loading p-gp inhibitor in SMEs is an effective strategy to overcome drug resistance.

In vivo pharmacokinetic (PK) studies of DC-SMEs
A comparative pharmacokinetic study was conducted to evaluate the oral absorption of free drugs and SMEs. After the oral dose of 20 mg/kg, the plasma concentration-time curves and the pharmacokinetic parameters of docetaxel, D-SMEs, and DC-SMEs were compared. As shown in Fig. 7, D-SMEs prolonged the in vivo half-life of the free docetaxel by 3.7-fold compared with free docetaxel, indicating the sustained release of SMEs. Compared with free docetaxel, the AUC 0-24 h of D-SMEs was increased by 3.1 times, which suggested the SMEs could improve the solubility and release of docetaxel, and thus improve its oral bioavailability. However, the efflux effect of P-gp significantly decreased the oral absorption of D-SMEs.
In contrast, the parameters AUC 0-24 and C max of DC-SMEs were increased by 1.6 times and 1.5 times compared to D-SMEs, respectively ( Table 1). The absolute bioavailabilities of docetaxel increased from 0.9% in the free drug to 2.9% in D-SMEs and 4.8% in DC-SMEs. These results suggested DC-SME was a potent platform to improve the oral bioavailability of docetaxel. In addition, the AUC 0-24 of curcumin was 784.7 ± 61.0 μg h L −1 , which indicated its excellent release from DC-SMEs in vivo. Combined with the results of in vitro release experiment, the cumulative release of curcumin in pH 6.8 buffer was more than 70%, and its release rate was superior to that of docetaxel. In addition, cremophor EL reduced the outflow of P-gpmediated substrate in the previous report [29]. Therefore, we proved that the co-delivery of curcumin in DC-SMEs could inhibit the p-gp protein, thus improving the oral bioavailability of docetaxel.
Oral chemotherapy with docetaxel is also restricted by its hepatic first-pass metabolism. As shown in Table 1, the t 1/2 of DC-SMEs was 1.7 times higher than that of D-SMEs, which indicated the co-loading of curcumin could limit the CYP metabolism of docetaxel [22]. Importantly, co-loaded DC-SMEs showed a long-term high plasma concentration of more than 120 ng/ml for about 25 h (Fig. 7). The results indicated that high oral bioavailability of DC-SMEs was related to the inhibitory effect of CYP 450.

Conclusion
In summary, docetaxel and curcumin were successfully coencapsulated into SMEs with a rapid self-nanoemulsifying rate. Optimized SMEs with a size of 30 nm showed excellent stability within 30 days and steady sequential release behavior. More importantly, the DC-SMEs significantly improved the solubility, permeability, and oral bioavailability of docetaxel by simultaneously limiting the efflux of P-gp and the first-pass metabolism of the liver. Therefore, DC-SMEs exhibited great potential in novel oral docetaxel development.
Author contribution These authors contributed equally to this work.
Funding This work was financially supported by the National Nature Science Foundation of China (No. 82060641), and the China postdoctoral fund (2019M653814XB).

Data availability
The data that support the findings of this study are available on request from the corresponding author.

Declarations
Ethics approval and consent to participate All the animal procedures were performed in accordance with the guidelines for the care and use of laboratory animals.

Consent for publication
All authors have approved that the submitted works are original, and the work has not been published and is not being considered for publication elsewhere.

Competing interests
The authors declare no competing interests. Table 1 Pharmacokinetic parameters of curcumin and docetaxel in plasma after oral administration of D-SMEs and DC-SMEs at a dose of 20 mg/kg and i.v. administration of docetaxel at a dose of 2 mg/kg, respectively AUC 0-t area under the plasma concentration-time profiles from time 0 to the last time point, t 1/2 elimination half-life, C max peak plasma concentration, T max time to reach peak plasma concentration *p < 0.01 compared with free docetaxel group PK parameters AUC 0-24 h /(μg h l −1 ) t