Construction and evaluation of a phospholipid-based phase transition in situ gel system for brexpiprazole

Construction and evaluation of a phospholipid-based phase transition in situ gel system for brexpiprazole | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Construction and evaluation of a phospholipid-based phase transition in situ gel system for brexpiprazole Ran Tao, Li Liu, Yingxin Xiong, Qianyu Zhang, Xiangyu Lv, Linbo He, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2363215/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 May, 2023 Read the published version in Drug Delivery and Translational Research → Version 1 posted 5 You are reading this latest preprint version Abstract The objective of this study was to develop phospholipid-based injectable phase transition in situ gels (PTIGs) for the sustained release of Brexpiprazole (Brex). Phospholipid (Lipoid S100, S100) and stearic acid (SA) were used as the gel matrix which was dissolved in biocompatible solvent medium-chain triglyceride (MCT), N -methyl pyrrolidone (NMP), and ethanol to obtain PTIGs solution. The Brex PTIG showed a solution condition of low viscosity in vitro and was gelatinized in situ in vivo after subcutaneous injection. Both in vitro release assay and in vivo pharmacokinetics study in SD rats displayed that Brex in PTIGs could achieve a sustained release, compared with brexpiprazole solution (Brex-Sol) or brexpiprazole suspension (Brex-Sus). The Brex-PTIGs had good degradability and biocompatibility in vivo with rare inflammation at the injection site. Among the three Brex-PTIG formulations, Brex-PTIG-3 with the SA in the formulation had the greatest gelation viscosity, the lowest initial release rate, and the most stable release profile with sustained release of up to 60 days. The above results indicated that, as a novel drug delivery system, the Brex-PTIGs offered a new option for the clinical treatment of patients with schizophrenia. Phospholipids In situ gel Long-acting injection Sustained-release Schizophrenia Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction As one of the main types of central nervous illness, schizophrenia has a prolonged course and a high relapse rate, making it necessary to take long-term medication [ 1 , 2 ]. Aripiprazole is the mainstream drug for schizophrenia but still suffers from problems such as high doses and poor tolerability [ 3 ]. Brexpiprazole (Brex) as the first multi-target antipsychotic that exerts agonism at dopamine receptors, partial agonism of 5-HT 1A receptor, and antagonism of 5-HT 2A receptor [ 4 ], is a new option for the treatment of schizophrenia [ 5 ]. Research data had shown that Brex was approximately 7.5 to 10 times more potent than aripiprazole. It has a higher affinity towards 5-HT receptors, and less activity on D 2 receptors, which means better tolerability performance and lower incidence of sedentary inability side effects [ 3 , 6 ]. Rexulti®, a brexpiprazole tablet, was approved by the FDA in 2015 [ 7 ]. Another orally disintegrating tablet of brexpiprazole was approved by PMDA in 2018 [ 8 ]. Both preparations of brexpiprazole should take once a day, which leads to poor compliance with long-term treatment [ 6 , 9 ]. Therefore, it is necessary to develop a long-acting formulation of brexpiprazole with good adherence. The drug depot is a long-acting, sustained-release drug delivery system that can last for days or even months with a single administration, it has drawn tremendous attention for its advantages [ 10 , 11 ]. Injectable in situ gels are drug depots that can undergo non-covalent cross-linking phase transitions at the injection site and become local drug delivery depots following administration due to changes in external environmental conditions (e.g., light, temperature, pH, hydrophobicity, ionic strength, etc.) [ 12 , 13 ]. In situ gel drug delivery systems offer more benefits, such as multiple routes of administration (subcutaneous, intramuscular, nasal, or ocular, etc.), simple manufacturing techniques, and low-cost manufacturing, than other drug depots such as suspensions, microspheres, or implants [ 14 ]. Currently, available gel injections included OncoGel®, Eligard®, AtridoxElyzol®, SomatulineDepot®, etc. All these gel injections provided a delayed release and prolonged the duration of action [ 15 – 17 ]. This strategy may also apply to enhancing treatment outcomes in schizophrenia. Most of the long-acting gel injectable products used poly (lactic-co-glycolic acid) (PLGA) gel technology (Atrigel®) owning advantages such as ease of minimally invasive administration and reduced dosing frequency [ 18 , 19 ]. However, some adverse effects hindered the practical use of PLGA systems, such as inflammation and potential toxicity [ 20 , 21 ]. In contrast, phospholipid-based phase transition in situ gels showed excellent clinical potential to address the deficiencies of PLGA gel [ 21 – 23 ]. As is well known, phospholipids have excellent biocompatibility and safety [ 24 ]. Xiang et al. developed a phospholipid-based phase transition in situ gelation system to treat hyperlipidemia [ 25 ]. This system could release pitavastatin slowly with good biocompatibility and a mild inflammatory response. Moreover, solid lipids have been widely applied in pharmaceutics for sustained-release drug delivery [ 26 , 27 ]. Zhang et al. developed an in situ hybrid implant composed of stearic acid (SA) and PLGA. The unique advantage of incorporating SA was the improved drug release profiles and reduced burst release caused by morphology altering and phase inversion of the implant [ 28 ]. Medium-chain triglycerides (MCTs) lacked the hydrophilic domains, but they could act as dopants to replace some molecules in the liquid crystal structure of phospholipids and form a better drug depot. [ 14 , 29 ]. This study aimed to develop phospholipid-based phase transition in situ gel systems of brexpiprazole (Brex-PTIGs) with good biocompatibility and improved medication adherence in patients with schizophrenia. Brex-PTIGs consist of phospholipid (S100), medium-chain triglyceride (MCT) or stearic acid (SA), and biocompatible solvents N -methyl pyrrolidone (NMP) and ethanol (Table 1 ). NMP is one of the FDA-approved solvents with a high safety profile and could dissolve brexpiprazole extremely well. The excipients in the Brex-PTIGs are biodegradable and have been approved by the FDA for approved marketing [ 30 ]. The Brex PTIG showed a solution condition of low viscosity in vitro and was gelatinized in situ in vivo after subcutaneous injection. The Brex-PTIGs were characterized by viscosity, in vitro release of brexpiprazole, in vivo biocompatibility, and biodegradability. In addition, the gelation conditions, rheological properties, and stability were also evaluated. The sustained-release preparations of brexpiprazole in this study have the potential for industrial production and clinical selection. 2. Materials And Methods 2.1. Materials Brexpiparzole (Brex) and Aripiprazole were supplied by Yaopharma Co., Ltd (Chongqing, China). Phospholipid (S100) was purchased from Lipoid Co. (Germany). Ethanol was supplied by Chengdu Kelong Chemical Co., Ltd (Chengdu, China). Medium-chain triglycerides (MCT) were purchased from Liaoning Xinxing Pharmaceutical Co. (Liaoning, China). N -methyl pyrrolidone (NMP) was supplied by Honeywell Co. LTD (USA). Other chemicals and reagents were of an analytical or higher grade. 2.2. Animals Healthy male SD rats, 170 ± 22 g in body mass, were provided by the Experimental Animal Centre of Chongqing Medical University. All rats were provided with standard housing conditions and maintained at 20℃ and 70% RH with a normal 12 h light/dark cycle starting 1 week before the experiment. The care of laboratory animal and the animal experimental operation had conforming to Chongqing Management Approach of Laboratory Animal (chongqing government order NO.195). 2.3. Preparation of the Brex-PTIG system Brex was dissolved in NMP, and different amounts of S100, SA, MCT, and ethanol were added sequentially to obtain a gel solution by stirring at 37°C for 30 min. The composition of the formulations was presented in Table 1 . To obtain samples in the gel state, different volumes of phosphate buffer (PBS, 0.01 M, pH 7.4) were added to the Brex-PTIGs solution and stirred for 10 min at room temperature [ 31 ]. 2.4. Determination of Brex The Brex in Brex-PTIGs or releasing medium were analyzed by high-performance liquid chromatography (HPLC) (Agilent 1260infinity, USA). The samples were diluted with methanol and separated on an Agilent Poroshell EC-C18 column (50 mm x 4.6mm, 2.7 µm). The mobile phase was potassium dihydrogen phosphate (pH 2.0)-methanol (35:65) at a flow rate of 1.0 ml/min. The detection wavelength was 213 nm. The concentration of Brex was calculated by the established standard curve (0.1–8.1 µg/ml). The Brex in plasma was analyzed by LC-MS (Agilent 1290 + 6460). 100 µl of plasma sample was transferred into a 2 ml centrifuge tube, 300 µl of precipitant acetonitrile was added, and centrifuged at 13000 rpm for 5 min. 50 µl of the supernatant was taken and 25 µl of internal standard solution (Aripiprazole, 240 ng/ml) and 150 µl acetonitrile were added. After vortexed for 1 min and then centrifuged at 13000 rpm for 3 min, the supernatant was collected for determination. A liquid chromatographic column (4.6 × 150 mm, 3 µm) (Luna PFP) was employed for the analytical separation. The mobile phases A and B were 0.5 vol% formic acid aqueous and formic acid - water – acetonitrile (0.5:15:85), respectively. The flow rate of the mobile phase was 0.5 ml/min, the injection volume was 20 µl, and the temperature of the chromatographic column was 40℃. For the mass spectroscopic measurements, an electrospray ionization source (ESI), positive ion mode detection, full ion scanning mode (100–500 m/z), and a capillary voltage of 7 V were used. The concentration of Brex was calculated by the established standard curve (10–180 ng/ml). 2.5. Characterization of the Brex-PTIG system The viscosities of Brex-PTIGs before and after gelation were determined at room temperature using a rotational viscometer (Shanghai Fangrui Instruments Co., Ltd.). The morphological images after gelation were also taken by scanning electron microscopy (Thermo). The rheological behavior of Brex-PTIGs was investigated using a rheometer (Anton Paar). The energy loss modulus G'' and storage modulus G' were enumerated from vibration measurements at the constant strain in the angular frequency range of 0.01-10 Hz [ 14 , 32 ]. 2.6. In vitro release The release of Brex from Brex-PTIGs was monitored according to the literature [ 33 ]. The release medium was PBS containing 30% ethanol (pH 6.5) [ 32 , 34 , 35 ]. Brex powder was dissolved in the release medium to a concentration of 30 mg/ml (Brex-Sol) which served as the control. 100 µl of Brex-PTIGs or Brex-Sol was added to dialysis cassettes (Thermo) with a cut-off molecular weight of 10,000, and each dialysis cassette was immersed in 300 ml of fresh release medium under magnetic stirring at a constant speed of 200 rpm at 37°C. At each time point, 50 ml of release medium was collected and the same amount of fresh release medium was replenished. The collected release medium was filtered through a 0.22 µm filter and then analyzed by HPLC according to the method mentioned in section 2.4. The cumulative release of Brex was calculated and the cumulative release-time profile was plotted. 2.7. In vivo pharmacokinetic studies in rats Brexpiprazole solution (Brex-Sol) and Brexpiprazole suspension (Brex-Sus) were prepared as controls. Brex powder was dissolved in NMP to form Brex-Sol at a concentration of 30 mg/ml. Brex-Sus was prepared by dissolving the drug in a mixture of pure water containing 25% NMP at a concentration of 30 mg/ml. The rats were divided into 6 groups randomly and administrated subcutaneously with saline, or Brex-PTIG-1, Brex-PTIG-2, Brex-PTIG-3, Brex-Sol, and Brex-Sus at a Brex dosage of 100 mg/kg, respectively. Blood was taken from the retroorbital plexus of the rat at each time point and collected into heparinized tubes. The plasma was obtained after centrifuging (5500 rpm, 5 min) at 4°C. The concentration of Brex in plasma was analyzed by LC-MS according to the method mentioned in section 2.4. The pharmacokinetic parameters of different formulations were fitted using DAS 2.0 software. 2.8. Biocompatibility and biodegradability studies Rats were shaved and randomly divided into 6 groups, injected subcutaneously with saline, Brex-PTIG-1, Brex-PTIG-2, Brex-PTIG-3, Brex-Sol, or Brex-Sus (100 mg/kg for Brex), respectively. The appearance of the rat’s skin in each group was photographed at 1, 7, 14, and 21 d after injection. A 2×2 cm area of skin around the injection site was cut after executing the rats. The gels maintained at the injection site were separated and weighed. 2.9. Histological analysis The skin removed was fixed in 4% paraformaldehyde for 48 hours. Paraffin sections were processed with hematoxylin-eosin (HE) staining and observed. 2.10. Data and statistical analysis All experiments were repeated at least three times and data are presented as mean ± standard deviation (SD). Differences between experimental groups were assessed using a two-way analysis of variance by the analysis software GraphPad InStat 3.3 following a normal distribution test. P < 0.05 was considered statistically different. [*, p < 0.05, *, p < 0.01 and *, p < 0.001] 3. Results And Discussion 3.1. Preparation of the Brex-PTIG system Brex is a novel multi-target antipsychotic drug for the treatment of schizophrenia, and its preparations on market are oral tablets [ 7 ]. To enhance medication adherence in patients with schizophrenia, there is an urgent need to develop psychotropic drug delivery systems that can deliver medication that lasts for months rather than days. Phospholipid-based organogels are of interest due to their non-toxicity, good biocompatibility, and injectability [ 36 ]. In this study, we designed in situ gel systems for Brex based on the phenomenon that high concentrations of phospholipids form in situ drug depots by precipitating, prolonging the duration of drug action. The Brex-PTIGs consist of S100, MCT or SA, NMP, and ethanol (Table 1 ). Brex-PTIGs were obtained only by mixing and stirring of various components, which makes the preparation process simple, cost-effective, and suitable for mass production. As shown in Fig. 1 , the obtained Brex-PTIGs solution were all clear yellow homogeneous solutions. After mixing with PBS, the Brex-PTIGs solution underwent a fast phase transition and convert to a semi-solid state (Fig. 1A 2 , 1B 2 , and 1C 2 ). Although extensive research was focusing on various in-situ implant delivery systems, only solvent diffusion-based in-situ polymer precipitation systems are commercially available, such as doxycycline (AtridoxElyzol®) for periodontal delivery and leuprolide (Eligard®) for the treatment of prostate cancer [ 33 , 34 ]. In this study, we used S100 as the main gel matrix, which is a biodegradable and biocompatible excipient with low toxicity that has been approved by the FDA for marketing [ 27 ]. Brexpiprazole was a weakly basic compound with very poor solubility, but it is well dissolved in NMP (an FDA-approved solvent for injection) [ 32 ]. A marketed in situ phase transition gel product, a long-acting injection for buprenorphine (SUBLOCADE®), contained up to 50% NMP [ 37 ]. Therefore, the content of 45%-50% NMP in Brex-PTIGs was considered to be safe. Ethanol is a good solvent in injections, but when injections contain high concentrations of ethanol, they often cause severe pain and even local necrosis at the injection site [ 14 , 33 ]. To improve tolerance and reduce adverse effects in patients with schizophrenia, we strictly control the ethanol dosage to less than 10%. Moreover, studies had shown that the higher concentration of ethanol in sustained-release gels, the more severer the initial burst release of drugs [ 36 ]. Meanwhile, MCT and SA were also used to regulate viscosity, improve the release profile of the Brex and alleviate the irritation response caused by ethanol [ 32 , 38 ]. Table 1 Percentage of materials for three brexpiprazole phospholipid-based phase transition in situ gels. Samples Brexpiprazole S100 SA MCT ethanol NMP Brex-PTIG-1 5%(w/v) 35%(w/v) \ 5%(v/v) 10%(v/v) 45%(v/v) Brex-PTIG-2 5%(w/v) 40%(w/v) \ \ 10%(v/v) 45%(v/v) Brex-PTIG-3 5%(w/v) 40%(w/v) 2%(w/v) \ 8%(v/v) 45%(v/v) 3.2. Characterization of the Brex-PTIGs Different volumes of PBS were added to investigate the effect of water on the gelation of the Brex-PTIGs. As shown in Fig. 2 A, the viscosity of the Brex-PTIGs increased with the addition of PBS when the PBS content was less than 7% (w/w). The insolubility of phospholipids in an aqueous solution may be the main reason for the increase in viscosity of the Brex-PTIGs [ 39 ]. Ethanol, NMP, and water are miscible, providing the basis for the rapid precipitation of S100 and phase separation of Brex-PTIGs. It was demonstrated that water would enter and diffuse into the gel after injection, thus triggering an exchange of solvent and water [ 38 ]. However, when the PBS content was higher than 7% (w/w), the dilution effect of PBS caused the viscosity of the Brex-PTIGs to decrease (Fig. 2 A). After gelation, the viscosity of the Brex-PTIGs increased significantly (p < 0.001, Fig. 2 B). Viscosity is an important factor in evaluating long-lasting injectable gels. The viscosity of injections needed to be less than 300 cP, while a higher viscosity is required for the gel to form a depot after injection for sustained release of the drug [ 32 , 40 ]. Therefore, the gel must undergo a significant viscosity shift before and after injection. As shown in Fig. 2 B, Brex-PTIGs were all suitable for injectable use, with viscosities below 300 cP in the solution state, and underwent effective phase transition after injection, with a significant increase of viscosity. Among the three Brex-PTIGs, BPPG-3 showed the greatest change in viscosity, increasing from a viscosity of 27.11 cP in a solution state to 1634.96 cP in a gelled state. The steep increase in viscosity suggested a shift in the state of the gel solution, which could help reduce the initial release of Brex and prolong its release time [ 41 ]. The surface morphologies of the gelation gels were photographed by scanning electron microscopy (Fig. 3 A and 3 B). Brex-PTIG-3 exhibited an angular-shaped surface, while the surface of Brex-PTIG-1 was sparse and porous, and Brex-PTIG-2 was smooth. This might be related to the viscosity of the gel after the phase transition [ 33 ]. The more gel matrix added to the PTIG of Brex, the more viscous the gel semi-solid was, and the less porous and more angular the gel surface was. The complex modulus of Brex-PTIGs after gelation was also measured (Fig. 4 ). The magnitude of the coefficient of storage elasticity (G') and the coefficient of loss elasticity (G'') was associated with the denaturing of the materials[ 42 , 43 ]. Viscoelastic behavior (G' > G'') was perceived for Brex-PTIGs following dynamic strain scan experiments, and the results indicated that all Brex-PTIGs were more elastic than viscous (solid-like). 3.3. Drug release studies in vitro Brex is insoluble in water and its release could be facilitated in release media containing ethanol. In addition, ethanol in the release medium increases the concentration-dependent diffusion outside the gel matrix, thus accelerating the degradation of the gel matrix [ 38 , 44 ]. We also adjusted the pH of the release medium to 6.5 [ 35 ]. The above conditions were set to mimic the biodegradation situation of in situ gels in vivo since there might be certain acids and enzymes at the injection site [ 14 , 35 ]. The release profiles of the Brex-PTIGs were shown in Fig. 5 . Brex in Brex-Sol was released rapidly, with a cumulative release percentage of over 90% within 1 h. In Brex-PTIG-1, Brex-PTIG-2, and Brex-PTIG-3, 29.26%, 15.59%, and 10.97% of Brex were released respectively, with significant differences compared to Brex-Sol (all p < 0.001). After 144 h, the cumulative release percentages of Brex in Brex-PTIG-1, Brex-PTIG-2, and Brex-PTIG-3 achieved 91.46%, 85.49%, and 80.62%, respectively, showing a significantly delayed release compared with Brex-sol. These results demonstrated the sustained release of Brex from Brex-PTIGs. The water-insoluble character of S100 might be the reason for the retarded release effect. The release profiles of the three Brex- PTIGs were generally similar (Fig. 5 ). However, among the three Brex-PTIGs, Brex-PTIG-3 had the slowest release rate. The release rate of Brex in Brex-PTIG-3 was significantly lower than that of Brex-PTIG-1 at 1 h (p < 0.05). This indicated the SA was necessary for improving the initial sudden release of Brex from Brex-PTIG. 3.4. Pharmacokinetic studies To investigate the pharmacokinetic properties of Brex-PTIGs in vivo , healthy SD rats were injected subcutaneously with Brex-Sol, Brex-Sus, Brex-PTIG-1, Brex-PTIG-2, or Brex-PTIG-3 with a single dose of Brex at 100 mg/kg. As shown in Fig. 6 , the plasma concentration of Brex in the Brex-Sol group showed a significant increase within 1 h after injection, with a C max of 875.52 ng/ml followed by rapid clearance and Brex could not undetectable after 24 h. Similarly, the concentration of Brex in the Brex-Sus group reached a peak quickly, with a C max of 548.68 ng/ml (Table 2 ), and then cleared quickly (Fig. 6 ), and Brex was undetected in plasma after 7 d. In contrast, as shown in Table 2 and Fig. 6 , the Brex-PTIGs groups had significantly delayed peaking, compared with Brex-sol and Brex-sus (all p < 0.001). The peak concentration of Brex was observed around 2 h post-injection for Brex-PTIG-1, Brex-PTIG-2, and Brex-PTIG-3, with the C max of 139.30 ng/ml, 113.83 ng/ml, and 89.45 ng/ml respectively, which were significantly lower than that of Brex-sol (875.52 ng/ml) and Brex-sus (548.68 ng/ml) (all p < 0.001). A major challenge in the clinical implementation of in situ molded implants was the control of the initial burst release of drugs, particularly for in situ precipitation systems. Compared to solution and suspension, the Brex-PTIGs provided a significantly improved initial burst release of Brex and were able to release Brex smoothly for two months. Undoubtedly, the decreased initial burst release was attributed to the formation of Brex-PTIGs gel depots. There was no significance between the three Brex-PTIGs in the area under the concentration-time curves (AUC (0−∞) ), indicating the same degree of absorption of the three Brex-PTIGs. All Brex-PTIGs released Brex stably for more than 60 d, which might facilitate medication adherence in patients with schizophrenia. Among the three Brex-PTIGs (Table 2 ), the C max of Brex-PTIG-3 was significantly lower than Brex-PTIG-1 (p < 0.01), and the t 1/2z of Brex-PTIG-3 was significantly longer than that of Brex-PTIG-1 (p < 0.05). This was correlated with the release assay of Brex-PTIGs in vitro , which indicated that although a sustained-release profile of Brex-PTIGs was observed, the composition of the gel matrix might have influenced the initial burst release. The results of in vitro release and pharmacokinetics assay suggested that MCT in Brex-PTIG-1 might lessen the initial burst release of phospholipid-based phase transition in situ gel, but it was not as effective as that of SA in Brex-PTIG-3, suggesting SA was a more suitable component for Brex-PTIGs. Table 2 Pharmacokinetic parameters after a single administration of three Brex-PTIGs (mean±SD, n=15). Parameter value Brex-Sol Brex-Sus Brex-PTIG-1 Brex-PTIG-2 Brex-PTIG-3 C max (ng/mL) 875.52±79.45 548.68±55.86 139.30±50.49 ***,### 113.83±20.59 ***,###,$ 89.45±22.50 ***,###.$$ T max (h) 0.96±0.17 0.96±0.09 1.92±0.38 ***,### 2.00±0.05 ***,### 2.36±0.21 ***,### t 1/2z (d) 0.45±0.06 1.21±0.08 12.48±2.69 ***,### 20.75±3.66 ***,###,$ 22.07±3.54 ***,###,$ AUC (0-∞) (μg·d/L) 358.50±85.94 770.42±79.65 3303±118.95 ***,### 3344.00±169.69 ***,### 3339.37±186.69 ***,### MRT (0-∞) (d) 0.28±0.13 1.23±0.20 26.64±3.61 ***,### 31.92±2.95 ***,### 42.47±1.98 ***,###,$ Abbreviations: *** p<0.001 compared to Brex-Sol; ### p<0.001 compared to Brex-Sus; $ p<0.05 and $$ p<0.01 compared to Brex-PTIG-1. 3.5 Biodegradability and biocompatibility of the Brex-PTIG system in vivo The state of the skin around the injection site was observed on days 0, 7, 14, and 21 after the injection. As shown in Fig. 7, on Day 0 an injection bulge could be seen in the Brex-PTIGs and Brex-sus groups, while the skin seemed smooth and flat with no discernable bulge in the Brex-sol group. On Day 7 after the injection, the volume of the injection bulge significantly decreased in all Brex-PTIGs groups. By Day 14, the skin at the injection site was almost flat in the Brex-PTIGs group. To further evaluate the degradation of Brex-PTIGs in vivo , the depots formed after subcutaneous injection of Brex-PTIGs were dissected from the rat skin and weighed on different days after injection. The weight change of the residual gel was displayed in Fig. 8A. The weight of the residual Brex-PTIG-1 and Brex-PTIG-2 increased during the 0-5 d after injection and then decreased. This was consistent with the previous study. In a comparative study of in situ gels performed by Zhang et. al., the gel formulation with the best performance (better viscosity and best sustained-release characteristics) showed a slow increase and degradation of the gel weight in vivo [14]. The residual weight of Brex-PTIGs at Day 60 all decreased to 10% of the initial weight. Organic solvents located in the in situ precipitation system may irritate the injection site. The inflammatory reaction is the main side effect after the local injection of in situ gel [45]. Thus, we examined the compatibility and inflammatory effect of Brex-PTIGs at the injection site after a single subcutaneous injection. At first, the skin around the injection site was dissected and observed on 7 d after injection. As shown in Fig. 8B, the yellowish gel-like depot of Brex-PTIGs was with smooth and flat edges and was covered by a transparent biofilm. However, in Brex-Sol and Brex-Sus groups, signs of bleeding and inflammations were observed. A partial white fatty accumulation at the injection site was also observed. Then, the skin tissues at the injection site were fixed, sliced, and stained with hematoxylin and eosin (HE) for histological analysis. As shown in Fig. 9, none of the Brex-PTIGs showed a severe inflammatory response at the injection site over the 21 d following administration. However, for Brex-Sol and Brex-Sus groups, on the first day after injection, the structure of adipocytes was severely disrupted with massive cell necrosis, in addition, infiltration of lymphocytes, granulocytes, and other inflammatory cells into the tissues was also observed on Day 7 after injection, with the formation of large amounts of granulation and fibrous connective tissue. On Day 21, the skin conditions of all groups recovered to normal. These results indicated the Brex-PTIGs were more biocompatible than solution and suspensions in vivo . The biocompatibility and mild inflammatory response of Brex-PTIGs might be owing to the biocompatible excipients and their reasonable adding amount. The matrix in Brex-PTIGs was all biocompatible, including S100 and SA. The solvents used in the Brex-PTIGs were MCT, ethanol, and NMP, which were seen as safe [45, 46]. As an FDA-approved solvent for injectable use, the safety of NMP could be effectively assured, and the low percentage of ethanol (less than 10%) might help reduce skin irritation, as demonstrated in previous studies [32]. The content of solvents in Brex-PTIGs was controlled strictly to ensure the safety of Brex-PTIGs. 4. Conclusions In this study, an injectable in situ gel of Brexpiprazole (Brex) based on phospholipid-based phase transition consisting of S100, MCT/SA, NMP, and ethanol was constructed. Brex-PTIGs in solution state were suitable for injection, and when got in contact with water in vivo , they rapidly transformed to a semi-solid gel state. The Brex-PTIGs had a good sustained release of Brex in vitro and in vivo . The preliminary safety profile was also confirmed in this study. This study for the first time confirmed the feasibility of injectable in situ gel based on phospholipid-based phase transition for the sustained delivery of Brex, which holds high potential in developing a valid, safe, easy-producing, and low-cost preparation of Brex for the long-term treatment of schizophrenia. Declarations Acknowledgements The study leading to the reported results was funded by the Program of Postgraduate Joint Training Base of Chongqing (Chongqing Medical University& Yaopharma Co., Ltd joint training base for postgraduate in pharmacy) (to Huali Chen and Yan Zhang), and the Science and Technology Research Program of Chongqing Municipal Education Commission (grant no. KJQN202000407; to Huali Chen). Author contributions Ran Tao: Conceptualization, Methodology, Investigation, Data curation, Writing an original draft, Visualization. Li Liu: Methodology, Investigation, Data curation, Visualization , Supervision, Writing - review & editing. Yingxin Xiong: Methodology, Investigation, Resources, Supervision. Qianyu Zhang: Methodology, Investigation , Writing - review & editing. Xiangyu Lv: Data curation, Formal analysis. Linbo He: Data curation, Formal analysis, Resources. Fang Ren: Validation, Methodology. Lu Zhou: Methodology, Software. BaoYan Chen: Formal analysis, Software. Kexin Wu: Validation, Visualization. Yan Zhang: Project administration, Resources, Supervision. Huali Chen: Funding acquisition, Conceptualization, Supervision, Writing - review & editing. Ethics approval The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of Chongqing Management Approach of Laboratory Animal (chongqing government order NO.195). Informed Consent Statement Not applicable. Data Availability Statement The data presented in this study are available upon request. Conflicts of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Mai, Y., et al., Topical formulation based on disease-specific nanoparticles for single-dose cure of psoriasis. J Control Release, 2022. 349 : p. 354-366. Xu, W., et al., Analysis of Factors Influencing Telemedicine-Based Psychiatric Extended Care and Care of Psychiatric Patients. J Healthc Eng, 2022. 2022 : p. 9434820. Ferraiolo, M., et al., Receptor density influences the recruitment bias of aripiprazole and brexpiprazole at the dopamine D2L receptor. Fundam Clin Pharmacol, 2022. Brexpiprazole for schizophrenia. Aust Prescr, 2017. 40 (5): p. 197-198. Orsolini, L., et al., A case report of clozapine-treatment-resistant schizophrenia successfully managed with brexpiprazole combination therapy. 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Ren, T., et al., Lipid emulsions in parenteral nutrition: current applications and future developments. Expert Opin Drug Deliv, 2013. 10 (11): p. 1533-49. Han, L., et al., An injectable, low-toxicity phospholipid-based phase separation gel that induces strong and persistent immune responses in mice. Biomaterials, 2016. 105 : p. 185-194. Li, H., et al., An in situ-forming phospholipid-based phase transition gel prolongs the duration of local anesthesia for ropivacaine with minimal toxicity. Acta Biomater, 2017. 58 : p. 136-145. Luo, J., et al., Efficient weapon for protracted warfare to malaria: A chondroitin sulfate derivates-containing injectable, ultra-long-lasting meshy-gel system. Carbohydr Polym, 2019. 214 : p. 131-141. Liang, Y., et al., Adhesive Hemostatic Conducting Injectable Composite Hydrogels with Sustained Drug Release and Photothermal Antibacterial Activity to Promote Full-Thickness Skin Regeneration During Wound Healing. Small, 2019. 15 (12): p. e1900046. Remenar, J.F., Making the leap from daily oral dosing to long-acting injectables: lessons from the antipsychotics. Mol Pharm, 2014. 11 (6): p. 1739-49. Wang, K., et al., Self-assembled L-alanine derivative organogel as in situ drug delivery implant: characterization, biodegradability, and biocompatibility. Drug Dev Ind Pharm, 2010. 36 (12): p. 1511-21. Yadav, S.K., G. Khan, and B. Mishra, Advances in patents related to intrapocket technology for the management of periodontitis. Recent Pat Drug Deliv Formul, 2015. 9 (2): p. 129-45. Zhang, T., et al., A high-efficiency, low-toxicity, phospholipids-based phase separation gel for long-term delivery of peptides. Biomaterials, 2015. 45 : p. 1-9. Wu, W., et al., A novel doxorubicin-loaded in situ forming gel based high concentration of phospholipid for intratumoral drug delivery. Mol Pharm, 2014. 11 (10): p. 3378-85. Xuan, J.J., et al., Rheological characterization and in vivo evaluation of thermosensitive poloxamer-based hydrogel for intramuscular injection of piroxicam. Int J Pharm, 2010. 395 (1-2): p. 317-23. Chen, H., et al., Characterization of pH- and temperature-sensitive hydrogel nanoparticles for controlled drug release. PDA J Pharm Sci Technol, 2007. 61 (4): p. 303-13. H, P.S.A., et al., A review on chitosan-cellulose blends and nanocellulose reinforced chitosan biocomposites: Properties and their applications. Carbohydr Polym, 2016. 150 : p. 216-26. Ning, Q., et al., Neurodegenerative changes and neuroapoptosis induced by systemic lipopolysaccharide administration are reversed by dexmedetomidine treatment in mice. Neurol Res, 2017. 39 (4): p. 357-366. Milak, S. and A. Zimmer, Glycerol monooleate liquid crystalline phases used in drug delivery systems. Int J Pharm, 2015. 478 (2): p. 569-87. Thakur, R.R., H.L. McMillan, and D.S. Jones, Solvent induced phase inversion-based in situ forming controlled release drug delivery implants. J Control Release, 2014. 176 : p. 8-23. Kempe, S. and K. Mader, In situ forming implants - an attractive formulation principle for parenteral depot formulations. J Control Release, 2012. 161 (2): p. 668-79. Supplementary Files Graphicalabstract.tif Cite Share Download PDF Status: Published Journal Publication published 09 May, 2023 Read the published version in Drug Delivery and Translational Research → Version 1 posted Editorial decision: Major Revisions Needed 29 Jan, 2023 Reviewers agreed at journal 30 Dec, 2022 Reviewers invited by journal 27 Dec, 2022 Editor assigned by journal 13 Dec, 2022 First submitted to journal 12 Dec, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2363215","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":163137718,"identity":"e5ec5424-c099-4f3d-8a45-d42ef1fa1332","order_by":0,"name":"Ran Tao","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ran","middleName":"","lastName":"Tao","suffix":""},{"id":163137719,"identity":"e847e925-915c-48a1-9459-61318f699b04","order_by":1,"name":"Li Liu","email":"","orcid":"","institution":"Chongqing Yaopharma co Ltd","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Liu","suffix":""},{"id":163137720,"identity":"e4df008d-01cf-4962-95cc-6a6f320c3278","order_by":2,"name":"Yingxin Xiong","email":"","orcid":"","institution":"Chongqing Yaopharma co ltd","correspondingAuthor":false,"prefix":"","firstName":"Yingxin","middleName":"","lastName":"Xiong","suffix":""},{"id":163137721,"identity":"7ae7488b-1436-4ef6-ac49-8c25753049d8","order_by":3,"name":"Qianyu Zhang","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qianyu","middleName":"","lastName":"Zhang","suffix":""},{"id":163137722,"identity":"f18026cf-f58f-4b73-84fc-c7e3af7bc3ae","order_by":4,"name":"Xiangyu Lv","email":"","orcid":"","institution":"Chongqing Yaopharma co 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00:29:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2363215/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2363215/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s13346-023-01349-0","type":"published","date":"2023-05-09T20:48:57+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":31036182,"identity":"b075f6bb-e75b-403c-81ca-cdf047f9e3f7","added_by":"auto","created_at":"2023-01-03 15:41:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3726349,"visible":true,"origin":"","legend":"\u003cp\u003eMorphology of Brex-PTIG-1, Brex-PTIG-2, Brex-PTIG-3 before (A1, B1, C1) and after (A2, B2, C2) phase transitions.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2363215/v1/23a100c709e26510014e21f3.png"},{"id":31036179,"identity":"7a8510db-1f25-437e-9c72-9c32477c61bd","added_by":"auto","created_at":"2023-01-03 15:41:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":606181,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The viscosity of Brex-PTIG-1, Brex-PTIG-2, and Brex-PTIG-3 with different PBS content (%, w/w). (B) Viscosity Changes of Gels at 25℃. Data represent mean ± SD (n = 3).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2363215/v1/33439745378f96bfbabf825e.png"},{"id":31036185,"identity":"089cbc69-8e3a-4f0a-9a10-a0a6bf02b71d","added_by":"auto","created_at":"2023-01-03 15:41:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":32464232,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscope (SEM) images of Brex-PTIGs magnified 500 × (A)( Scale bar: 200 μm) and 1000 × (B)( Scale bar: 100 μm).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2363215/v1/b7a69bdec2f70c42890adb06.png"},{"id":31036178,"identity":"b29e5a7a-0799-4fe1-aabb-b4ccb9135afe","added_by":"auto","created_at":"2023-01-03 15:41:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":367865,"visible":true,"origin":"","legend":"\u003cp\u003eComplex modulus of Brex-PTIG-1 (A), Brex-PTIG-2 (B), and Brex-PTIG-3 (C) after gelation. Data represent mean ± SD (n = 3).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2363215/v1/8d26ca708ecbb0ad2e50dce1.png"},{"id":31037470,"identity":"f331ce8b-b18d-4a4d-b2a8-4bc4de7d93a2","added_by":"auto","created_at":"2023-01-03 15:49:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":360350,"visible":true,"origin":"","legend":"\u003cp\u003eRelease profiles of the formulations PBS containing 30% ethanol (pH 6.5) over 168 h, Data represent mean ± SD (n = 3). *p\u0026lt;0.05 compared to Brex-Sol; #p\u0026lt;0.05 compared to Brex-PTIG-1. Abbreviations: Brex-Sol, Brexpiparzole solution.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-2363215/v1/39398668e945a3f48bf1f551.png"},{"id":31037469,"identity":"a1094fab-5253-41bd-aa76-0385fc9e9b62","added_by":"auto","created_at":"2023-01-03 15:49:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":508177,"visible":true,"origin":"","legend":"\u003cp\u003ePlasma concentration-time curves of Brex samples: 1-60 d (A); The first day (B). Data were presented as mean ±SD (n = 15). Abbreviations: Brex-Sol, Brexpiparzole solution; Brex-Sus, Brexpiparzole suspension.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-2363215/v1/3d08bd91fb6cf3f45a684b40.png"},{"id":31036184,"identity":"a2012a15-92ce-4efc-8001-7bf22791059d","added_by":"auto","created_at":"2023-01-03 15:41:53","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":6833868,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative photos of samples after a single subcutaneous injection in rats on days 0, 7, 14, and 21. Data were presented as mean ±SD(n = 4). Brex-Sol, Brexpiparzole solution; Brex-Sus, Brexpiparzole suspension.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-2363215/v1/d1f331c779498107c19bda31.png"},{"id":31036181,"identity":"d9599264-4311-4403-8f04-a411537f61fa","added_by":"auto","created_at":"2023-01-03 15:41:52","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2085858,"visible":true,"origin":"","legend":"\u003cp\u003eWeight of residual gel(%) \u003cem\u003ein vivo\u003c/em\u003e (A); State of local injection site on the 7th day of administration (B). Data were presented as mean ±SD (n = 4).\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-2363215/v1/3d523798bf34b2c4675094ac.png"},{"id":31037471,"identity":"a856d42e-7b28-4e6e-9731-ef05b6f9eac9","added_by":"auto","created_at":"2023-01-03 15:49:53","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":16560238,"visible":true,"origin":"","legend":"\u003cp\u003eHE staining results of saline, Brex-BTIG-1, Brex-BTIG-2, Brex-BTIG-3, Brex-Sol, and Brex-Sus, Scale bar: 100 μm.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-2363215/v1/3dfabfda28ba01f86cba7b8a.png"},{"id":44729122,"identity":"c16ccc1a-55f8-4e02-aa50-6a58632221a1","added_by":"auto","created_at":"2023-10-16 21:13:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7172070,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2363215/v1/772a8b5d-3d05-4724-ab74-2fd7c7c7ad7b.pdf"},{"id":31036187,"identity":"93831f2d-53e3-4cab-9407-bd09834cbaf0","added_by":"auto","created_at":"2023-01-03 15:41:53","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13797424,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-2363215/v1/2c379438fbed64aa2398d12a.tif"}],"financialInterests":"","formattedTitle":"Construction and evaluation of a phospholipid-based phase transition in situ gel system for brexpiprazole","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAs one of the main types of central nervous illness, schizophrenia has a prolonged course and a high relapse rate, making it necessary to take long-term medication [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Aripiprazole is the mainstream drug for schizophrenia but still suffers from problems such as high doses and poor tolerability [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Brexpiprazole (Brex) as the first multi-target antipsychotic that exerts agonism at dopamine receptors, partial agonism of 5-HT\u003csub\u003e1A\u003c/sub\u003e receptor, and antagonism of 5-HT\u003csub\u003e2A\u003c/sub\u003e receptor [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], is a new option for the treatment of schizophrenia [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Research data had shown that Brex was approximately 7.5 to 10 times more potent than aripiprazole. It has a higher affinity towards 5-HT receptors, and less activity on D\u003csub\u003e2\u003c/sub\u003e receptors, which means better tolerability performance and lower incidence of sedentary inability side effects [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Rexulti\u0026reg;, a brexpiprazole tablet, was approved by the FDA in 2015 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Another orally disintegrating tablet of brexpiprazole was approved by PMDA in 2018 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Both preparations of brexpiprazole should take once a day, which leads to poor compliance with long-term treatment [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, it is necessary to develop a long-acting formulation of brexpiprazole with good adherence.\u003c/p\u003e \u003cp\u003eThe drug depot is a long-acting, sustained-release drug delivery system that can last for days or even months with a single administration, it has drawn tremendous attention for its advantages [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Injectable in situ gels are drug depots that can undergo non-covalent cross-linking phase transitions at the injection site and become local drug delivery depots following administration due to changes in external environmental conditions (e.g., light, temperature, pH, hydrophobicity, ionic strength, etc.) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In situ gel drug delivery systems offer more benefits, such as multiple routes of administration (subcutaneous, intramuscular, nasal, or ocular, etc.), simple manufacturing techniques, and low-cost manufacturing, than other drug depots such as suspensions, microspheres, or implants [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Currently, available gel injections included OncoGel\u0026reg;, Eligard\u0026reg;, AtridoxElyzol\u0026reg;, SomatulineDepot\u0026reg;, etc. All these gel injections provided a delayed release and prolonged the duration of action [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This strategy may also apply to enhancing treatment outcomes in schizophrenia.\u003c/p\u003e \u003cp\u003eMost of the long-acting gel injectable products used poly (lactic-co-glycolic acid) (PLGA) gel technology (Atrigel\u0026reg;) owning advantages such as ease of minimally invasive administration and reduced dosing frequency [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, some adverse effects hindered the practical use of PLGA systems, such as inflammation and potential toxicity [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In contrast, phospholipid-based phase transition \u003cem\u003ein situ\u003c/em\u003e gels showed excellent clinical potential to address the deficiencies of PLGA gel [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. As is well known, phospholipids have excellent biocompatibility and safety [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Xiang et al. developed a phospholipid-based phase transition \u003cem\u003ein situ\u003c/em\u003e gelation system to treat hyperlipidemia [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This system could release pitavastatin slowly with good biocompatibility and a mild inflammatory response. Moreover, solid lipids have been widely applied in pharmaceutics for sustained-release drug delivery [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Zhang et al. developed an \u003cem\u003ein situ\u003c/em\u003e hybrid implant composed of stearic acid (SA) and PLGA. The unique advantage of incorporating SA was the improved drug release profiles and reduced burst release caused by morphology altering and phase inversion of the implant [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Medium-chain triglycerides (MCTs) lacked the hydrophilic domains, but they could act as dopants to replace some molecules in the liquid crystal structure of phospholipids and form a better drug depot. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study aimed to develop phospholipid-based phase transition \u003cem\u003ein situ\u003c/em\u003e gel systems of brexpiprazole (Brex-PTIGs) with good biocompatibility and improved medication adherence in patients with schizophrenia. Brex-PTIGs consist of phospholipid (S100), medium-chain triglyceride (MCT) or stearic acid (SA), and biocompatible solvents \u003cem\u003eN\u003c/em\u003e-methyl pyrrolidone (NMP) and ethanol (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). NMP is one of the FDA-approved solvents with a high safety profile and could dissolve brexpiprazole extremely well. The excipients in the Brex-PTIGs are biodegradable and have been approved by the FDA for approved marketing [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The Brex PTIG showed a solution condition of low viscosity in vitro and was gelatinized \u003cem\u003ein situ\u003c/em\u003e in vivo after subcutaneous injection. The Brex-PTIGs were characterized by viscosity, \u003cem\u003ein vitro\u003c/em\u003e release of brexpiprazole, \u003cem\u003ein vivo\u003c/em\u003e biocompatibility, and biodegradability. In addition, the gelation conditions, rheological properties, and stability were also evaluated. The sustained-release preparations of brexpiprazole in this study have the potential for industrial production and clinical selection.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eBrexpiparzole (Brex) and Aripiprazole were supplied by Yaopharma Co., Ltd (Chongqing, China). Phospholipid (S100) was purchased from Lipoid Co. (Germany). Ethanol was supplied by Chengdu Kelong Chemical Co., Ltd (Chengdu, China). Medium-chain triglycerides (MCT) were purchased from Liaoning Xinxing Pharmaceutical Co. (Liaoning, China). \u003cem\u003eN\u003c/em\u003e-methyl pyrrolidone (NMP) was supplied by Honeywell Co. LTD (USA). Other chemicals and reagents were of an analytical or higher grade.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Animals\u003c/h2\u003e \u003cp\u003eHealthy male SD rats, 170\u0026thinsp;\u0026plusmn;\u0026thinsp;22 g in body mass, were provided by the Experimental Animal Centre of Chongqing Medical University. All rats were provided with standard housing conditions and maintained at 20℃ and 70% RH with a normal 12 h light/dark cycle starting 1 week before the experiment. The care of laboratory animal and the animal experimental operation had conforming to Chongqing Management Approach of Laboratory Animal (chongqing government order NO.195).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Preparation of the Brex-PTIG system\u003c/h2\u003e \u003cp\u003eBrex was dissolved in NMP, and different amounts of S100, SA, MCT, and ethanol were added sequentially to obtain a gel solution by stirring at 37\u0026deg;C for 30 min. The composition of the formulations was presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. To obtain samples in the gel state, different volumes of phosphate buffer (PBS, 0.01 M, pH 7.4) were added to the Brex-PTIGs solution and stirred for 10 min at room temperature [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Determination of Brex\u003c/h2\u003e \u003cp\u003eThe Brex in Brex-PTIGs or releasing medium were analyzed by high-performance liquid chromatography (HPLC) (Agilent 1260infinity, USA). The samples were diluted with methanol and separated on an Agilent Poroshell EC-C18 column (50 mm x 4.6mm, 2.7 \u0026micro;m). The mobile phase was potassium dihydrogen phosphate (pH 2.0)-methanol (35:65) at a flow rate of 1.0 ml/min. The detection wavelength was 213 nm. The concentration of Brex was calculated by the established standard curve (0.1\u0026ndash;8.1 \u0026micro;g/ml).\u003c/p\u003e \u003cp\u003eThe Brex in plasma was analyzed by LC-MS (Agilent 1290\u0026thinsp;+\u0026thinsp;6460). 100 \u0026micro;l of plasma sample was transferred into a 2 ml centrifuge tube, 300 \u0026micro;l of precipitant acetonitrile was added, and centrifuged at 13000 rpm for 5 min. 50 \u0026micro;l of the supernatant was taken and 25 \u0026micro;l of internal standard solution (Aripiprazole, 240 ng/ml) and 150 \u0026micro;l acetonitrile were added.\u003c/p\u003e \u003cp\u003eAfter vortexed for 1 min and then centrifuged at 13000 rpm for 3 min, the supernatant was collected for determination. A liquid chromatographic column (4.6 \u0026times; 150 mm, 3 \u0026micro;m) (Luna PFP) was employed for the analytical separation. The mobile phases A and B were 0.5 vol% formic acid aqueous and formic acid - water \u0026ndash; acetonitrile (0.5:15:85), respectively. The flow rate of the mobile phase was 0.5 ml/min, the injection volume was 20 \u0026micro;l, and the temperature of the chromatographic column was 40℃. For the mass spectroscopic measurements, an electrospray ionization source (ESI), positive ion mode detection, full ion scanning mode (100\u0026ndash;500 m/z), and a capillary voltage of 7 V were used. The concentration of Brex was calculated by the established standard curve (10\u0026ndash;180 ng/ml).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Characterization of the Brex-PTIG system\u003c/h2\u003e \u003cp\u003eThe viscosities of Brex-PTIGs before and after gelation were determined at room temperature using a rotational viscometer (Shanghai Fangrui Instruments Co., Ltd.). The morphological images after gelation were also taken by scanning electron microscopy (Thermo).\u003c/p\u003e \u003cp\u003eThe rheological behavior of Brex-PTIGs was investigated using a rheometer (Anton Paar). The energy loss modulus G'' and storage modulus G' were enumerated from vibration measurements at the constant strain in the angular frequency range of 0.01-10 Hz [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. In vitro release\u003c/h2\u003e \u003cp\u003eThe release of Brex from Brex-PTIGs was monitored according to the literature [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The release medium was PBS containing 30% ethanol (pH 6.5) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Brex powder was dissolved in the release medium to a concentration of 30 mg/ml (Brex-Sol) which served as the control.\u003c/p\u003e \u003cp\u003e100 \u0026micro;l of Brex-PTIGs or Brex-Sol was added to dialysis cassettes (Thermo) with a cut-off molecular weight of 10,000, and each dialysis cassette was immersed in 300 ml of fresh release medium under magnetic stirring at a constant speed of 200 rpm at 37\u0026deg;C. At each time point, 50 ml of release medium was collected and the same amount of fresh release medium was replenished. The collected release medium was filtered through a 0.22 \u0026micro;m filter and then analyzed by HPLC according to the method mentioned in section 2.4. The cumulative release of Brex was calculated and the cumulative release-time profile was plotted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. In vivo pharmacokinetic studies in rats\u003c/h2\u003e \u003cp\u003eBrexpiprazole solution (Brex-Sol) and Brexpiprazole suspension (Brex-Sus) were prepared as controls. Brex powder was dissolved in NMP to form Brex-Sol at a concentration of 30 mg/ml. Brex-Sus was prepared by dissolving the drug in a mixture of pure water containing 25% NMP at a concentration of 30 mg/ml. The rats were divided into 6 groups randomly and administrated subcutaneously with saline, or Brex-PTIG-1, Brex-PTIG-2, Brex-PTIG-3, Brex-Sol, and Brex-Sus at a Brex dosage of 100 mg/kg, respectively. Blood was taken from the retroorbital plexus of the rat at each time point and collected into heparinized tubes. The plasma was obtained after centrifuging (5500 rpm, 5 min) at 4\u0026deg;C. The concentration of Brex in plasma was analyzed by LC-MS according to the method mentioned in section 2.4. The pharmacokinetic parameters of different formulations were fitted using DAS 2.0 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Biocompatibility and biodegradability studies\u003c/h2\u003e \u003cp\u003eRats were shaved and randomly divided into 6 groups, injected subcutaneously with saline, Brex-PTIG-1, Brex-PTIG-2, Brex-PTIG-3, Brex-Sol, or Brex-Sus (100 mg/kg for Brex), respectively. The appearance of the rat\u0026rsquo;s skin in each group was photographed at 1, 7, 14, and 21 d after injection. A 2\u0026times;2 cm area of skin around the injection site was cut after executing the rats. The gels maintained at the injection site were separated and weighed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Histological analysis\u003c/h2\u003e \u003cp\u003eThe skin removed was fixed in 4% paraformaldehyde for 48 hours. Paraffin sections were processed with hematoxylin-eosin (HE) staining and observed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Data and statistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were repeated at least three times and data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Differences between experimental groups were assessed using a two-way analysis of variance by the analysis software GraphPad InStat 3.3 following a normal distribution test. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically different. [*, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, *, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and *, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001]\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results And Discussion","content":"\u003cdiv class=\"Section2\" id=\"Sec14\"\u003e\n \u003ch2\u003e3.1. Preparation of the Brex-PTIG system\u003c/h2\u003e\n \u003cp\u003eBrex is a novel multi-target antipsychotic drug for the treatment of schizophrenia, and its preparations on market are oral tablets [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]. To enhance medication adherence in patients with schizophrenia, there is an urgent need to develop psychotropic drug delivery systems that can deliver medication that lasts for months rather than days. Phospholipid-based organogels are of interest due to their non-toxicity, good biocompatibility, and injectability [\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]. In this study, we designed \u003cem\u003ein situ\u003c/em\u003e gel systems for Brex based on the phenomenon that high concentrations of phospholipids form \u003cem\u003ein situ\u003c/em\u003e drug depots by precipitating, prolonging the duration of drug action. The Brex-PTIGs consist of S100, MCT or SA, NMP, and ethanol (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eBrex-PTIGs were obtained only by mixing and stirring of various components, which makes the preparation process simple, cost-effective, and suitable for mass production. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, the obtained Brex-PTIGs solution were all clear yellow homogeneous solutions. After mixing with PBS, the Brex-PTIGs solution underwent a fast phase transition and convert to a semi-solid state (Fig.\u0026nbsp;1A\u003csub\u003e2\u003c/sub\u003e, 1B\u003csub\u003e2\u003c/sub\u003e, and 1C\u003csub\u003e2\u003c/sub\u003e).\u003c/p\u003e\n \u003cp\u003eAlthough extensive research was focusing on various in-situ implant delivery systems, only solvent diffusion-based in-situ polymer precipitation systems are commercially available, such as doxycycline (AtridoxElyzol\u0026reg;) for periodontal delivery and leuprolide (Eligard\u0026reg;) for the treatment of prostate cancer [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. In this study, we used S100 as the main gel matrix, which is a biodegradable and biocompatible excipient with low toxicity that has been approved by the FDA for marketing [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eBrexpiprazole was a weakly basic compound with very poor solubility, but it is well dissolved in NMP (an FDA-approved solvent for injection) [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]. A marketed \u003cem\u003ein situ\u003c/em\u003e phase transition gel product, a long-acting injection for buprenorphine (SUBLOCADE\u0026reg;), contained up to 50% NMP [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]. Therefore, the content of 45%-50% NMP in Brex-PTIGs was considered to be safe.\u003c/p\u003e\n \u003cp\u003eEthanol is a good solvent in injections, but when injections contain high concentrations of ethanol, they often cause severe pain and even local necrosis at the injection site [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. To improve tolerance and reduce adverse effects in patients with schizophrenia, we strictly control the ethanol dosage to less than 10%. Moreover, studies had shown that the higher concentration of ethanol in sustained-release gels, the more severer the initial burst release of drugs [\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]. Meanwhile, MCT and SA were also used to regulate viscosity, improve the release profile of the Brex and alleviate the irritation response caused by ethanol [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e].\u0026nbsp;\u003c/p\u003e\u0026nbsp;\u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePercentage of materials for three brexpiprazole phospholipid-based phase transition in situ gels.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSamples\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBrexpiprazole\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eS100\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSA\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMCT\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eethanol\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNMP\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBrex-PTIG-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5%(w/v)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35%(w/v)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\\\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5%(v/v)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10%(v/v)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45%(v/v)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBrex-PTIG-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5%(w/v)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40%(w/v)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\\\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\\\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10%(v/v)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45%(v/v)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBrex-PTIG-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5%(w/v)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40%(w/v)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2%(w/v)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\\\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8%(v/v)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45%(v/v)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec15\"\u003e\n \u003ch2\u003e3.2. Characterization of the Brex-PTIGs\u003c/h2\u003e\n \u003cp\u003eDifferent volumes of PBS were added to investigate the effect of water on the gelation of the Brex-PTIGs. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, the viscosity of the Brex-PTIGs increased with the addition of PBS when the PBS content was less than 7% (w/w). The insolubility of phospholipids in an aqueous solution may be the main reason for the increase in viscosity of the Brex-PTIGs [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]. Ethanol, NMP, and water are miscible, providing the basis for the rapid precipitation of S100 and phase separation of Brex-PTIGs. It was demonstrated that water would enter and diffuse into the gel after injection, thus triggering an exchange of solvent and water [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]. However, when the PBS content was higher than 7% (w/w), the dilution effect of PBS caused the viscosity of the Brex-PTIGs to decrease (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). After gelation, the viscosity of the Brex-PTIGs increased significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\n \u003cp\u003eViscosity is an important factor in evaluating long-lasting injectable gels. The viscosity of injections needed to be less than 300 cP, while a higher viscosity is required for the gel to form a depot after injection for sustained release of the drug [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]. Therefore, the gel must undergo a significant viscosity shift before and after injection. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB, Brex-PTIGs were all suitable for injectable use, with viscosities below 300 cP in the solution state, and underwent effective phase transition after injection, with a significant increase of viscosity. Among the three Brex-PTIGs, BPPG-3 showed the greatest change in viscosity, increasing from a viscosity of 27.11 cP in a solution state to 1634.96 cP in a gelled state. The steep increase in viscosity suggested a shift in the state of the gel solution, which could help reduce the initial release of Brex and prolong its release time [\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eThe surface morphologies of the gelation gels were photographed by scanning electron microscopy (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). Brex-PTIG-3 exhibited an angular-shaped surface, while the surface of Brex-PTIG-1 was sparse and porous, and Brex-PTIG-2 was smooth. This might be related to the viscosity of the gel after the phase transition [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. The more gel matrix added to the PTIG of Brex, the more viscous the gel semi-solid was, and the less porous and more angular the gel surface was.\u003c/p\u003e\n \u003cp\u003eThe complex modulus of Brex-PTIGs after gelation was also measured (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The magnitude of the coefficient of storage elasticity (G\u0026apos;) and the coefficient of loss elasticity (G\u0026apos;\u0026apos;) was associated with the denaturing of the materials[\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e]. Viscoelastic behavior (G\u0026apos; \u0026gt; G\u0026apos;\u0026apos;) was perceived for Brex-PTIGs following dynamic strain scan experiments, and the results indicated that all Brex-PTIGs were more elastic than viscous (solid-like).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec16\"\u003e\n \u003ch2\u003e3.3. Drug release studies in vitro\u003c/h2\u003e\n \u003cp\u003eBrex is insoluble in water and its release could be facilitated in release media containing ethanol. In addition, ethanol in the release medium increases the concentration-dependent diffusion outside the gel matrix, thus accelerating the degradation of the gel matrix [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e]. We also adjusted the pH of the release medium to 6.5 [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]. The above conditions were set to mimic the biodegradation situation of \u003cem\u003ein situ\u003c/em\u003e gels \u003cem\u003ein vivo\u003c/em\u003e since there might be certain acids and enzymes at the injection site [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eThe release profiles of the Brex-PTIGs were shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e. Brex in Brex-Sol was released rapidly, with a cumulative release percentage of over 90% within 1 h. In Brex-PTIG-1, Brex-PTIG-2, and Brex-PTIG-3, 29.26%, 15.59%, and 10.97% of Brex were released respectively, with significant differences compared to Brex-Sol (all p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). After 144 h, the cumulative release percentages of Brex in Brex-PTIG-1, Brex-PTIG-2, and Brex-PTIG-3 achieved 91.46%, 85.49%, and 80.62%, respectively, showing a significantly delayed release compared with Brex-sol. These results demonstrated the sustained release of Brex from Brex-PTIGs. The water-insoluble character of S100 might be the reason for the retarded release effect.\u003c/p\u003e\n \u003cp\u003eThe release profiles of the three Brex- PTIGs were generally similar (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). However, among the three Brex-PTIGs, Brex-PTIG-3 had the slowest release rate. The release rate of Brex in Brex-PTIG-3 was significantly lower than that of Brex-PTIG-1 at 1 h (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This indicated the SA was necessary for improving the initial sudden release of Brex from Brex-PTIG.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec17\"\u003e\n \u003ch2\u003e3.4. Pharmacokinetic studies\u003c/h2\u003e\n \u003cp\u003eTo investigate the pharmacokinetic properties of Brex-PTIGs \u003cem\u003ein vivo\u003c/em\u003e, healthy SD rats were injected subcutaneously with Brex-Sol, Brex-Sus, Brex-PTIG-1, Brex-PTIG-2, or Brex-PTIG-3 with a single dose of Brex at 100 mg/kg. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, the plasma concentration of Brex in the Brex-Sol group showed a significant increase within 1 h after injection, with a C\u003csub\u003emax\u003c/sub\u003e of 875.52 ng/ml followed by rapid clearance and Brex could not undetectable after 24 h. Similarly, the concentration of Brex in the Brex-Sus group reached a peak quickly, with a C\u003csub\u003emax\u003c/sub\u003e of 548.68 ng/ml (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), and then cleared quickly (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e), and Brex was undetected in plasma after 7 d. In contrast, as shown in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, the Brex-PTIGs groups had significantly delayed peaking, compared with Brex-sol and Brex-sus (all p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The peak concentration of Brex was observed around 2 h post-injection for Brex-PTIG-1, Brex-PTIG-2, and Brex-PTIG-3, with the C\u003csub\u003emax\u003c/sub\u003e of 139.30 ng/ml, 113.83 ng/ml, and 89.45 ng/ml respectively, which were significantly lower than that of Brex-sol (875.52 ng/ml) and Brex-sus (548.68 ng/ml) (all p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). A major challenge in the clinical implementation of \u003cem\u003ein situ\u003c/em\u003e molded implants was the control of the initial burst release of drugs, particularly for \u003cem\u003ein situ\u003c/em\u003e precipitation systems. Compared to solution and suspension, the Brex-PTIGs provided a significantly improved initial burst release of Brex and were able to release Brex smoothly for two months. Undoubtedly, the decreased initial burst release was attributed to the formation of Brex-PTIGs gel depots. There was no significance between the three Brex-PTIGs in the area under the concentration-time curves (AUC \u003csub\u003e(0\u0026minus;\u0026infin;)\u003c/sub\u003e), indicating the same degree of absorption of the three Brex-PTIGs. All Brex-PTIGs released Brex stably for more than 60 d, which might facilitate medication adherence in patients with schizophrenia.\u003c/p\u003e\n \u003cp\u003eAmong the three Brex-PTIGs (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), the C\u003csub\u003emax\u003c/sub\u003e of Brex-PTIG-3 was significantly lower than Brex-PTIG-1 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and the t\u003csub\u003e1/2z\u003c/sub\u003e of Brex-PTIG-3 was significantly longer than that of Brex-PTIG-1 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This was correlated with the release assay of Brex-PTIGs \u003cem\u003ein vitro\u003c/em\u003e, which indicated that although a sustained-release profile of Brex-PTIGs was observed, the composition of the gel matrix might have influenced the initial burst release. The results of \u003cem\u003ein vitro\u003c/em\u003e release and pharmacokinetics assay suggested that MCT in Brex-PTIG-1 might lessen the initial burst release of phospholipid-based phase transition \u003cem\u003ein situ\u003c/em\u003e gel, but it was not as effective as that of SA in Brex-PTIG-3, suggesting SA was a more suitable component for Brex-PTIGs.\u003c/p\u003e\n \u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp style=\"text-align: center;\"\u003ePharmacokinetic parameters after a single administration of three Brex-PTIGs (mean\u0026plusmn;SD, n=15).\u003c/p\u003e\n \u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"595\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003eParameter value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.77310924369748%\"\u003e\n \u003cp\u003eBrex-Sol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.285714285714286%\"\u003e\n \u003cp\u003eBrex-Sus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.991596638655462%\"\u003e\n \u003cp\u003eBrex-PTIG-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.991596638655462%\"\u003e\n \u003cp\u003eBrex-PTIG-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.991596638655462%\"\u003e\n \u003cp\u003eBrex-PTIG-3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003eC\u003csub\u003emax\u003c/sub\u003e (ng/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.77310924369748%\"\u003e\n \u003cp\u003e875.52\u0026plusmn;79.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.285714285714286%\"\u003e\n \u003cp\u003e548.68\u0026plusmn;55.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.991596638655462%\"\u003e\n \u003cp\u003e139.30\u0026plusmn;50.49\u003csup\u003e***,###\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.991596638655462%\"\u003e\n \u003cp\u003e113.83\u0026plusmn;20.59\u003csup\u003e***,###,$\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.991596638655462%\"\u003e\n \u003cp\u003e89.45\u0026plusmn;22.50\u003csup\u003e***,###.$$\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003eT\u003csub\u003emax\u003c/sub\u003e (h)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.77310924369748%\"\u003e\n \u003cp\u003e0.96\u0026plusmn;0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.285714285714286%\"\u003e\n \u003cp\u003e0.96\u0026plusmn;0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.991596638655462%\"\u003e\n \u003cp\u003e1.92\u0026plusmn;0.38\u003csup\u003e***,###\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.991596638655462%\"\u003e\n \u003cp\u003e2.00\u0026plusmn;0.05\u003csup\u003e***,###\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.991596638655462%\"\u003e\n \u003cp\u003e2.36\u0026plusmn;0.21\u003csup\u003e***,###\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003et\u003csub\u003e1/2z\u003c/sub\u003e (d)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.77310924369748%\"\u003e\n \u003cp\u003e0.45\u0026plusmn;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.285714285714286%\"\u003e\n \u003cp\u003e1.21\u0026plusmn;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.991596638655462%\"\u003e\n \u003cp\u003e12.48\u0026plusmn;2.69\u003csup\u003e***,###\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.991596638655462%\"\u003e\n \u003cp\u003e20.75\u0026plusmn;3.66\u003csup\u003e***,###,$\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.991596638655462%\"\u003e\n \u003cp\u003e22.07\u0026plusmn;3.54\u003csup\u003e***,###,$\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003eAUC\u003csub\u003e(0-\u0026infin;)\u0026nbsp;\u003c/sub\u003e(\u0026mu;g\u0026middot;d/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.77310924369748%\"\u003e\n \u003cp\u003e358.50\u0026plusmn;85.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.285714285714286%\"\u003e\n \u003cp\u003e770.42\u0026plusmn;79.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.991596638655462%\"\u003e\n \u003cp\u003e3303\u0026plusmn;118.95\u003csup\u003e***,###\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.991596638655462%\"\u003e\n \u003cp\u003e3344.00\u0026plusmn;169.69\u003csup\u003e***,###\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.991596638655462%\"\u003e\n \u003cp\u003e3339.37\u0026plusmn;186.69\u003csup\u003e***,###\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003eMRT\u003csub\u003e\u0026nbsp;(0-\u0026infin;)\u003c/sub\u003e (d)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.77310924369748%\"\u003e\n \u003cp\u003e0.28\u0026plusmn;0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.285714285714286%\"\u003e\n \u003cp\u003e1.23\u0026plusmn;0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.991596638655462%\"\u003e\n \u003cp\u003e26.64\u0026plusmn;3.61\u003csup\u003e***,###\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.991596638655462%\"\u003e\n \u003cp\u003e31.92\u0026plusmn;2.95\u003csup\u003e***,###\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.991596638655462%\"\u003e\n \u003cp\u003e42.47\u0026plusmn;1.98\u003csup\u003e***,###,$\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp style=\"text-align: center;\"\u003eAbbreviations: \u003csup\u003e***\u003c/sup\u003ep\u0026lt;0.001 compared to Brex-Sol; \u003csup\u003e###\u003c/sup\u003ep\u0026lt;0.001 compared to Brex-Sus; \u003csup\u003e$\u003c/sup\u003ep\u0026lt;0.05 and \u003csup\u003e$$\u003c/sup\u003ep\u0026lt;0.01 compared to Brex-PTIG-1.\u003c/p\u003e\n \u003ch2\u003e3.5 Biodegradability and biocompatibility of the Brex-PTIG system in vivo\u003c/h2\u003e\n \u003cp\u003eThe state of the skin around the injection site was observed on days 0, 7, 14, and 21 after the injection. As shown in Fig. 7, on Day 0 an injection bulge could be seen in the Brex-PTIGs and Brex-sus groups, while the skin seemed smooth and flat with no discernable bulge in the Brex-sol group. On Day 7 after the injection, the volume of the injection bulge significantly decreased in all Brex-PTIGs groups. By Day 14, the skin at the injection site was almost flat in the Brex-PTIGs group. \u0026nbsp;\u003c/p\u003e\n \u003cp\u003eTo further evaluate the degradation of Brex-PTIGs \u003cem\u003ein vivo\u003c/em\u003e, the depots formed after subcutaneous injection of Brex-PTIGs were dissected from the rat skin and weighed on different days after injection. The weight change of the residual gel was displayed in Fig. 8A. The weight of the residual Brex-PTIG-1 and Brex-PTIG-2 increased during the 0-5 d after injection and then decreased. This was consistent with the previous study. In a comparative study of \u003cem\u003ein situ\u003c/em\u003e gels performed by Zhang et. al., the gel formulation with the best performance (better viscosity and best sustained-release characteristics) showed a slow increase and degradation of the gel weight \u003cem\u003ein vivo\u003c/em\u003e [14]. The residual weight of Brex-PTIGs at Day 60 all decreased to 10% of the initial weight.\u003c/p\u003e\n \u003cp\u003eOrganic solvents located in the\u003cem\u003e\u0026nbsp;in situ\u003c/em\u003e precipitation system may irritate the injection site. The inflammatory reaction is the main side effect after the local injection of \u003cem\u003ein situ\u003c/em\u003e gel\u0026nbsp;[45]. Thus, we examined the compatibility and inflammatory effect of Brex-PTIGs at the injection site after a single subcutaneous injection. At first, the skin around the injection site was dissected and observed on 7 d after injection. As shown in Fig. 8B, the yellowish gel-like depot of Brex-PTIGs was with smooth and flat edges and was covered by a transparent biofilm. However, in Brex-Sol and Brex-Sus groups, signs of bleeding and inflammations were observed. A partial white fatty accumulation at the injection site was also observed.\u003c/p\u003e\n \u003cp\u003eThen, the skin tissues at the injection site were fixed, sliced, and stained with hematoxylin and eosin (HE) for histological analysis. As shown in Fig. 9, none of the Brex-PTIGs showed a severe inflammatory response at the injection site over the 21 d following administration. However, for Brex-Sol and Brex-Sus groups, on the first day after injection, the structure of adipocytes was severely disrupted with massive cell necrosis, in addition, infiltration of lymphocytes, granulocytes, and other inflammatory cells into the tissues was also observed on Day 7 after injection, with the formation of large amounts of granulation and fibrous connective tissue. On Day 21, the skin conditions of all groups recovered to normal. These results indicated the Brex-PTIGs were more biocompatible than solution and suspensions \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003eThe biocompatibility and mild inflammatory response of Brex-PTIGs might be owing to the biocompatible excipients and their reasonable adding amount. The matrix in Brex-PTIGs was all biocompatible, including S100 and SA. The solvents used in the Brex-PTIGs were MCT, ethanol, and NMP, which were seen as safe [45, 46]. As an FDA-approved solvent for injectable use, the safety of NMP could be effectively assured, and the low percentage of ethanol (less than 10%) might help reduce skin irritation, as demonstrated in previous studies [32]. The content of solvents in Brex-PTIGs was controlled strictly to ensure the safety of Brex-PTIGs.\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eIn this study, an injectable \u003cem\u003ein situ\u003c/em\u003e gel of Brexpiprazole (Brex) based on phospholipid-based phase transition consisting of S100, MCT/SA, NMP, and ethanol was constructed.\u0026nbsp;Brex-PTIGs in solution state were suitable for injection, and when got in contact with water \u003cem\u003ein vivo\u003c/em\u003e, they rapidly transformed to a semi-solid gel state. The Brex-PTIGs had a good sustained release of Brex \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. The preliminary safety profile was also confirmed in this study. This study for the first time confirmed the feasibility of injectable \u003cem\u003ein situ\u003c/em\u003e gel based on phospholipid-based phase transition for the sustained delivery of Brex, which holds high potential in developing a valid, safe, easy-producing, and low-cost preparation of Brex for the long-term treatment of schizophrenia.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study leading to the reported results was funded by the Program of Postgraduate Joint Training Base of Chongqing (Chongqing Medical University\u0026amp; Yaopharma Co., Ltd joint training base for postgraduate in pharmacy) (to Huali Chen and Yan Zhang), and the Science and Technology Research Program of Chongqing Municipal Education Commission (grant no. KJQN202000407; to Huali Chen).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRan Tao: Conceptualization, Methodology, Investigation, Data curation, Writing an original draft, Visualization. Li Liu: Methodology, Investigation, Data curation, Visualization\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003eSupervision, Writing - review \u0026amp; editing. Yingxin Xiong: Methodology, Investigation,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eResources, Supervision. Qianyu Zhang: Methodology, Investigation\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003eWriting - review \u0026amp; editing. Xiangyu Lv: Data curation, Formal analysis. Linbo He: Data curation, Formal analysis, Resources. Fang Ren: Validation, Methodology. Lu Zhou: Methodology, Software. BaoYan Chen: Formal analysis, Software. Kexin Wu: Validation, Visualization. Yan Zhang: Project administration,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eResources, Supervision. Huali Chen: Funding acquisition, Conceptualization, Supervision,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eWriting - review \u0026amp; editing. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of Chongqing Management Approach of Laboratory Animal (chongqing government order NO.195).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data presented in this study are available upon request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMai, Y., et al., \u003cem\u003eTopical formulation based on disease-specific nanoparticles for single-dose cure of psoriasis.\u003c/em\u003e J Control Release, 2022. \u003cstrong\u003e349\u003c/strong\u003e: p. 354-366.\u003c/li\u003e\n\u003cli\u003eXu, W., et al., \u003cem\u003eAnalysis of Factors Influencing Telemedicine-Based Psychiatric Extended Care and Care of Psychiatric Patients.\u003c/em\u003e J Healthc Eng, 2022. \u003cstrong\u003e2022\u003c/strong\u003e: p. 9434820.\u003c/li\u003e\n\u003cli\u003eFerraiolo, M., et al., \u003cem\u003eReceptor density influences the recruitment bias of aripiprazole and brexpiprazole at the dopamine D2L receptor.\u003c/em\u003e Fundam Clin Pharmacol, 2022.\u003c/li\u003e\n\u003cli\u003e\u003cem\u003eBrexpiprazole for schizophrenia.\u003c/em\u003e Aust Prescr, 2017. \u003cstrong\u003e40\u003c/strong\u003e(5): p. 197-198.\u003c/li\u003e\n\u003cli\u003eOrsolini, L., et al., \u003cem\u003eA case report of clozapine-treatment-resistant schizophrenia successfully managed with brexpiprazole combination therapy.\u003c/em\u003e Asian J Psychiatr, 2022. \u003cstrong\u003e72\u003c/strong\u003e: p. 103121.\u003c/li\u003e\n\u003cli\u003eAladeen, T., et al., \u003cem\u003eThe use of brexpiprazole amongst individuals with insufficient outcomes with aripiprazole or bupropion: A case series.\u003c/em\u003e Perspect Psychiatr Care, 2018. \u003cstrong\u003e54\u003c/strong\u003e(4): p. 507-513.\u003c/li\u003e\n\u003cli\u003e\u003cem\u003eBrexpiprazole (Rexulti) for schizophrenia and depression.\u003c/em\u003e Med Lett Drugs Ther, 2015. \u003cstrong\u003e57\u003c/strong\u003e(1475): p. 116-8.\u003c/li\u003e\n\u003cli\u003eFrampton, J.E., \u003cem\u003eBrexpiprazole: A Review in Schizophrenia.\u003c/em\u003e Drugs, 2019. \u003cstrong\u003e79\u003c/strong\u003e(2): p. 189-200.\u003c/li\u003e\n\u003cli\u003eCrapanzano, C., et al., \u003cem\u003eBrexpiprazole 2 mg Starting Dose: A Case Series.\u003c/em\u003e Psychiatr Danub, 2022. \u003cstrong\u003e34\u003c/strong\u003e(2): p. 308-309.\u003c/li\u003e\n\u003cli\u003eChitkara, D., et al., \u003cem\u003eBiodegradable injectable in situ depot-forming drug delivery systems.\u003c/em\u003e Macromol Biosci, 2006. \u003cstrong\u003e6\u003c/strong\u003e(12): p. 977-90.\u003c/li\u003e\n\u003cli\u003eFakhari, A. and J. 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Mader, \u003cem\u003eIn situ forming implants - an attractive formulation principle for parenteral depot formulations.\u003c/em\u003e J Control Release, 2012. \u003cstrong\u003e161\u003c/strong\u003e(2): p. 668-79.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"drug-delivery-and-translational-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ddtr","sideBox":"Learn more about [Drug Delivery and Translational Research](https://www.springer.com/journal/13346)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ddtr/default.aspx","title":"Drug Delivery and Translational Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Phospholipids, In situ gel, Long-acting injection, Sustained-release, Schizophrenia","lastPublishedDoi":"10.21203/rs.3.rs-2363215/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2363215/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe objective of this study was to develop phospholipid-based injectable phase transition \u003cem\u003ein situ\u003c/em\u003e gels (PTIGs) for the sustained release of Brexpiprazole (Brex). Phospholipid (Lipoid S100, S100) and stearic acid (SA) were used as the gel matrix which was dissolved in biocompatible solvent medium-chain triglyceride (MCT), \u003cem\u003eN\u003c/em\u003e-methyl pyrrolidone (NMP), and ethanol to obtain PTIGs solution. The Brex PTIG showed a solution condition of low viscosity in vitro and was gelatinized \u003cem\u003ein situ\u003c/em\u003e in vivo after subcutaneous injection. Both \u003cem\u003ein vitro\u003c/em\u003e release assay and \u003cem\u003ein vivo\u003c/em\u003e pharmacokinetics study in SD rats displayed that Brex in PTIGs could achieve a sustained release, compared with brexpiprazole solution (Brex-Sol) or brexpiprazole suspension (Brex-Sus). The Brex-PTIGs had good degradability and biocompatibility \u003cem\u003ein vivo\u003c/em\u003e with rare inflammation at the injection site. Among the three Brex-PTIG formulations, Brex-PTIG-3 with the SA in the formulation had the greatest gelation viscosity, the lowest initial release rate, and the most stable release profile with sustained release of up to 60 days. The above results indicated that, as a novel drug delivery system, the Brex-PTIGs offered a new option for the clinical treatment of patients with schizophrenia.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Construction and evaluation of a phospholipid-based phase transition in situ gel system for brexpiprazole","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-01-03 15:41:47","doi":"10.21203/rs.3.rs-2363215/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2023-01-30T02:14:11+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2022-12-30T14:12:25+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-12-27T11:32:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-12-13T07:19:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Drug Delivery and Translational Research","date":"2022-12-12T10:13:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"drug-delivery-and-translational-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ddtr","sideBox":"Learn more about [Drug Delivery and Translational Research](https://www.springer.com/journal/13346)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ddtr/default.aspx","title":"Drug Delivery and Translational Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b7625819-86c2-4f8e-8552-f4214126abf3","owner":[],"postedDate":"January 3rd, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T20:59:48+00:00","versionOfRecord":{"articleIdentity":"rs-2363215","link":"https://doi.org/10.1007/s13346-023-01349-0","journal":{"identity":"drug-delivery-and-translational-research","isVorOnly":false,"title":"Drug Delivery and Translational Research"},"publishedOn":"2023-05-09 20:48:57","publishedOnDateReadable":"May 9th, 2023"},"versionCreatedAt":"2023-01-03 15:41:47","video":"","vorDoi":"10.1007/s13346-023-01349-0","vorDoiUrl":"https://doi.org/10.1007/s13346-023-01349-0","workflowStages":[]},"version":"v1","identity":"rs-2363215","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2363215","identity":"rs-2363215","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}