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. 1A2, 1B2, and 1C2).
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. 2A, 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. 2A). After gelation, the viscosity of the Brex-PTIGs increased significantly (p < 0.001, Fig. 2B).
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. 2B, 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. 3A and 3B). 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 Cmax 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 Cmax 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 Cmax 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 Cmax of Brex-PTIG-3 was significantly lower than Brex-PTIG-1 (p < 0.01), and the t1/2z 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
|
Cmax (ng/mL)
|
875.52±79.45
|
548.68±55.86
|
139.30±50.49***,###
|
113.83±20.59***,###,$
|
89.45±22.50***,###.$$
|
Tmax (h)
|
0.96±0.17
|
0.96±0.09
|
1.92±0.38***,###
|
2.00±0.05***,###
|
2.36±0.21***,###
|
t1/2z (d)
|
0.45±0.06
|
1.21±0.08
|
12.48±2.69***,###
|
20.75±3.66***,###,$
|
22.07±3.54***,###,$
|
AUC(0-∞) (μg·d/L)
|
358.50±85.94
|
770.42±79.65
|
3303±118.95***,###
|
3344.00±169.69***,###
|
3339.37±186.69***,###
|
MRT (0-∞) (d)
|
0.28±0.13
|
1.23±0.20
|
26.64±3.61***,###
|
31.92±2.95***,###
|
42.47±1.98***,###,$
|
Abbreviations: ***p<0.001 compared to Brex-Sol; ###p<0.001 compared to Brex-Sus; $p<0.05 and $$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.