Long-acting injectable in situ gel of rasagiline: a patented product development

Rasagiline has a certain potential in neuroprotection and delaying the progression of Parkinson’s disease (PD). However, the poor pharmacokinetics (PK) characteristics of conventional oral tablets and poor medication compliance limit the optimal efficacy of rasagiline. Based on this, we designed and optimized a sustained-release rasagiline in situ gel based on in vitro release and in vivo PK results. Among them, we found for the first time that aluminum hydroxide can effectively shorten the lag phase and promote early and late release, making the daily release more uniform. After subcutaneous administration of the optimized gel formulation at a monthly dose, the Cmax (64 ng/ml) was lower than that of free rasagiline (494 ng/ml) administered subcutaneously at a daily dose and comparable to that of oral administration of Azilect® (59.1 ng/ml) at a daily dose. In the meantime, the plasma concentration of rasagiline was mainly maintained at 5–10 ng/ml for about 1 month, and the active metabolite 1-aminoindane in plasma was also able to maintain a steady state. The rasagiline in situ gel has suitable viscosity and injectability, good repeatability of subcutaneous injection, and controllable impurities and can achieve sustained release in vivo with small burst release, which may have the clinical application advantages of maximizing the disease-modifying effect of rasagiline and improving medication compliance. The rasagiline in situ gel was optimized through the feedback of in vitro release and in vivo pharmacokinetics (PK), in which the addition of aluminum hydroxide had a modulating effect on uniform release. The gel has low burst release and maintains steady-state blood drug concentration for about 1 month.


Introduction
Parkinson's disease (PD) is a common neurodegenerative disease, and its main pathological change is the irreversible degeneration of dopaminergic neurons in the substantia nigra of the midbrain, resulting in a significant decrease in striatal dopamine content and causing disease. At the time of diagnosis of PD, patients have only 30% of dopaminergic neurons remaining, but about 50-60% of their axonal terminals have died [1]. The main clinical manifestations of PD are resting tremor, bradykinesia, rigidity, and postural gait disturbance. Currently, PD cannot be completely cured. Drugs are the main means of treating PD, and most patients need to take medicines for life. Dopamine (DA) replacement therapy with levodopa, DA agonists, and others has remained the standard of care for PD since the 1960s [2]. Although this drug treatment has significantly improved the quality of life of many PD patients, its efficacy diminishes over time and leads to serious side effects such as on-off effects and dyskinesias. Although there are alternative surgical treatments, such as deep-brain stimulation [3], both forms of treatment are symptomatic and do not prevent or modify disease progression. Thus, there is a significant unmet need to develop neuroprotective and disease-modifying therapies for PD.
Rasagiline is a selective irreversible monoamine oxidase inhibitor that increases DA levels and indirectly increases dopaminergic activity in the striatum, and rasagiline and its metabolite 1-aminoindane have potential neuroprotective effects [4]. Animal and cell studies concluded that rasagiline increases antioxidant capacity (SOD, catalase, bcl-2) and protects against the toxic effects of peroxynitrite, MPTP, and 6-OHDA [5][6][7]. In addition, rasagiline was observed to increase the survival of dopaminergic neurons in an in vitro culture study of human brain tissue [8]. In the clinical phase, TEMPO (TVP-1012 in Early Monotherapy for PD Outpatients Study) and its long-term results showed that subjects who received rasagiline for 12 months provided greater longterm benefit and less functional decline compared to subjects * Chia-Wen Liu liujiawen@simcere.com 1 State Key Laboratory of Translational Medicine and Innovative Drug Development, Jiangsu Simcere Pharmaceutical Co., Ltd., 210042 Nanjing, China who delayed treatment for 6 months [9,10]. Another ADA-GIO (Attenuation of Disease Progression with Azilect ® Given Once-daily) clinical study showed that early treatment with rasagiline 1 mg per day provided benefits consistent with disease-modifying effects, but early treatment with rasagiline 2 mg per day did not [11]. Despite the confusion, there is no doubt that rasagiline is still the clinical treatment drug with the most potential to delay the progression of PD. At present, there is only one dosage form of rasagiline mesylate tablets on the market. The original research company is Teva, and its trade name is Azilect ® . Frequent fluctuations in plasma concentrations are generally not beneficial for improvement in disease progression, and a durable steady-state pharmacokinetics (PK) profile may maximize the potential neuroprotective effects of rasagiline [12][13][14]. In addition, as a typical chronic disease of the elderly, the prevalence of PD has doubled in the elderly population [15,16]. Maintaining good medication compliance is the key to relieving symptoms of the disease; however, missed doses often occur in elderly patients. Research institutions have conducted a survey on medication compliance and persistence of PD patients (n = 29,682), and it was found that about half of the population had difficulty achieving continuous medication for 60 days [17]. Tarrants et al. [18] found that patients who persisted on antiparkinsonian medication were 67% less likely to have worsening PD symptoms. Therefore, rasagiline has a demand for the development of sustained-release formulations in terms of enhancing potential neuroprotection or improving medication compliance.
Herein, we designed a sustained-release rasagiline in situ gel and optimized the formulation through the feedback of in vitro release and in vivo PK. We found for the first time that the introduction of aluminum hydroxide had a uniform modulation of rasagiline release, and thus, we obtained a patented candidate formulation. In addition, a series of clinical application-related characterizations such as viscosity, injectability, and in vitro and in vivo gel solidification have also been carried out to ensure that the gel formulation can be applied to the human body. Finally, stability changes during in vitro release and long-term storage were also investigated to obtain products with controlled impurities. Overall, the in situ gel has a small burst release and maintains steady-state blood drug concentration for about 1 month, which has a promising application prospect in the continuous improvement of PD progression and medication compliance.

Preparation of in situ gel
Gel formulations were prepared according to the ingredients shown in Table 1. First, rasagiline mesylate or rasagiline and a polymer such as PLGA 752H, PLGA 753H, PLGA 85-2A, or PLA 202H were dissolved in the prescribed amount of NMP by a vortex mixer (Kylin-Bell, China). The solution was then transferred to one end of a dual-chamber syringe, and the prescribed amount of aluminum hydroxide or magnesium hydroxide was added to the other end. The final in situ gel was obtained by pushing and pulling the syringe back and forth to mix the matrix solution evenly.

In vitro drug release
The prepared in situ gel was added to the dialysis tubing (MWCO 3500 Da). Each tubing contains approximately 12.7 mg equivalents of rasagiline (n = 3). The dialysis tubing was placed in a 5 ml PBS (pH 7.4) release medium, and the drug release was investigated in a DSHZ-300A thermostatic water bath (Peiying Experimental Equipment Co. Ltd., China) at 37 °C and 100 rpm. At predetermined time points, the release medium was removed and replaced with an equal weight of fresh PBS (pH 7.4). The release of rasagiline was quantified using an Agilent 1260 HPLC system (Agilent, USA). An ACE Excel3 AR-C18 column (150 mm × 4.6 mm, 3 µm) (ACE, UK) was used with a mobile phase consisting of acetonitrile and 0.4% perchloric acid (20:80). The column temperature was maintained at 30 °C, the flow rate was set to 1 ml/min, and the detection wavelength was 265 nm.

In vivo pharmacokinetics
Animal experiments were conducted according to the Guide for the Care and Use of Laboratory Animals issued by the Institutional Animal Ethical Care Committee of Simcere Pharmaceutical Group Limited. Male Sprague-Dawley rats (220-250 g) were subcutaneously administered free rasagiline mesylate (1 mpk, rasagiline equivalent), free rasagiline mesylate (30 mpk, rasagiline equivalent), or in situ gels (30 mpk, rasagiline equivalent) after anesthesia (n = 3). At predetermined time points, blood samples were collected by the jugular vein, and the blood in EDTA tubes was centrifuged to obtain the plasma.
An aliquot of 50 µl of plasma sample, 25 µl of rasagiline-13 C3 mesylate (as internal standard), and 25 µl of 0.1 N sodium hydroxide were added to a vial of 96-well plate and vortex-mixed for 3 min. Three hundred microliters of ethyl acetate was added into the 96-well plate. After vortexing (3 min) and centrifugation (15 min, 5000 rpm, 4 °C), the supernatant was transferred to another 96-well plate and evaporated to dryness under a nitrogen stream at 40 °C. The remaining residue was reconstituted with 200 µl of 10 mM ammonium formate (pH 7.5)-acetonitrile (95:5) and injected into the LC-MS/MS. Chromatographic separation was achieved using a BEH C18 column (2.1 mm × 50 mm, 1.7 µm) (Waters, USA). The mobile phase consisted of acetonitrile (mobile phase B) and 10 mM ammonium formate (pH 7.5) (mobile phase A). The flow rate was maintained at 0.3 ml/min, and the column temperature was set at 40 °C. The gradient was as follows: 5% B from 0 to 0.3 min, 5% B-90% B from 0.3 to 1.5 min; 90% B from 1.5 to 2 min; 90% B-5% B from 2 to 2.5 min, and 5% B until 3.5 min for column equilibration prior to the next injection. Quantification was performed by multiple reaction monitoring (MRM) with transitions from m/z 172.142 to 117.100 for rasagiline, m/z 134.134 to 117.100 for 1-aminoindane, and m/z 175.3 to 117.100 for rasagiline-13 C3. PK parameters were obtained by data analysis using Phoenix WinNonlin software (Pharsight, USA).

Viscosity measurement and syringeability
The viscosity of the gel matrix was determined by using a DVNXRVCP digital viscometer (Brookfield, USA) equipped with a CPA-40Z spindle. The amount of gel added was 0.5 ml, and the measurement temperature was controlled at 25 °C (n = 3). In addition, the gel was pulled into the syringe, and the needles of different sizes (25,23,22,21,20, and 18G) were replaced to test and record the difficulty of the gel passing.

In vitro and ex vivo matrix solidification
0.25 ml of gel matrix was added to a tube containing 5 ml of PBS (pH 7.4) via a syringe. In addition, PBS was changed every 2 days. At 5, 15, and 30 min; 1, 3, and 6 h; and 1, 3, and 7 days of gel incubation at room temperature, the gel was removed, cut open, and photographed. For ex vivo solidification studies, the gel matrix was injected subcutaneously into fresh pork through a syringe equipped with a 20G needle (n = 3). After 1 day of incubation at room temperature, the pork was dissected, and the solidified gel was isolated and photographed.

Gel stability during in vitro release
The experimental procedure of gel stability during release was consistent with the previously mentioned in vitro release. At 0, 10, and 25 days of drug release, the gel was taken out to investigate the purity of rasagiline and the molecular weight change of PLA. For rasagiline purity determination, the gel was dissolved in DMSO by sonication, diluted with water, centrifuged, and analyzed by an Agilent 1260 HPLC system (Agilent, USA). An ACE Excel3 AR-C18 column (150 mm × 4.6 mm, 3 µm) (ACE, UK) was used with a mobile phase consisting of acetonitrile and 0.4% perchloric acid. Ratio change of acetonitrile are as follows: 0-5 min, 5%; 5-30 min, 5-40%; 30-45 min, 40-80%; 45-45.1 min, 80-5%; 45.1-55 min, 5%. The column temperature was maintained at 40 °C, the flow rate was set to 0.8 ml/min, and the detection wavelength was 265 nm. For the molecular weight analysis of PLA, the gel was dissolved in tetrahydrofuran and measured by an Agilent 1260 HPLC system (Agilent, USA) equipped with a refractive index detector. A Waters Styragel HR 4E THF column (300 mm × 7.8 mm) (Waters, USA) was used, and the mobile phase consisted of pure tetrahydrofuran. The column temperature was maintained at 30 °C, the flow rate was set to 0.5 ml/min, and the detection wavelength was 265 nm.

Storage stability of in situ gel
The long-term storage stability of six groups of mixtures was investigated. The prescribed amount of the mixture was packed in vials and placed in a 4 °C refrigerator. At 0, 1, and 3 months, the mixture was determined for rasagiline purity and PLA molecular weight. Both assays were consistent with the previously mentioned gel stability studies during in vitro release.

Optimization of sustained-release in situ gel via in vitro release combined with in vivo PK
We designed a series of formulations (Table 1) and optimized the rasagiline in situ gel through the feedback of in vitro release and in vivo PK. We first examined the in vitro release of formulations F1-F4, and as shown in Fig. 1a, the burst release of rasagiline mesylate formulations (F1 and F3) was higher than that of rasagiline formulations (F2 and F4). Among them, the burst release of F3 was about 60%, that of F1 was about 30%, and that of F2 and F4 was not higher than 20%. Correspondingly, as shown in Fig. 1b and Table 2, the C max of F1 (8473 ng/ml) was much higher than that of F2 and F4 (2620 ng/ml and 614 ng/ml). This result is consistent with literature reports that the salt form is inferior to the base form in controlling burst release due to better water solubility [19,20]. However, the C max of F2 and F4 was still higher compared to the rasagiline-free drug (FD)-1mpk group (494 ng/ml), indicating that the formulation still needs to be optimized to avoid possible C max -related side effects ( Fig. 1b and Table 2). In addition, the in vitro release slope of rasagiline formulations (F2 and F4) was faster than that of rasagiline mesylate formulations (F1 and F3), corresponding to maintaining higher steady-state plasma concentrations in vivo (Fig. 1a, b). However, no drug was detected in rat plasma from all groups after 11 or 14 days, suggesting a need for slower-release formulations (Fig. 1b). Therefore, our follow-up design of rasagiline formulations focused Fig. 1 a In vitro release of rasagiline from F1, F2, F3, and F4 formulations (n = 3). b Plasma concentration-time profiles of rasagiline in rats after subcutaneous injection of rasagiline-free drug (FD) (1mpk or 30mpk, rasagiline equivalent) or in situ gel (F1, F2, or F4) (30mpk, rasagiline equivalent) (n = 3). The inserted picture represents the PK profiles from 0 to 1 day on achieving two goals: (1) low burst release, with C max lower than that of FD-1mpk; (2) slow release, maintaining steady-state plasma concentration for 1 month.
As shown in Fig. 2a, the ratio of PLGA to NMP was unchanged at 1:1.5, and the in vitro burst release was significantly reduced when the ratio of rasagiline to PLGA was adjusted from 1:9 to 1:18 (F5 vs. F2). In addition, by adjusting the ratio of PLGA to NMP from 1:1.5 to 1:1, the burst release in vitro was further decreased (F6 vs. F5). The results showed that increasing the amount of polymer or reducing the amount of NMP could reduce the burst release well [21]. To achieve a slower release, as shown in Fig. 2b, the release of PLGA 85/15 or PLA formulations was investigated, as the higher the lactic acid (LA) ratio, the slower the polymer degradation [22]. Compared to PLGA 75/25, the release of the PLGA 85/15 formulation was somewhat slower (F7 vs. F6). Notably, the release of the PLA formulation (F7) was slower and more in line with our needs. The F7 formulation released 28% of rasagiline from 0 to 24 days, while 39% from 24 to 32 days, and finally only 10% from 32 to 40 days. Too long a lag phase in the early stage, uneven release, and too slow a release in the later stage limit the application of F7 formulation.
Tricalcium phosphate, calcium hydrogen phosphate, sodium hydrogen phosphate, and magnesium hydroxide have been reported to slow down PLGA/PLA hydrolysis due to acid neutralization [23][24][25][26], but it is unclear whether they can improve the release of rasagiline in situ gel. Thus, we chose magnesium hydroxide and the less irritating aluminum hydroxide to modulate the release behavior in vitro. Fig. 2 a In vitro release of rasagiline from F5 and F6 compared to F2 (n = 3). b In vitro release of rasagiline from F7 and F8 compared to F6 (n = 3). c In vitro release of rasagiline from F9 and F10 compared to F6 (n = 3). d In vitro release of rasagiline from F11 and F12 compared to F8 (n = 3). e The speculated mechanism concerns the difference between aluminum hydroxide and magnesium hydroxide in promoting or inhibiting the hydrolysis release of rasagiline in situ gel. The green circle represents lactic acid As shown in Fig. 2c, compared with the F6 formulation, the addition of aluminum hydroxide (F9) could effectively shorten the early lag phase and accelerate the release, while the addition of magnesium hydroxide (F10) did not change the early release. However, both aluminum hydroxide and magnesium hydroxide were able to accelerate the late release of the formulation. As shown in Fig. 2d, when aluminum hydroxide was added to the PLA formulation F8 (F11), the early lag phase was also shortened, and the release was accelerated, and the overall release remained uniform, while the addition of magnesium hydroxide (F12) even somewhat slowed the drug release. Therefore, in general, the introduction of aluminum hydroxide has a beneficial effect on the release of the formulation. This difference has not been reported in the previous literature. As shown in Fig. 2e, it is speculated that both aluminum hydroxide and magnesium hydroxide can play two roles. One is that the self-alkaline promotes the hydrolysis of PLGA/PLA and accelerates the release; the other is the self-alkaline neutralizes acidic degradation products and inhibits acidic autocatalytic hydrolysis. For weakly alkaline aluminum hydroxide (saturation pH 8.3), its acid neutralization ability is weak, thus slightly inhibiting the autocatalytic hydrolysis of acidic products. Conversely, self-alkaline promotes polymer hydrolysis, resulting in enhanced release overall. For more alkaline magnesium hydroxide (saturation pH 10.3), its acid neutralization ability is strong, and the autocatalytic hydrolysis of acidic products is significantly inhibited. In addition, the remaining magnesium hydroxide also promotes polymer hydrolysis, and the overall performance is that the release rate is unchanged or slightly inhibited. For the late release, the accumulation of acidic hydrolysis products is less, and both aluminum hydroxide and magnesium hydroxide can promote its late release. Of course, the speculated mechanism needs to be verified by later studies.
To verify the in vivo PK behavior of the aluminum hydroxide-regulated F11 formulation, we first adjusted its PLA to NMP ratio from 1:1 to 1:0.7 to obtain the F13 formulation to improve viscosity and subcutaneous retention. As shown in Fig. 3a, further reduction of NMP did not result in further reduction of burst release in vitro. However, in vivo C max could be decreased from 407 to 83 ng/ml, indicating that in vitro release does not fully reflect in vivo PK (Fig. 3b and Table 3). The magnesium hydroxide formulation (F14) Fig. 3 a In vitro release of rasagiline from F13 and F14 compared to F11 (n = 3). b Plasma concentration-time profiles of rasagiline in rats after subcutaneous injection of in situ gel (F11, F13, or F14) (30mpk, rasagiline equivalent) (n = 3). The inserted picture represents the PK profiles from 0 to 1 day Table 3 Pharmacokinetic parameters of rasagiline and 1-aminoindane (AI) in rats after subcutaneous injection of in situ gel (F11, F13, F14, or F15) (30mpk, rasagiline equivalent) (n = 3) "*" indicates that it is quoted from the original research data of rasagiline oral tablet (Azilect ® ), representing the pharmacokinetic parameters in rats after a single oral administration of rasagiline mesylate (1mpk, rasagiline equivalent) showed slow release in the early stage and fast release in the later stage. Correspondingly, the plasma drug concentration was low in the first 9 days, with a minimum of 1.85 ng/ml, and then increased with a maximum of 11.7 ng/ml (Fig. 3b). Such large fluctuations in plasma concentrations are unacceptable to avoid possible off periods and dyskinesia during PD treatment [27]. It is worth noting that the F13 formulation not only exhibited a small burst release in vivo but also a stable blood concentration, which is very suitable for our optimization goal ( Fig. 3b and Table 3). The fly in the ointment is that the sustained-release period in the body is greater than 1 month. In order to shorten the sustained-release time of F13, the ratio of rasagiline to PLA was adjusted from 1:18 to 1:12 under the constant ratio of PLA to NMP of 1:0.7, and the aluminum hydroxide still maintained 1% of PLA. That is to get the F15 formulation ( Table 1). As shown in Fig. 4a, the in vitro release of F15 was faster than that of F13. And the plasma concentration of F15 decreased significantly after 31 days and was only 0.45 ng/ml on the 35th day (Fig. 4b).
In addition, the C max of F15 was 64 ng/ml, which was much lower than 494 ng/ml of FD-1mpk and comparable to 59.1 ng/ml of Azilect ® -1mpk (Tables 2 and 3). Notably, 1-aminoindan, the active metabolite of rasagiline released by F15, also maintained steady-state concentrations well (Fig. 4b), which is critical for its potential neuroprotection [28]. In conclusion, the F15 formulation achieves the goal of low burst release and maintaining steady-state blood drug concentration for 1 month, which is the optimal formulation for product development.

Characterization of optimal in situ gel formulation
After obtaining the optimal formulation of F15, we performed some important characterizations relevant to clinical applications, including viscosity, injectability, and in vitro or ex vivo gel solidification. As shown in Table 4, the viscosity of the F15 gel matrix was determined to be approximately 2150 cp. Proper viscosity allows the gel to reside well subcutaneously, but a matrix that is too viscous can be difficult and painful to inject [21,29]. When we simulated the actual administration, we found that the 20G needle could ensure the smoothness of subcutaneous injection ( Fig. 5a and Table 4), and this type of needle is also commonly used for subcutaneous administration of other marketed gel preparations.
Next, we injected the F15 gel matrix into PBS (pH 7.4) to investigate the in vitro gel solidification properties. As shown in Fig. 5b, the gel matrix fixed its shape upon contact with the aqueous medium. With the solvent exchange of NMP with water, the surface shell thickness of the gel increased until the inside and outside were completely solidified. To date, among the different mechanisms of in situ forming implants, only in situ polymer precipitation systems based on solvent removal have been commercialized [30]. To simulate clinical application, the F15 gel matrix  was loaded into a syringe equipped with a 20G needle and injected subcutaneously into fresh pork, which is generally structurally similar to human skin [31]. As shown in Fig. 5c, the gel was dissected 1 day after injection, and it was found that the gel had obvious solid-state strength and showed a reproducible ellipsoid shape.

Stability study of optimal in situ gel formulation
It is well known that stability studies are critical for the development of new formulations. Here, we examined changes in the purity of rasagiline and the molecular weight of PLA during in vitro release from the gel. As shown in Table 5, the molecular weight of PLA in the optimal formulation F15 decreased significantly after 10 days, in which Mw was degraded from 18,928 to 5658, and Mn was degraded from 10,777 to 2794. Overall, the molecular weight decreased by about 70% compared with 0 days. On the 25th day, compared with the 10th day, the molecular weight of PLA decreased by only about 50%, indicating that the degradation rate of PLA in the gel was first fast and then slow.
With the degradation of PLA, the purity of rasagiline decreased from 99.75 to 94.27% at 10 days, presumably due to the interaction with PLA degradation products to generate some impurities (data not shown). While this change in purity is acceptable, on the one hand, the amount of impurities generated is small and controllable (about 5%). On the other hand, PLA is an FDA-approved excipient that is confirmed to be safe [32], and the impurities generated by PLA and rasagiline are theoretically not toxic to humans. Additionally, the purity of rasagiline in the gel returned to normal at day 25 as the PLA degradation rate decreased.
Next, as shown in Table 6, to determine the storage mode of the formulation, the storage stability of the F15 formulation and some of its compositions at 4 °C was examined. The choice   of temperature refers to the labels of Eligard ® and Atridox ® , in which PLGA and PLA can maintain molecular weight stability at 4 °C. As shown in the results in Table 7, with or without the addition of aluminum hydroxide, the mixing of rasagiline with the NMP solution of PLA could lead to a decrease in the molecular weight of PLA and the purity of rasagiline, indicating that the prepared preparation cannot be stored directly. In addition, the purity of rasagiline alone remained unchanged during 3 months of storage, while the addition of aluminum hydroxide resulted in a negligible decrease in purity. The NMP solution of PLA also showed good molecular weight stability, and the addition of aluminum hydroxide did not change its stability results. Therefore, the present results support a dual-chamber syringe storage model, in which one syringe is filled with rasagiline or a mixture of rasagiline and aluminum hydroxide, and the other is filled with a mixture of aluminum hydroxide and PLA in NMP or PLA in NMP alone. The final prepared preparation can be obtained by pushing and pulling the dual-chamber syringe back and forth before use.

Conclusion
In conclusion, we designed and optimized a long-acting rasagiline in situ gel formulation from the perspective of enhancing the potential neuroprotective and disease-modifying effects of rasagiline and improving medication compliance. Facts have proved that the addition of aluminum hydroxide to the gel formulation has the beneficial effect of regulating the uniform release of the gel matrix, and the effect is different from that of magnesium hydroxide. This difference is the first report, and we still need further research on its true mechanism in the later stage. The obtained formulation exhibited a small burst release and maintained steady-state plasma concentration for about 1 month, which was in line with the final optimization goal. The PK characteristics of a sustained release may be beneficial to strengthen the effect of rasagiline in delaying the progression of PD, of course, not only in theoretical mechanism but also needs to be verified in later clinical studies. In addition, each component of the designed rasagiline in situ gel is FDA-approved for human use, and safety and impurity control can be guaranteed. Relevant characterization results such as viscosity, injectability, solidification in vitro and in vivo, and injection reproducibility also support the clinical application of this formulation. We have applied for a patent for this product, and we believe that with further clarification of rasagiline neuroprotection, this sustained-release in situ gel can maximize the clinical advantages of rasagiline and is suitable for the majority of PD patients due to its good medication compliance.