CN114732789B - A compound long-acting drug delivery system for treating pulmonary hypertension and its preparation - Google Patents

Abstract

The invention belongs to the field of pharmaceutical preparations, and in particular relates to a eutectic-like long-acting pharmaceutical system and a preparation method thereof. The co-crystal is prepared by anti-solvent precipitation of tacrolimus and treprostinil, can be prepared by different preparation methods to obtain different particle sizes, can be kept stable for a long time at room temperature after freeze drying, and can be kept in a long-acting co-delivery system capable of being released for a long time after administration. Compared with the existing slow release delivery, the prepared quasi-co-crystal can simultaneously deliver two medicines, does not contain other auxiliary materials, does not need invasive means of operation or intravenous injection, and has high safety and high efficiency.

Description

A compound long-acting drug delivery system for treating pulmonary hypertension and its preparation

technical field

The invention belongs to the field of pharmaceutical preparations, in particular to the preparation and application of a co-crystal-like long-acting system.

Background technique

Pulmonary arterial hypertension (PAH) is a malignant cardiovascular disease characterized by progressive increase in pulmonary artery resistance, pulmonary vascular remodeling, right ventricular hypertrophy, and eventually right heart failure and even death caused by environmental, genetic and other factors. Clinically, the survival period has been prolonged in recent years, but it is still incurable. Among the currently approved therapeutic strategies, three important pathways of endothelial function are mainly targeted: prostacyclin pathway, nitric oxide (NO) pathway and endothelin pathway. In recent years, the progress in the treatment of PAH does not lie in the discovery of new pathways, but in the development of new combined treatment strategies. Clinically, PAH-targeted therapies, alone or in combination, improve functional capacity and hemodynamics, and reduce hospitalizations. However, these vasodilators do not target key features of PAH pathogenesis and have not been shown to reduce mortality, which remains approximately 50% at 5 years. The initiation and progression of PAH are closely related to endothelial dysfunction. Endothelial dysfunction is manifested in pulmonary vascular remodeling as impaired anticoagulant and repair functions, promotion of vasoconstriction, and increased release of cytokines and adhesion molecules, among others. It is now well established that aberrant BMPR2 signaling impairs endothelial barrier function, promotes accelerated cell proliferation, and may contribute to disease by enhancing PAEC sensitivity to apoptosis. Based on this, the present invention proposes to deliver the prostacyclin drug treprostinil which expands the pulmonary artery and tacrolimus which reverses endothelial dysfunction to achieve the therapeutic effect of treating both symptoms and root causes. At the same time, the target protein IP receptor of treprostinil will be significantly down-regulated in the inflammatory environment of PAH, and tacrolimus, as an anti-inflammatory drug, can significantly increase the expression of IP receptor, so as to achieve the purpose of synergistic treatment.

At the same time, as PAH is a chronic progressive disease, long-acting administration can effectively improve patient compliance. At the same time, for many drugs including tacrolimus and treprostinil, there are problems of short half-life and poor hydrophobicity. The current long-acting drug delivery strategy has some obvious disadvantages: low patient compliance with intravenous infusion, lung inhalation formulations can cause coughing and throat irritation, and subcutaneous osmotic pumps require invasive procedures and may cause inflammation. Nanocrystals are solid nanoparticles composed entirely of drugs, and the outer layer is covered by a stabilizer, which is suitable for delivering hydrophobic drugs. When administered by intramuscular or subcutaneous routes, the drug-loaded nanocrystals showed therapeutic effects for months or even up to a year. The reason for the long-lasting therapeutic effect is that the nanocrystals form a reservoir at the injection site, from which the hydrophobic drug is slowly released. However, the nanocrystals that have been on the market are all composed of nanomedicine crystals, which is difficult to solve the problem of multi-target pathogenesis of diseases. Co-crystals can deliver two or more drugs at the same time, and play a synergistic effect through multi-drug multi-target therapy, thereby improving the treatment effect of diseases. However, at present, the application of quasi-eutectic crystals is mostly used in the nanoscale field, which has problems such as poor stability, easy precipitation, and fast release speed.

Therefore, the present invention realizes the slow, stable and effective release of the two drugs through the intramuscular injection route through the preparation of the co-crystal, and realizes the long-acting treatment of pulmonary hypertension.

Contents of the invention

One of the objectives of the present invention is to construct a stable and easy-to-prepare co-crystal-like long-acting system. In the present invention, two kinds of hydrophobic medicines are dissolved in an organic phase, and co-crystals are formed by an anti-solvent precipitation method, so that the effective delivery of the two kinds of medicines can be realized. In order to improve the stability, bovine serum albumin is used as a freeze-drying protective agent, which is freeze-dried into powder for storage.

The second object of the present invention is to provide the application of the co-crystal in sustained release in vitro and pharmacokinetics in vivo.

The third object of the present invention is to provide the long-term therapeutic application of this type of co-crystal in the model rats suffering from pulmonary hypertension.

To achieve the above object, the present invention is realized through the following technical solutions:

The present invention provides a co-crystal-like long-acting system, characterized in that: the long-acting co-crystal-like co-crystal includes: two hydrophobic drugs or one hydrophobic drug + one water-soluble drug and a lyoprotectant albumin .

The described co-crystal is characterized in that: the hydrophobic drug is selected from tacrolimus, cyclosporine A, treprostinil, epoprostenol, iloprost, beraprost, macitentan Any one or both of , ambrisentan, vardenafil, pitavastatin, rosiglitazone; the water-soluble drug is selected from sildenafil, imatinib, dichloroacetate, celestine Vastatin.

The preparation method of the quasi-eutectic is characterized in that:

1) The mass ratio of the hydrophobic drug tacrolimus to treprostinil is 1:1 to 20:1;

2) The mass ratio of the lyoprotectant bovine serum albumin to tacrolimus is 1:1 to 5:1;

3) The size of the eutectic is 0.5-4 μm;

The present invention also provides a preparation method of the eutectic long-acting system, the steps are as follows:

(1) Add tacrolimus and treprostinil raw materials into organic reagents (methanol, ethanol, DMSO, acetone), and ultrasonically dissolve, preferably acetone as the organic phase.

(2) Pure as the water phase.

(3) Gradually drop the organic phase into the water phase under stirring, and stir at room temperature for 1 hour.

(4) Residual acetone was removed by evaporation under reduced pressure to obtain tacrolimus-treprostinil co-crystal.

(5) Bovine serum albumin is added to the eutectic preparation as a freeze-drying protective agent, and solid powder is obtained after vacuum freeze-drying.

The preparation method of the quasi-eutectic described in the present invention is characterized in that:

(1) The stirring condition described in step (3) is 500-2000 rpm, preferably 1200-1300 rpm.

(2) The bovine serum albumin described in step (5) is 0-5 mg/mL, preferably 1 mg/mL.

(3) The preparation described in step (5) was pre-frozen at -80° C. for 1 hour before lyophilization.

The key steps in the present invention include: formula screening and stability investigation in the preparation process of the eutectic, and determination of the method for preparing the eutectic by the anti-solvent co-precipitation method.

Co-crystal refers to the crystal formed by the combination of active pharmaceutical ingredient (Active pharmaceutical ingredient, API) and co-crystal former (CCF) under the action of hydrogen bond or other non-covalent bonds (CN108440456A); in the patent of the present invention , quasi-eutectics are defined as nanoscale crystals in which two or more hydrophobic small-molecule chemicals exist in the same lattice through hydrophobic interactions, and are called quasi-eutectics.

The key point of the present invention is that, through screening, it is found that the type of freeze-drying agent and the concentration of the freeze-drying agent are involved in the formation of the quasi-eutectic, forming TTLEs-1, which forms a sheet-like micron structure. Bovine serum albumin is not only a lyoprotectant, but also participates in the formation of co-crystals. The inventors compared without lyoprotectant (TTLEs-2) and lyoprotectant (TTLEs-1), and found that TTLEs-2 is a spherical nanocrystal (about 200nm), while TTLEs-1 is 2 μm Lamellar eutectic. The same method of preparation but lack of lyoprotectant prepared to obtain amorphous lyoprotectant TTLEs-3 compared with TTLEs-1, the results showed that the lyoprotectant in TTLEs-1 covered the drug peak, which can prove the lyoprotectant Played a protective role for the eutectic. The release results of tacrolimus in co-crystals showed that tacrolimus was completely released in 12h in TTLEs-1, 7h in TTLE-2, and only 4h in free drug. The release results of treprostinil in co-crystals showed that treprostinil was completely released at 24h in TTLEs-1, but only 4h in TTLE-2 and free drug. The results of the blood concentration of tacrolimus in rats showed that the tacrolimus in the eutectic TTLEs-1 could be continuously released for 14 days, the concentration was significantly higher than that in TTLEs-2, and the relative bioavailability was 2 times of TTLEs-2. The results of the blood concentration level of treprostinil in the body of co-crystals showed that treprostinil could be continuously released in the body for 14 days, and the concentration was significantly higher than that of TTLEs-2 and TRE-NPs. The half-life of TTLEs-1 is 10 times that of TTLEs-2 and 3.5 times that of TRE-NPs.

The results of in vivo experiments showed that compared with free drug group, co-crystal-like TTLEs-1 could significantly improve PAH-induced right heart hypertrophy. Compared with the free drug group, the mean pulmonary artery pressure of modeled rats treated with eutectic TTLEs-1 decreased significantly; compared with the normal saline group and TRE-NPs after modeling, eutectic TTLEs-1 could sustain Alleviate right ventricular free wall hypertrophy and pulmonary artery blood velocity increase caused by PAH.

Therefore, the present invention obtains a brand-new tacrolimus-treprostinil co-crystal through the choice of lyophilization agent and preparation process, which can be released continuously for 14 days in vivo, with a greatly extended half-life and significant the therapeutic effect.

Beneficial effect

No stabilizer is used in the preparation process, the production cost is reduced, and the safety is improved, and the tacrolimus and treprostinil are simultaneously encapsulated by an anti-solvent precipitation method to form a quasi-eutectic crystal. The system optimizes the type of lyoprotectant, the concentration of the lyoprotectant and the preparation method, and optimizes the prescription that can be stored stably at room temperature. At the same time, compared with the in vivo release behavior of the eutectic with small particle size (200 nanometers), the sustained release effect in the body of the micron-sized eutectic prepared by the present invention is more remarkable. Compared with micron-sized crystals with only one drug, co-crystals have better long-term therapeutic effects, reflecting the advantages of co-crystals in delivering two drugs. The system is simple to prepare, does not involve complex chemical synthesis, has high drug loading, low toxicity, and is stable under storage conditions at low temperature and room temperature. It can realize the slow release of two drugs at the same time, and realize long-term treatment of chronic diseases.

Description of drawings

Fig. 1 is a morphological diagram of eutectic TTLEs stored at room temperature for 14 days under an upright fluorescence microscope when the lyoprotectant of the present invention is bovine serum albumin or denatured bovine serum albumin.

Fig. 2 is a morphological diagram of eutectic TTLEs stored at room temperature for 14 days under an upright fluorescence microscope when the bovine serum albumin content is 0, 2, and 10 mg.

Fig. 3 is a morphological diagram of eutectic TTLEs stored at room temperature for 14 days under an upright fluorescence microscope under different preparation methods in the present invention.

Fig. 4 is a SEM image of eutectic TTLEs-1 in the present invention.

Fig. 5 is a particle size distribution diagram of eutectic TTLEs-2 in the present invention.

Fig. 6 is a TEM image of eutectic TTLEs-2 in the present invention.

Fig. 7 is a morphology diagram of nanocrystalline TRE-NPs under an upright fluorescent microscope in the present invention.

Fig. 8 is a morphological diagram of eutectic TTLEs-1 under an upright fluorescence microscope after being stored at room temperature for 30 days in the present invention.

Fig. 9 is a graph showing the X-ray diffraction results of the co-crystaloid in the present invention.

Fig. 10 is a graph showing the in vitro release results of tacrolimus and treprostinil in the co-crystal-like substance of the present invention.

Wherein A is the release curve of tacrolimus in vitro, and B is the release curve of treprostinil in vivo.

Fig. 11 is a diagram showing the results of blood concentration levels of tacrolimus in rats in the eutectic co-crystal of the present invention.

Fig. 12 is a graph showing blood concentration levels of Treprostinil in the eutectic co-crystal of the present invention in rats.

Figure 13 is the right heart hypertrophy index result graph of the pulmonary hypertension model rats treated with eutectics in the present invention (* represents the significant difference comparison with the normal saline group after modeling, # represents the significance with the free drug group difference comparison)

Fig. 14 is a graph showing the results of mean pulmonary artery pressure in pulmonary hypertension model rats treated with eutectics in the present invention.

Fig. 15 is a graph showing the ultrasound results of right heart function in pulmonary hypertension model rats after one week of eutectic treatment in the present invention.

Wherein A is the pulmonary artery blood flow acceleration time/ejection time of PAH rats after one week of administration and B is the free wall thickness of the right ventricle of PAH rats after one week of administration.

Fig. 16 is a graph showing the ultrasound results of right heart function in pulmonary hypertension model rats after two weeks of eutectic treatment in the present invention.

Wherein A is the pulmonary artery blood flow acceleration time/ejection time of the PAH rats after two weeks of administration and B is the free wall thickness of the right ventricle of the PAH rats after two weeks of administration.

Detailed ways

The raw materials or reagents used in the present invention are all commercially available.

The co-crystals were characterized by upright fluorescence microscope, transmission electron microscope, X-ray diffraction and other techniques.

Dialysis was used to investigate the release kinetics in vitro.

Healthy male rats were used.

The present invention will be further elaborated below in conjunction with specific implementation examples.

BSA: bovine serum albumin

TRE: Treprostinil

TAC: Tacrolimus

MCT: monocrotaline

PAH: pulmonary arterial hypertension

Embodiment 1: TTLEs-1 lyoprotectant type screening

prescription:

Preparation Process:

Place 1 or 5 mg/mL bovine serum albumin solution in a water bath at 90°C for 30 minutes to denature to obtain denatured bovine serum albumin.

Tacrolimus-treprostinil co-crystals (TTLEs-1) were prepared by anti-solvent precipitation method. Weigh 2.4 mg treprostinil and 2 mg tacrolimus and dissolve in 0.2 mL acetone as the organic phase. Measure 2 mL of pure water as the aqueous phase. While stirring (1200-1300 rpm), slowly drop the organic phase into the water phase with a syringe, and continue stirring at room temperature for 1 hour. The organic solvent was removed by evaporation under reduced pressure, and 2 mg of bovine serum albumin or denatured bovine serum albumin was added to the preparation as a freeze-drying protective agent. After pre-freezing at -80 ° C for 1 hour, vacuum freeze-drying was carried out to obtain fluffy and dry White solid powder. After standing at room temperature for 0, 7, and 14 days, the powder was reconstituted, dropped onto a glass slide coated with xanthan gum (10%, w/v), and placed under an upright fluorescence microscope to observe the eutectic morphology .

The results are shown in Table 1 and Figure 1, using BSA as a lyoprotectant is more stable than dBSA. If the freeze-dried powder is unstable, it can be observed under the microscope that the particle size becomes larger and aggregation occurs; if the freeze-dried powder is stable, it can be observed under the microscope that the particle size is the same as when it was just successfully prepared, the distribution is uniform, and the particle size does not change significantly. .

Table 1. Comparison of BSA and dBSA as Lyoprotectants

Embodiment 2: TTLEs-1 lyoprotectant concentration screening

prescription:

Treprostinil 2.4mg Tacrolimus 2mg pure water 2mL bovine serum albumin 0, 2, 10mg

Tacrolimus-treprostinil co-crystals (TTLEs-1) were prepared by anti-solvent precipitation method. Except using the BSA (0,2,10mg) of different concentrations, all the other steps are with embodiment 2.

The results are shown in Table 2 and Figure 2. When BSA is used as a lyoprotectant, the most stable concentration is 1 mg/mL. If the freeze-dried powder is unstable, it can be observed under the microscope that the particle size becomes larger and aggregation occurs; if the freeze-dried powder is stable, it can be observed under the microscope that the particle size is the same as when it was just successfully prepared, the distribution is uniform, and the particle size does not change significantly. .

Table 2. BSA Concentration Screening

Embodiment 3: TTLEs-1 preparation method screening

prescription:

Treprostinil 2.4mg Tacrolimus 2mg pure water 2mL bovine serum albumin 2mg

Tacrolimus-treprostinil-like co-crystals (TTLEs) were prepared by anti-solvent precipitation. Screen different preparation methods: After adding the organic phase to the water phase, stir at room temperature for 1 hour or sonicate for 5 minutes in an ice bath (50W, 2s on, 2s off) or stir in liquid nitrogen for 2 minutes. All the other steps are the same as in Example 1.

The results are shown in Table 3 and Figure 3, when stirred at room temperature for 1 h, the formulation was the most stable. If the freeze-dried powder is unstable, it can be observed under the microscope that the particle size becomes larger and aggregation occurs; if the freeze-dried powder is stable, it can be observed under the microscope that the particle size is the same as when it was just successfully prepared, the distribution is uniform, and the particle size does not change significantly. .

Table 3. Effect of different mixing methods on stability

Example 4 Preparation of TTLEs-1

prescription:

Preparation Process:

Tacrolimus-treprostinil co-crystals (TTLEs-1) were prepared by anti-solvent precipitation method. Weigh 2.4 mg treprostinil and 2 mg tacrolimus and dissolve in 0.2 mL acetone as the organic phase. Measure 2 mL of pure water as the aqueous phase. While stirring (1200-1300 rpm), slowly drop the organic phase into the water phase with a syringe, and continue stirring at room temperature for 1 hour. The organic solvent was removed by evaporation under reduced pressure, and 2 mg of bovine serum albumin was added to the preparation as a freeze-drying protective agent. After pre-freezing at -80° C. for 1 hour, vacuum freeze-drying was performed to obtain a fluffy and dry white solid powder. After the powder was reconstituted, it was dropped onto a glass slide coated with xanthan gum (10%, w/v), and placed under an upright fluorescence microscope to observe the eutectic morphology.

The results are shown in Figure 4, TTLEs-1 is a plate-shaped crystal with a particle size of about 2 μm.

Example 5 Preparation of TTLEs-2 (nanoscale eutectic preparation)

prescription:

Treprostinil 2.4mg Tacrolimus 2mg pure water 2mL

Preparation Process:

Tacrolimus-treprostinil co-crystals (TTLEs-2) were prepared by anti-solvent precipitation-ultrasonic method. Weigh 2.4 mg treprostinil and 2 mg tacrolimus and dissolve in 0.2 mL acetone as the organic phase. Measure 2 mL of pure water as the aqueous phase. Under the stirring state at 4°C (1200-1300 rpm), slowly drop the organic phase into the water phase with a syringe, transfer to the probe for ultrasonication, and sonicate for 10 minutes under ice bath conditions, and remove the organic solvent by vacuum distillation to obtain Tacrolimus-Treprostinil cocrystal (TTLEs-2).

The results are shown in Figure 5 and Figure 6, the particle size of TTLEs-2 is 192.83±6.48nm, the PDI is 0.267±0.131, and the shape is spherical

Example 6 Preparation of TRE-NPs (preparation of micron-sized crystals containing only treprostinil)

prescription:

Treprostinil 2.4mg pure water 2mL bovine serum albumin 2mg

Preparation Process:

Treprostinil nanocrystals (TRE-NPs) were prepared by anti-solvent precipitation method. Weigh 2.4mg treprostinil and dissolve it in 0.2mL acetone as the organic phase. Measure 2 mL of pure water as the aqueous phase. While stirring (1200-1300 rpm), slowly drop the organic phase into the water phase with a syringe, and continue stirring at room temperature for 1 hour. The organic solvent was removed by evaporation under reduced pressure, and 2 mg of bovine serum albumin was added to the preparation as a freeze-drying protective agent. After pre-freezing at -80° C. for 1 hour, vacuum freeze-drying was performed to obtain a fluffy and dry white solid powder. After the powder was reconstituted, it was dropped onto a glass slide coated with xanthan gum (10%, w/v), and placed under an upright fluorescence microscope to observe the eutectic morphology.

The results are shown in Figure 7, TRE-NPs are flake crystals with a particle size of about 1-3 μm.

Example 7 Investigating the Encapsulation Efficiency and Drug Loading Capacity of Tacrolimus-Treprostinil Co-Crystal

In order to investigate the encapsulation efficiency and drug loading capacity of tacrolimus-treprostinil co-crystal, the preparation and free drug were separated by ultra-high speed centrifugation, and the drug content was detected by high performance liquid chromatography. The specific steps are: use Example 1 and Example 2 to prepare TTLEs-1 and TTLEs-2, centrifuge at 16,000 r/min for 30 minutes at 4°C, take 20 μL of the supernatant for injection, and calculate the free drug content by peak area. Take 1mL of TTLEs-1 and TTLEs-2 respectively, add 4mL of acetonitrile to demulsify, ultrasonicate for 20min, cool to room temperature, add acetonitrile to the volume, filter through a 0.45μm microporous membrane, take 20μL of the filtrate for injection, and pass the peak area Calculate the total drug content of the preparation.

Encapsulation efficiency = content of free drug/total drug content in the preparation × 100% (1)

Drug loading=content of free drug/(mass of carrier+total drug content in preparation)×100% (2)

Calculate the encapsulation efficiency and drug loading of the preparation according to the formulas (1) and (2).

The results showed that the encapsulation efficiency of TAC in TTLEs-1 was 95.92%±4.43, and the drug loading was 49.80%±1.41; the encapsulation efficiency of TRE was 96.62%±1.74, and the drug loading was 50.20%±1.41. The encapsulation efficiency of TAC in TTLEs-2 was 99.52%±0.03, and the drug loading was 50.48±0.06; the encapsulation efficiency of TRE was 97.62±0.23, and the drug loading was 49.52±0.06.

Example 8 Investigating the Stability of Tacrolimus-Treprostinil Co-Crystal

In order to investigate the stability of the eutectic preparation at room temperature, TTLEs-1 was prepared by using Example 1, and the solid powder was stored at room temperature. After 30 days, it was redissolved with pure water, and TTLEs-1 was observed with an upright fluorescence microscope. .

The results shown in Figure 8 show that the morphology and particle size of TTLEs-1 did not change significantly, and the particles did not aggregate.

Example 9 Verifies the formation of tacrolimus-treprostinil-like co-crystal

prescription

Treprostinil 2.4mg Tacrolimus 2mg pure water 2mL

Preparation Process:

Tacrolimus-treprostinil co-crystals (TTLEs-3) were prepared by anti-solvent precipitation method. Weigh 2.4 mg treprostinil and 2 mg tacrolimus and dissolve in 0.2 mL acetone as the organic phase. Measure 2 mL of pure water as the aqueous phase. While stirring (1200-1300 rpm), slowly drop the organic phase into the water phase with a syringe, and continue stirring at room temperature for 1 hour. The organic solvent was removed by evaporation under reduced pressure, centrifuged at 16000 r/min at 4°C for 30 minutes, and the precipitate was vacuum-dried at 60°C for 2 hours to obtain TTLEs-3 as a white solid powder.

The difference between this example and Example 5 is that this example adopts stirring at room temperature without ultrasonic process. In order to study the crystal structures of the two drugs in the tacrolimus-treprostinil co-crystal, an X-ray diffractometer was used to investigate, and in order to reduce the masking of the drug peak by the lyoprotectant, no freeze-drying was performed.

The results are shown in Figure 9. The same drug characteristic peaks exist in the physical mixture in TTLEs-3, and because the two drugs exist in the form of heterocrystals, new diffraction peaks appear. For TTLEs-1 with added lyoprotectant, it can be found that the drug peak is covered by the amorphous lyoprotectant, which proves that the lyoprotectant has a protective effect on the co-crystal.

Example 10 Investigating the Sustained Release Behavior of Tacrolimus-Treprostinil Cocrystal in Vitro

In order to verify that the tacrolimus-treprostinil co-crystal has a sustained-release effect, the drug release kinetics of the formulation in vitro was investigated.

The specific steps are: use Example 1 and Example 2 to prepare TTLEs-1 and TTLEs-2, accurately measure 1 mL of TTLEs-1, TTLEs-2 and free TAC/TRE and put them into a dialysis bag (MWCO 3500Da), tie the dialysis bag Tightly, place in 30mL of PBS release medium (containing 20% SDS) at pH 7.2, shake at 120rpm in a water bath at 37°C, take out 1mL of release medium at 1h, 2h, 4h, 6h, 8h, 12h, 24h, 36h and 48h, and add Corresponding equal volume of fresh release medium. Taking the free co-soluble drug TAC/TRE as a control, the contents of TAC and TRE in the release medium were measured by high performance liquid chromatography-ultraviolet detection method (HPLC-UV), and the cumulative release percentage was calculated.

The release results of tacrolimus in co-crystals are shown in Figure 11. Tacrolimus was completely released in 12 hours in TTLEs-1, 7 hours in TTLE-2, and only 4 hours for the free drug.

The release results of Treprostinil in co-crystals are shown in Figure 10. Treprostinil was completely released in 24h in TTLEs-1, but only 4h in TTLE-2 and free drug.

Example 11 Investigating the In Vivo Sustained Release Effect of Tacrolimus-Treprostinil Co-Crystal

Using Example 1 to prepare micron-scale tacrolimus-treprostinil-like co-crystals (TTLEs-1), and Example 5 to prepare nano-scale tacrolimus-treprostinil-like co-crystals (TTLEs-2), implement Example 6 Nanoscale treprostinil crystals (TRE-NPs), to investigate the sustained release effect of the preparation in rats. 18 healthy male rats (200-250g) were randomly divided into three groups (n=6), numbered; intramuscular injection of TTLEs-1, TTLEs-2 and TAC/TRE physiological saline co-solution on the 0th day, respectively, On days 0.02, 0.04, 0.125, 0.208, 1, 3, 5, 9, and 14, blood was collected from the orbit, and the blood was centrifuged to obtain supernatant plasma for pretreatment. The pretreatment steps are: take 100 μL plasma, add 10 μL IS solution and 20 μL acetic acid, mix, add 300ul methanol, vortex for 5min and centrifuge (15000r/min, 10min), take 1μL sample, and use LC-MS/MS for quantitative detection .

The results of the blood concentration of tacrolimus in rats in eutectics are shown in Figure 11. Tacrolimus in eutectics TTLEs-1 can be released continuously for 14 days, and the concentration is significantly higher than that in TTLEs-2. The bioavailability is twice that of TTLEs-2.

The results of blood concentration levels of treprostinil in the body of co-crystals are shown in Figure 12. Treprostinil can be continuously released in vivo for 14 days, and its concentration is significantly higher than that of TTLEs-2 and TRE-NPs. The half-life of TTLEs-1 is 10 times that of TTLEs-2 and 3.5 times that of TRE-NPs.

Example 13: Investigating the in vivo long-acting therapeutic effect of tacrolimus-treprostinil co-crystal

Example 1 was used to prepare tacrolimus-treprostinil co-crystal, and the long-term therapeutic effect of the preparation on PAH model rats was investigated. 24 healthy male rats (200-250g) were randomly divided into four groups (n=6), numbered; the first three groups were subcutaneously injected with monocrotaline solution (60mg/kg), and the fourth group was subcutaneously injected with normal saline. After successful modeling in the third week, TTLEs-1, TAC/TRE normal saline co-solution and normal saline were injected intramuscularly. The mean pulmonary artery pressure (mPAP) and right heart hypertrophy index (RVHI) were measured in the second week after administration.

The results are shown in Figure 13, compared with the free drug group, co-crystal-like TTLEs-1 can significantly improve PAH-induced right heart hypertrophy.

The results are shown in FIG. 14 , compared with the free drug group, the mean pulmonary artery pressure of the model rats treated with eutectic TTLEs-1 decreased significantly.

Example 14: Investigating the in vivo long-acting therapeutic effect of tacrolimus-treprostinil co-crystal

Example 4 was used to prepare tacrolimus-treprostinil co-crystals, and Example 5 was used to prepare treprostinil nanocrystals, and the long-acting therapeutic effect of the preparations on PAH model rats was investigated. 24 healthy male rats (200-250g) were randomly divided into four groups (n=6), numbered; the first three groups were subcutaneously injected with monocrotaline solution (60mg/kg), and the fourth group was subcutaneously injected with normal saline. After successful modeling in the third week, TTLEs-1, TAC/TRE normal saline co-solution and normal saline were injected intramuscularly. The right heart function of the rats was detected with a small animal ultrasound imaging system in the first week and the second week after administration.

The results are shown in Figures 15 and 16. Compared with the normal saline group and TRE-NPs after modeling, the eutectic TTLEs-1 can continuously reduce the right ventricular free wall hypertrophy and pulmonary artery blood flow velocity induced by PAH.

Claims (4)

1. A co-crystal of the kind characterized by: comprises two hydrophobic drugs and a freeze-drying protective agent; the hydrophobic drugs are tacrolimus and treprostinil, and the lyoprotectant is bovine serum albumin; the mass ratio of tacrolimus to treprostinil is 1:1 to 20:1; the mass ratio of the freeze-drying protective agent bovine serum albumin to the tacrolimus is 1:1;

the quasi-eutectic is prepared by the following steps:

(1) Adding tacrolimus and treprostinil bulk drugs into acetone, and performing ultrasonic dissolution to obtain an organic phase;

(2) Pure water is used as an aqueous phase;

(3) Gradually dropwise adding the organic phase into the water phase under stirring, and stirring for 1 hour at room temperature;

(4) Removing residual acetone by a reduced pressure evaporation method to obtain tacrolimus-treprostinil eutectic;

(5) And adding bovine serum albumin into the eutectic-like preparation as a freeze-drying protective agent, and performing vacuum freeze-drying to obtain solid powder.

2. A co-crystal according to claim 1, characterized in that:

the stirring condition in the step (3) is 500-2000 rpm;

the bovine serum albumin in the step (5) is 0-5 mg/mL; the preparation is pre-frozen for 1 hour at-80 ℃ before freeze-drying.

3. A co-crystal according to claim 2, characterized in that:

the stirring condition in the step (3) is 1200-1300 rpm;

the bovine serum albumin in the step (5) is 1mg/mL.

4. Use of a co-crystal according to any one of claims 1-3 in the manufacture of a medicament for the treatment of pulmonary arterial hypertension.