CN114732789A - Compound long-acting drug delivery system for treating pulmonary hypertension and preparation thereof - Google Patents

Abstract

The invention belongs to the field of medicinal preparations, and particularly relates to a co-crystal-like medicament long-acting system and a preparation method thereof. The co-crystal is obtained by preparing tacrolimus and treprostinil by an anti-solvent precipitation method, can obtain different particle sizes by different preparation methods, can keep stable for a long time at room temperature after freeze drying, and can keep a long-acting co-delivery system released for a long time after administration. Compared with the existing slow release delivery, the prepared cocrystal can deliver two drugs simultaneously, does not contain other auxiliary materials, does not need an 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, and particularly relates to the preparation and application of a quasi-co-crystal long-acting system.

Background technique

Pulmonary arterial hypertension (PAH) is a malignant cardiovascular disease caused by environmental, genetic and other factors that cause progressive increase in pulmonary artery resistance, pulmonary vascular remodeling, right ventricular hypertrophy, and ultimately lead to right heart failure and even death. In recent years, clinical survival has been prolonged, but there is still no cure. The currently approved therapeutic strategies mainly target three important pathways of endothelial function: prostacyclin pathway, nitric oxide (NO) pathway and endothelin pathway. In recent years, the progress of PAH treatment is not the discovery of new pathways, but the development of new combination therapy strategies. Clinically, PAH-targeted therapy, alone or in combination, improves functional capacity and hemodynamics, and reduces hospitalizations. However, these vasodilators did not target key features of PAH pathogenesis, nor did they show a reduction in mortality, which was still about 50% at 5 years. The initiation and progression of PAH is closely related to endothelial dysfunction. Endothelial dysfunction is manifested in pulmonary vascular remodeling as impaired anticoagulation and repair functions, promotion of vasoconstriction, and increased release of cytokines and adhesion molecules. It is now well understood that aberrant BMPR2 signaling impairs endothelial barrier function, promotes accelerated cell proliferation, and may contribute to disease by enhancing PAEC susceptibility to apoptosis. Based on this, the present invention proposes the combined delivery of two drugs, the prostacyclin-type drug Treprostinil, which dilates pulmonary artery blood vessels, and tacrolimus, which reverses endothelial dysfunction, so as to achieve the therapeutic effect of treating both the symptoms and the symptoms. At the same time, the target protein IP receptor of treprostinil is significantly down-regulated in the inflammatory environment of PAH, and tacrolimus, as an anti-inflammatory drug, can significantly increase the expression of IP receptor, thus achieving the purpose of synergistic therapy.

Meanwhile, as a chronic progressive disease, long-acting administration of PAH can effectively improve patient compliance. At the same time, for many drugs including tacrolimus and treprostinil, there are problems with short half-life and poor hydrophobicity. Current long-acting drug delivery strategies have some significant disadvantages: patient compliance with intravenous infusion is low, pulmonary inhalation of formulations can cause coughing and throat irritation, and subcutaneous osmotic pumps require invasive procedures and may induce inflammation. Nanocrystals are solid nanoparticles composed entirely of drugs with an outer layer covered by stabilizers, suitable for the delivery of hydrophobic drugs. When administered via the intramuscular or subcutaneous route, 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 listed nanocrystals are all composed of nano-drug crystals, and it is difficult to solve the problem of multi-target pathogenesis of diseases. Cocrystal-like can deliver two or more drugs at the same time, and play a synergistic effect through multi-drug multi-target therapy, thereby improving the therapeutic effect of diseases. However, the applications of eutectic-like materials are mostly used in the nano-scale field at this stage, and there are 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 by preparing the quasi-co-crystal and through intramuscular injection, and realizes the long-term treatment of pulmonary hypertension.

SUMMARY OF THE INVENTION

One of the objectives of the present invention is to construct a stable and easy-to-prepare eutectic-like long-acting system. In the present invention, two kinds of hydrophobic drugs are dissolved in an organic phase, and a quasi-co-crystal is formed by an anti-solvent precipitation method, and the effective delivery of the two drugs can be realized. In order to improve the stability, bovine serum albumin was used as a lyoprotectant, and it was freeze-dried into powder for preservation.

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

The third purpose of the present invention is to provide the long-term therapeutic application of such co-crystals in model rats suffering from pulmonary arterial hypertension.

In order to achieve the above object, the present invention is realized through the following technical solutions:

The invention provides a kind of co-crystal long-acting system, which is characterized in that: the long-acting co-crystal-like system comprises: two kinds of hydrophobic drugs or one kind of hydrophobic drugs + one kind of water-soluble drugs and lyoprotectant albumin .

The described co-crystal is characterized in that: the hydrophobic drug is selected from tacrolimus, cyclosporine A, treprostinil, epoprostenol, iloprost, beraprost, macitentan , Ambesentan, vardenafil, pitavastatin, rosiglitazone, any one or two; water-soluble drugs are selected from sildenafil, imatinib, dichloroacetate, ciliate vastatin.

The described eutectic preparation method 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 lyophilized protective agent bovine serum albumin and tacrolimus is 1:1 to 5:1;

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

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

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

(2) Use pure as the water phase.

(3) The organic phase was gradually added dropwise to the aqueous phase with stirring, and stirred at room temperature for 1 hour.

(4) The residual acetone is removed by evaporation under reduced pressure to obtain a tacrolimus-treprostinil co-crystal.

(5) adding bovine serum albumin to the quasi-co-crystal preparation as a freeze-drying protective agent, and obtaining a solid powder after vacuum freeze-drying.

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

(1) The stirring conditions described in step (3) are 500-2000 rev/min, preferably 1200-1300 rev/min.

(2) The bovine serum albumin 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 of the eutectic-like preparation process, and determination of the method for preparing the eutectic-like crystal by an anti-solvent coprecipitation method.

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

The key point of the present invention is that, through screening, it is found that the type of lyophilizer and the concentration of lyophilizer are involved in the formation of eutectic-like, forming TTLEs-1, which forms a sheet-like microstructure. BSA is not only a lyoprotectant, but also participates in co-crystal formation. The inventors compared no lyoprotectant (TTLEs-2) and lyoprotectant (TTLEs-1), and found that TTLEs-2 is spherical nanocrystals (about 200nm), while TTLEs-1 is 2μm. Plate eutectic. Amorphous lyoprotectant TTLEs-3 was prepared by the same method but lacking lyoprotectant. Compared with TTLEs-1, the results showed that the lyoprotectant in TTLEs-1 masked the drug peak, which can prove that the lyoprotectant It has a protective effect on the eutectic. The release results of tacrolimus from co-crystals showed that tacrolimus was completely released in TTLEs-1 in 12h, in TTLE-2 in 7h, and free drug in 4h. The release results of treprostinil from co-crystals showed that the release of treprostinil in TTLEs-1 was complete in 24h, and only 4h was required in TTLE-2 and free drug. The blood concentration level of tacrolimus in co-crystal-like TTLEs-1 in rats showed that tacrolimus in co-crystal-like TTLEs-1 could be released continuously for 14 days, and the concentration was significantly higher than that of TTLEs-2, and the relative bioavailability was Twice the TTLEs-2. The results of blood concentration levels of Treprostinil in co-crystals show that Treprostinil can be continuously released in vivo for 14 days, and the 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.

The results of in vivo experiments showed that cocrystal-like TTLEs-1 could significantly improve PAH-induced right ventricular hypertrophy compared with the free drug group. Compared with the free drug group, the mean pulmonary artery pressure of the model rats treated with cocrystal-like TTLEs-1 was significantly decreased; compared with the normal saline group and TRE-NPs after modeling, the cocrystal-like TTLEs-1 could sustainably Relief of PAH-induced right ventricular free wall hypertrophy and increased pulmonary artery blood flow.

Therefore, the present invention obtains a brand-new tacrolimus-treprostinil co-crystal through the selection of lyophilizer and preparation process, which can be continuously released in vivo for 14 days, the half-life is greatly prolonged, and has 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 hecrolimus and treprostinil are encapsulated simultaneously to form a eutectic through an anti-solvent precipitation method. By choosing the type of lyoprotectant, the concentration of lyoprotectant and the preparation method, the system is preferably a formula that can be stored stably at room temperature. At the same time, compared with the in vivo release behavior of the eutectic-like crystal with small particle size (200 nanometers), the micron-scale eutectic-like crystal prepared by the present invention has a more significant sustained release effect in vivo. Compared with micron-scale crystals with only one drug, the co-crystal-like has better long-acting therapeutic effect, reflecting the advantage of co-crystal-like delivery of two drugs. The system is simple to prepare, does not involve complex chemical synthesis, has high drug loading, low toxicity, is stable under low temperature and room temperature storage conditions, can achieve simultaneous slow release of two drugs, and achieve 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 lyophilized protective agent is bovine serum albumin or denatured bovine serum albumin according to the present invention.

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

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

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

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

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

7 is a morphological diagram of nanocrystalline TRE-NPs under an upright fluorescence microscope in the present invention.

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

FIG. 9 is a graph showing the results of X-ray diffraction of eutectic-like crystals in the present invention.

Figure 10 is a graph showing the results of in vitro release of tacrolimus and treprostinil from the co-crystals of the present invention.

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

Figure 11 is a graph showing the results of blood concentration levels of tacrolimus in rats in the co-crystals of the present invention.

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

Fig. 13 is a graph showing the results of right ventricular hypertrophy index of pulmonary hypertension model rats treated with cocrystal-like treatment according to the present invention (* indicates the significant difference compared with the normal saline group after modeling, # indicates the significant difference with the free drug group difference comparison)

Figure 14 is a graph showing the mean pulmonary arterial pressure results of pulmonary hypertension model rats treated with cocrystal-like in the present invention.

Figure 15 is a graph showing the ultrasound results of the right heart function of a rat model of pulmonary hypertension after one week of cocrystal-like treatment according to the present invention.

Among them, A is the pulmonary artery blood flow acceleration time/ejection time of PAH rats after one week of administration; B is the right ventricular free wall thickness of PAH rats after one week of administration.

Figure 16 is a graph showing the ultrasound results of the right heart function of a rat model of pulmonary hypertension after two weeks of cocrystal-like treatment according to the present invention.

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

Detailed ways

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

The eutectic-like crystals were characterized by upright fluorescence microscopy, transmission electron microscopy, and X-ray diffraction.

In vitro release kinetics were investigated by dialysis.

Healthy male rats were used.

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

BSA: bovine serum albumin

TRE: Treprostinil

TAC: Tacrolimus

MCT: Lilycotine

PAH: Pulmonary Arterial Hypertension

Example 1: Screening of TTLEs-1 lyoprotectants

prescription:

Preparation Process:

The 1 or 5 mg/mL bovine serum albumin solution was placed in a 90°C water bath for denaturation for 30 minutes to obtain denatured bovine serum albumin.

Tacrolimus-Treprostinil-like co-crystals (TTLEs-1) were prepared by anti-solvent precipitation. 2.4 mg of Treprostinil and 2 mg of tacrolimus were weighed and dissolved in 0.2 mL of acetone as the organic phase. Measure 2 mL of purified water as the water phase. Under stirring (1200-1300 rpm), slowly drop the organic phase into the aqueous 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. White solid powder. After standing at room temperature for 0, 7, and 14 days, the powder was reconstituted and added dropwise to a glass slide coated with xanthan gum (10%, w/v), and the eutectic morphology was observed under an upright fluorescence microscope. .

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 that the particle size becomes larger and aggregation occurs under the microscope; 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 successfully prepared, the distribution is uniform, and the particle size does not change significantly. .

Table 1. Comparison of BSA and dBSA as lyoprotectants

Example 2: Screening of TTLEs-1 lyoprotectant concentration

prescription:

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

Tacrolimus-Treprostinil-like co-crystals (TTLEs-1) were prepared by anti-solvent precipitation. Except using different concentrations of BSA (0, 2, 10 mg), other steps are the same as in Example 2.

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

Table 2. BSA Concentration Screening

Example 3: Screening of the preparation method of TTLEs-1

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. Different preparation methods were screened: after adding the organic phase to the aqueous phase, stir at room temperature for 1 hour or sonicate for 5 minutes in an ice bath (50W, on for 2s, off for 2s) or stir in liquid nitrogen for 2 minutes. The remaining steps are the same as in Example 1.

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

Table 3. Effects of different mixing methods on stability

Example 4 Preparation of TTLEs-1

prescription:

Preparation Process:

Tacrolimus-Treprostinil-like co-crystals (TTLEs-1) were prepared by anti-solvent precipitation. 2.4 mg of Treprostinil and 2 mg of tacrolimus were weighed and dissolved in 0.2 mL of acetone as the organic phase. Measure 2 mL of purified water as the water phase. Under stirring (1200-1300 rpm), slowly drop the organic phase into the aqueous 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 added dropwise to a glass slide coated with xanthan gum (10%, w/v), and the eutectic morphology was observed under an upright fluorescence microscope.

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

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

prescription:

Treprostinil 2.4mg Tacrolimus 2mg pure water 2mL

Preparation Process:

Tacrolimus-Treprostinil-like co-crystals (TTLEs-2) were prepared by anti-solvent precipitation-sonication. 2.4 mg of Treprostinil and 2 mg of tacrolimus were weighed and dissolved in 0.2 mL of acetone as the organic phase. Measure 2 mL of purified water as the water phase. Under agitation at 4°C (1200-1300 rpm), slowly drop the organic phase into the aqueous phase with a syringe, transfer to the probe for sonication, sonicate for 10 minutes under ice bath conditions, and remove the organic solvent by vacuum distillation to obtain Tacrolimus-Treprostinil-like co-crystals (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. 2.4 mg of Treprostinil was weighed and dissolved in 0.2 mL of acetone to serve as the organic phase. Measure 2 mL of purified water as the water phase. Under stirring (1200-1300 rpm), slowly drop the organic phase into the aqueous 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 added dropwise to a glass slide coated with xanthan gum (10%, w/v), and the eutectic morphology was observed under an upright fluorescence microscope.

The results are shown in Fig. 7. The TRE-NPs are platelet-shaped crystals with a particle size of about 1-3 μm.

Example 7 Investigating the encapsulation efficiency and drug loading of tacrolimus-treprostinil co-crystals

In order to investigate the encapsulation efficiency and drug loading capacity of tacrolimus-treprostinil co-crystals, the preparation and free drug were separated by ultracentrifugation, and the drug content was detected by high performance liquid phase. The specific steps are as follows: using Example 1 and Example 2 to prepare TTLEs-1 and TTLEs-2, centrifuge at 16000 r/min for 30 minutes at 4°C, inject 20 μL of supernatant, and calculate the free drug content by peak area. Take 1 mL of TTLEs-1 and TTLEs-2 respectively, add 4 mL of acetonitrile to break the demulsification, ultrasonicate for 20 min, cool to room temperature, add acetonitrile to volume to the mark, filter through a 0.45 μm microporous membrane, and take 20 μL of the filtrate for injection, through the peak area Calculate the total drug content of the formulation.

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

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

According to formula (1) and (2), the encapsulation efficiency and drug loading capacity of the preparation were calculated respectively.

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-crystals

In order to investigate the stability of the eutectic-like preparation at room temperature, Example 1 was used to prepare TTLEs-1, the solid powder was stored at room temperature, reconstituted with pure water after 30 days, and the TTLEs-1 was observed with an upright fluorescence microscope. .

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

Example 9 Validation of the formation of tacrolimus-treprostinil co-crystals

prescription

Treprostinil 2.4mg Tacrolimus 2mg pure water 2mL

Preparation Process:

Tacrolimus-Treprostinil-like co-crystals (TTLEs-3) were prepared by anti-solvent precipitation. 2.4 mg of Treprostinil and 2 mg of tacrolimus were weighed and dissolved in 0.2 mL of acetone as the organic phase. Measure 2 mL of purified water as the water phase. Under stirring (1200-1300 rpm), the organic phase was slowly dropped into the aqueous phase with a syringe, and stirring was continued for 1 hour at room temperature. The organic solvent was removed by evaporation under reduced pressure, centrifuged at 16,000 r/min for 30 minutes at 4°C, 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 embodiment and Embodiment 5 is that this embodiment adopts room temperature stirring without ultrasonic process. In order to study the crystal structures of the two drugs in the tacrolimus-treprostinil-like co-crystal, X-ray diffractometer was used to investigate, and in order to reduce the masking of drug peaks by lyoprotectants, freeze-drying was not performed.

The results are shown in Figure 9. The characteristic peaks of the same drug present in the physical mixture exist in TTLEs-3. At the same time, since the two drugs exist in the form of heterocrystals, new diffraction peaks appear. For TTLEs-1 with lyoprotectant added, it can be found that the amorphous lyoprotectant masks the drug peak, which can prove that the lyoprotectant has a protective effect on the eutectic.

Example 10 Investigating the sustained release behavior of tacrolimus-treprostinil co-crystals in vitro

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

The specific steps are: using Example 1 and Example 2 to prepare TTLEs-1 and TTLEs-2, accurately measuring 1 mL of TTLEs-1, TTLEs-2 and free TAC/TRE into a dialysis bag (MWCO 3500Da), tying the dialysis bag Tightly, placed in 30 mL of PBS release medium (containing 20% SDS) pH 7.2, shaken at 120 rpm in a water bath at 37 °C, and taken out 1 mL of release medium at 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h and 48 h, and supplemented with A corresponding equal volume of fresh release medium was made. Taking the free co-soluble drug TAC/TRE as a control, the content of TAC and TRE in the release medium was determined by high performance liquid chromatography-ultraviolet detection (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 TTLEs-1 in 12 hours, 7 hours in TTLE-2, and only 4 hours in free drug.

Figure 10 shows the release results of treprostinil from co-crystals. Treprostinil is completely released in TTLEs-1 within 24 hours, and only takes 4 hours in TTLE-2 and free drug.

Example 11 To investigate the in vivo sustained-release effect of tacrolimus-treprostinil co-crystals

Application Example 1 was used to prepare micron-scale tacrolimus-treprostinil co-crystals (TTLEs-1), and Example 5 was to prepare nano-scale tacrolimus-treprostinil co-crystals (TTLEs-2), and implemented Example 6 Nano-sized Treprostinil crystals (TRE-NPs) were used to investigate the sustained-release effect of the preparation in rats. Eighteen healthy male rats (200-250g) were randomly divided into three groups (n=6), numbered; on day 0, TTLEs-1, TTLEs-2 and TAC/TRE normal saline co-solution were intramuscularly injected, respectively. Orbital blood was collected on days 0.02, 0.04, 0.125, 0.208, 1, 3, 5, 9, and 14, and the blood was centrifuged to collect supernatant plasma for pretreatment. The pretreatment steps are: take 100 μL of plasma, add 10 μL IS solution and 20 μL acetic acid, mix, add 300 μL methanol, vortex for 5 min, and then centrifuge (15000 r/min, 10 min), take 1 μL sample, and use LC-MS/MS for quantitative detection .

The blood concentration level of tacrolimus in co-crystal-like in rats is shown in Figure 11. Tacrolimus in co-crystal-like TTLEs-1 can be released continuously for 14 days, and the concentration is significantly higher than that of TTLEs-2. The bioavailability is 2 times that of TTLEs-2.

Figure 12 shows the blood concentration level of Treprostinil in cocrystal-like in vivo. Treprostinil can be continuously released in vivo for 14 days, and the 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 long-acting therapeutic effect of tacrolimus-treprostinil-like co-crystals in vivo

Application 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, injected After the third week, the model was successfully established, and TTLEs-1, TAC/TRE normal saline co-solution and normal saline were injected intramuscularly. Mean pulmonary arterial pressure (mPAP) and right ventricular hypertrophy index (RVHI) were measured in the second week after dosing.

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

The results are shown in Figure 14. Compared with the free drug group, the mean pulmonary artery pressure of the model rats treated with the cocrystal-like TTLEs-1 was significantly decreased.

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

Example 4 was used to prepare tacrolimus-treprostinil co-crystals, and Example 5 was used to prepare treprostinil nanocrystals, and the long-term 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, injected After the third week, the model was successfully established, and 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 and second weeks after administration.

The results are shown in Figures 15 and 16. Compared with the normal saline group and TRE-NPs after modeling, cocrystal-like TTLEs-1 can persistently alleviate PAH-induced right ventricular free wall hypertrophy and increased pulmonary artery blood flow.

Claims (8)

1. A co-crystal-like, characterized by: comprises two hydrophobic drugs or a hydrophobic drug and a water-soluble drug, and a freeze-drying protective agent albumin.

2. The co-crystal-like according to claim 1, wherein: the hydrophobic drug is selected from one or two of tacrolimus, cyclosporine A, treprostinil, epoprostenol, iloprost, beraprost, macitentan, ambrisentan, vardenafil, pitavastatin and rosiglitazone; the water-soluble drug is selected from sildenafil, imatinib, dichloroacetate and cerivastatin, and the albumin is bovine serum albumin or human serum albumin.

3. The co-crystal-like of claim 2, wherein the hydrophobic drugs are tacrolimus and treprostinil and the protem is bovine serum albumin.

4. A co-crystal-like preparation method according to claim 3, characterized in that:

1) the mass ratio of the hydrophobic drugs tacrolimus and treprostinil is 1:1 to 20: 1;

2) the mass ratio of the freeze-drying protective agent bovine serum albumin to the tacrolimus is 1:1 to 5: 1;

3) the size of the eutectic is 0.5-4 mu m.

5. The method of claim 3, wherein:

1) adding tacrolimus and treprostinil into an organic reagent, and ultrasonically dissolving, wherein the organic solvent is methanol, ethanol, DMSO or acetone

2) Taking the pure water as a water phase;

3) gradually dripping 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 cocrystal;

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

6. The method of claim 5, wherein:

the stirring condition in the step (3) is 500-2000 r/min, preferably 1200-1300 r/min;

the bovine serum albumin in the step (5) is 0-5mg/mL, preferably 1 mg/mL;

pre-freezing the preparation in the step (5) at-80 ℃ for 1 hour before freeze-drying.

7. The method of claim 6, wherein:

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

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

8. Use of a co-crystal-like according to any one of claims 1-3 in the manufacture of a medicament for the treatment of chronic cardiopulmonary disease.

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