Ruxolitinib intravenous formulations, methods of making and using the same

Abstract

The application belongs to the technical field of biotechnology, and specifically discloses a preparation method and application of a lucotinib intravenous preparation. One aspect relates to M@NP-R and a preparation method thereof, and also relates to application of the M@NP-R in preparation of an intravenous preparation for treating cytokine storm and treating hemophagocytic syndrome, and further relates to application of the M@NP-R in preparation of an intravenous preparation for treating hemophagocytic syndrome combined with multiple organ dysfunction syndrome. The M@NP-R of the application can be administered by intravenous injection, can effectively treat hemophagocytic syndrome, and also has obvious therapeutic effects on some complications of hemophagocytic syndrome, especially for treatment of severe hemophagocytic syndrome, avoids the problem that patients cannot orally take drugs, and can produce more beneficial effects through intravenous injection, especially for treatment of rapidly progressing hemophagocytic syndrome, which can greatly improve survival time.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and particularly relates to ruxolitinib intravenous preparations, their preparation methods, and applications. Background Technology

[0002] Hemophagocytic lymphohistiocytosis (HLH) is a group of clinical syndromes caused by the excessive activation and proliferation of the mononuclear macrophage system, leading to the production of large amounts of inflammatory factors. The main manifestations include persistent fever, hepatosplenomegaly, pancytopenia, and the presence of hemophagocytic cells in tissues such as the liver, spleen, and bone marrow. The disease progresses rapidly and has a high mortality rate. The main causes include primary factors such as genetic defects and secondary factors such as infections, tumors, or autoimmune diseases. Clinically, it progresses rapidly and has a poor overall prognosis. Currently, commonly used clinical treatments include induction therapy to control the cytokine storm, with main drugs including chemotherapy drugs (etoposide, doxorubicin liposomes) and hormone drugs, as well as etiological treatment such as aggressive treatment of the primary disease or bone marrow transplantation. However, current treatments are difficult to rapidly and effectively control the persistent activation of macrophages and the subsequent cytokine storm, resulting in limited efficacy and significant drug side effects. Therefore, there is an urgent need to design and develop novel treatment strategies to improve the treatment outcomes of HLH.

[0003] A vicious cycle of mutual promotion between cytokine storm and activated macrophages is the main pathophysiological feature driving the development of HLH. Targeted regulation of inflammation represents a new therapeutic direction for HLH. HLH is also accompanied by multi-organ dysfunction, leading to abnormalities in multiple organ indicators. Currently, there are no effective methods to alleviate these concurrent organ injuries. Monoclonal antibodies targeting certain types of inflammatory factors, such as the marketed interleukin-6 (IL-6) and interferon, are promising treatment options. ( Monoclonal antibodies have shown some efficacy in treating HLH. However, monoclonal antibodies can only target certain specific inflammatory factors, resulting in a single target and difficulty in efficiently controlling the cytokine storm involving multiple cytokines. The JAK / STAT pathway is a common pathway for intracellular signal transduction of multiple cytokines, mediating various biological responses such as cell proliferation, differentiation, migration, apoptosis, and immune regulation. It is a major pathogenic inflammatory factor in HLH. Ruxolitinib, a selective JAK1 / 2 inhibitor, effectively inhibits JAK / STAT pathway activation and reduces the expression of various inflammatory factors in HLH, demonstrating superior efficacy compared to monoclonal antibodies in animal studies. However, the clinical efficacy of ruxolitinib in treating HLH, especially in adult HLH patients, still requires further investigation. Furthermore, HLH patients are often critically ill, and oral administration of ruxolitinib is poorly tolerated and expensive, potentially limiting its clinical application. The lack of effective intravenous formulations for critically ill patients with poor oral compliance poses a significant challenge. While some researchers have recognized the need for direct intravenous administration of ruxolitinib to assess its efficacy, current reports indicate limited effectiveness of intravenous ruxolitinib monotherapy, particularly in combination therapy for hemophagocytic lymphohistiocytosis (HLH). Further research is needed to explore the optimal intravenous administration method and efficacy of ruxolitinib. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides an intravenous ruxolitinib preparation, its preparation method, and its application, primarily solving the current lack of an effective intravenous ruxolitinib preparation for treating hemophagocytic lymphohistiocytosis (HLH) and other conditions.

[0005] To solve the above problems, the present invention adopts the following technical solution:

[0006] The first aspect of this invention relates to M@NP-R and its preparation method.

[0007] The preparation method of M@NP-R includes the following steps:

[0008] S1. Inject acetone containing dissolved PLGA nanoscale carriers and ruxolitinib into water.

[0009] S2. After removing acetone, PLGA nanoparticles loaded with ruxolitinib were obtained.

[0010] S3. Add macrophage membrane proteins to prepare M@NP-R.

[0011] For the nanoscale carrier, PLGA is used as an example. Macrophage membrane vesicles are prepared using macrophage membranes. PLGA nanoparticles are then coated onto the macrophage membranes to form M@NP. PLGA and ruxolitinib are used to prepare ruxolitinib-loaded PLGA nanoparticles (NP-R). Macrophage membranes are then coated onto NP-R to prepare M@NP-R. PLGA nanoparticles can be prepared directly using existing materials or existing preparation methods. For small-scale preparation, the specific dosage can be referenced as follows: 500 μL of acetone containing 5 mg of PLGA polymer and 2 mg of ruxolitinib is rapidly injected into 1 mL of deionized water. After evaporating and removing the acetone using a vacuum pump, ruxolitinib-loaded PLGA nanoparticles are obtained. 1 mg of RAW264.7 macrophage membrane protein is added and mixed thoroughly. The mixture is then sonicated in an ice bath (200 W) for 5 minutes to obtain M@NP-R. For large-scale preparation, the dosage can be adjusted according to actual needs.

[0012] Some conditions in the preparation steps can be selected as follows, and any one of the sub-steps can also be selected individually:

[0013] In one of the steps, S2, after removing acetone by evaporation, PLGA nanoparticles loaded with ruxolitinib are obtained. The specific evaporation method can be vacuum pump evaporation.

[0014] Secondly, in step S3, macrophage membrane proteins are added and mixed, and then subjected to ice bath sonication (or other similar methods) to obtain M@NP-R;

[0015] Thirdly, in step S1, the ratio of ruxolitinib to PLGA is 5% to 40%. The acetone containing PLGA and ruxolitinib needs to be quickly injected into water. PLGA and ruxolitinib are first dissolved together in acetone to form a mixture.

[0016] Fourthly, in step S3, the macrophage membrane protein is any one of the following: RAW264.7 macrophage membrane protein, neutrophil cell membrane protein, lipopolysaccharide-stimulated and optimized macrophage membrane protein, hybrid membrane of different types of biological membranes, or gene-edited cell membrane protein. Generally, it can be directly extracted from various cell lines without special limitations.

[0017] The M@NP-R prepared by any of the aforementioned methods is non-toxic and has no side effects. M@NP-R is generally a regular sphere with a particle size of approximately 100 nm and a potential of approximately -40 mV, but is not limited to these characteristics. Any M@NP-R prepared by the aforementioned methods that can be identified using conventional material identification methods should be considered within the scope of this invention.

[0018] The second aspect of this invention relates to the application of the aforementioned M@NP-R in the preparation of intravenous formulations for treating cytokine storms; wherein the intravenous formulation is administered via intravenous injection. M@NP-R can adsorb inflammatory factors to a significant extent, thus mitigating cytokine storms. The cytokine receptors on the surface of M@NP-R can adsorb and remove already produced cytokines, and the ruxolitinib within it can be used to target and inhibit macrophage activation, reducing cytokine production to synergistically alleviate cytokine storm-related symptoms. Intravenous formulations are drugs that can be administered by injection in the conventional sense, which differs from oral administration. M@NP-R can also be used to prepare formulations that inhibit macrophage activation. These formulations are generally in vitro preparations and are non-diagnostic and therapeutic products; macrophage activation is mainly induced by cytokines, primarily at least one of IFN-γ, IL-6, TNF-α, and IL-1β; such reagents are mainly used in biological experiments, such as in cytokine-induced macrophage activation models to control variables and reduce macrophage activation levels. In other words, it can also be used in the preparation of formulations that promote the uptake of M@NP-R by macrophages. The cytokine can be IFN-γ, and this promoting effect is mainly relative to the ability of macrophages without added cytokines to take up M@NP-R.

[0019] A third aspect of this invention relates to the use of the aforementioned M@NP-R in the preparation of an intravenous formulation for treating hemophagocytic lymphohistiocytosis (HLH); wherein the intravenous formulation is administered via intravenous injection. HLH is a systemic inflammatory response, and simulation results at pH 6.8 indicate that the intravenous formulation exhibits better sustained-release efficacy in inflamed tissues.

[0020] Regarding applications, some cases where they can be listed in parallel should be specifically noted as follows:

[0021] The hemophagocytic syndrome is either primary or secondary; further, the secondary hemophagocytic syndrome is hemophagocytic syndrome induced by at least one of the following: severe infection, rheumatic disease, neoplastic disease, CAR-T therapy, or monoclonal antibody therapy. In this document, "at least" means that it can be any of the listed characteristics, or other currently known relevant features, and is not limited to the listed content.

[0022] In this case, hemophagocytic lymphohistiocytosis (HLH) is accompanied by a severe cytokine storm. High levels of cytokines help M@NP-R target macrophages to achieve its inhibitory effect, which makes M@NP-R more effective in treating this type of HLH. The presence of cytokines in this type of HLH can actually enhance the therapeutic effect of M@NP-R on HLH, and it can also alleviate the cytokine storm while treating HLH. M@NP-R has a significant therapeutic effect on HLH accompanied by a severe cytokine storm.

[0023] Specifically, the effects of M@NP-R include at least one of the following: inhibition of cytokine storm, inhibition of macrophage activity, and inhibition of hemophagocytosis. M@NP-R, in particular, has a significant inhibitory effect on cytokine storm in the treatment of hemophagocytic lymphohistiocytosis. The inhibitory effect of M@NP-R on cytokine storm is significantly superior to that produced by the same dose of ruxolitinib (Rux), especially its inhibitory effect on IFN-γ and IL-6 is extremely significant. This invention has discovered a synergistic effect between macrophage membrane-coated polyester nanoparticles and ruxolitinib in the preparation of an intravenous formulation. While the macrophage membrane-coated polyester nanoparticles primarily clear existing cytokines, their effect on reducing cytokine production is limited. This is because the clearance of cytokines by the polyester nanoparticles weakens the stimulation of macrophages, leading to inhibition of macrophage activation and thus reducing cytokines, albeit indirectly. In contrast, ruxolitinib loaded onto the polyester nanoparticles can directly inhibit macrophage activation and reduce cytokine production. Therefore, to a certain extent, the loading of polyester nanoparticles onto macrophage membranes can be considered a synergistic effect.

[0024] Based on the effects of M@NP-R in a lethal HLH mouse model, intravenous administration of M@NP-R showed better efficacy than direct administration of M@NP, and also demonstrated better therapeutic effects than the same dose of ruxolitinib, particularly significantly improving survival rates and even achieving zero mortality. This indicates that M@NP-R, as an intravenous preparation, significantly improved the efficacy of treating lethal hemophagocytic lymphohistiocytosis (HLH) compared to other groups, suggesting a synergistic effect of ruxolitinib loaded with M@NP. Therefore, in specific application scenarios, HLH can be interpreted as lethal HLH, such as lethal HLH complicating severe infectious inflammation (COVID-19 infection, EBV infection, etc.).

[0025] The fourth aspect of the present invention relates to the use of M@NP-R in the preparation of an intravenous formulation for treating hemophagocytic syndrome complicated with organ dysfunction syndrome; wherein the intravenous formulation is administered by intravenous injection.

[0026] Regarding applications, some cases where they can be listed in parallel should be specifically noted as follows:

[0027] M@NP-R also shows good alleviating effects for hemophagocytic lymphohistiocytosis (HLH) complicated with organ dysfunction. When organ function indicators are abnormal, M@NP-R can restore organ function, especially for the recovery of liver function, kidney function, and spleen damage (enlargement and / or inflammation), with significantly better effects than either M@NP or Rux alone. The M@NP particles in M@NP-R and the Rux they are loaded with produce a synergistic effect in the above treatments. M@NP-R also has a relatively significant therapeutic effect on HLH complicated with coagulation dysfunction and can be used as a therapeutic agent.

[0028] Among these, M@NP-R can essentially restore abnormal indicators to normal levels when alleviating renal dysfunction, splenic dysfunction, and dyslipidemia, especially showing a more prominent therapeutic effect on splenic inflammation and splenomegaly. Changes in various indicators in the study show that M@NP-R can restore splenic inflammation and splenomegaly to normal levels, demonstrating a highly significant therapeutic effect on splenic abnormalities (enlargement and / or inflammation), which M@NP and Rux cannot achieve. Analysis of the experimental results also shows that the efficacy of M@NP-R is superior to the sum of the efficacies of M@NP and Rux alone, especially in the improvement of splenic damage.

[0029] The fifth aspect of this invention relates to an intravenous preparation for treating hemophagocytic lymphohistiocytosis (HLH), comprising M@NP-R as the sole active ingredient, the remainder of which may be conventional adjuvants for intravenous preparations (such as PBS, water, etc.), but M@NP-R is the active ingredient for treating HLH; the HLH is either primary or secondary HLH, and the treatment of HLH is at least lethal HLH.

[0030] The beneficial effects of this invention are:

[0031] The M@NP-R of this invention, administered intravenously, can effectively treat HLH and also shows significant therapeutic effects on some complications of HLH, especially for the treatment of severe HLH. It avoids the problem of patients being unable to take oral medications, and intravenous injection can produce more beneficial effects. In particular, it can greatly improve survival time and achieve zero mortality in the treatment of rapidly progressive hemophagocytic lymphohistiocytosis. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the research project;

[0033] Figure 2 Preparation and characterization of M@NP-R; electron microscopy (A), particle size (B), potential (C), stability (D), in vitro release curve (E), drug loading and encapsulation efficiency (F) of M@NP-R; Western blot analysis of IFN-gR and IL-6R expression in M@NP-R (G); ELISA analysis of the adsorption capacity of M@NP-R for IFN-g and IL-6 (HK).

[0034] Figure 3 Evaluation of M@NP-R's inhibitory effect on macrophage activation in vitro. Flow cytometry analysis (AB) of macrophage activation and quantitative analysis of different types of cytokines (CG), qualitative (H) and quantitative analysis (I) of the uptake effect of M@NP-R in RAW 264.7 cells.

[0035] Figure 4 To improve the cytokine storm and related blood parameters in a CPG-induced HLH model using M@NP-R. Analysis included the expression levels of different types of cytokines (AD), blood cell markers (EH), and liver and kidney function and lipid levels (IL).

[0036] Figure 5 M@NP-R was used to improve macrophage activation in a CPG-induced HLH model. Gross (A) and wet weight (B) of spleen in different treatment groups; ratio (C), number (D), and quantification of pSTAT1 in macrophages (E); identification of hemophagocytic cells in different treatment groups (F), with arrows indicating hemophagocytic cells; immunohistochemical results of F4 / 80+ inflammatory cells (G), IFN-γ (H), and IL-6 (I) in spleen tissue sections.

[0037] Figure 6 Pharmacodynamic evaluation of M@NP-R treatment in a polyinosinic-polycytidylic acid combined with LPS-induced HLH model. Survival curves (A), coagulation function evaluation (BC), expression levels of different types of cytokines (DG), and identification of hemophagocytic cells (H) in different treatment groups of mice. Arrows indicate hemophagocytic cells. Detailed Implementation

[0038] The invention will be explained in detail below with reference to specific research projects.

[0039] Preparation and characterization of M@NP-R

[0040] Macrophage membrane vesicles were prepared using macrophage membranes. PLGA nanoparticles were then coated onto the macrophage membranes to form M@NPs. PLGA and ruxolitinib were used to prepare ruxolitinib-loaded PLGA nanoparticles (NP-R). NP-Rs were then coated onto macrophage membranes to prepare M@NP-Rs. Under cryo-electron microscopy, M@NP-Rs appeared as regular spherical shapes. Figure 2 (A), with a particle size of approximately 100 nm. Figure 2 (B), potential around -40mV ( Figure 2 M@NP-R can be stably dispersed in PBS solution for up to 7 days. Figure 2 (D). Under acidic and neutral conditions, 80% of ruxolitinib in M@NP-R is released, and at pH 6.8, M@NP-R releases it more rapidly in the initial stage. Figure 2 (E). When the ratio of ruxolitinib to PLGA input changed from 5% to 40%, its encapsulation efficiency stabilized at around 85.30%, and the drug loading gradually increased from 4.13% to 25.45%. Figure 2 Western blot results showed that the expression levels of IFN-γ and IL-6R in M@NP-R were comparable to those in the source cells and M@NP, indicating that M@NP-R can retain key molecules in the source cells (F). Figure 2 M@NP-R can adsorb IFN-g / IL-6 in a concentration-dependent manner. Figure 2 (Hong Kong).

[0041] Evaluation of M@NP-R's In vitro inhibition of macrophage activation

[0042] Bone marrow-derived macrophages (BMDM) were extracted and activated by IFN-γ stimulation. BMDM cells were then treated with PBS, M@NP, ruxolitinib, and M@NP-R, respectively. Flow cytometry results showed that M@NP-R reduced the proportion of F4 / 80+CD80+ cells, representing pro-inflammatory macrophages. Figure 3 (AB). Furthermore, compared to the IFN-γ stimulation group, the M@NP-R group showed significantly lower levels of IFN-γ, IL-6, TNF-α, and IL-1β. Figure 3 In CF, the protective cytokine IL-10 is elevated ( Figure 3 Compared with M@NP and Rux alone, M@NP-R further reduced the levels of most cytokines. While both had similar surface membranes and adsorption effects, M@NP-R still further reduced cytokine production, indicating a synergistic effect between the M@NP-R nanoparticles and the internally encapsulated drug. Fluorescence microscopy was used to analyze the effects. Figure 3 (H) and flow cytometry ( Figure 3(I) Evaluation of the uptake effect of activated macrophages on M@NP-R. Compared with unstimulated macrophages, IFN-γ-stimulated macrophages can take up more M@NP-R, indicating that the high level of cytokines present in the HLH mouse model helps M@NP-R target macrophages to achieve its inhibitory effect. Under inflammatory conditions, M@NP-R is more easily taken up by macrophages than under normal conditions. After being taken up by cells, it exerts an intracellular macrophage inhibitory effect, thereby reducing cytokine production.

[0043] M@NP-R improves cytokine storm and related blood parameters in a CPG-induced HLH model.

[0044] A mouse model of HLH was induced by intraperitoneal injection of CPG (50 μg / animal, every other day for a total of five times). The experiment was divided into four groups: normal mice (no treatment), HLH mice (CpG+PBS, CpG modeling, 100 μL PBS injected daily via tail vein as a negative control), M@NP treatment group (CpG+M@NP, CpG modeling, M@NP dose 2 mg / kg), Rux treatment group (CpG+Rux, CpG modeling, ruxolitinib dose 4 mg / kg), and M@NP-R treatment group (CpG+M@NP-R, CpG modeling, M@NP-R dose calculated based on cell membrane 2 mg / kg, dose calculated based on encapsulated ruxolitinib 4 mg / kg). All treatment groups received tail vein administration on days 1, 3, 5, 7, and 9. Results showed that M@NP-R treatment significantly alleviated the cytokine storm. Figure 4 (A-D), blood cell markers ( Figure 4 EH), liver function indicators (ALT and AST) Figure 4 (I&J), renal function indicator blood urea nitrogen (BUN) ( Figure 4 (K) and blood lipid indicators (TG) Figure 4 Both L) showed significant improvement.

[0045] M@NP-R improves macrophage activation in a CPG-induced HLH model.

[0046] A mouse model of HLH was induced by intraperitoneal injection of CPG (50 μg / animal, every other day for a total of five times). The experiment was divided into four groups: normal mice (no treatment), HLH mice (CpG+PBS, CpG modeling, 100 μL PBS injected daily via tail vein as a negative control), M@NP treatment group (CpG+M@NP, CpG modeling, M@NP dose 2 mg / kg), Rux treatment group (CpG+Rux, CpG modeling, ruxolitinib dose 4 mg / kg), and M@NP-R treatment group (CpG+M@NP-R, CpG modeling, M@NP-R dose calculated based on cell membrane 2 mg / kg, dose calculated based on encapsulated ruxolitinib 4 mg / kg). All treatment groups received tail vein administration on days 1, 3, 5, 7, and 9. The spleen in the M@NP-R group was significantly reduced in size. Figure 5 In the middle AB, the proportion and absolute number of pro-inflammatory macrophages were significantly reduced ( Figure 5 (C), consistent with immunohistochemical results ( Figure 5 (G). The M@NP-R group reduced the occurrence of hemophagocytosis (G). Figure 5 In the M@NP-R group, the level of cytokines in spleen tissue was significantly decreased (F). Figure 5 In H&I, pSTAT1 levels in macrophages were significantly reduced, almost matching normal levels. Figure 5 (E).

[0047] Pharmacodynamic evaluation of M@NP-R in treating polyinosinic acid combined with LPS-induced HLH model

[0048] A lethal HLH mouse model was established by intravenous injection of 10 mg / kg poly(I:C) via tail vein, followed by intraperitoneal injection of 5 mg / kg lipopolysaccharide 24 hours later. This model exhibited rapidly progressive HLH with significantly elevated systemic inflammation levels compared to the CPG model. M@NP, Rux, and M@NP-Rux (calculated as membrane protein 2 mg / kg, calculated as ruxolitinib 4 mg / kg) were administered intravenously every 2 hours for a total of 3 times. PBS was used as a negative control, and survival curves were plotted. The results showed that M@NP-R significantly improved the survival time of the model mice. Figure 6 (A) Coagulation and clotting indicators (PT and APTT) Figure 6 (BC), and inflammatory factor levels ( Figure 6 (DG) and reduced the occurrence of hemophagocytosis ( Figure 6 (H).

[0049] Those skilled in the art will appreciate that various modifications to the above embodiments can be made without departing from the overall spirit and concept of the present invention. All such modifications fall within the protection scope of the present invention. The protection scheme of the present invention is defined by the appended claims.

Claims

1. Application of M@NP-R in the preparation of intravenous formulations for the treatment of hemophagocytic lymphohistiocytosis; among which, The intravenous formulation is administered via intravenous injection, and M@NP-R is prepared by the following method: S1. Inject acetone containing dissolved PLGA nanoparticles and ruxolitinib into water. S2. After removing acetone, PLGA nanoparticles loaded with ruxolitinib were obtained. S3. Add macrophage membrane proteins to prepare M@NP-R.

2. The application according to claim 1; wherein, In S2, acetone was removed by evaporation to obtain PLGA nanoparticles loaded with ruxolitinib; in S3, macrophage membrane proteins were added and mixed, and then sonicated in an ice bath to obtain M@NP-R.

3. The application according to claim 2; wherein, In S1, the ratio of ruxolitinib input to PLGA input is 5%~40%; in S3, the macrophage membrane protein is any one of RAW264.7 macrophage membrane protein, neutrophil cell membrane protein, macrophage membrane protein optimized by lipopolysaccharide stimulation, hybrid membrane of different types of biological membranes, or gene-edited cell membrane protein.

4. The application according to claim 1; wherein, The hemophagocytic syndrome is either primary hemophagocytic syndrome or secondary hemophagocytic syndrome. The secondary hemophagocytic syndrome is hemophagocytic syndrome induced by at least one of the following: severe infection, rheumatic disease, neoplastic disease, CAR-T therapy, or monoclonal antibody therapy. Alternatively, the hemophagocytic syndrome may be lethal hemophagocytic syndrome. The effect of M@NP-R is manifested in at least one of the following: inhibiting cytokine storm, inhibiting macrophage activity, and inhibiting hemophagocytosis.

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