CN114504554A - Bortezomib liposome preparation - Google Patents

Abstract

The invention discloses a bortezomib liposome with the particle diameter of less than 300nm, which has higher liposome encapsulation efficiency. And a bortezomib liposome preparation consisting of the liposome and a water-based polymer, wherein the bortezomib liposome preparation can be used for treating cancers.

Description

Bortezomib liposome preparation

Technical Field

The invention relates to the field of medicines, and discloses a bortezomib liposome with the particle diameter smaller than 300nm, and a bortezomib liposome preparation consisting of the bortezomib liposome and a water-based polymer.

Background

Bortezomib (BTZ) is a potent and specific proteasome inhibitor, and binds to threonine residues in the proteasome active center through a boronic acid group to inhibit the proteasome of tumor cells, thereby achieving the purpose of treatment. Bortezomib has recently been well recognized for the treatment of tumors such as multiple myeloma and mantle cell lymphoma. However, bortezomib has the characteristics of non-specific binding of proteins, rapid clearance in the liver, poor tissue infiltration capacity, poor aggregation capacity and the like, has a limited inhibiting effect on solid tumors, and may cause serious toxic and side effects, thereby obviously limiting the wide clinical application of bortezomib.

At present, the clinical formulation of bortezomib is an injection, which was first developed by Millennium pharmaceuticals in the united states and is marketed under the name of "wu" (Velcade). The preparation is of a current marketed variety in China, the marketed specifications are 1mg and 3.5mg respectively, but the preparation is slow in redissolution speed and poor in clarity, and impurities of the preparation are increased continuously along with the prolonging of the standing time, so that potential safety hazards are brought to medication. In addition, bortezomib is poor in water solubility, sensitive to oxygen and easy to oxidize, and is a serious difficulty in the preparation process of the preparation. Therefore, the method for solving the problems of difficult dissolution and instability of the main ingredient of the bortezomib has important practical significance for improving the quality of the final bortezomib injection.

The liposome is a monolayer or multilayer vesicle composed of orderly arranged lipid bilayers, has a bilayer structure similar to a biological membrane, and has the main component of phospholipid, and the bilayer structure of the liposome can be used as a lipophilic and hydrophilic drug carrier. The liposome is used as a drug carrier for tumor treatment, has the characteristics of targeting property, lymph directionality and the like, and is more and more concerned because the liposome can increase the curative effect of the drug and reduce toxic and side effects.

The traditional liposome has low encapsulation efficiency on lipophilic drugs and is one of the main factors limiting the application of the liposome. Moreover, the fusion aggregation phospholipid component existing in the common liposome can exchange with apolipoprotein of high-density lipoprotein in blood, activate a complement system and cause the decomposition of the liposome and the leakage of encapsulated drugs. In addition, after entering blood circulation, the liposome is rapidly adhered by plasma proteins and then is identified and rapidly cleared by a mononuclear macrophage system (MPS), so that the retention time of the liposome in the blood circulation is reduced, and the medicine cannot effectively reach a target site to play a role.

Therefore, it is one of the approaches to research and search for liposomes to reduce the recognition and phagocytosis of drugs by MPS and prolong the circulation time in vivo. There is a need for modification and alteration of common liposomes to overcome the shortcomings of their short-term efficacy. Long circulating liposomes (also known as long-acting liposomes) are liposomes which are prepared by modifying the surface of common liposomes to achieve the functions of positioning, slow release and protection. The current long-circulating liposome modifiers include: gangliosides GMl, polyethylene glycol (PEG) and its lipid derivatives, and the like. The PEG and the lipoid derivatives thereof are commonly used long-circulating materials at present, and a protective layer can be formed on the surface of the liposome modified by the PEG, so that the recognition of MPS to the liposome can be reduced, the bioavailability and the treatment effect of the medicine are improved, and the damage to normal tissues and organs can be reduced.

The present invention therefore contemplates the encapsulation of bortezomib in a long-acting liposome carrier. More particularly, to solve the difficulties in efficiently loading bortezomib and retaining it in a liposome in a stably encapsulated form, the present invention relates to the preparation of liposomes which can improve the loading and retention components of bortezomib in liposomes, so that bortezomib is efficiently loaded and stably encapsulated, thereby achieving better drug therapeutic effects.

Disclosure of Invention

It is therefore an object of the present invention to provide a formulation comprising bortezomib stably encapsulated in liposomes. Another object is to provide liposomal suspensions with stable forms of boronates.

In one aspect, the invention provides liposomes having encapsulated bortezomib. In this section, the composition and preparation of liposomes will be described.

Methods suitable for bortezomib loading are designed to provide liposomal formulations in which bortezomib is encapsulated in liposomes in the form of boronates. Bortezomib freely permeates through the lipid bilayer in the external aqueous medium. Reacting with the polyol inside the liposome to form boronate, which gradually accumulates in the liposome by substantially failing to cross the lipid bilayer, thereby causing more bortezomib to permeate through the lipid bilayer from the external medium.

Aqueous polyol solutions are used for hydration of dry lipid membranes prepared from desired mixtures of vesicle-forming lipids, non-vesicle-forming lipids (e.g., cholesterol, DOPE, etc.), lipopolymers (e.g., mPEG-DSPE), and any other desired lipid bilayer components. A dry lipid film was prepared as follows: the selected lipids are dissolved in a suitable solvent (usually a volatile organic solvent) and the solvent is then evaporated to leave a dry film. The lipid film is hydrated with a solution containing a polyol adjusted to a pH greater than about 7.0 to form liposomes.

The pH gradient is formed inside and outside the liposome, the pH value gradient of the internal and external water phases is large, more bortezomib in the external water phase enters the internal water phase, and borate esters formed by the bortezomib and polyhydric alcohols in the internal water phase are accumulated in the liposome. Thus, it is preferred to prepare the liposomes as described below so as to be substantially free of polyols in the external aqueous phase.

The polyol compound is preferably a moiety having a plurality of hydroxyl functional groups and is classified into monomers or polymers containing alcoholic hydroxyl groups, and examples of the monomeric polyol include polysaccharides, glycerin, glycols, carbohydrates, aminosugars, sugar alcohols, desoxysorbitol, gluconic acid, tartaric acid, gallic acid, and the like. The polyhydric alcohols can be aliphatic compounds, cyclic diols, polyphenols, etc.

The blank liposomes in the examples consisted of egg yolk lecithin (PC), Cholesterol (CHOL) and polyethylene glycol derivatized distearoyl phosphatidyl ethanolamine (PEG-DSPE). Common phospholipid classes are egg yolk lecithin (PC), soy lecithin (SPC), hydrogenated soy lecithin (HSPC), in one embodiment, the molar mass ratio of each phospholipid is PC: CHOL: PEG-DSPE ═ 10:5: 1.

Among them, PEG-DSPE is a lipoid derivative, which is the most commonly used long-circulating material at present. There are a variety of PEG-DSPE molecular weights, such as 350, 550, 750, 1000, 2000, 3000 and 5000 daltons, with molecular weights of 2000 and 3000 daltons being common.

The liposome modified by PEG can form a protective layer on the surface, and can reduce the recognition of reticuloendothelial system macrophage (RES) to the liposome, thereby improving the bioavailability and the treatment effect of the medicine. PEG-derivatized liposomes can also be modified to link to other ligands to form lipid-polymer-ligand conjugates. The ligand may be a therapeutic molecule, such as a drug or a biomolecule having activity in vivo, may be a diagnostic molecule, such as a contrast agent or a biomolecule, or a targeting molecule having binding affinity for a binding partner, preferably a cell surface binding partner. Preferred ligands have binding affinity for the cell surface and assist in the entry of the liposome into the cytoplasm by cellular internalization. The ligands present in the liposome comprising the lipopolymer ligands are oriented outwardly from the liposome surface and thus can interact with their cognate receptors.

Methods for attaching ligands to lipopolymers are known, for example, end-functionalized PEG-lipid conjugates are also commercially available. The linkage between the ligand and the polymer may be a stable covalent linkage or a releasable linkage that is cleavable in response to a stimulus (e.g., a change in pH or the presence of a reducing agent).

The ligand may be a molecule having binding affinity for a cellular receptor or for a pathogen circulating in the blood. The ligands may also be therapeutic or diagnostic molecules, in particular molecules with a short blood circulation lifetime when administered in free form. In one embodiment, the ligand is a biological ligand, and is preferably a ligand having binding affinity for a cellular receptor. Exemplary biological ligands are molecules that have binding affinity for receptors for the following molecules: CD4, folic acid, insulin, LDL, vitamins, transferrin, asialoglycoprotein, lactoferrin, EGF, integrin HER2, and the like. Preferred ligands include proteins and peptides, including antibodies and antibody fragments, and may also be small molecule peptidomimetics. It is understood that cell surface receptors or fragments thereof may serve as ligands. Other exemplary targeting ligands include, but are not limited to, vitamin molecules (e.g., biotin, folic acid), oligopeptides, chitosan, oligosaccharides.

The preparation of liposomes can be divided into two main categories according to the carrying mode of the drug: passive drug loading and active drug loading. In general, passive loading is the hydration of a dry lipid membrane with an aqueous medium to form multilamellar vesicles that passively entrap the compound during liposome formation. The compound may be a lipophilic compound contained in the dry lipid film or a water-soluble compound contained in the hydration medium. For water soluble compounds, the encapsulation efficiency of this method is rather poor, typically only 5-20% of the total compound in the final liposome suspension is in encapsulated form. Common passive drug delivery methods include passive drug delivery methods including thin film dispersion, ultrasonic dispersion, freeze-drying, and freezingMelting method, multiple emulsion method, organic solvent dispersion method, reverse phase evaporation method, supercritical CO₂Fluid technology, calcium fusion method.

The commonly used active drug-loading methods mainly include a pH gradient method, an ammonium sulfate gradient method and a calcium acetate gradient method. Compound loading methods, as opposed to inside-out pH or electrochemical liposome gradients, have been demonstrated to be useful for loading ionizable compounds into liposomes. In theory, by using a suitable gradient (e.g., a pH gradient of 2-4 units) and by appropriate selection of initial loading conditions, very high loading efficiencies can be achieved. In this way, leakage of the compound from the liposomes occurs after the liposomes have lost the ionic gradient. Thus, as long as the ion gradient is maintained, the compound is stably maintained in a liposome-encapsulated form.

In the invention, the bortezomib liposome is prepared by adopting a pH gradient method.

In a multilamellar liposome the lipid bilayer membrane is composed of multiple lipid bilayers with spatial aqueous spaces. The pH of the liposome external medium is about 7.0, typically between 5.5 and 7.0, and more typically between 6.0 and 7.0. The pH of the internal aqueous phase chamber is preferably greater than about 7.0, more preferably 7.0-9.0, and still more preferably between 7.5 and 8.5. Experiments demonstrate that during encapsulation, the lower pH in the external suspension medium and the higher pH inside the liposomes, in combination with the polyol inside the liposomes, induce the conversion of bortezomib to boronate esters to accumulate in the aqueous internal compartment of the liposomes. This process is driven by pH, where the lower pH outside the liposome (e.g., pH6-7) and the slightly higher pH inside the liposome (pH7.5-8.5) with the presence of a polyol, facilitates drug accumulation and loading. Liposomes are prepared by having an internal higher/external lower polyol gradient, the pH of the aqueous polyol solution being greater than about 7.0.

Preparing liposome composed of lipid, egg yolk lecithin (PC), Cholesterol (CHOL) and polyethylene glycol-derivatized distearoyl phosphatidyl ethanolamine (PEG-DSPE). The lipid film is formed by dissolving each lipid in chloroform at a certain molar ratio and then evaporating the solvent. The lipid film is hydrated with an aqueous solution of polyvinyl alcohol (pH7.5) to form liposomes in which the polyol is entrapped.

After liposome formation, the liposomes can be sized to obtain liposomes of a substantially uniform size range, typically about 0.01 to 0.5 microns, preferably 0.01 to 0.03 microns.

After finishing, the non-encapsulated aqueous polyol is removed by suitable techniques such as diafiltration, dialysis, centrifugation, size exclusion chromatography or ion exchange to obtain a suspension of liposomes with high internal polyol concentration and preferably with little or no polyol at the exterior. After liposome formation, the liposome external phase is adjusted to a pH of less than about 7.0 by titration, dialysis, or the like. Bortezomib is then added to the liposome dispersion to allow active loading into the liposomes.

Bortezomib and blank liposomes are incubated at a temperature above the phospholipid phase transition temperature, and different incubation temperatures and times affect the efficiency of liposome-encapsulated drugs. At the end of this incubation step, the suspension may be further processed to remove free (unencapsulated) polyol.

Bortezomib is a reversible proteasome inhibitor that reversibly inhibits chymotrypsin/trypsin activity of the proteasome 26S subunit in mammalian cells by selectively binding to threonine at the proteasome active site. The 26S proteasome is a large intracellular protein complex that primarily degrades proteins bound to ubiquitin, which can affect intracellular cascade of multiple signals. Research has proved that in tumor cells, bortezomib can obviously reduce the degradation of inhibitor (I kappa B) of nuclear factor kappa B (NF kappa B) after specifically inhibiting the activity of proteasome 26S subunit, the activity of NF kappa B can be effectively inhibited after the I kappa B is combined with the NF kappa B, the expression of genes related to cell proliferation is inhibited, the secretion of myeloma cell growth factors such as IL-6 and the like and the expression of adhesion factors are reduced, and finally, the tumor cells are apoptotic.

Preclinical in vivo tumor model experiments demonstrated that bortezomib was able to delay the growth of tumor cells, including multiple myeloma, induce apoptosis and inhibit angiogenesis. Clinical studies prove that bortezomib has definite curative effects on relapsed and refractory multiple myeloma, and relatively mild and more tolerable adverse reactions.

Liposomal formulations comprising bortezomib are useful for the treatment of cancer, more specifically for the treatment of multiple myeloma.

In addition, the formulation can enhance the antitumor activity of drugs such as paclitaxel and doxorubicin when used in combination with conventional chemotherapeutic drugs.

General description of the invention

The present invention describes various embodiments in the context of lipid nanoparticles. Those skilled in the art will appreciate that the following detailed description of the embodiments is illustrative only and is not intended to be in any way limiting. Other embodiments will readily suggest themselves to such skilled persons having the benefit of this disclosure. References herein to "an embodiment," "an aspect," or "an example" indicate that the embodiment of the invention described may include a particular property, structure, or characteristic, but every embodiment may not necessarily include the particular property, structure, or characteristic. Moreover, repeated use of the phrase "in one embodiment" does not necessarily refer to the same embodiment, although it may.

Not all of the routine features of the implementations or methods described herein are shown and described. It will of course be appreciated that in the development of any such actual embodiment, numerous implementation-specific decisions should be made to achieve the specific goals of the invention, such as compliance with application-and business-related constraints, and that these specific goals will vary from one implementation to another and from one developer to another. Moreover, it will be appreciated that such a development effort might be complex and time-consuming, but would nevertheless be a routine undertaking for those of ordinary skill in the art having the benefit of this disclosure.

The borate ester compound refers to a compound which is formed by esterification of bortezomib and a polyol compound and is stably encapsulated by liposome.

The blank liposome refers to liposome which is composed of various lipids without medicine loading according to a certain proportion.

By "liposome-encapsulated" is meant a compound that is sequestered in the aqueous compartment in the center of the liposome, the aqueous space between the liposome lipid bilayers, or within the bilayers themselves.

Detailed Description

Example 1:

1.1 preparation of blank liposome:

PC, CHOL, PEG-DSPE (molecular weight 2000) were mixed and dissolved in chloroform at a molar ratio of 10:5:1, and the solvent was evaporated by thin film vacuum method. Polyvinyl alcohol (molecular weight 2000) was dissolved in water, the pH was adjusted to 7.5, the resulting lipid film was incubated in a polyvinyl alcohol solution with shaking, and the resulting dispersion was extruded under pressure through 2 membranes with a pore size of 0.2. mu.m. The external buffer was replaced with 0.14M NaCl (containing 5mM HEPES, pH6.5) by Sepharose CL-4B gel chromatography, while removing the unencapsulated polyvinyl alcohol.

1.2 loading of the drug bortezomib:

bortezomib was added to the blank liposomes prepared by the above method. The mixture was incubated overnight at 37 ℃ with shaking, treated with Dowex50WX4 ion resin, and equilibrated with NaCl-HEPES solution to remove unencapsulated bortezomib. The liposomes obtained were sterile filtered through a 0.2 μm filter. The particle size and encapsulation efficiency of the finally obtained liposome suspension were measured, and the results are shown in tables 1 and 2.

Example 2:

2.1 preparation of blank liposome:

dissolving mannitol in water, adjusting pH to 7.5, mixing PC, CHOL, and PEG-DSPE (molecular weight 2000) at a molar ratio of 10:5:1, dissolving in chloroform, and evaporating solvent by thin film vacuum method. The resulting lipid film was incubated in mannitol solution with shaking to form blank liposomes. The resulting dispersion was extruded under pressure through 2 membranes with a pore size of 0.2 μm.

2.2 loading of the drug bortezomib:

bortezomib was then added to the external NaCl-HEPES solution and the resulting mixture incubated overnight at 37 ℃ with shaking. Any unencapsulated compounds are then removed by filtration. The encapsulation efficiency of the finally obtained liposomal suspension was determined and the results are shown in tables 1 and 2.

Example 3:

3.1 preparation of blank liposomes:

dissolving aminosalactose in water, adjusting pH to 7.5, mixing PC, CHOL, PEG-DSPE (molecular weight 2000) at a molar ratio of 10:5:1, dissolving in chloroform, and evaporating solvent by thin film vacuum method. The resulting lipid film was hydrated with an amino lactose solution and incubated with shaking to form blank liposomes. The resulting dispersion was extruded under pressure through 2 membranes with a pore size of 0.2 μm.

3.2 loading of the drug bortezomib:

bortezomib was then added to the external NaCl-HEPES solution and the resulting mixture incubated overnight at 37 ℃ with shaking. Any unencapsulated compounds are then removed by filtration. The encapsulation efficiency of the finally obtained liposome suspension was measured and the results are shown in tables 1 and 2.

Example 4:

4.1 preparation of blank liposomes:

dissolving tromethamine in water, adjusting pH to 7.5, mixing PC, CHOL, PEG-DSPE (molecular weight 2000) at a molar ratio of 10:5:1, dissolving in chloroform, and evaporating solvent by thin film vacuum method. The resulting lipid film was incubated in tromethamine solution with shaking to form blank liposomes. The resulting dispersion was extruded under pressure through 2 membranes with a pore size of 0.2 μm. Loading the drug bortezomib:

4.2 loading of the drug bortezomib:

bortezomib was then added to the external NaCl-HEPES solution and the resulting mixture incubated overnight at 37 ℃ with shaking. Any unencapsulated compounds are then removed by filtration. The encapsulation efficiency of the finally obtained liposome suspension was measured and the results are shown in tables 1 and 2.

Example 5:

5.1 preparation of blank liposome:

sorbitol was dissolved in water, pH was adjusted to 7.5, PC, CHOL, PEG-DSPE (molecular weight 2000) -chitosan were mixed and dissolved in chloroform at a molar ratio of 10:5:1, and the solvent was evaporated in a thin film vacuum. The resulting lipid film was incubated in a sorbitol solution with shaking to form blank liposomes. The resulting dispersion was extruded under pressure through 2 membranes with a pore size of 0.2 μm.

5.2 loading of the drug bortezomib:

bortezomib was then added to the external NaCl-HEPES solution and the resulting mixture incubated overnight at 37 ℃ with shaking. Any unencapsulated compounds are then removed by filtration. The encapsulation efficiency of the finally obtained liposome suspension was measured and the results are shown in tables 1 and 2.

Particle size of the liposome preparation obtained in examples 1-5: the particle size of each sample was measured using a ZS90 nanometer particle sizer (malvern instruments ltd, uk) and the results are shown in table 1.

TABLE 1 measurement of particle size of Liposomal suspension

Batch number	Day 0	1 month	2 months old	3 months old
					Example 1: 0901	102.3nm	113.8nm	135.5nm	147.0nm
Example 2: 0902	115.7nm	117.2nm	128.0nm	140.1nm
					Example 3: 0903	144.8nm	154.7nm	169.4nm	188.8nm
Example 4: 0904	127.5nm	139.6nm	150.4nm	173.5nm
					Example 5: 0905	110.9nm	119.0nm	133.3nm	155.8nm

Drug encapsulation efficiency of the liposome preparations obtained in examples 1 to 5: the drug encapsulation efficiency was determined by size exclusion chromatography. Samples (100. mu.L) were loaded onto a Bio-Gel, P-6 column (0.5X30cm), eluted with 150mM NaCl (containing 5mM HEPES) solution at pH7.0, and fractions (1 mL/tube) were collected. The UV absorption at 270nm of the liposomes and free drug fractions were examined and combined separately and analyzed by HPLC to obtain the results shown in Table 2.

TABLE 2 encapsulation efficiency assay results for liposomal suspensions

Batch number	Day 0	25-1 month	25-2 months	25-3 months
					Example 1: 0901	94.3％	80.8％	72.6％	65.1％
Example 2: 0902	90.2％	84.7％	74.4％	70.5％
					Example 3: 0903	84.9％	75.3％	67.0％	57.5％
Example 4: 0904	87.2％	70.5％	65.8％	60.4％
					Example 5: 0905	92.6％	78.1％	77.3％	65.0％

From the results of the experimental examples and the encapsulation efficiency measurement results, the bortezomib liposome obtained by the scheme provided by the invention is a lipid nanoparticle with the diameter less than 300nm, and has high liposome encapsulation efficiency.

Claims (5)

1. A bortezomib liposome characterized in that the particle diameter is less than 300nm, a boronate compound is formed from bortezomib and a lipid composition, the bortezomib being stably encapsulated within the liposome.

2. The bortezomib liposomes according to claim 1 wherein the lipid content is predominantly phospholipids, cholesterol; wherein the phospholipid comprises egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin, phosphatidyl choline, and phosphatidyl ethanolamine.

3. A bortezomib liposome formulation, characterized by consisting of the bortezomib liposome of claim 1 and an aqueous polymer.

4. The bortezomib liposome formulation according to claim 3, wherein the aqueous polymer contained is a polyol, which can be aliphatic compounds, cyclic glycols, polyphenols, etc.

5. Use of a bortezomib liposome formulation according to claim 3 in a medicament for treating cancer in a subject.