RU2728976C2 - Liposomal composition and preparation thereof - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: group of inventions can be used to produce a medicinal agent based on a liposome composition for treating a condition associated with inflammation. Liposome contains a lipid ingredient and encapsulates the bisphosphonate. Liposome has weight ratio of said bisphosphonate to lipid ingredient from 1:5 to 1:8. Lipid ingredient contains distearoyl phosphatidyl choline (DSPC), distearoyl phosphatidyl glycerol (DSPG) and cholesterol in molar ratio of 3:1:2. Liposome has compressibility value less than 0.70 ml/g. Invention also discloses a liposome, a composition of liposomes and use of a formulation for preparing a medicinal agent.EFFECT: group of inventions enables to obtain a homogeneous and stable liposomal composition for treating a condition associated with inflammation.15 cl, 6 tbl, 5 dwg, 4 ex

Description

AREA OF TECHNOLOGY

The present invention relates to a novel liposome formulation and a process for preparing a liposome formulation.

LEVEL OF TECHNOLOGY

Liposomes are known in the art as vehicles for delivering therapeutic agents to target cells for the treatment of various medical conditions. In one application, liposomes can be prepared to encapsulate a pharmaceutical agent that can be selectively phagocytosed by macrophages. After phagocytosis, liposomes, among other effects, release the agent intracellularly, suppressing the inflammatory functions of macrophages.

Several methods for preparing such liposomes have been described in the art (see, for example, Mönkkönen, J. et al., 1994, J. Drug Target, 2: 299-308; Mönkkönen, J. et al., 1993, Calcif. Tissue Int ., 53: 139-145; Lasic DD., Liposomes Technology Inc., Elsevier, 1993, 63-105. (Chapter 3); Winterhalter M, Lasic DD, Chem Phys Lipids, 1993; 54 (1-3): 35 -43). In one such method, known as the Lipid Thin Film Hydration Method, liposomes are formed in stacks of crystalline bilayers that hydrate into hydrated lipid sheets that break off during agitation and close into multilayer vesicles (MLVs). After the formation of these particles, their size depends on the subsequent method used, for example, the use of ultrasonic energy (sonication) or mechanical energy (extrusion). The sonication produces small unilamellar vesicles (SUVs) and requires the use of bath and / or immersion type ultrasonic dispersers. Alternatively, lipid extrusion, in which a lipid suspension is forced under high pressure (up to 3.45 MPa (500 psi)) through a series of polycarbonate filters, usually membranes with pores of 0.8, 0.4, 02, and 0, 1 μm, particles with a size approximately equal to the pore diameter of the filter used are obtained. These methods are applicable only for small-scale production, mainly for research purposes. Moreover, high pressure extrusion processes are associated with high cost and long lead times. Thus, there is a need for a method for producing liposomes that is suitable for commercial scale production, reproducible and meets the requirements for quality control, stability, scalability and sterilization of liposome products.

In addition, liposome formulations known in the art are not substantially uniform in size and shape, which is a key property of a pharmaceutical composition to ensure product sterility and avoid potential toxic side effects from large aberrant liposomes. At present, it is difficult to obtain a liposome composition having liposomes of uniform size. During the process of extrusion of multilayer vesicles through a series of filters, including, for example, polycarbonate filters with pores of 100 nm, it is not possible to obtain a composition consisting of a population of essentially uniform liposomes of 100 nm in size. In fact, depending on the physical characteristics of the liposomes such as compressibility and / or stability, the average diameter of the extruded vesicles can vary greatly depending on the type and size of filters used. Thus, there is a need for a method suitable for producing substantially uniform in size and shape liposome formulations.

In addition, many physical characteristics of the liposome composition affect the cellular response to liposomes and affect the effectiveness of the liposome as a pharmaceutical composition. The method of obtaining largely influences the physical characteristics of the liposome composition. However, it is not disclosed in the art how the characteristics of liposomes can be controlled during the formulation process so as to improve the production efficiency and stability of the liposomes. Thus, there is a need to develop a cost-effective large-scale liposome formulation process that can control the characteristics of the liposomal formulation to provide uniform and clinically valid liposomes.

BRIEF DESCRIPTION OF THE INVENTION

The present invention describes new liposomes encapsulating therapeutic agents. The invention also relates to liposome formulations having a high degree of size uniformity, resulting in minimized side effects and increased reliability during sterilization. The invention also describes an efficient and cost-effective method for large-scale production of liposomes under low pressure extrusion conditions. The liposomes thus obtained have desirable characteristics and efficacious therapeutic properties.

Liposome composition is characterized by liposomes having the desired composition and physical characteristics. The liposome contains a lipid ingredient that encapsulates a therapeutic agent. In one aspect of the present invention, the lipid ingredient comprises distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylglycerol (DSPG), and cholesterol, preferably in a 3: 1: 2 molar ratio (DSPC: DSPG: cholesterol).

The liposome is composed of a lipid ingredient and a therapeutic agent, the ratio of the weight of the therapeutic agent to the weight of the lipid ingredient, called the drug: lipid ratio, is about 1: 5 to 1: 8 by weight, preferably 1: 6 to 1: 7. The drug: lipid ratio increases stability and efficacy, and also affects the rate of drug loss by liposomes and liposome integrity. These and other structural characteristics provide unexpected advantages to the composition of the present invention.

The liposomes described in the present invention have a size of 30-500 nm, preferably 70-120 nm, 100-300 nm, 100-180 nm and 70-150 nm, depending on the type of therapeutic agent and / or carrier used. In one preferred embodiment, the liposomes can be 80 ± 5 nm in size.

Other physical characteristics of the liposome composition also significantly affect its stability and effectiveness. For example, the electrical conductivity of liposomes can affect their selective uptake by phagocytic cells. In one embodiment, the conductivity value is 13.5-17.5 mS / cm. Also, the intrinsic and extrinsic osmolality of a formulation similarly affects its stability and effectiveness. It is preferred that the external osmolality coincides with that of the human body, with the internal osmolality remaining low enough to increase the stability of the formulation. In one embodiment, the intrinsic osmolality is 340-440 mO / kg. Another important parameter is the pH of the liposomes and / or liposome composition. For example, an internal pH of about 6.9 of the liposome formulation improves long-term stability as well as drug clearance rate as well as drug encapsulation ability.

The liposomes of the present invention are quite stiff compared to those known in the art because they have less compressible liposome membranes. The liposomes of the present invention have a compressibility of less than 0.7 ml / g. Due to the increased rigidity, the liposomes of the present invention have increased stability and shelf life.

The liposome composition also has new and useful characteristics. The liposome composition according to the present invention consists of liposomes that are substantially uniform in size and shape and are relatively rigid. The variation in size between liposomes of the presented composition is small. The uniformity of the liposomes, as measured by the Poly Diversity Index (“PDI”), is less than 0.075, preferably in the range of about 0.02-0.05, indicating a high degree of compositional uniformity. Accordingly, the liposome formulation of the present invention significantly reduces the potential for side effects associated with large liposomes and also allows sterilizing filtration to be performed more efficiently.

Another aspect of the present invention is a method for preparing a liposome composition. The preparation method includes the steps of (1) mixing the therapeutic agent with preselected lipids to form vesicles, (2) extruding the vesicles through a filter with one pore size in one step, and (3) ultrafiltration. After ultrafiltration, the product can be standardized to the desired final concentration. Since the extrusion in Step 2 is carried out in one stage and at low pressure, the present method reduces production costs and time and also increases product yield compared to extrusion at high pressure and using multiple stages. These production steps can be adapted for large-scale production.

In one embodiment, the formulation is prepared by (1) mixing the therapeutic agent with lipids containing DSPC, DSPG, and cholesterol in a molar ratio of 3: 1: 2 to form vesicles, the therapeutic agent to lipid weight ratio being from about 1: 5 to 1 : 8, (2) extrusion of the vesicles through a filter with a pore size of about 100 nm in one step, and (3) ultrafiltration.

Also, another aspect of the present invention is a liposome formulation prepared according to the preparation steps described above. The composition includes a plurality of liposomes composed of an amount of a lipid ingredient encapsulating a therapeutic agent. For example, such a lipid ingredient may contain DSPC, DSPG, and cholesterol in a 3: 1: 2 molar ratio, and the weight ratio of therapeutic agent to lipid ingredient may be from about 1: 5 to about 1: 8. The composition is prepared in the following steps: (1) mixing the therapeutic agent with preselected lipids to form vesicles, (2) extruding the vesicles through a filter with one pore size in one step, and (3) ultrafiltration. After ultrafiltration, the product can be standardized to the desired final concentration. Preferably, the composition obtained by this method has a PDI value of less than 0.075, more preferably in the range of 0.02 to 0.05. This process provides a liposome composition having novel and beneficial properties, including, for example, a ratio of lipid ingredients of 3: 1: 2, drug: lipid, PDI, hardness, pH, osmolality, and electrical conductivity.

BRIEF DESCRIPTION OF THE GRAPHIC MATERIALS

FIG. 1 is a schematic diagram of a process for preparing a liposome composition according to the present invention.

FIG. 2 shows a diagram describing the process of obtaining 1 L of liposomal alendronate for iv. infusion at a dosage of 5 mg / ml, and in this process the parameters described in one aspect of the present invention are controlled.

FIG. 3 shows a TEM image of liposomal alendronate in accordance with one aspect of the present invention.

FIG. 4 shows a graph comparing the specific compressibility of various liposome formulations.

FIG. 5A is a plot of the size distribution of liposomes in the formulation described in the present invention.

FIG. 5B shows a graph of the size distribution of liposomes in a prior art formulation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to new liposomes, a composition and a method for its preparation for use in the treatment of various diseases. The composition consists of a plurality of liposomes encapsulating a therapeutic agent, or "encapsulating agent". The physical characteristics of each liposome improve the stability and effectiveness of the liposome formulation. The composition is defined by liposomes that are substantially uniform in shape and size. Also, the present invention describes an efficient and cost-effective method for producing a liposome formulation while still being suitable for large scale production. Further, the invention relates to a liposome composition obtained by this method having new and useful characteristics.

A. Components of liposomes

The present invention relates to a novel and useful form of liposomes. Various liposome ingredients can be used to prepare the liposomes of the invention. Preferably the lipid ingredient is a non-toxic biocompatible lipid such as, for example, lipids derived from phosphatidyl choline, phosphoglycerol and / or cholesterol. In one embodiment, the lipid ingredient comprises distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylglycerol (DSPG), and cholesterol, preferably in a molar ratio of about 3: 1: 2 (DSPC: DSPG: cholesterol).

The lipid ingredient encapsulates the therapeutic agent, both having a predetermined weight. As used herein, the term "drug to lipid ratio" (or "drug: lipid ratio") refers to the ratio of the weight of the drug to the weight of the lipid ingredient that make up the liposomes and / or formulation. In one embodiment, the liposomes have a drug: lipid ratio of about 1: 5 to 1: 8, preferably between 1: 6 and 1: 7, by weight.

B. Physical properties of liposomes

Various physical parameters and characteristics of liposomes can affect the uniformity, stability and effectiveness of the formulation. Specified physical characteristics include (1) liposome osmolality (external and internal), (2) liposome conductivity (external and internal to the liposome), (3) drug to lipid ratio, (4) liposome pH (external and internal to to the liposome) and (5) the type of lipids and drugs used in the composition.

Osmolality is a measure of the concentration of solutes in a liposome composition. As used herein, the term "osmolality" refers to a measure of the concentration of solutes, defined as the number of dissolved molecules in osmols (osmol) of a solute per kilogram of solvent (osmol / kg). Internal osmolality refers to the concentration of solutes inside the liposome, while external osmolality refers to the concentration of solutes outside the liposome. In one embodiment, the liposomes have a low intrinsic osmolality. Intrinsic osmolality can be controlled by varying the amount of drug encapsulated in the liposome. The art describes liposomes containing as much drug as possible, resulting in liposomes with high osmolality (high encapsulating ratio). However, the present invention describes liposomes having a lower osmolality (or a lower encapsulating ratio) than those known in the art. The reduced osmolality (eg, about 340-440 mO / kg) described herein increases the stability and uniformity of liposomes in the liposome formulation. One way to obtain low osmolality is to reduce the amount of encapsulated therapeutic agent in the liposomal formulation. Another way of lowering intrinsic osmolality without requiring a change in the drug: lipid ratio is to use an uncharged agent such as certain polysaccharides or sugars known in the art.

External osmolality is preferably isotonic with respect to the body, which is particularly important for injectable formulations. Thus, the external osmolality is preferably relatively constant. Internal and external osmolality in the present invention results in a highly stable product, a low rate of drug loss, and reliable encapsulation ability in liposomes.

As used herein, the term "conductivity" refers to the ability of liposomes to conduct electricity due to the ionic composition of a solution. Conductivity refers to the ionic composition of liposomes and affects the stability, rate of drug loss by liposomes, and the ability to encapsulate the liposome composition. The conductivity of liposomes is approximately 13.5-17.5 mS / cm. In one embodiment of the present invention, the encapsulated drug is charged. It should be noted that the charged drug has a 1: 1 correlation between liposome conductance and osmolality. Therefore, changing the amount of encapsulated agent proportionally affects both characteristics. In an alternative embodiment, a neutral (uncharged) drug agent is used. When neutral agents such as, for example, polysaccharides are used to alter conductivity, osmolality becomes independent of drug concentration and thus can be controlled independently of drug concentration.

Another novel aspect of the liposomes according to the invention is the relative rigidity of the structure, that is, the stability of the liposomes under various environmental and intra-body conditions, and their susceptibility to degradation. Liposome stiffness is a measure of the strength of the liposome membrane and its ability to withstand shear forces and pressures, which can increase the storage time of liposomes. The rigidity of liposomes is inversely proportional to their compressibility. Liposomes with low compressibility are more rigid. One of the possible methods for determining the rigidity of liposomes is ultrasonic viscometry and densitometry. Methods for determining the hardness of liposomes are known in the art and are described, for example, by Cavalcanti, Leide P., et al., "Compressibility study of quaternary phospholipid blend monolayers", Colloids and Surfaces B: Biointerfaces 85 (2011) 153-160; and Hianik, Tibor et al., "Specific volume and compressibility of bilayer lipid membranes with incorporated Na, K-ATPase" General Physiology and Biophisics 30 (2011) 145-153, the contents of which are incorporated herein by reference in their entirety.

Liposomes have pH values in the external environment of the liposome (external pH) and in the inner encapsulated part of the liposome (internal pH). pH affects the stability, rate of drug loss by the liposome, and the encapsulating ability of the liposome formulation. In one embodiment, the composition has an internal pH of about 6.8-7.0. Internal pH 6.8-7.0 is an important condition for the stability of the composition. The pH of the dissolved therapeutic agent can be maintained, inter alia, by continuously titrating the solution to maintain a pH of 6.8-7.0, or maintained at a predetermined pH, for example, about pH 6.9, by dissolving the therapeutic agent in a known buffer. The external pH of the liposome may differ from the internal pH of the liposome. Different pH can be achieved by changing the pH of the solutions that make up the internal and external environment of the liposome.

C. Composition

The liposome formulation described in the present invention includes a plurality of liposomes having the characteristics described above and substantially uniform in size and shape, with little variation in size between liposomes. The uniformity of a composition is measured by its Poly Dipersity Index (PDI). PDI is measured on a non-linear scale of 0 to 1, where 0 means perfectly homogeneous composition, and composition having a PDI value of 1 is highly heterogeneous (heterogeneous). The composition according to the present invention has a PDI of less than 0.075, preferably between 0.02 and 0.05. The PDI value can be calculated as described in Zetasizer nano user manual, 2003, Malvern Instruments, pp 5.5-5.6 and Kazuba M., Nano Series and HPPS Training Manual Chapter 1, 2003, Malvern Instruments, pp 9, the contents of which are included in this application by reference. Moreover, the PDI value of the present formulation is close to that of measurement standards where the PDI value is less than 0.02. The formulations previously described in the prior art had a markedly different level of uniformity compared to the present invention, with a PDI of typically about 0.3. Since the PDI has a non-linear scale, the PDI value of the present invention is significantly different from those known in the art. Moreover, the low PDI value of the present invention advantageously reduces the toxic effects associated with large liposomes, and characterizes a formulation more suitable for filter sterilization.

The composition described in the present invention contains liposomes with increased rigidity and uniformity, which increases their stability and storage time. It can also be assumed that the compositions according to the present invention are effective. Banai, Shmuel, et al., "Targeted anti-inflammatory systemic therapy for restenosis: The Biorest Liposomeal Alendronate with Stent sTudy (BLAST) -a double blind, randomized clinical trial", Am Heart J. (2013) 165 (2): 234- 40.

Liposomes of the present composition are specially sized for uptake by macrophages and monocytes. Liposomes can be 30-500 nm in size. Although, depending on the type of agent and / or carrier used, these dimensions include, but are not limited to, 70-120 nm, 100-500 nm, 100-300 nm, 100-180 nm, and 80-120 nm. These ranges are, however, examples and other specific sizes suitable for absorption during phagocytosis will be understood without departing from the spirit and scope of the present invention. In one preferred embodiment, the size of the liposomes in the formulation is about 80 ± 5 nm.

The liposomes of the present invention are capable of encapsulating many kinds of therapeutic agents. After liposome phagocytosis, the therapeutic agent becomes a substance that can reduce or inhibit the activity and / or destroy a number of phagocytic cells in the patient's body. The therapeutic agent can be a chemical compound including small and large molecules, a mixture of chemical compounds, an organic or inorganic substance, biological macromolecules such as proteins, carbohydrates, peptides, antibodies, or nucleic acids. The therapeutic agent can be a natural product obtained from known organisms or a synthetic compound.

One type of useful therapeutic agent is bisphosphonates. Bisphosphonates (formerly called diphosphonates) are compounds characterized by two CP bonds. When both bonds are located on the same carbon (P-C-P), they are called geminal bisphosphonates. Bisphosphonates are analogs of endogenous inorganic pyrophosphates that are involved in the regulation and resorption of bone formation. Bisphosphonates can sometimes form polymer chains. Being highly hydrophilic and negatively charged, free bisphosphonates are practically unable to cross cell membranes.

In this document, the term "bisphosphonates" refers to both geminal and non-geminal bisphosphonates. A preferred bisphosphonate agent has the following formula (I):

where R ₁ is H, OH or a halogen atom;

R ₂ is halogen, linear or branched C ₁ -C ₁₀ alkyl, or C ₂ -C ₁₀ alkenyl optionally substituted with heteroaryl or heterocyclyl C ₁ -C ₁₀ alkyl amines or C ₃ -C ₈ cycloalkyl amines, where the amine can be primary, secondary or tertiary ; -NHY, where Y is hydrogen, C ₃ -C ₈ cycloalkyl, aryl or heteroaryl; or R ₂ is —SZ, where Z is chloro substituted phenyl or pyridinyl.

One example of a bisphosphonate agent is alendronate having the following formula (II):

Many bisphosphonates have alendronate-like activity and are useful as therapeutic agents for the present invention. Such bisphosphonates can be selected based on their ability to mimic the biological activity of aledronate. Such activity includes, for example: inhibitory activity in vitro in phagocytic cells, such as macrophages and fibroblasts, when entering these cells; inhibition of IL-2 and / or IL-6 and / or TNF-α secretion by macrophages; and in vivo activity, for example, the ability of the tested formulations to deplete or inactivate blood monocytes in animal models or in humans, or to treat myocardial infarction and reduce the infarction zone.

Bisphosphonates useful in the present invention include, without limitation, clodronate, tiludronate, 3- (N, N-dimethylamino) -1-hydroxypopane-1,1-diphosphonic acid, for example, dimethyl-APD; 1-hydroxy-ethylidene-1,1-bisphosphonic acid, for example etidronate; 1-hydroxy-3 (methylpentylamino) -propylidene bisphosphonic acid (ibandronic acid), for example ibandronate; 6-amino-1-hydroxyhexane-1,1-diphosphonic acid, for example amino-hexyl-BP; 3- (N-methyl-N-pentylamino) -1-hydroxypropane-1,1-diphosphonic acid, for example methyl-pentyl-APD; 1-hydroxy-2- (imidazol-1-yl) ethane-1,1-diphosphonic acid, for example zoledronic acid; 1-hydroxy-2- (3-pyridyl) ethane-1,1-diphosphonic acid (risedronic acid), for example risedronate; 3- [N- (2-phenylthioethyl) -N-methylamino] -1-hydroxypropane-1,1-bisphosphonic acid; 1-hydroxy-3- (pyrrolidin-1-yl) propane-1,1-bisphosphonic acid, 1- (N-phenylaminothiocarbonyl) methane-1,1-diphosphonic acid, for example FR 78844 (Fujisawa); 5-benzoyl-3,4-dihydro-2H-pyrazole-3,3-diphosphonic acid tetraethyl ester, for example U81581 (Upjohn); and 1-hydroxy-2- (imidazo [1,2-a] pyridin-3-yl) ethane-1,1-diphosphonic acid, for example YM 529.

In some embodiments, such as, for example, sodium alendronate encapsulated in DSPC, DSPG, and cholesterol, a drug: lipid weight ratio of 1: 5.7 can be used, which is about 1: 3 molar ratio. In the case of sodium clodronate, another therapeutic agent encapsulated in the same lipid ingredient, an approximate weight ratio of 1: 5.4 can be used, which is approximately equal to a molar ratio of 1: 3. Other therapeutic agents can be used in the present invention that inhibit or deplete phagocytic cells by stopping, slowing down proliferation, and / and downregulating the activity of phagocytic cells. Specific therapeutic agents include any agent that is cytotoxic or cytostatic, including but not limited to, for example, gallium, gold, silica, 5-fluurouracil, cisplatin, alkylating agents, mithramycin, and paclikaxel. When any of the above therapeutic agents are used, the drug / lipid ratio in the liposome is about 1: 5 to 1: 8, preferably between 1: 6 and 1: 7, by weight.

In one embodiment, the formulation includes an encapsulated therapeutic agent capable of selectively entering a cell through phagocytosis and selectively targeting macrophages and monocytes without affecting non-gocytic cells. Since macrophages and monocytes in their normal state collapse into damaged areas at the cellular level and cause inflammation beyond that caused by the disease or condition itself, inhibition and / or depletion of monocytes / macrophages can mitigate the condition of the damaged area. Upon entering a phagocytic cell, the agent is released and inhibits, inactivates, damages, kills and / or depletes monocytes and / or macrophages for the treatment of various diseases involving a phagocytic immune response, such as, for example, ischemic reperfusion injury or inflammatory damage such as, for example , myocardial infarction, or to reduce the infarction zone in the final stage and enhance the recovery of the heart after acute myocardial infarction. Liposomes included in the composition described in the present invention have special characteristics, including size, charge, pH, conductivity and osmolality, which facilitate direct absorption through phagocytosis.

After being absorbed by monocytes / macrophages, the agent exhibits prolonged inhibitory activity towards monocytes / macrophages. This prolonged activity is sufficient to alter the inflammatory activity of monocytes / macrophages. Thus, sustained release of the agent is not required for sustained inhibition of activity. Accordingly, the method of treating certain diseases by inhibiting monocytes / macrophages, for example using an encapsulated agent, is preferably a systemic therapy in which circulating monocytes and macrophages are the target of the therapeutic composition. Phagocytic cells can exhibit different responses depending on the type of encapsulated therapeutic agent used. For example, liposome-encapsulated alendronate induces apoptosis, while liposome-encapsulated clodronate induces necrosis. Nonphagocytic cells are comparatively incapable of absorbing the composition due to the specific physicochemical properties of the liposomal composition.

Moreover, the liposomes of the present invention not only retain the therapeutic agent for a time sufficient for the agent not to be released into the body fluid, but also effectively release it within the target cell. The liposomes described in the present invention deliver an effective amount of the agent to the target cells. The term "effective amount" means an amount of a formulation that is effective to achieve the desired therapeutic result, for example, treatment of endometriosis, restenosis, ischemic reperfusion injury (IRI), myocardial infarction, or other related diseases. For example, reducing the number and / or activity of activated macrophages and monocytes decreases the infarction area and / or improves remodeling when the disease is associated with myocardial injury. The effective amount also depends on a number of factors, including, but not limited to: the weight and gender of the patient being treated; the method of administration of the composition (namely, whether it is administered systemically or directly into the affected area); therapeutic regimen (for example, is the composition administered once a day, several times a day, once every several days, or once); clinical signs of inflammation; clinical factors influencing the rate of development of the underlying disease, such as smoking, hypercholesterolemia, pro-inflammatory status, kidney disease; and the type of dosage of the composition. Successful delivery and use of a liposome formulation depends on its stability and effectiveness. Important factors determining the therapeutic index of a product are the number of therapeutic agent molecules encapsulated in each liposome (payload) and the level of free unencapsulated material.

D. Dosage and Administration

The liposome formulation can be administered in any manner that effectively transfers the liposomes to the appropriate or desired site of action. Preferred types of administration include intravenous (IV) or intra-arterial (VA) (particularly suitable for on-line administration) administration. Other suitable routes of administration include intramuscular (IM), subcutaneous (PC), and intraperitoneal (IP) routes of administration. Such routes of administration can be bolus injections or infusions. Another route of administration may be perivascular administration. The introduction of the composition can be carried out immediately directly or after dilution. Any combination of the methods of administration described above can be used according to the present invention. Any route of administration can be used that brings the particles into contact with phagocytic cells (eg, circulating monocytes or abdominal macrophages).

Pharmaceutical compositions for use according to the present invention can be prepared from one or more physiologically acceptable carriers, including excipients and auxiliaries known in the art, which facilitate the conversion of active ingredients into formulations that can be used in pharmaceuticals.

The amount and frequency of dosing can be individually set to provide plasma levels sufficient to induce or suppress a biological effect (minimum effective concentration of "MEK"). The MEK value varies from drug to drug, but can be determined from in vitro data. The dosage required to achieve IEC will depend on the patient's condition and route of administration. Detection methods can be used to determine the concentration of an agent in plasma.

Depending on the severity of the patient's condition and his responsiveness to treatment, the dosage may be administered on a one-off basis, or over multiple administrations, with a treatment period ranging from several hours to several weeks, or until treatment has taken effect. The frequency of administration may depend on the conditions and the severity of the disease. In one embodiment, the composition is administered intermittently. The dosage can be any amount or volume required for the desired treatment. For example, dosages of either 1 mcg or 10 mcg may be prescribed to patients undergoing intravascular procedures, such as stent implantation, to prevent further proliferation and severity of late stent lumen loss. As a further example, the dosage can be at least 100 mcg. In such a case, the composition can be administered once, several times, and / or continuously, for example, by means of continuous infusion (s) over a period of time. For example, doses can be administered according to the methods described in Banai, Am Heart J. (2013), supra .

E. Method for producing liposomes

Another aspect of the present invention relates to a method for producing liposomes and liposome formulations for the commercial production of a product. This method of preparation allows control of the physical characteristics described above, as well as control of several process parameters, including the ratio of water to solvent, solvent composition and solvent ratios, vesicle preparation temperature, vesicle shear index, extrusion temperature, extrusion pressure and membrane size and membrane type. used for extrusion.

Obtaining a liposome composition includes the steps of (1) mixing the therapeutic agent with preselected lipids to form vesicles, (2) extruding the vesicles through a filter with one pore size in one step, and (3) ultrafiltration. The phrase "filter extrusion in one step" means that the extrusion step refers to the step of filtering through a filter with one pore size. It can include multiple passes through a filter with the same pore size, but does not require multiple and / or sequential passes through filters with different pore sizes, as is known in the art, for example, sequential passes through membranes with pores of 1.0, 0, 8, 0.6, 0.4 and 0.2 microns. After ultrafiltration, the product can be standardized to the desired final concentration. Since the extrusion in step (2) is carried out in one stage and at low pressure, the present method reduces production costs and time, as well as increases product yield compared to extrusion at high pressure and using multiple stages. Figure 1 shows the steps in the process of obtaining a liposome formulation.

The first step involves mixing the therapeutic agent and preselected lipid ingredients in a solution to form vesicles. In many cases, the therapeutic agent and the lipid ingredient are solubilized as a solution of the therapeutic agent and a solution of the lipid ingredient prior to mixing with each other. In this embodiment, the therapeutic agent solution has several physical characteristics, including pH, osmolality, and conductivity. The therapeutic agent solution comprises the internal environment of the liposome composition. Therefore, the pH, conductivity and osmolality of the therapeutic agent solution affect the internal pH, conductivity and osmolality of the liposome composition. The preferred internal osmolality is in the range of 340-440 mO / kg, while the preferred external liposomal osmolality is 270-340 mO / kg. The pH of the solution is intended to maintain the pH of the therapeutic agent solution within the desired pH range. Thus, if an acidic therapeutic agent such as a bisphosphonate is used, the alkaline pH of the solution can be used to maintain the pH of the solution between 6.8 and 7.0. For example, a therapeutic agent solution can be prepared from sodium alendronate dissolved in a NaOH solution to give a final pH of the alendronate solution of about 6.8 and a conductivity of about 18.0 mS / cm. The solution can be additionally heated during the process. In an alternative embodiment, sodium clodronate or alendronate monohydrate can be used. The solution can be heated to a temperature that improves the dissolution of the bisphosphonate in the alkaline solvent, in the range from 55 ° to 75 ° C, for example up to 70 ° C. Depending on the amount of therapeutic agent and carrier dissolved, the solution may have an osmolality, which will become the intrinsic osmolality of the liposome formulation, in the range of 340-400 mO / kg. Also, depending on the amount of therapeutic agents and carriers, the solution may have a conductivity that becomes intrinsic conductivity in the range of 14.0-21.0 mS / cm. Internal pH, osmolality, conductivity are factors that influence the stability and effectiveness of the liposome formulation. In one embodiment, the concentration of the therapeutic agent solution may be in the range of 20-120 g / L during step (1) of the preparation process. The therapeutic agent solution can be prepared separately and / or prior to the described preparation process.

The lipid ingredients can be presented in the form of a solution containing the desired initial amounts of the lipid ingredient, dissolved in the volume of one or more lipid solvents. Any suitable lipid ingredient and lipid solvent can be used. For example, prior to liposome formation, the lipid ingredient may contain DSPC, DSPG, and cholesterol in a molar ratio of 3: 1: 2, respectively. The liposome obtained from this combination of lipids may also have a molar ratio of DSPC, DSPG and cholesterol of 3: 1: 2. Further, the lipid solvent may, for example, contain tert-butanol, ethanol and water in a volume ratio of 77/77/6, respectively. Other lipid solvents suitable for the formation of liposomes according to the invention include chloroform and methanol. Lipid ingredients dissolve in a lipid solvent. To obtain a lipid solution, the lipid solvent can be heated to a temperature that facilitates dissolution of the lipid ingredients, which can range from 55 ° to 57 ° C, for example 70 ° C. The concentration of dissolved lipids in solution can take values in the range of 50-350 g / l. The lipid solution can be prepared separately and / or before the described preparation process.

Mixing the therapeutic agent solution and the lipid solution results in the formation of multilamellar vesicles (MLV). The drug: lipid ratio in the vesicle can be controlled by varying the amount of the therapeutic agent solution or lipid solution. Moderate heating of both solutions can additionally be used to facilitate mixing of the solutions. This process results in efficient encapsulation of the therapeutic agent in multilamellar vesicles. In one example, the lipid solution can be added to the therapeutic agent in a ratio of 5.3 parts lipid to 1 part therapeutic agent. This drug: lipid ratio (by weight) increases the stability of the liposome composition without significantly impairing the delivery of the therapeutic agent. However, this process produces drug: lipid ratio values in the range of 1: 4 to 1: 8 by weight, preferably in the range of 1: 5 to 1: 6. Further, prior to the extrusion step, the solvent concentration can be adjusted without compromising the integrity of the liposomes.

The next step in the method for preparing the composition includes extrusion of vesicles through a filter with one pore size in one step. Extrusion of the vesicles reduces the size of the multilayer vesicles described above. The method includes a one-stage low pressure extrusion, which makes it possible to obtain liposomes that are highly uniform in size and shape. The extrusion process can involve the use of a high pressure homogenizer (microfluidizer) or a traditional homogenizer. Homogenizers use shear energy to break large liposomes into smaller ones. Homogenizers suitable for use in the present process include high pressure homogenizers available from a variety of manufacturers such as Microfluidics in Boston, MA. The resulting particle size distribution can be monitored using conventional laser beam particle size determination. An effective way to reduce the size of liposomes to obtain a relatively clear size distribution is the extrusion of liposomes through a polycarbonate membrane or an asymmetric ceramic membrane. The preferred extrusion temperature is above the transition temperature of the liposomes to facilitate size reduction. For example, in the case of DSPG, DSPC and cholesterol, the desired temperature should be about 55 ° C. When using other lipids, the transition temperature of such lipids will be the desired temperature for the extrusion step. It may be necessary to repeat the extrusion to achieve the desired homogeneity and size of the vesicles. During this step, samples can be analyzed in real time to assess the desired vesicle size and microbial count.

The extrusion process is carried out using a one-stage low pressure extrusion technique. During extrusion, MLVs are processed in one step by extrusion directly through a single membrane under low pressure, rather than by creating small single-layer liposomes of multi-step extrusion, where larger liposomes are passed through successively smaller membranes using extruders at high pressure (as used in methods according to the prior art). In one embodiment, the one-step extrusion process is carried out directly with a polycarbonate or ceramic membrane with 0.1 µm pores and a low pressure of 414-620 kPa (60-90 psi) is applied to form 100 nm liposomes. Also, the present method uses a lower pressure of 414-620 kPa (60-90 psi) compared to the higher pressures (up to 3447 kPa (500 psi)) used in high pressure extrusion.

The present method has a number of advantages due to the elimination of the need for multiple membranes and low pressure extrusion. Primarily, the single-stage extrusion process has an advantage over the multi-stage extrusion process because it takes less time. Further, the absence of the need for high pressure extrusion reduces the costs associated with such extrusion equipment. One example of extruders suitable for use in the present invention is the LIPEX ^™ Extruder available from Northern Lipids, Inc. Moreover, materials used for low pressure extruders, such as compressed air, generally cost less than materials used for high pressure extruders, such as nitrogen. Further, reducing the plurality of extrusion steps to one causes a corresponding reduction in waste and a higher product yield.

The last step of the present method involves ultrafiltration of the extruded vesicles into a buffered solution. While the extrusion process results in liposomes that are uniform in size and shape, the ultrafiltration process involves applying pressure to the formulation and passing it through a membrane to separate the encapsulated liposomes from the non-encapsulated therapeutic agent, solvents and lipids. This step is preferably carried out at a temperature below the transition temperature of the lipids used in the formulation, for example, in most cases below 45 ° C. Various types of filtration membranes can be used during the ultrafiltration process. One embodiment of the ultrafiltration step involves the use of hollow fiber membranes, in which the composition is forced through the open cavities of the fiber, and micromolecules (e.g., solvent, non-encapsulated bisphosphonate, lipids) are filtered through the outer membrane of the fiber, while larger liposomes remain inside the fiber. As a result of ultrafiltration, a composition is obtained with a proportion of homogeneous encapsulated agents greater than or equal to 96%.

The ultrafiltration step may further comprise a dialysis step, wherein the formulation is dialyzed against a volume of buffered solution. One example of a buffered solution is phosphate-sodium buffer (PBS), but any buffer that has a balance of positive and negative ions maintained at physiological osmolality can be used. Other buffering additives known in the art may include, for example, sucrose, glycine, sodium succinate and / or albumin. The buffered solution should preferably reflect the external environment of the final formulation, therefore it is important to carefully monitor the pH and conductivity values. Preferably, the buffered solution is isotonic and non-toxic to cells. The buffered solution can be filtered to reduce contamination levels, or the buffered solution can be prepared in advance. The dialysis endpoint is defined as the composition reaching the desired pH and conductivity.

At the end of the ultrafiltration step, the formulation can be further filtered for sterilization, for example, to maintain the required bioburden level. In one embodiment of the process, the sterilization filter is associated with a pressure vessel containing the liposome formulation. On the other hand, the sterilization filter is connected to a sterile collection container. Under pressure, the liposome composition is passed through a sterilization filter. Further at this stage, a sample can be taken to assess the content of bacteria. The pore sizes of sterilization filters can range from 0.2 to 0.45 μm. Since the liposomes of the present liposome composition are substantially homogeneous and no large liposomes are present, sterilization filtration can proceed relatively without complications.

Once obtained, the liposome composition can be standardized. Final standardization allows a series of liposome formulations to be prepared at a standard concentration. In the course of standardization, product yield, liposome size, lipid composition, encapsulated and free therapeutic agent content and product concentration are analyzed. Concentration analysis can be performed using methods known in the art, for example, HPLC or spectrophotometric determination of orthophosphate. After confirming the concentration of the ultrafit product, a certain volume of the buffered solution is used to dilute the liposome formulation to a standard concentration. Final standardization also includes sterile filtration of the liposome product. For example, after ultrafiltration, the bisphophonate encapsulating formulation can be diluted with a certain amount of the buffered solution to a final concentration. Likewise, by using different amounts of the buffered solution diluted to produce several portions of different concentration, the ultrafiltration product can be divided into several portions for use at different final concentrations. Samples can be taken to confirm the concentration of the therapeutic agent obtained.

This production process has the advantage that it allows you to control, change and reproduce the physiological and chemical properties of liposomes. For example, the external pH of the liposome can be controlled by changing the composition of the solution in which the therapeutic agent is dissolved. Intrinsic osmolality can be adjusted in a similar way by varying the amount of the therapeutic agent, or, for example, depending on whether the agent is charged or uncharged. The drug: lipid ratio can be adjusted by selecting the lipid ingredients that make up the liposome or by the amount of lipids added to the dissolved active agent. The increased amount of the lipid ingredient decreases the drug: lipid ratio and vice versa. The low drug: lipid ratio also reduces the intrinsic osmolality of the liposome composition. The external pH and osmolality of the composition depends in part on the composition of the buffered solution that contains the final product. Conductivity can be controlled by altering the nature of the therapeutic agent and other adjuvants encapsulated in the liposome. Other factors, including but not limited to viscosity, quality of excipient, sterility, compatibility with saline, infusion kits and syringes (for injectable preparations), and compatibility with formulation equipment are also independently controlled during the liposome formulation process described herein. invention.

As described above, in one preferred embodiment of the preparation process, the formulation is prepared by (1) mixing a solution containing a therapeutic agent with a solution containing lipids including DSPC, DSPG and cholesterol in a molar ratio of 3: 1: 2 to form vesicles, the weight ratio of therapeutic agent to lipids being from about 1: 5 to 1: 8, (2) extrusion of the vesicles through a filter with a pore size of about 100 nm in one step, and (3) ultrafiltration.

Another aspect of the present invention is a liposome formulation prepared according to the above preparation steps, the formulation comprising DSPC, DSPG and cholesterol in a molar ratio of 3: 1: 2 to form vesicles, the therapeutic agent to lipid weight ratio being from about 1: 5 up to 1: 8 and is carried out during the following stages: mixing the solution containing the therapeutic agent with the solution containing lipids to form vesicles, (2) extrusion of vesicles through a filter with one pore size nm in one stage, and (3) ultrafiltration. After ultrafiltration, the product can be standardized to the desired concentration. The composition obtained according to the present method has a PDI value of less than 0.075, preferably in the range between 0.02 and 0.05. This method provides a liposome composition having the new and beneficial qualities described above, including, for example, a ratio of lipid ingredients of 3: 1: 2, a ratio of drug: lipid between 1: 5 and 1: 8. Moreover, it allows independent control of individual liposomal characteristics, including pH, osmolality, conductivity and hardness.

The following examples are intended to illustrate and explain various aspects of the implementation of the present invention and are not intended to limit the scope of the invention in any way. The present invention is further described by way of examples with reference to the accompanying drawings. With particular reference to the accompanying, in particular, drawings, it is emphasized that particular cases are shown by way of example and solely for the purpose of illustrating the preferred implementation options, which are considered the most useful and understandable description of the principles and ideas of the present invention. This description is to be read in conjunction with the drawings to make it clear to a person skilled in the art how several forms of the present invention may be practiced.

Example 1 - Obtaining a batch of liposome composition

In accordance with the above process, an illustrative liposome composition batch was prepared. It should be understood that the lot size may vary depending on industrial production requirements. In this example, a one-liter batch of liposomal alendronate was prepared, encapsulated in liposomes containing cholesterol, DSPC and DSPG and dispersed in sodium phosphate buffer. For convenience of clinical use, liposomal alendronate can be presented in two concentrations: 5 mg / ml and 0.5 mg / ml as a whitish, sterile liposome dispersion. These concentrations can be further mixed to obtain the desired amount of therapeutic agent in any particular volume. Lipid ingredients were composed of cholesterol, DSPC and DSPG. The dispersion also contained a solution of phosphate buffer in saline to control the pH level, stability of the infusion and to maintain isotonicity. At least 96% of the drug was encapsulated in liposomes. For administration, the contents of the ampoule (or, if necessary, part of it) were diluted with saline and then infused.

The formula for obtaining a batch, including the quantity and quality of the components used in the production, as well as their quantity per 1 liter of the batch, are shown in Table 1 below.

Table 1 - Liposomal Alendronate for four infusions, formula for 1 L batch.

Component amount Quality Alendronate sodium trihydrate 68-80.75 g 100.5% NaOH 6.8-8.0 g High purity Cholesterol 10 ± 0.2 g ≥99% DSPC 30 ± 0.4 g ≥99% DSPG 10 ± 0.2 g ≥99% Ethanol 77 ± 1.1 ml Absolute, high purity Tert-butanol 77 ± 1.1 ml Analytical Water for injections 850 ± 13ml
6 ± 0.1 ml USP PBS pH 7 ≈ 6000 ml Na ₂ HPO ₄ * H ₂ O 13.86 g ± 1.5% High purity NaH ₂ PO ₄ * H ₂ O 6.54 g ± 1.5% High purity NaCL 50.82 g ± 1.5% High purity Water for injections 6000 ml USP

The content and quantitative composition of liposomal alendronate for the four infusions, obtained from the 1 liter batch described in Table 1, are recorded in Table 2 below. It should be noted that in this batch the molar ratio of DSPC: DSPG: cholesterol was 3: 1: 2. Also, the drug: lipid ratio was determined to be about 1% 5.7 ± 1.5 by volume.

Table 2 - Composition of liposomal alendronate for IV infusion

Component Content 0.5 mg / ml 5 mg / ml Medicinal substance Alendronate sodium trihydrate 0.5 ± 0.05 mg / ml
= 0.0015 mmol / ml 5.0 ± 0.05 mg / ml
= 0.015 mmol / ml Liposomal lipids Cholesterol 0.6 ± 0.1 mg / ml
= 0.0015 mmol / ml 5.2 ± 0.8 mg / ml
= 0.013 mmol / ml DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) 1.7 ± 0.3 mg / ml
= 0.0022 mmol / ml 15.65 ± 2.35
= 0.020 mmol / ml DSPG (1,2-distearoyl-sn-glycero-3-phospho-rac-glycerin) 0.6 ± 0.1 mg / ml
= 0.0007 mmol / ml 5.2 ± 0.8 mg / ml
= 0.006 mmol / ml Buffer solution (pH 7) Na ₂ HPO ₄ * H ₂ O 1.09 mg / ml 1.09 mg / ml NaH ₂ PO ₄ * H ₂ O 2.31 mg / ml 2.31 mg / ml NaCL 8.47 mg / ml 8.47 mg / ml Water for injection (WFI) 1 ml 1 ml

The composition of the liposomal alendronate for IV infusion, obtained by the above-described new method of production, is shown in Table 3 below.

Table 3 - Description of the dosage form with a concentration of 5 mg / ml

Index Description Appearance Whitish dispersion Identification Compliant Alendronate Test (HPLC) 5.0 ± 0.2 ml / ml Encapsulation of alendronate ≥ 96% DSPC test (HPLC) 13.3-18.0 mg / ml DSPG Test (HPLC) 4.4-6.0 mg / ml Cholesterol Test (HPLC) 4.4-6.0 mg / ml Drug: lipid ratio (including cholesterol) 1: 5.3 ± 1.0 Vesicle size Average particle size 80 ± 5 nm pH 6.7-7.3 Ethanol <0.5% Tert-butanol <0.5% Osmolality 270-340 mO / kg Sterility Sterile Pyrogen content Satisfactory

Figure 2 shows a schematic diagram summarizing the process for producing one liter of liposomal alendronate for IV injection at a dosage of 5 mg / ml, and the process for monitoring the characteristics of liposomal alendronate. The preparation route for a dosage of 0.5 mg / ml remains the same as for 5 mg / ml. The only difference is the method of obtaining the various final concentrations for administration during the final standardization step. For a person skilled in the art it will be clear that according to the present method, different dosages of the preparation can be obtained without the need for further experiments.

The therapeutic agent solution (alendronate solution) and the lipid solution were prepared according to the following:

Alendronate solution . NaOH (6.8-8.0 g) was weighed and dissolved in 850 ml of water for injection (WFI) at a temperature of 70 ± 3 ° C, and a stirring speed of 600 ± 150 rpm. Complete dissolution was confirmed by visual inspection. Sodium alendronate (68-80.75 g) was dissolved in a NaOH solution at a temperature of 70 ± 3 ° C, and a stirring speed of 600 ± 150 rpm. Complete dissolution was confirmed by visual inspection (for example, for the transparency of the solution), the conductivity and pH values were confirmed (pH = 6.8 ± 0.3. Conductivity = 18.0 ± 1 mS / cm).

Lipid solution . Lipid blend comprising 30 g DSPC (37.9 mmol), 10 g DSPG (12.5 mmol) and 10 g cholesterol (25.8 mmol) (DSPC / DSPG / cholesterol; 3/1/2 mol / mol / mol ) were weighed and dissolved in a 250 ml beaker using a heating magnetic stirrer in 160 ml of tert-butanol / ethanol / water (77/77/6, v / v) at 70 ± 3 ° C to obtain a lipid solution with a concentration at 312 mg / ml. A clear yellow solution was obtained when the required temperature was reached.

MLV roster . The lipid solution was added to the alendronate solution (one part of the drug, 5.3 parts of the lipid) with stirring at 600 ± 150 rpm and a constant temperature of 70 ± 3 ° C. After at least 5 minutes, 100 ml WFI (10% of total volume) was added to reduce the concentration of the solvent after extrusion. The composition was mixed for an additional 10 minutes.

Extrusion . For extrusion, the composition was placed in a heated stainless steel extruder with a volume of 1.2 liters, with a ceramic membrane mounted in it with a pore diameter of 0.14 μm or two polycarbonate membranes (for example, with pores of 0.2 μm for prefiltration and with pores in 0.1 μm) under a pressure of 620 ± 207 kPa (90 ± 30 psi) and a temperature of 68 ± 5 ° C in order to reduce the size of the vesicles and increase the degree of homogeneity of the vesicles. The process requires 12-18 passes to obtain vesicles at 80-100 nm. During the extrusion passes, samples were collected to determine the size of the vesicles prior to ultrafiltration. Liposome size analysis was analyzed using a Malvern Nano ZS analyzer (acceptable particle size for analysis: 95 ± 20 nm). The extrudate samples were also analyzed for aerobic bacteria content (bioburden control). The acceptable limit was <100 CFU / ml.

Ultrafiltration and diafiltration . First, the composition was left to cool to <45 ° C before subsequent ultrafiltration. Ultrafiltration was performed in an Amersham QuixStand system with a 500K hollow fiber membrane. The composition was concentrated using an inlet pressure not exceeding 172 kPa (25 psi). When the minimum volume was reached, the composition was dialyzed with a 10-fold initial volume (≈7 L) of a sodium phosphate buffer (PBS) solution. A PBS solution was prepared by dissolving 145 mM NaCl, 13 mM Na ₂ HPO ₄ * H ₂ O and 7 mM NaH ₂ PO ₄ * H ₂ O in 10 L of WFI. The PBS had a pH of about 6.9 and a conductivity of about 16.9 mS / cm. The PBS was filtered through a 0.2 µm filter. When the required pH and conductivity values were reached, diafiltration was stopped. The composition discharged from the ultrafiltration system did not exceed 120% of the initial volume (≈1.2 liters). Samples were taken for general analysis and for the content of aerobic bacteria (bioburden control). The acceptable limit was <100 CFU / ml.

At the end of dialysis, the formulation was filtered through a 0.2 nm filter to maintain the desired bioburden level. The pressure vessel containing the composition was connected to a 0.2 μm sterile filter (Sartorius Sartobran P). The filter was pre-assembled with a sterile receiving vessel. Filtration was carried out at a pressure of 34-207 kPa (5-30 psi) in a vessel containing the composition. Samples were taken for general analysis and for the content of aerobic bacteria (bioburden control). The acceptable limit was <100 CFU / ml.

Ultimate standardization . HPLC analyzed the liposome size of the formulation, the lipid / therapeutic agent content in the liposomes, the drug and free therapeutic agent content. The expected yield in this example was one liter of formulation containing about 6 mg / ml of encapsulated alendronate and 35 mg / ml of lipids. Based on the results of the analysis of the concentration of alendronate, the necessary dilution was calculated to obtain about a liter of formulation with a final concentration of 5 mg / ml or 0.5 mg / ml. To obtain a formulation with a concentration of 5 mg / ml, about 900 ml of liposomal alendronate after ultrafiltration was diluted with about 100 ml of PBS, prepared as described above. Additionally, to obtain a formulation with a concentration of 0.5 mg / ml, about 100 ml of liposomal alendronate after ultrafiltration was diluted with about 900 ml of PBS. Samples were taken to determine the resulting concentration of alendronate.

After obtaining the standard concentrations of the formulation, the bottle containing the formulation was attached to two successive sterile 0.2 μm filters (Sartorius Sartobran P) located in a sterility class 100 room or in a sterile laminar. The filters were pre-assembled with a pre-sterilized receiving bag or bottle. Filtration was performed using a peristaltic pump or under compressed nitrogen pressure. The pressure should not exceed 69 kPa (10 psi). At the end of the filtration, the filter was checked for integrity.

Liposomal alendronate for IV infusion obtained in the example above has a number of desirable characteristics, for example (i) three years of stable storage of at least alendronate and lipids at 5 ° C (range 2-8 ° C); (ii) the average diameter of the vesicles is 80 ± 5 without particulate material; (iii) alendronate sodium concentration up to 5 mg / ml (in the range of 0.1-5.0 mg / ml); (iv) alendronate encapsulation equal to or greater than 96%; (v) a lipid content in a molar ratio of distearoylphosphatidylcholine / distearoylphosphatidylglycerol / cholesterol (DSPC / DSPG / cholesterol) of 3/1/2; (vi) physiological osmolality of 270-340 mO / kg, (vii) a viscosity index close to that of water, that is, a dynamic viscosity of about 10 mPa · s at 20 ° C; (viii) a pH value of 6.8 (in the range of 6.8-7.0); (ix) worldwide acceptance for carrier quality in accordance with the United States Pharmacopoeia (USP) or Europe (EP); (x) meeting USP requirements for sterility and pyrogenicity as published in USP 24-NF 19; (xi) compatibility (when used) with saline, infusion sets and syringes; and (xii) compatibility (upon receipt) with filters, stainless steel pipes, and glass.

FIG. 3 shows a TEMS image of alendronate liposomal prepared in the above example. A good level of homogeneity and homogeneity of the liposomal population can be observed, and liposomes are between 40 and 120 nm in size.

Example 2 - Liposome Hardness Testing

In the liposome stiffness assay, four samples of large unilamellar liposomes (approximately 100 nm diameter) were analyzed. Empty liposomes dissolved in PBS prepared according to the present invention were labeled as LPO. Liposomal formulations containing 5.0 mg / ml alendronate prepared according to the present invention were labeled LSA. Two other liposome samples, labeled KS and HU and dissolved in HEPES, were prepared according to a method known in the art and described in Epstein-Barash, Hila et al., ʺPhysicochemical parameters affecting liposomal bisphosphonates bioactivity for restoring therapy: Internalization, cell inhibition, activation of cytokines and complement, and mechanism of cell deathʺ, J. Controlled Release 146 (2010) 182-195.

Each sample was analyzed to determine the specific volumetric compressibility of the liposomes. Using ultrasonic viscometry, the elastic properties of liposomes were evaluated based on the following ratio:

where β _S , ρ and u denote the adiabatic compressibility, density and sonic viscosity of the suspension, respectively. Hianik, T., Haburcak, M., Lohner, K., Prenner, E., Paltauf, F., & Hermetter, A. (1998). Compressibility and density of lipid bilayers composed of polyunsaturated phospholipids and cholesterol. Coll. and Surf. A 139 (2), 189-197. Hianik, T., Rybár, P., Krivánek, R., Petríková, M., Roudna, M., & Apell, HJ (2011). Specific volume and compressibility of bilayer lipid membranes with incorporated Na, K-ATPase. Gen. Physiol. Biophys. 30, 145-153. In this way, changes in compressibility can be determined by measuring changes in sonic viscosity and density.

Ultrasonic viscosity is measured using a differential viscometer with constant capillary length consisting of two nearly identical cavity resonators (Sarvazyan, AP (1991). Ultrasonic velocimetry of biological compounds. Annu. Rev. Biophys. Biophys. Chem. 20, 321-342; Sarvazyan , AP, Chalikian, TV (1991). Theoretical analysis of an ultrasonic interferometer for precise measurements at high pressures. Ultrasonics 29, 119-124.) Using frequencies of about 7.2 MHz. The resonant frequencies of the cells were measured using a computer network analyzer (USAT, USA). The sample volume was 0.7 ml. The resonator cells were equipped with magnetic stirrers to ensure homogeneous distribution of the samples during the measurement. One resonator contained a liposome formulation with a phospholipid concentration of 10 mg / ml, while the other resonator contained the same solution (PBS or HEPES) as a control without the addition of vesicles. Before carrying out a series of measurements, the resonance frequencies of both resonators were first compared by measuring both cells filled with the same test liquid. Since the energy density of the ultrasonic signal in the volume was small (the amplitude of the ultrasonic wave pressure was less than 103 Pa), it was possible to avoid the effect of the sound wave on the structural properties of the vesicles. In general, ultrasonic viscometry allows the determination of sound density [u] or its variation with concentration (Sarvazyan, AP (1982). Development of methods of precise ultrasonic measurements in small volumes of liquids. Ultrasonics 20, 151-154) as defined in equation:

where "c" is the concentration of the solute in mg / ml and the subscript "0" refers to the solvent (buffer). The value of [u] can be directly derived from the changes in the resonant frequencies f and f ₀ in both resonators (where f is the resonant frequency of the sample and f ₀ is the resonant frequency of the control-buffer):

(coefficient γ satisfies the condition γ << 1 and may not be taken into account in the calculations)

High precision densitometric system (DMA 60 with two sample chambers DMA 602 M, Anton Paar KG, Graz, Austria) using the vibrating tube principle (Kratky, O., Leopold, H., & Stabinger, H. (1973). The determination of the partial specific volume of proteins by the mechanical oscillator technique. Methods in enzymology (Ed. E. Grell), vol. 27, pp. 98-110), was used to determine the density (ρ) of a vesicle solution. Partial specific volumes (ϕV) were calculated from density data using the equation:

where the subscript 0 refers to the reference solute and [ρ] = (ρ- ρ ₀ ) / (ρ ₀ c) denotes concentration-related density changes. The temperature of the cells was controlled with an accuracy of ± 0.02 ° C using a Lauda RK 8 CS ultra-thermostat (Lauda, Germany).

Determination of specific volumes in addition to the change in sonic viscosity depending on concentration made it possible to calculate the reduced specific apparent compressibility, ϕ _K / β ₀ , of vesicles using the following equation:

where β ₀ is the compressibility factor and ρ ₀ is the buffer density (Sarvazyan 1991). The ϕ _K / β ₀ value means the volumetric compressibility of the liposomes as compared to the buffer. A high value of ϕ _K / β ₀ means high compressibility (as well as low rigidity) of liposomes.

In order to determine the specific volumetric compressibility of liposomes, the change in ultrasonic viscosity was measured as a function of concentration, [u] and density, ρ. Then determined the specific volume, ϕV and specific apparent compressibility, ϕ _K / β ₀ by formulas (4.5).

Figure 4 shows that the liposomes of the present invention are substantially rigid compared to other liposome formulations and empty liposomes. As shown in FIG. 4, the specific compressibility (the inverse of the stiffness) of LSA liposomes is significantly lower than that of empty liposomes obtained using conventional methods known in the art. The compressibility of LSA liposomes was about 0.70, while the same value for empty liposomes was about 0.90. The specific compressibility of the KS and HU liposomes was 0.75 and was significantly different from the other liposomes tested. This example demonstrates the sensitivity of the mechanical properties of liposomes to the composition of the liposomes and to the presence of a drug within the liposomes.

Example 3 - Liposome Stability Assay

The stability of the liposome formulation prepared according to Example 1 is illustrated in Tables 4 and 5. Table 4 shows that the liposomes described in the present invention are stable when stored at 4 ° C for at least 36 months, and that the formulation meets all necessary requirements.

Table 4 - stability at 4 ° C

Character
history Observation start 1 month 7 month 12 month 24 month 36 month Appearance Whitish dispersion Compliant Compliant Compliant Compliant Compliant Compliant Alendronate (mg / ml) 0.5 ± 0.05 0.49 0.53 0.54 0.51 0.52 0.52 Encapsulation percentage (%) > 96 98 98 96 98 99 99 DSPC (mg / ml) 1.4-2.0 1.9 1.8 2 1.6 1.8 1.8 DSPG (mg / ml) 0.5-0.7 0.6 0.6 0.6 0.5 0.6 0.6 Cholesterol (mg / ml) 0.5-0.7 0.55 0.59 0.56 0.58 0.6 0.61 Drug: lipid ratio 1: 5.7 ± 1.0 1: 6.2 1: 5.6 1: 5.9 1: 5.3 1: 5.8 1: 5.8 Vesicle size (nm) diameter 100 ± 30 92 92 92 92 91 91 pH 6.7-7.3 6.9 6.9 6.9 7 7 6.9 Osmolality (mO / kg) 270-340 309 - 317 316 312 312

Table 4 shows that the liposomes described in the present invention are stable when stored at 25 ° C for at least 7 months, and that the formulation meets all the necessary requirements.

Character
history Observation start 1 month 2 month 7 month Appearance Whitish dispersion Compliant Compliant Compliant Compliant Alendronate (mg / ml) 0.5 ± 0.05 0.49 0.52 0.51 0.51 Encapsulation percentage (%) > 96 98 98 > 99 98 DSPC (mg / ml) 1.4-2.0 1.9 1.9 1.9 1.9 DSPG (mg / ml) 0.5-0.7 0.6 0.6 0.6 0.6 Cholesterol (mg / ml) 0.5-0.7 0.55 0.59 0.58 0.57 Drug: lipid ratio 1: 5.7 ± 1.0 1: 6.0 Vesicle size (nm) diameter 100 ± 30 92 92 92 93 pH 6.7-7.3 6.9 7 6.9 6.9 Osmolality (mO / kg) 270-340 309 - - 315

Example 4 - Liposome Uniformity Assay

Liposome uniformity analysis was performed using a Malvern Nano ZS particle size measuring system. This method consists in determining the uniformity of the size of the vesicles of the liposome composition using Dynamic Light Scattering (DLS) and characterizes the size distribution of particles suspended in a liquid medium. This method is very sensitive to the 10-1000 nm size range and is applicable to a variety of particles, including liposome emulsions, nanoparticles, and synthetic polymers. This method can be used both to determine the average diameter of a homogeneous liposome composition, and to determine the heterogeneity of the composition. After standardizing the Malvern Nano ZS measuring system according to the instructions for use, liposome samples at a concentration of 0.5 mg / ml were diluted 1/3 in PBS. λmax of UV radiation at 600 nm of the diluted solution was determined using an Ultraspec 2100 pro UV / VIS spectrophotometer. The liposomal sample was again diluted to reach an optical density (OD) of 0.10 ± 0.02. Then, it was determined that the λmax of UV radiation at 600 nm of the second dilution was also 0.10 ± 0.02 OD. Then, 1.0-1.5 ml of the diluted sample was placed in a culture tube and the size of the vesicles was analyzed using a Malvern Nano ZS measuring system. The analysis was carried out at room temperature (23 ° ± 2 ° C), a fixed angle of 173 ° and a laser wavelength of 633 nm. The data was accumulated to reach 150-500 Kcps (thousands of measurements per second).

Table 6 shows the measurement data of the composition described in the present invention. The average liposome size was 80.41 nm, with a PDI of 0.04.

Table 6

Measurement, no. Size, nm PDI 1 80.28 0.042 2 80.49 0.024 3 80.29 0.052 4 80.82 0.022 five 80.69 0.041 6 79.68 0.042 7 80.57 0.043 8 80.56 0.030 nine 80.33 0.028 Mean 80.41 0.04 Mean square deviation 0.330 0.010 Relative standard deviation,% 0.41 28.50

The HU liposomes, which are the prior art liposomes described in Example 2, were analyzed for homogeneity according to the method described above. Z-Average size of HU liposomes was 176.5 nm.

FIG. 5A and 5B show the size distribution of liposomes prepared according to the present invention and HU liposomes, respectively. FIG. 5A shows that the liposome formulation of the present invention has an average particle diameter of about 88 nm, with particle diameters ranging substantially from 69 to 107 nm. In contrast, in FIG. 5B shows that the HU liposome formulation has an average particle diameter of about 201 nm with a particle diameter of 80 nm to 500 nm. The PDI value of the composition in FIG. 5A was 0.025. The PDI value of the HU composition in FIG. 5B was 0.118.

The contents of all published articles, books, support guides, and abstracts described herein are incorporated by reference in their entirety in order to fully describe the prior art to which the present invention relates.

Since it is possible to carry out the above with various modifications without departing from the scope and spirit of the present invention, it is intended that all information contained in the description above or defined in the appended claims are to be construed solely as a description and illustration of the present invention. In light of the above, various modifications and variations of the present invention are possible.

Claims (15)

1. Use of a liposome for the preparation of a medicament for the treatment of a condition associated with inflammation, wherein the liposome containing a lipid ingredient and encapsulating a bisphosphonate has a weight ratio of said bisphosphonate to lipid ingredient from 1: 5 to 1: 8, and the lipid ingredient contains distearoyl phosphatidylcholine ( DSPC), distearoyl phosphatidylglycerol (DSPG) and cholesterol in a molar ratio of 3: 1: 2, and said liposome has a compressibility value of less than 0.70 ml / g. 2. Use of the composition for the preparation of a medicament for therapeutic use in inflammation, wherein the composition contains a plurality of liposomes, wherein said liposomes contain a lipid ingredient and a bisphosphonate, and the lipid ingredient contains distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylglycerol (DSPG) and cholesterol in molar a ratio of 3: 1: 2, and the composition has a polydispersity index (PDI) value of less than 0.070. 3. Liposome containing a lipid ingredient and encapsulating a bisphosphonate having a weight ratio of said bisphosphonate to lipid ingredient of about 1: 5 to 1: 8, the lipid ingredient comprising distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylglycerol (DSPG) in a molar ratio and : 1: 2, and said liposome has a compressibility value of less than 0.70 ml / g. 4. A composition comprising a plurality of liposomes, wherein said liposomes comprise a lipid ingredient and a bisphosphonate, the lipid ingredient comprising distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylglycerol (DSPG) and cholesterol in a molar ratio of 3: 1: 2, and the composition has a polydispersity index value ( PDI) less than 0.070. 5. Application, liposome or composition according to any one of paragraphs. 1, 2, 3, or 4, where the liposomes are negatively charged. 6. Application, liposome or composition according to any one of claims. 1, 2, 3, or 4, where the liposomes have an osmolality internal to the liposome of 340-440 mO / kg. 7. Application, liposome or composition according to any one of claims. 1, 2, 3 or 4, where the liposomes have a conductivity internal to the liposome of 13.5-17.5 mS / cm. 8. Application, liposome or composition according to any one of claims. 1, 2, 3 or 4, where the liposomes have an average size of 80 ± 5 nm. 9. Application, liposome or composition according to any one of claims. 1, 2, 3, or 4, where the bisphosphonate is alendronate. 10. Use, a liposome or formulation according to claim 9, wherein the concentration of the bisphosphonate within the liposome is from 0.5 to 5 mg / ml. 11. Application, liposome or composition according to any one of claims. 1, 2, 3, or 4, where the internal liposomal pH is 6.9. 12. The use or formulation of claim 2 or 4, wherein at least 96% of the bisphosphonate in said formulation is encapsulated. 13. Use or formulation according to claim 2 or 4, wherein the weight ratio of bisphosphonate to said lipid ingredient is from 1: 5 to 1: 8. 14. Use or formulation according to claim 2 or 4, wherein the liposomes have a compressibility value of not more than 0.70 ml / g. 15. Use or formulation according to claim 2 or 4, wherein the PDI is from 0.02 to 0.05.

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