JP2019218392A - Liposome and formulation containing liposome - Google Patents

Abstract

To provide: a liposome used in producing pharmaceutical compositions used in treating or preventing symptoms including endometriosis, ischemia-reperfusion injury, myocardial infarction, and restenosis substantially.SOLUTION: A liposome has a lipid ingredient and an encapsulated therapeutic agent, where the mass ratio of the therapeutic agent to the lipid is about 1:5 to 1:8, where the lipid ingredient comprises DSPC, DSPG and cholesterol in a molar ratio of about 3:1:2, and where the liposome has compressibility less than 0.70 ml/g. Also provided is a use of the liposome where the therapeutic agent is a bisphosphonate. Further provided are a liposomal formulation containing the therapeutic agent and a process for producing the formulation.SELECTED DRAWING: Figure 4

Description

[Field of the Invention]
The present invention relates to a novel liposome, a formulation containing the liposome, and a method for producing a liposome formulation.

[Background of the Invention]
It is known in the art that liposomes function as carriers in delivering therapeutic agents to target cells for treating various medical conditions. In some applications, the liposomes can be in a drug-encapsulated formulation that is selectively phagocytosed by macrophages. The phagocytosed liposomes release the drug into the cells and, inter alia, inhibit the inflammatory function of macrophages.

  The art describes several methods for producing such liposomes (eg, Monkonen, J. et al., 1994, J. Drug Target, 2: 299-308; Monkonen, J. et al., 1993, Calcif. Tissue Int., 53: 139-145; Classic DD., Liposomes Technology Inc., Elsevier, 1993, 63-105. (Chapter 3); Winterhalter M, Lasic DD, 93; 3): 35-43). In one of these methods, in forming a liposome, a two-layered liquid crystal laminate is formed, which is hydrated to form a hydrated lipid sheet, which is further broken off and self-closed by stirring to produce large multilamellar vesicles (large). multilamellar vehicles (MLV) are formed. This method is known as a lipid thin film hydration method. If such particles are formed, the particle size of the particles will depend on the method used in the subsequent steps of the method (sonic energy (sonication), mechanical energy (extrusion)). In the case of sonication, usually small unilamellar, vesicles SUVs are produced and a bath and / or probe tip sonicator is required. On the other hand, a lipid extrusion method in which the lipid suspension is forced through a series of polycarbonate filters (typically using 0.8, 0.4, 0.2, and 0.1 μm membranes at high pressure (500 psi or less) In (2), particles having a diameter close to the pore size of the filter used are generated. These methods are limited to small batch production, which is mainly used for research purposes. In the case of high-pressure extrusion, there is a problem that operation costs are higher and time is required. Thus, this technique provides a method for producing liposomes that is reproducible, can address problems such as quality control, stability, scalability of production scale, and sterilization in liposome production, and can be used for industrial-scale production. is needed.

  Also, liposome formulations known in the art provide a substantially uniform particle size and shape that is essential for pharmaceutical compositions in producing sterile products and preventing the potential for harmful side effects from unusually large liposomes. Do not have the nature. At present, it is difficult to produce liposome formulations with a uniform particle size. Extrusion methods in which multilayer vesicles are extruded through a series of filters (including, for example, a 100 nm pore size polycarbonate filter) cannot stably produce formulations containing 100 nm particle size substantially uniform liposomes. In fact, depending on the physical properties of the liposome, such as compressibility and / or stability, the average vesicle size of the extruded vesicles will vary considerably depending on the type and pore size of the filter used. Thus, there is a need for a method of production that can produce liposomes that are substantially uniform in particle size and shape.

  In addition, many physical properties of liposome formulations affect the response of cells to liposomes and affect the effectiveness of liposomes as a pharmaceutical composition. The physical properties of liposome formulations are affected in many aspects by the method of manufacture. However, the art has not shown a method of controlling the characteristics of the liposome during the production process to control the production efficiency or the stability of the liposome. Accordingly, there is a need for a method that can produce cost effective, substantially uniform liposomes that can be used for large-scale production where the properties of the liposome formulation can be controlled and that are suitable for clinical use. .

[Summary of the Invention]
In the present invention, a novel liposome encapsulating a therapeutic agent is described. The present invention also relates to liposome formulations with high particle size uniformity, low side effects, and improved reliability of the sterilization process. The present invention also describes an efficient and cost-effective method for producing liposomes under low pressure extrusion conditions. Liposomes formulated in this way have the desired properties and effective therapeutic properties.

  Liposomal formulations are characterized in that the liposomes have the desired composition and physical properties. Liposomes consist of lipid components that encapsulate a therapeutic agent. In one aspect of the invention, the lipid component comprises diastearoyl phosphatidylcholine (DSPC), diastearoyl phosphatidylglycerol (DSPG), cholesterol in a molar ratio of 3: 1: 2 (DSPC: DSPG: chol).

  Liposomes are composed of a lipid component and a therapeutic agent, wherein the ratio of the mass of the therapeutic agent to the mass of the lipid component, referred to as the drug: lipid ratio, is from about 1: 5 to 1: 8, preferably 1: 6 to 1, by weight. 1: 7. When the drug: lipid ratio is in this range, stability and efficiency are enhanced, and drug release and liposome integrity are also increased. These structural properties have realized the unexpected advantages of the formulations of the present invention.

  The particle size range of the liposome of the present invention is 30-500 nm, preferably 70-120 nm, 100-300 nm, 100-180 nm, 70-150 nm, depending on the type of therapeutic agent and / or excipient used. It is. In a preferred embodiment, the particle size of the liposome can be 80 ± 5 nm.

  Other physical properties of the liposome composition also contribute significantly to stability and efficacy. For example, the conductivity of liposomes may affect selective uptake into phagocytes. In one aspect, the conductivity ranges from 13.5 to 17.5 ms / cm. Similarly, the external and internal osmolality of the formulation affects stability and efficacy. It is preferred that the external osmolality be matched to the osmolality in the human body and that the internal osmolality be sufficiently low to stabilize the formulation. In one aspect of the invention, the internal osmolality ranges from 340 to 440 mOsm / kg. Another preferred variable is the pH of the liposome and / or liposome formulation. For example, an internal pH of the liposome formulation of about 6.9 has a beneficial effect on long-term stability as well as drug leakage and encapsulation capacity.

  The liposomes of the present invention are relatively rigid compared to liposomes in the art, as also characterized by having a liposome membrane that is difficult to compress. The liposomes of the present invention have a compressibility of less than 0.7 ml / g. Due to such rigidity, the liposome of the present invention has improved stability and storage stability.

  This liposome formulation also has new and useful features. The liposome formulation of the present invention comprises liposomes that are not only rigid but also have a substantially uniform particle size and shape distribution. This formulation has almost no variation in particle size between liposomes. The homogeneity 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, from which a highly homogenous composition as well. Is shown. Therefore, the liposome formulation of the present invention is preferable because it can reduce the number of undesirable events associated with large liposomes and can efficiently perform filtration through a sterilizing filter.

  Another aspect of the present invention is a method for producing a liposome formulation. The method comprises (1) mixing the therapeutic agent with a preselected lipid to form vesicles, (2) extruding the vesicles in a single stage through a single size filter, and (3) And filtering. After ultrafiltration, the product may be standardized, if necessary, to the desired final concentration. Since the extrusion step (2) is carried out by extrusion in a single stage under low pressure, the method of the present invention can reduce the operating cost and time and reduce the yield as compared with extrusion in a high-pressure multi-stage process. Can be mentioned. Such a manufacturing process can also be adapted for large-scale manufacturing.

  In one embodiment of the production method, the formulation is mixed with (1) the therapeutic agent and a lipid comprising DSPC, DSPG, and cholesterol in a molar ratio of about 3: 1: 2, and the mass ratio of the therapeutic agent to the lipid is about 1: 1. : 5 to 1: 8 to form vesicles, (2) extruding the vesicles in a single stage through a filter with a pore size of about 100 nm, and (3) performing ultrafiltration.

  Yet another aspect of the present invention is a liposome formulation manufactured according to the manufacturing process described above. The formulation consists of a plurality of liposomes composed of an amount of a therapeutic agent encapsulated lipid component. For example, the lipid component can include DSPC, DSPG, cholesterol in a molar ratio of about 3: 1: 2, and the mass ratio of therapeutic agent to lipid component can be from about 1: 5 to 1: 8. The formulation comprises the following steps: (1) mixing the therapeutic agent with a preselected lipid to create vesicles; (2) extruding the vesicles in a single stage through a single size filter; (3) Produced by ultrafiltration. After ultrafiltration, the product may be standardized, if necessary, to the desired final concentration. Preferably, formulations made according to this method have a PDI of less than 0.075, more preferably in the range of 0.02-0.05. This method produces liposome formulations with new and useful features such as the 3: 1: 2 ratio of lipid components described above, drug: lipid ratio, PDI, stiffness, pH, osmolality, and conductivity.

It is a flowchart of the manufacturing method of the liposome formulation of this invention. 1 is a flowchart summarizing a method for producing 1 liter of liposome-enclosed alendronate for intravenous injection (volume strength, 5 mg / ml) and process control variables based on one aspect of the present invention. 4 is a TEM image of liposome-encapsulated alendronate based on one aspect of the present invention. 5 is a graph comparing the specific compressibility of various liposome formulations. 1 is a graph of the particle size distribution of a liposome formulation of the present invention. 1 is a graph of the particle size distribution of a prior art liposome formulation.

[Detailed description of the invention]
The present invention relates to a novel liposome formulation used for treating various diseases and a method for producing the same. The formulation consists of a plurality of therapeutic agent-encapsulated liposomes, ie, a plurality of liposomes containing an "encapsulated therapeutic agent". The physical properties of each liposome achieve the stability and effect of the liposome formulation. The formulation is characterized in that the particle size and shape of the liposomes are substantially uniform. The present invention also describes a method for producing liposome formulations that is efficient, cost-effective and meets the needs of large-scale production. The present invention also relates to a formulation having novel and useful properties produced by this production method.

A. The present invention relates to novel and useful forms of liposomes. In producing the liposome of the present invention, various liposome components can be used. Preferably, the lipid component is a non-toxic, biocompatible lipid, such as a lipid made from phosphatidylcholine, phosphoglycerol, and / or cholesterol. In one aspect of the invention, the lipid component comprises diastearoyl phosphatidylcholine (DSPC), diastearoyl phosphatidylglycerol (DSPG), and cholesterol, preferably about 3: 1: 2 molar non- (DSPC: DSPG: chol). Contained in.

  The lipid component encapsulates the therapeutic agent, where both components have a predetermined mass. As used herein, "drug to lipid ratio" (ie, drug: lipid ratio) refers to the relative mass ratio of drug to lipid components that make up the liposome and / or formulation. In one aspect of the invention, the liposomes have a drug: lipid weight ratio of about 1: 5 to 1: 8, preferably 1: 6 to 1: 7.

B. Physical Properties of Liposomes Various physical variables and properties of liposomes can affect homogeneity, stability, and the efficacy of formulations. These properties include (1) osmolarity inside and outside liposomes, (2) conductivity (inside and outside liposomes), (3) drug: lipid ratio, (4) (inside and outside liposomes). pH, and (5) the type of lipid and drug used in the composition.

  Osmolality is a measure for the concentration of solutes in a liposome formulation. As used herein, the term "osmolarity" is a measure of the concentration of a solute, defined as the number of solute molecules per kilogram of solvent (mOsm) (mOsm / kg). The internal osmolality is the concentration of the solute inside the liposome, and the external osmolality is the concentration of the solute outside the liposome. In one aspect of the invention, the liposome has a low liposome internal osmolarity. The internal osmolality can be adjusted by changing the amount of drug encapsulated in the liposome. The liposomes described in the art are liposomes that encapsulate as much of the drug as possible and consequently have a high osmolality (high encapsulation ratio). However, the liposomes disclosed in the present invention are liposomes with lower osmolality (or lower encapsulation ratio) than liposomes used in the art. Using a lower osmolality as disclosed herein, ie, about 340-440 mOsm / kg, improves the stability of the liposomes in the formulation and the homogeneity of the liposomes. One way to reduce internal osmolality is to reduce the amount of therapeutic agent encapsulated in the liposome formulation. Another way to reduce the internal osmolality without changing the drug: lipid ratio is to use non-charged substances such as certain polysaccharides or sugars known in the art.

  The external osmolarity is preferably made isotonic with the osmolarity in the body, and particularly in the case of an injectable formulation, it is preferably made isotonic with the osmolarity in the body. Therefore, it is preferable to make the external osmolality relatively constant. The internal and external osmolality of the present invention achieves high stability, low drug leakage, and the ability of liposomes to properly encapsulate drugs.

  As used herein, the term "conductive" is the electrical conductivity of liposomes based on the ionic content of the solution. Conductivity is related to the ionic content of the liposome and affects the stability, drug leakage and drug encapsulation of the liposome formulation. The conductivity of the liposomes ranges from about 13.5 to 17.5 ms / cm. In one aspect of the invention, the encapsulated drug is charged. Charged drugs have a one-to-one correlation with liposome conductivity and osmolarity. Therefore, if the amount of the charged substance to be enclosed is changed, both characteristics are affected in a proportional manner. In another embodiment, a neutral (non-electrically charged) drug substance can be used. When a substance such as a polysaccharide is used as a neutral substance for adjusting the conductivity, it can be controlled independently of the concentration of the osmolality drug and independently of the drug concentration.

  Another novel aspect of the liposomes of the present invention is the relative stiffness of the composition, ie, the stability and fragility of the liposomes under different conditions in the environment and the body. The liposome stiffness is a measure of liposome membrane strength, which leads to improved liposome preservation, as well as resistance to shear and pressure. Liposomal stiffness is inversely proportional to compressibility. Liposomes with low compressibility are more rigid. One example of a method for measuring the stiffness of liposomes is to use ultrasonic velocimetry and densitometry. Methods for measuring the stiffness of liposomes are well known in the art and are described, for example, in Leide P. et al. , Et al. , "Compressibility study of quaternary phospholipid blend monolayers", Colloids and Surfaces B: Biointerfaces 85 (2011) 153, 160; , "Specific volume and Compresibility of bilayer lipid membranes with incorporated Na, K-ATPase", General Physiology and Biophysics on Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical and Biophysical products. These are hereby incorporated by reference in their entirety.

  For liposomes, there is a pH for the solvent outside the liposome (external pH) and a pH for the encapsulated portion inside the liposome (internal pH). pH affects stability, drug leakage from liposomes, and drug encapsulation of liposome formulations. In one aspect of the formulation, the internal pH ranges from about 6.8 to 7.0. An internal pH between 6.8 and 7.0 is advantageous for formulation stability. The pH of the solvent of the therapeutic agent may be, for example, a specific value when the pH range is maintained at 6.8 to 7.0 while continuously titrating the solution or when the therapeutic agent is dissolved using a known buffer. The pH can be maintained, for example, by maintaining the pH at about 6.9. The internal pH of the liposome may be different from the external pH of the liposome. In order to make the pH different, the pH of the solution constituting the internal and external environments of the liposome may be changed.

C. Formulation The liposome formulation of the present invention has the above-mentioned characteristics, and is composed of a plurality of liposomes having a substantially uniform particle size and shape, that is, almost no variation in particle size between liposomes. Formulation uniformity is measured by the polydispersity index (PDI). PDI is measured on a non-linear scale of 0 to 1 (a value of 0 is a perfectly homogeneous formulation, a composition with a PDI of 1 is large (non-uniform)). The PDI of the formulation of the present invention is less than 0.075, preferably in the range of 0.02-0.05.
The value of PDI is described in Zetasizer nano series user manual, 2003, Malvern Instruments, pp. 5.5-5.6 and Kazuba M.S. , Nano Series and HPPS Training Manual Chapter 1, 2003, Malvern Instruments, pp. 9, and the contents of these references are incorporated herein by reference. In fact, the PDI of the formulation resembles a particle size standard with a PDI of less than 0.02. Formulations known in the art typically have a PDI on the order of 0.3 and have a uniformity substantially different from that of the present invention. Because PDI is on a non-linear scale, the PDI of the present invention is substantially different from the PDI of formulations in the art. Indeed, the low PDI of the present invention advantageously prevents the harmful effects associated with large liposomes and produces a formulation suitable for filter sterilization.

  The formulations of the present invention comprise liposomes with increased stiffness and homogeneity, thereby improving stability and storage. The formulations of the present invention are also believed to be 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.)

  The liposomes of the present formulation have a particular particle size so that they are taken up by macrophages and monocytes. Liposomes can be of a size ranging from 30 to 500 nm. However, depending on the type of drug or carrier used, the range of the particle size of the liposome can be 70 to 120 nm, 100 to 500 nm, 100 to 300 nm, 100 to 180 nm, or 80 to 120 nm. However, these ranges are given by way of example, and other specific particle sizes suitable for uptake by phagocytic cells could be found without departing from the spirit and scope of the present invention. In one preferred aspect, the formulation has a liposome particle size of about 80 ± 5 nm.

  Various therapeutic agents can be encapsulated in the liposome of the present invention. A therapeutic agent is a substance that can reduce or inhibit activity and / or reduce the amount of phagocytic cells in a patient when liposomes are incorporated. The therapeutic agent can be any chemical, and can be large or small molecules, mixtures of chemical compounds, inorganic or organic compounds, biological macromolecules such as proteins, carbohydrates, peptides, antibodies, nucleic acids, etc. Can be. The therapeutic agent can be a natural product derived from a known organism or a synthetic compound.

  One type of therapeutic agent useful in the present invention is a bisphosphonate. Bisphosphonates (formerly phosphonates) are compounds characterized by having two CP bonds. Bisphosphonates are called geminal bisphosphonates when the two bonds are on the same carbon atom (P-C-P). Bisphosphonates are analogous to endogenous inorganic pyrophosphates that are involved in regulating bone formation and resorption. Bisphosphonates may form polymer chains in some cases. Since bisphosphonates are highly hydrophilic and negatively charged, it is almost completely impossible to pass through the cell membrane in its free form.

  The term bisphosphonate as used herein refers to both geminal and non-geminal bisphosphonates. Suitable bisphosphonates have the following formula (I):

  Here, R1 is H, OH, or a halogen atom, and R2 is halogen, heteroaryl or heterocyclic C1-C10 alkylamino or C3-C8 cycloalkylamino (amino is primary, secondary or tertiary). Linear or branched C1-C10 alkyl or C2-C10 alkenyl, -NHY (where Y is hydrogen), C3-C8 cycloalkyl, aryl or heteroaryl, or -SZ (wherein Z is chloro-substituted phenyl or pyridinyl).

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

  Many bisphosphonates have activity similar to alendronate and are useful as therapeutic agents of the present invention. These bisphosphonates can be chosen depending on how close to the biological activity of alendronate. The biological activity of alendronate includes, for example, in vitro activity that inhibits the activity of phagocytic cells such as macrophages and fibroblasts when entering the cells, IL-1 and / or Inhibition of IL-6 and / or TNF-α secretion, in vitro activity, eg, where the tested formulation treats animal models or humans with the ability to deplete or disable blood monocytes, or to treat myocardial infarction. And the ability to reduce the area of infarction.

  Bisphosphonates applicable in the present invention include clodronate, tiludronate, 3- (N, N-dimethylamino) -1-hydroxypropane-1,1-diphosphonic acid such as dimethyl-APD; 1-hydroxy-ethylidene-1, 1-bisphosphonic acid such as etidronate; 1-hydroxy-3 (methylpentylamino) -propylidene-bisphosphonic acid, (ibandronic acid) such as 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, such as methyl-pentyl-APD; 1-hydroxy-2- (imidazole-1- Il) ethane-1,1-diphosphonic acid For example, zoledronic acid; 1-hydroxy-2- (3-pyridyl) ethane-1,1-diphosphonic acid (risedronic acid) such as 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 acids, such as FR 78844 (Fujisawa); 5-benzoyl-3,4-dihydro-2H-pyrazole-3,3-diphosphonic acid tetraethyl ester, such as U81581 (Upjohn); and 1-hydroxy-2- (imidazo [1 , 2-a] pyridine-3-yl) ethane-1,1-diphosphonic acid such as YM 529, but is not limited thereto.

  In certain embodiments, for example, where sodium alendronate is encapsulated in DSPC, DSPG, and cholesterol, a drug: lipid mass ratio of 1: 5.7 (about 1: 1 for sodium alendronate). : 3 molar ratio). If another therapeutic agent, disodium clodronate, is encapsulated in the same lipid component, a mass ratio of about 1: 5.4 (equivalent to a molar ratio of 1: 3) can be used. In the present invention, other therapeutic agents that inhibit or reduce phagocytic cells by inhibiting or delaying proliferation and / or down-regulating the activity of phagocytic cells may be encapsulated. Specific therapeutic agents include any cytotoxic or cytostatic agents, such as gallium, gold, silica, 5-fluorouracil, cisplatin, alkylating agents, mitramycin, paclitaxol. However, the present invention is not limited to these. In any of the above therapeutic agents, the drug: lipid ratio (by weight) of the liposome is from about 1: 5 to 1: 8, preferably from 1: 6 to 1: 7.

  In one embodiment, the formulation comprises a liposome-encapsulated therapeutic agent that enters cells through phagocytosis and targets macrophages and monocytes without affecting non-phagocytic cells. Inhibition and / or deficiency of monocytes / macrophages can cause damage to normal macrophages and monocytes as they are directed to the damaged area of cells and promote inflammation beyond the level of inflammation caused by the disease or condition. The adverse effect on the affected area. When taken inside phagocytes, the drug is released, including ischemia-reperfusion injury or inflammatory injury, such as myocardial infarction, reduction of the final area of infarction, cardiac repair after acute myocardial infarction and improved outcomes Inhibiting, inactivating, dysfunctional, killing, and / or deficient monocytes and / or macrophages to treat various conditions involving an phagocytic immune response. The liposomes of the formulation have specific properties such as particle size, charge, pH, conductivity, particle size, charge, pH, conductivity, and osmolality that promote uptake primarily through phagocytosis.

  After being taken up by monocytes / macrophages, the formulation exerts a sustained inhibitory activity on monocytes / macrophages. This sustained activity is sufficient to attenuate the inflammatory effects of monocytes / macrophages, and therefore does not require sustained release of the formulation to retain the inhibitory effect. Thus, methods of treating certain diseases, for example, by inhibiting monocytes / macrophages using liposome-encapsulated formulations, require a systemic approach where the formulation targets circulating monocytes and macrophages. Preferably, it is a treatment. Depending on the type of encapsulated therapeutic agent, the response of the phagocyte can be different. For example, liposomes encapsulating alendronate cause apoptosis, whereas liposomes enclosing clodronate cause necrosis. Cells other than phagocytes are relatively less likely to be taken up by the liposome formulation due to the inherent physiological properties of the liposome formulation.

  In addition, the liposomes of the present invention not only prevent the therapeutic agent from being released in body fluids by holding the therapeutic agent for a sufficient time, but also efficiently release the formulation within target cells. The liposomes of the present invention deliver an effective amount of the formulation to target cells. The term "effective amount" refers to a prescription agent that is effective in achieving the desired therapeutic result, eg, endometriosis, restenosis, ischemia-reperfusion injury (IRI), myocardial infarction or a related condition. Means the amount of For example, if the disorder is associated with myocardial damage, reducing the number and / or activity of activated macrophages and monocytes will narrow the area of infarction and / or improve repair. The effective amount will depend on a number of factors, including the weight and gender of the patient to be treated, the mode of administration of the formulation (ie, systemic or direct site administration), the treatment regimen (eg, one day per day of administration of the therapeutic agent). Dose, several times a day, every other day, single dose, etc.), clinical indices of inflammation, clinical factors affecting the rate of deterioration of the symptoms to be treated, smoking, hypercholesterolemia , Proinflammatory condition, renal disease, composition dosage form, etc., but the factors are not limited thereto. Successful delivery of a liposome formulation depends on the stability and efficacy of the liposome formulation. The number of therapeutic molecules to be encapsulated in each liposome (payload), vesicle size, and free non-encapsulated material are important variables that determine the therapeutic index of the product.

D. Dosage and Administration Liposomal formulations can be administered by any route, provided that the liposomes can be effectively transported to the appropriate or desired site of action. Preferred modes of administration include intravenous (IV) and intra-arterial (IA) administration (particularly suitable for in-line administration). Other suitable dosage forms include intramuscular (IM), subcutaneous (SC), and intraperitoneal (IP) administration. Such administration can be by rapid injection or infusion. Another mode of administration can be by perivascular delivery. The formulation may be administered directly or after dilution. In accordance with the present invention, any of the above administration routes may be used in combination. Any route of administration can be used provided that the particles come into contact with phagocytes (circulating monocytes or peritoneal macrophages).

  The pharmaceutical composition used according to the invention facilitates the processing of the active ingredient into a pharmaceutical preparation and is a pharmaceutically usable physiologically acceptable carrier consisting of excipients and auxiliaries known in the art. It can be prescribed by using the above.

  Dosage amount and interval can be adjusted individually to provide plasma levels of the formulation that are sufficient to induce or suppress a biological effect (minimum effective concentration, MEC). . The MEC will vary with the particular formulation, but can be determined from in vitro data. The volume required to achieve MEC will depend on individual characteristics and route of administration. Various detection assays can be used to measure plasma concentration.

  Depending on the severity and responsiveness of the condition to be treated, the administration can be a single or multiple administrations, and the treatment period can be from several hours to several weeks, or until the treatment is completed. The frequency of administration can also vary depending on the condition and severity. In one aspect, the formulation can be administered on a regular basis. Dosage can be formulated in any amount or volume required for the desired treatment. For example, a dose of 1 microgram or 10 micrograms may be administered to a patient undergoing an endovascular procedure, such as a stent, to reduce the subsequent occurrence and severity of in-stent late loss within the stent. Can be prevented. In another example, the dose can be 100 micrograms or more. In this sense, the formulations may be administered in a single dose, multiple doses, and / or in a continuous infusion over a period of time, for example. For example, capacities are described in Banai, Am Heart J., supra. It can be administered according to the description in (2013).

E. FIG. Another aspect of the present invention is a method for producing liposomes and liposome formulations suitable for commercial production of products. This manufacturing method can manipulate the physical properties as described above, including water: solvent ratio, solvent composition and solvent ratio, vesicle preparation temperature, vesicle preparation shear rate, extrusion temperature, extrusion pressure. Specific process variables, such as extruded film particle size, film type, etc., can also be controlled.

  The production of liposome formulations involves (1) mixing the therapeutic agent with a preselected lipid to form vesicles, and (2) extruding the vesicles in a single stage through a single size filter. Step (3) Ultrafiltration step is included. Here, the expression “the step of extruding using a filter in a single step” means that “in the extrusion step, a filtration step having a single pore size” is used. That is, a single size filter may be passed multiple times, but different size filters must be passed multiple times and / or consecutively as is known in the art (eg, 1. 0, 0.8, 0.6, 0.4, 0.2 micrometer continuous passage). After ultrafiltration, the product can be standardized to the desired final concentration. Since the extrusion in step (2) is carried out by a single stage extrusion at low pressure, using the method of the present invention reduces the operating costs and time and increases the yield compared to multistage high pressure extrusion. . FIG. 1 illustrates each step of the manufacturing method.

  The first step comprises mixing the therapeutic agent and a preselected lipid component in a solution to form vesicles. In many cases, the therapeutic agent and the lipid component are solubilized in the form of a therapeutic agent solution and a lipid component solution, and then the two solutions are mixed. In this aspect, the therapeutic agent solution has certain physical properties, including pH, osmolality, conductivity. The therapeutic solution forms the internal environment of the liposome formulation. Thus, the pH, conductivity, and osmolarity of a therapeutic solution affects the internal pH, conductivity, and osmolarity of a liposome formulation. The internal osmolarity of the liposomes is preferably in the range of 340-440 mOsm / kg, while the external osmolarity of the liposomes is in the range of 270-340 mOsm / kg. The pH of the solution is such that the pH of the therapeutic solution is in the desired pH range. Thus, when using an acidic therapeutic agent, such as a bisphosphonate, a basic pH solution is used to keep the pH of the solution in the range of 6.8-7.0. For example, a therapeutic agent solution can be prepared from a solution of sodium alendronate in a NaOH solution, and the resulting alendronate solution has a pH of about 6.8 and a conductivity of about 18.0 ms /. cm. The solution may be heated during this process if necessary. In another embodiment, sodium clodronate or alendronate monohydrate can be used. The solution can be heated to a temperature of 55-75 ° C, such as 70 ° C, to facilitate solubilization of the bisphosphonate in a basic solvent. Depending on the amount of therapeutic agent and other excipients dissolved, the solution may have an osmolality of 340-400 mOsm / kg, which is the internal osmolality. The solution can also have a conductivity in the range of 14.0 to 21.0 ms / cm, depending on the amount of therapeutic agent and excipient, and this conductivity becomes the internal conductivity. Internal pH, osmolality, and conductivity are factors that affect the stability and efficacy of liposome formulations. In one aspect, the concentration of the therapeutic agent solution in step (1) of the manufacturing process can be 20-120 g / L. The therapeutic agent solution can be prepared separately from and / or prior to the manufacturing process discussed herein.

  The lipid component can be in the form of a solution containing the desired starting amount of the lipid component in one or more lipid solvents. Any suitable lipid component and lipid solvent can be used. For example, the lipid component may consist of a 3: 1: 2 molar ratio of DSPC, DSPG, cholesterol prior to liposome formation. Liposomes obtained by combining lipids with this combination may have a molar ratio of DSPC, DSPG, and cholesterol of 3: 1: 2. Further, the lipid solvent can be composed of, for example, t-butanol, ethanol, and water in a v / v / v ratio of 77/77/6. Other lipid solvents that can be used when formulating the liposomes of the present invention include chloroform or methanol. The lipid component dissolves in the lipid solvent. The lipid solvent can be heated to a temperature that promotes solubilization of lipid components in the range of 55-75 ° C, for example, 70 ° C, to form a lipid solution. The concentration of the dissolved lipid solution can range from 50 to 350 g / L. The lipid solution can be prepared separately from and / or prior to the manufacturing process discussed herein.

  By mixing the therapeutic agent solution and the lipid solution, multilamellar vesicles (MLV) are formed. The drug: lipid ratio can be controlled by varying the amount of the lipid or therapeutic solution. The two solutions may be gently heated, if desired, to facilitate mixing of the solutions. This process allows the therapeutic agent to be efficiently encapsulated in the multilamellar vesicles. In one example, the lipid solution can be added to the therapeutic agent solution at a ratio of 5.3 parts lipid to 1 part therapeutic agent. This lipid to drug (weight) ratio increases the stability of the liposome formulation without significantly sacrificing delivery. In fact, the drug: lipid ratio in this process can vary from 1: 4 to 1: 8 by weight, preferably from 1: 5 to 1: 6. Also, the solvent concentration can be adjusted before the extrusion step without sacrificing liposome integrity.

  The next step in the method of preparing the formulation involves extruding the vesicles through a single size filter in a single step. Extruding the vesicles reduces the particle size of the multilayer vesicles described above. This method uses a single extrusion step and low pressure, resulting in the production of liposomes with a high uniformity in particle size and shape. In the process of extrusion, a microfluidizer or other conventional homogenizer can be used. Homogenizers use shear energy to split large liposomes into small liposomes. Homogenizers suitable for use in the present invention include microfluidizers manufactured by various manufacturers, such as Microfluidics, Inc. of Boston, Mass., USA. The particle size distribution can be monitored by discriminating the particle size with a normal laser beam. Filtration of liposomes through polycarbonate membranes or asymmetric ceramic membranes is an effective way to limit liposome particle size to a relatively well-defined particle size distribution. The extrusion temperature is preferably higher than the transition phase temperature of the liposome in order to reduce the particle size. For example, when DSPG, DSPC, or cholesterol is used, the temperature is preferably set to about 55 ° C. Other lipids can be used, and a known transition phase temperature can be used as an indicator of the desired temperature of the extrusion process. Multiple passes may be required to achieve the desired vesicle particle size, uniformity and homogeneity. During this step, the sample can be analyzed in real time to assess vesicle size and bacterial count.

  The extrusion process is performed by a single stage low pressure extrusion process. In the extrusion process, small unilamellar liposomes are formed by multi-stage extrusion in which large liposomes are sequentially passed from a large pore membrane to a small pore membrane using a high pressure extruder (as has been done in the conventional method). Instead, processing is performed in a single step by extruding the MLV directly through one type of membrane while applying low pressure. In one aspect, the single-step extrusion process is performed directly using a 0.1 micrometer pore size polycarbonate or ceramic membrane at a low pressure of 60-90 psi to obtain liposomes with a particle size of 100 nm. This process also uses a low pressure of 60-90 psi as opposed to the high pressure (500 psi) in high pressure extrusion.

  The method of the present invention of extruding at low pressure so that no multilayer film is formed has a number of advantages. First, a single-stage extrusion process can perform the extrusion process in less time than a multi-stage process. Also, the elimination of the need for high-pressure extrusion also avoids the high costs associated with extrusion equipment. Low pressure extruders are significantly less expensive. One example of a suitable extruder of the present invention is LIPEX® Extruder sold by Northern Lipids, Inc. Further, the materials used in low pressure extruders, such as compressed air, are also generally less expensive than the materials used in high pressure extruders, such as nitrogen. Further, by changing the multi-stage extrusion to a single-stage extrusion, waste was similarly reduced and the yield increased.

  The last step of the present invention consists of ultrafiltration of the extruded vesicles into a buffer. The liposomes formed during the extrusion process are uniform in particle size and shape, but in ultrafiltration, pressure is applied to the formulation to pass through the membrane, and the encapsulated liposomes are removed from the unencapsulated therapeutic agent and solvent. , Separated from lipids. This step is preferably carried out at a temperature below the transition phase temperature of the lipid used in the formulation, for example typically below 45 ° C. In the ultrafiltration process, various filtration membranes can be used. In one embodiment of ultrafiltration, a hollow fiber membrane is used to apply the formulation under pressure through the hollow interior of the fiber so that relatively large liposomes remain within the fiber while small molecules ( That is, the solvent, unencapsulated bisphosphonate, lipid) is filtered through the membrane outside the fiber. This ultrafiltration results in a formulation that contains more than 96% of the therapeutic agent.

  The ultrafiltration step may further include a dialysis step of dialyzing the formulation against a specific amount of buffer. One example of a buffer is phosphate buffered saline (PBS), but any buffer maintained at physiological osmotic pressure that contains a balance of positive and negative ions can be used. Other buffer additives are known in the art and may include, for example, sucrose, glycine, sodium succinate, and / or albumin. Preferably, the buffer carefully monitors the pH and conductivity of the solution to reflect the external environment of the final formulation. The buffer is preferably an isotonic solution and non-toxic to cells. The buffer can be filtered to further reduce contaminants or prepared prior to the manufacturing process. The final point of dialysis is when the desired pH and conductivity of the formulation has been reached.

  At the end of the ultrafiltration step, the formulation may be filtered, if necessary, for sterilization, ie for controlling biological contamination. In one aspect of this process, a sterile filter is connected to a pressure vessel containing the liposome formulation, and the sterile filter is connected to a further sterilized receiving tank. Applying pressure to the liposome formulation causes the formulation to pass through a sterile filter. In this step, another sample can be taken to determine the bacterial content. The pore size of the sterile filter can range from 0.2 to 0.45 micrometers. Since the liposomes of the formulation are substantially uniform and do not contain large liposomes, sterile filtration can be performed without any particular complication.

  After manufacture, the formulation can be standardized. Final normalization results in a liposome formulation having a standard concentration. Standardization analyzes for yield, particle size, lipid composition, drug and free drug content, and product concentration. Analysis of the concentration can be performed by methods known in the art, for example, analysis of phosphate using a HPLC, spectrophotometer. Once the concentration of the ultrafiltration product has been determined, the formulation is diluted to a standard concentration using the specified amount of buffer. Final standardization also involves sterile filtration of the liposome product. For example, the encapsulated bisphosphonate formulation after ultrafiltration can be diluted with a suitable amount of buffer to a final concentration. Similarly, the ultrafiltration product can be used at different final concentrations by splitting into multiple batches and using different amounts of buffer as a diluent in each batch. A sample may be obtained and the concentration of the formulation at that time may be measured.

  This production method has the advantage that the physiological and chemical characteristics of the liposome can be controlled, monitored and reproduced. For example, the internal pH of the liposome can be controlled by the composition of the solution that dissolves the therapeutic agent. The internal osmolality can be similarly manipulated by varying the amount of the therapeutic agent, for example, depending on whether the therapeutic agent is charged. The drug: lipid ratio can be controlled by selecting the lipid components that make up the liposome, ie, the amount of lipid added to the dissolved active therapeutic agent. Increasing the amount of the lipid component decreases the drug: lipid ratio, and vice versa. Also, lower drug: lipid ratios reduce the internal osmolality of liposome formulations. The external pH and external osmolarity of the composition are affected, in part, by the composition of the buffer containing the final product. Conductivity can be controlled by the nature of the therapeutic agent and other excipients encapsulated in the liposome. Other factors such as viscosity, excipient quality, sterility, saline solutions, infusion kits, compatibility with syringes (in the case of injectable formulations), compatibility with processing equipment are also provided by the method of the invention. Each can be controlled independently.

  In view of the above description, in a preferred embodiment of the production method, the formulation is prepared by mixing (1) a solution containing a therapeutic agent with a solution containing DSPC, DSPG, and cholesterol in a molar ratio of about 3: 1: 2, Forming vesicles such that the mass ratio of the therapeutic agent to lipid is about 1: 5 to 1: 8; (2) extruding the vesicles in a single step through a filter having a pore size of about 100 nm; Manufactured by external filtration.

  Another aspect of the present invention is a liposome formulation prepared according to the above process, wherein the formulation comprises DSPC, DSPG, cholesterol in a molar ratio of about 3: 1: 2, and comprises a therapeutic agent to a lipid. The mass ratio of the components can be from about 1: 5 to 1: 8, the following steps: (1) mixing the solution containing the therapeutic agent and the solution containing the lipid component to form vesicles; (2) Produced by extruding vesicles through a single size filter in a single stage and (3) ultrafiltration. After ultrafiltration, the product can be standardized to the desired final concentration. Formulations prepared according to this method have a PDI of less than 0.075, preferably in the range of 0.02 to 0.05. This method produces liposome formulations with new and useful characteristics as described above, including a 3: 1: 2 lipid component ratio, a 1: 5 to 1: 8 drug: lipid ratio. In addition, individual liposome properties, including pH, osmolality, conductivity, and stiffness, can be separately controlled as described above.

  The following examples are provided for illustratively describing various viewpoints in practicing the present invention, and do not limit the present invention. In the following embodiments, the present invention will be described in the form of embodiments only with reference to the drawings. When the drawings are specifically referred to, the details of the respective parts shown in the drawings are shown only for the purpose of carrying out the preferred embodiments of the present invention in a specific form, and the principles and conceptual aspects of the present invention are described. For the purpose of explanation, we have provided concrete examples of what seems to be the most useful and easy to understand. The description should be considered in conjunction with the drawings, which make apparent to those skilled in the art how some forms of the invention may be embodied in practice.

Example 1: Batch production of liposome formulations Test batches were prepared according to the method described above. Of course, the particle size of the batch can be varied as desired for commercial production. In this example, a 1 liter batch of phosphate buffered saline was prepared in which liposomes containing alendronate were dispersed in liposomes containing cholesterol, DSPC, and DSPG. In view of clinical convenience, liposome-encapsulated alendronate can be produced as a turbid liposome dispersion sterilized at two concentrations of 5 mg / ml and 0.5 mg / ml. With respect to these concentrations, a prescribed amount of the therapeutic agent can be obtained in a specific amount by appropriately formulating the therapeutic agent. The lipid component was composed of cholesterol, DSPC, and DSPG. The dispersion also contained a phosphate buffer in order to control the pH, drip appropriately and maintain isotonicity. More than 96% of the drug in the final product was encapsulated in liposomes. For administration, the contents of the vial (or a portion thereof, if necessary) were diluted with a saline solution and administered by infusion.

  Table 1 shows the recipes in batches, including the amounts and qualities of the components used in the manufacturing process. The amount indicates the amount per liter of the batch.

  Table 2 below shows the content and quantitative composition of intravenous liposome-encapsulated alendronate produced in a 1-liter batch of Table 1. In this batch, the molar ratio of DSPC: DSPG: cholesterol was 3: 1: 2. The drug: lipid ratio was calculated to be approximately 1: 5.7 ± 1.5 (w: w).

  Table 3 below shows intravenous liposome-encapsulated alendronate formulations actually produced by the novel production method described in this specification.

  FIG. 2 is a flowchart summarizing a method for producing 1 liter of alendronate encapsulating liposome for intravenous injection for an active ingredient content of 5 mg / ml, and process control parameters. The process for producing an active ingredient content of 0.5 mg / ml is the same as that used for an active ingredient content of 5 mg / ml. Only the final standardization step differs in having different final formulation concentrations for administration. One skilled in the art will appreciate that other dosages can be produced in this manner without undue experimentation.

  The therapeutic agent solution (alendronate solution) and lipid solution were prepared as follows.

  Alendronate solution NaOH (6.8-8.0 g) was weighed and dissolved in 850 ml of water for injection (WFI). It was confirmed by visual inspection that it was completely dissolved. Sodium alendronate (68-80.75 g) was dissolved in the NaOH solution at 70 ± 3 ° C. with stirring at 600 ± 150 RPM. It was confirmed by visual inspection that it was completely dissolved (ie, a transparent solution), and the conductivity and the measured pH value were confirmed (pH = 6.8 ± 0.3, conductivity = 18.0 ± 1 ms / cm). .

Lipid solution 30 g of DSPC (37.9 mmoles), 10 g of DSPG (12.5 mmoles), 10 g of cholesterol (25.8 mmoles) (DSPC / DSPG / Cholesterol; 3/1/2 mol / mol / mol) are weighed. And dissolved in 160 ml of t-butanol / EtOH / H ₂ O (77/77/6, v / v / v) in a 250 ml beaker at a temperature of 70 ± 3 ° C. using a heated magnetic stirrer. , A lipid solution with a concentration of 312 mg / ml was formed. When the temperature was within the range, a clear yellow solution was formed.

Formation of MLV The lipid solution was added to the alendronate solution while mixing at 600 + 150 RPM and keeping the temperature at 70 ± 3 ° C. (1 part drug: 5.3 parts lipid). After more than 5 minutes, 100 ml of water for injection (10% of the total volume) was added to reduce the solvent concentration before extrusion. The formulation was mixed for an additional 10 minutes.

Extrusion The formulation was heated to 1.2 L of heated stainless steel with one 0.14 micrometer ceramic membrane or two polycarbonate membranes (eg, a 0.2 micrometer prefilter and a 0.1 micrometer membrane). Extrusion was performed using an extruder at 90 ± 15 psi pressure and 68 ± 5 ° C. temperature to reduce vesicle particle size and increase vesicle homogeneity. This process required 12-18 passes to bring the vesicles to 80-100 nm. The extrudate was sampled in situ, checked for vesicle particle size, and then subjected to ultrafiltration. The particle size of the liposome formulation was analyzed using a Malvern Nano ZS analyzer (acceptance limit: 95 ± 20 nm). Extruded samples were also counted for aerobic bacterial counts (biofouling control). The pass limit was less than 100 CFU / ml.

Ultrafiltration and Diafiltration First, before performing ultrafiltration, the formulation was allowed to cool to less than 45 ° C. Ultrafiltration was performed using Amersham's QuixStand system with a 500K hollow fiber membrane. The formulation was concentrated such that the inlet pressure was 25 psi or less. When the minimum volume was reached, the formulation was dialyzed against 10 times the initial volume (about 7 L) of a phosphate buffered saline (PBS) solution. PBS solution was prepared by dissolving NaCl of 145 mM, the _{Na 2 HPO 4, NaH 2 PO} 4 of 7mM of 13mM in water for injection 10L. The pH of PBS was about 6.9, and the conductivity was about 16.9 ms / cm. The PBS was filtered through a 0.2 micrometer filter. The end point of diafiltration was determined by measuring the pH and conductivity of the dialyzed formulation. Formulations not exceeding 120% (about 1.2 liters) of the initial volume were discharged from the ultrafiltration system. Samples were taken for general analysis and for aerobic bacterial counts (control of biological contamination). The pass limit was less than 1,000 CFU / ml.

  Upon completion of the dialysis, the formulation was filtered through a 0.2 micron filter to maintain control over biological contamination. The pressure vessel containing the formulation was connected to a sterile 0.2 micrometer filter (Sartorius Sartobran P). The filter was previously connected to the sterile receiving tank. Filtration was performed by applying a pressure of 5-30 psi to the pressure vessel. Samples were taken for general analysis and for aerobic bacterial counts (control of biological contamination). The pass limit was less than 1,000 CFU / ml.

Final Standardization Formulation particle size, liposome-injected lipid / therapeutic composition, drug and free therapeutic content were analyzed by HPLC. The expected yield in this example was that one liter of the formulation contained about 6 mg / ml liposome-encapsulated alendronate and 35 mg / ml lipid. Based on the results for the alendronate concentration, the dilution required to bring the concentration of about 1 liter of the final formulation to 5 mg / ml or 0.5 mg / ml was calculated. After ultrafiltration, about 900 ml of liposome-encapsulated alendronate was diluted with about 100 ml of PBS prepared as described above to prepare a 5 mg / ml formulation. To prepare a 0.5 mg / ml formulation, after ultrafiltration, about 100 ml of liposome-encapsulated alendronate was diluted with about 900 ml of PBS to a concentration of 0.5 mg / ml. A sample was taken to determine the actual concentration of alendronate.

  After preparing a standard concentration formulation, the vial containing the formulation was connected to two 0.2 micrometer sterile filters (Sartorius Sartobran P) placed consecutively in a class 100 room or in a sterile biological hood. did. The filters were previously connected to sterile disposable receiving bags or bottles. Filtration was performed using a peristaltic pump or pressurized nitrogen. Pressure should not exceed 10 psi. At the end of the filtration, the integrity of the filter was tested.

  The intravenous liposome-encapsulated alendronate produced in the above example has several desirable properties, such as (i) storage for at least alendronate and lipids at 5 ° C (range 2-8 ° C) for 3 years. Gender, (ii) average vesicle diameter 80 ± 5 (no particulate matter), (iii) sodium alendronate concentration in the range of 5 mg / ml or less (0.1-5.0 mg / ml), (iv) 96% Above alendronate encapsulation rate, (v) 3/1/2 (mol / mol / mol) diastearoyl phosphatidylcholine / diastearoyl phosphatidylglycerol / cholesterol (DSPC / DSPG / CHOL) lipid composition, (vi) 270-340 mOsm Osmolarity / kg, (vii) viscosity similar to water, ie 1.0 mPa.s at 20 ° C. s kinematic viscosity, (viii) pH 6.8 (range 6.8-7.0), (ix) globally acceptable US or European Pharmacopoeia compliant excipient quality, (x) USP (Xi) Salt solution, infusion kit, syringe compatibility (use), (xii) filters, stainless steel and glass tubing It had compatibility (method).

  FIG. 3 is a TEMS image of alendronate of the liposome produced in the above example. Good homogeneity and uniformity were observed for the liposome group, and the particle size of the liposomes in the image ranged from 40 to 120 nm.

Example 2 Test on stiffness of liposome Four kinds of large unilamellar liposomes (about 100 nm in diameter) were examined for stiffness. That is, the PBS suspension of hollow liposomes manufactured according to the present invention was referred to as LPO, and the liposome formulation containing 5.0 mg / ml alendronate manufactured according to the present invention was referred to as LSA. Samples of two other liposomes (KS and HU) dissolved in HEPES are described in Epstein-Barash, Hila, et al. "Physicochemical parameters effecting liposomal bisphosphonates bioactivity for resentosis therapy: Internationalization, cell internation, activitation, activation Manufactured according to the prior art method described in Controlled Release 146 (2010) 182-195.

  Each sample was examined for specific volume compressibility of the liposomes. Using ultrasonic velocimetry, the elastic properties of the liposomes were evaluated based on the following relationship:

  ΒS, ρ, and u in the equation are the adiabatic compressibility, density, and sound velocity 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. Surf. A 139, 189-197; Hianik T .; , Rybar P .; , Krivanek R .; Petrikova M. , Milena Roudna M., et al. Hans-Jurgen Apell, HJ. (2011): Specific volume and Comprehensiveness of bilayer lipid membranes with incorporated Na, K-ATPase. Gen. Physiol. Biophys. 30, 145-153. Therefore, by measuring changes in sound speed and density, changes in compression ratio can also be determined.

  Supersonic velocities were measured using a fixed path differential velocimeter consisting of two nearly identical sonic cavity resonators operating at a frequency of about 7.2 MHz (Sarvazyan AP (1991): Ultrasonic velocimetry of biological Compounds). , Annu Rev. Biophys Biophys Chem 20, 321-342; Sarvazyan A.P., Chalikian T.V. (1991):.... Theoretical analysis of an ultrasonic interferometer for precise measurements at high pressures, Ultrasonics 29, 119- 124). The cell resonance frequency was measured (USAT, USA) using a computer controlled network analyzer (USAT, USA). The volume of the sample was 0.7 ml. The resonator cell was equipped with a magnetic stirrer so that the sample was homogeneously dispersed during the measurement. One resonator contained the liposome solution at a concentration of 10 mg / ml relative to the phospholipid, and the other was filled with the same buffer without vesicles (PBS or HEPES) as a control. . At the start of a series of measurements, the resonance frequencies of both resonators were first compared by measuring both cells with the same reference liquid. The energy density of the sonic signal was small throughout (ultrasonic pressure amplitude less than 103 Pa), thus preventing any effect of the sonic wave on the structural properties of the vesicles. In general, as shown in the following equation, the velocity of sound [u], or rather the concentration dependent increment of the velocity of sound [u], can be measured by performing ultrasonic velocity measurements. (Sarvazian AP (1982): Development of methods of precision measurements in small volumes of liquids, Ultrasonics 20, 151-154).

Here, c is the concentration of the solute (mg / ml), and the subscript “0” relates to the solvent (buffer solution). The value [u] is the change in the resonance frequencies f and f _{0 of} both resonators (f is the resonance frequency of the sample, f ₀ is the resonance frequency of the reference buffer):

(This coefficient satisfies the condition of γ << 1 and can be ignored in the calculation)
Can be determined directly from

  The density (ρ) of the vesicle solution is determined by the principle of a vibrating tube (Kratky O., Leopold H., Stabinger H. (1973): The determination of the specific volume of the molecular mechanism of the biosynthesis of the vesicle solution). Ed. E. Grell), vol. 27, pp. 98-110, Academic Press, London; high-precision densitometer system (two sample chambers of DMA60, DMA602M, Anton Paar KG, Graz, Austria) Measured using The apparent specific partial volume (φV) was calculated from the density data using the following equation.

  Here, the subscript 0 indicates the reference solvent, and [ρ] = (ρ-ρ0) / (ρ0c) is the density increment of the density. Cell temperature was controlled using Lauda RK 8 CS ultra-thermostat (Lauda, Germany) in the range of ± 0.02 ° C.

  By determining the specific volume in addition to the increment of the sonic velocity concentration, it became possible to predict φK / β0 of the reduced apparent specific compressibility of vesicles based on the following equation.

In the equation, β ₀ is a coefficient of the compressibility, and ρ ₀ is the density of the buffer solution (Sarvazyan 1991). The value of φK / β0 indicates the volume compression ratio of the liposome to the buffer. The higher the value of φK / β0, the higher the compression ratio of the liposome (that is, the lower the rigidity).

  That is, in order to determine the specific volume compressibility of the liposome, the concentration increment [u] of the ultrasonic velocity and the density ρ are measured, and then the specific volume φV and the apparent specific compressibility φK / β0 are calculated by the formula (4). 5).

  FIG. 4 shows that the liposomes of the present invention are substantially more rigid than other liposome formulations and hollow liposomes. As shown in FIG. 4, the specific compressibility of LSA liposomes (inversely proportional to stiffness) is significantly lower than that of hollow liposomes made with conventional prior art formulations. The specific compressibility of hollow liposomes is about 0.90, while the compressibility of LSA liposomes is about 0.70. The specific compressibility of KS and HU liposomes differs significantly at 0.75. This example shows that the mechanical properties depend on the formulation and the presence or absence of the drug inside the liposome.

Example 3: Analysis of liposome stability Tables 4 and 5 show the stability of the liposome formulations prepared according to Example 1. As shown in Table 4, the liposomes of the present invention are stable for at least 36 months when stored at 4 ° C, and the formulation meets all required specifications.

  As shown in Table 5, the liposomes of the present invention are stable for up to at least 7 months when stored at 25 ° C. and the formulation meets all required specifications.

Example 4: Analysis for liposome homogeneity The liposome formulation homogeneity was measured using a Malvern Nano zs particle sizer. In this method, the particle size uniformity of liposomal formulation vesicles is measured by dynamic light scattering (DLS) to examine the particle size distribution of particles suspended in a liquid medium. This technique can measure particle size distributions in the range of 10-1000 nm and is useful for particle groups such as liposome emulsions, nanoparticles, and synthetic polymers. This technique can be used to obtain an indication of the average diameter of the homogeneous liposome formulation as well as the heterogeneity of the formulation. After standardizing the results of measurement with a Malvern Nano-zs particle size analyzer according to the operation instructions, a 0.5 mg / ml liposome sample was first diluted 1/3 with PBS. The UV λmax 600 nm of the diluted solution was checked on an Ultraspec 2100pro UV / VIS spectrophotometer. The liposome sample was diluted again until the optical density (OD) value was 0.10 ± 0.02. Again, it was confirmed that the UV λmax 600 nm of the second diluted solution was 0.10 ± 0.02 in terms of the optical density. Next, 1.0 to 1.5 ml of the diluted sample was placed in a culture tube, and the particle size of the vesicle was measured using a Malvern Nano-zs particle size analyzer. The analysis is performed at ambient temperature (23 ° C. ± 2 ° C.) with a fixed angle of 173 ° and a laser wavelength of 633 nm. Data was accumulated until 150-500 Kcps (kilo counts / second).

  Table 6 shows the measurement results for the formulations prepared according to the invention. The average particle size of the liposome was 80.41 nm, and the PDI was 0.04.

  The homogeneity of the HU liposomes and the prior art liposomes of Example 2 above was analyzed according to the procedure described above. The HU liposome had a Z-average particle size of 176.5 nm.

  5A and 5B show the particle size distribution of liposomes and HU liposomes, respectively, made according to the present invention. FIG. 5A shows that the liposome formulation of the present invention has an average diameter of about 88 nm, and the particle diameter falls within a narrow range of 69 to 107 nm. In contrast, FIG. 5B shows that the HU liposome formulation has an average diameter of about 201 nm, and the liposome diameter ranges from a small diameter of 80 nm to a large diameter of 500 nm. The PDI of the formulation of FIG. 5A was 0.025. The PDI of the HU formulation of FIG. 5B was 0.118.

  In order to more fully describe the state of the art to which this invention pertains, the content of all published articles, books, reference manuals, and summaries cited herein are hereby incorporated by reference in their entirety. Shall be incorporated into the certificate.

  Since various changes can be made in the subject matter described above without departing from the scope and spirit of the invention, all the subject matter included in the above description, and defined in the claims, explain the present invention. , Should be interpreted as illustrative. Many modifications and variations can be made to the present invention in light of the above teachings.

[Field of the Invention]
The present invention relates to a formulation containing a novel liposomes, and liposomes.

Claims (29)

Use of liposomes for the manufacture of a pharmaceutical composition for the treatment or prevention of conditions including substantially endometriosis, ischemia-reperfusion injury, myocardial infarction, and restenosis,
The liposome has a lipid component and an encapsulated therapeutic agent, the mass ratio of therapeutic agent to lipid is about 1: 5 to 1: 8, and the lipid component is DSPC, DSPG, cholesterol at a molar ratio of about 3: 1. Use of the liposome, wherein the compression ratio of the liposome is less than 0.70 ml / g.   The use of the liposome according to claim 1, wherein the liposome is negatively charged.   The use of the liposome according to claim 1, wherein the liposome has an internal osmolarity of liposome in the range of 340 to 440 mOsm / kg.   The use of the liposome according to claim 1, wherein the liposome has a liposome internal conductivity in the range of 13.5 to 17.5 ms / cm.   The use of a liposome according to claim 1, wherein the therapeutic agent is a bisphosphonate.   The use of the liposome according to claim 5, wherein the concentration range of the bisphosphonate in the liposome is in the range of 0.5 to 5 mg / ml.   The liposome according to claim 1, wherein the internal pH of the liposome is in the range of 6.8 to 7.0.   The use of the liposome according to claim 1, wherein the condition is related to a stenting procedure. Substantially endometriosis, ischemia-reperfusion injury, myocardial infarction, and a prescription agent used for treating or preventing symptoms including restenosis,
The formulation contains a plurality of liposomes, the liposome has a lipid component and a therapeutic agent, the lipid component contains DSPC, DSPG, and cholesterol in a molar ratio of about 3: 1: 2, and the PDI of the formulation is 0,1. 075 or less.   The formulation according to claim 9, wherein the liposome is negatively charged.   The formulation according to claim 9, wherein the liposome has an liposome internal osmolarity of 340 to 440 mOsm / kg.   The formulation according to claim 9, wherein the liposome has a liposome internal conductivity in the range of 13.5 to 17.5 ms / cm.   The formulation according to claim 9, wherein the therapeutic agent is a bisphosphonate.   14. The formulation according to claim 13, wherein the concentration of bisphosphonate in the liposome is in the range of 0.5 to 5 mg / ml.   The formulation according to claim 9, wherein the internal pH of the liposome is in the range of 6.8 to 7.0.   The formulation according to claim 9, wherein the average particle size of the liposome is 80 ± 5 nm.   The formulation according to claim 9, wherein 96% or more of the therapeutic agent in the formulation is encapsulated.   10. The formulation of claim 9, wherein the weight ratio of therapeutic agent to lipid component is from about 1: 5 to 1: 8.   The formulation according to claim 9, wherein the compression ratio of the liposome is 0.70 ml / g or less.   The formulation according to claim 9, wherein PDI is 0.02 to 0.05.   10. The prescription of claim 9, wherein the condition is related to a stent placement procedure. Substantially endometriosis, ischemia-reperfusion injury, myocardial infarction, and a method of producing a formulation for use in the treatment or prevention of conditions including restenosis,
The formulation comprises a plurality of liposomes containing a certain amount of a lipid component and a therapeutic agent, wherein the lipid component contains DSPC, DSPG, and cholesterol in a molar ratio of about 3: 1: 2, and the mass ratio of the therapeutic agent to the lipid is About 1: 5 to 1: 8, wherein the production method is
(A) A solution containing a therapeutic agent is mixed with a lipid solution containing DSPC, DSPG, and cholesterol at a molar ratio of about 3: 1: 2, so that the mass ratio of the therapeutic agent to the lipid is about 1: 5 to 1: 8. Forming vesicles so that
(B) substantially extruding a vesicle by repeatedly extruding a single filter having a pore size of about 100 nm through a plurality of times;
(C) ultrafiltration of vesicles;
A method comprising:   23. The method of claim 22, further comprising the step of: (d) diluting the formulation with a phosphate buffer to a final therapeutic agent concentration of the formulation.   The method according to claim 22, wherein the extrusion in the step (b) is performed at a temperature in the range of 55C to 75C.   23. The method of claim 22, wherein the extruding in step (b) is performed at a pressure in the range of 60 to 90 psi.   The method according to claim 22, wherein the ultrafiltration is performed after repeating the extrusion of the step (b) 10 to 18 times.   23. The method according to claim 22, wherein the internal pH of the liposome of the formulation is in the range of 6.8 to 7.0.   23. The method of claim 22, wherein the formulation has a PDI of less than 0.075. 23. The method of claim 22, wherein the condition is related to a stenting procedure.