Method for manufacturing liposomal drug formulations

Abstract

The present invention provides a method for mass production of liposomal drug formulations containing aminoglycosides, such as amikacin, having favorable lipid / drug characteristics. This method utilizes a specific relative flow rate ratio of lipid to drug streams to obtain liposomes having high aminoglycoside encapsulation efficiency. The obtained liposomal drug formulation advantageously comprises a total lipid-to-drug weight ratio of less than 1:1.

Description

Technology Field

[0001] Cross-references for related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 665,564 filed on May 2, 2018, the full text of which is incorporated herein by reference for all purposes. Background Technology

[0003] Liposomal drug formulations enable the targeting of active agents at specific disease sites and enhance their absorption. These formulations have been developed to treat various lung disorders, including those caused by lung infections, where their characteristics make them an ideal choice for the inhaled delivery of anti-infectives.

[0004] Amikacin, a single anti-infective in liposomal form, has been studied in adult patients in several clinical trials for the treatment of refractory non-tuberculosis mycobacterial (NTM) lung disease induced by Mycobacterium avium complex (MAC). In a recent Phase 3 study of amikacin liposomal inhalation suspension (ALIS), the addition of ALIS to guideline-based therapy (GBT) resulted in the elimination of evidence of MAC-induced NTM lung disease from sputum in 29% of patients by 6 months, compared to 9% of patients treated with GBT alone.

[0005] It is widely known that although liposomes containing a relatively high amikacin-to-lipid ratio have been manufactured on a bench scale (see, e.g., KR 10-1358660), scaling up this process to a commercial manufacturing scale to produce liposomal formulations in which parameters such as drug concentration, amount of lipid in the formulation, lipid-to-drug ratio, captured volume, drug leakage, viscosity, and particle size are consistently maintained within specifications for clinical and / or commercial use is not a routine issue.

[0006] The present invention addresses the need for a repeatable large-scale process for producing liposomes containing an aminoglycoside antibiotic, e.g., amikacin, having a high aminoglycoside-to-lipid weight ratio (and also, a low lipid-to-aminoglycoside weight ratio) and excellent encapsulation efficiency.

[0007] In one embodiment, the present invention provides a method for mass production of a liposomal aminoglycoside formulation comprising lipids and an aminoglycoside (e.g., amikacin) having a high aminoglycoside loading relative to the lipid concentration (i.e., a high relative weight ratio of aminoglycoside to lipid). In particular, the lipid-to-aminoglycoside (e.g., amikacin) weight ratio (also referred to as "L / D weight ratio") in the liposomal suspension prepared according to the method of the present invention is less than 1:1, e.g., about 0.5:1 to about 0.9:1, at the completion of the process. In one embodiment, the lipid-to-aminoglycoside weight ratio of the liposomal suspension prepared according to the method of the present invention is about 0.7:1 (lipid:aminoglycoside) at the completion of the manufacturing process.

[0008] In another aspect, the present invention relates to a method for mass production of a liposomal drug formulation comprising lipids and an aminoglycoside (e.g., amikacin), wherein the aminoglycoside is contained within the liposome with high encapsulation efficiency (e.g., at least about 40% encapsulation efficiency before washing to remove free aminoglycosides from the formulation).

[0009] In one embodiment, the method comprises mixing a first stream containing lipids with a second stream containing aminoglycosides to form a combined lipid-aminoglycoside stream. The combined lipid-aminoglycoside stream comprises an aminoglycoside encapsulated in liposomes, which, in one embodiment, is formed at the intersection of the lipid stream and the aminoglycoside stream. In a further embodiment, the lipid-aminoglycoside stream is mixed with an aqueous saline solution in a reaction vessel (e.g., see FIG. 1). In one embodiment, the aminoglycoside is present in an aqueous solution prior to the mixing step. In another embodiment, the lipid is present in an alcohol solution, e.g., an ethanol solution, prior to the mixing step. In a further embodiment, the lipid comprises phospholipids and cholesterol. In one embodiment, the relative flow rate ratio of the second stream containing aminoglycosides to the first stream containing lipids is about 1.5:1 (aminoglycoside stream:lipid stream) to about 2:1 (aminoglycoside stream:lipid stream). In additional embodiments, lipids include dipalmitoylphosphatidylcholine (DPPC) and cholesterol.

[0010] In one embodiment, the aqueous saline is added to the reaction vessel via a third stream. In a further embodiment, the third stream is added to the reaction vessel simultaneously with the combined lipid-aminoglycoside stream. In another embodiment, the third stream is added to the reaction vessel before the addition of the combined lipid-aminoglycoside stream to the reaction vessel. In another embodiment, the aqueous saline is present at approximately room temperature before entering the reaction vessel. In one embodiment, the aqueous saline is approximately 1.5% aqueous sodium chloride.

[0011] In one particular embodiment, the present invention provides a method for mass production of a liposomal drug formulation comprising a lipid and an aminoglycoside (e.g., amikacin), wherein, prior to a washing step, the aminoglycoside is encapsulated within or complexed with the liposome with an encapsulation efficiency of at least 40%. In one embodiment, after a washing step performed via tangential flow filtration, the weight ratio of lipid to aminoglycoside in the liposomal aminoglycoside formulation is less than 1:1, e.g., about 0.5:1 to about 0.9:1 (e.g., about 0.7:1). In one embodiment of this method, the aminoglycoside is amikacin. In further embodiments, the amikacin is present as amikacin sulfate.

[0012] In one embodiment, a first stream containing lipids is mixed with a second stream containing aminoglycosides to form a combined lipid-aminoglycoside stream (e.g., lipid-amikacin stream) containing liposomal aminoglycosides. In one embodiment, the liposomal aminoglycoside formulation is formed at the intersection of the two streams, i.e., at the time of forming the combined lipid-aminoglycoside stream. In a further embodiment, the flow rate of the first stream containing lipids is about 0.5 kg / min to about 1.5 kg / min, and the flow rate of the second stream containing aminoglycosides is about 1 kg / min to about 2 kg / min. In a further embodiment, the flow rate of the first stream containing lipids is about 3 kg / min to about 4 kg / min, and the flow rate of the second stream containing aminoglycosides is about 5 kg / min to about 7 kg / min. In another embodiment, the relative flow rate ratio of a second stream containing aminoglycosides to a first stream containing lipids is about 1.5:1 (aminoglycoside stream flow rate: lipid stream flow rate) to about 2:1 (aminoglycoside stream flow rate: lipid stream flow rate). In yet another embodiment, the lipids include dipalmitoylphosphatidylcholine (DPPC) and cholesterol.

[0013] In one embodiment, a method for mass production of a liposomal drug formulation comprises mixing a first stream containing lipids with a second stream containing aminoglycosides to form a combined lipid-aminoglycoside stream, and adding the combined lipid-aminoglycoside stream to a vessel containing aqueous saline. In one embodiment, the aqueous saline is added to a reaction vessel via a third stream (see, for example, FIG. 1).

[0014] In a further embodiment, when the flow rate of the first stream containing lipids is about 0.5 kg / min to about 1.5 kg / min and the flow rate of the second stream containing aminoglycosides is about 1 kg / min to about 2 kg / min, the flow rate of the third stream is about 1.0 L / min to about 1.5 L / min, including about 0.5 L / min to about 2.0 L / min, for example, about 1.0 L / min to about 2.0 L / min, for example, about 1.25 L / min. In another embodiment, when the flow rate of the first stream containing lipids is about 3 kg / min to about 4 kg / min and the flow rate of the second stream containing aminoglycosides is about 5 kg / min to about 7 kg / min, the flow rate of the third stream is about 4.5 L / min to about 5.5 L / min, including about 3 L / min to about 6 L / min, for example, about 4 L / min to about 6 L / min, for example, about 5 L / min.

[0015] Unless otherwise specified, the term “aminoglycoside” as used herein is intended to include aminoglycoside free bases and any pharmaceutically acceptable salts thereof. For example, the term “amikacin” is intended to include amikacin free bases and any pharmaceutically acceptable salts thereof (e.g., amikacin sulfate).

[0016] In one embodiment, a method for mass-producing a liposomal aminoglycoside (e.g., amikacin) formulation comprises mixing a first stream containing lipids, including phospholipids, with a second stream containing aminoglycosides (e.g., amikacin) to form a combined lipid-aminoglycoside stream. In a further embodiment, the lipid-aminoglycoside stream is mixed with an aqueous saline solution in a reaction vessel. In one embodiment, the phospholipid is phosphatidylcholine. In a further embodiment, the phosphatidylcholine is DPPC. In another embodiment, the lipid comprises phospholipids and sterols. In a further embodiment, the sterol is cholesterol. In one embodiment, the lipid comprises DPPC and cholesterol.

[0017] In one embodiment, a method for mass-producing a liposomal aminoglycoside formulation comprises mixing a first stream containing lipids with a second stream containing aminoglycosides, wherein the first stream is mixed with the second stream to form a combined lipid-aminoglycoside stream. The combined lipid-aminoglycoside stream comprises liposomal aminoglycosides. In one embodiment, the liposomal aminoglycoside is formed at the intersection of the two streams, for example, at the mixing of the first stream and the second stream. In a further embodiment, the combined lipid-aminoglycoside stream is added to a reaction vessel and mixed with an aqueous saline solution. In a further embodiment, the aminoglycoside stream and the lipid stream are each maintained at a temperature of about 30°C to about 50°C prior to mixing. In additional embodiments, the aminoglycoside stream and the lipid stream are each maintained at a temperature of about 35°C to about 45°C, for example, about 38°C to about 42°C, before mixing. In one embodiment, the combined lipid-aminoglycoside stream is cooled upon entering the reaction vessel. In another embodiment, the combined lipid-aminoglycoside stream is cooled in the reaction vessel by an aqueous saline solution. In one embodiment, the reaction vessel is maintained at a temperature of about 25°C to about 40°C, for example, about 27°C to about 35°C. In another embodiment, the reaction vessel is maintained at a temperature of about 30°C. In another embodiment, the reaction vessel is maintained at a temperature of about 33°C.

[0018] In another aspect of the present invention, a liposomal aminoglycoside formulation is prepared in bulk according to the method provided herein. In one embodiment, the concentration of the aminoglycoside (e.g., amikacin) present in the liposomal drug formulation prepared accordingly is about 50 g / L to about 100 g / L, including about 10 g / L or more, e.g., about 60 g / L to about 80 g / L and about 65 g / L to about 75 g / L (e.g., about 20 g / L, about 30 g / L, about 40 g / L, about 50 g / L, about 60 g / L, about 70 g / L, or about 80 g / L). In additional embodiments, the concentration of lipids present in the liposomal drug formulation prepared accordingly is about 10 g / L to about 100 g / L, including about 20 g / L to about 80 g / L and about 40 g / L to about 60 g / L (e.g., about 50 g / L). In other embodiments, the L / D ratio of the liposomal drug formulation prepared in bulk according to the method provided herein is less than 1:1, e.g., about 0.5:1 to about 0.8:1 (e.g., about 0.7:1).

[0019] In another embodiment, a liposomal drug formulation produced in bulk according to the method provided herein comprises liposomal particles having an average particle size (i.e., average diameter) of about 200 nm to about 500 nm, for example, about 200 nm to about 400 nm (for example, about 250 nm to about 350 nm). Brief explanation of the drawing

[0020] Fig. 1 This illustrates one embodiment of the present invention for preparing a liposomal aminoglycoside formulation. Fig. 2 This shows the effect of relative lipid / amikacin flow rate on the final L / D ratio of various liposomal amikacin formulations. Specific details for implementing the invention

[0021] In one embodiment, the invention described herein relates to a method for producing liposomal aminoglycoside formulations in bulk. In one embodiment, the method comprises mixing a first stream containing lipids (also referred to herein as the “lipid stream”) with a second stream containing an aminoglycoside such as amikacin (also referred to herein as the “drug stream”) to form a combined lipid-aminoglycoside stream, wherein the lipid-aminoglycoside stream is mixed with an aqueous saline solution in a reaction vessel. In some embodiments, the aqueous saline solution enters the reaction vessel through a third stream.

[0022] The mixing of lipid and drug streams is performed to create turbulence when forming a combined lipid-aminoglycoside stream. Turbulence is conveniently achieved using an appropriate T-shaped or Y-shaped injection module for "in-line" mixing of the lipid and drug streams.

[0023] The term “bulk” means the use of at least about 5 kg of aminoglycoside-based starting material in the drug stream (calculated as at least about 5 kg of aminoglycoside-based if a pharmaceutically acceptable salt is used). In one embodiment, about 5 kg to about 50 kg of aminoglycoside-based starting material, e.g., about 5 kg to about 35 kg of aminoglycoside-based starting material is used. In one embodiment, at least about 8 kg of aminoglycoside-based starting material is used. In another embodiment, at least about 30 kg of aminoglycoside-based starting material is used. In one embodiment, the aminoglycoside is amikacin (e.g., amikacin sulfate).

[0024] The aminoglycoside used in the method provided herein may exist as a pharmaceutically acceptable salt or as a free base. As provided above, in one embodiment, the aminoglycoside is amikacin, e.g., amikacin sulfate.

[0025] In other embodiments, the aminoglycoside is amikacin, aframycin, arbekacin, astromycin, capreomycin, dibekacin, pramycetin, gentamicin, hygromycin B, isepamycin, kanamycin, neomycin, netilmycin, paromomycin, rodesstreptomycin, ribostamycin, sisomycin, spectinomycin, streptomycin, tobramycin, verdamycin, or a combination thereof.

[0026] In another embodiment, the aminoglycoside is AC4437, amikacin, aframycin, arbekacin, astromycin, becanamycin, boholmycin, brulamycin, capreomycin, dibekacin, dactymycin, etymycin, pramycetin, gentamicin, H107, hygromycin, hygromycin B, inosamycin, K-4619, isepamycin, KA-5685, kanamycin, neomycin, netilmycin, paromomycin, plazomycin, ribostamycin, sisomycin, rhodestreptomycin, sorbistine, spectinomycin, sporarisin, streptomycin, tobramycin, verdamycin, vertilmycin, or a combination thereof.

[0027] "Pharmaceuticalally acceptable salts" include both acid addition salts and base addition salts. Pharmaceutically acceptable addition salts retain the biological effects and properties of free bases and are not biologically desired or otherwise desired, and are inorganic acids, e.g., non-limitingly, hydrochloric acid (HCl), hydrobromide, sulfuric acid, nitric acid, phosphoric acid, etc., and organic acids, e.g., non-limitingly, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphor acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactar acid, gentisic acid, glucoheptonic acid, Gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphate, glycolic acid, hipfuric acid, isobutyric acid, lactic acid (e.g., as lactate), lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muxic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, parmoic acid, propionic acid, pyroglutamic acid, pyruvate, salicylic acid, 4-aminosalicylic acid, sebactic acid, stearic acid, succinic acid, acetic acid (e.g., as acetate), tartaric acid, thiocyanate, p-toluenesulfonic acid, It refers to a salt formed of trifluoroacetic acid (TFA), undecylenic acid, etc. In one embodiment, the pharmaceutically acceptable salt is HCl, TFA, lactate, or acetate. In one embodiment, the pharmaceutically acceptable salt is a sulfate salt, for example, amikacin sulfate.

[0028] "Liposome aminoglycoside formulations" relate to lipid-aminoglycoside formulations in which lipids have the form of liposomes and aminoglycosides are encapsulated by a liposomal bilayer or complexed with a liposomal bilayer. A liposome is a completely closed lipid bilayer membrane containing a trapped aqueous volume. The liposome may be a monolayer vesicle (having a single membrane bilayer) or a multilayer vesicle (an onion-like structure characterized by a multilayer bilayer, each separated from the next layer by an aqueous layer), or a combination thereof. The bilayer consists of two lipid monolayers having a hydrophobic "tail" region and a hydrophilic "head" region. The structure of the membrane bilayer is such that the hydrophobic (non-polar) "tail" of the lipid monolayer is oriented toward the center of the bilayer and the hydrophilic "head" is oriented toward the aqueous phase.

[0029] In one embodiment, a lipid-aminoglycoside formulation is prepared through a method comprising a two-stream infusion process. In one embodiment, the method comprises mixing a first lipid stream with a second aminoglycoside stream in a T-shaped infusion module or a Y-shaped infusion module. As used herein, the terms “T-shaped infusion module” and “Y-shaped infusion module” refer to a T-shaped or Y-shaped chamber in which two or more streams are combined, for example, a lipid stream and a drug stream are combined to form a single lipid-aminoglycoside stream [see, for example, the diagram in FIG. 1]. It will be recognized that the infusion module will have an appropriate bore size for the desired ratio of lipid and drug streams used. Examples of appropriate bore sizes include, but are not limited to, 3 / 16" and 3 / 8".

[0030] In one embodiment, the first stream (lipid stream) comprises an alcohol (e.g., ethanol) lipid solution. In one embodiment, the second stream (aminoglycoside stream) comprises an aqueous aminoglycoside solution (e.g., amikacin aqueous solution). In one embodiment, the first stream and the second stream are mixed to form a combined lipid-aminoglycoside stream. In one embodiment, each of the first stream and the second stream enters an injection module, and the first stream and the second stream are mixed in the injection module. In a further embodiment, the combined lipid-aminoglycoside stream is discharged from the injection module and subsequently enters a reaction vessel (see FIG. 1).

[0031] In one embodiment, the combined lipid-aminoglycoside stream is mixed with an aqueous saline solution in the same reaction vessel into which the combined lipid-aminoglycoside stream is introduced after being discharged from the injection module, for example. In one embodiment, the aqueous saline solution comprises an aqueous solution of sodium chloride of about 0.5 to 2% (e.g., about 1.5%). In one embodiment, the saline solution is added to the reaction vessel before the combined lipid-aminoglycoside stream. In another embodiment, the saline solution is added to the reaction vessel simultaneously with or approximately simultaneously with the combined lipid-aminoglycoside stream. In a further embodiment, the saline solution is added to the reaction vessel via a third stream. Accordingly, in some embodiments, the lipid-aminoglycoside formulation is prepared by a method comprising a three-stream injection process. In some embodiments, the third stream is added to the reaction vessel separately from the combined lipid-aminoglycoside stream.

[0032] In one embodiment, the combined lipid-aminoglycoside stream comprises a liposomal aminoglycoside (e.g., amikacin), wherein the encapsulation efficiency of the aminoglycoside within the liposome (or complexed in the liposome) is at least about 40%. As used herein, “encapsulation efficiency” refers to the amount of aminoglycoside encapsulated or complexed in the liposome prior to a filtration step, e.g., tangential flow filtration of the liposomal aminoglycoside formulation to remove free aminoglycosides. For example, an encapsulation efficiency of about 40% to about 70% (e.g., about 45% to about 55%) can be achieved by mixing the lipid stream and the aminoglycoside (e.g., amikacin) stream according to the method of the present invention described herein.

[0033] In one embodiment, the aminoglycoside stream and the lipid stream are each maintained at a temperature of about 30°C to about 50°C before mixing the two streams. In a further embodiment, the aminoglycoside stream and the lipid stream are each maintained at a temperature of about 35°C to about 45°C, e.g., about 38°C to about 42°C, before mixing. In another embodiment, the combination of lipid and aminoglycoside solutions exhibits exothermic behavior. In one embodiment, the temperature of the combined lipid-aminoglycoside stream is about 40°C to 55°C. In a further embodiment, the temperature of the combined lipid-aminoglycoside stream is about 45°C to about 50°C. In another embodiment, the combined lipid-aminoglycoside stream is mixed with an aqueous saline solution in a reaction vessel, wherein the aqueous saline solution is maintained at room temperature before mixing with the combined lipid-aminoglycoside stream. In another embodiment, an aqueous saline solution is added to the reaction vessel via a third stream, wherein the third stream is maintained at room temperature before mixing with the combined lipid-aminoglycoside stream. In one embodiment, the combined lipid-aminoglycoside stream is cooled upon entering the reaction vessel. In another embodiment, the combined lipid-aminoglycoside stream is cooled by the aqueous saline solution in the reaction vessel. In another embodiment, the combined lipid-aminoglycoside stream is cooled upon entering the reaction vessel. In one embodiment, the reaction vessel is maintained at a temperature of about 25°C to about 40°C, for example, about 27°C to about 35°C. In another embodiment, the reaction vessel is maintained at a temperature of about 30°C. In another embodiment, the reaction vessel is maintained at a temperature of about 33°C.

[0034] In one embodiment, the lipid component of the liposomal drug formulation prepared by the method provided herein comprises an electrically net neutral lipid, a positively charged lipid, a negatively charged lipid, or a combination thereof. In another embodiment, the lipid component comprises an electrically net neutral lipid. In a further embodiment, the lipid component essentially comprises an electrically net neutral lipid. In yet another embodiment, the lipids are DPPC and cholesterol.

[0035] The lipids used in the preparation of the liposomal formulation of the present invention may be synthetic, semi-synthetic, or natural lipids comprising one or more of phospholipids, tocopherols, sterols, fatty acids, negatively charged lipids, and cationic lipids. In one embodiment, the lipid component consists of electrically neutral lipids, for example, sterols and phospholipids.

[0036] In one embodiment, at least one phospholipid is present in the liposomal drug formulation. In one embodiment, the phospholipid is phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidic acid (PA), soybean phosphatidylcholine (SPC), soybean phosphatidylglycerol (SPG), soybean phosphatidylserine (SPS), soybean phosphatidylinositol (SPI), soybean phosphatidylethanolamine (SPE), and soybean phosphatidic acid (SPA); Hydrogenated egg and soybean counterparts (e.g., hydrogenated egg phosphatidylcholine and hydrogenated soybean phosphatidylcholine), phospholipids consisting of ester bonds of fatty acids at positions 2 and 3 of glycerol containing different head groups at position 1 of glycerol and chains of 12 to 26 carbon atoms, including choline, glycerol, inositol, serine, and ethanolamine, as well as corresponding phosphatidic acids. In these fatty acids, the carbon chains may be saturated or unsaturated, and the phospholipids may consist of fatty acids of different chain lengths and different degrees of unsaturation.

[0037] In one embodiment, the lipid component of the liposomal drug formulation prepared by the method provided herein comprises phosphatidylcholine. For example, in one embodiment, the lipid component in the liposomal drug formulation comprises dipalmitoylphosphatidylcholine (DPPC). In one embodiment, the lipid component of the liposomal drug formulation comprises DPPC and sterols, for example, DPPC and cholesterol. Alternatively, the lipid essentially comprises DPPC and cholesterol, or consists of DPPC and cholesterol. In additional embodiments, DPPC and cholesterol have a molar ratio ranging from about 19:1 (DPPC:cholesterol) to about 1:1 (DPPC:cholesterol), or about 9:1 (DPPC:cholesterol) to about 1:1 (DPPC:cholesterol), or about 4:1 (DPPC:cholesterol) to about 1:1 (DPPC:cholesterol), or about 2:1 (DPPC:cholesterol) to about 1:1 (DPPC:cholesterol). In another embodiment, DPPC and cholesterol have a molar ratio of about 2:1 (DPPC:cholesterol).

[0038] Other examples of lipid components of liposomal drug formulations prepared by the method provided herein include, but are not limited to, dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), dioleylphosphatidylethanolamine (DOPE), mixed phospholipids, e.g., palmitoylstearoylphosphatidylcholine (PSPC), and single acylated phospholipids, e.g., mono-oleoylphosphatidylethanolamine (MOPE).

[0039] Examples of sterol compounds in liposomal drug formulations prepared by the method provided herein include, but are not limited to, cholesterol, esters of cholesterol including cholesterol semi-succinate, salts of cholesterol including cholesterol hydrogen sulfate and cholesterol sulfate, ergosterol, esters of ergosterol including ergosterol semi-succinate, salts of ergosterol including ergosterol hydrogen sulfate and ergosterol sulfate, lanosterol, esters of lanosterol including lanosterol semi-succinate, salts of lanosterol including lanosterol hydrogen sulfate and lanosterol sulfate, and tocopherol. Tocopherol includes tocopherol, esters of tocopherol including tocopherol semi-succinate, and salts of tocopherol including tocopherol hydrogen sulfate and tocopherol sulfate. The term "sterol compound" includes sterols, tocopherols, etc. Tocopherols and their water-soluble derivatives are used to form liposomes [see, for example, PCT Publication No. 87 / 02219].

[0040] In one embodiment, the concentration of lipids in the first stream is about 10 g / L to about 50 g / L, or about 10 g / L to about 30 g / L, or about 15 g / L to about 25 g / L. In one embodiment, the concentration of lipids in the first stream is about 17 g / L, about 18 g / L, about 19 g / L, about 20 g / L, about 21 g / L, about 22 g / L, about 23 g / L, about 24 g / L, or about 25 g / L. In one embodiment, the concentration of lipids in the first stream is about 20 g / L.

[0041] In one embodiment, the concentration of the aminoglycoside in the second stream (aminoglycoside stream) is about 10 g / L to about 100 g / L; or about 20 g / L to about 70 g / L; or about 30 g / L to about 60 g / L; or about 40 g / L to about 50 g / L. In one embodiment, the concentration of the drug in the second stream is about 41 g / L, about 42 g / L, about 43 g / L, about 44 g / L, about 45 g / L, about 46 g / L, about 47 g / L, about 48 g / L, about 49 g / L, or about 50 g / L. In one embodiment, the concentration of the aminoglycoside in the second stream is about 45 g / L. In a further embodiment, the aminoglycoside is amikacin.

[0042] In one embodiment of the present invention, the pH of the aminoglycoside stream is 6 to about 7, or about 6.5 to about 7.0. In a further embodiment, the pH of the aminoglycoside stream is about 6.7. The pH of the aminoglycoside stream can be adjusted to an appropriate pH using a suitable base, for example, an alkali or alkaline earth metal hydroxide, for example, sodium hydroxide.

[0043] In another embodiment, the aqueous saline solution comprises about 0.5% sodium chloride to about 3% sodium chloride, for example, about 0.75%, about 1.0%, about 1.25%, about 1.5%, about 1.75%, about 2.0%, or about 2.5% sodium chloride. In one embodiment, the aqueous saline solution comprises about 1.5% sodium chloride.

[0044] In one embodiment, the flow rate of the lipid stream is about 0.5 kg / min to about 1.5 kg / min, and the flow rate of the aminoglycoside stream is about 1 kg / min to about 2 kg / min. In a further embodiment, the flow rate of the lipid stream is about 3 kg / min to about 4 kg / min, and the flow rate of the drug stream is about 5 kg / min to about 7 kg / min. In another embodiment, the relative flow rate ratio of the aminoglycoside stream to the lipid stream is about 1.5:1 (aminoglycoside stream flow rate: lipid stream flow rate) to about 2:1 (aminoglycoside stream flow rate: lipid stream flow rate).

[0045] In one embodiment, when the flow rate of the lipid stream is about 0.5 kg / min to about 1.5 kg / min and the flow rate of the aminoglycoside stream is about 1 kg / min to about 2 kg / min, the flow rate of the third stream containing aqueous saline is about 0.5 L / min to about 2.0 L / min, for example, about 1.0 L / min to about 2.0 L / min, for example, about 1.0 L / min to about 1.5 L / min, and includes about 1.25 L / min. In another embodiment, when the flow rate of the lipid stream is about 3 kg / min to about 4 kg / min and the flow rate of the aminoglycoside stream is about 5 kg / min to about 7 kg / min, the flow rate of the third stream containing aqueous saline is about 3 L / min to about 6 L / min, for example, about 4 L / min to about 6 L / min, for example, about 4.5 L / min to about 5.5 L / min, and includes about 5 L / min.

[0046] In one embodiment of the present invention, both the lipid solution and the aminoglycoside (e.g., amikacin) solution are filtered, for example, through one or more (e.g., two in series) filters of about 0.2 μm before being mixed into a combined stream. FIG. 1 illustrates two filters in series, but it should be noted that this number may be changed according to the preference of the user of the method. For example, one to five filters may be used to initially filter the lipid stream and the aminoglycoside stream.

[0047] In another embodiment, an aqueous saline solution (e.g., 1.5% saline solution) is also filtered, for example, through one or more (e.g., two in series) filters of about 0.2 µm before mixing with the lipid-aminoglycoside combined stream in the reaction vessel. In a further embodiment, a liposomal suspension comprising liposomes formed at the intersection of the lipid stream and the aminoglycoside stream and / or in the combined lipid-aminoglycoside stream is concentrated within the reaction vessel using a recirculating filtration system, such as a volumetric filtration. As provided above, the term "encapsulation efficiency" as used herein refers to the amount of aminoglycoside encapsulated or complexed into liposomes before a filtration step to remove free aminoglycosides, for example, tangential flow filtration of the liposomal aminoglycoside formulation. For example, an encapsulation efficiency of about 40% to about 70% (e.g., about 45% to about 55%) can be achieved by mixing a lipid stream and an aminoglycoside (e.g., amikacin) stream according to the method of the present invention described herein.

[0048] In another embodiment, the obtained concentrated liposome suspension is treated (i.e., "washed") with additional aqueous saline (e.g., filtered 1.5% saline) and further filtered using a recirculating filtration system, such as volume filtration, until the liposome suspension contains an appropriate final aminoglycoside concentration and substantially all free aminoglycosides are removed. In a further embodiment, three or more washes (e.g., 3, 4, 5, or 6 washes) are performed to achieve an appropriate final aminoglycoside concentration.

[0049] In one embodiment, after washing, the concentration of aminoglycoside present in a liposomal aminoglycoside formulation prepared in bulk according to the method provided herein is about 10 g / L or more. In a further embodiment, the aminoglycoside is present in the formulation at a concentration of about 20 g / L or more. In a further embodiment, the aminoglycoside is present in the formulation at a concentration of about 30 g / L or more. In a further embodiment, the aminoglycoside is present in the formulation at a concentration of about 40 g / L or more. In a further embodiment, the aminoglycoside is present in the formulation at a concentration of about 50 g / L or more. In a further embodiment, the aminoglycoside is present in the formulation at a concentration of about 60 g / L or more. In a further embodiment, the aminoglycoside is present in the formulation at a concentration of about 70 g / L or more. In another embodiment, the aminoglycoside is present in the formulation at a concentration of about 10 g / L to about 100 g / L. In additional embodiments, the aminoglycoside is amikacin. In one embodiment, the aminoglycoside is present in the formulation at a concentration of about 50 g / L to about 100 g / L. In additional embodiments, the aminoglycoside is amikacin. In one embodiment, the aminoglycoside is present in the formulation at a concentration of about 60 g / L to about 80 g / L. In additional embodiments, the aminoglycoside is amikacin. In another embodiment, the aminoglycoside is present in the formulation at a concentration of about 65 g / L to about 80 g / L. In additional embodiments, the aminoglycoside is amikacin. In another embodiment, the aminoglycoside is present in the formulation at a concentration of about 65 g / L to about 75 g / L. In additional embodiments, the aminoglycoside is amikacin. In another embodiment, amikacin is present in the formulation at a concentration of about 70 g / L. In an additional embodiment, the aminoglycoside is amikacin.

[0050] In additional embodiments, after washing, the concentration of lipids present in the liposomal drug formulation prepared in bulk according to the method provided herein is about 10 g / L to about 100 g / L, including about 20 g / L to about 80 g / L and about 40 g / L to about 60 g / L (e.g., about 50 g / L).

[0051] In another embodiment, after volume filtration, the lipid-to-aminoglycoside weight ratio in a liposomal drug formulation prepared in bulk according to the method provided herein is less than 1:1, e.g., about 0.5:1 (lipid:aminoglycoside) to about 0.8:1 (lipid:aminoglycoside) (e.g., about 0.5:1 (lipid:aminoglycoside) or 0.6:1 (lipid:aminoglycoside) or 0.7:1 (lipid:aminoglycoside) or 0.8:1 (lipid:aminoglycoside)). In one embodiment, the lipid-to-aminoglycoside weight ratio is about 0.7:1 (lipid:aminoglycoside).

[0052] Liposomal aminoglycoside formulations prepared in bulk according to the method provided herein comprise liposomal particles having an average particle size (i.e., average diameter) of about 200 nm to about 500 nm, e.g., about 200 nm to about 400 nm (e.g., about 250 nm to about 350 nm). The liposomal diameter can be measured using commercially available light scattering techniques, for example, using quasi-elastic light scattering with a Nicomp™ 380 submicron particle sizer (Nicomp, Santa Barbara, California USA).

[0053] The present invention is further illustrated with reference to the following examples. However, it should be noted that such examples, as well as the specific embodiments and aspects described above, are illustrative and should not be interpreted in any way as limiting the scope of the invention.

[0054] Examples

[0055] Example 1: Manufacturing process and process control for liposomal amikacin

[0056] The preparation of liposomal amikacin sulfate was carried out using an aseptic process comprising preparing three sterile solution streams, mixing the lipid stream and the amikacin sulfate stream at an appropriate flow rate through a T-connector injection module, collecting the combined lipid-amikacin sulfate stream containing liposomes with encapsulated amikacin sulfate in a sterile volumetric filtration (reaction) vessel, adding a stream of 1.5% aqueous sodium chloride at an appropriate flow rate to the volumetric filtration vessel, and subsequently volumetric filtration (including washing) and concentration of the obtained liposomal dispersion to form the final product.

[0057] a) Solution preparation A sufficient amount of the following three solutions were prepared.

[0058] ● Amikacin sulfate solution: pH adjusted to 6.6 to 6.8 with amikacin sulfate and sodium hydroxide in water for injection (WFI).

[0059] ● Lipid solution: DPPC / cholesterol in ethanol (2:1 w / w).

[0060] ● 1.5% sodium chloride solution: 1.5% sodium chloride in WFI, pH adjusted to 6.6 to 6.8.

[0061] The solution must be used within 24 hours of preparation.

[0062] b) Injection / Initial Concentration The amikacin sulfate solution and lipid solution were heated and passed through a separate sterile filter before being passed through an in-line T-connector injection module at a controlled addition rate. The mixed stream was collected in a pre-sterile reactor vessel. At the same time, a 1.5% aqueous sodium chloride solution was passed through a sterile filter and introduced into the reactor vessel as a stream at an appropriate flow rate. At this stage, the solution can be sampled according to the level of amikacin encapsulation.

[0063] c) Jeong Yong-yeogwa Volume filtration was performed. This step serves to remove ethanol from the bulk solution and wash away any "untrapped" or free amikacin sulfate.

[0064] d) Final Concentration Using the results of the in-process test, the bulk solution was concentrated to an appropriate concentration level of amikacin sulfate. After the concentration was completed, a verification test for the amikacin sulfate concentration and L / D ratio can be performed.

[0065] Table 1 describes experiments (A) and (B) performed according to the general method of Example 1. In (A), a 3 / 8" T-connector injection module is used. In (B), a 3 / 16" T-connector injection module is used.

[0066]

[0067] In additional experiments, generally after the process of Example 1, the lipid and amikacin stream (flow) rates were varied, and the final concentrations of lipids and amikacin in the liposomal formulation were measured. The L / D ratio for each test was calculated, and the results were Fig. 2 The results were presented in [document]. The results provide guidelines for the optimal relative lipid / amikacin flow rate to achieve a desirable L / D ratio.

[0068] * * * * * * *

[0069] All literature, patents, patent applications, publications, product descriptions, and protocols cited throughout this application are incorporated herein by reference in their entirety for all purposes.

[0070] The embodiments exemplified and discussed in this specification are intended merely to teach those skilled in the art the best manner known to the inventors for making and using the invention. Modifications and changes to the embodiments described above are possible without departing from the invention, as recognized by those skilled in the art in the context of the above teaching. Accordingly, it is understood that within the claims and their equivalents, the invention may be practiced differently from as described in detail.

Claims

Claim 1 A method for mass-producing a liposomal amikacin sulfate formulation, wherein the liposomal amikacin sulfate formulation comprises amikacin sulfate encapsulated in lipids including dipalmitoylphosphatidylcholine (DPPC) and cholesterol, and (a) a step of mixing a first stream containing an alcoholic solution of said lipids with a second stream containing an aqueous solution of amikacin sulfate to form a combined lipid-amikacin sulfate stream, wherein the concentration of lipids in the first stream is 15 g / L to 25 g / L and the concentration of amikacin sulfate in the second stream is 40 g / L to 45 g / L, and the second stream contains an amount of amikacin sulfate equivalent to 5 kg to 50 kg of amikacin; (b) a step of mixing the lipid-amikacin sulfate stream of step (a) with an aqueous saline solution in a reaction vessel; and (c) a step of washing the product of step (b) comprising the liposomal amikacin sulfate formulation to remove unencapsulated amikacin sulfate and providing a washed liposomal amikacin sulfate formulation having a total ratio of lipid weight to amikacin equivalent weight of 0.5 (lipid) : 1 (amikacin) to 0.8 (lipid) : 1 (amikacin), wherein the relative flow rate ratio of the second stream to the first stream is 1.5:1 to 2:

1. Claim 2 A method according to claim 1, wherein the concentration of lipids in the first stream is 20 g / L. Claim 3 A method according to claim 1, wherein the concentration of amikacin sulfate in the second stream is 40 g / L, 41 g / L, 42 g / L, 43 g / L, 44 g / L, or 45 g / L. Claim 4 A method according to claim 1 in which an aqueous saline solution is added to a reaction vessel through a third stream. Claim 5 A method according to paragraph 4 in which the third stream is added to the reaction vessel simultaneously with the lipid-amikacin sulfate stream. Claim 6 A method according to any one of claims 1 to 5, wherein the flow rate of the first stream is 0.5 kg / min to 1.5 kg / min and the flow rate of the second stream is 1 kg / min to 2 kg / min. Claim 7 A method according to any one of claims 1 to 5, wherein the flow rate of the first stream is 3 kg / min to 4 kg / min and the flow rate of the second stream is 5 kg / min to 7 kg / min. Claim 8 A method according to any one of claims 1 to 5, wherein the aqueous saline solution is 0.5-2 weight% or 1.5 weight% sodium chloride aqueous solution. Claim 9 A method according to any one of claims 1 to 5, wherein a second stream comprising an aqueous solution of amikacin sulfate and a first stream comprising an alcoholic solution of lipids are each maintained at a temperature of 30°C to 50°C before mixing. Claim 10 A method according to any one of claims 1 to 5, wherein after step (b), liposomes are produced with an amikacin sulfate encapsulation efficiency of at least 40%. Claim 11 A method according to any one of claims 1 to 5, wherein the washed liposomal amikacin sulfate formulation has a total ratio of lipid weight to amikacin equivalent weight of 0.5 (lipid) : 1 (amikacin). Claim 12 A method according to any one of claims 1 to 5, wherein the washed liposomal amikacin sulfate formulation has a total ratio of lipid weight to amikacin equivalent weight of 0.6 (lipid) : 1 (amikacin). Claim 13 A method according to any one of claims 1 to 5, wherein the washed liposomal amikacin sulfate formulation has a total ratio of lipid weight to amikacin equivalent weight of 0.7 (lipid) : 1 (amikacin). Claim 14 A method according to any one of claims 1 to 5, wherein the washing step (c) is performed using a 1.5 wt% aqueous sodium chloride solution. Claim 15 A method according to claim 14, wherein the washing step (c) is repeated and the product is concentrated to provide a liposomal amikacin sulfate formulation having amikacin sulfate present at a concentration of 60 g / L to 80 g / L. Claim 16 In claim 15, a method in which amikacin sulfate is present at a concentration of 70 g / L in a liposomal amikacin sulfate formulation. Claim 17 In claim 15, a method in which lipids are present in a liposomal amikacin sulfate formulation at a concentration of 40 g / L to 60 g / L. Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete Claim 29 delete Claim 30 delete Claim 31 delete Claim 32 delete Claim 33 delete Claim 34 delete Claim 35 delete

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