Liposomal doxorubicin preparations, methods for manufacturing liposomal doxorubicin preparations, and use of liposomal doxorubicin preparations as pharmaceuticals.

Abstract

To provide a liposomal doxorubicin formulation, a method for producing a liposomal doxorubicin formulation, and a liposomal doxorubicin formulation for use as a medicament, in particular for use in the treatment of cancer, uterine leiomyosarcoma and adnexal skin cancer.SOLUTION: The present invention provides a liposomal doxorubicin formulation, where the lipid bilayer of the liposomes comprises at least phosphatidylcholine, cholesterol, and polyethyleneglycol-lipid conjugate, where the encapsulated doxorubicin crystals have an average fiber width of 5-15 nm and / or an average fiber length of 15-40 nm.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] The present invention relates to a liposomal doxorubicin preparation, a method for producing a liposomal doxorubicin preparation, and a liposomal doxorubicin preparation for use as a drug.

Background Art

[0002] Liposomes are spherical vesicles having at least one lipid bilayer. The liposome may be a multilamellar liposome in which one vesicle contains one or more vesicles smaller than it. A liposome has an aqueous solution at the center, which is surrounded by a hydrophobic membrane in the form of a lipid bilayer.

[0003] For various drugs, particularly parenterally administered drugs, the use of liposomes for drug delivery has been proposed. Liposomes can release the administered drug in a controlled "depot" manner over a long period of time, and can reduce the side effects of the drug by limiting the blood concentration of the free drug. In addition, liposomes can change the distribution and uptake of drugs in tissues to be advantageous for treatment, and can further increase the convenience of therapy by reducing the number of drug administrations. For example, liposomes can directly transport the encapsulated active ingredient to disease sites including tumor cells and inflammatory sites. The active ingredient can be directly released from the liposome at the treatment site. As a result, the required dosage of the active ingredient can be reduced, and as a result, side effects are limited.

[0004] Liposomes can move the active ingredient to the site of action. Since the membrane of the liposome is similar in structure to the biological membrane, the liposome can integrate with the cell membrane. When integrated, the contents of the liposome flow out into the cell, where the active ingredient can act. By using liposomes as a drug carrier system, side effects associated with the administration of each active ingredient and related to the high systemic absorption of the active ingredient can be reduced. The active ingredient can be accumulated at the desired target site. The components of the liposomal bilayer can be metabolized in the liver and / or spleen.

[0005] The development of drug delivery systems for treating cancer is particularly important because many drugs used in cancer treatment are either cell proliferation inhibitors or cytotoxic. Preventing their release into healthy tissue is highly desirable.

[0006] Liposome compositions are used to deliver encapsulated therapeutic agents. For example, Doxil® (Caelyx® in Europe) is a PEGylated liposome formulation containing doxorubicin, used to treat cancers such as ovarian cancer. Weakly amphiphilic bases like doxorubicin can be packed into liposomes using ion gradients across the membrane (see, for example, Nichols et al. (1976) Biochim. Biophys. Acta 455:269-271; Cramer et al (1977) Biochemical and Biophysical Research Communications 75(2):295-301). This packing method is generally called remote packing and is typically ionized The liposomes are filled by adding a drug containing a sex amine group to a liposome suspension prepared to have an ionic gradient (often a pH gradient) that is low on the inside and high on the outside.

[0007] Regarding the processes governing drug release after these liposome-filled PLDs (PEGylated liposomal doxorubicin) have been extravasated into the interstitial fluid, little is known. These processes include the gradual loss of the ammonium / proton gradient that holds the drug, enzymatic degradation of the liposome's phospholipids by phospholipases, and / or scavenging. Endocytosis by macrophages, which act as drug release agents, is thought to potentially contribute to drug release (Barenholz, (2012) J Control Release. 160(2): 117-34).

[0008] Liposome-encapsulated doxorubicin has been proven effective in treating cancer. However, tumor accumulation, cytotoxicity, and tumor weight reduction efficiency could be further improved.

[0009] Liposomal-encapsulated doxorubicin has been shown to be less cardiotoxic than unencapsulated doxorubicin. However, liposomal-encapsulated doxorubicin, as is well known in the art, can cause severe side effects such as palmar-plantar erythrodysesthesia (PPE), commonly known as hand-foot syndrome (see, for example, Gabizon et al (1994) Cancer Research 54:987-992; Solomon et al. (2008) Clinical Lymphoma and melanoma 1:21-32). This can cause redness, tenderness, and peeling of the skin, which may lead to discomfort and even pain. In a clinical trial where 50 mg / m² was administered every four weeks, 50.6% of patients treated with Doxil® developed hand-foot syndrome. Due to this adverse event incidence, the available dose of Doxil® is limited compared to free doxorubicin in the same treatment regimen. Furthermore, liposomal-encapsulated doxorubicin, which is well known in the art, still causes hematological side effects such as neutropenia.

[0010] Several doxorubicin-filled liposomes are known from Hong et al. (Clin Cancer Res (1999) 5:3645-3652) and US9895313. However, these liposomes were obtained by different manufacturing processes, one by a combination of thin-film hydration and extrusion, and the other by mixing a lipid solution in a water-miscible organic solvent with an aqueous solution in a specially designed multi-port mixing chamber. Comparative tests conducted for liposome preparation according to Hong's instructions showed that the indicated particle size of 65–75 nm was not reproducible. Instead, a particle size of approximately 114 nm was obtained. Furthermore, neither disclosure describes the roundness of the liposomes, their size distribution, or the shape and size of the filled doxorubicin crystals. However, the instructions for the preparation of the studied liposomes indicate an ammonium sulfate concentration of 250 mM, which, according to the publication by Wei et al. (Wei et al. (2018) ACS Omega 3:2508-2517), suggests liposomes with a significant aspect ratio. According to the same publication, the minimum intraliposomal ammonium sulfate concentration required to facilitate stable nanocrystallization in PEGylated liposomal doxorubicin (PLD) is 200 mM, and PLD lacking such crystals or with insufficient crystallinity exhibits rapid and biphasic doxorubicin release.

[0011] Therefore, there is still a need for liposomal formulations for doxorubicin delivery that are chemically and physically stable and have improved tumor accumulation, cytotoxicity, and tumor weight reduction efficiency. Furthermore, there is still a need to reduce undesirable side effects such as PPE without compromising therapeutic efficacy. In addition, there is a need for highly efficient, reliable, and cost-effective methods for producing suitable liposomal formulations. [Overview of the project] [Problems that the invention aims to solve]

[0012] Therefore, an object of the present invention is to address these needs and provide an improved liposomal doxorubicin formulation for the treatment of cancer. Another object of the present invention is to provide a method for producing such a liposomal doxorubicin formulation and to provide the use of such liposomal doxorubicin as a pharmaceutical agent. [Means for solving the problem]

[0013] The above-mentioned problem concerns liposomal doxorubicin preparations having the characteristics described in the independent section, liposomal doxorubicin preparations This issue was resolved by the method for manufacturing liposomal doxorubicin preparations and the use of liposomal doxorubicin preparations as pharmaceuticals.

[0014] The present invention relates to a liposomal doxorubicin formulation, wherein the lipid bilayer of the liposome is • Phosphatidylcholine, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), ·cholesterol, • A polyethylene glycol-lipid conjugate, preferably comprising at least DSPE-PEG2000, where, Liposomes have an average diameter of 30-70 nm, preferably 40-65 nm, as measured by DLS, or The present invention relates to a liposomal doxorubicin formulation in which the liposomes have an average diameter of 20-50 nm, preferably 30-40 nm, as measured based on images obtained by Cryo-TEM.

[0015] "Liposomal doxorubicin preparations" refer to compositions containing doxorubicin encapsulated in liposomes. In particular, these preparations contain doxorubicin hydrochloride. Doxorubicin is an anthracycline poisomerase II inhibitor well known in the art for its use in treating cancer. Its chemical name is (8S,10S)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyl)oxy]-8-glycolyl-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12-naftacendione hydrochloride. Diseases that can be treated with liposomal doxorubicin preparations include certain cancers, including acute leukemia, breast cancer, Hodgkin's disease, non-Hodgkin's lymphoma, and sarcomas. Particularly preferred, this drug is intended for the treatment of metastatic breast cancer, advanced ovarian cancer, Kaposi's sarcoma, and multiple myeloma.

[0016] One of the main factors determining stability and the location and rate of drug release from liposomes is the composition of the liposomal lipid membrane. Other factors include the size and morphology of the liposomes in the composition, as well as the morphology of the doxorubicin crystals encapsulated within the liposomes.

[0017] For the purposes of the present invention, the phosphatidylcholine in the lipid bilayer may be any of DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, and DEPC, or a mixture thereof. Most preferably, it is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0018] Caelyx® and Doxil® are essentially based on fully hydrogenated soy phosphatidylcholine (HSPC). HSPC has the following structural formula.

[0019] [ka]

[0020] Where m and n are 14 or 16. Since it is obtained from a natural product (soybean), HSPC , compared with a fully synthetic molecule, has low structural homogeneity. Therefore, HSPC is not relatively suitable for high-density packing of fatty acid chains during arrangement in a lipid bilayer, and thus is not relatively suitable for the formation of liposomes of a desired size. The same applies to liposome preparations, because the amount of phospholipids in the lipid bilayer consists of a mixture of different phosphatidylcholines, for example, a mixture of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Therefore, for the purposes of the present invention, it is particularly preferred that at least 95% by weight, preferably at least 99% by weight, more preferably 100% by weight of the total amount of phosphatidylcholine (PC) used for the lipid bilayer is DSPC.

[0021] Liposomes containing cholesterol in addition to phosphatidylcholine have improved circulation life, pharmacokinetics, and therapeutic characteristics. These are biocompatible and biodegradable.

[0022] The polyethylene glycol-lipid conjugate is preferably methoxypolyethylene glycol (MPEG) (more specifically N-(carbonyl-methoxypolyethylene glycol 2000))-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt (MPEG2000-DSPE). It is known that modifying the membrane surface based on a polyethylene glycol (PEG) conjugate lipid improves blood circulation ability by, for example, avoiding liposome capture by phagocytes in the liver and spleen.

[0023] The liposomes in the liposomal doxorubicin preparation according to the present invention have an average diameter measured by dynamic light scattering DLS of 30 to 70 nm, preferably 40 to 65 nm, and / or the average diameter of the liposomes measured by cryo-TEM is 20 to 50 nm, preferably 30 to 40 nm.

[0024] "Measurement by dynamic light scattering (DLS)" means that DLS was performed on a sample with a lipid concentration of 20-30 mg / ml, diluted 19-fold in PBS or MQ H2O to reach the instrument's decay constant of approximately 6. DLS was measured using a Malvern Zetasizer Nano device at 25°C and a scattering angle of 0°. Instrument control and data analysis were performed using Malvern Zetasizer software (version 7.11). Particle size (hydrodynamic diameter) was determined using the following Stokes-Einstein equation.

[0025]

number

[0026] In the equation, k is Boltzmann's constant, T is absolute temperature, η is the viscosity of the dispersant, and D is the diffusion coefficient. The viscosity was determined using Zetasizer software and was 0.8872 cP. The refractive index of the dispersant was 1.330. D was obtained by fitting the autocorrelation function to an appropriate algorithm. Cumulant analysis is a simple method for obtaining the mean particle size (Z-ave) and polydispersity index (PDI) by analyzing the autocorrelation function produced by DLS experiments. The calculation is defined in ISO 13321 (1996) and ISO 22412 (2008). The primary result from the DLS experiment is the intensity distribution of particle size. The intensity distribution is naturally weighted according to the scattering intensity. The size distribution is shown with logarithmically spaced intervals. The results are shown as a plot of the relative intensity of scattered light by particles (Y-axis) against various size classes (X-axis). A transparent, disposable zeta cell with a path length of 10 mm was used for the measurements.

[0027] "Measurement based on images acquired by Cryo-TEM" means that the sample was subjected to cryogenic transmission electron microscopy. Liposome samples were appropriately diluted and vitrified and prepared on-grid (Formvar and carbon) at an acc voltage of 200kV. Images were acquired using a cryoTEM JEOL JEM-2100F and a TVIPS TemCam F415MP camera at magnifications of 20,000x, 40,000x, and 80,000x. Particle identification and size determination were performed by semi-automated image processing using Vironova Analyzer Software (Vironova, Sweden). Briefly, multiple images were imported randomly at the same magnification. Only liposome particles whose entire body was contained within the image boundary and whose membrane was clearly defined were detected. The identified objects were analyzed for sphere diameter, roundness, and unilamellarity. All images were processed in batches using the same threshold and settings, and 5 to 18 images corresponding to 1560 to 1178 analyte particles were accumulated for each sample. The standard deviation of the mean values ​​was approximately 10 nm.

[0028] While not always the case, the liposomes in the formulation according to the present invention are usually within the size range described above, regardless of the measurement method. The diameter size measured by cryo-TEM is generally smaller than the diameter size measured by DLS. There are other factors involved, but in particular, this is due to the fact that PEG chains are not visible in cryo-TEM images and that the hydrodynamic radius affects DLS but not cryo-TEM imaging.

[0029] Compared to prior art disclosures, it has been found that the liposomal doxorubicin formulations disclosed herein remarkably form stable and highly uniform doxorubicin crystalline fibers even at significantly lower intraliposomal ammonium sulfate concentrations than the former.

[0030] While not bound by theory, it is currently believed that higher ammonium sulfate concentrations allow for the packing of larger amounts of doxorubicin, resulting in larger doxorubicin crystals within the liposomes. These doxorubicin crystals, due to their size and rigidity, expand within the liposomes, ultimately causing the liposome's shape to deviate significantly from a perfect sphere. Furthermore, the varying expansion of the particles results in the appearance of long and short axes, which can be observed, for example, in cryo-TEM images of such PEGylated liposomal doxorubicin particles. Needless to say, such particles do not exhibit high homogeneity, i.e., a narrow particle size distribution and / or high circularity. However, these characteristics positively impact the pharmacokinetic and adverse event profiles of the liposomal doxorubicin formulations disclosed herein, as will be described in further detail below.

[0031] The liposomes according to the present invention, having the composition and size described above, were shown to be more stable than those known in the art. Liposomes with such small diameters are opsonized more slowly and to a lesser degree compared to larger ones, and are also removed more slowly by the reticuloendothelial system. Furthermore, the larger the liposome, the higher the likelihood of it fusing or interacting with other liposomes or particles.

[0032] As a result, the liposomal doxorubicin formulation according to the present invention was demonstrated to be more effective in preventing tumor growth. More doxorubicin accumulates in the tumor and is more easily accumulated and removed in the liver. Furthermore, cytotoxicity in in vitro assays is also increased. The liposomal doxorubicin formulation according to the present invention is Doxil® Compared to the Caelyx® formulation, it exhibits less serum leakage but good cellular uptake. The serum half-life in humans is significantly higher than that of liposomal doxorubicin formulations known in the art. This formulation provides a higher drug exposition at a given dose in the relevant target region. By using the formulation according to the present invention instead of Doxil® and Caelyx®, adverse effects such as PPE and neutropenia can be significantly reduced.

[0033] The advantages, such as improved efficacy and reduced adverse effects, are described in the following examples.

[0034] Preferably, in the liposomal doxorubicin formulations described above, the lipid bilayer consists essentially of synthetic phosphatidylcholine, preferably a structurally homogeneous form of synthetic phosphatidylcholine, cholesterol, and DSPE-PEG. Particularly preferably, the lipid bilayer consists essentially of 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, and DSPE-PEG. In particular, at least 95% by weight, preferably at least 99% by weight, and more preferably 100% of the lipid bilayer consists solely of synthetic phosphatidylcholine, preferably a structurally homogeneous form of synthetic phosphatidylcholine, cholesterol, and DSPE-PEG. As described above, a homogeneous lipid bilayer composition is advantageous for high-density packing of fatty acid moieties. Therefore, a homogeneous lipid bilayer improves vesicle stability.

[0035] Preferably, the weight ratio of phosphatidylcholine to cholesterol in the lipid bilayer of liposomes in the formulation is 50:50 to 70:30, preferably 55:45 to 65:35, and more preferably 60:40.

[0036] Preferably, in a liposomal doxorubicin formulation, the liposomes have an average relative circularity of at least 0.99, as measured based on images obtained by Cryo-TEM. The 10th percentile is at least 0.98, Preferably, the 5th percentile is at least 0.98. More preferably, the 5th percentile is at least 0.98 and the 2nd percentile is at least 0.96.

[0037] The vesicle morphology of liposomes is determined as described above (see "Measurement based on images obtained by cryo-TEM"). Circularity is calculated using the following formula.

[0038]

number

[0039] A high average roundness value of liposomes in the formulation further supports vesicle stability and reduces side effects such as PPE and neutropenia associated with doxorubicin therapy. While not theoretically bound, an elliptical shape, combined with its size and lipid composition, could lead to premature release of the drug at untarget locations. Such elliptical shapes are characteristic of the approved drugs Caelyx® and Doxil®. be.

[0040] In a preferred embodiment, the liposomal doxorubicin formulation has a polydispersity index of 0.15 or less, preferably 0.10 or less, and more preferably 0.09 or less, as measured by DLS. Therefore, such liposomes are essentially monodisperse. The measurement is performed as described above (see "Measurement by Dynamic Light Scattering"). A polydispersity index of 0.15 or less is superior to the polydispersity indices of liposomal formulations known in the art. Liposomal formulations obtained by processes known in the art, such as extrusion, homogenization, and sonication, typically have a polydispersity index of 0.2 to 0.4 (Gim Ming Ong et al., Evaluation of Extrusion Technique for Nanosizing Liposomes, Pharmaceutics 2016 (8) 36, p. 5). Liposome formulations, being inherently monodisperse, are beneficial for reproducibility purposes and industrial-scale manufacturing and comply with marketing authorization standards.

[0041] Preferably, the liposomes are unilamellar and have one internal compartment. Preferably, at least 90%, more preferably at least 97%, of the liposomes in the liposome formulation according to the present invention are unilamellar. The homogeneous size, circular shape, and unilamellar shape of the liposome dispersion allow for a controlled and industrially scalable manufacturing process.

[0042] In the formulations described above, the polyethylene glycol-lipid conjugate, preferably DSPE-PEG, may be located essentially only on the outer layer of the lipid bilayer.

[0043] In the context of the present invention, "essentially only on the outer layer" means that the amount of polyethylene glycol-lipid conjugate, preferably DSPE-PEG, in the inner layer of the lipid bilayer of the liposome in the formulation is less than 0.1 mol%, preferably even less than 0.01 mol%, and more preferably 0.0 mol%. The essential absence of polyethylene glycol-lipid conjugate can be confirmed by applying the post-modification PEGylation method described below.

[0044] PEG-lipids located on the inner surface of liposomes are ineffective, unnecessarily increasing the size of liposomes, and their hydrolysates can increase membrane permeability.

[0045] Preferably, the relative amount of polyethylene glycol-lipid conjugate, preferably DPE-PEG, in the lipid bilayer is at least 2 mol%, preferably at least 3 mol%, and more preferably 4 mol% to 6 mol%. Because the inner layer is essentially free of polyethylene glycol-lipid conjugate, the relative amount of polyethylene glycol-lipid conjugate, preferably DSPE-PEG, on the outer layer of the liposomes in the formulation is up to 12 mol%. A higher relative amount of polyethylene glycol-lipid conjugate in the outer surface has been shown to be effective in further improving the blood circulation stability and biocompatibility of the vesicles. Surface-modified liposomes are known to be less easily metabolized or removed.

[0046] Notably, liposomal doxorubicin formulations containing a relatively large amount of polyethylene glycol-lipid conjugate in the lipid bilayer, where the polyethylene glycol-lipid conjugate, preferably DSPE-PEG, is essentially located only on the outer layer of the lipid bilayer, are an advantageous concept in themselves, independently of the size and morphology of the liposomes. Therefore, this embodiment can be applied in combination with the above-described features, or even on its own.

[0047] Therefore, one aspect of the present invention is a liposomal doxorubicin formulation, wherein liposome The lipid bilayer, • Phosphatidylcholine, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), ·cholesterol, • A polyethylene glycol-lipid conjugate, preferably comprising at least DSPE-PEG2000, where, This invention relates to a liposomal doxorubicin formulation in which the relative amount of polyethylene glycol-lipid conjugate in the lipid bilayer is at least 2 mol%, preferably at least 3 mol%, more preferably 4 mol% to 6 mol%, and the polyethylene glycol-lipid conjugate is essentially located only on the outer layer of the lipid bilayer.

[0048] Furthermore, as described in the paragraphs above, liposomal doxorubicin formulations are also preferred, which contain a relatively large amount of polyethylene glycol-lipid conjugate in the lipid bilayer, essentially only on the outer layer of the lipid bilayer, and which also have any other features or combinations of advantageous features described herein, in particular features relating to the size and / or morphology of liposomes.

[0049] In a preferred embodiment of the present invention, the average diameter of the liposomes in the liposomal doxorubicin formulation according to the present invention, after being stored for 6 months, preferably 12 months, after production, as measured by dynamic light scattering, is 30 to 70 nm, preferably 40 to 65 nm, and / or, the average diameter of the liposomes, as measured based on images obtained by cryo-TEM, is 20 to 50 nm, preferably 30 to 40 nm.

[0050] Particularly preferably, the average diameter of liposomes in the formulation 6 months, preferably 12 months, after manufacturing is essentially the same as the average diameter of liposomes in the formulation immediately after manufacturing. The diameter variation measured by DLS does not exceed ±5 nm, preferably ±2 nm, and particularly preferably ±1 nm, for 12 months after manufacturing. The polydispersity index variation measured by DLS does not exceed ±0.05, preferably ±0.02, and particularly preferably ±0.01.

[0051] Therefore, the liposomes according to the present invention are particularly stable. The controllability and long-term persistence of the liposome size are beneficial for purposes such as manufacturing, storage, shelf life, and patient safety.

[0052] The formulation may have a drug-to-total lipid weight ratio of 0.01 to 0.10, preferably 0.03 to 0.07. For comparison, Doxil® has a total lipid content of approximately 16 mg / mL and a doxorubicin concentration of 2 mg / mL. As a result, the drug-to-lipid ratio of Doxil® is 0.138:1 (weight:weight). A lower drug-to-lipid ratio is advantageous in that it allows for the production of smaller and more spherical liposomes. Such a morphology also contributes to the reduction of adverse effects. In addition, as shown in the examples, the patient's drug exposure at a given dose is maintained or even increased.

[0053] Preferably, the encapsulated doxorubicin crystals have an average length of 15–40 nm, preferably 18–37 nm, more preferably 25–35 nm, and / or an average crystal width of 5–15 nm, preferably 6–12 nm, more preferably 7–11 nm. The crystal length and width are measured manually from a series of high-magnification images obtained by cryo-TEM. These dimensions are smaller than those of similar compositions well known in the art.

[0054] More preferably, the encapsulated doxorubicin has an average number of fibers per liposome of 1 to 6, preferably 2 to 5, more preferably 3 to 4. The number of fibers is Cry The number of individual fibers (high-density nodes) per crystal was determined manually from a series of high-magnification images obtained by o-TEM. It was possible to derive the number of individual fibers per crystal. Notably, doxorubicin crystals have a helical conformation, and the number of individual fibers per rotation can vary. To obtain an accurate presentation, one measurement was taken for each rotation of the doxorubicin crystal. This value is smaller than that of similar compositions well known in the art.

[0055] The small crystal size and low number of fibers per crystal are advantageous in that liposomes can be manufactured to be smaller and more spherical. Such a morphology also contributes to the reduction of adverse effects. In addition, as shown in the examples, the patient's drug exposure at a given dose is maintained or even increased.

[0056] Notably, liposomal doxorubicin formulations, in which the width and length of the doxorubicin crystal fibers encapsulated within the liposomes are relatively small, are an advantageous concept in themselves, independently of the size and morphology of the liposomes.

[0057] Therefore, one aspect of the present invention, which can be applied in combination with the above-described content but can also be applied by itself, is a liposomal doxorubicin preparation in which the lipid bilayer of the liposome is • Phosphatidylcholine, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), ·cholesterol, • A polyethylene glycol-lipid conjugate, preferably comprising at least DSPE-PEG2000, where, The present invention relates to a liposomal doxorubicin formulation in which the average fiber width of the encapsulated doxorubicin crystals is 5 to 15 nm, preferably 6 to 12 nm, more preferably 7 to 11 nm, and / or the average fiber length is 15 to 40 nm, preferably 18 to 37 nm, more preferably 25 to 35 nm.

[0058] Furthermore, as described in the paragraphs above, liposomal doxorubicin formulations are also preferred, having relatively small fiber width and length, and further possessing any other features or combinations of advantageous features described herein, in particular features relating to the size and / or morphology of liposomes.

[0059] Preferably, the formulation is dispersed in HEPES buffer at a concentration of 10 mM, resulting in a pH of 6.8. This solution may contain 0.9% NaCl. Liposome formulations known in the art are dispersed in buffers based on histidine and sugars (myocet lactose, doxil sucrose). In this embodiment, the formulation can be sucrose-free, thereby reducing the risk of bioburden. Furthermore, sterile filtration after manufacturing is possible. As described below, this embodiment promotes cost-effective manufacturing.

[0060] It may be possible to encapsulate active ingredients other than doxorubicin in the formulation. For example, active ingredients that exhibit a synergistic effect upon release may be included or encapsulated. Alternatively, at least one active ingredient may be contained within the liposome bilayer, and at least one other active ingredient may be encapsulated within the same liposome. The term "active ingredient" may include pharmacologically active drugs and prodrugs. A prodrug is a drug or compound that is metabolized after administration to become a pharmacologically active drug.

[0061] The active ingredient may be selected from the group consisting of small or large organic or inorganic molecules, nucleic acids, nucleic acid analogs and derivatives, peptides, peptide mimes, proteins, antibodies and their antigen-binding fragments, monosaccharides, disaccharides, trisaccharides, oligosaccharides, lipids, glycosaminoglycans, extracts obtained from biomaterials, and any combination thereof.

[0062] Liposomes, whether filled or not, may themselves be active ingredients.

[0063] According to another aspect of the present invention, which can be applied in combination with the above-described information but can also be applied on its own, it is particularly advantageous to administer a liposomal docetaxel preparation together with a liposomal doxorubicin preparation. Docetaxel is a chemotherapeutic agent used in the treatment of cancer, particularly breast cancer, non-small cell lung cancer, prostate cancer, gastric adenocarcinoma, and head and neck cancer. Docetaxel is marketed under the name Taxotere, which contains docetaxel as a trihydrate in a solvent. The chemical name of docetaxel is [(1S,2S,3R,4S,7R,9S,10S,12R,15S)-4-acetyloxy-1,9,12-trihydroxy-15-[(2R,3S)-2-hydroxy-3-[(2-methylpropan-2-yl)oxycarbonylamino]-3-phenylpropanoyl]oxy-10,14,17,17-tetramethyl-11-oxo-6-oxatetracyclo[11.3.1.03,10.04,7]heptadeca-13-en-2-yl]benzoate.

[0064] In the art, it has been suggested that doxorubicin and docetaxel be administered in combination to treat certain types of cancer. However, the present inventors have now found that the combined administration of liposomal doxorubicin and liposomal docetaxel formulations has a positive effect. Furthermore, it has been found that this effect is even more pronounced when liposomal formulations are administered in which each liposome simultaneously contains these two drug substances. In such embodiments, each liposome encapsulates hydrophilic doxorubicin in its internal aqueous compartment, and docetaxel (which has hydrophobic properties) is located between the layers of the lipid bilayer.

[0065] Therefore, particularly preferably, The aqueous internal compartment contains doxorubicin, Liposomal doxorubicin preparations contain individual liposomes, each containing docetaxel, within a lipid bilayer.

[0066] Particularly preferred, the formulation consists of individual liposomes that essentially contain doxorubicin in an aqueous internal compartment and docetaxel in a lipid bilayer, i.e., the amount of liposomes having a combination of active substances is at least 80%, preferably at least 90%.

[0067] Preferably, liposomal doxorubicin formulations comprising individual liposomes containing doxorubicin in an aqueous internal compartment and docetaxel in a lipid bilayer have the advantageous properties described herein, namely size, roundness, bilayer composition, stability, surface modification, etc. Furthermore, such liposomal doxorubicin formulations are preferably used as pharmaceuticals, preferably as pharmaceuticals in the treatment of medical conditions as described herein. However, it should be noted that the combination of doxorubicin and docetaxel in individual liposomes is an advantageous concept in itself.

[0068] A further aspect of the present invention is a method for producing liposomes, preferably the liposomes described above. The method is: a) A process that provides phosphatidylcholine, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and cholesterol in an organic solvent. To what extent, b) A step of adding an aqueous liquid, c) A step of inducing liposome formation by sonication, d) Optionally, a step of separating liposomes by filtration, e) A step of modifying liposomes by PEGylation, f) The step of filling liposomes with doxorubicin, preferably by remote filling technology, Step c) is, The average diameter of the liposomes, as measured by dynamic light scattering, is 30-70 nm, preferably 40-65 nm, and / or The procedure is characterized by the fact that the average diameter of the liposomes, as measured by cryo-TEM, is 20-50 nm, preferably 30-40 nm.

[0069] This method yields liposomes with significantly improved stability, bioavailability, and reduced toxicity. Empty liposomes are produced using a gentle sonication process, where lipids naturally form liposomes. These liposomes remain stable for extended periods in their natural form without the addition of stabilizers. The size distribution of the liposomes was measured at various points in time and was found to be essentially constant. This small size was also found to be constant over time.

[0070] Preferably, the organic solvent used in step a) is selected from the group consisting of ethanol, methanol, chloroform, and mixtures thereof. Most preferably, a high-purity organic solvent is used, such as anhydrous ethanol or methanol with a purity of over 99.99%. More preferably, thin-film hydration is not required.

[0071] The lipids used have good solubility in these organic solvents. By using high-purity organic solvents, contamination of liposomes with impurities is avoided.

[0072] The aqueous liquid used in step b) can be selected from the group consisting of water, aqueous buffer, and aqueous glycine solution. Preferably, an aqueous buffer with physiological saline concentration, such as PBS (10 mM phosphate, pH 7.2-7.4, 0.9% NaCl), can be used. Aqueous buffers such as 150 mM ammonium sulfate, 150 nM calcium acetate, 150 mM magnesium acetate, 150 mM manganese acetate, 150 mM iron chloride, or 150 mM copper sulfate can also be used.

[0073] It is preferable to use an aqueous solution of ammonium sulfate, preferably 140-160 mM, and more preferably 150 mM. Within the above numerical range of concentrations, controlled remote filling in step f) is possible, and it is suitable for delivering liposomal doxorubicin formulations with a drug-to-total lipid weight ratio of 0.01-0.1, preferably 0.04-0.6. Physiological salt concentration can be provided to bring the inside of the liposome closer to the physiological conditions in the body.

[0074] The ultrasonic treatment in step c) is preferably performed with an amplitude of at least 60 μm for at least 1 hour. The ultrasonic treatment may be performed for a maximum of 24 hours.

[0075] If separation step d) is performed, it may be carried out by centrifugation, filtration, field flow fractionation (FFF), dialysis, chromatography, preferably gel permeation chromatography.

[0076] Liposomes are formed from residual substances in a mixture, such as organic solvents, salts, and / or surfactants. They are separated.

[0077] The liposome distribution is preferably such that at least 95% are unilamellar, preferably at least 97%, and more preferably at least 98% are unilamellar. Preferably, the liposomes have an average relative circularity of at least 0.99 as measured by cryo-TEM, with the 10th percentile at at least 0.98, and preferably the 5th percentile at at least 0.98. Even more preferably, the 5th percentile is at least 0.98 and the 2nd percentile is at least 0.96. Both unilamellarity and circularity are measured as described above. In the liposome formulation according to the present invention, the ratio of spherical liposomes to broken particles and / or aggregates as measured by cryo-transmission electron microscopy is greater than 9:1 by weight.

[0078] In step e), the liposome dispersion is modified by PEGylation. "PEGylation" refers to polyethylene glycol modification, which is carried out as a post-modification method. Conventionally, phosphatidylcholine, cholesterol, and PEG-lipids are dissolved in a mixture to form crude liposomes, which are then reduced in size by extrusion. This method is called pre-modification. In contrast, when applying post-modification, empty liposomes composed of phosphatidylcholine and cholesterol are prepared in a solvent and then extruded through a suitable membrane. In a modified post-modification method, PEG-derivativeized phospholipids are added to a dilute suspension of pre-formed liposomes at a temperature close to the melting temperature of the liposome components. This technique is also called "post-insertion," and the insertion of PEG-derivativeized phospholipids is mainly driven by hydrophobic interactions between the membrane lipids and the hydrophobic sites of the PEG-derivativeized phospholipids. Preferably, PEGylation is carried out at 60°C to 70°C, preferably 65°C. This method is described in detail in Nakamura, K. (Comparative studies of polyethylene glycol-modified liposomes prepared using different PEG-modification methods; Biochim Biophys Acta, 1818 (2012) 2801-2807). Preferably, DSPE-MPEG2000 is used. In another modification of the post-modification method, PEG modification of the liposome surface is performed by covalently bonding a PEG moiety containing a reactive group (which can react with complementary reactive groups present on the liposome components). Various different methods for attaching moieties such as PEG to the surface of pre-formed liposomes are well known, and these include crosslinking of primary amines with glutaraldehyde, carbonyl-amine bond formation, amide bond formation by reaction of activated esters with primary amines, disulfide bond formation, thioester bond formation by maleimide-thiol addition reaction, and hydrazine bond formation.Furthermore, various biorthogonal approaches exist, often broadly categorized as "click" chemistry, which enable chemoselectivity, mild reaction conditions in aqueous media, and good yields with little to no by-products. Examples of click chemistry reactions used to modify liposome surfaces include copper(I)-catalyzed hysgene 1,3-dipolar cycloaddition (CuAAC), copper-free click reactions facilitated by ring distortion, Staudinger ligation, and tetrazine / trans-cyclooctene reverse electron-demanded Diels-Alder cycloaddition (IEDDA), details of which are described below: Nag, OK; Surface Engineering of Liposomes for Stealth Behavior; Pharmaceutics; 5 (2013) 542-569. In any modification method, the addition of PEG lipids is performed only after the liposome formation / minimization step, which is preferably carried out in aqueous solution. Therefore, PEGylated liposomes are obtained in which DSPE-PEG is essentially located only on the outer layer of the lipid bilayer.

[0079] This method has the advantage of producing small, homogeneous liposomes with an average diameter of less than 50 nm, high circularity, and a strong tendency towards stability. A more preferred embodiment of the present invention is the reduction of manufacturing costs and the number of manufacturing steps, thereby promoting mass production.

[0080] In step f), doxorubicin is filled into liposomes using remote filling technology. "Remote loading" refers to an approach for loading doxorubicin into the aqueous phase within liposomes. Remote loading is applied to pre-formed liposomes. In such liposomal formulations, an ammonium sulfate gradient is established across the membrane, with the concentration of [(NH4)2SO4] in the aqueous portion inside the liposome being significantly higher than that in the outer medium. This gradient acts as a driving force for moving amphiphilic weakly basic drugs such as doxorubicin to the opposite side of the liposomal bilayer (where crystalline precipitates form). This approach was first created by Barenholz. This is well known in the field (see: Barenholz, YC; Doxil(R) - The first FDA-approved nano-drug: Lessons learned; J. Control. Release 2012, 160, 117-134).

[0081] Preferably, the method does not include an extrusion step or a thin-film hydration step. "Extrusion" refers to a conventional liposome preparation technique in which a liposome formulation is passed through a membrane having a specified pore size. The extrusion process is considered the optimal method for liposome production in this field (Gim Ming Ong et al., Evaluation of Extrusion Technique for Nanosizing Liposomes, Pharmaceutics 2016 (8) 36; Perrie et al., Manufacturing Methods for Liposome Adjuvants, in; Vaccine Adjuvants: Methods and Protocols, Methods in Molecular Biology, vol. 1494, 2017). However, the extrusion process is costly and results in low vesicle morphology values, leading to lower liposome quality.

[0082] "Thin-film hydration" refers to a conventional liposome preparation method that involves creating a thin lipid film by removing an organic solvent, for example, in a round-bottom flask. Heterogeneous liposomes are formed by adding a dispersion medium and stirring. Typically, to obtain a more homogeneous formulation consisting of smaller liposomes, extrusion through a polycarbonate membrane is performed after thin-film hydration. Thin-film hydration is even more costly than extrusion.

[0083] The liposome formulation according to the present invention, manufactured by ultrasonic treatment, has been found to have lower polydispersity, be more stable, and be less prone to degradation than liposomes obtained by conventional techniques. Furthermore, these liposomes have a smaller diameter and higher circularity compared to those obtained by extrusion processes.

[0084] Preferably, one more step, step g), is performed following the steps of the method described above. Step g) includes sterilization by filtration.

[0085] "Sterilization by filtration" means passing the completed liposome formulation through a sterilization filter with a pore size of 0.22 μm or less, which is necessary to retain any impurities and bacterial cells that may be present.

[0086] Sterilization by filtration enables the production of liposomal doxorubicin formulations in accordance with GMP standards, meaning that the processes prior to sterilization filtration do not necessarily have to be carried out under sterile conditions. This is particularly cost-effective. However, in order to perform sterilization by filtration, it is a prerequisite that the formulation be dispersed in a storage buffer that does not contain sugars, such as dispersed in HEPES buffer.

[0087] The liposomal doxorubicin formulations described above may be used as agents specifically for the treatment of cancer, and more specifically for the treatment of solid tumors, metastatic breast cancer, advanced ovarian cancer, Kaposi's sarcoma, and multiple myeloma.

[0088] The liposomal doxorubicin preparations described above may be used, in particular, in the treatment of uterine leiomyosarcoma.

[0089] The liposomal doxorubicin preparations described above may be used, in particular, in the treatment of adnexal skin cancer.

[0090] Liposomal doxorubicin preparations can be administered intravenously in the treatment of cancer. For intravenous injection, the liposomes may be in a dissolved or suspended form. The amount of the formulation is 1 ml. 2 The amount may be in the range of 1 to 100 ml per (body surface) and is dose-dependent. The injection solution may contain further components such as stabilizers. It may also contain physiologically compatible components such as salts, especially sodium chloride, or alcohol, preferably ethanol.

[0091] A further aspect of the present invention is the liposome described above, which can be obtained by the method described above.

[0092] The present invention will be further illustrated by the following examples. These examples are not intended to limit the scope of the present invention. [Brief explanation of the drawing]

[0093] [Figure 1a] The morphology and size distribution of the liposomal doxorubicin formulation according to the present invention, as measured by cryo-TEM, are shown. [Figure 1b] The morphology and size distribution of the liposomal doxorubicin formulation according to the present invention, as measured by cryo-TEM, are shown. [Figure 2a] This shows the morphological and size distribution of Caelyx® formulations as measured by cryo-TEM. [Figure 2b] This shows the morphological and size distribution of Caelyx® formulations as measured by cryo-TEM. [Figure 3]The size distributions measured by DLS for different embodiments of the liposomal doxorubicin formulation according to the present invention are shown in comparison with the Caelyx® formulation. [Figure 4a] The circularity distribution of the liposomal doxorubicin formulation (4a) according to the present invention, as measured by cryo-TEM, is shown in comparison with that of the Caelyx® formulation (4b). [Figure 4b] The circularity distribution of the liposomal doxorubicin formulation (4a) according to the present invention, as measured by cryo-TEM, is shown in comparison with that of the Caelyx® formulation (4b). [Figure 5a] The measured widths of doxorubicin crystals (5a, 5b) according to the present invention, as measured by cryo-TEM, are shown in comparison with those of the Caelyx® formulation (5c). [Figure 5b] The measured widths of doxorubicin crystals (5a, 5b) according to the present invention, as measured by cryo-TEM, are shown in comparison with those of the Caelyx® formulation (5c). [Figure 5c] The measured widths of doxorubicin crystals (5a, 5b) according to the present invention, as measured by cryo-TEM, are shown in comparison with those of the Caelyx® formulation (5c). [Figure 6a] The measured lengths of doxorubicin crystals (6a, 6b) according to the present invention, as measured by cryo-TEM, are shown in comparison with those of the Caelyx® formulation (5c). [Figure 6b] The measured lengths of doxorubicin crystals (6a, 6b) according to the present invention, as measured by cryo-TEM, are shown in comparison with those of the Caelyx® formulation (5c). [Figure 6c] The measured lengths of doxorubicin crystals (6a, 6b) according to the present invention, as measured by cryo-TEM, are shown in comparison with those of the Caelyx® formulation (5c). [Figure 7a]The number of doxorubicin fibers per liposome, measured based on cryo-TEM imaging, is shown by class. Figure 7a shows the doxorubicin formulation according to the present invention, and Figure 7b shows the Caelyx® formulation. [Figure 7b] The number of doxorubicin fibers per liposome, measured based on cryo-TEM imaging, is shown by class. Figure 7a shows the doxorubicin formulation according to the present invention, and Figure 7b shows the Caelyx® formulation. [Figure 8] For comparison, the accumulation of doxorubicin over time in the liver and tumors for three different formulations (CALYX, TLD-1, and free DXR) administered is shown. [Figure 9] This paper presents in vitro cytotoxicity studies based on MTS absorption for three doxorubicin preparations (CALYX, TLD-1, and free DXR) for comparison. [Figure 10a] This paper presents in vitro cytotoxicity studies based on luciferase luminescence for three doxorubicin preparations (CALYX, TLD-1, and free DXR) for comparison. [Figure 10b] This paper presents in vitro cytotoxicity studies based on luciferase luminescence for three doxorubicin preparations (CALYX, TLD-1, and free DXR) for comparison. [Figure 10c] This paper presents in vitro cytotoxicity studies based on luciferase luminescence for three doxorubicin preparations (CALYX, TLD-1, and free DXR) for comparison. [Figure 11] The results of a comparative study on tumor growth over time (MDA-MB231) under administration of the liposomal doxorubicin preparation of Example 1 are shown. [Figure 12a] The results of a comparative study on tumor growth (A2780) over time under administration of the liposomal doxorubicin preparation of Example 1 are shown. [Figure 12b] The results of a comparative study on tumor growth (A2780) over time under administration of the liposomal doxorubicin preparation of Example 1 are shown. [Figure 13] The results of a comparative study on tumor growth over time (4T1) under administration of the liposomal doxorubicin preparation of Example 1 are shown. [Figure 14] The results of in vivo survival studies in mice for three doxorubicin formulations for comparison, including the one used in Example 1, are shown. [Figure 15a] The results regarding size and polydispersity of the liposomal doxorubicin preparation of Example 1, measured over time (12 months), are shown below. [Figure 15b] The results regarding size and polydispersity of the liposomal doxorubicin preparation of Example 1, measured over time (12 months), are shown below. [Modes for carrying out the invention]

[0094] Example 1: Production of TLD-1 1,2-Distearoyl-sn-glycero-3-phosphocholine and cholesterol were provided in a weight ratio of 60:40 and dissolved in over 99.99% anhydrous ethanol. This solution was hydrated at 68°C in a 150 mM aqueous solution of ammonium sulfate in sterile water. The solution was sonicated at an amplitude of 60 μm for 24 hours to obtain crude liposomes. Next, an aqueous solution of PSPE-MPEG2000 was added to the liposome suspension and heated at 65°C for 30 minutes to obtain PEGylated liposomes with a desired PEG-lipid content of 5 mol% (corresponding to a PEG-lipid content of 10 mol% in the outer layer of the lipid bilayer). Doxorubicin HCl was packed into the liposomes by remote packing technology to achieve a DXR / total lipid weight ratio of 0.05. Unpacked DXR was removed by gravity precipitation and filtration. The liposome dispersion was washed by tangential flow filtration, followed by buffer exchange to achieve dispersion of liposomes in 10 mM HEPES buffer containing 0.9% by weight of NaCl.

[0095] The liposomal doxorubicin obtained as described in this embodiment may hereafter be referred to as "TLD," "TLD-1," or "Talidox."

[0096] Whenever free doxorubicin is used as a comparative formulation, it may be referred to as "Doxo," "DXR," "DX," or "doxorubicin" in the examples and figures.

[0097] Comparative Example I purchased commercially available Caelyx®. For cryo-TEM measurement, I diluted Caelyx® 10-fold in HEPES buffer (NaCl, pH 6.8).

[0098] Example 2: Size Measurement The liposome sizes obtained by the methods described above were measured using cryo-TEM and DLS, and the results were compared with corresponding measurements for commercially available Caelyx® formulations.

[0099] Figure 1a shows a representative image of the formulation obtained in Example 1 at high magnification (80,000x). Figure 1b shows the measured size distribution histogram. Figure 2a shows a representative image of the Caelyx® formulation used for comparison at high magnification (80,000x). Figure 2b shows the measured size distribution histogram.

[0100] CryoTEM measurements were performed as follows: Liposome samples from Example 1 and the Comparative Example were vitrified. Samples were prepared on-grid (Formvar and carbon) at an acc voltage of 200kV. Images were acquired using a cryoTEM JEOL JEM-2100F device and a TVIPS TemCam F415MP camera at a magnification of 40,000x. Particle identification and size determination were performed by semi-automatic image processing using Vironova Analyzer Software (Vironova, Sweden). Briefly, multiple images were imported randomly at the same magnification. Only liposome particles whose entire structure was contained within the image boundary and whose membranes were clearly defined were detected. The identified objects were analyzed for sphere diameter, roundness, and unilamellarity. All images were processed in batches with the same threshold and settings, and 5 to 18 images corresponding to 1560 to 1178 analyte particles were accumulated for each sample. The standard deviation of the mean was approximately 10 nm.

[0101] Figure 1b shows the size distribution of the liposome formulation of Example 1. Five images were analyzed. The number of particles analyzed was 1560. The average diameter was 35.61 nm, and the standard deviation was 7.42 nm. The smallest measured diameter was 24.81 nm, and the largest measured diameter was 103.35 nm. A homogeneity Z test yielded a score of 1.01 as a measure of sampling homogeneity, indicating that all images included in the analysis contained groups of particles with the same average size.

[0102] Figure 2b shows the size distribution of the comparative liposome formulation. Eighteen images were analyzed. A total of 1178 particles were analyzed. The average diameter was 70.26 nm, and the standard deviation was 13.41 nm. The smallest measured diameter was 32.52 nm, and the largest measured diameter was 159.09 nm. A homogeneity Z-test yielded a score of 3.15 on the sampling homogeneity scale, indicating that not all images included in the analysis contained groups of particles with the same average size.

[0103] Furthermore, cryoTEM analysis of the liposome formulation in Example 1 showed that the number of broken particles was less than 10%, and no particle aggregates or clusters were observed.

[0104] Figure 3 shows the sizes of the liposome formulations of Example 1 and the Comparative Example as measured by dynamic light scattering (DLS). Both samples were diluted 10-fold in PBS or MQ H2O and multiply by... Measurements were performed using a Levain zetasizing device at 25°C and a scattering angle of 0°. TLD-1 (Example 1) had an average diameter of 60.5 (±4.7 nm) nm and a polydispersity index of 0.084 ± 0.038. Caelyx® had an average diameter of 85.0 nm in DLS measurements. Notably, the values ​​measured by dynamic light scattering were slightly higher than those obtained by cryoTEM imaging, because the PEGylated surface is undetectable by cryoTEM but is included in DLS as part of the liposome's hydrodynamic radius.

[0105] Example 3: Measurement of circularity The roundness of the liposome formulations of Example 1 and the Comparative Example was measured by Cryo-TEM. The results for Example 1 are shown in Figure 4a, and the results for the Comparative Example are shown in Figure 4b. Sample preparation and measurement were carried out as previously described.

[0106] For Example 1, the mean circularity of the particles was 0.99, the relative standard error was 0.03%, and the mean standard deviation was 0.01. The 50th percentile was measured at 1.00, the 10th percentile at 0.98, the 5th percentile at 0.98, and the 2nd percentile at 0.96. A homogeneity Z-test yielded a score of 1.19 as a measure of sample homogeneity, indicating that all images included in the analysis contained groups of particles with the same mean size.

[0107] For the comparative example, the mean circularity of the particles was 0.99, the relative standard error was 0.06%, and the mean standard deviation was 0.02. The 50th percentile was measured at 1.00, the 10th percentile at 0.97, the 5th percentile at 0.95, and the 2nd percentile at 0.92. A homogeneity Z-test yielded a score of 6.10 as a measure of sample homogeneity, indicating that not all images included in the analysis contained groups of particles with the same mean size.

[0108] Therefore, commercially available Caelyx® has low roundness of liposomes in its formulation. For example, 10% of the liposomes in Caelyx® have a roundness of at best 0.97 or less.

[0109] Furthermore, cryoTEM measurements of the liposome formulation in Example 1 showed a filling rate (filling with doxorubicin) of at least 80% and a unilamellar rate of 98%.

[0110] Example 4: Crystal dimensions and number of fibers per crystal The dimensions of the liposome formulations of Example 1 and the Comparative Example were measured by Cryo-TEM. The width measurement results are shown in Figures 5a and 5b for Example 1 and in Figure 5c for the Comparative Example. The length measurement results are shown in Figures 6a and 6b for Example 1 and in Figure 6c for the Comparative Example. Sample preparation and measurement were carried out as previously described. The length and width of the crystals were measured manually from a series of high-magnification images obtained by Cryo-TEM.

[0111] For Example 1, the average crystal width was 9.57 nm with a standard deviation of 2.78 nm (140 measurements, 12 images analyzed), and the average crystal length was 27.36 nm with a standard deviation of 9.15 nm (289 measurements, 5 images analyzed). For the Comparative Example, the average crystal width was 17.45 nm with a standard deviation of 4.60 nm (60 measurements, 21 images analyzed), and the average crystal length was 47.77 nm with a standard deviation of 15.33 nm (105 measurements, 5 images analyzed).

[0112] The amount of fiber per liposome can be determined from a series of high-magnification images obtained by cryo-TEM. We were able to manually determine the number of individual fibers (high-density nodes) per liposome. Because doxorubicin crystals have a helical conformation and the number of individual fibers per rotation can vary, we took one measurement per rotation to obtain an accurate representation.

[0113] The class ratio for Example 1 is shown in Figure 7a. The class ratio for the comparative example is shown in Figure 7b. The x-axis represents the class number (1 to 12), which indicates the number of individual fibers in the doxorubicin crystal.

[0114] In Example 1, the most common conformation was a number of fibers per crystal of 3. No crystals with 1, 7, or more individual fibers were observed. The average spacing between individual fibers measured for all doxorubicin crystals included in the dataset was 2.6 nm.

[0115] In the comparative examples, the most common conformation was seven fibers per crystal. No crystals with one, two, three, twelve, or more individual fibers were observed. The average spacing between individual fibers measured for all doxorubicin crystals included in the dataset was 2.7 nm.

[0116] Therefore, commercially available Caelyx® has low roundness of liposomes in its formulation. For example, 10% of the liposomes in Caelyx® have a roundness of at best 0.97 or less.

[0117] Furthermore, cryoTEM measurements of the liposome formulation in Example 1 showed a filling rate (filling with doxorubicin) of at least 80% and a unilamellar rate of 98%.

[0118] Example 5: Tumor accumulation in mice The liposomal doxorubicin preparation of Example 1 ("TLD-1"), commercially available Caelyx® ("CAELYX"), and free doxorubicin (Adliblastin "Free Doxorubicin") were administered to mice (athymic nude Foxn1) at a dose of 3.5 mg / kg. nu The drug was administered to mice. After 4 or 16 hours, the mice were sacrificially killed to detect the total amount of doxorubicin by HPLC analysis.

[0119] Figure 8 shows the time course of doxorubicin accumulation in the liver and tumors for the three formulations administered for comparison. The bars represent the mean and standard deviation (n=3).

[0120] TLD-1 accumulation in tumors was approximately four times greater than that of free doxorubicin and twice as great as that of CAELYX®. The serum half-lives up to 16 hours were comparable for both CAELYX and TLD-1. However, TLD-1 showed higher efficiency in liver accumulation and clearance.

[0121] Example 6: In vitro cytotoxicity The in vitro cytotoxicity of TLD-1, CAELYX, and free doxorubicin ("DX") was measured in A2780 cells seeded at 10,000 cells / ml (100 ml / well) in 96-well plates. Cells were treated with different concentrations of doxorubicin 24 hours after seeding.

[0122] Figure 9 shows the results when cells were treated with concentrations of 10, 50, 100, 500, 1000, 5000, and 10,000 ng / ml. 72 hours after treatment, the MTS colorimetric analysis component ((3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethylmethyl Live cells were stained with TLD-1 (Ciphenyl)-2-(4-sulfophenyl)-2H-tetrazolium ("MTS"). After 3 hours, the absorbance was measured. As can be seen from the figure, at all concentrations added, the absorbance of the samples treated with TLD-1 was clearly lower than that of the samples treated with CAELYX. Therefore, the cytotoxicity of TLD-1 is higher and is more similar to that of free doxorubicin.

[0123] Figures 10a to 10c show the results when concentrations of 5, 50, 500, 5000, 25,000, and 50,000 ng / ml were added. Cells were treated in the presence of a luciferase substrate, and the luminescence of living cells was measured over time (6, 24, 48, and 73 hours after treatment). As can be seen from the chart, at 48 hours, the luminescence of samples treated with TLD-1 was significantly lower than that of samples treated with CAELYX. This effect became even more pronounced at 72 hours. Therefore, the cytotoxicity of TLD-1 is higher and more similar to that of free doxorubicin.

[0124] Serum Leakage Study: Experiments were conducted to investigate the extent to which TLD-1 and Caelyx® release free doxorubicin over time into RPMI (cell medium) + / -10% FCS medium. TLD-1 and CAELYX were incubated in RPMI medium + / -10% FBS at 37°C in a metal bead bath, shielded from light, for 72 hours. Free doxorubicin in the medium was then measured. Free doxorubicin and liposomal doxorubicin were detected at 478 nm by HPLC size exclusion chromatography (to avoid protein-derived background absorption). Because liposomal doxorubicin is conjugated and aggregated, its peak appears later than that of free doxorubicin. This was confirmed by comparing it with values ​​from a free DX (adlibrastin) control sample. A comparative analysis of the area under the peak curve was performed (liposomal doxo vs. free doxo). Experiments revealed that both TLD-1 and Caelyx remained stable after 72 hours of exposure at 37°C in the culture medium used in the in-vitro experiments. The percentage of free DX in solution was less than 4% in the TLD-1 incubation and less than 6% in the Caelyx® incubation. Generally, leakage of TLD-1 was less than leakage of Caelyx. This suggests that the high efficacy observed in the in-vitro cytotoxicity assay was due to increased cell uptake, not leakage of free doxorubicin into the culture medium.

[0125] Example 7: Tumor Growth The effects of placebo, TLD-1, and CAELYX on tumor growth in vivo were investigated by administering them to mice and measuring their effects on tumor size over time.

[0126] Figure 11 shows the results of this study. Mice (5 mice / formulation) injected with the MDA-MB231 cell line were regularly administered empty liposomes, free doxorubicin, Caelyx®, and TLD-1. Each arrow indicates an injection at a dose of 3.5 mg / kg body weight. The effect of TLD-1 (measured as an increase in tumor weight, μg) was already clearly better than that of Caelyx® after 3 days from the start of treatment and remained good over 26 days.

[0127] Figures 12a and 12b show the results of a similar alternative test setup. Mice injected with the A2780 cell line (5 mice / formulation) were regularly administered PBS, free doxorubicin, Caelyx®, and TLD-1. In Figure 12a, a formulation similar to that in Example 1 was administered, but unlike in Example 1, sonication was performed, resulting in an average liposome diameter of 86.78 nm and a polydispersity index of 0.117, as measured by DLS. The tests were conducted only under the specified conditions. In Figure 12b, the formulation from Example 1, i.e., liposomes with an average diameter of 64.87 nm and a polydispersity index of 0.168 as measured by DLS, were administered. Each arrow indicates an injection at a dose of 3.5 mg / kg body weight. During the tests presented in Figure 12a, the effect of TLD-1 with an out-of-specification average diameter (greater than 70 nm) on tumor growth was found to be inferior to that of treatment with Caelyx®. The effect of TLD-1 in accordance with specifications was already clearly better than that of Caelyx 19 days after the start of treatment, and remained better for a further 12 days (measured as an increase in tumor weight, μg).

[0128] Figure 13 shows the results of a similar alternative test setup. Mice implanted with 4T1 tumors (5 mice / product) were regularly administered saline, Caelyx®, and TLD. The effect of TLD-1 was clearly superior to that of Caelyx 12 days after the start of treatment (measured as an increase in tumor size, mm²). 3 ).

[0129] Figure 14 shows in-vivo survival studies of mice treated with physiological saline, Caelyx®, or TLD-1 (5 mice / formulation as in Example 1). 60% of the mice treated with TLD-1 survived until day 30, and 40% survived until day 34. In contrast, 100% of the mice treated with Caelyx® had already died by day 26.

[0130] Example 8: Other Research Further comparative studies were conducted on the liposomal doxorubicin formulation (TLD-1) of Example 1 and Caelyx®. These included adverse event studies and efficacy studies in animal models. For Example 1, in addition to adverse event studies, studies on serum half-life and area under the curve (AUC) in humans were also included.

[0131] Serum half-life studies have been conducted in human serum, and Caelyx has been recorded to have a half-life of 74 hours in human serum. Currently, the estimated serum half-life of TLD-1 in humans is approximately 100 hours, based on data from five patients. Furthermore, the area under the curve (AUC) of the serum half-life data for TLD-1 has been found to be larger than that of Caelyx® at the corresponding dose, which means that a higher drug exposure is achieved for a given dose. The drug exposure in patients treated with TLD-1 (30 mg / m2) was higher than that in patients treated with Caelyx® (37 mg / m2), despite the difference in dose.

[0132] Adverse effects studies have been conducted in rats. In toxicological studies conducted in rats, skin toxicity, particularly PPE (e.g., hand-foot syndrome), was investigated. Administration of TLD-1, even at a high concentration of 6 mg / kg (data from males and females were pooled together due to no statistically significant difference), did not promote skin toxicity, particularly PPE. Similarly, neutropenia was also investigated in toxicological studies conducted in rats (by neutrophil count). When TLD-1 was administered, neutrophil counts did not change significantly, even at a high concentration of 6 mg / kg (data from males and females were pooled together due to no statistically significant difference).

[0133] Example 9: Stability Results Figures 15a and 15b show the time-dependent size and polydispersity stability of the liposome formulation according to the present invention, as measured by DLS. The liposome formulation was obtained by the method described above (Example 1). The liposome formulation was stored in HEPES buffer at a pH of 6.5-6.8 and a temperature of 4°C. The size variation did not exceed ±1 nm over a 12-month period from preparation. The variation in the polydispersity index, as measured by DLS, did not exceed ±0.01.

[0134] Example 10: Results of the clinical trial A clinical study is currently underway, in which the liposomal doxorubicin formulation of Example 1 (TLD-1) has so far been used in 12 patients with advanced solid tumors (conducted by a Swiss group for Clinical Cancer Research, study number SAKK65 / 16). The study was designed as an open-label, single-arm, multicenter, first-in-human, phase 1 trial. The primary objective of this study was to identify the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) for TLD-1 in patients with advanced solid tumors. Further objectives of this study were to evaluate the safety, preliminary antitumor activity, and pharmacokinetics of TLD-1.

[0135] According to this interim report, TLD-1 was administered at a dose of 45 mg / m² every three weeks to patients with advanced, pre-treated solid tumors. 2 This dosage can be safely administered up to this upper limit. This dosage is higher than that of Caelyx®, the latter having an MTD of 50 mg / m² every four weeks. 2 Furthermore, the frequency and severity of undesirable side effects were lower with TLD-1 than with Caelyx®. Specifically (TLD-1 vs. Caelyx®), clinically insignificant nausea (<8.3% vs. 38.5%), vomiting (<8.3% vs. 24.3%), alopecia (0% vs. 13.4%), or cardiotoxicity were observed, while myelosuppression was rare and mild (8.3% vs. 25.6%). No unexpected toxicities were reported.

[0136] As a hypothesis (though not limited to this), the reason why fewer side effects were observed with TLD-1 compared to conventional doxorubicin liposomal formulations including Caelyx® is thought to be because the doxorubicin-filled liposomes administered to patients have a relatively small liposome size and high homogeneity, in particular because the doxorubicin crystalline fibers within the liposomes have remarkable circularity, low polydispersity, and high uniformity (length and width).

Claims

1. A liposomal doxorubicin preparation wherein the lipid bilayer of the liposome is Phosphatidylcholine, ·cholesterol, • Containing at least polyethylene glycol-lipid conjugate, wherein the encapsulated doxorubicin crystals are - The average fiber width is 5 to 15 nm, and / or, - A liposomal doxorubicin preparation with an average fiber length of 15-40 nm.

2. The liposomal doxorubicin preparation according to claim 1, wherein the lipid bilayer comprises synthetic phosphatidylcholine, cholesterol, and DSPE-PEG.

3. The liposomes have an average relative circularity of at least 0.99 as measured by Cryo-TEM. The 10th percentile is at least 0.98, where the degree of circularity is given by the following formula [Math 1] A liposomal doxorubicin preparation according to claim 1 or 2, calculated by...

4. The formulation according to any one of claims 1 to 3, wherein the polyethylene glycol-lipid conjugate is located only on the outer layer of the lipid bilayer.

5. The formulation according to any one of claims 1 to 4, wherein the relative amount of polyethylene glycol-lipid conjugate in the lipid bilayer is at least 2 mol%.

6. The formulation according to any one of claims 1 to 5, wherein the drug-to-total lipid weight ratio is 0.01 to 0.

10.

7. The formulation according to any one of claims 1 to 6, wherein the liposomes are dispersed in a HEPES buffer.

8. - The liposomes have an average diameter of 30-70 nm as measured by dynamic light scattering, and / or - The formulation according to any one of claims 1 to 7, wherein the liposomes have an average diameter of 20 to 50 nm, as measured based on images obtained by cryo-TEM.

9. A method for producing a liposomal doxorubicin preparation according to any one of claims 1 to 8, a) A step of providing phosphatidylcholine and cholesterol in an organic solvent, b) A step of adding an aqueous liquid, c) A step of inducing liposome formation by sonication, e) A step of modifying liposomes by PEGylation, f) The process includes filling the liposomes with doxorubicin, preferably by remote filling technology, where, The aqueous liquid added in step b) is ammonium sulfate in a concentration of 140 to 160 mM. Process f) is The enclosed doxorubicin crystals - The average fiber width is 5 to 15 nm, and / or, - The average fiber length is 15-40 nm. A method characterized by being carried out in such a manner.

10. The method according to claim 9, wherein the method does not include an extrusion step or a thin film hydration step.

11. The method according to claim 9 or 10, wherein step f) is followed by step g) sterilization by filtration.

12. A preparation according to any one of claims 1 to 8, for use as a pharmaceutical agent.

13. The preparation according to claim 12 for use as a drug in the treatment of uterine leiomyosarcoma.

14. The formulation according to claim 12 for use as a drug in the treatment of skin adnexal cancer.

15. A preparation according to any one of claims 1 to 8, for use in the treatment of a medical indication according to claims 13 to 14, wherein the treatment includes intravenous administration of the preparation.

16. The liposomal doxorubicin preparation according to any one of claims 1 to 8, wherein the polydispersity index measured by DLS is 0.15 or less.

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