Liposomal doxorubicin formulation, method for producing liposomal doxorubicin formulation, and use of liposomal doxorubicin formulation as medicament

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 liposomal doxorubicin formulations, methods for producing liposomal doxorubicin formulations, and liposomal doxorubicin formulations for use as medicaments. [Background technology]

[0002] Liposomes are spherical vesicles with at least one lipid bilayer. Liposomes may be multivesicular, with each vesicle containing one or more smaller vesicles. Liposomes have an aqueous solution in the center, which is surrounded by a hydrophobic membrane in the form of a lipid bilayer.

[0003] The use of liposomes for drug delivery has been proposed for a variety of drugs, especially those administered parenterally. Liposomes can provide controlled "depot" release of administered drugs over an extended period of time, and can reduce drug side effects by limiting the blood concentration of free drug. Liposomes can also alter the distribution and uptake of drugs in tissues in favor of treatment, and can increase the convenience of therapy by reducing the frequency of drug administration. For example, liposomes can transport encapsulated active ingredients directly to disease sites, including tumor cells and sites of inflammation. The active ingredients can be released directly from the liposomes at the treatment site. As a result, a lower dosage of the active ingredient is required, which in turn limits side effects.

[0004] Liposomes can transport active ingredients to the site of action. Liposome membranes are structurally similar to biological membranes, so liposomes can integrate with cell membranes. Once integrated, the contents of the liposomes are released into the cell, where the active ingredients can act. The use of liposomes as a drug carrier system can reduce the side effects associated with the administration of each active ingredient and related to the high systemic absorption of the active ingredients. The active ingredients can accumulate at the desired target site. The components of the liposome 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 of the drugs in cancer treatments are cytostatic or cytotoxic, and it is desirable to prevent their release into healthy tissue.

[0006] Liposomal compositions have been used to deliver encapsulated therapeutic agents. For example, Doxil® (Caelyx® in Europe) is a PEGylated liposomal formulation encapsulating doxorubicin used to treat cancers such as ovarian cancer. Weakly amphiphilic bases such as doxorubicin can be loaded into liposomes by utilizing ion gradients across the membrane (see, e.g., Nichols et al. (1976) Biochim. Biophys. Acta 455:269-271; Cramer et al (1977) Biochemical and Biophysical Research Communications 75(2):295-301). This loading method is commonly referred to as remote loading and typically involves the use of ionized bases. Drugs with reactive amine groups are used and loaded by adding them to a suspension of liposomes that have been prepared to have a low inside, high outside ion gradient (often a pH gradient).

[0007] After drug-loaded PLD (pegylated liposomal doxorubicin) is extravasated into the interstitial fluid by these liposomes, little is known about the processes governing drug release. These include gradual loss of the ammonium / proton gradient that holds the drug in place, enzymatic degradation by phospholipases of liposomal phospholipids, and / or scavenging. It is thought that endocytosis by macrophages, the primary receptor for drug release, may contribute to drug release (Barenholz, (2012) J Control Release. 160(2): 117-34).

[0008] Liposomal doxorubicin has been proven effective in the treatment of cancer. However, tumor accumulation, cytotoxicity, and tumor weight reduction efficiency can be further improved.

[0009] Liposomal doxorubicin has been shown to be less cardiotoxic than non-encapsulated doxorubicin. However, liposomal doxorubicin, as known in the art, causes severe side effects, such as palmar-plantar erythrodysesthesia (PPE), commonly known as hand-foot syndrome (see, e.g., Gabizon et al (1994) Cancer Research 54:987-992; Solomon et al. (2008) Clinical Lymphoma and melanoma 1:21-32). PPE The skin may become red, tender, and peeling, which may cause discomfort or even pain. In clinical trials administered at 50 mg / m every 4 weeks, 50.6% of patients treated with Doxil® developed hand-foot syndrome. Due to this side effect incidence, the administrable dose of Doxil® is limited compared to free doxorubicin in the same treatment regimen. Also, liposomal doxorubicin, which is known in the art, still causes hematological side effects such as neutropenia.

[0010] Some doxorubicin-loaded liposomes are known by Hong et al. (Clin Cancer Res (1999) 5:3645-3652) and US9895313. However, these liposomes were obtained by different manufacturing processes, namely, on the one hand, by a combination of thin film hydration and extrusion, and on the other hand, by mixing lipid solutions in water-miscible organic solvents with aqueous solutions in a specially designed multi-port mixing chamber. Comparative tests carried out for liposome preparation according to Hong's teachings showed that the indicated particle size of 65-75 nm was not reproducible. Instead, a particle size of about 114 nm was obtained. Also, neither disclosure describes the circularity of the liposomes, their size distribution, or the shape and size of the loaded doxorubicin crystals. However, the instructions for the preparation of the liposomes studied indicate an ammonium sulfate concentration of 250 mM, which suggests liposomes with a significant aspect ratio according to the publication by Wei et al. (2018) ACS Omega 3:2508-2517. According to the same publication, the minimum intraliposomal ammonium sulfate concentration required to support stable nanocrystallization in PEGylated liposomal doxorubicin (PLD) is 200 mM, and PLDs without such crystals or with poorly crystalline crystals show rapid and biphasic doxorubicin release.

[0011] Therefore, there is still a need for a liposomal formulation for doxorubicin delivery that is chemically and physically stable and has improved tumor accumulation, cytotoxicity and tumor weight reduction efficiency.There is also still a need to reduce undesirable side effects such as PPE without compromising therapeutic efficacy.In addition, there is also a need for a highly efficient, reliable and cost-effective method for producing suitable liposomal formulations. Summary of the Invention [Problem to be solved by the invention]

[0012] It is therefore an object of the present invention to address this need and provide improved liposomal doxorubicin formulations for the treatment of cancer. Another object of the present invention is to provide a method for the preparation of such liposomal doxorubicin formulations and to provide the use of such liposomal doxorubicin as a medicament. [Means for solving the problem]

[0013] The above-mentioned problems are solved by the liposomal doxorubicin formulation, liposomal doxorubicin, having the characteristics described in the independent claims. The problem is solved by a method for producing a liposomal doxorubicin formulation and the use of a liposomal doxorubicin formulation as a medicine.

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

[0015] "Liposomal doxorubicin formulation" refers to a composition that contains doxorubicin encapsulated in liposomes. In particular, the formulation contains doxorubicin hydrochloride. Doxorubicin is an anthracycline topoisomerase II inhibitor that is well known in the art for the treatment of 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-naphthacenedione hydrochloride. Diseases that can be treated with liposomal doxorubicin formulations are certain cancers, including acute leukemia, breast cancer, Hodgkin's disease, non-Hodgkin's lymphoma, and sarcoma. Particularly preferably, the medicament is intended for the treatment of metastatic breast cancer, advanced ovarian cancer, Kaposi's sarcoma, and multiple myeloma.

[0016] One of the main factors that determine the 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 shape of the liposomes in the composition, and the morphology of the doxorubicin crystals encapsulated in the liposomes.

[0017] For 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, and most preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0018] Caelyx® and Doxil® are essentially based on fully hydrogenated soy phosphatidylcholine HSPC, which has the following structural formula:

[0019] [ka]

[0020] In the formula, m and n are 14 or 16. Since it is obtained from a natural product (soybean), HSPC is HSPC has a lower structural homogeneity than fully synthetic molecules. Therefore, HSPC is less suitable for dense packing of fatty acid chains during arrangement in the lipid bilayer and therefore for forming liposomes of the desired size. The same applies to liposome formulations, since 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, it is particularly preferred for the purposes of the present invention 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. They 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 membrane surface modification based on polyethylene glycol (PEG)-conjugated lipid improves blood circulation capacity, for example by avoiding liposome capture by phagocytes in the liver and spleen.

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

[0024] "Measured by dynamic light scattering (DLS)" means that DLS was performed on samples with lipid concentrations of 20-30 mg / ml diluted 19-fold in PBS or MQ H2O to reach the instrument's attenuation 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 calculated using the Stokes-Einstein equation:

[0025]

number

[0026] where k is the Boltzmann constant, T is the absolute temperature, η is the dispersant viscosity, and D is the diffusion coefficient. The viscosity was determined using the Zetasizer software and was 0.8872 cP. The dispersant refractive index was 1.330. D was obtained by fitting the autocorrelation function to an appropriate algorithm. Cumulant analysis is a simple method to obtain the mean particle size (Z-ave) and polydispersity index (PDI) by analyzing the autocorrelation function generated by a DLS experiment. The calculations are defined in ISO 13321 (1996) and ISO 22412 (2008). The primary result from a DLS experiment is the intensity distribution of particle sizes. The intensity distribution is naturally weighted according to the scattering intensity. The size distribution is shown to be logarithmically spaced. The results are shown as plots of the relative intensity of light scattered by particles (Y-axis) against various size classes (X-axis). Measurements were made using a clear disposable Zeta cell with a 10 mm path length.

[0027] "Measured based on images acquired by Cryo-TEM" means that the samples were subjected to cryogenic transmission electron microscopy. Liposome samples were appropriately diluted, vitrified and prepared on-grid (Formvar and carbon) at an acc voltage of 200 kV. 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 that were entirely contained inside the image boundary and had a clear membrane were detected. Identified objects were analyzed for sphericity, circularity and unilamellarity. All images were processed collectively with the same threshold and settings, and 5–18 images were accumulated for each sample, corresponding to a number of particles analyzed ranging from 1560 to 1178. The standard deviation of the average was approximately 10 nm.

[0028] Typically, but not necessarily, liposomes in the formulations of the present invention will be within the size ranges set forth above, whether measured by any method. Diameter sizes measured by cryo-TEM are generally smaller than diameter sizes measured by DLS. Among other factors, this is due to the invisibility of PEG chains in cryo-TEM images and the hydrodynamic radius, which affects DLS but not cryo-TEM imaging.

[0029] In comparison with the disclosures from the prior art, it has surprisingly been found that the liposomal doxorubicin formulations disclosed herein result in the formation of stable and highly uniform doxorubicin crystal fibers already at significantly lower intraliposomal ammonium sulfate concentrations than the former.

[0030] Without being bound by theory, it is currently believed that higher ammonium sulfate concentrations allow for greater loading of doxorubicin, resulting in larger doxorubicin crystals within the liposomes. These doxorubicin crystals expand the interior of the liposome due to their size and rigidity, ultimately resulting in the liposomes' shape deviating significantly from a perfect sphere. The different particle expansions also result in the appearance of major and minor axes, which can be observed, for example, in cryo-transmission electron microscopy (cryo-TEM) images of such PEGylated liposomal doxorubicin particles. And, of course, such particles are not characterized by a high degree of homogeneity, i.e., narrow particle size distribution and / or high circularity. However, these characteristics have a positive impact on the pharmacokinetics and side effect profile of the liposomal doxorubicin formulations disclosed herein, as described in more detail below.

[0031] Liposomes according to the present invention, having the composition and size as described above, have been shown to be more stable than those known in the art. Liposomes with such small diameters are opsonized slower and less extensively than larger ones, and are removed more slowly by the reticuloendothelial system. Larger liposomes are also more likely to fuse or interact with other liposomes or particles.

[0032] As a result, the liposomal doxorubicin formulation of the present invention has been demonstrated to be more effective in preventing tumor growth; it accumulates more doxorubicin in the tumor, is more easily accumulated and cleared in the liver, and has higher cytotoxicity in in vitro assays. The liposomal doxorubicin formulation of the present invention is more effective than Doxil® and It has less serum leakage but better cellular uptake compared to Doxil® and Caelyx® formulations. Its serum half-life in humans is significantly higher than that of liposomal doxorubicin formulations known in the art. The formulation provides higher drug exposition at a given dose in the relevant target area. Adverse effects such as PPE and neutropenia can be significantly reduced by using the formulation of the present invention instead of Doxil® and Caelyx®.

[0033] The advantages of improved efficacy and reduced adverse effects are illustrated in the examples below.

[0034] Preferably, in the liposomal doxorubicin formulation as described above, the lipid bilayer is essentially composed of synthetic phosphatidylcholine, preferably synthetic phosphatidylcholine of structurally uniform type, cholesterol and DSPE-PEG. Particularly preferably, the lipid bilayer is essentially composed 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, more preferably 100% of the lipid bilayer is exclusively composed of synthetic phosphatidylcholine, preferably synthetic phosphatidylcholine of structurally uniform type, cholesterol and DSPE-PEG. As described above, homogeneous lipid bilayer composition is favorable for dense packing of fatty acid moieties. Thus, homogeneous lipid bilayer improves the stability of vesicles.

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

[0036] Preferably, in the 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 the liposomes is determined as described above (see "Measurement based on images obtained by cryo-TEM"). The circularity is calculated by the following formula:

[0038]

number

[0039] The high mean circularity value of liposomes in the formulation further supports the stability of the vesicles and reduces side effects such as PPE and neutropenia from doxorubicin therapy. Without being bound by theory, the ellipsoidal shape, combined with the size and lipid composition, may lead to premature release of the drug at non-targeted locations. Such an ellipsoidal shape is 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, more preferably 0.09 or less, as measured by DLS. Such liposomes are therefore essentially monodisperse. Measurement is performed as described above (see "Measured by Dynamic Light Scattering"). A polydispersity index of 0.15 or less is superior to the polydispersity index of liposomal formulations known in the art. Liposomal formulations obtained by the processes of extrusion, homogenization, and sonication known in the art typically have a polydispersity index of 0.2-0.4 (Gim Ming Ong et al., Evaluation of Extrusion Technique for Nanosizing Liposomes, Pharmaceutics 2016 (8) 36, p. 5). Liposomal formulations, being essentially monodisperse, are advantageous for reproducibility purposes and industrial-scale manufacturing, and comply with marketing approval criteria.

[0041] Preferably, the liposomes are unilamellar and have one internal compartment. The liposomes of the liposome formulation of the present invention are preferably at least 90%, more preferably at least 97% unilamellar. The homogeneous size, circularity and unilamellar nature of the liposome dispersion allows 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" refers to 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%, 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 interior surface of the liposome are ineffective, unnecessarily increase the size of the liposomes, and their hydrolysis products 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%, more preferably 4 mol% to 6 mol%. The inner layer is essentially free of polyethylene glycol-lipid conjugate, resulting in a relative amount of polyethylene glycol-lipid conjugate, preferably DSPE-PEG, on the outer layer of the liposome in the formulation of up to 12 mol%. To further improve the blood circulation stability and biocompatibility of the vesicle, a high relative amount of polyethylene glycol-lipid conjugate in the outer surface has been proven to be effective. It has been found that surface-modified liposomes are less likely to be metabolized or eliminated.

[0046] It is worth noting that the liposomal doxorubicin formulation, which contains a relatively large amount of polyethylene glycol-lipid conjugate in lipid bilayer, and the polyethylene glycol-lipid conjugate, preferably DSPE-PEG, is essentially located only on the outer layer of lipid bilayer, is an advantageous concept in itself, independent of the size and shape of liposome.Therefore, this embodiment can be applied in combination with the description above, but also by itself.

[0047] Thus, one aspect of the present invention is a liposomal doxorubicin formulation, comprising: The lipid bilayer of phosphatidylcholine, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), ·cholesterol, at least a polyethylene glycol-lipid conjugate, preferably DSPE-PEG2000, wherein The present invention relates to liposomal doxorubicin formulations, wherein 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 wherein the polyethylene glycol-lipid conjugate is essentially located only on the outer layer of the lipid bilayer.

[0048] Also preferred are liposomal doxorubicin formulations which contain relatively large amounts of polyethylene glycol-lipid conjugates in the lipid bilayer, essentially only on the outer layer of the lipid bilayer, as described in the paragraphs above, and further have any other characteristics or combinations of advantageous characteristics described herein, in particular those relating to the size and / or morphology of the liposomes.

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

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

[0051] Thus, the liposomes of the present invention are particularly stable. Controllable and long-lasting liposome size is beneficial for purposes of manufacturing, storage, shelf life, and patient safety.

[0052] The formulation may have a drug to total lipid weight ratio of 0.01-0.10, preferably 0.03-0.07. By comparison, Doxil® has a total lipid content close to 16 mg / mL and a doxorubicin concentration of 2 mg / mL. This results in a drug to lipid ratio of 0.138:1 (weight:weight) for Doxil®. ​​A lower drug to lipid ratio is advantageous in that it allows liposomes to be made smaller and more spherical. This morphology also contributes to reduced adverse effects. In addition, as shown in the examples, the drug exposition of the patient 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 lengths and widths are measured manually from a series of high magnification images obtained by cryo-TEM. The dimensions are smaller than similar compositions 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 determined by the Cry It was determined manually from a series of high magnification images obtained by o-TEM. The number of individual fibers (high density nodes) per crystal could be derived. It is noteworthy that doxorubicin crystals have a helical conformation, and the number of individual fibers per turn can vary. To obtain an accurate representation, one measurement was taken per turn of doxorubicin crystal. This number is smaller than that of similar compositions known in the art.

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

[0056] Notably, a liposomal doxorubicin formulation in which the fiber width and length of the doxorubicin crystals encapsulated in the liposomes are relatively small is an advantageous concept in itself, independent of the size and morphology of the liposomes.

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

[0058] Also preferred are liposomal doxorubicin formulations having relatively small fiber widths and lengths, as described in the preceding paragraphs, and further having any other characteristic or combination of advantageous characteristics described herein, particularly those relating to liposome size and / or morphology.

[0059] Preferably, the formulation is dispersed in a HEPES buffer, preferably at a concentration of 10 mM, resulting in a pH value of 6.8. The solution may contain 0.9% NaCl. Liposomal formulations known in the art are dispersed in a buffer based on histidine and a group of sugars (myocet lactose, doxil sucrose). In this embodiment, the formulation may be sucrose-free, thus reducing the risk of bioburden. It also allows for post-production sterile filtration. As described below, this aspect facilitates cost-effective production.

[0060] It is possible that active ingredients other than doxorubicin can be encapsulated in the formulation. For example, active ingredients that exhibit synergistic effects upon release can be included or encapsulated. Also, at least one active ingredient may be included in the liposome bilayer and at least another active ingredient may be encapsulated in the same liposome. The term "active ingredient" may include pharmacologically active drugs and prodrugs. Prodrugs are drugs or compounds that are metabolized to pharmacologically active drugs after administration.

[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, peptidomimetics, proteins, antibodies and antigen-binding fragments thereof, monosaccharides, disaccharides, trisaccharides, oligosaccharides, lipids, glycosaminoglycans, extracts obtained from biological materials, and any combination thereof.

[0062] The liposomes, whether loaded or unloaded, may themselves be the active ingredient.

[0063] According to another aspect of the present invention, which can be applied in combination with the above-mentioned description but also by itself, it is particularly advantageous to administer a liposomal docetaxel formulation together with a liposomal doxorubicin formulation.Docetaxel is a chemotherapeutic drug used in the treatment of cancer, particularly breast cancer, non-small cell lung cancer, prostate cancer, gastric adenocarcinoma, head and neck cancer.Docetaxel is sold under the name Taxotere, which contains docetaxel as 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]heptadec-13-en-2-yl]benzoate.

[0064] The art suggests combining doxorubicin and docetaxel for the treatment of certain types of cancer. However, the present inventors have now found that the combined administration of liposomal doxorubicin and docetaxel formulations has a positive effect. Furthermore, the effect is even more pronounced when a liposomal formulation is administered in which each liposome contains the two drug substances simultaneously. In such an embodiment, each liposome encapsulates hydrophilic doxorubicin in its internal aqueous compartment, and docetaxel (having hydrophobic properties) is located between the layers of the lipid bilayer.

[0065] Therefore, particularly preferably containing doxorubicin in an aqueous internal compartment, The liposomal doxorubicin formulation comprises individual liposomes that contain docetaxel in the lipid bilayer.

[0066] Particularly preferably, the formulation consists essentially of individual liposomes containing doxorubicin in the aqueous inner compartment and docetaxel in the lipid bilayer, i.e. the amount of liposomes carrying the active substance combination is at least 80%, preferably at least 90%.

[0067] Preferably, the liposomal doxorubicin formulation, which comprises individual liposomes containing doxorubicin in the aqueous internal compartment and docetaxel in the lipid bilayer, has advantageous properties as described herein, i.e., size, circularity, bilayer composition, stability, surface modification, etc. Also preferably, such liposomal doxorubicin formulations are used as medicines, preferably as medicines 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 as described above. The method comprises the steps of: a) providing a phosphatidylcholine, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and cholesterol in an organic solvent; With b) adding an aqueous liquid; c) inducing liposome formation by sonication; d) optionally isolating the liposomes by filtration; e) modifying the liposomes by PEGylation; f) loading doxorubicin into the liposomes, preferably by remote loading technique; Step c) and / or so that the liposomes have an average diameter, as measured by dynamic light scattering, of 30 to 70 nm, preferably 40 to 65 nm. The method is characterized in that the liposomes have an average diameter of 20 to 50 nm, preferably 30 to 40 nm, as measured by cryo-TEM.

[0069] This method can obtain liposomes with significantly improved stability values, significantly improved bioavailability, and reduced toxicity. Empty liposomes are produced in a gentle sonication process, where lipids naturally become liposomes. These liposomes are stable in their natural shape for long periods of time without the addition of stabilizers. The size distribution of liposomes is measured at various time points and is essentially constant. The small size is 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, for example, more than 99.99% absolute ethanol or methanol.Even more preferably, thin film hydration is not required.

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

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

[0073] It is preferable to use an aqueous solution of ammonium sulfate, preferably an aqueous solution of 140-160 mM ammonium sulfate, more preferably an aqueous solution of 150 mM ammonium sulfate. A concentration within the above numerical range allows controlled remote loading in step f) and is suitable for delivering a liposomal doxorubicin formulation with a drug to total lipid weight ratio of 0.01-0.1, preferably 0.04-0.6. A 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 carried out for at least 1 hour with an amplitude of at least 60 μm. The ultrasonic treatment is carried out for a maximum period of 24 hours.

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

[0076] Liposomes are encapsulated in a liquid that is free of residual substances from the mixture, such as organic solvents, salts, and / or surfactants. It is separated from the

[0077] The liposome distribution is preferably at least 95% unilamellar, preferably at least 97% unilamellar, more preferably at least 98% unilamellar. Preferably, the liposomes have a mean relative circularity of at least 0.99, with a 10th percentile of at least 0.98, and preferably a 5th percentile of at least 0.98, as measured by cryo-TEM. 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 liposomal dispersion is modified by PEGylation. "PEGylation" means polyethylene glycol modification, which is carried out as a post-modification method. Conventionally, phosphatidylcholine, cholesterol, and PEG-lipid are dissolved in a mixture to give crude liposomes, which are then reduced in size by extrusion. This method is called pre-modification. In contrast, when applying the post-modification method, empty liposomes composed of phosphatidylcholine and cholesterol are prepared in a solvent and then extruded through a suitable membrane. In a modification of the post-modification method, PEG-derivatized phospholipids are added to a dilute suspension of preformed liposomes at a temperature close to the melting temperature of the liposomal components. This technique is also called "post-insertion", and the insertion of PEG-derivatized phospholipids is mainly driven by hydrophobic interactions between membrane lipids and the hydrophobic moieties of the PEG-derivatized phospholipids. Preferably, PEGylation is carried out at 60°-70°C, preferably at 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 achieved by covalently attaching a PEG moiety containing a reactive group that can react with a complementary reactive group present on the liposome component. A variety of different methods are known for attaching moieties such as PEG to the surface of preformed liposomes, including crosslinking of primary amines with glutaraldehyde, carbonyl-amine bond formation, reaction of activated esters with primary amines to form amide bonds, disulfide bond formation, maleimide-thiol addition reaction to form thioester bonds, and hydrazine bond formation.Furthermore, there are various biorthogonal approaches, such as those often summarized under the term "click" chemistry, which allow for chemical selectivity, mild reaction conditions in aqueous media, and good yields with little or no by-products. Examples of click chemistry reactions that have been utilized to modify liposome surfaces include copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition (CuAAC), copper-free click reaction promoted by ring distortion, Staudinger ligation, and tetrazine / trans-cyclooctene inverse electron demand Diels-Alder cycloaddition (IEDDA), which are described in detail in: Nag, OK; Surface Engineering of Liposomes for Stealth Behavior; Pharmaceutics; 5 (2013) 542-569. In both modification methods, the addition of PEG lipids is only performed after the liposome formation / minimization step, which is preferably carried out in aqueous solution. Thus, PEGylated liposomes are obtained in which the DSPE-PEG is essentially located only on the outer layer of the lipid bilayer.

[0079] The method has the advantage of producing small, uniform liposomes with an average diameter of less than 50 nm, high circularity, and a strong tendency to be stable. A further preferred aspect of the present invention is the reduction in production costs and the reduction in the number of production steps, which facilitates mass production.

[0080] In step f), doxorubicin is loaded into the liposomes by remote loading technique. "Remote loading" refers to an approach to loading doxorubicin into the aqueous phase within liposomes. Remote loading is applied to preformed liposomes. In such liposomal formulations, a transmembrane ammonium sulfate gradient is established, with the [(NH4)2SO4] concentration in the aqueous interior of the liposome being significantly higher than that in the external medium. This gradient serves as a driving force for the movement of amphiphilic weakly basic drugs such as doxorubicin to the opposite side of the liposomal bilayer, where it forms crystal-like precipitates. This approach was first pioneered by Barenholz. and is well known in the art (see: Barenholz, YC; Doxil® - 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 and does not include a thin film hydration step. "Extrusion" refers to a conventional liposome preparation technique in which a liposome formulation is passed through a membrane with a defined pore size. The extrusion process is considered in the art to be the method of choice for liposome manufacturing (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, which in turn results in low quality liposomes.

[0082] "Thin film hydration" refers to the traditional method of liposome preparation, which involves the creation of a thin lipid film by removing organic solvent, for example in a round-bottom flask. Addition of dispersion medium and stirring results in the formation of heterogeneous liposomes. Typically, thin film hydration is followed by extrusion through a polycarbonate membrane to obtain a more homogeneous preparation consisting of smaller liposomes. Thin film hydration is even more costly than extrusion.

[0083] The liposome formulations according to the invention produced by ultrasonication were found to be less polydisperse, more stable and less prone to degradation than liposomes obtainable by prior art techniques, and also have smaller diameters and greater circularity compared to those obtained by extrusion processes.

[0084] Preferably, the above described method steps are followed by one further step g), which comprises sterilization by filtration.

[0085] "Sterilization by filtration" means passing the completed liposome formulation through a sterile filter with a pore size of 0.22 μm or less to retain possible impurities and bacterial cells.

[0086] Sterilization by filtration allows the production of liposomal doxorubicin formulations in accordance with GMP, whereby the steps prior to sterile filtration do not necessarily have to be carried out under sterile conditions, which is particularly cost-effective. However, a prerequisite for sterilization by filtration is that the formulation is dispersed in a storage buffer that does not contain sugar groups, for example in a HEPES buffer.

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

[0088] The liposomal doxorubicin formulations described above may be used specifically in the treatment of uterine leiomyosarcoma.

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

[0090] Liposomal doxorubicin formulations can be administered intravenously in the treatment of cancer. For intravenous injection, the liposomes may be in dissolved or suspended form. The amount of the formulation is 2 The volume may be in the range of 1-100 ml per (body surface), depending on the dose. 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 a liposome as described above, obtainable by the method as described above.

[0092] The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. [Brief description of the drawings]

[0093] [Figure 1a] FIG. 1 shows the morphology and size distribution of liposomal doxorubicin formulations according to the present invention as measured by cryo-TEM. [Figure 1b] FIG. 1 shows the morphology and size distribution of liposomal doxorubicin formulations according to the present invention as measured by cryo-TEM. [Figure 2a] FIG. 1 shows the morphology and size distribution of Caelyx® formulations as determined by cryo-TEM. [Figure 2b] FIG. 1 shows the morphology and size distribution of Caelyx® formulations as determined by cryo-TEM. [Diagram 3]FIG. 1 shows the size distribution, as measured by DLS, of different embodiments of liposomal doxorubicin formulations according to the present invention compared to the Caelyx® formulation. [Figure 4a] FIG. 4 shows the circularity distribution, as measured by cryo-TEM, of a liposomal doxorubicin formulation according to the invention (4a) compared to the Caelyx® formulation (4b). [Figure 4b] FIG. 4 shows the circularity distribution, as measured by cryo-TEM, of a liposomal doxorubicin formulation according to the invention (4a) compared to the Caelyx® formulation (4b). [Figure 5a] FIG. 1 shows width measurements, as measured by cryo-TEM, of doxorubicin crystals according to the invention (5a, 5b) in comparison with the Caelyx® formulation (5c). [Figure 5b] FIG. 1 shows width measurements, as measured by cryo-TEM, of doxorubicin crystals according to the invention (5a, 5b) in comparison with the Caelyx® formulation (5c). [Figure 5c] FIG. 1 shows width measurements, as measured by cryo-TEM, of doxorubicin crystals according to the invention (5a, 5b) in comparison with the Caelyx® formulation (5c). [Figure 6a] FIG. 1 shows length measurements, as determined by cryo-TEM, of doxorubicin crystals according to the invention (6a, 6b) in comparison with the Caelyx® formulation (5c). [Figure 6b] FIG. 1 shows length measurements, as determined by cryo-TEM, of doxorubicin crystals according to the invention (6a, 6b) in comparison with the Caelyx® formulation (5c). [Figure 6c] FIG. 1 shows length measurements, as determined by cryo-TEM, of doxorubicin crystals according to the invention (6a, 6b) in comparison with the Caelyx® formulation (5c). [Figure 7a]The number of doxorubicin fibers per liposome as measured based on cryo-TEM imaging is shown sorted by class in FIG. 7a for a doxorubicin formulation according to the invention and in FIG. 7b for the Caelyx® formulation. [Figure 7b] The number of doxorubicin fibers per liposome as measured based on cryo-TEM imaging is shown sorted by class in FIG. 7a for a doxorubicin formulation according to the invention and in FIG. 7b for the Caelyx® formulation. [Figure 8] Shown is the accumulation of doxorubicin over time in the liver and tumor for three comparatively administered formulations (CALYX, TLD-1, and free DXR). [Figure 9] FIG. 1 shows an in vitro cytotoxicity study based on MTS absorption for three comparative doxorubicin formulations (CALYX, TLD-1, free DXR). [Figure 10a] FIG. 1 shows an in vitro cytotoxicity study based on luciferase luminescence for three comparative doxorubicin formulations (CALYX, TLD-1, free DXR). [Figure 10b] FIG. 1 shows an in vitro cytotoxicity study based on luciferase luminescence for three comparative doxorubicin formulations (CALYX, TLD-1, free DXR). [Figure 10c] FIG. 1 shows an in vitro cytotoxicity study based on luciferase luminescence for three comparative doxorubicin formulations (CALYX, TLD-1, free DXR). [Figure 11] 1 shows the results of a comparative study on tumor growth (MDA-MB231) over time under administration of the liposomal doxorubicin formulation of Example 1. [Figure 12a] 1 shows the results of a comparative study on tumor growth (A2780) over time under administration of the liposomal doxorubicin formulation of Example 1. [Figure 12b] 1 shows the results of a comparative study on tumor growth (A2780) over time under administration of the liposomal doxorubicin formulation of Example 1. [Figure 13] 1 shows the results of a comparative study of tumor growth (4T1) over time upon administration of the liposomal doxorubicin formulation of Example 1. [Figure 14] 1 shows the results of an in-vivo survival study in mice for three comparative doxorubicin formulations, including that of Example 1. [Figure 15a] FIG. 1 shows the size and polydispersity results of the stability measurements over time (12 months) for the liposomal doxorubicin formulation of Example 1. [Figure 15b] FIG. 1 shows the size and polydispersity results of the stability measurements over time (12 months) for the liposomal doxorubicin formulation of Example 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0094] Example 1: Generation of TLD-1 1,2-distearoyl-sn-glycero-3-phosphocholine and cholesterol were provided in a weight ratio of 60:40 and dissolved in >99.99% absolute ethanol. The solution was hydrated in a 150 mM aqueous solution of ammonium sulfate in sterile water at 68°C. The solution was sonicated at an amplitude of 60 μm for 24 hours to obtain crude liposomes. The liposome suspension was then added with an aqueous PSPE-MPEG2000 solution and heated at 65°C for 30 minutes to obtain PEGylated liposomes with the desired PEG-lipid amount of 5 mol% (corresponding to a PEG-lipid amount of 10 mol% in the outer layer of the lipid bilayer). Doxorubicin HCl was loaded into the liposomes by remote loading technique to achieve a DXR / total lipid weight ratio of 0.05. Unloaded DXR was removed by gravity settling and filtration. The liposome dispersion was washed by tangential flow filtration and buffer exchange was performed to achieve dispersion of the liposomes in 10 mM HEPES buffer containing 0.9 wt % NaCl.

[0095] Liposomal doxorubicin obtained as described in this example may hereinafter be referred to as "TLD", "TLD-1", or "Talidox".

[0096] Free doxorubicin, whenever applied as a comparative formulation, may be referred to in the examples and figures as "Doxo", "DXR", "DX" or "doxorubicin".

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

[0098] Example 2: Size Measurement Size measurements of liposomes obtained by the methods described above were performed by cryo-TEM and DLS and the results were compared with the corresponding measurements for the commercial Caelyx® formulation.

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

[0100] CryoTEM measurements were performed as follows: liposome samples from Example 1 and Comparative Example were vitrified. Samples were prepared on-grid (Formvar and carbon) at an acc voltage of 200 kV. Images were acquired using a cryoTEM JEOL JEM-2100F device and a TVIPS TemCam F415MP camera at 40,000x magnification. Particle identification and sizing 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 that were entirely contained inside the image border and had a clear membrane were detected. Identified objects were analyzed for sphericity, circularity, and unilamellarity. All images were processed collectively with the same thresholds and settings, and 5-18 images were accumulated for each sample, corresponding to a number of particles analyzed of 1560-1178. The standard deviation of the mean value is about 10 nm.

[0101] Figure 1b shows the size distribution of the liposomal formulation of Example 1. The number of images analyzed was 5. The number of particles analyzed was 1560. The mean diameter was 35.61 nm with a standard deviation of 7.42 nm. The smallest diameter measured was 24.81 nm and the largest diameter measured was 103.35 nm. The homogeneity Z test gave a value of 1.01 as a measure of the homogeneity of the sampling, indicating that all images included in the analysis contained a population of particles with the same average size.

[0102] Figure 2b shows the size distribution of the comparative liposomal formulation. The number of images analyzed was 18. The number of particles analyzed was 1178. The mean diameter was 70.26 nm with a standard deviation of 13.41 nm. The smallest diameter measured was 32.52 nm and the largest diameter measured was 159.09 nm. The homogeneity Z test gave a value of 3.15 as a measure of the homogeneity of the sampling, indicating that not all images included in the analysis contained a group of particles with the same average size.

[0103] The liposome formulation of Example 1 further demonstrated less than 10% broken particle counts and no particle aggregates or clusters in cryoTEM analysis.

[0104] FIG. 3 shows the size of the liposome formulations of Example 1 and Comparative Example as measured by dynamic light scattering (DLS). All samples were diluted 10-fold in PBS or MQ H2O and Measurements were performed at 25°C and a scattering angle of 0° using a Luverne Zetasizer device. TLD-1 (Example 1) had a mean diameter of 60.5 (±4.7 nm) nm with a polydispersity index of 0.084 ±0.038. Caelyx® had a mean diameter of 85.0 nm as measured by DLS. It is noteworthy that the values ​​measured by dynamic light scattering are slightly higher than those obtained by cryoTEM imaging, because the PEGylated surface is not detectable by cryoTEM but is included in DLS as part of the hydrodynamic radius of the liposomes.

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

[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 as 1.00, the 10th percentile as 0.98, the 5th percentile as 0.98, and the 2nd percentile as 0.96. The homogeneity Z test gave a value of 1.19 as a measure of the homogeneity of the sampling, indicating that all images included in the analysis contained a group of particles with the same average size.

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

[0108] Thus, commercially available Caelyx® has low circularity of liposomes in the formulation, for example, 10% of the liposomes in Caelyx® have at most a circularity of 0.97 or less.

[0109] The liposome formulation of Example 1 further had a loading fraction (loading with doxorubicin) of at least 80% and a unilamellar fraction of 98% as measured by cryoTEM.

[0110] Example 4: Crystal size and number of fibers per crystal The dimensions of the liposome formulations of Example 1 and Comparative Example were measured by Cryo-TEM. The results of the width measurements are presented in Figures 5a and 5b for Example 1 and in Figure 5c for Comparative Example. The results of the length measurements are presented in Figures 6a and 6b for Example 1 and in Figure 6c for Comparative Example. Sample preparation and measurements were performed as described previously. 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 fibers per liposome was determined from a series of high-magnification images taken by Cryo-TEM. The number of individual fibers (dense nodes) per liposome could be derived manually. Because doxorubicin crystals have a helical conformation and the number of individual fibers per turn can vary, one measurement per turn was taken to obtain an accurate representation.

[0113] The class ratios for Example 1 are shown in Figure 7a. The class ratios for the Comparative Example are shown in Figure 7b. The x-axis shows the class number (1-12), which indicates the number of individual fibers in the doxorubicin crystal.

[0114] In Example 1, three fibers per crystal was the most common conformation. No crystals were observed with one, seven, or more individual fibers. The average spacing between individual fibers measured for all doxorubicin crystals in the data set was 2.6 nm.

[0115] In the comparative examples, 7 fibers per crystal was the most common conformation. No crystals were observed with 1, 2, 3, 12 or more individual fibers. The average spacing between individual fibers measured for all doxorubicin crystals in the data set was 2.7 nm.

[0116] Thus, commercially available Caelyx® has low circularity of liposomes in the formulation, for example, 10% of the liposomes in Caelyx® have at most a circularity of 0.97 or less.

[0117] The liposome formulation of Example 1 further had a loading fraction (loading with doxorubicin) of at least 80% and a unilamellar fraction of 98% as measured by cryoTEM.

[0118] Example 5: Tumor accumulation in mice The liposomal doxorubicin formulation of Example 1 ("TLD-1"), commercially available Caelyx® ("CAELYX"), and free doxorubicin (Adlibrastine "free doxorubicin") were administered at 3.5 mg / kg to mice (athymic nude Foxn1 nu After 4 or 16 hours, the mice were sacrificed for detection of total doxorubicin by HPLC analysis.

[0119] Figure 8 shows doxorubicin accumulation in liver and tumor over time for three comparatively administered formulations. Bars represent mean and standard deviation (n=3).

[0120] Accumulation of TLD-1 in tumors was approximately four times higher than that of free doxorubicin and twice that of CAELYX®. Serum half-lives of up to 16 hours are comparable for CAELYX and TLD-1. However, hepatic accumulation and clearance are more efficient for TLD-1.

[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. 24 hours after seeding, cells were treated with different concentrations of doxorubicin formulations.

[0122] FIG. 9 shows the results when cells were treated with concentrations of 10, 50, 100, 500, 1000, 5000, and 10'000 ng / ml. After 72 hours of treatment, the MTS colorimetric component ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethox-1,3-diyl)- The viable cells were stained by adding MTS (2-(4-diphenyl)-2H-tetrazolium) ester. After another 3 hours, the absorbance was measured. As can be seen, the absorbance of the samples treated with TLD-1 was obviously lower than that of the samples treated with CAELYX at all added concentrations. Therefore, the cytotoxicity of TLD-1 is higher and more similar to that of free doxorubicin.

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

[0124] Serum leakage study: Experiments were performed to determine the extent to which TLD-1 and Caelyx® release free doxorubicin into RPMI (cell culture medium) + / - 10% FCS medium over time. Free doxorubicin in the medium was measured after incubating TLD-1 and CAELYX, respectively, in RPMI medium + / - 10% FBS at 37° in a metal bead bath for 72 hours in the dark. Free and liposomal doxorubicin were detected by HPLC size exclusion chromatography at 478 nm (to avoid background absorption from proteins). Liposomal doxorubicin appears after the free doxorubicin peak due to conjugation and aggregation. This was confirmed by comparing with the values ​​from the free DX (adliblastin) control sample. A comparative analysis of the peak area under the curve was performed (liposomal doxo vs. free doxo). The experiments showed that both TLD-1 and Caelyx were stable over 72 hours of exposure at 37° in the medium used in the in-vitro experiments. The percentage of free DX in solution was less than 4% in TLD-1 incubations and less than 6% in Caelyx incubations. In general, leakage of TLD-1 was less than that of Caelyx. This indicates that the increased activity seen in the in-vitro cytotoxicity assays is due to increased cellular uptake and not leakage of free doxorubicin into the medium.

[0125] Example 7: Tumor Growth The effect on tumor growth in vivo was examined by administering a placebo formulation, TLD-1, and CAELYX to mice and measuring the effect 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 administered empty liposomes, free doxorubicin, Caelyx®, and TLD-1 at regular intervals. Each arrow indicates an injection at a dose of 3.5 mg / kg body weight. The effect of TLD-1 (measured as increase in tumor weight, μg) was clearly better than that of Caelyx® already 3 days after the start of treatment and remained better for 26 days.

[0127] Figures 12a and 12b show the results of a similar but different test setup. Mice (5 mice / formulation) injected with the A2780 cell line were administered with PBS, free doxorubicin, Caelyx®, and TLD-1 periodically. For Figure 12a, a formulation similar to that in Example 1 was administered, but unlike Example 1, sonication resulted in an average liposome diameter of 86.78 nm measured by DLS, as well as a polydispersity index of 0.117. For Fig. 12b, the formulation of Example 1 was administered, i.e. liposomes with a mean diameter of 64.87 nm and a polydispersity index of 0.168, measured by DLS. Each arrow indicates an injection at a dose of 3.5 mg / kg body weight. During the study presented in Fig. 12a, it was found that TLD-1 with an out-of-specification mean diameter (more than 70 nm) had an inferior effect on tumor growth compared to treatment with Caelyx®, the effect of TLD-1 according to the specification being clearly better than that of Caelyx already 19 days after the start of treatment and for a further 12 days (measured as increase in tumor weight, μg).

[0128] Figure 13 shows the results of a similar but different test setup. Mice implanted with 4T1 tumors (5 mice / formulation) were administered saline, Caelyx®, and TLD at regular intervals. The effect of TLD-1 was clearly better than that of Caelyx after 12 days of treatment initiation (measured as an increase in tumor size, mm 3 ).

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

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

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

[0132] Adverse effects studies have been performed in rats. Toxicology studies performed in rats have investigated dermal toxicity, especially PPE (e.g. hand-foot syndrome). Administration of TLD-1 did not promote dermal toxicity, especially PPE, even at a high concentration of 6 mg / kg (male and female data pooled together due to lack of statistical significance). Similarly, toxicology studies performed in rats have investigated neutropenia (by neutrophil count). Administration of TLD-1 did not significantly alter neutrophil count, even at a high concentration of 6 mg / kg (male and female data pooled together due to lack of statistical significance).

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

[0134] Example 10: Clinical trial results 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 the 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 study. The primary objective of the study was to identify the maximum tolerated dose (MTD) and the recommended phase 2 dose (RP2D) of TLD-1 in patients with advanced solid tumors. Further objectives of the study were to evaluate the safety, preliminary antitumor activity, and pharmacokinetics of TLD-1.

[0135] Interim results from this study indicate that TLD-1 is being evaluated at 45 mg / m every 3 weeks in patients with advanced, previously treated solid tumors. 2 This dose is higher than that of Caelyx®, the MTD of which is 50 mg / m2 every 4 weeks. 2 Furthermore, the number and severity of undesirable side effects of TLD-1 were lower than those of Caelyx®. Specifically (TLD-1 vs. Caelyx®), clinically non-significant nausea (<8.3% vs. 38.5%), vomiting (<8.3% vs. 24.3%), alopecia (0% vs. 13.4%), or cardiotoxicity were observed, and myelosuppression was rare and mild (8.3% vs. 25.6%). No unexpected toxicities were reported.

[0136] It is hypothesized (without being limited thereto) that the fewer side effects observed with TLD-1 compared to conventional liposomal formulations of doxorubicin, including Caelyx®, are due to the relatively small liposome size and high homogeneity of the doxorubicin-loaded liposomes administered to patients, in particular because the doxorubicin crystal fibers in the liposomes have pronounced circularity, low polydispersity, and high uniformity (length and width).

Claims

1. A liposomal doxorubicin formulation, comprising a lipid bilayer of the liposome comprising: phosphatidylcholine, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine, ·cholesterol, - comprising at least a polyethylene glycol-lipid conjugate, preferably DSPE-PEG2000, wherein the liposomes have an average diameter, as measured by dynamic light scattering, of 30 to 70 nm, preferably 40 to 65 nm; and / or A liposomal doxorubicin formulation, wherein said liposomes have an average diameter of 20-50 nm, preferably 30-40 nm, as measured based on images obtained by cryo-TEM.

2. The liposomal doxorubicin formulation of claim 1, wherein the lipid bilayer consists essentially of synthetic phosphatidylcholine, preferably of structurally uniform type, 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; Preferably, the 5th percentile is at least 0.98; More preferably, the liposomal doxorubicin formulation of any one of the preceding claims, wherein the 5th percentile is at least 0.98 and the 2nd percentile is at least 0.

96.

4. 10. The formulation of any one of the preceding claims, wherein the polyethylene glycol-lipid conjugate is located essentially only on the outer layer of the lipid bilayer.

5. 10. The formulation according to any one of the preceding claims, wherein the relative amount of polyethylene glycol-lipid conjugate in the lipid bilayer is at least 2 mol%, preferably at least 3 mol%, more preferably between 4 mol% and 6 mol%.

6. A formulation according to any one of the preceding claims, wherein the drug to total lipid weight ratio is from 0.01 to 0.10, preferably from 0.03 to 0.

07.

7. 2. The liposomal doxorubicin formulation according to any one of the preceding claims, wherein the encapsulated doxorubicin crystals have an average fibre width of 5-15 nm, preferably 6-12 nm, more preferably 7-11 nm and / or an average fibre length of 15-40 nm, preferably 18-37 nm, more preferably 25-35 nm.

8. 2. The formulation of any one of the preceding claims, wherein the liposomes are dispersed in a HEPES buffer.

9. A method for the preparation of a liposomal doxorubicin formulation, preferably a liposomal doxorubicin formulation according to any one of the preceding claims, comprising the steps of: a) providing a phosphatidylcholine, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and cholesterol in an organic solvent; b) adding an aqueous liquid; c) inducing liposome formation by sonication; d) optionally isolating the liposomes by filtration; e) modifying the liposomes by PEGylation; f) loading doxorubicin into said liposomes, preferably by remote loading technique; Step c) is and / or such that the liposomes have an average diameter, as measured by dynamic light scattering, of between 30 and 70 nm, preferably between 40 and 65 nm; and / or A method characterized in that it is carried out in such a way that the liposomes have an average diameter, measured by cryo-TEM, of 20 to 50 nm, preferably 30 to 40 nm.

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

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

12. The formulation according to any one of claims 1 to 8 for use as a medicament, in particular for use in the treatment of cancer, more particularly for use in the treatment of solid tumors, metastatic breast cancer, advanced ovarian cancer, Kaposi's sarcoma and multiple myeloma.

13. 13. A formulation as claimed in claim 12 for use as a medicament in the treatment of uterine leiomyosarcoma.

14. 13. A formulation as claimed in claim 12 for use as a medicament in the treatment of skin adnexal cancer.

15. A formulation according to any one of claims 1 to 8 for use in the treatment of a medical indication according to claims 12 to 14, wherein said treatment comprises intravenous administration of said formulation.

16. A formulation according to any one of claims 1 to 8, obtainable by a method according to any one of claims 9 to 11.

17. The liposomal doxorubicin formulation according to any one of claims 1 to 8, wherein the liposomal doxorubicin formulation has a polydispersity index as measured by DLS of less than or equal to 0.15, preferably less than or equal to 0.10, more preferably less than or equal to 0.09.

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