A composition comprising trabectedin and an amino acid

Abstract

The present invention relates to a stable pharmaceutical composition comprising trabectedin. The present invention also provides a lyophilized formulation and an intravenous injection solution comprising trabectedin and an amino acid.

Description

Technical Field

[0001] Description

[0001] The present invention relates to a composition, a lyophilized formulation and an intravenous injection comprising trabectedin and an amino acid.

Background Art

[0002]

[0002] Trabectedin (ecteinascidin or ET-743) is a tetrahydroisoquinoline alkaloid first isolated from the marine tunicate Ecteinascidia turbinata and having antitumor activity.

[0003]

[0003] Trabectedin has limited water solubility and thermal stability, presenting challenges for the development of formulations for medical purposes. Trabectedin is particularly effective in the treatment of sarcoma and ovarian cancer.

[0004]

[0004] WO2000069441 discloses the use of trabectedin in the preparation of a pharmaceutical composition for the treatment of cancer in humans. The pharmaceutical composition is formulated as a lyophilized product containing mannitol as a bulking agent and a phosphate buffer at pH 4 to stabilize trabectedin. The formulation is administered by intravenous drip infusion.

[0005]

[0005] WO2006046079 discloses that disaccharides can stabilize ecteinascidin formulations. WO2006046079 describes a composition comprising ecteinascidin, such as ET-743, and a disaccharide, such as sucrose, which composition is lyophilized, reconstituted and administered by intravenous drip infusion.

[0006]

[0006] Trabectedin is currently formulated as a sterile lyophilized product provided in the form of vials containing 0.25 mg of trabectedin, 100 mg of sucrose and 2 mg of potassium in the form of potassium dihydrogen phosphate, as well as potassium hydroxide and phosphoric acid for pH adjustment.

[0007]

[0007] WO2017133544A1 relates to a stable trabectedin formulation which may include glucose as a first excipient and hydroxyethyl starch, dextran, or sodium carboxymethyl cellulose or hydroxypropyl-β-cyclodextrin as a second excipient.

[0008]

[0008] IN201741041173 discloses a stable pharmaceutical composition of trabectedin. A formulation containing trabectedin and L-arginine in a ratio of 1:400 is exemplified. Such a large amount of amino acid results in a high solution volume for lyophilization and a continuously longer drying cycle. Furthermore, a large amount of auxiliary additives may have an adverse effect on patients.

[0009]

[0009] Considering the importance of trabectedin as a chemotherapeutic agent, there is a further need for an improved formulation of trabectedin that is biocompatible, stable, and suitable for medical purposes.

Summary of the Invention

Problems to be Solved by the Invention

[0010]

[0010] The object of the present invention is to provide a stable formulation of trabectedin that is suitable for medical purposes.

Means for Solving the Problems

[0011]

[0011] This object is solved by the claimed subject matter and is as disclosed herein.

[0012] The present invention provides a composition comprising trabectedin and at least one amino acid, and the weight ratio (w / w) of trabectedin to amino acid is 1:10 to 1:250.

[0012]

[0013] According to one embodiment, the amino acid is selected from the group consisting of arginine, histidine, lysine, phenylalanine and isoleucine, methionine, acetylcysteine, cysteine, citrulline, or any combination thereof.

[0013]

[0014] In one embodiment, the amino acid is L-arginine.

[0015] In one embodiment, the weight ratio (w / w) of trabectedin to amino acid is 1:10 to 1:250, or 1:50 to 1:100, or about 1:70.

[0014]

[0016] In one aspect, the composition described herein includes a buffer. The buffer may be selected from the group consisting of citric acid, phosphoric acid, acetic acid, basic amino acids and sodium hydroxide, or any mixture thereof, or a mixture of citric acid, phosphoric acid, and optionally basic amino acids.

[0015]

[0017] In one aspect, the composition described herein includes additional substances selected from the group consisting of a complexing agent such as ethylenediaminetetraacetic acid (EDTA); an antioxidant such as monothioglycerol; a surfactant such as polysorbate; mannitol; and ascorbic acid.

[0016]

[0018] In the formulation according to the invention, the primary source of pH control is the buffer. Usually, the buffer exists as an acid or a base and its conjugate base or acid, respectively. In one embodiment, the range of the buffer salt is between 1 and 100 mM, preferably between 5 and 50 mM, and most preferably about 10 mM (in the solid formulation, the amount of the buffer solution is selected to produce this concentration after reconstitution / dilution). The concentration of the buffer solution and the pH of the solution are preferably selected to obtain an optimal balance of solubility and stability. Examples of suitable buffer solutions include mixtures of weak acids and alkali metal salts of the conjugate bases of weak acids (e.g., sodium, potassium), such as a mixture of sodium citrate and disodium hydrogen phosphate.

[0017]

[0019] In one embodiment, the composition is in the form of a lyophilized formulation. Accordingly, the present invention also provides a lyophilized formulation comprising trabectedin and at least one amino acid, wherein the weight ratio (w / w) of trabectedin to amino acid is from 1:10 to 1:250. In a further aspect, the amino acid is selected from the group consisting of arginine, histidine, lysine, phenylalanine, isoleucine, methionine, acetylcysteine, cysteine, citrulline, or any combination thereof. In a particular embodiment, the amino acid is L-arginine. According to a further embodiment, the lyophilized formulation comprises trabectedin and the amino acid is in a weight ratio (w / w) of 1:50 to 1:100. In a particular embodiment, the weight ratio (w / w) of trabectedin to amino acid is about 1:70.

[0018]

[0020] In a further embodiment, the lyophilized formulation comprises a buffer.

[0021] In one aspect, the amino acid in the lyophilized formulation is L-arginine and the buffer is citric acid or phosphoric acid, or a mixture thereof.

[0019]

[0022] In another aspect, the lyophilized formulation is provided in the form of a vial. In an exemplary embodiment, the vial contains 0.1 - 1 mg of trabectedin, 10 - 60 mg of L-arginine, 0.5 - 8 mg of citric acid, and 5 - 40 mg of phosphoric acid. In a particular embodiment, the vial contains 0.25 mg of trabectedin, 17.4 mg of L-arginine, 1.9 mg of citric acid, and 10 - 15 mg of phosphoric acid.

[0020]

[0023] The lyophilized formulation is suitable for preparing an intravenous drip solution by reconstitution in an aqueous medium. Accordingly, the present invention further provides an intravenous drip solution comprising trabectedin, an amino acid, a buffer, and water for injection.

[0021]

[0024] In one aspect, the intravenous drip solution is used in the treatment of cancer. The cancers that can be treated can be sarcomas selected from the group of leiomyosarcoma, liposarcoma, osteosarcoma, ovarian cancer, breast cancer, melanoma, colorectal cancer, mesothelioma, renal cancer, endometrial cancer and lung cancer, or any combination thereof.

[0022]

[0025] In a further embodiment, the intravenous drip described herein is used for the treatment of adult patients with progressive soft tissue sarcoma.

[0026] In one embodiment, the intravenous drip solution is used for the treatment of patients with recurrent platinum-sensitive ovarian cancer. The treatment may be in combination with pegylated liposomal doxorubicin (PLD).

Brief Description of the Drawings

[0023]

Figure 1

[0027] It is a diagram showing the purity of short-term stability samples stored at 5 °C (upper figure) and 22 °C (lower figure) until 29 h.

Figure 2

[0028] Stability of lyophilized product at 2 - 8 °C: A diagram showing the content (%) of trabectedin (A285nm) by RP-HPLC of the reference (0.25 mg / mL).

Figure 3

[0029] Stability of lyophilized product at 2 - 8 °C: A diagram showing the purity of the sample as the relative peak area of the trabectedin peak by RP-HPLC analysis (A285nm).

Mode for Carrying Out the Invention

[0024]

[0030] The present invention relates to a composition containing trabectedin and at least one amino acid. This formulation contains trabectedin as an active substance and can be formulated as a lyophilized formulation, which can be reconstituted to obtain an intravenous drip.

[0025]

[0031] In the context of the present invention, trabectedin can be of natural, semi-synthetic or synthetic origin, including combinations of their origins.

[0032] Known formulations of trabectedin use saccharides (e.g., WO2017133544A1) to obtain stable formulations. However, saccharides can exhibit some undesirable properties when used in pharmaceutical formulations. First, glucose and sucrose cannot be administered to diabetic patients without considering additional carbohydrate intake. Diabetic patients need to control the intake of carbohydrates that produce glucose and glucose-lowering drug therapy to maintain their blood glucose levels within certain boundaries. Therefore, drug formulations containing glucose or sucrose are not optimal for this specific patient group.

[0026]

[0033] Furthermore, saccharides are also a source for certain degradation products of trabectedin that would be formed. All saccharides capable of forming a hemiacetal structure (i.e., all "reducing" sugars) in solution can react with a hydroxy group to form a stable acetal and water (Jerry March, Advanced Organic Chemistry, 3rd Edition, pages 789 - 790; John Wiley & Sons). The reaction products, acetal and water, are in equilibrium with the drug compound, e.g., trabectedin and its hydroxy groups. Under removal of water (e.g., after freeze-drying), the equilibrium strongly shifts towards the acetal side and thus forms this reaction product in relevant amounts.

[0027]

[0034] Therefore, trabectedin, the structure of which exhibits such hydroxy groups, tends to form an acetal with a reducing sugar such as glucose or lactose during freeze-drying.

[0028]

[0035] This specific degradation pathway has been observed in the freeze-dried formulation of trabectedin containing glucose. When such a formulation is freeze-dried, the formation of trabectedin-glucose acetal has been observed. Nevertheless, this acetal hydrolyzes during the reconstitution of the lyophilizate and it still persists for several hours in the reconstituted solution (e.g., after 6 hours at a relative level of about 0.5%). It can be detected by HPLC. The HPLC method used is the same as that described in the formulation screening section herein. The degradation eventually equilibrates at a rather low but detectable level of acetal impurities. Since a portion of the trabectedin dose is lost in the acetal and is not available for therapy, applying a formulation containing unknown toxic degradation products to a patient can be problematic not only from the perspective of toxicity but also from the perspective of dosing accuracy.

[0029]

[0036] This side effect of trabectedin formulations containing reducing sugars (e.g., glucose or lactose) can be overcome by simply waiting after the preparation of the infusion until most of the acetal has hydrolyzed before the infusion is started. However, in medical practice, this is a time-consuming, undesirable and avoidable procedure. Therefore, formulations that do not exhibit this type of degradation product are advantageous.

[0030]

[0037] Also, the formulations disclosed in IN201741041173 mainly use monosaccharides in combination with sugar alcohols for the preparation of stable pharmaceutical compositions of trabectedin. Alternatively, amino acids can also be used as excipients. A formulation containing trabectedin and L-arginine in a ratio of 1:400 is exemplified.

[0031]

[0038] The inventors have surprisingly found that even lower amounts of amino acids stabilize trabectedin compositions without producing trabectedin degradation products. In this context, the term "stability" is understood to mean the definition developed by the Working Group for Pharmaceutical Process Engineering (APV), according to which "stability" means the quality of the investigational medicinal product as specified by the manufacturer until the end of that period. The quality of a drug is determined by the content and purity of the active substance and its physicochemical and microbiological properties that can be perceived sensorially, whereby the content of the active substance should not decrease to less than 90% of the published value by the end of that period.

[0032]

[0039] The formulations produced according to the invention have a stability (or, in other words, shelf life or working time) of at least 3 months, preferably at least 6 months, more preferably at least 12 months, even more preferably at least 24 months, and most preferably at least 36 months at a temperature of 2 to 8 °C, whereby the trabectedin content by the end of that period does not decrease to less than 90%, preferably less than 95% of the trabectedin originally used.

[0033]

[0040] The term "amino acid" refers to all of natural amino acids, synthetic amino acids, and amino acid analogs, including proteinogenic and non-proteinogenic amino acids, and in the case where such stereoisomeric forms are possible, their D and L stereoisomeric forms. Amino acids include nonpolar, polar, basic, and acidic amino acids. Nonpolar proteinogenic amino acids are glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), tryptophan (Trp), phenylalanine (Phe), and methionine (Met). Polar proteinogenic amino acids include serine (Ser), threonine (Thr), tyrosine (Tyr), asparagine (Asn), glutamine (Gln), and cysteine (Cys). Basic proteinogenic amino acids include arginine (Arg), histidine (His), and lysine (Lys). Acidic proteinogenic amino acids include aspartic acid (Asp) and glutamic acid (Glu). Synthetic amino acids include acetylcysteine. Non-proteinogenic amino acids include citrulline.

[0034]

[0041] In connection with the present invention, the amino acid can be a nonpolar, polar, basic or acidic amino acid, or any combination thereof. According to one embodiment, the amino acid is selected from the group of nonpolar proteinogenic amino acids, particularly L-phenylalanine or L-isoleucine, or any combination thereof.

[0035]

[0042] In a further embodiment, the amino acid is selected from the group of basic amino acids, such as L-arginine, L-lysine or L-histidine. Specifically, the amino acid is L-arginine. In one aspect, L-arginine may be combined with one or more further amino acids, such as L-phenylalanine or L-isoleucine.

[0036]

[0043] In another embodiment, the amino acid is citrulline. In yet another embodiment, the amino acid is acetylcysteine.

[0044] In the embodiments described herein, the ratio of trabectedin to amino acid is determined according to the solubility of the amino acid and, if the formulation is freeze-dried, also according to the freeze-dryability of the amino acid. The weight ratio (w / w) of trabectedin to amino acid (w / w) can be about 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:150, 1:200, or 1:250. In some embodiments, the weight ratio is in the range of 1:10 to 1:250, or in the range of 1:50 to 1:100. In certain embodiments, the weight ratio is about 1:70.

[0037]

[0045] As used in the present invention, the term "about" refers to a value within the range of + / - 10% of the recited value.

[0046] In certain aspects, the compositions described herein further comprise a buffer. The buffer enables the maintenance of the pH of the system within a range that supports the stability of trabectedin. Typically, the pH will be in the range of pH 2 to pH 5. Suitable buffers are, for example, citrate buffer, phosphate buffer, citrate / phosphate buffer, lactate buffer, ascorbic acid buffer, tartrate / citrate buffer, bicarbonate / hydrochloric acid buffer, acetate or acetic acid buffer, succinate buffer, glycine / hydrochloric acid buffer. In some embodiments, an amino acid-based buffer may be used, for example, a buffer based on an amino acid already present in the composition. A mixture of the aforementioned buffers may also be used.

[0038]

[0047] According to one embodiment, the buffer is selected from the group consisting of citric acid, phosphoric acid, acetic acid, basic amino acids, and sodium hydroxide, or any mixture thereof. In a further embodiment, the buffer is a mixture of citric acid and phosphoric acid (citrate / phosphate buffer). Specifically, the citrate / phosphate buffer may further contain a basic amino acid, for example, L-arginine, as a buffer. Additionally, sodium hydroxide may be used for pH adjustment.

[0039]

[0048] Other components may be included in the composition, for example, complexing agents such as ethylenediaminetetraacetic acid (EDTA); antioxidants such as monothioglycerol; surfactants; mannitol; and ascorbic acid. Examples of surfactants include polysorbate, polyoxyethylene (20) sorbitan monooleate or polyoxyl stearate, phospholipids such as lecithin; polyoxyethylene-polyoxypropylene copolymers such as Pluronic surfactants; polyoxyethylene esters of 12-hydroxystearic acid such as Solutol surfactants; ethoxylates of cholesterol such as diacylglycerol, dialkylglycerol; bile salts such as sodium cholate, sodium deoxycholate, etc.; sucrose monolaurate, sucrose esters such as sucrose monolaurate; polyvinylpyrrolidone (PVP); or polyvinyl alcohol (PVA).

[0040]

[0049] The compositions described herein may be in the form of lyophilized formulations. The term "lyophilized formulation" as used in the present invention refers to a formulation prepared by lyophilizing (freeze-drying) a mixture containing a pharmaceutically effective amount of trabectedin. The inventors have surprisingly found that the use of one or more amino acids as bulking agents in the lyophilization process improves the storage conditions and enables the long-term storage of the lyophilized formulation over a wide temperature range, including refrigerated conditions and room temperature.

[0041]

[0050] Accordingly, the present invention provides a lyophilized formulation comprising trabectedin, at least one amino acid, a buffer, and optionally a bulking agent such as mannitol. The amino acid may act as a bulking agent.

[0042]

[0051] As used herein, the term "bulking agent" refers to a compound that, when added to a lyophilized (freeze-dried) mixture of a mass, contributes to the physical structure of the lyophilized cake (e.g., facilitates the production of an essentially uniform lyophilized cake that maintains a pore structure). In addition to providing a pharmaceutically elegant cake, the bulking agent can also impart useful qualities with respect to adjusting the collapse temperature, providing cryoprotection, and enhancing stability against long-term storage. Exemplary bulking agents include mannitol, glycine, lactose, sucrose, dextrose, and hydroxyethyl starch. The bulking agent can be crystalline (e.g., glycine, or mannitol) or amorphous (e.g., dextran, or hydroxyethyl starch).

[0043]

[0052] In certain embodiments, the amino acids in the lyophilized formulations described herein are nonpolar, polar, basic or acidic amino acids, or any combination thereof. The amino acids can be nonpolar amino acids, such as, for example, L-phenylalanine or L-isoleucine, or basic amino acids, such as, for example, L-arginine, L-lysine or L-histidine, or any combination thereof. Specifically, the amino acid is L-arginine. In one aspect, L-arginine may be combined with one or more additional amino acids, such as, for example, L-phenylalanine or L-isoleucine. In another embodiment, the amino acid is citrulline. In yet another embodiment, the amino acid is acetylcysteine.

[0044]

[0053] In one embodiment, the amino acid in the lyophilized formulation is L-arginine and the buffer is a mixture of citric acid and phosphoric acid (citric acid / phosphate buffer). The amino acid, together with the base, can further act as a buffer.

[0045]

[0054] The lyophilized formulation according to the present invention can be prepared by freeze-drying the composition described herein in the form of a solution before lyophilization. Described herein is a method for preparing a lyophilized formulation, which includes the step of freeze-drying the composition in the form before lyophilization. The solution before lyophilization contains trabectedin at a concentration of 0.1-1 mg / mL, L-arginine at a concentration of 10-30 mg / mL, citric acid at a concentration of 0.5-4 mg / mL, and phosphoric acid at a concentration of 5-20 mg / mL. The pH of the solution before lyophilization is in the range of pH 2 to pH 5, specifically in the range between pH 4 and pH 5, and more specifically, the pH is 4.8. The method for preparing the lyophilized formulation may further include adjusting the pH to the desired value, for example, by adding phosphoric acid and / or sodium hydroxide.

[0046]

[0055] In one aspect, the lyophilized formulation is provided in the form of a vial. According to one embodiment, the vial is a molded glass vial or a tubular glass vial. However, the present invention is not limited by the specific form or design of the container, provided that the container is acceptable for its intended use and standards.

[0047]

[0056] The lyophilized formulation is usually indicated in a vial containing a specified amount of trabectedin. For example, the amount of trabectedin in the vial is 0.25 mg or 1 mg. To provide a vial containing the lyophilized formulation, the solution before lyophilization is added to the vial and freeze-dried. The volume before lyophilization added to the vial is in the range of 1 mL to 5 mL, or 1 to 4 mL.

[0048]

[0057] In one embodiment, the vial contains 0.1-1 mg of trabectedin, for example, 0.25 mg of trabectedin, and 10-30 mg of L-arginine, for example, 17.4 mg of L-arginine, and 0.5-4 mg of citric acid, for example, 1.9 mg of citric acid, and 5-20 mg of phosphoric acid, or 10-15 mg of phosphoric acid.

[0049]

[0058] Providing a formulation lyophilized in the form of a vial enables simple transport and handling, as well as direct reconstitution into a formulation that can be easily administered to patients in need thereof.

[0050]

[0059] The lyophilized formulation can be reconstituted and diluted to obtain a composition in the form of a solution for intravenous injection.

[0060] As used in the present invention, the term "reconstitute" or "reconstitution" refers to the process by which a lyophilized formulation is converted to a liquid form by adding and mixing it with a pharmaceutically acceptable aqueous reconstitution solution, such as water for injection, sodium chloride solution or glucose solution.

[0051]

[0061] The present invention described herein provides an intravenous drip containing trabectedin, amino acids, a buffer and water for injection. The present invention further provides a method for preparing an intravenous drip, comprising the steps of preparing a lyophilized formulation, reconstituting the lyophilized formulation in an aqueous system, and diluting the reconstituted formulation to a concentration suitable for intravenous drip using an aqueous infusion medium.

[0052]

[0062] The actual amount of the reconstitution solution does not limit the features of the embodiments of the present invention. By way of non-limiting example, embodiments of the lyophilized formulation according to the present invention are reconstituted with a volume of water. Most such volumes do not exceed about 20 mL, and preferred volumes range from about 1 mL to about 15 mL, or from about 1 mL to about 10 mL, or about 5 mL. The reconstituted solution in such embodiments contains a concentration of trabectedin of about 0.05 mg / mL, about 0.1 mg / mL, or about 0.15 mg / mL.

[0053]

[0063] The reconstituted solution may, if desired, be further diluted. This further dilution can be carried out with an aqueous infusion medium, which is usually 0.9% sodium chloride or 5% glucose. The reconstituted solution will be diluted depending on the concentration in the reconstituted solution and the desired concentration in the diluent.

[0054]

[0064] In one aspect, the intravenous drip infusion is used in the treatment of cancer. In one aspect, the cancer is selected from the group of sarcomas, such as leiomyosarcoma, liposarcoma, osteosarcoma; ovarian cancer, breast cancer, melanoma, colorectal cancer, mesothelioma, renal cancer, endometrial cancer and lung cancer, or any combination thereof, and conditions having a plurality of such forms of cancer. It is understood that in this context "treatment" refers to an act that brings about remission of the cancerous state(s). Embodiments of the formulation according to the invention can also advantageously be used for the treatment of treatment-resistant cancerous states that do not respond to other treatments. In one embodiment, the intravenous drip infusion is used for the treatment of adult patients with progressive soft tissue sarcoma.

[0055]

[0065] Trabectedin can be used in combination with another drug. For example, trabectedin can be administered together with another anti-tumor drug. Examples of such other drugs include doxorubicin, cisplatin, paclitaxel, carboplatin, pegylated liposomal doxorubicin, docetaxel, capecitabine, and gemcitabine. Drugs having other mechanisms of action can be used, including dexamethasone. The administration of other drugs can be before, during or after the administration of trabectedin.

[0056]

[0066] In another embodiment, the intravenous drip infusion described herein is used for the treatment of patients with recurrent platinum-sensitive ovarian cancer, and the treatment is combined with pegylated liposomal doxorubicin (PLD).

[0057]

[0067] Specifically, the intravenous drip infusion described in this specification is administered intravenously based on cycles, for example, 1 to 20 cycles. This cycle usually includes a phase of injecting trabectedin and also a phase of not injecting trabectedin. Usually, this cycle is carried out on a weekly basis. Therefore, this cycle generally includes a phase of injecting trabectedin for 1 or several weeks and 1 or several weeks to complete this cycle. A 3-week cycle is preferred, but alternatively, this can be 1 to 6 weeks. The injection phase itself can be, for example, a single administration per cycle of 1 to 72 h, more usually about 1, 3 or 24 h; or daily injection in an injection phase of a cycle of 1 to 5 h, particularly 1 or 3 h; or injection on a weekly basis in an injection phase of a cycle of 1 to 3 h, particularly 2 or 3 h. A single administration at the start of each cycle is preferred. Specifically, the injection time is about 1, 3 or 24 h.

[0058]

[0068] Exemplary dosing protocols for intravenous trabectedin formulations are described, for example, in WO2006046079. Such dosing protocols include the following. a) Administered as an intravenous drip infusion over 24 h at 3-week intervals between cycles at a body surface area of about 1.5 mg / m 2 ; b) Administered as an intravenous drip infusion over 3 h at 3-week intervals between cycles at a body surface area of about 1.3 mg / m 2 ; c) Administered weekly as an intravenous drip infusion over 3 h for 3 weeks, with a 1-week rest. 2

Example

[0059]

[0069] The following examples are described to assist in understanding the present invention, but are not intended to limit the scope of the present invention in any way and should not be construed as limiting. The examples do not include a detailed description of conventional methods, for example, quantification by high performance liquid chromatography (HPLC). Such methods are well known to those skilled in the art. Example 1 - Variants of the formulation

[0070] Variants of the formulation for travethedine were shown as shown in Table 1. Different bulking agents were used. For example, "0.1M Arg" refers to L-arginine at a concentration of 0.1 mol / L.

[0060]

Table 1

[0061] Example 2 - Short-term stability of the solution before lyophilization

[0071] In total, 9 solutions before lyophilization were prepared. Variants of the formulation were prepared by pre-weighing approximately 10 mg of travethedine in a 50 mL glass container and adding the calculated amount of solvent to reach the target concentration of travethedine of 0.25 mg / mL. The solution before lyophilization was mixed with a roller mixer at room temperature (22 °C) for approximately 30 minutes. The solution was filtered using a 0.2 μm syringe filter (Millipore, Fluorodyne® II membrane) and filled into clean and depyrogenized 10R glass vials (1 mL per vial).

[0062]

[0072] The stability of a liquid formulation of trabectedin (0.25 mg / mL) was tested in short-term (0 h and 24 h) stability experiments at 2 - 8 °C and 25 °C. Some dispersions in the trabectedin content were observed by RP-HPLC analysis between variants of different formulations (see Table 4 for parameters), but the differences between stressed (at 2 - 8 °C or 25 °C for 24 h) and non-stressed (0 h) samples were minor (see Table 2). Table 2 shows the results of the trabectedin stability assay. The content of trabectedin, as determined by RP-HPLC (detected at 285 nm), is shown as % of the reference. Purity is analyzed as relative peak area. The amount of impurities shown in Table 2 was determined. The amount was determined as relative peak area (%). Most impurities were detected only in minor amounts (Table 2). The temperature dependence of trabectedin stability could be observed. The low pH variants seemed to be significantly more stable compared to their high pH counterparts.

[0063]

Table 2

[0064] Example 3 - Lyophilization

[0073] The filled vials were stoppered in the lyophilization position, loaded into a stainless steel tray, and wrapped in a LyoProtect® bag (a bag with a steam-permeable PTFE membrane to prevent contamination of the lyophilizer). Two pilot lyophilizers (Hof Sonderanlagenbau, Lohra, Germany) were used (GT2, shelf area 0.36 m 2 and GF3, shelf area 0.25 m 2 ). For several variants of different formulations freeze-dried in one run of lyophilization (Run 1: Formulation variants 1 to 4, Run 2: Formulation variants 8 to 9), general conservative lyophilization parameters were selected (Table 3).

[0065]

Table 3

[0066]

[0074] All variants of the formulations were suitable for lyophilization. Most variants showed some increase in impurities during lyophilization (especially RRT 1.32 (A285nm)), indicating sensitivity to trabectedin.

[0067]

[0075] To further distinguish the variants of the formulations, the stability of the lyophilized samples of variants 1 - 4 and 8 - 9 of all formulations was evaluated in an accelerated stability test. Example 4 - Short - term stability test of lyophilized samples

[0076] Samples obtained in feasible lyophilization were subjected to stability tests at three temperatures: 2 - 8 °C, 25 °C, and 40 °C. The samples were analyzed for appearance, reconstitution rate, degradation profile by RP - HPLC, and residual water content using the general Karl Fischer oven method (for T0 only) immediately after lyophilization (T0), 1 month (T1), 2 months (T2), 3 months (T3), and 6 months (T6) of the storage period.

[0068]

[0077] Samples of each variant of the formulation were stored and analyzed. All variants of the formulations formed solid cakes, which remained visually stable during the test period. Reconstitution in 1 mL of water for injection was rapid for all variants at all time points and occurred spontaneously within 10 seconds. Content and purity of the lyophilized product

[0078] The reconstituted lyophilized product was analyzed by RP - HPLC (see Table 4 for parameters) to determine the trabectedin content and impurity profile. The results for storage at 2 - 8 °C are summarized in Figures 2 and 3.

[0069]

Table 4

[0070]

[0079] The formulation containing L-arginine in citric acid containing H3PO4 for pH adjustment to pH 4.8 (Formulation No. 4, see Tables 1 and 5) shows very good stability results even at a storage temperature of 40°C.

[0071]

Table 5

[0072] Example 5 - 3-Month Stability Test

[0080] Variants of three different formulations (arginine phosphate pH 3.0, pH 3.4, and pH 3.8, see Tables 1 and 5) were prepared, lyophilized, and analyzed over time with respect to assay and impurity profile. Details of the experiment

[0081] For the stability test, lyophilized samples of variants of the trabectedin formulation were stored at 2 - 8°C, 25°C, and 30°C. A placebo solution without trabectedin was stored at 25°C and used as a reference sample for HPLC analysis. The analysis time points were after 1 month and 3 months (see Tables 6 and 7).

[0073]

Table 6

[0074]

Table 7

[0075] Example 6 - 6-Month Stability Test Preparation of the lyophilized solution

[0082] Twelve different variants of the formulation (see Table 8) were prepared, lyophilized, and analyzed over time with respect to trabectedin content and purity profile. Each formulation contained 0.25 mg / mL of trabectedin before lyophilization.

[0076]

Table 8

[0077]

[0083] Variants of the selected formulation were prepared by pre-weighing approximately 12.5 mg of trabectedin in a 50 mL glass vial and adding the calculated amount of solvent to reach a target concentration of trabectedin of 0.25 mg / mL.

[0078]

[0084] Each of the 12 formulation variants (buffer without API) was prepared by individually weighing the corresponding substances into a beaker, dissolving them in 90% of the specified volume of purified water, adjusting the pH with sodium hydroxide solution (30%) or hydrochloric acid solution (25%) in the case of variant 7, and ortho-phosphoric acid (85%) in the case of the other variants, and then adding purified water to the final volume. Subsequently, the calculated amount of solvent (buffer) was added to the pre-weighed trabectedin in 50 mL glass vials to give a target concentration of trabectedin of 0.25 mg / mL. The buffer volume for dilution was adapted to the mass actually weighed for 0.25 mg / mL for each variant. The solutions were stirred with a magnetic stirrer at room temperature (22 °C) for approximately 30 minutes until completely dissolved. The dissolved solutions were filtered using a 0.2 μm syringe filter (Millipore, Fluorodyne® II membrane) and filled into clean and pyrogen-free 10R glass vials (1 mL per vial). Lyophilization

[0085] The filled vials were partially stoppered in the lyophilization position, placed in a stainless steel tray, and wrapped and sealed in a LyoProtect® bag (a bag with a steam-permeable PTFE membrane to prevent contamination of the lyophilizer). The vials were loaded into a lyophilizer and lyophilized.

[0079]

[0086] After lyophilization, the vials were degassed with nitrogen up to 750 mbar and the vials were closed. After removal, the vials were crimp-sealed and decontaminated with 70% isopropanol / water. Storage

[0087] The lyophilized samples were stored at 25 °C for up to 6 months, · Appearance, · Reconstitution behavior; · Transparency of the solution (turbidity in nephelometric analysis); · pH measurement after reconstitution; · Degradation profile by RP-HPLC; and · Residual water content using the general Karl Fischer oven method (T0 only) were analyzed at immediately after lyophilization (T0), 1 month (T1m), 3 months (T3m) and 6 months (T6m) after the storage period. Reconstitution

[0088] The co-stopper was removed from the vial and the lyophilized product was reconstituted with 5.0 mL of purified water using a pipette; the reconstitution rate and behavior were monitored. Analysis of residual water content by the general Karl Fischer oven method

[0089] The remaining moisture of the sample was determined by Karl Fischer titration using a 756 Karl Fischer coulometer equipped with a 774 oven sample processor (Metrohm).

[0080]

[0090] For each measurement, one lyophilized product of each sample (approximately 20 - 50 mg) was transferred to a Karl Fischer vial. The exact weight was recorded. The vial was sealed with a crimp cap and transferred to the oven of a Karl Fischer coulometer heated to 110 °C. The septum of the crimp cap was pierced with a syringe needle and the generated water vapor was directly transferred to the titration chamber by dry nitrogen. An empty glass vial was used as a blank correction. The sample was analyzed twice. pH measurement

[0091] The pH of the sample was measured directly in the vial after reconstitution (5 mL of purified water) using a general pH meter. pH meter: SevenMulti with a microelectrode, Mettler Toledo.

[0081] The pH meter was calibrated at pH 4.0, pH 7.0 and pH 9.0 before use. Content and purity by RP-HPLC

[0092] RP-HPLC was performed according to Table 4.

[0082]

[0093] The detection of impurities was carried out at the wavelengths of two different detectors (285 nm and 255 nm) to include all possible impurities. It was observed that some impurities showed higher absorption at one of the wavelengths than the other. Therefore, the evaluation approaches at two different wavelengths support the complete scope of all relevant impurities. Sample preparation:

[0094] For RP-HPLC analysis, the lyophilized product was reconstituted with 1 mL of purified water. No further sample dilution for HPLC analysis was required. Content and purity of the lyophilized product

[0095] The reconstituted lyophilized product was analyzed by RP-HPLC to determine the trabectedin content and purity profile. These results are summarized in Figures 13 and 14. Figure 13 shows the content of trabectedin at T0, T1m, T3m and T6m measured by reference RP-HPLC (%). All samples showed a stable trabectedin content over that period. Figure 14 shows the purity of the samples as the relative peak area of trabectedin analyzed by RP-HPCL. All samples showed a high purity profile over that period. Impurity profile of the sample

[0096] The samples were stored at 25 °C and tested after 1, 3 and 6 months. 25 °C takes into account the accelerated test conditions, thereby enabling the evaluation of the stability of the formulation. The long-term storage conditions are most likely to be storage in a refrigerator (2 - 8 °C).

[0083]

[0097] The impurities of the samples were analyzed and the results are shown in the following table.

[0084]

Table 9

[0085]

Table 10

[0086]

Table 11

[0087]

Table 12

[0088]

Table 13

[0089]

Table 14

[0090]

Table 15

[0091]

Table 16

[0092]

Table 17

[0093]

Table 18

[0094]

Table 19

[0095]

Table 20

Claims

**Claim 1** A composition comprising trabectedin and an amino acid, wherein the weight ratio of trabectedin to amino acid (w / w) is from 1:50 to 1:100, and the amino acid is L-arginine. **Claim 2** The composition according to claim 1, wherein the weight ratio of trabectedin to amino acid (w / w) is about 1:

70. **Claim 3** The composition according to claim 1 or 2, further comprising a buffering agent. **Claim 4** The composition according to claim 3, wherein the buffering agent is selected from the group consisting of citric acid, phosphoric acid, acetic acid, basic amino acids, and sodium hydroxide, or any mixture thereof, or a mixture of citric acid, phosphoric acid, and optionally basic amino acids. **Claim 5** The composition according to any one of claims 1 to 4, further comprising a further substance selected from the group consisting of a complexing agent such as ethylenediaminetetraacetic acid (EDTA); an antioxidant such as monothioglycerol; a surfactant such as polysorbate; and ascorbic acid. **Claim 6** A lyophilized formulation comprising trabectedin and an amino acid, wherein the weight ratio of trabectedin to amino acid (w / w) is from 1:50 to 1:100, and the amino acid is L-arginine. **Claim 7** The lyophilized formulation according to claim 6, wherein the weight ratio of trabectedin to amino acid (w / w) is about 1:

70. **Claim 8** The lyophilized formulation according to claim 6 or 7, wherein the composition further comprises a buffering agent. **Claim 9** The lyophilized formulation according to claim 8, wherein the buffering agent is selected from the group consisting of citric acid, phosphoric acid, acetic acid, basic amino acids, and sodium hydroxide, or any mixture thereof, or a mixture of citric acid, phosphoric acid, and optionally basic amino acids. **Claim 10** The lyophilized formulation according to any one of claims 6 to 9, wherein the composition further comprises a further substance selected from the group consisting of a complexing agent such as ethylenediaminetetraacetic acid (EDTA); an antioxidant such as monothioglycerol; a surfactant such as polysorbate; and ascorbic acid. **Claim 11** The lyophilized formulation according to any one of claims 8 to 10, wherein the buffering agent is citric acid or phosphoric acid, or a mixture thereof. **Claim 12** The lyophilized formulation according to any one of claims 6 to 11, provided in the form of a vial. **Claim 13** The vial contains 0.1 to 1 mg of trabectedin, 10 to 60 mg of L-arginine, 0.5 to 8 mg of citric acid, and 5 to 40 mg of phosphoric acid, and is the lyophilized formulation according to claim 12.

14. The vial contains 0.25 mg of trabectedin, 17.4 mg of L-arginine, 1.9 mg of citric acid, and 10 to 15 mg of phosphoric acid, and is the lyophilized formulation according to claim 13.

15. An intravenous drip solution containing trabectedin, an amino acid, a buffer, and water for injection, wherein the weight ratio (w / w) of trabectedin to the amino acid is 1:50 to 1:100, and the amino acid is L-arginine.

16. The intravenous drip solution according to claim 15, for use in the treatment of cancer.

17. The intravenous drip solution according to claim 16, wherein the cancer is a sarcoma selected from the group consisting of leiomyosarcoma, liposarcoma, and osteosarcoma, ovarian cancer, breast cancer, melanoma, colorectal cancer, mesothelioma, renal cancer, endometrial cancer, lung cancer, or any combination thereof.

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