Composition comprising trabectedin and amino acids

Abstract

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

Description

Technical Field

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

[0002] Trabectedin (ecteinascidin or ET-743) is a tetrahydroisoquinoline alkaloid originally isolated from the Caribbean tunicate Ecteinascidia turbinata with antitumor activity.

[0003] Trabectedin has limited aqueous solubility and thermal stability, posing challenges in developing formulations for medical purposes. Trabectedin is particularly effective in the treatment of sarcomas and ovarian cancer.

[0004] WO 2000069441 discloses the use of trabectedin in the preparation of a pharmaceutical composition for treating human cancer. The pharmaceutical composition is formulated as a lyophilized product containing mannitol as a bulking agent and a pH 4 phosphate buffer to stabilize trabectedin. The formulation is administered by intravenous infusion.

[0005] WO 2006046079 discloses disaccharide-stabilized ecteinascidin formulations. WO 2006046079 describes a composition comprising an ecteinascidin (such as ET-743) and a disaccharide (such as sucrose) which is lyophilized and reconstituted to obtain an intravenous infusion solution.

[0006] Trabectedin is currently formulated as a sterile lyophilized product supplied in 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] WO 2017133544 A1 relates to a stable trabectedine formulation, which may contain glucose as a first excipient, and hydroxyethyl starch, dextran, sodium carboxymethyl cellulose, or hydroxypropyl β-cyclodextrin as a second excipient.

[0008] IN 201741041173 discloses a stable trabectedin pharmaceutical composition. A formulation containing trabectedin and L-arginine at a ratio of 1:400 is exemplified. Such a high amino acid content results in a higher solution volume for lyophilization, leading to a longer drying cycle. Furthermore, high levels of auxiliary additives may lead to negative effects on patients.

[0009] Given the importance of trabectedin as a chemotherapeutic agent, there remains a need for improved formulations of trabectedin that are biocompatible, stable, and suitable for medical purposes. Summary of the Invention

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

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

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

[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.

[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.

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

[0017] In one aspect, the compositions described herein comprise additional substances selected from the group consisting of complexing agents such as ethylenediaminetetraacetic acid (EDTA); antioxidants such as monothioglycerol; surfactants such as polysorbates; mannitol; and ascorbic acid.

[0018] In the preparation according to the present invention, the main source of pH control is buffer.Usually, buffer exists in the form of acid or alkali and its conjugate base or acid respectively.In one embodiment, the scope of buffer salt is 1-100mM, preferably 5-50mM, most preferably about 10mM (in solid preparation, the amount of buffer is selected to produce this concentration after reconstruction / dilution).The concentration of buffer and the pH value of solution are advantageously selected to provide the best balance of solubility and stability.The example of suitable buffer comprises the mixture of the alkali metal salt (such as sodium, potassium) of weak acid and weak acid conjugate base, such as sodium citrate and disodium hydrogen phosphate.

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

[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.

[0022] In another aspect, the lyophilized formulation is provided in a vial. In an exemplary embodiment, 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. In a specific embodiment, 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.

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

[0024] In one aspect, the intravenous infusion solution is used to treat cancer. The cancer to be treated can be a sarcoma selected from the group consisting of leiomyosarcoma, liposarcoma, osteosarcoma, ovarian cancer, breast cancer, melanoma, colorectal cancer, mesothelioma, renal cancer, endometrial cancer, and lung cancer, or any combination thereof.

[0025] In a further embodiment, the intravenous infusion described herein is used to treat an adult patient with advanced soft tissue sarcoma.

[0026] In one embodiment, the intravenous solution is used to treat patients with recurrent platinum-sensitive ovarian cancer. This treatment can be combined with pegylated liposomal doxorubicin (PLD) BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : Purity of short-term stability samples stored at 5°C (upper panel) and 22°C (lower panel) for up to 29 hours.

[0028] Figure 2 : Stability of the lyophilizate at 2-8°C: Trabectedin content (A285 nm) determined by RP-HPLC, expressed as % of the reference substance (0.25 mg / mL).

[0029] Figure 3 : Stability of the lyophilizate at 2-8°C: Purity of the samples expressed as the relative peak area of ​​the trabectedin peak by RP-HPLC analysis (A285 nm). DETAILED DESCRIPTION

[0030] The present invention relates to a composition comprising trabectedin and at least one amino acid. The preparation comprises trabectedin as an active substance and can be formulated into a lyophilized preparation that can be reconstituted to obtain an intravenous infusion solution.

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

[0032] Known formulations of trabectedin use sugars (e.g., WO 2017133544 A1) to obtain stable formulations. However, when used in pharmaceutical formulations, sugars may exhibit some unfavorable properties. First, glucose and sucrose cannot be administered to diabetic patients without taking into account additional carbohydrate intake. Diabetic patients need to control their intake of glucose-producing carbohydrates and their hypoglycemic drugs to keep their blood sugar levels within certain limits. Therefore, pharmaceutical formulations containing glucose or sucrose are not optimal for this particular patient population.

[0033] In addition, saccharides are also the source of specific degradation products of trabectedin to be formed. All saccharides that can form hemiacetal structures in solution (i.e., all "reducing" sugars) can react with hydroxyl groups to form stable acetals and water (Jerry March, Advanced Organic Chemistry, 3rd edition. Pages 789-790; John Wiley & Sons). The reaction product acetal and water maintain equilibrium with pharmaceutical compounds, such as trabectedin and its hydroxyl groups. When water is removed (e.g., after freeze-drying), the equilibrium moves to the acetal side, thereby forming the reaction product of the relevant amount.

[0034] Trabectedin, whose structure shows such hydroxyl groups, is therefore susceptible to forming acetals with reducing sugars such as glucose or lactose during freeze-drying.

[0035] This specific degradation pathway was observed in a lyophilized formulation of trabectedin containing glucose. When the formulation was freeze-dried, the formation of trabectedin-glucose acetal was observed. Although the acetal was hydrolyzed when the lyophilized material was reconstituted, it still existed in the reconstituted solution for several hours (for example, the relative level was about 0.5% after 6 hours). It can be detected by HPLC. The HPLC method used is the same as the method described in the preparation screening section. Degradation ultimately leads to a balance with a fairly low but still detectable acetal impurity level. The application of drugs containing degradation products of unknown toxicological properties to patients may be problematic from a toxicological point of view as well as from a dosage accuracy point of view, because part of the trabectedin dose is lost in the acetal and cannot be used for treatment.

[0036] This side effect of trabectedine formulations containing reducing sugars (e.g., glucose or lactose) can be overcome by simply waiting after the infusion solution is prepared until most of the acetal has been hydrolyzed before starting the infusion. However, in hospital practice, this is a time-consuming and less than ideal process that should be avoided. Therefore, formulations that do not show such degradation products are advantageous.

[0037] Furthermore, the formulation disclosed in IN 201741041173 primarily utilizes a combination of monosaccharides and sugar alcohols to prepare a stable trabectedin pharmaceutical composition. Alternatively, amino acids can also be used as excipients. A formulation comprising trabectedin and L-arginine at a ratio of 1:400 is exemplified.

[0038] The inventors were surprised to find that even small amounts of amino acids can stabilize the trabectedin composition without producing trabectedin degradation products. In this article, the term "stability" should be understood as defined by the Pharmaceutical Process Engineering Working Group (APV), according to which "stability" refers to the quality of the drug product as specified at the end of the manufacturer's specified period. The quality of the drug is determined by the content and purity of the active substance, and the perceived physicochemical and microbiological properties, so the content of the active substance should not be less than 90% of the claimed value at the end of the period.

[0039] The preparations produced according to the present invention have a stability (or in other words, shelf life or run 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 at the end of the period is not less than 90%, preferably 95%, of the trabectedin initially used.

[0040] The term "amino acid" refers to naturally occurring amino acids, including proteinogenic and non-proteinogenic amino acids, synthetic amino acids, and amino acid analogs, which, if their structure allows for stereoisomerization, are all D and L stereoisomers. Amino acids include non-polar, polar, basic, and acidic amino acids. Non-polar 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.

[0041] In the context of the present invention, the amino acid may be a non-polar, polar, basic or acidic amino acid, or any combination thereof. According to one embodiment, the amino acid is selected from the group of non-polar proteinogenic amino acids, in particular L-phenylalanine or L-isoleucine, or any combination thereof.

[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 can be combined with one or more other amino acids, such as L-phenylalanine or L-isoleucine.

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

[0044] In embodiments described herein, the ratio of trabectedin to amino acid is determined according to the solubility of amino acid, and when the formulation is freeze-dried, it is also determined according to the freeze-drying ability of 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 certain 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 a specific embodiment, the weight ratio is about 1: 70.

[0045] As used herein, the term "about" refers to a value that is within + / - 10% of the stated value.

[0046] In specific aspects, the compositions described herein also include a buffer. The buffer allows the pH value of the system to be maintained in a range that is conducive to 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, ascorbate buffer, tartaric acid / citrate buffer, bicarbonate / hydrochloric acid buffer, acetate or acetate buffer, succinate buffer, glycine / hydrochloric acid buffer. In certain embodiments, an amino acid-based buffer can be used, for example, a buffer based on an amino acid already present in the composition. A mixture of the buffers can also be used.

[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 as a buffer, such as L-arginine. In addition, sodium hydroxide can be used for pH adjustment.

[0048] The composition may include other ingredients such as chelating agents, such as ethylenediaminetetraacetic acid (EDTA); antioxidants, such as monothioglycerol; surfactants; mannitol; and ascorbic acid. Examples of surfactants include polysorbates, polyoxyethylene (20) sorbitan monooleate or polyoxyethylene stearate, phospholipids (such as lecithin); polyoxyethylene-polyoxypropylene copolymers, such as Pluronic surfactants; polyoxyethylene esters of 12-hydroxystearic acid, such as Solutol surfactants; cholesterol ethoxylates, such as diacylglycerols, dialkylglycerols; bile salts, such as sodium cholate, sodium deoxycholate; sucrose esters, such as sucrose monolaurate, sucrose monooleate; polyvinylpyrrolidone (PVP); or polyvinyl alcohol (PVA).

[0049] The compositions described herein may be in the form of a lyophilized preparation. The term "lyophilized preparation" as used herein refers to a preparation prepared by lyophilizing (freeze drying) a mixture comprising a pharmaceutically effective amount of trabectedin. The inventors were surprised to find that using one or more amino acids as fillers during the lyophilization process improves storage conditions and allows the lyophilized preparation to be stored for a long time in a wide temperature range including refrigerated conditions and room temperature.

[0050] Therefore, the present invention provides a lyophilized formulation comprising trabectedin, at least one amino acid, a buffer and optionally a bulking agent such as mannitol. Amino acids can serve as bulking agents.

[0051] The term "filler" as used herein refers to a compound that adds mass to the freeze-dried (freeze-dried) mixture and contributes to the physical structure of the freeze-dried mass (e.g., promoting the production of substantially uniform freeze-dried masses that maintain an open-pore structure). In addition to providing pharmaceutically excellent masses, fillers can also impart useful qualities in terms of changing the disintegration temperature, providing freeze-thaw protection, and enhancing stability for long-term storage. Exemplary fillers include mannitol, glycine, lactose, sucrose, glucose, and hydroxyethyl starch. Fillers can be crystalline (e.g., glycine or mannitol) or amorphous (e.g., dextran or hydroxyethyl starch).

[0052] In specific embodiments, the amino acids in the lyophilized formulations described herein are non-polar, polar, basic, or acidic amino acids, or any combination thereof. The amino acid can be a non-polar amino acid, such as L-phenylalanine or L-isoleucine; or a basic amino acid, such as L-arginine, L-lysine, or L-histidine; or any combination thereof. Specifically, the amino acid is L-arginine. In one aspect, L-arginine can be combined with one or more other amino acids, such as L-phenylalanine or L-isoleucine. In another embodiment, the amino acid is citrulline. In another embodiment, the amino acid is acetylcysteine.

[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 (citrate / phosphate buffer). Amino acids can also act as buffers together with bases.

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

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

[0056] Lyophilized preparations are usually contained in vials containing a specific amount of trabectedin. For example, the amount of trabectedin in the vial is 0.25 mg or 1 mg. In order to provide a vial containing a lyophilized preparation, a pre-lyophilized solution is added to the vial and freeze-dried. The volume of the pre-lyophilized material added to the vial is in the range of 1 mL to 5 mL, or 1 to 4 mL.

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

[0058] Providing the lyophilized formulation in a vial allows for simple shipping and handling, as well as direct reconstitution into a formulation that can be easily administered to a patient in need thereof.

[0059] The lyophilized formulation can be reconstituted and diluted to provide a composition in the form of a solution that is easily injectable intravenously.

[0060] The term "reconstitute" or "reconstitution" as used herein refers to the process of converting a lyophilized formulation into liquid form by adding and mixing with a pharmaceutically acceptable aqueous reconstitution solution, such as water for injection, sodium chloride solution or glucose solution.

[0061] The invention described herein provides an intravenous infusion solution comprising trabectedin, an amino acid, a buffer, and water for injection. The invention further provides a method for preparing the intravenous infusion solution, comprising providing a lyophilized formulation, reconstituting the lyophilized formulation in an aqueous system, and diluting the reconstituted formulation with an aqueous infusion medium to a concentration suitable for intravenous infusion.

[0062] The actual amount of the reconstituted solution is not a limiting feature of the embodiments of the present invention. By way of illustration, but not limitation, embodiments of the lyophilized formulations according to the present invention are reconstituted with a volume of water. Most such volumes do not exceed about 20 mL, with preferred volumes ranging from about 1 mL to about 15 mL, or from about 1 mL to about 10 mL, or about 5 mL. In such embodiments, the reconstituted solution contains trabectedin at a concentration of about 0.05 mg / mL, about 0.1 mg / mL, or about 0.15 mg / mL.

[0063] If necessary, the reconstituted solution may be further diluted. This further dilution can be performed with an aqueous infusion medium, typically 0.9% sodium chloride or 5% dextrose. The reconstituted solution will be diluted based on the concentration in the reconstituted solution and the desired concentration in the dilution solution.

[0064] In one aspect, the intravenous infusion is used to treat cancer. In one aspect, the cancer is a sarcoma selected from the group consisting of leiomyosarcoma, liposarcoma, osteosarcoma; ovarian cancer, breast cancer, melanoma, colorectal cancer, mesothelioma, renal cancer, endometrial cancer, and lung cancer, or any combination thereof, as well as conditions having multiple such forms of cancer. It should be understood that "treating" herein refers to the act of causing an improvement in the cancer condition. Embodiments of the formulations according to the present invention can also be used to treat refractory cancer conditions that have not responded well to other treatments. In one embodiment, the intravenous infusion is used to treat adult patients with advanced soft tissue sarcoma.

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

[0066] In another embodiment, the intravenous infusion described herein is used to treat a patient with recurrent platinum-sensitive ovarian cancer, wherein the treatment is combined with pegylated liposomal doxorubicin (PLD).

[0067] Specifically, the intravenous infusion described herein is administered intravenously on a cycle basis, for example, 1 to 20 cycles. The cycle includes a phase in which the trabectedin infusion solution is infused, and typically also includes a phase in which trabectedin is not infused. Typically, the cycle is carried out in weeks, so the cycle typically includes one or more weeks of trabectedin infusion phases, and one or more weeks to complete the cycle. A 3-week cycle is preferred, but alternatively can also be 1 to 6 weeks. The infusion phase itself can be a single dose in each cycle, for example 1 to 72h, more typically about 1, 3 or 24h; or 1 to 5h, particularly 1 or 3h, infused daily during the infusion phase of the cycle; or 1 to 3h, particularly 2 or 3h, infused weekly during the infusion phase of the cycle. Preferably, a single dose is administered at the beginning of each cycle. Specifically, the infusion time is about 1, 3 or 24h.

[0068] For example, an exemplary dosing regimen for intravenous trabectedine is described in WO 2006046079. This dosing regimen includes:

[0069] a) About 1.5 mg / m 2 body surface area, administered as an intravenous infusion over 24 hours with three-week intervals between cycles;

[0070] b) About 1.3 mg / m 2 body surface area, administered as an intravenous infusion over 3 hours with three-week intervals between cycles;

[0071] c) about 0.580 mg / m 2 body surface area, with weekly intravenous infusions for 3 hours during 3 weeks and 1 week of rest.

[0072] Example

[0073] The following examples are intended to help understand the present invention and are not intended to nor should be construed as limiting the scope of the present invention in any way. The examples do not include a detailed description of conventional methods, such as quantitative analysis by high pressure liquid chromatography (HPLC). These methods are well known to those of ordinary skill in the art.

[0074] Example 1 - Formulation Variants

[0075] Formulation variations of trabectedin are provided as shown in Table 1. Different bulking agents are used. For example, "0.1 M arginine" refers to L-arginine at a concentration of 0.1 mol / L.

[0076] Table 1: Formulation variants

[0077] # Fillers buffer pH adjustment pH 1 0.1M arginine, 0.1M phenylalanine 10 mM citric acid <![CDATA[H3PO4]]> 4.8 2 0.1M arginine, 0.1M isoleucine 10 mM citric acid <![CDATA[H3PO4]]> 4.8 3 0.1 M arginine 10 mM citric acid L-Aspartic Acid 4.8 4 0.1 M arginine 10 mM citric acid <![CDATA[H3PO4]]> 4.8 5 0.1 M arginine 10 mM citric acid <![CDATA[H3PO4]]> 3.8 6 0.1 M arginine 10 mM citric acid <![CDATA[H3PO4]]> 3.4 7 0.1 M arginine 10 mM citric acid <![CDATA[H3PO4]]> 3.0 8 0.1M arginine, 0.1M phenylalanine 10 mM citric acid NaOH 3.0 9 0.1M arginine, 0.1M phenylalanine 10 mM citric acid NaOH 3.8

[0078] Example 2 - Short-term stability of pre-lyophilized solution

[0079] A total of 9 pre-lyophilized solutions were prepared. The formulation variants were prepared by pre-weighing approximately 10 mg of trabectedin in a 50 mL glass container and adding a calculated amount of solvent to obtain a target concentration of 0.25 mg / mL of trabectedin. The pre-lyophilized solutions were mixed on a drum mixer at room temperature (22° C.) for approximately 30 minutes. The lyophilized solutions were filtered using a 0.2 μm syringe filter (Millipore, II membrane) and filled (1 mL per bottle) in clean and depyrogenated 10R glass bottles.

[0080] In the short-term (0 hour and 24 hour) stability experiment at 2-8 ℃ and 25 ℃, the stability of the trabectedin liquid formulation (0.25mg / mL) was tested. Some differences in the content of trabectedin between different formulation variants were observed by RP-HPLC analysis (parameters are shown in Table 4), but the difference between the stressed (2-8 ℃ or 25 ℃, 24 hours) and unstressed (0 hour) samples was small (see Table 2). Table 2 shows the results of the stability analysis of trabectedin. The content of trabectedin measured by RP-HPLC (detected at 285nm) is given as % of the reference value. Purity analysis is relative peak area. The amount of impurities listed in Table 2 was determined. Amount is determined as relative peak area %. Most impurities were only detected in trace amounts (Table 2). The temperature dependence of the stability of trabectedin can be observed. Compared with the variants of high pH values, the variants of low pH values ​​seem to be more stable.

[0081] Table 2: Results of stability test: Contents of trabectedin and impurities determined by HPLC

[0082]

[0083]

[0084]

[0085] Example 3 - Freeze-drying

[0086] At the lyophilization location, filled vials are stoppered, loaded into stainless steel trays, and packaged in Bags (bags with a vapor permeable PTFE membrane to prevent contamination of the freeze dryer). Two pilot freeze dryers (Hof Sonderanlagenbau, Lohra, Germany) (GT2, 0.36 m 2 Storage area and GF3, 0.25m 2 Since several different formulation variants were freeze-dried in one freeze-drying run (Run 1: Formulation variants #1-#4, Run 2: Formulation variants #8-#9), general conservative freeze-drying parameters were selected (Table 3).

[0087] Table 3: Feasibility freeze-drying parameters

[0088]

[0089] *Primary drying was performed overnight without operator involvement. Recorded drying times ranged from 14 to 16 hours. Note that for process development purposes, shorter primary drying times may be sufficient.

[0090] All formulation variants were suitable for lyophilization. Most variants showed an increase in impurities during lyophilization (especially RRT 1.32 (A285 nm)), indicating the sensitivity of trabectedin.

[0091] To further differentiate between formulation variants, the stability of lyophilized samples of all formulation variants #1-4 and #8-9 were evaluated in an accelerated stability study.

[0092] Example 4 - Short-term stability study of freeze-dried samples

[0093] The samples obtained by feasibility lyophilization were subjected to stability testing at three temperatures: 2-8° C., 25° C., and 40° C. The samples were analyzed for visual appearance, reconstitution rate, RP-HPLC degradation profile, and residual moisture content using the universal Karl Fischer oven method (T0 only) immediately after lyophilization (T0), one month (T1), two months (T2), three months (T3), and six months (T6) of storage.

[0094] Samples of each formulation variant were stored and analyzed. All formulation variants formed a solid mass that remained visually stable during testing. Reconstitution in 1 mL of water for injection was rapid and spontaneous within 10 seconds for all variants at all time points.

[0095] Content and purity of lyophilized material

[0096] The reconstituted lyophilisate was analyzed by RP-HPLC (parameters see Table 4) to determine the trabectedin content and impurity profile. Figure 2 and Figure 3 Results for storage at 2-8°C are summarized.

[0097] Table 4: HPLC measurement parameters

[0098]

[0099] The formulation with L-arginine, citric acid (containing H3PO4 to adjust the pH to pH 4.8) (Formulation #4, see Tables 1 and 5) showed very good stability results even at a storage temperature of 40°C.

[0100] Table 5: Composition of Formulation #4 (100 mM L-Arginine)

[0101]

[0102]

[0103] Example 5 - 3-month stability study

[0104] Three different formulation variants (arginine-phosphate pH 3.0, pH 3.4 and pH 3.8, see Tables 1 and 5) were prepared and lyophilized and analyzed for assay and purity profiles over time.

[0105] Experimental details

[0106] In the stability study, lyophilized samples of the trabectedine formulation variants 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 1 month and 3 months later (see Tables 6 and 7).

[0107] Table 6: Impurities detected at 285 nm after storage at 2-8°C for 3 months

[0108] impurities #7 (Arginine pH 3.0) #6 (Arginine pH 3.4) #5 (Arginine pH 3.8) RRT 0.12[%] 0.16 0.29 0.36 RRT 1.16[%] 0.23 0.46 0.49 RRT 1.28[%] 0.10 0.15 0.16 RRT 0.73[%] 0.11 0.12 0.15

[0109] Table 7: Impurities detected at 285 nm after storage at 25°C for 3 months

[0110] impurities #7 (Arginine pH 3.0) #6 (Arginine pH 3.4) #5 (Arginine pH 3.8) RRT 0.12[%] 0.42 0.52 0.60 RRT 1.16[%] 0.22 0.42 0.47 RRT 1.28[%] 0.12 0.19 0.20 RRT 0.73[%] 0.64 0.44 0.48

[0111] Example 6 - 6-month stability study

[0112] Preparation of lyophilized solution

[0113] Twelve different formulation variants were prepared and lyophilized (see Table 8) and analyzed for trabectedin content and purity profiles over time. Each formulation contained 0.25 mg / mL trabectedin prior to lyophilization.

[0114] Table 8: Formulation variants

[0115]

[0116]

[0117] Selected formulation variants were prepared by pre-weighing approximately 12.5 mg of trabectedin in a 50 mL glass vial and adding a calculated amount of solvent to achieve a target concentration of 0.25 mg / mL of trabectedin.

[0118] Each of the 12 formulation variants (without API buffer) was prepared by weighing the corresponding substance into a beaker, dissolving in 90% of the specified volume of pure water, adjusting the pH with sodium hydroxide solution (30%) or hydrochloric acid solution (25%) for variant #7, and with orthophosphoric acid (85%) for the other variants, followed by adding pure water to the final volume. The calculated amount of solvent (buffer) was then added to the pre-weighed amount of trabectedin in a 50 mL glass bottle to obtain a target concentration of 0.25 mg / mL of trabectedin. The volume of buffer used for dilution was adapted to the actual weighed mass of 0.25 mg / mL for each variant. The solution was stirred on a magnetic stirrer at room temperature (22°C) for approximately 30 minutes until complete dissolution. The filtration was performed using a 0.2 μm syringe filter (Millipore, II membrane) and filled (1 mL per bottle) into clean and depyrogenated 10R glass bottles.

[0119] Freeze-dried

[0120] The filled vials are partially stoppered, loaded into stainless steel trays, packaged and sealed at the lyophilization location. The vials were placed in a freeze dryer and lyophilized.

[0121] After lyophilization, the vials were vented with nitrogen to 750 mbar and the vials were closed. After unloading, the vials were crimped and purged with 70% isopropanol / water.

[0122] store

[0123] The lyophilized samples were stored at 25°C for up to six months and analyzed immediately after lyophilization (T0), one month (T1m), three months (T3m), and six months (T6m) of storage period:

[0124] ■Visual appearance,

[0125] ■Refactoring behavior;

[0126] ■ Solution clarity (nephelometric turbidity);

[0127] ■ pH measurement after reconstitution;

[0128] ■ Analysis of degradation profiles by RP-HP1C; and

[0129] ■ Determination of residual moisture content using the universal Karl Fischer oven method (T0 only).

[0130] Refactoring

[0131] The stoppers were removed from the vials and the lyophilisate was reconstituted with 5.0 mL of purified water using a pipette; the reconstitution speed and behavior were monitored.

[0132] Residual moisture analysis by the universal Karl Fischer oven method

[0133] The residual moisture of the samples was determined by Karl Fischer titration using a 756 Karl Fischer coulometer equipped with a 774 oven sample processor (Metrohm).

[0134] For each measurement, transfer a lyophilized portion of each sample (≈20-50 mg) to a Karl Fischer vial. Record the exact mass. Seal the vial with a gland and transfer it to the oven of a Karl Fischer coulometer heated to 110°C. Use a syringe to penetrate the gland's septum, and the resulting water vapor is transferred directly to the titration chamber using dry nitrogen. An empty glass vial serves as a blank. Samples are analyzed in duplicate.

[0135] pH measurement

[0136] The pH of the samples was measured directly in the vials after reconstitution (5 mL purified water) using a common pH meter.

[0137] pH meter: Mettler Toledo, SevenMulti with microelectrode.

[0138] The pH meter was calibrated at pH 4.0, pH 7.0, and pH 9.0 before use.

[0139] RP-HPLC analysis of content and purity

[0140] RP-HPLC was performed according to Table 4.

[0141] Impurity detection was performed at two different detector wavelengths (285 nm and 255 nm) to cover all possible impurities. It was observed that some impurities showed higher absorption at one wavelength than at the other. Therefore, the evaluation method at two different wavelengths supports complete coverage of all relevant impurities.

[0142] Sample preparation:

[0143] For RP-HPLC analysis, reconstitute the lyophilizate with 1 mL of purified water. No further dilution of the sample is required for HPLC analysis.

[0144] Content and purity of lyophilized material

[0145] The reconstituted lyophilisates were analyzed by RP-HPLC to determine the content and purity profile of trabectedin. The results are summarized in Figures 13 and 14. Figure 13 shows the content of trabectedin measured by RP-HPLC at T0, T1m, T3m and T6m, expressed as % of reference value. Over time, all samples showed stable trabectedin content. Figure 14 shows the sample purity expressed as the relative peak area of ​​trabectedin analyzed by RP-HPLC. Over time, all samples showed a high purity profile.

[0146] Impurity profile of the sample

[0147] The samples were stored at 25°C and tested after 1, 3, and 6 months. 25°C is considered an accelerated testing condition, which allows for the assessment of formulation stability. Long-term storage conditions are most likely refrigerated storage (2–8°C).

[0148] The samples were analyzed for impurities and the results are listed in the table below.

[0149] Table 9 - Impurity Profile of #1 (0.1M arginine, 10mM citric acid, H3PO4)

[0150]

[0151] If >0.1%, report the average of two injections

[0152] Table 10 - Impurity Profile of #2 (0.1 M arginine, 35 mg / ml mannitol, 10 mM citric acid, H3PO4)

[0153]

[0154]

[0155] If >0.1%, report the average of two injections

[0156] Table 11 - Impurity Profile of #3 (0.1M arginine, 10mM sodium acetate, H3PO4)

[0157]

[0158] If >0.1%, report the average of two injections

[0159] Table 12 - Impurity Profile of #4 (0.1M L-acetylcysteine, 10mM citric acid, NaOH)

[0160]

[0161]

[0162] If >0.1%, report the average of two injections

[0163] Table 13 - Impurity Profile of #5 (0.1 M arginine, 0.2% (w / V) L-ascorbic acid, 10 mM citric acid, NaOH)

[0164]

[0165] If >0.1%, report the average of two injections

[0166] Table 14 - Impurity Profile of #6 (0.1M Valine, 10mM Citric Acid, H3PO4)

[0167]

[0168]

[0169] If >0.1%, report the average of two injections

[0170] Table 15 - Impurity Profile of #7 (0.1M L-Aspartic Acid, Lysine)

[0171]

[0172]

[0173] If >0.1%, report the average of two injections

[0174] Table 16 - Impurity Profile of #8 (0.1M L-citrulline, 10mM citric acid, NaOH)

[0175]

[0176] If >0.1%, report the average of two injections

[0177] Table 17 - Impurity Profile of #9 (0.1M arginine, 10mM monothioglycerol, 10mM citric acid, H3PO4)

[0178]

[0179] If >0.1%, report the average of two injections

[0180] Table 18 - Impurity Profile of #10 (0.1M Arginine, 0.2% (w / V) L-Methionine, 10mM Citric Acid, H3PO4)

[0181]

[0182] If >0.1%, report the average of two injections

[0183] Table 19 - Impurity Profile of #11 (0.1M Arginine, 0.2% (w / V) Tween 20, 10mM Citric Acid, H3PO4)

[0184]

[0185]

[0186] If >0.1%, report the average of two injections

[0187] Table 20 - Impurity Profile of #12 (0.1M Arginine, 0.1% (w / V) EDTA, 10mM Citric Acid, H3PO4)

[0188] .

Claims

1. A composition comprising trabectedin and an amino acid, wherein the weight ratio (w / w) of trabectedin to the amino acid is 1:50 to 1:100, wherein the amino acid is L-arginine.

2. The composition according to claim 1, wherein the weight ratio (w / w) of trabectedin to amino acid is 1:

70.

3. The composition of claim 1, wherein the composition further comprises a buffer.

4. The composition according to claim 3, wherein the buffer is selected from the group consisting of citric acid, phosphoric acid, acetic acid, a basic amino acid and sodium hydroxide, or any mixture thereof, or a mixture of citric acid, phosphoric acid and optionally a basic amino acid.

5. The composition according to any one of claims 1 to 4, wherein the composition comprises other substances selected from the group consisting of a complexing agent ethylenediaminetetraacetic acid (EDTA); an antioxidant monothioglycerol; a surfactant polysorbate; and ascorbic acid.

6. A lyophilized formulation comprising trabectedin and an amino acid, wherein the weight ratio (w / w) of trabectedin to the amino acid is 1:50 to 1:100, wherein the amino acid is L-arginine.

7. The lyophilized preparation according to claim 6, wherein the weight ratio (w / w) of trabectedin to amino acid is 1:

70. The lyophilized formulation according to claim 6 , wherein the lyophilized formulation further comprises a buffer.

9. The lyophilized formulation according to claim 8, wherein the buffer is selected from the group consisting of citric acid, phosphoric acid, acetic acid, a basic amino acid and sodium hydroxide, or any mixture thereof, or a mixture of citric acid, phosphoric acid and optionally a basic amino acid.

10. The lyophilized formulation according to claim 6, wherein the lyophilized formulation comprises other substances selected from the group consisting of a complexing agent ethylenediaminetetraacetic acid (EDTA); an antioxidant monothioglycerol; a surfactant polysorbate; and ascorbic acid. The lyophilized formulation according to claim 9 , wherein the buffer is citric acid or phosphoric acid or a mixture thereof.

12. The lyophilized formulation according to any one of claims 6 to 11, wherein the lyophilized formulation is provided in a vial.

13. The lyophilized formulation according to claim 12, wherein 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.

14. The lyophilized formulation according to claim 13, wherein 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.

15. A solution for intravenous infusion, comprising 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, wherein the amino acid is L-arginine.

16. Use of the solution for intravenous infusion according to claim 15 in the preparation of a medicament for treating cancer.

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

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