Pharmaceutical compositions containing p-boronated phenylalanine

Abstract

A pharmaceutical composition is disclosed, which may include boronated phenylalanine (BPA) or a pharma- ceutically acceptable salt thereof and a 2-hydroxyamine compound (2HA) or a pharma- ceutically acceptable salt thereof.

Description

[Technical field]

[0001] The present disclosure relates to pharmaceutical compositions and methods of producing the same. [Background technology]

[0002] Boron Neutron Capture Therapy (BNCT) is a form of non-invasive treatment for malignant tumors. In BNCT, a patient is injected with a drug capable of localizing to the tumor and carrying non-radioactive boron-10 atoms. When the drug is irradiated with low-energy thermal neutrons, it emits biologically destructive alpha particles and lithium-7 nuclei.

[0003] BNCT requires agents such as boronated phenylalanine (hereinafter sometimes referred to as "BPA") that can specifically localize to tumors. Such agents need to be stable, soluble, safe, and easily produced. However, the provision of such agents is complicated by, for example, the failure of certain chemistries to function with boron-10 containing compounds.

[0004] In order to facilitate administration of boron atoms to patients and facilitate delivery of boron atoms to cancer cells, it is necessary to prepare an aqueous solution of a suitable boron-containing compound. BPA has extremely low solubility in water at physiological pH, but can be solubilized in the presence of either a strong acid or a strong base. Various methods have been attempted to improve the solubility of BPA in water. In particular, it is known to solubilize BPA by increasing the pH with a base (such as sodium hydroxide), add a sugar (particularly fructose) to form a BPA-fructose complex, and then readjust the pH of the mixture with an acid.

[0005] However, the solubility of the BPA-fructose complex in water may be insufficient. For example, when preparing an aqueous solution of BPA complexed with fructose at room temperature for a patient weighing 60 kg, the volume of the solution may be at least about 1 L. Injecting such a large volume would impose a large physical burden on the patient, and it would take a long time to infuse the solution into the patient before neutron irradiation.

[0006] It would also be desirable for the BPA formulation to be freeze-dried and available to medical facilities as a powder. However, freeze-dried powders of the BPA-fructose complex have extremely low solubility in water at room temperature. Furthermore, it is known that the stability of the BPA-fructose complex is poor, and that aqueous solutions of BPA and fructose have a shelf life of only a few days.

[0007] Thus, there is a need for pharmaceutical compositions that have improved properties compared to known BPA compositions and formulations. Summary of the Invention

[0008] A pharmaceutical composition is disclosed, which may comprise boronated phenylalanine (BPA) or a pharma- ceutically acceptable salt thereof and a 2-hydroxyamine compound (2HA) or a pharma- ceutically acceptable salt thereof, wherein the 2HA is selected from a compound represented by any one of formulas I-II or a pharma- ceutically acceptable salt thereof, or any combination or mixture thereof: [ka] wherein R1 and R2 are each independently H or selected from hydroxy-C1-C6-alkyl, 2-hydroxyethyl, 2,3,4,5,6-pentahydroxyhexyl, carboxy-C1-C6-alkyl, acetyl, C1-C6-alkyl, sulfo-C1-C6-alkyl, 3-sulfopropyl, 2-hydroxy-3-sulfopropyl, 1-sulfo-2-propanyl, 2-sulfoethyl, 3-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]-C1-C6-alkyl, and 3-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]propyl; [ka] where n is 0 or 1; When n is 1, R3 is absent; when n is 0, R3 is selected from H, hydroxy-C1-C6-alkyl, 2-hydroxyethyl, carboxy-C1-C6-alkyl, acetyl, C1-C6-alkyl, sulfo-C1-C6-alkyl and 2-sulfoethyl. [Brief description of the drawings]

[0009] [Figure 1] Comparison of biodistribution of BPA-T and BPA-F in tumor-bearing mice 2 hours after administration. The graph shows box plots of 10B concentrations in all sampled tissues and scatter plots for each individual mouse for BPA-F at 30 g / L (F, n=6) and BPA-T at 30 g / L (T, n=6). 10B concentrations are shown on the y-axis (μg / kg=ppm, parts per million). Grey boxes indicate interquartile ranges (IQR) from the 25th percentile (Q1) to the 75th percentile (Q3). Whiskers indicate minimum (Q1-1.5xIQR) and maximum (Q3+1.5xIQR). Solid lines within boxes indicate median and dashed lines indicate mean. Muscle=buccinator. Saliv.gland=salivary gland. [Diagram 2]Tumor:tissue 10B ratios for BPA-T and BPA-F in tumor mice 2 hours post-dose. The graph shows box plots of tumor:tissue 10B ratios in all tissues sampled and scatter plots for each individual mouse for 30 g / L BPA-F (F, n=6) and 30 g / L BPA-T (T, n=6). Box plots are depicted as in Figure 1. Muscle=buccinator. Saliv.gland=salivary gland. [Diagram 3] Time series of BPA-T and BPA-F 1-8 hours post-dose. 10B concentrations decreased similarly as a function of time after both BPA-F and BPA-T administration in tumor, buccal muscle, and blood samples. Error bars indicate standard deviation (STD). n=3 for each time point. [Figure 4] Comparison of biodistribution 2 hours after administration of BPA-T (T, n=9), BPA-F (F, n=9), BPA-mannitol (M, n=6), BPA-sorbitol (S, n=6) (all at 30 g / L), and BPA-T at 120 g / L (T120, n=6). The graph shows box plots of tumor:blood and tumor:muscle 10B ratios, as well as scatter plots for each individual mouse. Box plots are depicted as in Figure 1. Muscle=buccinator. Saliv.gland=salivary gland. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] A pharmaceutical composition is disclosed.

[0011] The pharmaceutical compositions disclosed herein are suitable for use in boron neutron capture therapy.

[0012] For purposes of this specification, the term "boron neutron capture therapy" (BNCT) may be understood to refer to targeted radiation therapy, in which non-radioactive boron-10 is irradiated with low-energy thermal neutrons to produce biologically destructive alpha particles and lithium-7 nuclei. The non-radioactive boron-10 may be targeted by being incorporated into a tumor-localizing agent. Examples of such tumor-localizing agents include tumor-localizing complexes and boronated phenylalanine.

[0013] The pharmaceutical composition may comprise boronated phenylalanine (BPA) or a pharma- ceutically acceptable salt thereof and a 2-hydroxyamine compound (2HA) or a pharma- ceutically acceptable salt thereof.

[0014] In the context of this specification, the term "2-hydroxyamine compound" or "2HA" may be understood to refer to any 2-hydroxyamine compound disclosed herein, or one or more of the 2-hydroxyamine compounds disclosed herein. The 2HA may have one amine group and at least two hydroxyl groups (located at the β-position relative to the amine group). Unless expressly indicated otherwise, the term "2-hydroxyamine compound" or "2HA" may also be understood to refer to a pharmaceutically acceptable salt of the 2HA. For example, a 2HA compound that is a tertiary amine may form a pharmaceutically acceptable salt.

[0015] The amine groups may be primary, secondary, or tertiary amine groups.

[0016] For purposes of this specification, unless expressly stated to the contrary, the term "BPA" may also be understood to refer to pharma- ceutically acceptable salts of BPA.

[0017] Without being limited by theory, it is believed that the arrangement of the hydroxyl and amine groups in 2HA enhances the aqueous solubility of BPA: 2HA can form complexes with the boronic acid groups of BPA through one or more of its hydroxyl and / or amine groups, thereby promoting the aqueous solubility of BPA.

[0018] The 2HA may be selected from a compound set forth in any one of formulas I-II, or a pharma- ceutically acceptable salt thereof, or any combination or mixture thereof: [ka] wherein R1 and R2 are each independently H or selected from hydroxy-C1-C6-alkyl, 2-hydroxyethyl, 2,3,4,5,6-pentahydroxyhexyl, carboxy-C1-C6-alkyl, acetyl, C1-C6-alkyl, sulfo-C1-C6-alkyl, 3-sulfopropyl, 2-hydroxy-3-sulfopropyl, 1-sulfo-2-propanyl, 2-sulfoethyl, 3-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]-C1-C6-alkyl, and 3-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]propyl; [ka] where n is 0 or 1; When n is 1, R3 is absent; when n is 0, R3 is selected from H, hydroxy-C1-C6-alkyl, 2-hydroxyethyl, carboxy-C1-C6-alkyl, acetyl, C1-C6-alkyl, sulfo-C1-C6-alkyl and 2-sulfoethyl.

[0019] The 2HA may be selected from a compound set forth in any one of formulas I-II, or a pharma- ceutically acceptable salt thereof, or any combination or mixture thereof: [ka] wherein R1 and R2 are each independently H or selected from hydroxy-C1-C6-alkyl, 2,3,4,5,6-pentahydroxyhexyl, carboxy-C1-C6-alkyl, acetyl, C1-C6-alkyl, sulfo-C1-C6-alkyl, 2-hydroxy-3-sulfopropyl, and 3-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]-C1-C6-alkyl; [ka] where n is 0 or 1; When n is 1, R3 is absent; when n is 0, R3 is selected from H, hydroxy-C1-C6-alkyl, carboxy-C1-C6-alkyl, acetyl, C1-C6-alkyl, and sulfo-C1-C6-alkyl.

[0020] The hydroxy-C1-C6-alkyl may be, for example, 2-hydroxyethyl.

[0021] The sulfo-C1-C6-alkyl may be, for example, 3-sulfopropyl, 1-sulfo-2-propanyl, or 2-sulfoethyl.

[0022] The 3-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]-C1-C6-alkyl may be, for example, 3-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]propyl.

[0023] For purposes of this specification, the term "BPA" may be understood to refer to boronated phenylalanine (including any isomers thereof). The term "BPA" may include isomers of boronated phenylalanine or pharma- ceutically acceptable salts thereof. BPA may be selected from the group consisting of p-boronated phenylalanine, Lp-boronated phenylalanine, Dp-boronated phenylalanine, m-boronated phenylalanine, Lm-boronated phenylalanine, Dm-boronated phenylalanine, o-boronated phenylalanine, Lo-boronated phenylalanine, Do-boronated phenylalanine, and any combination or mixture thereof. The BPA may be selected from the group consisting of p-boronated phenylalanine, Lp-boronated phenylalanine, Dp-boronated phenylalanine, m-boronated phenylalanine, Lm-boronated phenylalanine, Dm-boronated phenylalanine, and any combination or mixture thereof.

[0024] The BPA may comprise Lp-boronated phenylalanine or a pharma- ceutically acceptable salt thereof, or may be the same.

[0025] Lp-boronated phenylalanine has the following structure: [ka]

[0026] The BPA may additionally or alternatively be or include Dp-boronophenylalanine, or a mixture of Lp-boronophenylalanine and Dp-boronophenylalanine, or a pharma- ceutically acceptable salt thereof.

[0027] Additionally or alternatively, BPA may be or include m-boronophenylalanine (3-boronophenylalanine), or a mixture of p-boronophenylalanine and m-boronophenylalanine, or a pharma- ceutically acceptable salt thereof.

[0028] Additionally or alternatively, BPA may be or include Dm-boronophenylalanine (3-boronophenylalanine), or a mixture of p-boronophenylalanine and m-boronophenylalanine, or a pharma- ceutically acceptable salt thereof.

[0029] Additionally or alternatively, BPA may be or include Lm-boronophenylalanine (3-boronophenylalanine), or a mixture of p-boronophenylalanine and m-boronophenylalanine, or a pharma- ceutically acceptable salt thereof.

[0030] Lm-boronated phenylalanine has the following structure: [ka]

[0031] Alternatively, fluorine-18-BPA, i.e. 18 The pharmaceutical composition may contain, as an alternative to or in addition to BPA, [F]BPA. 18 The BPA may include fluorine-18-BPA, i.e., [F]BPA. 18 F]BPA or may contain the same. 18 F]BPA is useful for tumor imaging by positron emission tomography (PET), which may be performed prior to BNCT. 18 F]BPA is 4-boron-2- 18 F-fluoro-L-phenylalanine.

[0032] The BPA may have a normal isotopic distribution of about 20% boron-10 and 80% boron-11, or the BPA may be enriched with boron-10. For BNCT, boron-10 enriched BPA may be preferred. In one embodiment, the BPA has at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% boron-10. In this regard, the percentage (%) value may be understood as the proportion (%) of boron-10 atoms among the total boron atoms in the BPA. In one embodiment, the BPA is essentially pure with respect to boron-10. The BPA may be considered essentially pure with respect to boron-10 when at least 99.5% or about 100% of the boron atoms of the BPA are boron-10.

[0033] It has now been found that BPA can be dissolved in high concentrations in an aqueous solution of a 2-hydroxyamine compound (2HA) and that by adding an acid to the aqueous solution, BPA can remain in solution, even at relatively high concentrations and when the pH is adjusted to a substantially physiological pH. By drying the aqueous solution of the pharmaceutical composition, it will be possible to obtain a dry formulation, such as a powder, of the pharmaceutical composition. Moreover, the powder obtained by freeze-drying the neutralized aqueous solution can be easily dissolved when mixed with water to obtain an aqueous solution containing BPA in its original (high) concentration.

[0034] In one embodiment, in the (dissolved) aqueous solution, the BPA is distributed uniformly throughout the solution. In other words, the aqueous solution is a homogeneous solution; the BPA is dissolved in the aqueous solution so that the solution is completely homogeneous throughout. In one embodiment, the solution in which BPA is dissolved can be filtered without change in BPA concentration and can be safely administered to patients. In one embodiment, the dissolved BPA in the (dissolved) aqueous solution can effectively localize to tumors and not accumulate in normal tissues (at least not in undesirable amounts, or accumulates in lesser amounts in normal tissues than in tumors), which is advantageous in generating tumor:tissue and tumor:blood gradients for the safe and effective administration of neutron irradiation to patients.

[0035] In one embodiment, BPA may be considered dissolved when at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, or essentially 100% of the BPA is in the dissolved phase. The dissolution of BPA may be analyzed by a variety of methods known in the art. For example, undissolved BPA can be removed by filtering or centrifuging the solution, and the percentage of BPA in the solution phase (% dissolved) can then be analyzed, for example, by UV (ultraviolet) spectroscopy, infrared spectroscopy, NMR (nuclear magnetic resonance) spectroscopy or chromatography (for suitable analytical methods, see, for example, Pierro et al., 2000, Anal. Biochem. 284:301-6; and Heikkinen et al., 2011, J. Radiat. Res. 52:360-4), or by direct measurement of boron content (e.g., ICP-MS as described in Verlinden et al., 2021, J. Anal. At. Spectrom. 36:598-606). In one embodiment, the BPA can be considered dissolved if the aqueous solution is clear upon visual inspection.

[0036] The 2HA may be selected from a compound as set forth in formula I, a pharma- ceutically acceptable salt thereof, or any combination or mixture thereof: [ka] wherein R1 and R2 are each independently H or selected from hydroxy-C1-C6-alkyl, 2-hydroxyethyl, 2,3,4,5,6-pentahydroxyhexyl, carboxy-C1-C6-alkyl, acetyl, C1-C6-alkyl, sulfo-C1-C6-alkyl, 3-sulfopropyl, 2-hydroxy-3-sulfopropyl, 1-sulfo-2-propanyl, 2-sulfoethyl, 3-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]-C1-C6-alkyl, and 3-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]propyl.

[0037] The 2HA may be selected from a compound represented by formula II, a pharma- ceutically acceptable salt thereof, or any combination or mixture thereof: [ka] where n is 0 or 1; and In the case of n=R3 is absent, and in the case of n=0 R3 is selected from H, hydroxy-C1-C6-alkyl, 2-hydroxyethyl, carboxy-C1-C6-alkyl, acetyl, C1-C6-alkyl, sulfo-C1-C6-alkyl and 2-sulfoethyl.

[0038] The 2HA may be selected from the following compounds, pharma- ceutically acceptable salts thereof, and any mixtures and combinations thereof: Tris(hydroxymethyl)aminomethane (Tris), 2-[Bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol (Bis-Tris), (2-hydroxyethyl)amino-tris(hydroxymethyl)methane, N-(tris(hydroxymethyl)methyl)glycine (tricine), 2-(Dimethylamino)-2-(hydroxymethyl)propane-1,3-diol (N,N-Dimethyl-tris), 1-deoxy-1-{[1,3-dihydroxy-2-(hydroxymethyl)-2-propanyl]amino}hexitol, 2-{[1,3-dihydroxy-2-(hydroxymethyl)-2-propanyl]amino}-1-propanesulfonic acid, [Tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS), 3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid (TAPSO), 2-{[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}ethane-1-sulfonic acid (TES), Bis-trispropane (BTP), Diethanolamine (DEA), Triethanolamine (TEA), [Bis(2-hydroxyethyl)amino]acetic acid (bicine), and N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES).

[0039] The 2HA may be selected from a compound according to any one of formulas Ia-IId, a pharma-ceutically acceptable salt thereof, any mixtures and combinations thereof: Tris(hydroxymethyl)aminomethane (Tris; also referred to as TRIS, tris base, trizma, trisamine, THAM, tromethamine, trometamol, tromethane, or trisaminol) according to formula Ia: [ka] 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol (bis-tris) according to formula Ib: [ka] (2-Hydroxyethyl)amino-tris(hydroxymethyl)methane according to formula Ic: [ka] N-(tris(hydroxymethyl)methyl)glycine (tricine) according to formula Id: [ka] 2-(Dimethylamino)-2-(hydroxymethyl)propane-1,3-diol (N,N-dimethyl-tris) according to formula Ie: [ka] 1-Deoxy-1-{[1,3-dihydroxy-2-(hydroxymethyl)-2-propanyl]amino}hexitol according to the formula If: [ka] 2-{[1,3-dihydroxy-2-(hydroxymethyl)-2-propanyl]amino}-1-propanesulfonic acid according to formula Ig: [ka] [Tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS) according to formula Ih: [ka] 3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid (TAPSO) according to formula Ii: [ka] 2-{[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}ethane-1-sulfonic acid (TES) according to formula Ij: [ka] Bis-trispropane (BTP) according to the formula Ik: [ka] Diethanolamine (DEA) according to formula IIa: [ka] Triethanolamine (TEA) according to formula IIb: [ka] [Bis(2-hydroxyethyl)amino]acetic acid (bicine) according to formula IIc: [ka] N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) according to formula IId: [ka] The 2HA may be or include tris(hydroxymethyl)aminomethane (Tris) or a pharma- ceutically acceptable salt thereof. Tris(hydroxymethyl)aminomethane (Tris) is represented by the chemical formula Ia: [ka] The 2HA may be or include 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol (bis-tris) or a pharma- ceutically acceptable salt thereof. 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol (bis-tris) is represented by the chemical formula Ib: [ka] The 2HA may be or include (2-hydroxyethyl)amino-tris(hydroxymethyl)methane or a pharma- ceutically acceptable salt thereof. (2-hydroxyethyl)amino-tris(hydroxymethyl)methane is represented by the chemical formula Ic: [ka] The 2HA may be or include N-(tris(hydroxymethyl)methyl)glycine (tricine), i.e., {[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}acetic acid, or a pharma- ceutically acceptable salt thereof. N-(tris(hydroxymethyl)methyl)glycine (tricine) has the chemical formula Id: [ka] The 2HA may be or include 2-(dimethylamino)-2-(hydroxymethyl)propane-1,3-diol (N,N-dimethyl-tris) or a pharma- ceutically acceptable salt thereof. 2-(Dimethylamino)-2-(hydroxymethyl)propane-1,3-diol (N,N-dimethyl-tris) is represented by the chemical formula Ie: [ka] The 2HA may be or include 1-deoxy-1-{[1,3-dihydroxy-2-(hydroxymethyl)-2-propanyl]amino}hexitol or a pharma- ceutically acceptable salt thereof. 1-deoxy-1-{[1,3-dihydroxy-2-(hydroxymethyl)-2-propanyl]amino}hexitol has the chemical formula If: [ka] The 2HA may be or include 2-{[1,3-dihydroxy-2-(hydroxymethyl)-2-propanyl]amino}-1-propanesulfonic acid or a pharma- ceutically acceptable salt thereof. 2-{[1,3-dihydroxy-2-(hydroxymethyl)-2-propanyl]amino}-1-propanesulfonic acid has the formula Ig: [ka] The 2HA may be or include [tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS) or a pharma- ceutically acceptable salt thereof. [Tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS) is represented by the chemical formula Ih: [ka] The 2HA may be or include 3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid (TAPSO) or a pharma- ceutically acceptable salt thereof. 3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid (TAPSO) is represented by the chemical formula Ii: [ka] The 2HA may be or include 2-{[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}ethane-1-sulfonic acid (TES) or a pharma- ceutically acceptable salt thereof. 2-{[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}ethane-1-sulfonic acid (TES) is represented by the chemical formula Ij: [ka] The 2HA may be or include bis-trispropane (BTP) or a pharma- ceutically acceptable salt thereof. Bis-trispropane (BTP) has the formula Ik: [ka] The 2HA may be or include diethanolamine (DEA) or a pharma- ceutically acceptable salt thereof. Diethanolamine has the formula IIa: [ka] The 2HA may be or include triethanolamine (TEA) or a pharma- ceutically acceptable salt thereof. Triethanolamine is represented by the chemical formula IIb: [ka] The 2HA may be or include [bis(2-hydroxyethyl)amino]acetic acid (bicine) or a pharma- ceutically acceptable salt thereof. [Bis(2-hydroxyethyl)amino]acetic acid (bicine) is represented by the chemical formula IIc: [ka] The 2HA may be or include N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) or a pharma- ceutically acceptable salt thereof. N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) is represented by the chemical formula IId: [ka]

[0040] The molar ratio of 2HA:BPA in the pharmaceutical composition may be in the range of 0.5-3, about 0.5, in the range of 0.5-1, about 1, in the range of 1-1.5, in the range of 1-2, about 2, in the range of 2-3, or about 3.

[0041] When the molar ratio of 2HA:BPA is at least 1, the dissolution of BPA can be improved.

[0042] In embodiments where Tris is the 2HA, the molar ratio of Tris:BPA in the pharmaceutical composition may be in the range of 0.5-3, about 0.5, in the range of 0.5-1, about 1, in the range of 1-1.5, in the range of 1-2, about 2, in the range of 2-3, or about 3.

[0043] In embodiments where Tris is the 2HA and Lp-boronated phenylalanine is the BPA, the molar ratio of tris(hydroxymethyl)aminomethane (Tris):Lp-boronated phenylalanine can be in the range of 0.5-3, about 0.5, in the range of 0.5-1, about 1, in the range of 1-1.5, in the range of 1-2, about 2, in the range of 2-3, or about 3.

[0044] The pharmaceutical composition may be an aqueous solution. In other words, the pharmaceutical composition may be in the form of an aqueous solution. Therefore, the BPA (and the 2HA) may be dissolved in the aqueous solution.

[0045] The pH of the aqueous solution may be in the range of 6.5 to 8.5.

[0046] The pH of the aqueous solution may be in the range of 7 to 8, in the range of 7.3 to 7.5, in the range of 7.35 to 7.45, about pH 7.4, or at physiological pH or substantially physiological pH.

[0047] For purposes of this specification, the term "substantially neutral pH" may be understood to refer to a pH range of about 6.5 to 7.5, or a pH of about 7.

[0048] For purposes of this specification, the term "substantially physiological pH" may be understood to refer to a pH range of about 6.5 to 8.5. For purposes of this specification, the term "physiological pH" may be understood to refer to a pH range of about 7.35 to 7.45, or a pH of about 7.4.

[0049] The aqueous solution may have a BPA concentration of at least 30 g / L.

[0050] The BPA concentration of the aqueous solution is not particularly limited and can be set to an appropriate concentration. However, it is generally desirable that the concentration is higher than about 30 g / L of the conventional BPA-fructose complex aqueous solution. The reason is that the volume of the aqueous solution administered to the patient can be reduced in this case (compared to the volume of the BPA-fructose complex aqueous solution). Thus, the BPA concentration may be, for example, at least 30 g / L, or more than 30 g / L, more than 35 g / L, more than 40 g / L, more than 45 g / L, more than 50 g / L, more than 55 g / L, or more than 60 g / L. The concentration of BPA in the aqueous solution may be in the range of 30 to 300 g / L, or in the range of 30 to 100 g / L, or in the range of 100 to 300 mg / ml, or in the range of 30 to 60 g / L, or in the range of 30 to 45 g / L, or about 30 g / L, or 30 g / L.

[0051] The aqueous solution may be isotonic or substantially isotonic.

[0052] The aqueous solution may be hypertonic.

[0053] In one embodiment, the osmolality of the aqueous solution is suitable for intravenous administration of the aqueous solution to the subject.

[0054] In one embodiment, the osmotic pressure of the aqueous solution is 310 mOsm / L or more, or 320 mOsm / L or more, In one embodiment, the osmotic pressure of the aqueous solution is in the range of 250 to 350 mOsm / L, or in the range of 280 to 330 mOsm / L, or in the range of about 300 to 310 mOsm / L.

[0055] In one embodiment, the osmotic pressure of the aqueous solution is in the range of 310 to 900 mOsm / L, 310 to 600 mOsm / L, 310 to 500 mOsm / L, 310 to 400 mOsm / L, 400 to 600 mOsm / L, 400 to 500 mOsm / L, or 500 to 600 mOsm / L. In one embodiment, the osmotic pressure of the aqueous solution is 900 mOsm / L or less, 800 mOsm / L or less, 700 mOsm / L or less, 600 mOsm / L or less, 500 mOsm / L or less, or 400 mOsm / L or less. A relatively high osmotic pressure may be suitable for intravenous administration.

[0056] The pharmaceutical composition may be a dry formulation. In other words, the pharmaceutical composition may be in the form of a dry formulation. For example, the dry formulation may be a powder.

[0057] The dry formulation, e.g., powder, can be obtained by drying, e.g., freeze-drying the aqueous solution.The dry formulation can be easily dissolved when mixed with water.When water is added, i.e., mixed with water, the dry formulation can provide an aqueous solution containing BPA at its original (high) concentration.

[0058] The dry formulation may be capable of forming an aqueous solution having a pH range of 6.5 to 8.5 upon addition of water. When the dry formulation is dissolved upon addition of water, an aqueous solution having a pH range of 6.5 to 8.5 may be formed. For example, the dry formulation may be capable of forming an aqueous solution having a pH range of 6.5 to 8.5 upon addition of 1 L of water (pure water, e.g., distilled water or deionized water) to at least 30 g of BPA, or to a dry formulation containing 30 g of BPA.

[0059] The dry formulation may be capable of forming an aqueous solution having a pH in the range of 7 to 8, in the range of 7.3 to 7.5, in the range of 7.35 to 7.45, at about pH 7.4, or at physiological pH or substantially physiological pH.

[0060] For purposes of this specification, the term "water" may be understood to refer to, for example, distilled water, deionized water, or other forms of water that are considered pure water, but may additionally or alternatively refer to, for example, saline, buffer solutions, buffered saline, or other aqueous solutions that contain water.

[0061] Since the aqueous solution may be mixed with an infusion fluid and administered to a subject, it is not always necessary to adjust the pH to a strictly neutral or physiological pH, since the buffering effect of a buffer contained in the infusion fluid may correct any deviation from the neutral or physiological pH.

[0062] Aqueous solutions of BPA and 2HA are highly stable at essentially physiological pH and almost perfectly stable at substantially neutral pH, and therefore the pH of the aqueous solution may be appropriately set to be convenient for use as a product, for example at substantially neutral pH, slightly alkaline pH, or essentially physiological pH.

[0063] The pharmaceutical composition may further comprise additional ingredients or excipients.

[0064] In other words, the pharmaceutical composition does not necessarily exclude ingredients other than BPA, water and 2HA, so long as such ingredients do not have a substantial adverse effect on the stability (solubility and storage stability) of the pharmaceutical composition. Usually, the combined base of the acid added when adjusting the pH (in the case of hydrochloric acid, Cl - ions) may be considered to be contained in the pharmaceutical composition. In addition to the above, a component derived from a pH adjuster (such as sodium bicarbonate) (which can be used for fine adjustment of the pH during preparation of the pharmaceutical composition) may be contained. The pH adjuster of the pharmaceutical composition may be, for example, an acid. The pH adjuster of the pharmaceutical composition may be, for example, an alkali, such as sodium hydroxide, potassium hydroxide and / or sodium carbonate.

[0065] The pharmaceutical composition may further comprise at least one of hydrochloric acid, sodium chloride, acetic acid, sodium acetate, polyethylene glycol, a polyol, a sugar, fructose, mannitol, or sorbitol.

[0066] The pharmaceutical composition may further comprise at least one of sodium hydroxide, potassium hydroxide, or sodium carbonate.

[0067] In one embodiment, the pharmaceutical composition does not contain or is free of polyethylene glycol, polyols, sugars, fructose, mannitol, and sorbitol.

[0068] In one embodiment, the pharmaceutical composition comprises a polyol.

[0069] In the context of this specification, the term "polyol" may be understood to refer to an organic compound containing multiple hydroxyl groups. In one embodiment, the polyol is selected from the group of diols, triols, tetrols, pentols, and hexols. In one embodiment, the polyol is a reducing sugar, such as sorbitol, mannitol, etc. In one embodiment, the polyol is a non-reducing sugar, such as saccharose, trehalose, etc. In the context of this specification, "a polyol" or "polyol" may refer to one or more polyols. In other words, the polyol may include one polyol or a mixture of two or more polyols.

[0070] The polyol may include or be mannitol, which is a suitable polyol for the pharmaceutical composition.

[0071] In one embodiment, the pharmaceutical composition consists of BPA, the 2HA and water.

[0072] In one embodiment, the pharmaceutical composition comprises or consists of BPA, the 2HA, a polyol and water. In one embodiment, the molar ratio of BPA, 2HA and polyol is such that the sum of the molar amounts of 2HA and polyol is greater than the molar amount of BPA (i.e., [2HA]+[polyol]>[BPA]). With respect to the above molar ratio, it can be understood that [2HA] is the molar amount of 2HA, [polyol] is the molar amount of polyol (i.e., the (combined) molar amount of one or more polyols), and [BPA] is the molar amount of BPA. In other words, when the polyol comprises two or more different polyols or is a mixture thereof, [polyol] is the sum of the molar amounts of two or more different polyols. As an example, when [BPA] is 1 mol, [2HA]+[polyol]>1 mol.

[0073] The term "molar amount" may be understood to refer to the number of moles, i.e., the number of moles in the pharmaceutical composition.

[0074] In one embodiment, the molar ratios and / or amounts of the BPA, the 2HA, and the polyol are such that the sum of the molar amounts of BPA and polyol is about equal to or greater than the molar amount of BPA.

[0075] In the embodiment described herein, [2HA] is the molar amount of 2HA, [polyol] is the molar amount of polyol, [BPA] is the molar amount of BPA, and x is a multiplication symbol.In other words, 3x[BPA] can be understood to refer to three times the molar amount of BPA.When the polyol comprises two or more different polyols or is a mixture thereof, [polyol] is the sum of the molar amounts of two or more different polyols.

[0076] In one embodiment, [2HA] + [polyol] > 1.5 x [BPA]. In one embodiment, [2HA] + [polyol] > 2 x [BPA]. In one embodiment, [BPA] < [2HA] + [polyol] < 3 x [BPA]. In one embodiment, [BPA] < [2HA] + [polyol] < 2.5 x [BPA]. In one embodiment, [BPA] < [2HA] + [polyol] < 2 x [BPA].

[0077] In one embodiment, [2HA] + [polyol] > 0.8 x [BPA]. In one embodiment, [2HA] + [polyol] > 0.9 x [BPA]. In one embodiment, [2HA] + [polyol] > 1.0 x [BPA]. In one embodiment, [2HA] + [polyol] > 1.5 x [BPA]. In one embodiment, [2HA] + [polyol] > 2.0 x [BPA].

[0078] In one embodiment, 0.8x[BPA]≦[2HA]+[polyol]≦5.0x[BPA]. In one embodiment, 0.9x[BPA]≦[2HA]+[polyol]≦4.0x[BPA]. In one embodiment, 1.0x[BPA]≦[2HA]+[polyol]≦3.0x[BPA]. In one embodiment, 1.5x[BPA]≦[2HA]+[polyol]≦2.5x[BPA]. In one embodiment, 2.0x[BPA]≦[2HA]+[polyol]≦2.2x[BPA].

[0079] In one embodiment, [2HA]+[polyol]=2.1×[BPA].

[0080] In one embodiment, the pharmaceutical composition comprises or consists of ingredients derived from BPA, the 2HA, water, and an agent to adjust the pH of the pharmaceutical composition.

[0081] In one embodiment, the pharmaceutical composition comprises or consists of ingredients derived from BPA, the 2HA, a polyol, water and an agent to adjust the pH of the pharmaceutical composition.

[0082] In one embodiment, the pharmaceutical composition comprises Lp-boronated phenylalanine. In other words, the BPA may be Lp-boronated phenylalanine.

[0083] In one embodiment, the pharmaceutical composition comprises tris(hydroxymethyl)aminomethane (Tris). In other words, the 2HA may be tris(hydroxymethyl)aminomethane.

[0084] In one embodiment, the pharmaceutical composition comprises mannitol, ie, the polyol may be mannitol.

[0085] In one embodiment, the pharmaceutical composition comprises Lp-boronated phenylalanine as the BPA, tris(hydroxymethyl)aminomethane (Tris) as the 2HA, and mannitol as the polyol.

[0086] In one embodiment, the pharmaceutical composition comprises Lp-boronated phenylalanine, tris(hydroxymethyl)aminomethane (Tris), and mannitol.

[0087] The molar ratio of tris(hydroxymethyl)aminomethane (Tris):Lp-boronated phenylalanine may be in the range of 0.5-3, about 0.5, in the range of 0.5-1, about 1, in the range of 1-1.5, in the range of 1-2, about 2, in the range of 2-3, or about 3.

[0088] In the embodiments below, [Tris] is the molar amount of Tris, [Mannitol] is the molar amount of mannitol, and [BPA] is the molar amount of Lp-boronated phenylalanine.

[0089] In one embodiment, [Tris] + [mannitol] > [Lp-boronated phenylalanine], where [Tris] is the molar amount of Tris, [mannitol] is the molar amount of mannitol, and [Lp-boronated phenylalanine] is the molar amount of Lp-boronated phenylalanine. In one embodiment, [Tris] + [mannitol] > 1.5 x [Lp-boronated phenylalanine]. In one embodiment, [Tris] + [mannitol] > 2 x [Lp-boronated phenylalanine]. In one embodiment, [Tris] + [mannitol] < 3 x [Lp-boronated phenylalanine]. In one embodiment, [Tris] + [mannitol] < 2.5 x [Lp-boronated phenylalanine]. In one embodiment, [Tris] + [mannitol] < 2 x [Lp-boronated phenylalanine].

[0090] In one embodiment, the pharmaceutical composition comprises Lp-boronated phenylalanine, Tris, and mannitol in a molar ratio of 10:10:11.

[0091] In one embodiment, the pharmaceutical composition comprises Lp-boronated phenylalanine, Tris, and mannitol in a molar ratio of 10:10:11, and the pH of the composition is 6.5-7.5, or the pH is about 7.4 or a physiological pH or a substantially physiological pH. In one embodiment, [Tris] + [mannitol] is ≧0.8×[BPA]. In one embodiment, [Tris] + [mannitol] is ≧0.9×[BPA]. In one embodiment, [Tris] + [mannitol] is ≧1.0×[BPA]. In one embodiment, [Tris] + [mannitol] is ≧1.5×[BPA]. In one embodiment, [Tris] + [mannitol] is ≧2.0×[BPA].

[0092] In one embodiment, 0.8x[BPA]≦[Tris]+[mannitol]≦5.0x[BPA]. In one embodiment, 0.9x[BPA]≦[Tris]+[mannitol]≦4.0x[BPA]. In one embodiment, 1.0x[BPA]≦[Tris]+[mannitol]≦3.0x[BPA]. In one embodiment, 1.5x[BPA≦[Tris]+[mannitol]≦2.5x[BPA]. In one embodiment, 2.0x[BPA]≦[Tris]+[mannitol]≦2.2x[BPA].

[0093] In one embodiment, [Tris] + [mannitol] = 2.1 x [BPA].

[0094] In the above embodiments where molar ratios and / or amounts are given, the pharmaceutical composition may be in the form of an aqueous solution or a dry formulation.

[0095] The pharmaceutical composition may be provided as a product in the form of an aqueous solution. Alternatively or additionally, the pharmaceutical composition may be provided as a solid preparation, i.e., a dry composition. In such a solid preparation, the aqueous solution is dried. The dry product may be sealed in a package that prevents melting. For the purpose of drying the aqueous solution, it may be convenient to use lyophilization, but there is no particular limitation on the drying method. The drying may be achieved by using a method that can provide a sterile formulation suitable for injection.

[0096] The pharmaceutical composition has high stability over time even in the form of a lyophilized product, and can be easily restored to the form of an aqueous solution, for example, by adding distilled water for injection before use.

[0097] The pharmaceutical composition may be stable for a period of up to 24 months, or up to 12 months, or at least 1 month, or at least 2 years, or at least 3 years.

[0098] The pharmaceutical composition may be stable at a temperature of -90 to +40°C for such a period. In one embodiment, the pharmaceutical composition may be stable at a temperature of -90 to 0°C, -60 to 0°C, -30 to 0°C, 0 to 10°C, 2 to 8°C, 2 to 6°C, 0 to 30°C, or 10 to 25°C for such a period. For example, the pharmaceutical composition may be stable at a temperature of 2 to 6°C for a period of up to 24 months, or up to 12 months, or at least 1 month, or at least 2 years, or at least 3 years. In one embodiment, the pharmaceutical composition may be stable at normal room temperature of 20 to 24°C for a period of up to 24 months, or up to 12 months, or at least 1 month, or at least 2 years, or at least 3 years. The pharmaceutical composition may be considered stable if it remains clear without the presence of precipitate or if it remains clear after dissolution without the presence of precipitate.

[0099] It has been found that the main decomposition pathway of BPA contained in the pharmaceutical composition can be the formation of phenylalanine (Phe) from BPA.When the pharmaceutical composition is stored for a certain period (for example, for a period of up to 24 months, or up to 12 months, or at least 1 month, or at least 2 years or at least 3 years), and the amount of Phe formed during storage is less than about 5 w-%, less than 4 w-%, less than 3 w-%, less than 2 w-%, less than 1 w-%, less than 0.5 w-%, less than 0.2 w-%, less than 0.1 w-% or about 0 w-% based on the total amount of BPA and Phe in the pharmaceutical composition, the pharmaceutical composition can be considered stable after storage.The amount of Phe and BPA can be measured, for example, by RP-HPLC or other methods in the art.

[0100] Disclosed are pharmaceutical compositions according to one or more of the embodiments described herein for use in therapy.

[0101] A pharmaceutical composition according to one or more of the embodiments described herein is disclosed for use in treating cancer.

[0102] The cancer may be head and neck cancer.

[0103] The cancer may be selected from the group consisting of head and neck cancer, brain cancer, glioma, skin cancer, melanoma, leukemia, lymphoma, breast cancer, prostate cancer, ovarian cancer, colorectal cancer, gastric cancer, squamous cell carcinoma, small cell lung cancer, multidrug resistant cancer and testicular cancer.

[0104] A pharmaceutical composition according to one or more of the embodiments described herein is disclosed for use in boron neutron capture therapy (BNCT).

[0105] The BPA may be or may include fluorine-18-BPA; fluorine-18-BPA is, i.e., 18 F]BPA(4-boron-2- 18 F-fluoro-L-phenylalanine, etc. In such an embodiment, the pharmaceutical composition may be administered to a subject having a tumor; the tumor may be imaged by positron emission tomography (PET); and the subject may then be treated by BNCT. In one embodiment, the [ 18 F]BPA is 4-boron-2- 18 F-fluoro-L-phenylalanine.

[0106] Disclosed is the use of a pharmaceutical composition according to one or more of the embodiments described herein in the manufacture of a therapeutic medicament.

[0107] Also disclosed is the use of a pharmaceutical composition according to one or more of the embodiments described herein in the manufacture of a therapeutic medicament for the treatment of cancer and / or boron neutron capture therapy.

[0108] Disclosed is a method for preparing a pharmaceutical composition according to one or more of the embodiments described herein. The method may include: mixing BPA with 2HA in the form of an aqueous solution of BPA and 2HA; Optionally mixing a polyol with the BPA and 2HA or adding a polyol to the aqueous solution; and The pH of the aqueous solution is adjusted to a pH range of 6.5 to 8.5.

[0109] In one embodiment, the method may include: mixing BPA with 2HA in the form of an aqueous solution of BPA and 2HA; Optionally mixing a polyol with the BPA and 2HA or adding a polyol to the aqueous solution; adjusting the pH of the aqueous solution with an alkali to completely dissolve the BPA; and The pH of the aqueous solution is adjusted to a pH range of 6.5 to 8.5. The pH of the aqueous solution may be adjusted to a range of 6.5 to 8.5 using an acid.

[0110] The aqueous solution of 2HA may be mixed with BPA to form the aqueous solution of BPA and 2HA, or BPA, the 2HA and water may be mixed to form the aqueous solution of BPA and 2HA.

[0111] BPA may be mixed into the aqueous solution of the 2HA and the polyol to form an aqueous solution of the BPA, the 2HA, and the polyol. Alternatively, the BPA, the 2HA, the polyol, and water may be mixed to form an aqueous solution of the BPA, the 2HA, and the polyol. Alternatively, BPA may be mixed with the aqueous solution of the 2HA to form an aqueous solution of the BPA and the 2HA, and the polyol may be added to the aqueous solution.

[0112] Thus, the pharmaceutical composition prepared by the method may be an aqueous solution.

[0113] The aqueous solution may be prepared, for example, by mixing BPA and the 2HA in water, adding an acid to the resulting alkaline aqueous solution, and adjusting the pH of the resulting alkaline aqueous solution to, for example, a substantially neutral pH or a substantially physiological pH. In one embodiment, the aqueous solution may be prepared, for example, by mixing BPA, the 2HA, and optionally a polyol in water, and adding an acid to the resulting alkaline aqueous solution to adjust the pH of the resulting alkaline aqueous solution to, for example, a substantially neutral pH or a substantially physiological pH. In one embodiment, before adjusting the pH, an alkali may be added to completely dissolve the BPA. The temperature at which the aqueous solution is prepared is not particularly limited, and it can simply be performed at room temperature. Heating (for example, heating to 60°C) as in the preparation of a BPA-fructose complex solution is not required. The aqueous solution may be further sterilized by filter as necessary.

[0114] The pH of the aqueous solution may be adjusted with an alkali to a higher pH, for example, a pH in the range of 7.5 to 8, in order to completely dissolve the BPA. The need to adjust the pH of the aqueous solution to completely dissolve the BPA may depend on the exact composition of the aqueous solution.

[0115] The pH of the aqueous solution may be adjusted with a suitable agent, acid, or the like to a pH in the range of 6.5 to 8.5.

[0116] The acid to be added after mixing BPA with water and 2HA for the purpose of adjusting the pH of the aqueous solution is not particularly limited, so long as it is an acid suitable for the purpose and can be used for medical purposes, i.e., is a pharma- ceutically acceptable acid. The acid may be, for example, hydrochloric acid.

[0117] The alkali to be added for the purpose of completely dissolving the BPA is not particularly limited, so long as it is an alkali suitable for the purpose and can be used for medical purposes, i.e., is a pharma- ceutically acceptable alkali. The alkali may be, for example, sodium hydroxide, potassium hydroxide, and / or sodium carbonate.

[0118] The molar ratio of 2HA:BPA may be in the range of 0.5-3, about 0.5, in the range of 0.5-1, about 1, in the range of 1-1.5, in the range of 1-2, about 2, in the range of 2-3, or about 3.

[0119] In one embodiment, the molar ratio of 2HA:BPA is 1.

[0120] For the method, the molar ratios and / or amounts of the 2HA, the BPA, and optionally the polyol can be any of the molar ratios and / or amounts described for the pharmaceutical composition.

[0121] In one embodiment, the molar ratio and / or molar amounts of the BPA, the 2HA, and the polyol are such that the sum of the molar amounts of BPA and the polyol is approximately equal to or greater than the molar amount of BPA.

[0122] In the embodiment described herein, [2HA] is the molar amount of 2HA, [polyol] is the molar amount of polyol, [BPA] is the molar amount of BPA, and x is a multiplication symbol.In other words, 3x[BPA] can be understood to refer to three times the molar amount of BPA.When the polyol comprises two or more different polyols or is a mixture thereof, [polyol] is the sum of the molar amounts of two or more different polyols.

[0123] In one embodiment, [2HA] + [polyol] > 1.5 x [BPA]. In one embodiment, [2HA] + [polyol] > 2 x [BPA]. In one embodiment, [BPA] < [2HA] + [polyol] < 3 x [BPA]. In one embodiment, [BPA] < [2HA] + [polyol] < 2.5 x [BPA]. In one embodiment, [BPA] < [2HA] + [polyol] < 2 x [BPA].

[0124] In one embodiment, [2HA] + [polyol] > 0.8 x [BPA]. In one embodiment, [2HA] + [polyol] > 0.9 x [BPA]. In one embodiment, [2HA] + [polyol] > 1.0 x [BPA]. In one embodiment, [2HA] + [polyol] > 1.5 x [BPA]. In one embodiment, [2HA] + [polyol] > 2.0 x [BPA].

[0125] In one embodiment, 0.8x[BPA]≦[2HA]+[polyol]≦5.0x[BPA]. In one embodiment, 0.9x[BPA]≦[2HA]+[polyol]≦4.0x[BPA]. In one embodiment, 1.0x[BPA]≦[2HA]+[polyol]≦3.0x[BPA]. In one embodiment, 1.5x[BPA]≦[2HA]+[polyol]≦2.5x[BPA]. In one embodiment, 2.0x[BPA]≦[2HA]+[polyol]≦2.2x[BPA].

[0126] In one embodiment, [2HA]+[polyol]=2.1×[BPA].

[0127] In one embodiment, the aqueous solution and / or the pharmaceutical composition is free of polyethylene glycol, polyols, sugars, fructose, mannitol, or sorbitol, in other words, the pharmaceutical composition is free of polyethylene glycol, polyols, sugars, fructose, mannitol, and / or sorbitol.

[0128] In one embodiment, a polyol is added to the aqueous solution and / or the pharmaceutical composition.

[0129] In one embodiment, the polyol is mannitol.

[0130] In one embodiment, the molar ratio of polyol:BPA may be in the range of 0.5-3, about 0.5, in the range of 0.5-1, about 1, about 1.1, in the range of 1-1.5, in the range of 1-2, about 2, in the range of 2-3, or about 3.

[0131] In one embodiment, the molar ratio of polyol:BPA is 11:10.

[0132] In one embodiment, the molar ratio of BPA:2HA:polyol is 10:10:11.

[0133] The molar ratio of tris(hydroxymethyl)aminomethane (Tris):Lp-boronated phenylalanine may be in the range of 0.5-3, about 0.5, in the range of 0.5-1, about 1, in the range of 1-1.5, in the range of 1-2, about 2, in the range of 2-3, or about 3.

[0134] In the embodiments below, [Tris] is the molar amount of Tris, [Mannitol] is the molar amount of mannitol, and [BPA] is the molar amount of Lp-boronated phenylalanine.

[0135] In one embodiment, [Tris] + [mannitol] > [Lp-boronated phenylalanine], where [Tris] is the molar amount of Tris, [mannitol] is the molar amount of mannitol, and [Lp-boronated phenylalanine] is the molar amount of Lp-boronated phenylalanine. In one embodiment, [Tris] + [mannitol] > 1.5 x [Lp-boronated phenylalanine]. In one embodiment, [Tris] + [mannitol] > 2 x [Lp-boronated phenylalanine]. In one embodiment, [Tris] + [mannitol] < 3 x [Lp-boronated phenylalanine]. In one embodiment, [Tris] + [mannitol] < 2.5 x [Lp-boronated phenylalanine]. In one embodiment, [Tris] + [mannitol] < 2 x [Lp-boronated phenylalanine].

[0136] In one embodiment, the pharmaceutical composition comprises Lp-boronated phenylalanine, Tris, and mannitol in a molar ratio of 10:10:11.

[0137] In one embodiment, the pharmaceutical composition comprises Lp-boronated phenylalanine, Tris, and mannitol in a molar ratio of 10:10:11, and the pH of the composition is in the range of 6.5 to 7.5, or about pH 7.4, or physiological pH, or substantially physiological pH. In one embodiment, [Tris] + [mannitol] ≧ 0.8 x [BPA]. In one embodiment, [Tris] + [mannitol] ≧ 0.9 x [BPA]. In one embodiment, [Tris] + [mannitol] ≧ 1.0 x [BPA]. In one embodiment, [Tris] + [mannitol] ≧ 1.5 x [BPA]. In one embodiment, [Tris] + [mannitol] ≧ 2.0 x [BPA].

[0138] In one embodiment, 0.8x[BPA]≦[Tris]+[mannitol]≦5.0x[BPA]. In one embodiment, 0.9x[BPA]≦[Tris]+[mannitol]≦4.0x[BPA]. In one embodiment, 1.0x[BPA]≦[Tris]+[mannitol]≦3.0x[BPA]. In one embodiment, 1.5x[BPA]≦[Tris]+[mannitol]≦2.5x[BPA]. In one embodiment, 2.0x[BPA]≦[Tris]+[mannitol]≦2.2x[BPA].

[0139] In one embodiment, [Tris] + [mannitol] = 2.1 x [BPA].

[0140] In the above embodiments where the molar ratios and / or amounts are indicated, the pharmaceutical composition may be in the form of an aqueous solution or a dry formulation.

[0141] In one embodiment, the method may include: mixing BPA with 2HA to form an aqueous solution of said BPA and said 2HA; Optionally adding said polyol to said aqueous solution; and The pH of the aqueous solution is adjusted to a pH range of 6.5 to 8.5.

[0142] In one embodiment, the method may include: Mixing Lp-boronated phenylalanine and tris(hydroxymethyl)aminomethane (Tris) to form an aqueous solution; mixing mannitol with the BPA and the 2HA or adding mannitol to the aqueous solution; Optionally, a pH adjuster such as an alkali is added to the aqueous solution to completely dissolve the BPA; and Adjusting the pH of the aqueous solution to about 7.4, or to a physiological pH or substantially a physiological pH, with an acid.

[0143] In one embodiment, the alkali is sodium hydroxide and / or the acid is hydrochloric acid.

[0144] In one embodiment, the method has a BPA concentration in the aqueous solution in the range of 30-120 g / L and the pH of the aqueous solution is about 7.4 or a physiological pH or substantially a physiological pH.

[0145] The method may further comprise drying the aqueous solution, thereby obtaining a dry formulation of the pharmaceutical composition.

[0146] Pharmaceutical compositions obtainable by one or more embodiments of the method are also disclosed.

[0147] Further disclosed is a method of making the pharmaceutical composition as an aqueous solution according to one or more embodiments described herein. The method may include: providing the pharmaceutical composition as a dry formulation (such as a dry formulation according to one or more embodiments described herein); mixing the pharmaceutical composition as a dry formulation with water and optionally one or more compounds selected from hydrochloric acid, sodium chloride, acetic acid, sodium acetate, polyethylene glycol, polyols, sugars, fructose, mannitol, and sorbitol, thereby obtaining the pharmaceutical composition as an aqueous solution.

[0148] The concentration of BPA in the aqueous solution obtainable by the method of preparing the pharmaceutical composition as an aqueous solution may be in the range of 30 to 120 g / L.The pH of the aqueous solution may be about 7.4, or a physiological pH or a substantially physiological pH.

[0149] In this aspect, the composition may then be ready for use in therapy.The dry formulation may then be reconstituted by mixing with water and optionally one or more compounds.The method according to this aspect may, for example, be carried out before administration to a subject.

[0150] In one embodiment, no polyethylene glycol, polyol, sugar, fructose, mannitol, or sorbitol is added to the aqueous solution and / or the pharmaceutical composition.

[0151] In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of BPA.

[0152] The term "therapeutically effective amount" or "effective amount" of the conjugate can be understood to refer to a dosage regimen that exerts a therapeutic effect. The therapeutically effective amount can be selected according to various factors, including the age, weight, sex, diet, and medical condition of the patient; the severity of the disease, and the pharmacological considerations (such as activity, efficacy, pharmacokinetics, and toxicity profile) of the specific conjugate used. The therapeutically effective amount can also be determined by referring to standard medical textbooks (such as Physicians Desk Reference 2004). The patient can be male or female, and can be an infant, a child, or an adult.

[0153] The terms "treatment" or "treat" are used in the conventional sense to mean the attendance, care, and nursing of a patient for the purpose of combating, alleviating, attenuating, or relieving a disease or health condition, and for the purpose of improving the living conditions impaired by the disease, such as a cancer disease.

[0154] The pharmaceutical composition may be or may include a composition, e.g., a composition for intravenous, intraarterial, and / or intratumoral (it) administration, or for direct injection into tissue. The administration of the pharmaceutical composition may be performed by different methods, e.g., intravenous, intraperitoneal, intraarterial, or intratumoral administration.

[0155] Also disclosed is the use of a 2-hydroxyamine compound (2HA) or a pharma- ceutically acceptable salt thereof according to one or more embodiments described herein in dissolving BPA, thereby forming a pharmaceutical composition comprising BPA in an aqueous solution.

[0156] The 2HA or a pharma- ceutically acceptable salt thereof may be used to dissolve BPA in the absence of polyethylene glycol, polyols, sugars, fructose, mannitol and / or sorbitol.

[0157] In one embodiment, the 2HA or a pharma- ceutically acceptable salt thereof may be used to dissolve BPA in the presence of polyethylene glycol, polyols, sugars, fructose, mannitol and / or sorbitol.

[0158] In one embodiment, the 2HA or a pharma- ceutically acceptable salt thereof may be used to dissolve BPA in the presence of mannitol.

[0159] For this use, the pharmaceutical composition may be the pharmaceutical composition according to one or more embodiments described herein.For this use, the BPA, the 2HA, the polyol, and the molar amount and / or molar ratio are the same as those described above for the pharmaceutical composition and method.

[0160] Working Example All of the BPA used in Examples 1 to 5 was synthesized by a conventional method using boron; 10The proportion of B is about 20%, and the boron isotopes do not affect the results obtained in the examples, so 10 Even BPA containing B can be used.

[0161] Example 1: Solubilization of BPA BPA (Lp-boronated phenylalanine, Katchem, Prague, Czech Republic) was weighed out at 15 mg (72 μmol) into each of a series of test tubes. At room temperature (RT), 0.5 ml of each of the various aqueous solutions listed below was added in parallel to the test tubes and vigorously stirred. After 2 hours, visual inspection for BPA dissolution and pH were as follows: water Deionized Water - Non-dissolving strong base 0.5M NaOH - completely dissolved Organic alcohols 96% ethanol (v / v) - insoluble 0.5M Propylene Glycol - Undissolved Neutral solution 0.12M L-histidine (neutral pH) - insoluble 0.5M L-Serine (neutral pH) - insoluble 0.5M Sodium-Potassium Tartrate (neutral pH) - partially soluble Basic Compounds 0.5M L-Arginine (pH above 11) - Partially soluble Strong base followed by hydroxyamino acid 0.25 ml of 0.5M NaOH followed by 0.5 ml of 0.5M L-serine - 1. Complete dissolution 2-Hydroxyamine compounds (2HA) 0.5M Tris (trisma base, pH greater than 10) - complete lysis 0.5M Tris-HCl (pH8) - partial lysis

[0162] Example 2: Aqueous BPA Formulation at Physiological pH BPA-Tris-HCl (pH 8) 15 mg of BPA was dissolved in 0.45 ml of 0.5 M Trizma base at room temperature (BPA was completely dissolved). After adding 20 μl of 5 M HCl, the pH was adjusted to about 8 to completely dissolve BPA.

[0163] BPA-Tris-HCl (pH 7.5) 15 mg of BPA was dissolved in 0.45 ml of 0.5 M Trizma base at room temperature (BPA was completely dissolved). After adding 20 μl of 5 M HCl and 20 μl of 1 M HCl, the pH was adjusted to about 7.5 to completely dissolve BPA.

[0164] Example 3: BPA Formulations with Fructose BPA-Tris-HCl-fructose (pH 8) To the above BPA-Tris-HCl-fructose formulation (pH 8), 0.5 ml of 0.5 M fructose was added to completely dissolve the BPA.

[0165] BPA-Tris-HCl-fructose (pH 7.5) To the above BPA-Tris-HCl-fructose formulation (pH 7.5), 0.5 ml of 0.5 M fructose was added to completely dissolve the BPA.

[0166] Example 4: Freeze-dried BPA formulation Lyophilized BPA-Tris-HCl (pH8) The BPA-Tris-HCl formulation (pH 8) was lyophilized. For lyophilization, 355 μl of MQ was added before BPA was completely dissolved.

[0167] Lyophilized BPA-Tris-HCl (pH7.5) The BPA-Tris-HCl formulation (pH 7.5) was lyophilized. For lyophilization, 355 μl of MQ was added after which BPA was completely dissolved.

[0168] Example 5: Freeze-dried BPA formulations containing fructose Freeze-dried BPA-Tris-HCl (pH 8) containing fructose The BPA-Tris-HCl formulation (pH 8) was lyophilized. For lyophilization, 355 μl of MQ and 145 μl of 0.5 M fructose were added after which BPA was completely dissolved.

[0169] Freeze-dried BPA-Tris-HCl (pH 7.5) containing fructose The BPA-Tris-HCl formulation (pH 7.5) was lyophilized. For lyophilization, BPA was completely dissolved after adding 355 μl MQ and 145 μl 0.5 M fructose.

[0170] All BPA used in Examples 6 and thereafter below was 10 B( 10 BLp-boronated phenylalanine; hereafter referred to as BPA or 10 It is a fortified version of EPA (BPA is used as a synonym). 10 B was obtained from Interpharma Praha (Prague, Czech Republic). All other reagents were from Sigma unless otherwise stated. RT = room temperature, approximately 20-23 °C.

[0171] Example 6: Comparison of BPA-Tris-mannitol and BPA-Tris-fructose formulations at physiological and slightly alkaline pH At 37°C, 10A 30 mg aliquot of BPA was dissolved in 0.5 M aqueous Tris base (Sigma) at a 2:1 Tris:BPA molar ratio and, after complete dissolution, the pH was adjusted to either pH 7.4 or pH 8.0 using dilute hydrochloric acid. The aliquot was then lyophilized. In order to compare mannitol and fructose for their potential to keep lyophilized BPA-Tris solutions mannitol- and fructose-free, the aliquot was dissolved at a concentration of 30 g BPA / L in an aqueous sugar solution (containing either mannitol or fructose at an 11:10 sugar:BPA molar ratio) and incubated at room temperature. The next day, the pH 7.4 formulation containing fructose began to precipitate. After 4 days, the pH 8.0 formulation containing fructose also began to precipitate, whereas the mannitol-containing formulation maintained a clear solution free of precipitate at both pH 7.4 and pH 8.0.

[0172] Contains either fructose or mannitol (11:10 sugar:BPA molar ratio) 10 A 30 mg aliquot of BPA was dissolved in 0.9 ml of 0.5 M aqueous Tris base (Sigma) at +37° C. (3:1 Tris:BPA molar ratio). After complete dissolution, the pH was adjusted to 7.4 with dilute hydrochloric acid. The aliquot was then lyophilized. In order to compare mannitol and fructose for their potential to keep BPA-Tris-sugar solutions mannitol- and fructose-free, the aliquot was dissolved in water at a concentration of 30 g BPA / L and incubated at room temperature. The next day, the pH 7.4 formulation containing fructose started to precipitate. In contrast, the formulation containing mannitol was still a clear solution without precipitate even after 15 days.

[0173] Therefore, mannitol is superior to fructose in maintaining BPA-tris formulations for extended periods without precipitation in aqueous solutions at pH 7.4-8.0.

[0174] Example 7: BPA Formulations with Bis-Tris and Triethanolamine 0.9 ml of either 0.5 M aqueous Bis-Tris base (Sigma) or 0.5 M aqueous triethanolamine (Sigma) and 30 mg 10 The BPA aliquots were combined. Both Bis-Tris:BPA and Triethanolamine:BPA were in a 3:1 molar ratio. The pH of the aliquots was adjusted to pH>8 using 3M NaOH and then incubated at +37°C for 15-30 minutes to allow complete dissolution of the BPA. The pH of the different aliquots was then adjusted to either 7.4 or 8.0 using dilute HCl and then the aliquots were freeze-dried. The aliquots were dissolved in water to 30 g BPA per L and incubated at room temperature. After 2 hours, the pH 7.4 formulation containing Triethanolamine started to precipitate. In contrast, all other formulations (BPA-Bis-Tris(pH 7.4), BPA-Bis-Tris(pH 7.4), and BPA-Bis-Tris(pH 7.4)) still maintained clear solutions without precipitate even after 15 days.

[0175] 30mg 10 The BPA aliquots were combined with different amounts of aqueous Bis-Tris base (Sigma) to give 3:1, 2:1, and 1:1 molar ratios of Bis-Tris:BPA. They were also either mannitol-free or contained mannitol at a molar ratio of 11:10 mannitol:BPA. The aliquots were adjusted to pH>8 with 3M NaOH and incubated at +37°C until complete dissolution. The pH was then adjusted to 7.4 with dilute hydrochloric acid, the volume was adjusted to 30 g BPA per liter with water, and the solution was incubated at room temperature. After 7 days, the 1:1 Bis-Tris:BPA pH 7.4 formulation began to precipitate. In contrast, all other formulations (BPA-Bis-Tris(pH 7.4), BPA-Bis-Tris(pH 7.4), and BPA-Bis-Tris(pH 7.4)) remained clear solutions without precipitate.

[0176] In conclusion, BPA could be formulated into aqueous solutions that remained in solution for long periods without precipitation, including various forms of 2HA, with Bis-Tris at pH 7.4-8.0, and with triethanolamine at pH 8.0. Bis-Tris:BPA molar ratios ranging from 1:1 to 3:1 allowed for BPA solubility at pH 7.4, and the addition of mannitol at a molar ratio of 1:1 to the formulations helped to keep BPA in solution at pH 7.4 for long periods without precipitation.

[0177] Example 8: BPA-tris-mannitol formulations at different pH Multiple aliquots of BPA-tris-mannitol were prepared; each contained 288 micromolar at +37°C. 10 BPA, 288 micromolar Tris base, 318 micromolar mannitol (molar ratio of BPA:Tris:mannitol = 10:10:11), and 600 micromolar NaOH were combined and completely dissolved in water. The pH was adjusted to either 7.2, 7.5, or 8.0 with dilute hydrochloric acid and the volume was adjusted with water to 30 g BPA per liter. Parallel aliquots of each pH were either incubated directly at room temperature or lyophilized and redissolved in water to 30 g BPA per liter and then incubated at room temperature. The pH 7.2 formulation began to precipitate both before and after lyophilization and upon reconstitution, whereas the other formulations at pH 7.5 and pH 8.0 maintained clear solutions without precipitate.

[0178] In contrast, BPA-mannitol formulations prepared similarly to above but without Tris base precipitated after adjustment to either pH 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8. These aliquots had a 288 micromolar 10 It was prepared by combining BPA, 318 micromolar mannitol (molar ratio of BPA:mannitol = 10:11), and 600 micromolar NaOH in water and dissolving completely, followed by pH adjustment with dilute hydrochloric acid.

[0179] In conclusion, the BPA-Tris-mannitol formulation could be easily adjusted to pH values ​​above pH 7.2, whereas a similar BPA-mannitol formulation without Tris could not be adjusted to pH values ​​in the range of pH 7.3 to 7.8.

[0180] Example 9: High BPA Concentration Formulations with Tris Using the same procedure as above, BPA-Tris-mannitol lyophilisate and solutions with concentrations of 60 g BPA / L and 90 g BPA / L at pH 7.4 (molar ratio of BPA:Tris:mannitol = 10:10:11) were prepared. High concentration BPA-Tris-mannitol lyophilisate and a solution with a concentration of 120 g BPA / L at pH 7.4 were further prepared as described in Example 11 below. All these formulations were completely soluble at both +4°C and room temperature without precipitation. In contrast, in the absence of Tris, BPA-sorbitol and BPA-fructose formulations with 120 g BPA / L could not be prepared by the method described below.

[0181] 240 mg (1153 μmol) in a 2 mL volume to give 120 g BPA / L 10 An attempt was made to prepare high concentration BPA-sorbitol by combining BPA and 252 mg of sorbitol (1383 micromoles; molar ratio of BPA:sorbitol = 10:12) and adding water with 600 micromoles of NaOH at +37° C. However, BPA-sorbitol was insoluble at this concentration and remained as a precipitate.

[0182] 240 mg (1153 μmol) in a volume of approximately 2 mL to achieve 120 g BPA / L 10 An attempt was made to prepare high concentration BPA-fructose by combining BPA and 229 mg of fructose (1268 micromoles; molar ratio of BPA:fructose=10:11) and mixing with aqueous NaOH at +37° C. until the pH was greater than 10. However, the BPA-fructose was insoluble at this concentration and remained as a precipitate.

[0183] Example 10: BPA-Tris-sorbitol formulation 60 mg (288 μmol) at +37°C 10 BPA-Tris-sorbitol was prepared by combining BPA, 288 micromolar Tris base, 317 micromolar Sorbitol (molar ratio of BPA:Tris:sorbitol=10:10:11) and 225 micromolar NaOH and completely dissolving in water. The pH was adjusted to 7.4 with dilute hydrochloric acid and the volume was adjusted with water to 30 g BPA (2 ml) per L. This formulation remained a clear solution without precipitate at both +4°C and room temperature.

[0184] Example 11: BPA formulations containing fructose, mannitol, tris-mannitol, or sorbitol at pH 7.4-8 GMP grade from Interpharma Praha (Prague, Czech Republic) 10 I purchased BPA. Other essential reagents were:

[0185] [Table 1]

[0186] The standard BPA-fructose formulation is called BPA-F. The BPA-tris-mannitol formulation with a molar ratio of 1:1:1.1 is called BPA-T. Compound 2 is BPA-T (30 g BPA per L) pH 7.4, and compound 5 is BPA-T (120 g BPA per L) pH 7.6.

[0187] Compound 1 (BPA-F, pH 7.4) was prepared essentially according to the method of van Rij et al. (Am. J. Health-Syst. Pharm., 2005, 62:2608-10) at 30 g BPA per L. Two lots were prepared so that fresh BPA-F was available for two experiments.

[0188] Lot 1 149.9 mg of BPA was added to 2.5 mL of 0.54 M NaOH. 30 μL of 2 M NaOH was added and stirred until completely dissolved and clear, after which 142.8 mg of fructose was added and stirred. At this point, the pH was 10.45. 320 μL of 2 M HCl was added to bring the pH to 7.4. 2.15 mL of endotoxin-free water was added to bring the volume to 5 mL, and the product was then sterile filtered.

[0189] Lot 2 90.4 mg of BPA was added to 1.5 mL of 0.54 M NaOH. 20 μL of 2 M NaOH was added and stirred until completely dissolved and clear, after which 85.2 mg of fructose was added and stirred. At this point, the pH was 10.49. 192 μL of 2 M HCl was added to bring the pH to 7.43. 1.288 mL of endotoxin-free water was added to bring the volume to 3 mL, and the product was then sterile filtered.

[0190] Compound 2 (BPA-T, pH 7.4) was prepared at 30 g BPA per L (two lots for two experiments).

[0191] Lot 1 90 mg BPA and 86.5 mg mannitol were dissolved in 2.022 mL endotoxin-free water containing 865 μL 0.5 M Tris base and 113 μL 3 M NaOH to give a solution with a pH of 8.71. 158 μL 2 M HCl was added to give a pH of 7.42. The product was sterile filtered, frozen and lyophilized. The finished product was obtained by dissolution in 3 mL endotoxin-free water and sterile filtration.

[0192] Lot 2 149.8 mg BPA and 144.6 mg mannitol were dissolved in 3.375 mL endotoxin-free water containing 1.44 mL 0.5 M Tris base and 188 μL 3 M NaOH to give a solution with a pH of 8.63. 265 μL 2 M HCl was added to give a pH of 7.42. The product was sterile filtered, frozen and lyophilized. The finished product was obtained by dissolution in 5 mL endotoxin-free water and sterile filtration.

[0193] Compound 3 (BPA-sorbitol, pH 7.4) was prepared essentially according to JP2009051766 at 30 g BPA per L. To a solution of 2.4 mL endotoxin-free water, 450 μL 1M NaOH, and 15 μL 3M NaOH, 90.4 mg BPA and 94.3 mg sorbitol were added and stirred. The mixture took 51 minutes to completely dissolve at 37° C., at which point the pH was 8.68. 8 μL 2M HCl and 52 μL 1M HCl were added to bring the pH to 7.43. 77 μL endotoxin-free water was added to bring the volume to 3 mL, and the product was sterile filtered.

[0194] Compound 4 (BPA-mannitol, pH 8) was prepared essentially according to Halbert et al. (Eur. J. Pharm. Sci. 2013, 48:735-9) at 30 g BPA per L. 90.1 mg BPA and 99.2 mg mannitol were added to 2 mL endotoxin-free water and stirred. After adding 185 μL 3M NaOH and stirring at 37° C. until complete dissolution, the pH was 9.01. 50 μL 2M HCl was added to a pH of 7.93. 765 μL endotoxin-free water was added to bring the volume to 3 mL, and the product was sterile filtered, frozen and lyophilized. The finished product was obtained by dissolution in 3 mL endotoxin-free water and sterile filtration.

[0195] Compound 5 (BPA-T, pH 7.6) was prepared at 120 g BPA per L. 120 mg BPA and 115.3 mg mannitol were dissolved in 700 μL of endotoxin-free water containing 1152 μL of 0.5 M Tris base and 125 μL of 3 M NaOH to give a solution with a pH of 8.37. 148 μL of 2 M HCl was added to give a pH of 7.63. The product was sterile filtered, frozen and lyophilized. The finished product was obtained by dissolving in 1 mL of endotoxin-free water and sterile filtering.

[0196] The ready-to-test test materials were stored at room temperature until use. The final composition of these materials is shown in the table below.

[0197] [Table 2]

[0198] Based on the BPA substance composition table, several advantages of the BPA-T formulation are found compared to other BPA formulations.

[0199] BPA concentration and dose volume BPA-T could be prepared in both the standard concentration of 30 g BPA / L and a four-fold higher concentration of 120 g BPA / L for successful intravenous administration in mice. The four-fold higher concentration allowed for intravenous administration in four-fold smaller volumes. This resulted in lower infusion volume stress for intravenous recipients with BPA-T, which has a higher concentration of BPA, compared to other BPA formulations.

[0200] Amount of sodium The BPA-T formulations contained the lowest amount of sodium (Na ) of all BPA formulations, ranging from 2.2 to 2.6 g of sodium per 30 g of BPA, compared with 3.8 to 6.5 g of sodium per 30 g of BPA in the BPA-F, BPA-sorbitol, and BPA-mannitol formulations. + This resulted in lower sodium stress in intravenous recipients with BPA-T compared to other BPA formulations.

[0201] Sugar:BPA ratio In particular, the BPA-T formulation contained the least amount of sugars, 1.1 moles of mannitol per mole of BPA, compared with 1.2-1.3 moles of sugar per mole of BPA in the BPA-sorbitol and BPA-mannitol formulations.

[0202] pH BPA-T was formulated at a physiological pH of 7.4, showing excellent solubility of 30-120 g BPA / L; on the other hand, BPA-mannitol in particular could not be formulated in a soluble form at a pH below 7.9. This results in a lower pH stress for intravenous recipients with BPA-T, especially compared to BPA-mannitol.

[0203] Example 12: In vivo biodistribution of BPA formulations in tumor xenografted mice In vivo studies were initiated to evaluate whether BPA-T could be safely administered to clinically relevant mice and whether tumor localization and biodistribution of BPA-T in tumor xenografted mice following intravenous (iv) administration was similar to that of BPA-F, BPA-mannitol, and BPA-sorbitol.

[0204] Head and neck cancer HSC-2 cells were obtained from the JCRB Cell Bank (JCRB; https: / / cellbank.nibiohn.go.jp / english / ). This cell line was established from a 69-year-old male patient with oral squamous cell carcinoma (Momose et al., 1989, J. Oral Pathol. Med. 18:391-5). The cells were cultured by standard procedures according to the instructions of the cell bank. On the day of cell implantation, cells were harvested and inoculated subcutaneously (sc) into mice at 2 million cells / mouse in 100 μl of 50% Matrigel.

[0205] The experiments were performed at the Turku Center for Disease Modeling (TCDM; Turku, Finland) under the approval of the ethical committee. The tumor formation rate was excellent (100%). The mean tumor size was 200 mm 3 Tumor growth was assessed by palpation until tumor growth reached 0.01 mg / kg / day. From this point onwards, biodistribution experiments were performed on two dates.

[0206] (1) Time-course study of BPA-F and BPA-T The first 24 tumor-bearing mice were divided into groups of 3 mice (n=3, mean group tumor size 208 mm3 , group average range 203-219mm 3 , tumor size range 124-385mm 3 ) in the 10-day dose. Half of the mice received BPA-F and the other half received BPA-T by bolus intravenous injection (all at 30 g BPA / L in a volume of 200 μl). The average weight of the mice in this study was 20.9 g (range 18.5-22.3 g) and the average dose was 287 mg BPA / kg (range 269-324 mg / kg). At 1, 2, 4, and 8 hours, three mice per group from both the BPA-F and BPA-T cohorts were sacrificed for tissue samples.

[0207] (2) Biodistribution of BPA-F, BPA-T (30 g / L and 120 g / L), BPA-mannitol, BPA-sorbitol, and vehicle (PBS) 2 hours after administration. The remaining 36 tumor-bearing mice were divided into groups of 6 mice (n=6, mean group tumor size 203 mm 3 , group average range 196-218mm 3 , tumor size range 100-414 mm 3 ) were administered intravenously in a bolus of test compound or vehicle in a volume of 200 μl, except for the 120 g / L BPA-T group, where the administration volume was 50 μl. The average body weight of the mice in this experiment was 21.4 g (range 18.4-24.9 g), and therefore the average dose was 280 mg BPA / kg (range 241-326 mg / kg). At 2 hours, all groups were sacrificed and tissue samples were taken.

[0208] The following tissue samples were prepared and weighed from all mice: tumor, blood, buccal muscle, skin, brain, liver, and salivary glands. Samples were weighed and stored frozen until analysis.

[0209] The in vivo tests were carried out as planned, and no adverse events, abnormalities or signs of toxicity were observed for any of the test substances.

[0210] In selected mouse tissue samples10 B and 11 The localization of B was analyzed at ALS Scandinavia (Løvere, Sweden). 212 samples were included in this analysis. All tissue samples were subjected to biodistribution study analysis at 2 h for BPA-F and BPA-T, whereas in all other study groups only tumor, blood and buccal muscle samples were analyzed. All samples were successfully analyzed by ICP-SFMS method (inductively coupled plasma mass spectrometry; https: / / www.alsglobal.se / en / isotope-analysis / laboratory). Control samples from mice receiving only the BPA-free vehicle (PBS) showed concentrations of 3–22 μg (equivalent to 3–22 ppb) per gram of tissue. 10 B and 13–91 μg per g of tissue 11 B. Average 10 B: 11 The B ratio is 0.238, which is 10 The published natural abundance of B corresponds to 19.2%. 10 B abundance (19.3%, Bentley and Hamer, 1958, Nature 182:1156). In contrast, samples from mice receiving BPA ranged from 1368 to 31211 μg / g tissue. 10 B (1368–31211 ppb, or approximately equal to 1–31 ppm) and 30–457 μg per gram of tissue. 11 B, which means that 10 This indicates that B accumulation has occurred. 10 B showed the expected results for the highly enriched BPA doses.

[0211] Figure 1 shows the concentrations of BPA-T and BPA-F in all tissues sampled 2 hours after administration to tumor-bearing mice. 10The B concentration in all tissues was highest in the tumor, with BPA-F at 9.5-20.6 μg / kg (mean 12.9 μg / kg) and BPA-T at 10.3-22.8 μg / kg (mean 16.1 μg / kg). The brain was lowest, with BPA-F at 2.8 μg / kg and BPA-T at 3.1 μg / kg. In tumors and tissues, 10 The distribution of B concentrations was similar for both BPA-T and BPA-F.

[0212] absolute 10 Such levels of B concentration were clinically relevant. Furthermore, the BPA dose (roughly 300 mg / kg) was the average value typically used in animal studies, whereas higher doses (up to 400-500 mg / kg) are routinely used in clinical BNCT (Kankaanranta et al., 2011, Int. J. Radiat. Oncol. Biol. Phys. 80:369-76; Kankaanranta et al., 2011, Int. J. Radiat. Oncol. Biol. Phys. 82:e67-75).

[0213] Figure 2 shows the concentrations of BPA-T and BPA-F in all tissues sampled 2 hours after administration to tumor-bearing mice. 10 B Comparison of tumor:tissue ratios. A t-test showed no significant difference between BPA-T and BPA-F. 10 No statistically significant difference in tumor:tissue ratio was observed. Thus, the tissue distribution of BPA-T was similar to that of BPA-F in tumor xenograft mice.

[0214] Figure 3 shows the results for BPA-T and BPA-F. 10 The graph shows the time series of B concentration from 1 to 8 hours after administration of BPA-F and BPA-T in this study. 10 The tumor:blood ratio of BPA-T averaged 5.3 (range 4.7-6.2) over follow-up, and the tumor:muscle ratio averaged 3.2 (range 2.9-3.6).

[0215] In summary, no significant differences were observed in the spatial or temporal in vivo biodistribution of BPA-F and BPA-T in tumor xenograft mice.

[0216] FIG. 4 shows the tumor:blood and tumor:muscle profiles of BPA-T, BPA-F, BPA-mannitol, and BPA-sorbitol at 30 g BPA / L formulations, and of the BPA-T formulation at 120 g BPA / L. 10 Comparison of B ratios is shown. No significant differences were found between BPA-T and other BPA formulations as assessed by the Kruskal-Wallis test.

[0217] In conclusion, this study demonstrated that BPA-T had similar biodistribution, tumor localization, and tissue clearance rates compared to BPA-F, BPA-mannitol, and BPA-sorbitol.

[0218] Example 13: Stability of BPA formulations containing tris-mannitol Aliquots were prepared from a BPA-Tris-mannitol formulation with a molar ratio of 10:10:11 BPA:Tris:mannitol and pH 7.4 using the methods described above and were lyophilized and stored at -20°C or +4°C, or as solutions at +4°C or +37°C (higher temperatures for degradation studies). For analysis at various time points, lyophilized aliquots were dissolved in water to a solution concentration of 30 g BPA / L.

[0219] RP-HPLC analysis was performed on a Gemini C18 column using a gradient of 100% A (water with 0.1% trifluoroacetic acid) to 100% B (acetonitrile) as follows: 5 min at 100% A, 19.5 min at 0–13% B, 10 min at 13–40% B, and 5 min at 40–100% B. Samples equivalent to 50 nmol of amino acids were injected into A.

[0220] Standard compounds were analyzed to determine the elution times for both BPA and its potential degradants (L-phenylalanine and L-tyrosine). BPA eluted at 17.5-18.0 ml; L-phenylalanine eluted at 21.0-22.0 ml; and L-tyrosine eluted at 16.5-17.0 ml. Thus, with respect to relative quantification, the above analytical method allowed the separation of these components. No tyrosine was observed in the BPA-tris-mannitol preparation, but an increased amount of phenylalanine was detected after prolonged incubation at the higher temperature of +37°C. The elution times for the standard compounds were: 1 Further differential confirmation of BPA and phenylalanine was performed using H-NMR spectroscopy. Thus, it was determined that the degradation of the BPA-tris-mannitol formulation results in the production of phenylalanine but not tyrosine. Phenylalanine quantification was used as a measure of stability in the stability studies. BPA was quantified based on absorbance at 256 nm, and phenylalanine was quantified against standards at 214 nm. The BPA standards eluted in the same position as BPA in the BPA-tris-mannitol, BPA-mannitol, BPA-fructose, and BPA-sorbitol formulations.

[0221] Lyophilized formulations stored at -20°C and +4°C for 1 month were both 100% BPA with no detectable amounts of phenylalanine; the solution stored at +4°C was 99.7% BPA with 0.3% phenylalanine (mol %). Thus, both the lyophilized and solution BPA-tris-mannitol formulations exhibited excellent stability, with the lyophilized formulation being completely stable at these temperatures.

[0222] It is obvious to those skilled in the art that with the advancement of technology, the basic concept can be implemented in various ways. Therefore, the embodiments are not limited to the above examples; rather, there may be various modifications within the scope of the claims.

[0223] The embodiments described herein above may be used in any combination. Multiple embodiments may be combined to form further embodiments. The products, methods, or uses disclosed herein may include at least one of the embodiments described hereinafter. It will be understood that the benefits and advantages described above may relate to one embodiment or to multiple embodiments. The embodiments are not limited to those that solve any or all of the problems mentioned or have any or all of the benefits and advantages mentioned. It will be further understood that the expression "an" or "an" item refers to one or more of the items. The term "comprising" is used herein to mean including the feature or function following "comprising" without excluding the presence of one or more additional features or functions.

Claims

1. A pharmaceutical composition comprising boronated phenylalanine (BPA) or a pharmaceutically acceptable salt thereof and a 2-hydroxyamine compound (2HA) or a pharmaceutically acceptable salt thereof, wherein the 2HA is a compound represented by any one of formulas I-II: 【Chemistry 1】 (R １ and R ２ are each independently H or hydroxy-C １ ~C ６ -Alkyl, 2-hydroxyethyl, 2,3,4,5,6-pentahydroxyhexyl, carboxy-C １ ~C ６ -Alkyl, acetyl, C １ ~C ６ -Alkyl, sulfo-C １ ~C ６ -alkyl, 3-sulfopropyl, 2-hydroxy-3-sulfopropyl, 1-sulfo-2-propanyl, 2-sulfoethyl, 3-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]-C １ ~C ６ -alkyl, and 3-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]propyl. 【Chemistry 2】 wherein n is 0 or 1; When n is 1, R ３ is absent; if n is 0, then R ３ is H, hydroxy-C １ ~C ６ -Alkyl, 2-hydroxyethyl, carboxy-C １ ~C ６ -Alkyl, acetyl, C １ ~C ６ -Alkyl, sulfo-C １ ~C ６ 2-sulfoethyl. or a pharmaceutically acceptable salt thereof, or any combination or mixture thereof; Pharmaceutical compositions.

2. 2. The pharmaceutical composition of claim 1, wherein the BPA is L-p-boronophenylalanine or a pharmaceutically acceptable salt thereof.

3. 2. The pharmaceutical composition of claim 1, wherein the 2HA is: Tris(hydroxymethyl)aminomethane (Tris), 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol (bis-tris), (2-hydroxyethyl)amino-tris(hydroxymethyl)methane, N-(tris(hydroxymethyl)methyl)glycine (tricine), 2-(dimethylamino)-2-(hydroxymethyl)propane-1,3-diol (N,N-dimethyl-tris), 1-deoxy-1-{[1,3-dihydroxy-2-(hydroxymethyl)-2-propanyl]amino}hexitol, 2-{[1,3-dihydroxy-2-(hydroxymethyl)-2-propanyl]amino}-1-propanesulfonic acid, [tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS), 3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid (TAPSO), 2-{[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}ethane-1-sulfonic acid (TES), Bis-tris propane (BTP), diethanolamine (DEA), triethanolamine (TEA), [bis(2-hydroxyethyl)amino]acetic acid (bicine), and N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), and any mixtures and combinations thereof, Pharmaceutical compositions.

4. 2. The pharmaceutical composition of claim 1, wherein the 2HA is or comprises tris(hydroxymethyl)aminomethane (Tris) or a pharmaceutically acceptable salt thereof.

5. 10. The pharmaceutical composition of claim 1, wherein the molar ratio of 2HA:BPA is in the range of 0.5 to 3, about 0.5, in the range of 0.5 to 1, about 1, in the range of 1 to 1.5, in the range of 1 to 2, about 2, in the range of 2 to 3, or about 3.

6. 2. The pharmaceutical composition of claim 1, further comprising a polyol, wherein the molar ratio and / or amount of the BPA, the 2HA, and the polyol is such that the sum of the molar amounts of the BPA and the polyol is approximately equal to or greater than the molar amount of the BPA.

7. 2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition further comprises a polyol, and the molar ratios and / or amounts of the BPA, the 2HA, and the polyol are: [2HA] + [polyol] ≥ 0.8 x [BPA]; or [2HA] + [polyol] ≥ 0.9 x [BPA]; or [2HA] + [polyol] ≥ 1.0 x [BPA]; or [2HA] + [polyol] ≥ 1.5 x [BPA]; or [2HA] + [polyol] ≥ 2.0 x [BPA]; or 0.8 x [BPA] ≤ [2HA] + [polyol] ≤ 5.0 x [BPA]; or 0.9 x [BPA] ≤ [2HA] or 1.0 x [BPA] < [2HA] + [polyol] < 4.0 x [BPA]; or 1.5 x [BPA] < [2HA] + [polyol] < 2.5 x [BPA]; or 2.0 x [BPA] < [2HA] + [polyol] < 2.2 x [BPA]; or [2HA] + [polyol] = 2.1 x [BPA].

8. 10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition further comprises at least one of hydrochloric acid, sodium chloride, acetic acid, sodium acetate, polyethylene glycol, a polyol, a sugar, fructose, mannitol, or sorbitol.

9. 9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition comprises L-p-boronated phenylalanine, tris(hydroxymethyl)aminomethane (Tris), and mannitol; and wherein, in terms of the molar amounts of L-p-boronophenylalanine, tris(hydroxymethyl)aminomethane, and mannitol, [Tris] + [mannitol] > [L-p-boronophenylalanine]; or [Tris] + [mannitol] > 1.5 x [L-p-boronophenylalanine]; or [Tris] + [mannitol] > 2 x [L-p-boronophenylalanine]; or [Tris] + [mannitol] < 3 x [L-p-boronophenylalanine]; or [Tris] + [mannitol] < 2.5 x [L-p-boronophenylalanine]; or [Tris] + [mannitol] < 2 x [L-p-boronophenylalanine]; or [Tris] + [mannitol] > 0.8 x [BPA]; or [Tris] + [mannitol] > 0.9 x [BPA]; or [Tris] + [mannitol] [Tris] + [mannitol] ≥ 1.0 x [BPA]; or [Tris] + [mannitol] ≥ 1.5 x [BPA]; or [Tris] + [mannitol] ≥ 2.0 x [BPA]; or 0.8 x [BPA] ≤ [Tris] + [mannitol] ≤ 5.0 x [BPA]; or 0.9 x [BPA] ≤ [Tris] + [mannitol] ≤ 4.0 x [BPA]; or or 1.0 x [BPA] < [Tris] + [mannitol] < 3.0 x [BPA]; or 1.5 x [BPA] < [Tris] + [mannitol] < 2.5 x [BPA]; or 2.0 x [BPA] < [Tris] + [mannitol] < 2.2 x [BPA]; or [Tris] + [mannitol] = 2.1 x [BPA]. Pharmaceutical compositions.

10. 10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is an aqueous solution, and wherein the pH of the aqueous solution is in the range of 7 to 8, in the range of 7.3 to 7.5, in the range of 7.35 to 7.45, about pH 7.4, or at physiological pH or substantially physiological pH.

11. 11. The pharmaceutical composition of claim 10, wherein the concentration of the BPA in the aqueous solution is at least 30 g / L.

12. 10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is a dry formulation, and wherein the dry formulation is optionally capable of becoming an aqueous solution having a pH in the range of 6.5 to 8.5 when water is added.

13. 10. The pharmaceutical composition of claim 1, wherein the BPA has at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% boron-10 atoms of the total boron atoms of the BPA.

14. 14. The pharmaceutical composition according to any one of claims 1 to 13, for use in therapy or for use in boron neutron capture therapy (BNCT).

15. 10. A method for preparing the pharmaceutical composition of claim 1, comprising: mixing BPA with the 2HA to form an aqueous solution of the BPA and the 2HA; Optionally, mixing a polyol with the BPA and the 2HA or adding a polyol to the aqueous solution; and adjusting the pH of the aqueous solution to a range of 6.5 to 8.5; A method comprising:

16. 16. The method of claim 15, comprising: Mixing L-p-boronated phenylalanine and tris(hydroxymethyl)aminomethane (Tris) to form an aqueous solution; mixing mannitol with the BPA and the 2HA or adding mannitol to the aqueous solution; Optionally, a pH adjuster such as an alkali is added to the aqueous solution to completely dissolve the BPA; and adjusting the pH of the aqueous solution to about 7.4, or to a physiological pH or substantially physiological pH, with an acid; A method comprising:

17. 16. The method of claim 15, wherein the molar ratio of 2HA:BPA is in the range of 0.5 to 3, about 0.5, in the range of 0.5 to 1, about 1, in the range of 1 to 1.5, in the range of 1 to 2, about 2, in the range of 2 to 3, or about 3. method.

18. 16. The method of claim 15, wherein the molar ratio of tris(hydroxymethyl)aminomethane (Tris):L-p-boronophenylalanine is in the range of 0.5 to 3, about 0.5, in the range of 0.5 to 1, about 1, in the range of 1 to 1.5, in the range of 1 to 2, about 2, in the range of 2 to 3, or about 3.

19. 16. The method of claim 15, wherein the alkali is sodium hydroxide and the acid is hydrochloric acid.

20. 16. The method of claim 15, wherein the method further comprises drying the aqueous solution, thereby obtaining a dry formulation of the pharmaceutical composition.

21. 10. A method for producing the pharmaceutical composition of claim 1, wherein the method comprises: providing the pharmaceutical composition as a dry formulation; and mixing the pharmaceutical composition as a dry formulation with water, and optionally with one or more compounds selected from hydrochloric acid, sodium chloride, acetic acid, sodium acetate, polyethylene glycol, polyols, sugars, fructose, mannitol, and sorbitol, thereby obtaining the pharmaceutical composition as an aqueous solution; A method comprising:

22. 22. The method of claim 21, wherein the concentration of the BPA in the aqueous solution ranges from 30 to 120 g / L, and the pH of the aqueous solution is about 7.4, or a physiological pH or a substantially physiological pH.

23. 10. Use of a 2-hydroxyamine compound (2HA) or a pharmaceutically acceptable salt thereof as defined in claim 1 or 3 in dissolving boronated phenylalanine (BPA) or a pharmaceutically acceptable salt thereof, thereby forming a pharmaceutical composition comprising BPA as an aqueous solution.

Images