Pharmaceutical compositions containing thrombolytic peptide-tetrahydroisoquinoline conjugates

Abstract

The present invention provides a pharmaceutical composition comprising the binary conjugate DC009, which is a conjugate of a thrombolytic peptide (Pro-Ala-Lys) and a tetrahydroisoquinoline compound having two C1-4 alkyl groups via the lysine linking arm, and a pharmaceutically acceptable carrier. The composition has a pH of less than 6.5, preferably between about pH 2 and 5.5. The composition may also contain a pharmaceutically acceptable excipient such as mannitol, sorbitol, sucrose, lactose, or trehalose.

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition comprising a thrombolytic peptide-tetrahydroisoquinoline conjugate and a pharma- ceutically acceptable carrier having a pH of less than pH 6.5. Summary of the Invention [Means for solving the problem]

[0002] Disclosed herein is a pharmaceutical composition comprising DC009 or a pharma- ceutically acceptable salt thereof and a pharma- ceutically acceptable carrier. DC009 is also abbreviated as LT3001, and has the chemical name 3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys (Pro-Ala-Lys) or L-lysine N6-(L-prolyl-L-alanyl-L-lysyl)-N2-[[(3S)-1,2,3,4-tetrahydro-6,7-dihydroxy-1,1-dimethyl-3-isoquinolinyl]carbonyl] (CAS Registry Number: 1639303-73-3). The structure of DC009 is shown in Figure 1A, and the amide bond between the lysine linking arm and the Pro-Ala-Lys peptide is shown in Figure 1B. DC009 is a binary conjugate that may be formed by linking a thrombolytic peptide (Pro-Ala-Lys) to a tetrahydroisoquinoline compound having two C1-4 alkyl groups via a lysine linking arm. The preparation of the DC009 compound is disclosed in Example 63 of US Patent Publication No. 2016-0083423, which is incorporated herein by reference. Pharmaceutically acceptable salts of DC009 include any pharma-ceutically acceptable salt, such as the hydrochloride salt, i.e., L-lysine N6-(L-prolyl-L-alanyl-L-lysyl)-N2-[[(3S)-1,2,3,4-tetrahydro-6,7-dihydroxy-1,1-dimethyl-3-isoquinolinyl]carbonyl]-hydrochloride (1:3) (CAS Registry Number: 2419930-71-3). [Brief description of the drawings]

[0003] [Figure 1A] FIG. 1A shows the chemical structure of DC009. [Figure 1B] FIG. 1B shows the chemical structure of DC009 detailing NH-Lys-Ala-Pro. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0004] The term "pharmaceutically acceptable" means biologically or pharmacologically compatible for in vivo use in animals or humans. In some embodiments, "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, or more particularly, in humans.

[0005] In general, pharmaceutical compositions are prepared by uniformly and intimately associating an active ingredient with a liquid carrier or finely divided solid carrier or both, and then, if necessary, shaping the product into a desired formulation. In a pharmaceutical composition, the active object compound is contained in an amount sufficient to produce the desired effect on a disease process or condition. Thus, in some embodiments, the pharmaceutical composition of the present invention encompasses any composition made by mixing 0.1-50% of an active ingredient with a pharma-ceutically acceptable carrier. In yet other embodiments, the pharmaceutical compositions of the present invention include any composition made by mixing 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of the active ingredient with a pharma- ceutically acceptable carrier. In certain embodiments, the pharmaceutical compositions of the present invention include any composition made by mixing 0.2%-5% of the active ingredient with a pharma- ceutically acceptable carrier. In other embodiments, the pharmaceutical compositions of the present invention include any composition made by mixing 0.5%-5% of the active ingredient with a pharma- ceutically acceptable carrier.

[0006] Pharmaceutically acceptable carriers for the inactive ingredients can be selected by those skilled in the art using conventional criteria, including, but not limited to, non-aqueous based solutions, suspensions, emulsions, microemulsions, micellar solutions, gels, and ointments. Pharmaceutically acceptable carriers include saline and aqueous electrolyte solutions; ionic and non-ionic osmotic agents such as sodium chloride, potassium chloride, glycerol, and dextrose; pH adjusting agents and buffers such as hydroxides, phosphates, citrates, acetates, borates, and other salts; trolamine; antioxidants such as bisulfite, sulfite, metabisulfite, thiosulfite, ascorbic acid, acetylcysteine, cysteine, glutathione, butylated hydroxyanisole, butylated hydroxytoluene, tocopherol, salts of ascorbyl palmitate, acids and / or bases; surfactants such as phospholipids, including, but not limited to, lecithin, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol; poloxamers and poloxamers; The composition may also include, but is not limited to, ingredients including polysorbates such as polysorbate 80, polysorbate 60, and polysorbate 20, polyvinyls such as polyvinyl alcohol and povidone; cellulose derivatives such as methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, carboxymethylcellulose, and hydroxypropylmethylcellulose and their salts; petroleum derivatives such as mineral oil, white petrolatum; fats such as lanolin, peanut oil, palm oil, soybean oil; mono-, di-, and triglycerides; acrylic acid polymers such as carboxypolymethylene gels, and hydrophobically modified crosslinked acrylate copolymers; polysaccharides and glycosaminoglycans such as sodium hyaluronate. Such pharma- ceutically acceptable carriers may be preserved against bacterial contamination using well-known preservatives, including, but not limited to, benzalkonium chloride, ethylenediaminetetraacetic acid and its salts, benzethonium chloride, chlorhexidine, chlorobutanol, methylparaben, thimerosal and phenylethyl alcohol, and may be formulated as unpreserved preparations for single or multiple use.

[0007] In some embodiments, the pharmaceutical composition comprises DC009 and a pharma- ceutically acceptable carrier, and the composition has a pH<7 or pH<6. In some embodiments, the composition has a pH of about pH1 to pH6. In some embodiments, the composition has a pH of about pH2 to pH5.5. In some embodiments, the composition has a pH of about pH3 to pH6. In some embodiments, the composition has a pH of about pH3.5 to pH5.5. In some embodiments, the composition has a pH of about pH3.5, pH4, pH4.5, pH5, or pH5.5. In some embodiments, the composition has a pH of about 4.5.

[0008] As used in this application, "about" refers to ±5% of the stated value.

[0009] In some embodiments, the pharmaceutical composition comprises DC009 and a pharma- ceutically acceptable excipient, wherein the pharma- ceutically acceptable excipient is a disaccharide or a sugar alcohol.

[0010] In some embodiments, the sugar alcohol is mannitol, sorbitol, ethylene glycol, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, or lactitol. In some embodiments, the sugar alcohol is arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, or inositol. In some embodiments, the sugar alcohol is arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, or inositol. The preferred sugar alcohol is mannitol or sorbitol.

[0011] In some embodiments, the disaccharide is sucrose, lactose, trehalose (β,β-trehalose, α,β-trehalose), lactulose, maltose, cellobiose, chitobiose, kojibiose, nigerose, sophorose, laminaribiose, gentiobiose, turanose, maltulose, palatinose, gentiobiulose, mannobiose, melibiose, melibiulose, rutinose, rutinulose, or xylobiose. In some embodiments, the disaccharide is sucrose, lactulose, lactose, trehalose, cellobiose, or chitobiose. The preferred disaccharide is sucrose, lactose, or trehalose.

[0012] In the present application, the weight ratio of DC009 to pharma- ceutically acceptable excipient (DC009 / excipient ratio) may be expressed, for example, as either 9:1 or 9. For example, a DC009 / excipient ratio expressed as 0.5:1 is the same as a DC009 / excipient ratio expressed as 0.5. The range of DC009 / excipient ratios may be expressed, for example, as 1:1 to 9:1.

[0013] In some embodiments, the pharmaceutical composition comprises DC009 and a pharma- ceutically acceptable excipient, where the pharma- ceutically acceptable excipient is mannitol, sorbitol, sucrose, lactose, or trehalose, and the weight ratio of DC009 to pharma- ceutically acceptable excipient is 1:1 to 1:9. If the ratio is less than 1:1, the volume is too small for lyophilization, and if the ratio is greater than 1:9, the osmolality is too high for intravenous use.

[0014] In some embodiments, the ratio of DC009 to pharma- ceutically acceptable excipient is 1:9. In one embodiment, the pharmaceutical composition comprises about 1% DC009 and about 9% pharma- ceutically acceptable excipient. For example, the pharma- ceutically acceptable excipient is sucrose, lactose, sorbitol, or trehalose.

[0015] In some embodiments, the pharmaceutical composition comprises DC009 and a pharma- ceutically acceptable excipient, the pharma- ceutically acceptable excipient being mannitol, and the weight ratio of DC009 to mannitol is 1:1 to 1:7, preferably 1:1 to 1:5. For example, the weight ratio of DC009 to mannitol is about 1:1, 1:1.3, 1:3.8, or 1:5. In various embodiments, the weight ratio of DC009 to mannitol is 1:5. In one embodiment, the pharmaceutical composition comprises about 1% DC009 and about 5% mannitol.

[0016] In various embodiments, the ratio of DC009 to pharma- ceutically acceptable excipient is about 1:3.8. In one embodiment, the pharmaceutical composition comprises about 1% DC009 and about 3.8% mannitol. In one embodiment, the pharmaceutical composition comprises about 4% DC009 and about 14.8% mannitol.

[0017] In various embodiments, the ratio of DC009 to pharma- ceutically acceptable excipient is about 1:1.3, hi some embodiments, the pharmaceutical composition comprises about 2% DC009 and about 2.6% mannitol.

[0018] In some embodiments, the pharmaceutical composition comprises about 1% DC009 and about 3.8% mannitol, and the composition has a pH of about pH 3.5 to pH 5.5, more preferably, the composition has a pH of about pH 4.5.

[0019] In some embodiments, the pharmaceutical composition comprises about 2% DC009 and about 2.6% mannitol, and the composition has a pH of about pH 3.5 to pH 5.5, more preferably, the composition has a pH of about 4.5.

[0020] The following examples further illustrate the invention. These examples are intended merely to be illustrative of the invention and are not to be construed as limiting. EXAMPLES

[0021] Example 1. Preparation and Purity Measurement of DC009 DC009 was prepared according to US Patent Publication No. 2016-0083423. To increase the purity of the final product, it was repeatedly washed with DCM, THF, and ethyl ether several times. The concentration and purity of DC009 in the solution were determined by HPLC under the following conditions. Column: Alkyl reversed phase stationary phase column Mobile phase (MP): MP A: 0.1% by volume TFA in deionized (DI) water / MP B: 0.1% by volume TFA in acetonitrile Detection wavelength: 214nm and 280nm Column temperature: 20~25℃ Sample temperature: 10~15℃ Injection volume: 10-20μL Gradient: MP A and MP B

[0022] In Examples 2-6, the stability of each formulation is presented as "assay recovery" or "relative purity level." Samples at each time point were analyzed by HPLC as described above. For "assay recovery," the percentage is calculated by dividing the DC009 concentration at that time point by the DC009 concentration at T=0. For "relative purity level," the percentage is calculated as (DC009 peak area % at Tn / DC009 peak area % at T0) x 100%.

[0023] Example 2. Stability of formulations in different pH solutions Seven formulations shown in Table 1 were prepared containing 10 mg / mL DC009, which was dissolved in each buffer and adjusted to the indicated pH with NaOH and HCl.

[0024] [Table 1]

[0025] These formulations were stored at 40° C. and 60° C. and assay recovery and total impurities were evaluated. Appearance and stability at each storage condition are recorded in Tables 2 and 3. No precipitate was observed in any of the samples.

[0026] [Table 2]

[0027] [Table 3]

[0028] In accelerated stability studies, the assay recoveries of T-1 (pH 4) and T-2 (pH 5) were greater than 98% at 4 days at 40°C and 95% at 60°C. The assay recoveries of T-1 and T-2 at 7 days were 95% and 96% at 40°C and both 90% at 60°C (data not shown). On the other hand, the assay recoveries of the pH 7-8 formulations (T-4-T-7) dropped to less than 90% after 4 days of storage at 60°C, indicating a large increase in impurity %. These data suggest that DC009 was stable in acidic conditions and very stable at pH 4-5. 5 mM sodium acetate is sufficient to keep a 10 mg / mL DC-009 solution stable at pH 4-5.

[0029] Example 3. Stability of formulations in solutions of different pH In a separate, independent experiment, the nine formulations shown in Table 4 were prepared containing 10 mg / mL DC009, which was dissolved in each buffer and adjusted to the indicated pH with NaOH and HCl.

[0030] [Table 4]

[0031] These formulations were stored at 60°C and the stability of the samples at different pH was evaluated. The relative purity levels of DC009 in all formulations are recorded in Table 5, with the purity of DC009 defined as 100% at T=0. The data suggests that DC009 was stable in all acidic conditions (pH<7) and maintained purity levels above 95% for 10 days at 60°C. After one month of storage at 60°C (day 38), DC009 was stable at pH 1 to pH 6 and maintained purity levels above 86%. The best stability was demonstrated at pH 2.0 to 5.5.

[0032] [Table 5]

[0033] Example 4A. Stability of formulations in different excipients Six formulations containing 10 mg / mL DC009 and different amounts of excipients (in mg / g) were prepared and each formulation was adjusted to pH 4.5 with NaOH and HCl, as shown in Table 6. The formulations were stored at 40° C. and 60° C. and assay recovery and total impurities were evaluated. The stability data are summarized in Table 7.

[0034] [Table 6]

[0035] [Table 7]

[0036] In accelerated stability studies, assay recoveries for E2-E5 were 90% or greater at 60°C for 2 weeks (Table 7). The E1 glycine excipient significantly reduced stability compared to the control E6, whereas mannitol, trehalose, sucrose, and lactose improved stability compared to the control E6.

[0037] Example 4B. Stability of formulations in different excipients In another experiment, different excipients, as shown in Table 8, were tested for their effect on the stabilization of DC009. For formulations S1-S10, 10 mg / mL DC009 was prepared in 5 mM sodium acetate buffer, with the concentration of each excipient being 3.8%, and the pH of the formulations was adjusted to pH 4.5. These formulations are stored at 60° C. for 2 weeks. The relative purity levels of DC009 in all formulations are recorded in Table 8, and the purity of DC009 was defined as 100% at T=0. The purity levels of S1 and S3 were greater than 80% at 60° C. for 2 weeks. The results indicate that mannitol and sorbitol provided better stability than other excipients.

[0038] [Table 8]

[0039] Example 5. Stability in formulations containing mannitol Four formulations were prepared containing 10 mg / mL DC009, as shown in Table 9, and each formulation was adjusted to pH 4.5 with NaOH and HCl. M1-M3 contained 50 mg / g D-mannitol, and M4 contained no D-mannitol. These formulations were stored at 40° C. and 60° C. and assay recovery was evaluated. The stability results at each storage condition are recorded in Table 10.

[0040] [Table 9]

[0041] [Table 10]

[0042] In accelerated stability studies, assay recoveries of M1-M3 were greater than 90 at 60°C for 2 weeks. The data suggest that the addition of mannitol improved the stability of the formulations compared to the M4 formulation without mannitol.

[0043] Example 6. Formulation Stability Testing Lipid-Based Formulations Two formulations were prepared as shown in Table 11. Each formulation was lyophilized in a lyophilizer, stored at 25° C. and 40° C., and assay recovery was evaluated. The stability results at each storage condition are summarized in Table 12.

[0044] [Table 11]

[0045] [Table 12]

[0046] Two stable solution-based intravenous lyophilized formulations (F-1 and F-1H) were developed at 10 mg / mL and 20 mg / mL, respectively, for DC009. Based on the stability studies performed, F-1 and F-1H were physically and chemically stable for 3 months at 25° C. and 40° C. The lyophilized formulations can be reconstituted with normal saline prior to intravenous injection.

[0047] The invention, and the methods and processes for making and using it, are now described in such full, clear, concise and exact terms as to enable any person skilled in the art to which it pertains, to make and use the same. The foregoing describes the preferred embodiments of the invention, it being understood that changes may be made therein without departing from the scope of the invention as defined in the appended claims. To particularly point out and distinctly claim the subject matter regarded as invention, the following claims conclude this specification.

Claims

1. (a) a compound of the following formula or a pharma- ceutically acceptable salt thereof; 【Chemistry 1】 (b) a pharma- ceutically acceptable excipient selected from the group consisting of mannitol, sorbitol, sucrose, lactose, and trehalose, said composition having a pH between pH 2 and pH 5.5, and a weight ratio of said compound to said pharma-ceutically acceptable excipient between 1:1 and 1:

9.

2. The pharmaceutical composition of claim 1, wherein the pharma- ceutically acceptable excipient is mannitol.

3. The pharmaceutical composition according to claim 2, having a pH of from pH 3.5 to pH 5.

5.

4. The pharmaceutical composition of claim 2 having a pH of pH 4.

5.

5. The pharmaceutical composition of claim 2, wherein the pharma- ceutically acceptable salt is a hydrochloride salt.

6. The pharmaceutical composition of claim 1 having a pH of pH 3.5 to pH 5.

5.

7. The pharmaceutical composition of claim 1 having a pH of 4.

5.

8. 2. The pharmaceutical composition of claim 1, wherein the pharma- ceutically acceptable salt is a hydrochloride salt.

9. The pharmaceutical composition of claim 1, wherein the weight ratio of the compound to the pharma- ceutically acceptable excipient is from 1:1.3 to 1:

5.

10. The pharmaceutical composition of claim 1, wherein the pharma- ceutically acceptable excipient is sorbitol.

11. The pharmaceutical composition of claim 1, wherein the pharma- ceutically acceptable excipient is sucrose.

12. The pharmaceutical composition of claim 1, wherein the pharma- ceutically acceptable excipient is lactose.

13. The pharmaceutical composition of claim 1, wherein the pharma- ceutically acceptable excipient is trehalose.

14. (a) a compound of the following formula or a pharma- ceutically acceptable salt thereof; 【Chemistry 2】 (b) mannitol, wherein the weight ratio of the compound to mannitol is from 1:1.3 to 1:3.8, and the pH of the composition is 4.5 after reconstitution with saline to 10 mg / mL to 20 mg / mL.

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