Pharmaceutical compositions comprising thrombolytic peptide-tetrahydroisoquinoline conjugates

Abstract

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

Description

[0001] This application is a divisional application of application number 202080064177.1 (PCT / US2020 / 052514), application date September 24, 2020, and invention name “Pharmaceutical composition comprising a thrombolytic peptide-tetrahydroisoquinoline conjugate”. Technical Field

[0002] The invention relates to a pharmaceutical composition comprising a thrombolytic peptide-tetrahydroisoquinoline conjugate and a pharmaceutically acceptable carrier, and the pH value of the pharmaceutical composition is less than pH 6.5. BRIEF DESCRIPTION OF THE DRAWINGS

[0003] Figure 1A The chemical structure of DC009 is shown.

[0004] Figure 1B The chemical structure of DC009 with NH-Lys-Ala-Pro details is shown. DETAILED DESCRIPTION

[0005] Disclosed herein is a pharmaceutical composition comprising DC009 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. DC009 is also abbreviated as LT3001, and its chemical name is 3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-formyl-Lys (Pro-Ala-Lys), or N6-(L-prolyl-L-alanyl-L-lysyl)-N2-[[(3S)-1,2,3,4-tetrahydro-6,7-dihydroxy-1,1-dimethyl-3-isoquinolyl]carbonyl]-L-lysine (CASRN: 1639303-73-3). The structure of DC009 is shown in Figure 1A The amide bond between the lysine linker and the Pro-Ala-Lys peptide is shown in Figure 1B . DC009 is a binary conjugate that can be formed by coupling a thrombolytic peptide (Pro-Ala-Lys) and a tetrahydroisoquinoline compound having two C1-4 alkyl groups via a lysine linker. The preparation of the DC009 compound is disclosed in Example 63 of US2016-0083423, which is incorporated herein by reference. The pharmaceutically acceptable salt of DC009 includes any pharmaceutically acceptable salt; for example, a hydrochloride, i.e., N6-(L-prolyl-L-alanyl-L-lysyl)-N2-[[(3S)-1,2,3,4-tetrahydro-6,7-dihydroxy-1,1-dimethyl-3-isoquinolyl]carbonyl]-L-lysine hydrochloride (1:3) (CAS RN: 2419930-71-3).

[0006] 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 regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, particularly humans.

[0007] Typically, the pharmaceutical composition is prepared by uniformly and closely combining the active ingredient with a liquid carrier and / or a uniformly finely chopped solid carrier, and then, if necessary, molding the product into the desired formulation. In the pharmaceutical composition, the content of the active target compound is an amount sufficient to produce the desired effect on the process or condition of the disease. Therefore, in some embodiments, the pharmaceutical composition of the present invention includes any composition made by mixing 0.1% to 50% of the active ingredient with a pharmaceutically acceptable carrier. In other embodiments, the pharmaceutical composition of the present invention includes 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 pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition of the present invention includes any composition made by mixing 0.2% to 5% of the active ingredient with a pharmaceutically acceptable carrier. In other embodiments, the pharmaceutical composition of the present invention includes any composition made by mixing 0.5% to 5% of the active ingredient with a pharmaceutically acceptable carrier.

[0008] Pharmaceutically acceptable carriers as inactive ingredients can be selected by those skilled in the art using conventional criteria. Pharmaceutically acceptable carriers include, but are not limited to, non-aqueous solutions, suspensions, emulsions, microemulsions, micellar solutions, gels, and ointments. Pharmaceutically acceptable carriers may also contain the following ingredients, including, but not limited to: saline and aqueous electrolyte solutions; ionic and non-ionic osmotic agents, such as sodium chloride, potassium chloride, glycerol, and dextrose; pH regulators and buffers, such as salts of hydroxide, phosphate, citrate, acetate, borate, and triethanolamine; antioxidants, such as acidic sulfurous acid, sulfurous acid, pyrosulfite, thiosulfate, ascorbic acid, acetylcysteine, cysteine, glutathione, butylated hydroxyanisole, butylated hydroxytoluene, tocopherol, and salts, acids, and / or bases of ascorbyl palmitate; surfactants, such as lecithin, phospholipids, including but not limited to phosphatidylcholine, phosphatidyl Ethanolamine and phosphatidylinositol; poloxamers and ploxamines, 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 and white petrolatum; fats, such as lanolin, peanut oil, palm oil, soybean oil; mono-, di- and triglycerides; polymers of acrylic acid, such as carboxypolymethylene gel and hydrophobically modified cross-linked acrylate copolymers; polysaccharides and glycosaminoglycans, such as sodium hyaluronate. Such pharmaceutically acceptable carriers may be preserved to prevent bacterial contamination with well-known preservatives including, but not limited to, benzalkonium chloride, ethylenediaminetetraacetic acid and its salts, benzethonium chloride, chlorhexidine, chlorobutanol, methylparaben, thimerosal, and phenylethyl alcohol, or may be formulated unpreserved for single or multiple uses.

[0009] In some embodiments, the pharmaceutical composition comprises DC009 and a pharmaceutically acceptable carrier, wherein the pH of the composition is <7 or pH <6.5. In some embodiments, the pH of the composition is about pH 1 to pH 6. In some embodiments, the pH of the composition is about pH 2 to pH 5.5. In some embodiments, the pH of the composition is about pH 3 to pH 6. In some embodiments, the pH of the composition is about pH 3.5 to pH 5.5. In some embodiments, the pH of the composition is about pH 3.5, pH 4, pH 4.5, pH 5, or pH 5.5. In some embodiments, the pH of the composition is about pH 4.5.

[0010] As used in this application, "about" means ± 5% of the recited value.

[0011] In some embodiments, the pharmaceutical composition comprises DC009 and a pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient is a disaccharide or a sugar alcohol.

[0012] In some embodiments, sugar alcohol is mannitol, sorbitol, ethylene glycol, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, galactitol, fucitol, iditol, inositol, wolemitol, isomalt, maltitol or lactitol. In some embodiments, sugar alcohol is arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol or inositol. In some embodiments, sugar alcohol is arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol or inositol. Preferred sugar alcohol is mannitol or sorbitol.

[0013] 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, gentiodiulose, mannobiose, melibiose, plantagobiose, rutinose, rutinulose or xylobiose. In some embodiments, the disaccharide is sucrose, lactulose, lactose, trehalose, cellobiose or chitobiose. Preferred disaccharides are sucrose, lactose or trehalose.

[0014] In the present application, the weight ratio of DC009 to a pharmaceutically acceptable excipient (DC009 / excipient ratio) can be expressed as, for example, 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. For example, a DC009 / excipient ratio can range from 1:1 to 9:1.

[0015] In some embodiments, the pharmaceutical composition comprises DC009 and a pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient is mannitol, sorbitol, sucrose, lactose or trehalose, and wherein the weight ratio of DC009 to the pharmaceutically acceptable excipient is 1: 1 to 1: 9. If the ratio is less than 1: 1, the volume is too small to be lyophilized; if the ratio is greater than 1: 9, the osmotic pressure is too high to be used for intravenous use.

[0016] In some embodiments, the ratio of DC009 to the pharmaceutically acceptable excipient is 1:9. In one embodiment, the pharmaceutical composition comprises about 1% DC009 and about 9% pharmaceutically acceptable excipient. For example, the pharmaceutically acceptable excipient is sucrose, lactose, sorbitol or trehalose.

[0017] In some embodiments, the pharmaceutical composition comprises DC009 and a pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient is 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.

[0018] In various embodiments, the ratio of DC009 to pharmaceutically 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.

[0019] In various embodiments, the ratio of DC009 to pharmaceutically acceptable excipient is about 1: 1.3. In some embodiments, the pharmaceutical composition comprises about 2% DC009 and about 2.6% mannitol.

[0020] In some embodiments, the pharmaceutical composition comprises about 1% DC009 and about 3.8% mannitol, and wherein the pH of the composition is about pH 3.5 to pH 5.5, more preferably, the pH of the composition is about pH 4.5.

[0021] In some embodiments, the pharmaceutical composition comprises about 2% DC009 and about 2.6% mannitol, and wherein the pH of the composition is about pH 3.5 to pH 5.5, more preferably, the pH of the composition is about 4.5.

[0022] The following examples further illustrate the present invention. These examples are intended only to illustrate the present invention and should not be construed as limiting.

[0023] Example

[0024] Example 1. Preparation and purity determination of DC009

[0025] DC009 was prepared according to US2016-0083423. The final product was washed repeatedly with DCM, THF and ether several times to improve the purity of the final product. The concentration and purity of DC009 in the solution were determined by HPLC under the following conditions.

[0026] Column: Alkyl reverse phase bonded phase column

[0027] Mobile phase (MP): MP A: 0.1 vol% TFA in DI water / MP B: 0.1 vol% TFA in acetonitrile

[0028] Detection wavelength: 214 and 280 nm

[0029] Column temperature: 20-25℃

[0030] Sample temperature: 10-15°C

[0031] Injection volume: 10-20 μL

[0032] Gradient: MP A and MP B.

[0033] In Examples 2 to 6, the stability of each formulation is shown as "Sample Recovery" or "Relative Purity Value". Samples at each time point were analyzed by HPLC as described above. For "Sample Recovery", the % value is calculated by dividing the DC009 concentration at the time point by the DC009 concentration at T = 0. For "Relative Purity Value", the % value is calculated as follows: (peak area % of DC009 at Tn / peak area % of DC009 at T0)*100%.

[0034] Example 2. Stability of formulations in solutions of different pH

[0035] Seven formulations listed in Table 1 containing 10 mg / mL DC009 were prepared, DC009 was dissolved in each buffer and adjusted to the indicated pH with NaOH and HCl.

[0036] Table 1. Formulations T-1 to T-7

[0037] Preparation ID pH Buffer T-1 4 5 mM sodium acetate T-2 5 5 mM sodium acetate T-3 6 5 mM sodium citrate T-4 7 50 mM sodium phosphate T-5 7 DI water T-6 7 0.9% NaCl T-7 8 5 mM sodium phosphate

[0038] These formulations were stored at 40°C and 60°C to evaluate sample recovery and total impurities. The appearance and stability under each storage condition are recorded in Tables 2 and 3. No precipitation was observed in any of the samples.

[0039] Table 2. Appearance of Formulations T-1 to T-7

[0040]

[0041] Table 3. Stability of Formulations T-1 to T-7

[0042]

[0043]

[0044] In the accelerated stability study, the sample recoveries of T-1 (pH 4) and T-2 (pH 5) were ≥98% on the 4th day at 40°C and 95% at 60°C. On the 7th day at 40°C, the sample recoveries of T-1 and T-2 on the 7th day were 95% and 96%, and both were 90% at 60°C (data not shown). However, at pH 7 to 8 (T-4 to T-7), the sample recoveries of the formulations after 4 days of storage at 60°C dropped below 90%, indicating a significant increase in the % impurities. These data indicate that DC009 is stable under acidic conditions and is very stable at pH between 4 and 5. For a 10 mg / mL DC-009 solution, 5 mM sodium acetate is sufficient to maintain a stable pH between pH 4 and pH 5.

[0045] Example 3. Stability of formulations in solutions of different pH

[0046] In another independent experiment, nine formulations listed in Table 4 containing 10 mg / mL DC009 were prepared, DC009 was dissolved in each buffer and adjusted to the indicated pH with NaOH and HCl.

[0047] Table 4. Formulations P1 to P6

[0048] sample pH Buffer P1 pH 7.0 5 mM sodium phosphate P2 pH 6.5 5 mM sodium phosphate P3 pH 6.0 5 mM sodium citrate P4 pH 5.5 5 mM sodium citrate P5 pH 5.0 5 mM sodium acetate P6 pH 4.0 5 mM sodium acetate P7 pH 3.5 5 mM sodium acetate P8 pH 2.0 5 mM sodium acetate P9 pH 1.0 5 mM sodium acetate

[0049] These formulations were stored at 60°C to evaluate the stability of the samples at different pH. The relative purity values ​​of DC009 in all formulations are recorded in Table 5, with the purity of DC009 defined as 100% at T=0. The data show that DC009 is stable under all acidic conditions (pH<7) and maintains a purity of>95% for 10 days at 60°C. After being stored at 60°C for one month (day 38), DC009 is stable at pH 1 to pH 6 and maintains a purity value of>86%. The best stability is shown at pH 2.0 to 5.5.

[0050] Table 5. Stability of formulations P1 to P7 (relative purity values)

[0051]

[0052]

[0053] *Relative purity value: (peak area % of DC009 at Tn / peak area % of DC009 at T0)*100%

[0054] Example 4A. Formulation stability in different excipients

[0055] Six formulations containing 10 mg / mL DC009 and different amounts of excipients (mg / g) listed in Table 6 were prepared, each adjusted to pH 4.5 with NaOH and HCl. These formulations were stored at 40°C and 60°C to evaluate sample recovery and total impurities. Stability data are summarized in Table 7.

[0056] Table 6. Formulations E1 to E6

[0057] Ingredients (mg / g) E1 E2 E3 E4 E5 E6 DC-009 10 10 10 10 10 10 Anhydrous sodium acetate 5 mM 5mM 5mM 5mM 5mM 5mM <![CDATA[EDTA2Na 2H2O]]> 0.055 0.055 0.055 0.055 0.055 0.055 Glycine 22 lactose 90 Mannitol 50 Trehalose dihydrate 90 sucrose 90

[0058] Table 7. Stability of Formulations E1 to E5

[0059]

[0060] In the accelerated stability study, sample recoveries of E2 to E5 were ≥90% at 60°C for 2 weeks (Table 7). Glycine excipient of E1 significantly reduced stability compared to control E6, while mannitol, trehalose, sucrose, and lactose improved stability compared to control E6.

[0061] Example 4B. Formulation stability in different excipients

[0062] In a separate experiment, the DC009 stabilization effect of different excipients listed in Table 8 was tested. For formulations S1 to S10, 10 mg / mL DC009 was prepared in 5 mM sodium acetate buffer, the concentration of each excipient was 3.8%, and the pH of the formulation was adjusted to pH 4.5. These formulations were stored at 60°C for two weeks. The relative purity values ​​of DC009 in all formulations are recorded in Table 8, and the DC009 purity at T=0 is defined as 100%. The purity levels of S1 and S3 at 60°C for 2 weeks were >80%. The results show that mannitol and sorbitol provide better stability than other excipients.

[0063] Table 8. Stability of formulations S1 to S10 (relative purity values, %)

[0064] sample excipient Day 0 Day 6 Day 10 Day 14 S1 Mannitol 100.00 90.33 89.01 88.92 S2 maltose 100.00 84.43 81.58 69.91 S3 Sorbitol 100.00 88.71 85.57 81.47 S4 Arginine 100.00 81.96 68.01 56.00 S5 glucose 100.00 82.91 61.12 46.77 S6 Raffinose 100.00 87.72 67.89 48.89 S7 Ethylene glycol 100.00 72.68 59.53 50.83 S8 Dextrose 100.00 83.16 62.49 45.60 S9 Maltitol 100.00 88.77 83.75 74.74 S10 Histidine 100.00 88.76 78.86 69.85

[0065] Example 5. Stability of formulations containing mannitol

[0066] Prepared 4 kinds of preparations containing 10mg / mL DC009 listed in Table 9, each preparation was adjusted to pH 4.5 with NaOH and HCl.M1 to M3 contained 50mg / g D-mannitol, and M4 did not contain D-mannitol.These preparations were stored at 40 ℃ and 60 ℃ to evaluate sample recovery.Table 10 records the stability results under each storage condition.

[0067] Table 9. Formulations M1 to M4

[0068]

[0069] Table 10. Stability of Formulations M1 to M4

[0070]

[0071] In the accelerated stability study, sample recoveries of M1 to M3 were ≥90% at 60°C for 2 weeks. The data showed that the addition of mannitol improved formulation stability compared to the M4 formulation without mannitol.

[0072] Example 6. Study on the stability of freeze-dried formulations

[0073] Two formulations listed in Table 11 were prepared. Each formulation was lyophilized in a lyophilizer and stored at 25°C and 40°C to assess sample recovery. Table 12 summarizes the stability results under each storage condition.

[0074] Table 11. Lyophilized formulations

[0075] Element F-1 F-1H DC009 1 2 Mannitol 3.8 2.6 DI water Make up 100 Make up 100 1N NaOH / HCl to pH 4.5 + + DC009 / mannitol ratio 1:3.8 1:1.3

[0076] Table 12. Stability of lyophilized formulations

[0077]

[0078] Two stable intravenous solution lyophilized formulations (F-1 and F-1H) of 10 mg / mL and 20 mg / mL DC009 were developed, respectively. According to the stability tests performed, F-1 and F-1H were physically and chemically stable for 3 months at 25°C and 40°C. These lyophilized formulations can be reconstituted with normal saline before intravenous injection.

[0079] The present invention and the manner and process of making and using the same have now been described in full, clear, concise and accurate terms to enable one skilled in the art to make and use the invention. It should be understood that the foregoing describes preferred embodiments of the invention and that modifications may be made thereto without departing from the scope of the invention as set forth in the claims. In order to particularly point out and clearly claim the subject matter regarded as the invention, the following claims summarize the specification.

Claims

1. A pharmaceutical composition comprising: A compound having the formula or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, in, The pH of the composition is < 6.

5.

2. The pharmaceutical composition according to claim 1, wherein The pharmaceutically acceptable carrier is saline, a pH regulator or a pH buffer.

3. The pharmaceutical composition according to claim 2, wherein The saline solution is physiological saline.

4. The pharmaceutical composition according to claim 2, wherein The pH regulators are NaOH and HCl regulators.

5. The pharmaceutical composition according to claim 2, wherein The pH buffer is one or more of hydroxide salt, phosphate salt, citrate salt, acetate salt, borate salt and triethanolamine.

6. The pharmaceutical composition according to claim 5, wherein The acetate salt is sodium acetate.

7. The pharmaceutical composition according to claim 5, wherein The phosphate salt is sodium phosphate.

8. The pharmaceutical composition according to claim 5, wherein The citrate salt is sodium citrate.

9. The pharmaceutical composition according to claim 1, wherein The pH of the composition is about pH 1 to pH 6.

10. The pharmaceutical composition according to claim 1, wherein The pH of the composition is about pH 2 to pH 6.

11. The pharmaceutical composition according to claim 1, wherein The pH of the composition is about pH 2 to pH 5.

5.

12. The pharmaceutical composition according to claim 1, wherein The pH of the composition is about pH 3.5 to pH 5.

5.

13. The pharmaceutical composition according to claim 12, wherein The pH of the composition is about pH 4.

5.

14. The pharmaceutical composition according to claim 1, wherein The pharmaceutically acceptable salt is hydrochloride.

15. A pharmaceutical composition comprising: A compound having the formula or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, in, The pH of the composition is pH<6.5, Wherein, the pharmaceutically acceptable excipient is mannitol, sorbitol, sucrose, lactose or trehalose.

16. The pharmaceutical composition according to claim 15, wherein The pH of the composition is 2.0-5.

5.

17. The pharmaceutical composition according to claim 15, wherein The pharmaceutically acceptable excipient is mannitol.

18. The pharmaceutical composition according to claim 15, wherein The weight ratio of the compound to the pharmaceutically acceptable excipient is about 1:1 to 1:

9.

19. The pharmaceutical composition according to claim 18, wherein The weight ratio of the compound to the pharmaceutically acceptable excipient is about 1:1 to 1:

7.

20. The pharmaceutical composition according to claim 18, wherein The weight ratio of the compound to the pharmaceutically acceptable excipient is about 1:1 to 1:

5.

21. The pharmaceutical composition according to claim 20, wherein The pharmaceutically acceptable excipient is mannitol.

22. The pharmaceutical composition according to claim 21, wherein The weight ratio of the compound to the pharmaceutically acceptable excipient is about 1:1.

3.

23. The pharmaceutical composition according to claim 21, wherein The weight ratio of the compound to the pharmaceutically acceptable excipient is about 1:3.

8.

24. The pharmaceutical composition according to claim 21, wherein The pH of the composition is about pH 3.5 to pH 5.

5.

25. The pharmaceutical composition of claim 24, wherein The pH of the composition is about 4.

5.

26. The pharmaceutical composition of claim 24, wherein The composition is a lyophilized preparation.

27. The pharmaceutical composition of claim 15, wherein The pharmaceutically acceptable salt is hydrochloride.

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