Containing thrombin inhibitor aqueous solution for intravenous injection, stable during storage

 

(57) Abstract:

The invention relates to the preparation, intended for intravenous administration. It can be used for treatment of diseases associated with hypercoagulation. The aqueous solution has a pH of 1.0-2.5 and contains thrombin inhibitor of formula noos-CH2-Y, where Y-(R) Cha-Pic-Nag or (R)-Cgl-Aze-Pab, in the form of a salt or free base. The preferred concentration of the inhibitor is 0.001 to 300 mg/ml. the Invention allows to obtain the drug, stable when stored at room temperature for at least two years. After this period is not less than 95% of the active compounds. 7 C.p. f-crystals, 13 tables.

The invention relates to new, improved, stable in storage, ready forms of drugs inhibitors of thrombin for intravenous infusion, especially peptide inhibitors of thrombin HOOC-CH2-(R)Cha-Pic-Nag and HOOC-CH2-(R)Cgl-Aze-Pab, respectively, to a method for obtaining this form and to a method of treatment by introducing these forms of drugs.

I believe that thrombin inhibitors are effective medicines in a number of diseases characterized by hypercoagulation. Their therapeutic use can be facilitated, the EU is the temperature.

In WO 93/111528, Example P1, disclosed an aqueous solution for parenteral administration, and this form is a solution in acetate buffer HOOC-CH2-(R)Cha-Pic-Nag 2HBr for parenteral use with pH 3-7, which is a physiologically acceptable pH range, suitable for injection directly into the body. This solution also includes additives such as sodium chloride, to obtain isotonic.

The active compound HOOC-CH2-(R)-Cgl-Aze-Pab disclosed in PCT/SE 94/00535, filed July 2, 1994, not yet published. Example P2 in this document discloses a solution for parenteral administration, namely an aqueous solution of active compound and sodium chloride, which was adjusted with NaOH to pH 3-9, preferably 5-7.

Stability of peptides is usually a problem in the pharmaceutical industry. Pharmaceutical form, in particular with a low concentration of peptides, often lose their activity during storage.

Each peptide is different from the point of view of pharmacological properties. In the preparation of pharmaceutical forms specific peptide into account must be taken not only pharmacological properties, but that is ashenia with her for the hospital staff and security for the patient. The results of these aspects unpredictable when testing various finished forms and for each peptide has its own special solution with regard to stability.

It is well known that peptides are typically more sensitive to degradation in the acidic and basic environment than in a neutral solution. However, unexpectedly it was found that peptide thrombin inhibitor HOOC-CH2-(R) Cha-Pic-Nag and HOOC-CH2-(R)Cgl-Aze-Pab their storage stability is improved in acidic aqueous solutions.

The scope of this invention encompasses stable when stored as a concentrated aqueous solution for intravenous infusion of active thrombin inhibitor having the formula

HOOC-CH2-Y

where Y is (R)-Cha-Pic-Nag or (R)-Cgl-Aze-Pab pH in the range from 0.1 to 2.5, which does not require special storage conditions and can be stored at room temperature, i.e. in the range of 15-30oC, preferably 18-20oC, in particular, for example, at 25oC.

Thus, a stable aqueous solution of the present invention, which is a concentrated aqueous solution of thrombin inhibitor, bring physiologically acceptable organic or inorganic acid, PNA acid and so on, to obtain the desired pH. Hydrochloric acid is most preferred. This finished form is a simple form without any stabilizing additives.

In acidic solution the solubility of two different drugs increases and it is possible to obtain a more concentrated solution, which means that you can use small containers that are easy to handle.

It is important that this finished form easy to handle and easy to dose. So this form should be placed in containers for parenteral use, such as plastic or glass containers, such as vials, ampoules or pre-filled syringes, and add in bottles for infusion or bottles, i.e. stable when stored concentrated aqueous solution must be diluted before administration to patients.

An additional improvement is that the solutions do not require additives such as buffer salts, isotonic salt or co-solvents, because of their stability. However, a stable solution can also contain preservatives, i.e., to prevent microbiological growth. Solution purpose is ality, glucose, mannitol, dextran etc. or their combinations, and for parenteral administration.

The solution can be prepared with the active peptide thrombin inhibitors, respectively, HOOC-CH2-(R) Cha-Pic-Nag and HOOC-CH2-(R)Cgl-Aze-Pab in the form commonly used salts or in the form of the base.

The solution of the present invention are sufficiently stable for storage at room temperature for long periods of time. A commercial product should preferably have a shelf life of at least two years, and at the end of this period must be at least 95% of active compound, i.e. the total amount of degradation products should be less than 5%.

The preferred concentration of HOOC-CH2-(R) Cha-Pic-Nag can be varied in a wide range between 0.001 to 200 mg/ml, more preferably 0.1 to 100 mg/ml, most preferably 10-100 mg/ml Preferred concentration HOOC-CH2-(R)Cgl-Aze-Pab can be varied in a wide range 0.001 to 300 mg/ml, preferably 0.1 to 200 mg/ml, most preferably 1-100 mg/ml

This invention relates also to a method for producing storable aqueous solution, which comprises dissolving in water the active thrombin hinge to 2.5, preferably from 1.5 to 2.0, or to a particular desired value, for example, 1,0, 1,5, 2,0, 2,5. Appropriate volumes when using ampoules, vials or syringes are 0.1-100 ml, preferably 1-10 ml

Both active thrombin inhibitor HOOC-CH2-(R)Cha-Pic-Nag and HOOC-CH2-(R)Cgl-Aze-Pab, respectively, must be able to dissolve in water at room temperature.

Obtaining compounds in which Y is (R)-Cgl-Aze-Pab

Mass spectra were recorded on a triple quadrupole mass spectrometer Finnigan MAT TSQ 700, with electrospray system input.

Measurements of the spectra of1H NMR and13C NMR was performed on a spectrometer BRUKER AC-P 300 and BRUKER AM 500 and first worked on1H frequency 500,14 MHz and13C frequency 125,76 MHz, and the second on1H and13C frequency 300,13 MHz and 75,46 MHz, respectively.

The samples consisted of about 10-50 mg, dissolved in 0.6 ml of one of the following solvent: CDCl3(isotopic purity >99.8 per cent), CD3OD (isotopic purity >99.95 per cent) or D2O (isotopic purity >of 99.98%). All solvents were purchased from a company Dr. Glaser AG, Basel.

The magnitude of chemical shift1H and13C in CDCl3and CD3OD, soothe aunesty with a sodium salt of 3-(trimethylsilyl)-d4-propanoic acid, and chemical shifts13C and D2O associated with 1,4-dioxane (67,3 M. D.), both taken as an external standard. Calibration with an external standard in some cases may cause slight differences in the shift in comparison with the internal standard, however, the difference in1H chemical shift is less than 0.02 M. D., and13C the shift is less than 0.1 M. D.

Range1H NMR peptide sequences containing Proline or similar to Proline" remnant, often gives two series of resonances. This corresponds to the existence of two conformers, which contribute relative rotation around the amide bond, when Proline contains N-the part of the amide bond. These conformers are called CIS and TRANS. In compounds containing such sequences, the sequence (R)Cha-Aze and (R)Cha-Pic - often lead to the CIS-TRANS equilibrium with one conformera as the dominant conformer (>90%). In these cases presents data1H chemical shifts only the main rotamer. Only in cases of clear resolution signals secondary rotamer these data are included in the NMR spectral data. The same criterion is valid for the NH signals in CDCl3only with the otons, reported for some of the intermediate compounds is less than the number of protons expected based on the chemical formula.

Flash chromatography was performed on Merck Silica Gel 60 (40-63 mm, 230-400 mesh mesh) at atmospheric pressure.

The lyophilization was carried out on the installation Leydold-Heraeus, model Lyovac GT2.

4-Aminomethyl-1-(N-benzyloxycarbonylamino)-benzene(H-Pab(Z)

(i) 4-cyanobenzoate

To 49,15 g (251 mmol) of 4-cyanobenzaldehyde in 200 ml of DMF at room temperature was added a solution on 20, 23 g (0.31 mol) of sodium azide in 50 ml of water. Was exothermic reaction, and after 1.5 h the reaction mixture was diluted with 200 ml of toluene (note: to avoid extracting potentially explosive azide compounds should be added to the reaction mixture, the toluene before adding water) and 500 ml of water. The aqueous phase was extracted with additional CH ml of toluene. The combined organic extracts were washed h ml of water and saturated saline and then dried (MgSO4) and filtered. The solution was used as such in the next stage.

1H-NMR (300 MHz, CDCl3); and 4.4 (s, 2H), and 7.4 (d, 2H), 7.7 (d, 2H).

(ii) 4-amidinotransferase

In a mixture of 250 ml of absolute ethanol and the solution with the result storage for 24 h at 8oC and evaporation of the greater part of the solvent, followed by precipitation by addition of anhydrous ether was obtained white crystals, which were isolated by filtration and was dissolved in 1.8 liters of alcoholic ammonia solution. After 48 h the bulk of the solvent was removed and added to 200 ml of 3.75 M NaOH solution, after which 4-amidinotransferase was deposited in the form of colorless crystals. The crystals were isolated by filtration. At this point, the output 4-amidinotransferase 22.5 g (51%).

Hydrochloride ethylimidazole;

1H-NMR (500 MHz, CD3OD); 1,6 (t, 3H), and 4.5 (s, 2H) and 4.65 (q, 2H), 4,8 (user.s, 2H), and 7.6 (d, 2H) and 8.1 (d, 2H).

4-amidinotransferase:

1H-NMR (500 MHz, CDCl3); the 4.3 (s, 2H), 5,7 (user.s, 3H), and 7.3 (d, 2H), and 7.6 (d, 2H).

13C-NMR (125 MHz, CDCl3): carbon amidine: 165,5.

(iii) 4-(benzyloxycarbonylamino)benzylated

The crystals obtained in stage (ii), as described above, was dissolved in 500 ml of methylene chloride and the resulting solution was dried (K2CO3), filtered and added 27 ml (194 mmol) of triethylamine. To a stirred solution was slowly added to 25 ml of benzylchloride upon cooling, this reaction mixture in an ice bath. After 30 min DOPOLNITEL phase was brought to pH 7 using 2M HCl. The organic phase was dried (MgSO4) and the solvent was removed in vacuum. In conclusion, from a mixture of ether/methylene chloride/hexane allocated 4-(benzyloxycarbonylamino)benzylated in the form of colorless crystals.

1H-NMR (500 MHz, CDCl3); and 4.4 (s, 2H), 5,3 (s, 2H), 6,3-7,0 (user. s, 1H), 7.3 to 7.4 (m, 5H), 7.5 (d, 2H), 7,9 (d, 2H), and 9.3, and 9.6 (user. s, 1H).

13C-NMR (125 MHz, CDCl3): carbon amidine: 167,5.

(iv) 4-aminomethyl-1-(N-benzyloxycarbonylamino)benzene(H-Pab(Z))

To 4-(benzyloxycarbonylamino)benserazide from step (iii) above, dissolved in 160 ml of THF at room temperature was added to 26.3 g (100 mmol) of triphenylphosphine. After 16 h was added 6.6 g (25 mmol) of triphenylphosphine and the solution was allowed to stand for 4 h before removing the solvent in vacuo. The residue was dissolved in methylene chloride and was extracted with 2 M HCl. The aqueous phase was washed with methylene chloride and ether and then podslushivaet 3.75 M solution of sodium hydroxide. As a result of extraction with methylene chloride followed by drying (K2CO3) and removal of solvent in vacuo received 20 g (total yield in the calculation of the original cyanobenzeneboronic amounted to 28%) yellow oil, which solidifies upon maturation.

1H-NMR (500 MHz, CDCl3); 1,2-2: carbon of amidine and carbonyl: 164,6 and 168,17.

H-Aze-OMe HCl

Received in accordance with the procedure described by D. Seebach Et al., Liebigs Ann. Chem., p. 687, 1990.

Boc-(R)Cgl-OH

Boc-(R)-Pgl-OH, 32 g (0.13 mol), was dissolved in 300 ml of methanol was added 5 g of Rh/AI2O3. The solution was first made when 5,2 - 2,8 MPa for 3 days. After filtration and evaporation of solvent analysis NMR showed the presence of about 25% methyl ester specified in the connection header. The crude material was dissolved in 500 ml of THF and 300 ml of water was added 20 g of LiOH. The mixture was stirred over night and the THF evaporated. The remaining aqueous phase was acidified KHSO4and three times were extracted with ethyl acetate. The combined organic layer was washed with water, dried (Na2SO4) and evaporated to obtain 28,3 g (83%) of the desired product.

1H-NMR (300 MHz, CDCl3): 0,9-1,7 (m, 20H), 4,0-4,2 (m, 1H), and 5.2 (d, 1H).

Boc-(R)Cgl-Aze-OH

(i) Boc-(R)Cgl-Aze-OMe

To stir the mixture 3,86 g (15 mmol) Boc-(R)Cgl-OH, and 2.27 g (15 mmol) of H-Aze-OMe HCl and 2.75 g (to 22.5 mmol) DMAP in 40 ml of CH3CN in 5oC was added, and 3.16 g (16.5 mmol) of EDC. The reaction mixture was stirred at room temperature for 48 hours, the Solvent evaporated and the residue was dissolved in 150 ml EtOAc and 20 ml of H2O. the Separated organic layer was washed 2 20 ml of 0.5 M KHSO4, 2 x 10 ml NaHSO

1H NMR (500 MHz, CDCl3, 0.1 g/ml); the main rotamer, 0,83-of 1.35 (m, 5H), to 1.38 (s, 9H), 1,47-of 1.84 (m, 6H), 2,18-of 2.27 (m, 1H), 2,50-2,62 (m, 1H), and 3.72 (s, 3H), 3,94-4,06 (m, 1H), 4,07-to 4.15 (m, 1H), 4,39-4,47 (m, 1H), and 4.68 (DD, 1H), 5,09 (d, 1H). Resolved peaks from secondary rotamer, and 2.27 to 2.35 (m, 1H), of 3.77 (s, 3H), 3,80-a 3.87 (m, 1H), 3,88-3,95 (m, 1H), 4.92 in (d, 1H), total of 5.21 (DD, 1H).

(ii) Boc-(R)Cgl-Aze-OH

Hydrolysis of Boc-(R)Cgl-Aze-OMe was carried out according to the procedure described for Boc-(R)Cha-Pic-OEt (see below). The product was led from EtOH/acetone/water (1/1/3,95), yield 80%.

1H-NMR (500 MHz, CDCl3): 0,85-1,3 (m, 5H), of 1.40 (s, 9H), of 1.5-1.9 (m, 6H), 1,95-2,2 (m, 2H), 3,92 (m, 1H), 4.09 to (m, 1H), 4,35 (m, 1H), 4.95 points (m, 1H), 5,16 (user.d, 1H).

The connection is HOOC-CH2-(R)Cgl-Aze-Pab

(i) Boc-(R)Cgl-Aze-Pab(Z)

To stir the mixture 3,40 g (10 mmol) Boc-(R)Cgl-Aze-OH (see obtaining raw materials) and to 5.13 g of DMAP (42 mmol) in 120 ml of CH3CN was added 3,18 g H-Pab(Z) HCl (see Getting the source materials). After stirring for two hours at room temperature the mixture was cooled to -8oC and added a 2.01 g (10.5 mmol) of EDC. The reaction mixture was allowed to reach room temperature and stirring continued for another 47 hours, the Solvent evaporated and the residue was dissolved in 200 ml EtOAc. Orgy and dried. After evaporation of solvent received a total of 5.21 g (86%) specified in the connection header.

1H-NMR (500 MHz, CDCl3): 0,8-1,9 (m, 20H; this of 1.30 (s, 9H)), 2,35 of 2.6 (m, 2H), 3,74 (user.t, 1H), 4,10 (m, 1H), 4,25-4,4 (m, 2H), 4,45-4,6 (m, 1H, rotamer), 4.75 V to 5.0 (m, 1H, rotamer), 5,08 (user.d, 2H), further 5.15 (s, 2H), 7,15-to 7.35 (m, 5H), 7,41 (d, 2H), to 7.77 (d, 2H), 8,21 (m, 1H).

(ii) H-(R)Cgl-Aze-Pab(Z)

To a cold solution (the temperature of the ice bath) 18,8 g HCl(g)in 195 ml of EtOAc was added 4,69 g (7,743 mmol) Boc-(R)Cgl-Aze-Pab(Z) together with 40 ml of EtOAc. The reaction mixture was allowed to reach room temperature and was stirred for 30 minutes To transparent solution was added 140 ml of Et2O, after which the formed precipitate. The reaction mixture was left to stand at room temperature for another 1 h 40 min the Precipitate was filtered, quickly washed with 150 ml of Et2O and dried in vacuum. The precipitate was dissolved in 50 ml of water and podslushivaet 15 ml of 2 M NaOH. The alkaline aqueous phase was extracted with 1 x 100 + 1 x 50 ml CH2Cl2. The combined organic phase was washed 1 x 20 ml water, 1 x 20 ml of saturated saline solution and dried (MgSO4). Evaporation of the solvent gave 3,44 g (88%) specified in the connection header.

1H-NMR (500 MHz, CDCl3): 0,8-2,0 (m, 11H), of 2.51 (m, 1H), to 2.67 (m, 1H), of 3.07 (d, 1H), 4,11 (m, 1H), 4,18 (m, 1H), 4,43 (l is ol) H-(R)Cgl-Aze-Pab(Z) 0.9 g (2.6 mmol) benzyl-2-(ortho nitrobenzenesulfonate)acetate ((2-NO2)Ph-SO2-OCH2-COOBn) (see Getting the source materials) 0,99 g (5.6 mmol) of K2CO3and 113 ml of CH3CN was mixed and heated in an oil bath at 60oC for 3 hours the Solvent is evaporated in vacuum. Added EtOAc and the mixture was washed with water, the organic phase was extracted with 1 M KHSO4and this aqueous phase is washed with EtOAc. An acidic aqueous phase was podslushivaet 1H. NaOH to pH>8 and was extracted with EtOAc. The organic phase is washed with water, dried (Na2SO4), filtered and evaporated in vacuum to obtain 1,17 g of residue, which was twice subjected to flash chromatography using as eluents first CH2Cl2/MeOH (NH3-rich) 95/5, and then diethyl ether/MeOH (NH3-rich) 9/1, obtaining 0,525 g (36%) specified in the connection header.

Alkylation was also carried out using benzyl-2-(para-nitrobenzenesulfonate)acetate ((4-NO2)Ph-SO2-OCH2-COOBn) (see Obtaining raw materials), using the same procedure as above, obtaining specified in the connection header with 52% yield.

1H-NMR (300 MHz, CDCl3): 0,85-of 2.15 (m, 11H), 2,48 (m, 1H), 2.63 in (m, 1H) 8,42 (m, 1H).

(iva) HOOC-CH2-(R)Cgl-Aze-Pab 2HCl

BnOOC-CH2-(R)Cgl-Aze-Pab(Z), 20 mg (0,031 mmol), was dissolved in 5 ml of methanol. Added a few drops of chloroform and 5% Pd/C and the mixture was first made at atmospheric pressure for 1 h After filtration and evaporation the product liofilizirovanny of water to obtain 11 mg (72%) specified in the connection header.

1H-NMR (500 MHz, D2O): 1.0 to 2.0 (m, 11H), 2,10 (m, 1H), 2,44 (m, 1H), 2,82 (m, 1H), 3,90 (s, 2H), 4.09 to (d, 1H), 4,4-4,55 (m, 2H), of 4.66 (s, 2H), 5,08 (m, 1H), 7,65 (d, 2H), 7,89 (d, 2H).

13C-NMR (75.5 MHz, D2O): the carbon of amidine and carbonyl: 167,3, 167,9, 169,9 and 172,4.

(ivb) HOOC-CH2-(R)Cgl-Aze-Pab

BnOOC-CH2-(R)Cgl-Aze-Pab(Z) was dissolved in EtOH (99%) and was first made over 5% Pd/C at atmospheric pressure for 5 hours Filtration of the catalyst through cellit and evaporation of the solvent gave specified in the title compound with yield of 97%.

1H-NMR (500 MHz, CD3OD, mixture of two rotamers): main rotamer: 1,00-1,12 (m, 1H), 1,13-of 1.34 (m, 4H), 1,55-1,70 (m, 3H), 1,73-of 1.85 (m, 2H), 1,94-2,02 (user. d, 1H), 2,32-to 2.42 (m, 1H), 2,54-of 2.64 (m, 1H), 2.95 and-3,10 (AB-system plus d, 3H), 4,18-4,25 (user.kV, 1H), 4,28-4,32 (user.kV, 1H), 4,43-4,60 (AB-system, 2H), 4.80 to around 4.85 (DD, 1H), of 7.48-rate of 7.54 (d, 2H), 7,66-7,71 (d, 2H). Resolved signals from the minor rotamer appear when 0,95 (m), USD 1.43 (m), 2,24 (m) 2,84 (d), of 3.96 (m), a 4.03 (m), 7,.

Examples

Analytical methods used in the following examples,

Stability studies

For HOOC-CH2-(R)Cha-Pic-Nag and HOOC-CH2-(R)Cgl-Aze-Pab, respectively, a method of indicating the stability, using liquid chromatography with reversed-phase ion-pair method and UV-detection at 210 and 238 nm.

A standard solution of thrombin inhibitors 0,018-0,022 mg/ml receive in the mobile phase. The sample solution is diluted to a concentration of about 0.02 mg/ml mobile phase. Column counterbalance the mobile phase until then, until you get a stable baseline. The standard and the sample is injected with the appropriate intervals. Retention time for the active thrombin inhibitor HOOC-CH2-(R)Cha-Pic-Nag and HOOC-CH2-(R)Cgl-Aze-Pab is for these methods respectively about 4 and 12 minutes

pH

the pH of the solutions was measured potentiometric without prior dilution, using a Radiometer PHM Reference pH meter with a glass electrode (GK2401C electrode).

The following examples illustrate, but in no way limit the invention.

Example 1. This example presents the results of the stability study at 25oC previously investintech and having a pH of 5.0 in the beginning of the study.

(i) HOOC-CH2-(R)Cha-Pic-Nag 2HBr buffer solution (0.04 mg/ml), pH 5

Composition:

HOOC-CH2-(R)Cha-Pic-Nag 2HBr - 0.04 mg

Sodium hydroxide 1 M to pH 5 - as needed

Acetate buffer 0.05 M (including NaCl, 9 mg/ml) To 1 ml

Stability (see table. 1)

(ii) HOOC-CH2-(R)Cha-Pic-Nag 2HBr buffer solution (9 mg/ml), pH 5

Composition:

HOOC-CH2-(R)Cha-Pic-Nag 2HBr - 9 mg

Sodium hydroxide 1 M to pH 5 - as needed

Acetate buffer 0.05 M (including NaCl, 9 mg/ml) To 1 ml

Stability (see tab. 2).

Conclusion

This study storage indicates that storage at room temperature, 25oC, within 2 years may not be acceptable due to the significant destruction active compounds.

Example 2. This example presents the results of the stability studies at 25oC HOOC-CH2-(R)Cha-Pic-Nag in the form of free base in the same concentration as in the above example 1 (ii), and the initial pH value of about 5. The purpose of this study was to compare the stability of HOOC-CH2-(R)Cha-Pic-Nag in the form of a free base and its salts.

HOOC-CH2-(R)Cha-Pic-Nag buffer solution (9 mg/ml), pH 5

Composition:

HOOC-CH2-(R)Cha-Pic-Nag as its the,05 M (including NaCl, 9 mg/ml) To 1 ml

Stability (see tab. 3).

Conclusion

This study shows that there is a big difference in stability when stored at room temperature, 25oC, when using HOOC-CH2-(R)Cha-Pic-Nag in the form of a salt or free base (cf. example 1).

Example 3. This example reports the results of the stability studies at 25oC buffer solutions HOOC-CH2-(R)Cha-Pic-Nag at different pH between 3 and 9.

(i) HOOC-CH2-(R)Cha-Pic-Nag buffer solution (9 mg/ml) at different pH

Composition:

HOOC-CH2-(R)Cha-Pic-Nag in the form of a free base - 9 mg

Hydrochloric acid (1 M) to pH as needed

Acetate buffer of 0.05 M (incl. NaCl, 9 mg/ml) To 1 ml

Stability (see tab. 4).

(ii) HOOC-CH2-(R)Cha-Pic-Nag 2HCl buffer solutions (1 mg/ml) at different pH

Composition:

HOOC-CH2-(R)Cha-Pic-Nag 2HCl - 9 mg

Buffer solution*)To 1 ml

*)(acetate buffer with pH 4, 5 and 6, phosphate buffer pH 8, for carbonate buffer with a pH of 9)

Stability (see tab. 5).

Conclusion

This study shows that the destruction of HOOC-CH2-(R)Cha-Pic-Nag decreases at lower pH values, regardless of the route stability at 25oC HOOC-CHa-(R)Cha-Pic-Nag in two different concentrations at a pH of about 1.5, in particular from 1.5 to 1.6.

HOOC-CH2-(R)Cha-Pic-Nag aqueous solution (0.4 mg/ml), pH of 1.5

Composition:

HOOC-CH2-(R)Cha-Pic-Nag in the form of a free base - 0.4 mg

Hydrochloric acid 1 M to pH 1.5 to How you want

Water Up to 1 ml

Stability (see tab. 6).

(ii) HOOC-CH2-(R)Cha-Pic-Nag aqueous solution (66 mg/ml), pH of 1.5

Composition:

HOOC-CH2-(R)Cha-Pic-Nag in the form of a free base - 66 mg

Hydrochloric acid 1 M to pH 1.5 to How you want

Water Up to 1 ml

Stability (see tab. 7).

Conclusion

This study shows that the destruction of HOOC-CH2-(R)Cha-Pic-Nag is reduced at the specified low pH values.

Example 5. This example reports the results of stability studies at 25oC HOOC-CH2-(R)Cha-Pic-Nag in two different concentrations at pH of 2.0. The purpose of the study is to compare the fracture at different pH values in the range from 1.5 to 2.5 by comparing (i) with Example 4 (i).

(i) HOOC-CH2-(R)Cha-Pic-Nag aqueous solution (0.4 mg/ml), pH 2

Composition:

HOOC-CH2-(R)Cha-Pic-Nag in the form of a free base - 0.4 mg

Hydrochloric acid 1 is-Nag aqueous solution (26 mg/ml), pH 2

Composition:

HOOC-CH2-(R)Cha-Pic-Nag in the form of a free base - 26 mg

Hydrochloric acid 1 M to pH 2 - as needed

Water Up to 1 ml

Stability (see tab. 9).

Conclusion

This study shows that the fracture is reduced at this low pH.

Example 6. This example presents the results of the stability studies at 25oC HOOC-CH2-(R)-Cgl-Aze-Pab in the form of free base.

HOOC-CH2-(R)-Cgl-Aze-Pab buffer solution (0.4 mg/ml), pH 6

Composition:

HOOC-CH2-(R)-Cgl-Aze-Pab in the form of a free base - 0.04 mg

Sodium hydroxide (1 M) to pH 6 - as needed

Acetate buffer (0.05 M) (incl. NaCl 9 mg/ml) To 1 ml

Stability (see tab. 10).

Conclusion

This study storage shows that prolonged storage at 25oC this form with a pH of 6 leads to a significant destruction.

Example 7. This example reports the results of stability studies at 25oC HOOC-CH2-(R)-Cgl-Aze-Pab in the form of a buffer solution and aqueous solution, respectively, at different pH.

(i) HOOC-CH2-(R)-Cgl-Aze-Pab buffer solutions (12.9 mg/ml) at different pH

It is up to pH As needed

Acetate buffer 0.05 M (incl. NaCl, 9 mg/ml) To 1 ml

Stability (see tab. 11).

(ii) HOOC-CH2-(R)-Cgl-Aze-Pab aqueous solutions (12.9 mg/ml) at different pH

Composition:

HOOC-CH2-(R)-Cgl-Aze-Pab in the form of a free base - 12.9 mg

Hydrochloric acid 1 M to pH as needed

Water Up to 1 ml

Stability (see tab. 12).

Conclusion

This study shows that the destruction of HOOC-CH2-(R)-Cgl-Aze-Pab decreases at a lower value of

pH, with or without buffer systems.

Example 8. This example reports the results of stability studies at the 50oC HOOC-CH2-(R)-Cgl-Aze-Pab in aqueous solutions at different pH.

HOOC-CH2-(R)-Cgl-Aze-Pab aqueous solutions (12.0 mg/ml) at different pH

Composition:

HOOC-CH2-(R)-Cgl-Aze-Pab in the form of free base to 12.0 mg

Hydrochloric acid 1 M NaOH until a specified pH - as needed

Water Up to 1 ml

Stability (see tab. 13).

Conclusion

This study shows that at elevated temperatures the destruction of HOOC-CH2-(R)-Cgl-Aze-Pab also decreases at lower pH values.

Final conclusions

Wiseup inhibitor HOOC-CH2-(R)Cha-Pic-Nag and HOOC-CH2-(R)-Cgl-Aze-Pab, respectively, ke decreases at lower pH values, regardless of whether they are in the form of a base or in salt form, and regardless of the concentration of active compounds.

In addition, studies show that aqueous solutions of these thrombin inhibitors having a pH in the range from 1.0 to 2.5, are more stable when stored at room temperature for extended periods of time.

1. Aqueous solution for intravenous injection, stable during storage, having a pH of 1.0 to 2.5, containing the active thrombin inhibitor in a concentration of 0.001 to 300 mg/ml and having the formula HOOC-CH2-Y, where Y is (R)-Cha-Pic-Nag or (R)-Cgl-Aze-Pab, in the form of its salt or free base.

2. Aqueous solution under item 1, in which thrombin inhibitor is HOOC-CH2-(R) Cha-Pic-Nag in the form of free base.

3. Aqueous solution under item 1, in which thrombin inhibitor is HOOC-CH2-(R)-Cgl-Aze-Pab in the form of free base.

4. Aqueous solution under item 1, in which pH is 1.5 - 2.0.

5. An aqueous solution according to any one of paragraphs.1, 2, or 4, in which the concentration of active thrombin inhibitor is 0.001 - 20 HOOC-CH2-(R) Cha-Pic-Nag, water and inorganic acids.

7. An aqueous solution according to any one of paragraphs.1, 3, or 4, consisting of active thrombin inhibitor HOOC-CH2-(R)-Cgl-Aze-Pab, water and inorganic acids.

8. Aqueous solution under item 6 or 7, in which the inorganic acid is hydrochloric acid.

 

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