MXPA03008545A - Pharmaceutical compositions containing human growth hormone. - Google Patents

Abstract

Pharmaceutical composition containing human growth hormone (HGH) and a stabilizing amount of sucrose, as well as a procedure for the preparation thereof.

Description

PHARMACEUTICAL COMPOSITIONS CONTAINING HUMAN GROWTH HORMONE.

The present invention relates to pharmaceutical compositions containing the human growth hormone (HGH). More precisely, it refers to compositions of human growth hormone stabilized with sucrose. It is known that highly purified proteins are unstable over time and are stabilized, for example, in admixture with saccharides, such as lactose and mannitol, or also with proteins and amino acids, such as albumin and glycine. Human growth hormone is secreted in the human pituitary. In its mature form it consists of 191 amino acids, has a molecular weight of 22,000 and is, therefore, more than three times larger than insulin. This hormone is a linear polypeptide that contains two bisulfide bridges between chains. Until the advent of recombinant DNA technology, HGH could be obtained only by painstaking extraction from a limited source: the pituitary glands of human cadavers. The consequent shortage of substance limited its application to the treatment of hypopituitary dwarfism even though it had been considered effective in the treatment of burns, wound healing, dystrophy, bone welding, diffuse gastric hemorrhages and pseudoarthrosis. HGH can be produced in a recombinant host cell, in amounts that would be suitable for treating pituitary dwarfism and other disorders for which it is effective. The most important biological effect of HGH is to promote growth. Affected organ systems include the skeleton, connective tissue, muscles, and viscera such as the liver, intestine, and kidneys. Growth hormone exerts its action through its interaction with specific receptors on cell membranes. Compositions of lyophilized proteins are described in M. J. Pikal, Biopharm, October 1990, 25-30. Examples of growth hormone formulations have been reported with stabilizing excipients such as mannitol, glycine, arginine and lactose. In particular, lyophilization is described in the presence of several substances in their amorphous state, such as sugars, which increase the collapse temperature and allow shorter lyophilization times to be obtained. However, it is not feasible, according to the author, to provide a standard formulation for all proteins, and the choice of the best formulation requires a remarkable selection work. German Patent DE 3520228 discloses bioactive proteins, including growth hormone, in formulations that are stabilized by means of polysaccharides comprising repetitive units of maltotriose. WO 89/09614 discloses formulations of human growth hormone stabilized with glycine, mannitol and a buffer, in which the molar ratio of the human growth hormone: glycine is 1: 50-200. U.S. Pat. US 5122367 describes a controlled release system for the administration of growth hormones, comprising the protein and a polysaccharide incorporated within a polymeric matrix. Patent Application EP 210039 describes a controlled release implant for subcutaneous administration to an animal of bovine hormone or growth porcine, in the form of a matrix containing 40% sucrose. According to the present invention, HGH can be either natural or synthetic, that is, produced on the basis of recombinant DNA technology, with the latter being preferred. Injectable formulations of human growth hormone are obtained by a process that includes their lyophilization in order to obtain a dry powder. Human growth hormone is highly susceptible to denaturation during the lyophilization process and it is desirable to obtain stable formulations to maintain a longer life cycle when stored at room temperature. In order for materials such as HGH to be provided for the health care of staff and patients, these materials should be prepared as pharmaceutical compositions. The compositions must maintain their activity for appropriate periods of time, must be acceptable in their own right in terms of their easy and rapid administration to humans, and should be easy to manufacture. In many cases the pharmaceutical formulations are provided in frozen or lyophilized form. In this case, the composition must be thawed or reconstituted prior to its use. The frozen or lyophilized form is often used - to maintain the biochemical integrity and bioactivity of the medicinal agent contained in the compositions in a wide variety of storage conditions, as it is recognized by those skilled in the art that freeze-dried preparations they often maintain their activity better than their equivalent liquids. The lyophilized preparations are reconstituted prior to use by the addition of a suitable pharmaceutically acceptable diluent or diluents, such as sterile water for injection or sterile physiological saline, and the like. Alternatively, the composition can be provided in liquid form suitable for immediate use. A liquid formulation that maintains its activity during long-term storage is desirable. The current formulation of HGH loses activity due to the formation of higher order dimers and aggregates (macro-interval) during the processing of the formulation as well as during storage and reconstitution. Other chemical changes, such as deamidation and oxidation may also occur during storage. The human growth hormone is on the market in stabilized formulations, for example with Mannitol Saizen® and Grorm® by Serono. It has now been found that sucrose confers improved stability to the HGH formulation and in particular to the form of this glycoprotein that has been prepared with the recombinant DNA technique. It has also been found that sucrose unexpectedly prevents the formation of a precipitate when the reconstituted solutions are stirred. The main object of the present invention is to provide pharmaceutical compositions comprising a solid intimate mixture of human growth hormone and a stabilizing amount of sucrose alone. A further object is to provide a process for the preparation of the pharmaceutical composition, comprising the step of lyophilizing an aqueous solution of the components in the containers. Another object is to provide a form of presentation of the pharmaceutical composition comprising the aforementioned solid mixture sealed under sterile conditions in a container suitable for storage before use and suitable for reconstitution of the mixture of injectable substances.

A different object is to provide a solution for the solid mixture reconstituted in an injectable solution. In order to evaluate the effect of the excipient on the stability of the active ingredients, various formulations of recombinant HGH containing 5 or 10 mg per vial have been prepared with various excipients: sucrose, glycine, mannitol, sucrose plus mannitol and mannitol plus glycine . The compositions of the various formulations that have been prepared are shown in Tables 1 and 4. The preparation of the lyophilisate was carried out by diluting the HGH mass with solutions containing the stabilizers all of which in buffers at pH 7.5. The obtained solutions were filtered, taken to their final volume, poured into the various glass vials and lyophilized. The study of the stability of the formulations stored at 4 ° C, 25 ° C, 37 ° C and 50 ° C for 24 weeks, was determined through: Reverse phase HPLC (RP-HPLC) according to the method described by RM Riggin et al., Anal. Biochem., 167: 199-209, 1987, and size exclusion HPLC (HPSEC) according to US Pharmacopeia Preview, November-December 1990, pages 1253-1261. The results are shown in Tables 2-3 and 5-6 in which the measurement is expressed as percent recovery of HGH in the various formulations. The methodology of the chromatographic assay to evaluate the percentage of HGH recovery was performed as described by Pikal in Pharmaceutical Research 8, page 428"Assays". In the pre-formulation phase, the effect of pH and buffer on the stability of rHGH in lyophilized form was evaluated by evaluating the stability at 50 ° C. The tests were carried out in different buffer systems prepared with acetic acid, phosphoric acid, 0.01 M succinic acid at pH 6.00, 7.00 and 8.00 with NaOH. The results showed that the stability of rHGH was not affected by the buffer, and that the formulations were in any case more stable at pH 8.00, approximately. The pH selected for the compositions was 7.5. Next, seven lyophilized formulations were prepared with concentrations of gr. of 5 Mg. / vial, using both phosphate buffer and succinct to pp. 7.5 to test the compatibility of the active drug with different excipients (sucrose 68.4 Mg./ vial, mannitol 36.4 Mg./ vial, mannitol / glycine 25 + 4 Mg./ vial, mannitol / sucrose 32 + 7, 5 g / vial), the amount of excipients was selected in order to have an isotonic solution after reconstitution with a bacteriostatic solvent. The filling volume was from 1 to. Samples prepared under sterile conditions were stored at 50 ° C, 37 ° C, 25 ° C and 4 ° C for 24 weeks and tested by HPSEC, and reverse phase HPLC. The pH and the moisture content were determined. The stability of the solutions reconstituted with 0.3% m-cresol and 0.9% benzyl alcohol at 4 ° C and 25 ° C was also studied. The HPSEC and RP-HPLC were performed as described above. The pH was determined by a pH measuring device on a vial reconstituted with 1 ml of water for injection. To determine the moisture content of the lyophilized vials, the composition of a vial was suspended in 1 ml of 2-isopropanol, filtered through a 0.22 μm (Mere.) Anotop 10 disposable filter. Injected into a Coulometer Meter. The stability results, tested by RP-HPLC (Riggin's method), are shown in Table 2. The chromatographic profiles of the sucrose-containing formulations (HGH / 3 and HGH / 7 of Table 1) after 24 weeks at 50 ° C are not different from those obtained at zero time. At the same temperature a purity decrease of 13-22% was found in the formulations containing mannitol and mannitol + glycine. The data shown in Table 3 refer to the results obtained through the analyzes by HPSEC. No decrease in the purity percentage of rHGH was found in all the formulations tested. No significant variation in moisture content was observed during the study in all the lyophilized formulations tested. A decrease in pH was observed at 37 ° C and 50 ° C for the batches HGH / 5 and HGH / 7 of Table 1. The stability of the reconstituted solutions was also studied through the analyzes by RP-HPLC (Rigen method) and HPSEC. With the procedure by RP-HPLC, after five weeks at 25 ° C the purity decrease was found to be in the range of 30% -50% for the samples reconstituted with both vinyl alcohol and m-crucol. After seven weeks at 4 ° C the variation was about 14% in the presence of vencillic alcohol and 4% -8% with m-crucol. No variation was observed at 4 ° C with the procedure by HPSEC; on the contrary, a decrease in the purity of the g was found. about 5% at 25 ° C for all formulations in the presence of both vinyl alcohol and m-crucible. The results showed that the formulations containing sucrose and sucrose + mannitol presented a better stability profile when compared with the other formulations. On the basis of the results obtained with the 5 Mg compositions, sucrose and mannose were chosen for the preparation of five lyophilized formulations (Table 4) containing 10 Mg. of HGH / vial using phosphate buffer and sucinto to pp. Of 7.5 adjusted with 2.5 M Year. One formulation contained 68.4 Mg. / vial of sucrose (filling volume 1 ml) in phosphate buffer only, the others contained 102.6 Mg. / vial of sucrose (filling volume 1.5 ml) and mannitol + sucrose 130 + 40 Mg. / vial (filling volume 1.5 ml), both with phosphate buffer and succinct. The optimal ratio between sucrose and the manitos and the volume of filling to obtain a product with good physical characteristics was adjusted based on preliminary lyophilization tests. The optimum ratio of mannitol / sucrose in terms of resistance of the lyophilized cake at elevated temperature was 3: 1 and the maximum volume to be lyophilized was 1.5 ml. The formulations were subjected to stability tests by storing samples at 50 ° C, 37 ° C, 25 ° C and 4 ° C for 24 weeks. The samples were subjected to the following analytical controls. HPSEC, RP-HPLC (Riggin method), pp. And moisture content. The stability of the reconstituted solutions with 0.3% m-crucible and 0.9% benzyl alcohol at 4 ° C was observed for 4 weeks. The samples were subjected to the same controls carried out on the 5 Mg dose. as described above. The analyzes showed the following results: The formulations containing 68.4 Mg. / vial and 102.6 Mg. / vial of sucrose in conical buffer assayed by RP-HPLC analysis, showed no decrease in purity after 24 weeks of storage at all temperatures tested. The results are shown in Table 5. The formation of degradation products was observed in the other formulations even after 4/6 weeks of storage at 50 ° C. No decrease in the percentage of purity of the gr. in all the formulations tested by analysis by HPSEC, see Table 6. During the study, no variation in pH or in moisture content was observed in all the formulations tested. Studies were also carried out on the reconstituted solutions containing only sucrose by analysis by RP-HPLC (Riggin method) and HPSEC. After 4 weeks at 4 ° C, with the RP-HPLC method, the decrease in purity was found to be about 12% in the presence of vinyl alcohol and 4% with m-crucol. No decrease in the purity of the gr. at 4 ° C, with the HPSEC method, both in the presence of benzyl alcohol and m-cresol. To evaluate the efficacy of the antimicrobial protection, vials of the HGH / 3 formulation of Table 1 were reconstituted with 1 ml of bacteriostatic solvent (0.3% m-cresol or 0.9% benzyl alcohol). They were tested according to the European Pharmacopoeia until 21 days from sowing. The results are shown in Tables 7 and 8. The minimum acceptable efficacy (minimum criteria) was reached for both protective solutions. The results obtained in time zero, in which the microorganisms were counted after inactivation both in saline (NaCl 0.9%) and bacteriostatic, seem to indicate a higher efficacy of m-cresol against benzyl alcohol mainly for Staphylococcus and Pseudomonas that were reduced immediately after inactivation of 90,000 to 25,000 and 78,000 to 8,000 CFU / ml, respectively (Table 7). In addition, Aspergillus disappeared in m-cresol after 14 days from sowing (Table 8) and Pseudomonas after 6 hours. The above results indicate that the formulation containing 68.4 mg of sucrose, phosphate buffer at pH 7.5, filling volume of 1 ml reconstituted with 0.3% meta-cresol is the only one that guarantees the best stability of rHGH both in an amount of 5 and 10 mg.

EXAMPLE OF PHARMACEUTICAL MANUFACTURE Materials: pure sucrose Ph Eur, BP, Nord, NF (Merck); H3P04 Suprapur (Merck); NaOH for analytical use (Merck); water for your injection. As containers, DIN 2R vials (borosilicate glass type I), rubber closures (Pharmagummi W1816 V50) and aluminum rings and ejection capsules (Pharma-Metal GmbH) have been used.

Preparation of the rHGH solution containing sucrose (for 1000 vials each containing 10 mg of HGH). Sucrose (68.4 g), and H3PO4 (1.96 g) are dissolved in water for use as injectables (800 ml) in order to obtain the starting sucrose solution. The mass of the HGH (10 g) is added to the sucrose solution which, after the pH has been adjusted to 7.5 by means of 2.5 M NaOH, is brought to the final volume of 1000 ml. The solution is filtered through a 0.22 μm Durapore sterile filter (Millipore). During the procedure the temperature of the solution is maintained between 4 and 8 ° C. Solutions containing different excipients or a different active drug dosage have been prepared in a similar manner.

Filling and lyophilization The vials are filled with 1 ml of sterile HgH solution, transferred to the lyophilizer and chilled at 45 ° C for at least 6 hours.The lyophilization is started at a temperature of -45 ° C with a vacuum of 7 Pa. The heating is carried out according to the following scheme: +10 ° C for 12 hours, then +35 ° C until the end of the cycle. or TaDla COMPOSITION OF THE 5 mg VIAL Components: HGH / 1 HGH / 2 HGH / 3 HGH / 4 HGH / 5 HGH / 6 HGH / 7 Sucrose mg / vial 7.5 - 68.4 - 7.5 - 68, + Mannitol mg / vial 32 36.4 _ 25 32 36, + Glycine mg / vial _ - _ 5 - - ~ Buffer: Phosphoric acid mg / vial 0.98 0 ^ 98 0.98 0.98 Succinic acid mg / vial - - - - t > 18 V8 V8 NaOH cs. at pH 7.5 7.5 7.5 7.5 7.5 7.5 7.5 Filling volume 1 mi 1 mi 1 mi 1 mi 1 m 1 mi 1 mi Reconstitution volume 1 mi i mi 1 mi 1 mi 1 mi 1 mi 1 mi OR SAIZEN 5 mg CHROMATOGRAPHIC PURITY OF r-HGH THROUGH THE RIGGIN METHOD LIOFILIZED FORMULATIONS M / S | »Manthol + Sucrose U to Mannitol U / G - Mannitol + Glycine S Sucrose W per week or Table 3 CHROMATOGRAPHIC PURITY OF THE rHGH THROUGH LITHUATED FORMULATIONS Mannitol + Sucrose Mannitol Mannitol + Glycine Sucrose Week N3 O Table 4 COMPOSITION THE VIAL OF 10 mg Components: S10 / S / F / 1/01 S10 / S / F / 01 S10 / S / S / 01 S10 / S / F / 01 S10 / SM / S / 0 ' r-HGH mg / viol 10 10 10 10 10 Loten0 PG R9201D2 Sucrose mg / vial 68.4 102.6 102.6 40 40 Mannitol mg / vial 130 130 Buffer: Phosphoric acid mg / vial 1.98 1 > 98 1.98 Succinic acid mg / vial 2.36 2.36 NaOH is. at pH 7.5? 7.5 7.5 75 Filling volume 1 mi 1.5 mi 1; S mi 1-5 mi 15 mi Reconstitution volume 2 mi 2 mi 2 mi 2 mi 2 mi or < ~ ° TABLE 5 CHROMATOGRAPHIC PURITY OF rHGH BY RP-HPLC (METHOD OF RIGGIN'S) LIOFILIZED FORMULATIONS S10 / S / S / 01 SACROSE / SUCCINATE (FILLING VOLUME 1.5 ml) S10 / SA 1/01 - SACAROSA / PHOSPHATE (FILLING VOLUME 1 ml) S10 / S / r "/ 01 - SACAROSE / PHOSPHATE (VOLUME FILLING 1.5 ml) SO / SU / S / Ol, SACROSE + MANITOUSUCCINATE (FILLING VOLUME 1.5 ml) S 0 / St-l / r / 0. SACKAGE + MANITOUPHOSPHATE (FILLING VOLUME, 5 ml) Table 6 CHROMATOGRAPHIC PURITY OF THE rHGH THROUGH HPSEC LIOFILIZED FORMULATIONS S10 / S / S / 01 Sucrose / succinate (filling volume 1.5 ml) sto / sA / i / oi Sucrose / Phosphate (filling volume 1.0 ml) S10 / S / F / 01 Sucrose / Phosphate (volume filling 1.5 ml) S10 / SU / S / 0 Sucrose + Mannitol / Succinate (filling volume 1.5 ml) S10 / SU / F / 01 Sucrose + Manito / Phosphate (filling volume 1.5 mi) t O Table 7 Efficacy of antimicrobial protection. 0.9% Benzyl alcohol was used as an antimicrobial protector (PRES) in vials formulated with HGH (SAIZEN). The test was carried out in accordance with the European Pharmacopoeia and was followed until 21 days after planting. Log (logarithmic) reduction was calculated against the CFU counted at time Zero (2T) in the protective solution.

H.T. - not rehearsed Table 8 Efficacy of antimicrobial protection. M-Cresol 0.3% was used as antimicrobial protector (PRES) in vials formulated with HGH (SAIZEN). The test was carried out in accordance with the European Pharmacopoeia and was followed until 21 days after planting. Log (logarithmic) reduction was calculated against the CFU counted at time Zero (ZT) in the protective solution.

Claims (14)

1. R E I V I N D I C C O N S 1. - Pharmaceutical composition comprising a solid intimate mixture of human growth hormone (HGH) and a stabilizing amount of sucrose alone.
2. 2. - Pharmaceutical composition according to claim 1, wherein the solid intimate mixture is a lyophilized.
3. 3. - Pharmaceutical composition according to claim 1, wherein the HGH is recombinant.
4. 4. - Pharmaceutical composition according to any of claims 1 to 3, containing 5 or 10 mg / vial of HGH.
5. 5. Process for preparing a pharmaceutical composition according to any of claims 1 to 4, comprising the preparation of an aqueous solution of the components, the distribution in the containers and freeze-drying in the containers.
6. 6. The process according to claim 5, wherein the aqueous solution is a buffer solution selected from acetate buffer, succinate buffer and phosphate buffer.
7. 7. The method according to claim 6, wherein the buffer solution is phosphate buffer.
8. 8. - Process according to claim 6 or 7, wherein the pH of the solution is within the range of 6.0 to 8.0.
9. 9. - Pharmaceutical composition according to any of claims 6 to 8, wherein the pH of the solution is 7.5.
10. 10. - Forms of presentation of the pharmaceutical composition comprising the solid mixture according to any of claims 1 to 4, hermetically sealed under sterile conditions in a suitable container for storage before use and for the reconstitution of the mixture in a solvent or in a solution for injection.
11. 11. - Solution comprising the solid mixture according to claim 10, reconstituted in a solvent or solution for injection.
12. 12. Reconstituted solution according to claim 11, comprising 5 or 10 mg / vial of HGH, 68.4 mg / vial of sucrose and phosphate buffer at pH 7.5.
13. 13. The solution according to claim 12, wherein the solvent is a bacteriostatic solvent.
14. 14. The solution according to claim 13, wherein the bacteriostatic solvent is 0.3% m-cresol. SUMMARIZES Pharmaceutical composition based on human growth hormone (HGH) and a stabilizing amount of sucrose alone and a process for preparing that composition.