JP2005232177A - hGH CONTAINING PHARMACEUTICAL COMPOSITION - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means to stabilize a lyophilisate of recombinant human growth hormone (hGH). <P>SOLUTION: This invention relates to a pharmaceutical composition comprising a solid complete mixture of human growth hormone (hGH) and a stabilizing amount of saccharose, alone or in combination with other excipients. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a pharmaceutical composition containing human growth hormone (hGH). More particularly, it relates to a sucrose stabilized human growth hormone composition. Highly purified proteins are time labile and are known to be stabilized in admixture with saccharides such as lactose and mannitol, or other proteins and amino acids such as albumin and glycine.

  Human growth hormone is secreted in the human pituitary gland. In its mature form, it consists of 191 amino acids and has a molecular weight of 22,000, which is more than three times greater than insulin. This hormone is a linear polypeptide containing two intrachain disulfide bridges. Until the advent of recombinant DNA technology, hGH could only be obtained by a limited source: difficult extraction from the pituitary glands of human cadaver. The resulting deficiency of the substance is deficient in hypophyseal dwarfism even though it has been proposed to be effective in the treatment of burns, wound healing, dystrophies, bone fusion, diffuse gastric hemorrhage and false joints. Was limited to treatment. HGH can be produced in recombinant host cells in an amount that would be appropriate to treat hypopituitar dwarfism and other conditions for which it is effective.

  The main biological effect of hGH is to promote growth. Affected organ systems include the skeleton, connective tissue, muscles, and internal organs such as the liver, intestines and kidneys. Growth hormone exerts its action through interaction with specific receptors on the cell membrane.

  The composition of the lyophilized protein is described in M.J. Pikal, Biopharm, October 1990, 25-30. Examples of growth hormone formulations containing stabilizing excipients such as mannitol glycine, arginine and lactose have been reported.

  In particular, lyophilization in the presence of various substances in their amorphous state such as sugars that increase the disintegration temperature and allow for shorter lyophilization times has been described. However, according to the authors, it is not possible to predict a standard formulation for all proteins, and selection of the best formulation requires significant selection effort.

  German Patent DE 3520228 describes bioactive proteins containing growth hormone in a formulation stabilized by means of polyzacaride containing repetitive maltotriose units.

  WO89 / 09614 describes a preparation of human growth hormone stabilized with glycine, mannitol and a buffer with a human growth hormone: glycine molar ratio of 1: 50-200.

  The US5122367 patent describes a controlled release system for the administration of growth hormone comprising a protein and polysaccharide incorporated within a polymer matrix.

  The EP210039 patent application describes a controlled release implant for subcutaneous administration of bovine or porcine growth hormone to animals in the form of a matrix containing 40% saccharose.

  According to the invention, hGH can be either natural or synthetic, i.e. it can be produced on the basis of recombinant DNA technology, the latter being preferred.

  Injectable preparations of human growth hormone are obtained by treatment including their lyophilization to obtain a dry powder. Human growth hormone is very susceptible to denaturation during the lyophilization process, and maintaining a longer cycle of bioremediation than storing them at room temperature is required to obtain a stable formulation.

  Since hGH-like materials should be provided to health-conscious people and patients, these materials must be prepared as pharmaceutical compositions. Such compositions must remain active for an appropriate period of time, must be readily and quickly acceptable for human administration, and must be readily manufacturable. Often the pharmaceutical formulation is provided in frozen or lyophilized form. In this case, the composition must be thawed or reconstituted before use. Since it is recognized by those skilled in the art that lyophilized preparations often maintain superior activity over their liquids, frozen or lyophilized forms can be incorporated into compositions under a wide variety of storage conditions. Is often used to maintain the biochemical integrity and biological activity of the medical agents contained therein. These lyophilized preparations are reconstituted prior to use by the addition of a suitable pharmaceutically acceptable diluent such as sterile water for injection or sterile physiological saline solution.

  Alternatively, the composition can be provided in a liquid form suitable for immediate use. What is needed is a liquid formulation that maintains its activity during long-term storage.

  Current hGH formulations lose activity due to dimer and higher aggregation (macrorange) formulations during the formulation process and during storage and reconstitution. Other chemical changes such as deamidation and oxidation can also occur upon storage.

  Human growth hormone is found on the market, for example in formulations stabilized with mannitol Saizen® and Grorm®, Serono.

  We have now discovered that saccharose provides greater stability to hGH formulations, particularly to this glycoprotein form prepared by recombinant DNA technology. It has also been discovered that saccharose prevents the formation of a precipitate when the reconstituted solution is shaken.

  The main object of the present invention is to provide a pharmaceutical composition comprising a complete mixture of human growth hormone and a stabilizing amount of saccharose solids, alone or in combination with other stabilizers.

  A further object is to provide a method for the preparation of said pharmaceutical composition comprising the step of lyophilizing the aqueous solution of the composition in the container. Another object is to provide a form of providing said pharmaceutical composition comprising said solid mixture sealed in sterile conditions in a container suitable for storage prior to use and suitable for reconstitution of the mixture for injectable substances. It is.

  Another object is to provide a solution for the solid mixture reconstituted in an injectable solution. In order to estimate the effect of excipients on the stability of the active ingredient, various formulations of recombinant hGH containing 5 or 10 mg pro-vials have various excipients: saccharose, glycine, mannitol, saccharose + mannitol and Prepared with mannitol + glycine.

  The compositions of the various formulations prepared are reported in Tables 1 and 4. The lyophilizate was prepared by diluting the bulk of hGH with a solution containing a stabilizer, all buffered at pH 7.5. The resulting solution was filtered to the final volume, filled into various glass bottles and lyophilized.

  A study of the stability of such formulations stored at 4 ° C, 25 ° C, 37 ° C and 50 ° C for 24 weeks was performed as follows: RM Riggin et al., Anal. Biochem., 167: 199-209, 1987 Reversed phase HPLC (RP-HPLC) according to the method described by, and size exclusion HPLC (HPSEC) according to US Pharmacopeia Preview Nov-Dec 1990 pag. 1253-1261. The results are reported in Tables 2-3 and 5-6. Here, this measurement is expressed as a percentage of the recovery of hGH in the various formulations.

A chromatographic assay method for determining the percent recovery of hGH was performed as described by Pikal in Pharmaceutical Research 8 , pag. 428 “Assays”.

  In the pre-dispensing stage, the effect of pH and buffer on the stability of rhGH in lyophilized form was tested by measuring the stability at 50 ° C. Tests were carried out in different buffer systems prepared with acetic acid, phosphoric acid and succinic acid adjusted to pH 6.00, 7.00 and 8.00 with NaOH.

  This result indicated that rhGH stability was not affected by the buffer and that the formulation was more stable at about pH 8.00.

  The pH chosen for the composition was 7.5.

  7 dried formulations at a rhGH concentration of 5 mg / vial were then mixed with different excipients (saccharose 68.4 mg / vial, mannitol 36.4 mg / vial, mannitol / glycine 25 + 4 mg / vial, mannitol / saccharose 32 + 7.5 mg / vial) Was prepared using both phosphate and succinate buffers at pH 7.5. The amount of excipient was selected to have an isotonic solution after reconstitution with a bacteriostatic solution. The filling amount was 1 ml.

  Samples prepared under sterile conditions were stored for 24 weeks at 50 ° C., 37 ° C., 25 ° C. and 4 ° C. and tested by HPSEC, reverse phase HPLC. The pH and moisture content were measured.

  The stability of solutions reconstituted with 0.3% m-cresol and 0.9% benzyl alcohol at 4 ° C and 25 ° C was also studied.

  HPSEC and RP-HPLC were performed as previously described.

  The pH was measured with a pH meter in one vial reconstituted with 1 ml water for injection. To determine the moisture content of the lyophilized vial, one vial of composition was suspended in 1 ml of 2-isopropanol, filtered through an Anotop 10, 0.22um Disposable filter (Merck) and injected in a Metrohm Coulometer.

  The stability results (Riggin's method) tested by RP-HPLC are reported in Table 2. The chromatographic profile of formulations containing saccharose after 24 weeks at 50 ° C. (HGH / 3 and HGH / 7 in Table 1) is not different from that obtained at time zero.

  At the same temperature, a 13-22% decrease in purity was found in formulations containing mannitol and mannitol + glycine.

  The data reported in Table 3 show that no reduction in rHGH purity percentage, indicating the results obtained by HPSEC analysis, was found in all tested formulations. No significant change in water content was observed during the study in all lyophilized tested formulations.

  A decrease in pH was observed at 37 ° C. and 50 ° C. for lots HGH / 5 and HGH / 7 in Table 1.

  The stability of the reconstituted solution was also studied through RP-HPLC (Riggin's method) and HPSEC analysis.

  In the RP-HPLC method, after 5 weeks at 25 ° C., a decrease in purity was seen in the range of 30% to 50% in samples reconstituted both with benzyl alcohol and with m-cresol. After 7 weeks at 4 ° C., the mutation was about 14% in the presence of benzyl alcohol and 4% -8% with m-cresol.

  No mutation was observed with HPSEC at 4 ° C; on the other hand, about 5% reduction in rHGH purity was found at 25 ° C for all formulations in the presence of both benzyl alcohol and m-cresol. It was.

  The results showed that formulations containing saccharose and saccharose + mannitol showed a better stability profile compared to other formulations.

  Based on the results obtained with the 5 mg composition, sucrose and mannitol were prepared in 5 lyophilized formulations containing 10 mg hGH / vial using a pH 7.5 phosphate and succinate buffer adjusted with NaOH 2.5M. Selected for the standard in Table 4). One formulation contains 68.4 mg / vial of saccharose (filling volume 1 ml) in phosphate buffer only, the other 102.6 mg / vial of saccharose (filling volume) in both phosphate and succinate buffer. 1.5 ml) and mannitol + saccharose 130 + 40 mg / vial (filling volume 1.5 ml). The optimum ratio between sucrose and mannitol and the filling amount to obtain the product with good physical properties were adjusted based on preliminary freeze-drying tests. The optimum ratio mannitol / sucrose in terms of freeze-drying-cake resistance to high temperature was 3: 1 and the maximum volume to be freeze-dried was 1.5 ml.

  The formulation was tested for stability by storing samples at 50 ° C, 37 ° C, 25 ° C and 4 ° C for 24 weeks. Samples were subjected to the following analytical controls: HPSEC, RP-HPLC (Riggin's method), pH and moisture content.

  The stability of the reconstituted solution with 0.3% m-cresol and 0.9% benzyl alcohol at 4 ° C. was monitored for 4 weeks. Samples were subjected to the same controls performed at 5 mg dose as previously described. This analysis showed the following results: Formulations containing 68.4 mg / vial and 102.6 mg / vial of saccharose in succinate buffer tested by RP-HPLC analysis were for 24 weeks at all tested temperatures. There was no decrease in purity after storage. The results are reported in Table 5. Even after 4/6 weeks storage at 50 ° C., formation of degradation products was observed in other formulations.

  No decrease in rHGH purity percentage was found in all tested formulations by HPSEC analysis. See Table 6. During the study, no changes in pH and water content were observed in all tested formulations.

  Studies based on reconstituted solutions containing only sucrose were also performed by RP-HPLC (Riggin's method) and HPSEC analysis.

  After 4 weeks at 4 ° C., a reduction in purity of about 12% in the presence of benzyl alcohol and 4% with m-cresol was found in the RP-HPLC method. In the HPSEC method, no decrease in rHGH purity was observed at 4 ° C. in the presence of both benzyl alcohol and m-cresol.

  To evaluate the efficacy of antimicrobial storage, vials of the HGH / 3 formulations in Table 1 were reconstituted with a bacteriostatic solvent (m-cresol 0.3% or benzyl alcohol 0.9%). They were tested according to European Pharmacopeia from day 21 after inoculation. Results are reported in Tables 7 and 8.

  The minimum acceptable efficacy was reached in both stock solutions. The results obtained at 0 hours when microorganisms were counted after spiking in both saline (NaCl 0.9%) and bacteriostatic solutions were reduced immediately after spiking 90,000-25,000 and 78,000-8,000 UFC / ml, respectively It appears to show a higher efficacy of m-cresol versus benzyl alcohol for mainly Staphylococcus and Pseudomonas (Table 7).

  Furthermore, Aspergillus disappeared in m-cresol 14 days after inoculation (Table 8) and Pseudomonas disappeared 6 hours later.

  The previous results show that the formulation with 1 ml filling volume reconstituted with 68.4 mg saccharose, phosphate buffer at pH 7.5, meta-cresol 0.3% is the best stability of both 5 and 10 mg concentrations of r-HGH It shows that it is what guarantees sex.

Examples of pharmaceutical manufacture Materials: Pure saccharose Ph Eur, BP, Nord, NF (Merck); H3PO4 Suprapur (Merck); NaOH for analytical use (Merck); Water for injection.

  Containers include vial DIN 2R (borosilicate glass type I), rubber stopper (Pharmagummi W1816V50), aluminum ring and flip-off cap (Pharma-Metal GmbH).

Preparation of rHGH containing saccharose (for 1000 vials each containing 10 mg hGH).
To obtain the starting sucrose solution, saccharose (68.4 g), H3PO4 (1.96 g) are dissolved in water for injectables (800 ml). A bulk of hGH (10 g) is added to the saccharose solution and the pH is adjusted to 7.5 with 2.5 M NaOH to a final volume of 1000 ml. The solution is filtered through a 0.22 μm Durapore sterile filter (Millipore). During this process, the solution temperature is maintained at 4-8 ° C. Solutions containing different excipients or different active agent dosages are similarly prepared.

Fill and lyophilize Fill vials with 1 ml of HGH sterilization solution, transfer to lyophilizer and cool to −45 ° C. for at least 6 hours. Freeze-drying is started at a temperature of −45 ° C. with a vacuum of 0.07 mBar. Heating is performed according to the following scheme: 12 hours, + 10 ° C .; then + 35 ° C. until the end of the cycle.

Claims (16)

  A pharmaceutical composition comprising a complete mixture of solids of human growth hormone (hGH) and a stabilizing amount of saccharose, alone or in combination with other excipients.   2. The pharmaceutical composition according to claim 1, wherein the complete mixture of solids is a lyophilizate.   The pharmaceutical composition according to claim 1, wherein the hGH is recombinant.   The pharmaceutical composition according to any one of claims 1 to 3, wherein the stabilizer is only sucrose.   The pharmaceutical composition according to any one of claims 1 to 4, wherein the stabilizer is saccharose combined with mannitol.   6. A pharmaceutical composition according to any of claims 1 to 5, characterized in that it contains 5 or 10 mg / vial of hGH.   The pharmaceutical composition according to any one of claims 1 to 6, comprising a buffer solution selected from an acetate buffer solution, a succinate buffer solution and a phosphate buffer solution.   The pharmaceutical composition according to any one of claims 1 to 7, wherein the buffer solution is a phosphate buffer solution.   The pharmaceutical composition according to any one of claims 1 to 8, wherein the pH of the solution is in the range of 6.0 to 8.0.   The pharmaceutical composition according to any one of claims 1 to 9, wherein the pH of the solution is 7.5.   11. The pharmaceutical composition according to any one of claims 1 to 10, comprising 5 or 10 mg / vial of hGH, 68.4 mg / vial of saccharose and a phosphate buffer at pH 7.5.   12. A method for preparing a pharmaceutical composition according to any of claims 1 to 11, comprising the preparation of an aqueous solution of the composition, dispersion in a container and lyophilization in the container.   A solid mixture according to any of claims 1 to 11, sealed in sterile conditions in a container suitable for storage prior to use and reconstitution of the mixture in a solvent or solution for injectables. Provided form of said pharmaceutical composition comprising.   14. A solution comprising a solid mixture according to claim 13, reconstituted in a solvent or solution for the injectable.   15. The solution according to claim 14, wherein the solvent is a bacteriostatic solvent.   16. The solution according to claim 15, wherein the bacteriostatic solvent is m-cresol 0.3%.