MXPA06007308A - A pharmaceutical composition comprising an active principle and sulphobetaine - Google Patents

Abstract

The present invention relates to the pharmaceutical composition comprising an active agent and a non-detergent sulphobetaine (NDSB).

Description

PHARMACEUTICAL COMPOSITION COMPRISING AN ACTIVE PRINCIPLE AND SULFOBETAINE FIELD OF THE INVENTION The present invention relates to a pharmaceutical composition comprising a non-detergent sulfobetaine (NDSB).

STATE OF THE ART Pharmaceutical compositions comprising active pharmaceutical ingredients are well known. The common pharmaceutical compositions described comprise several pharmaceutically acceptable excipients which, with their different properties (for example stabilization of the active pharmaceutical ingredient, adjustment and / or maintenance of the pH, have an effect on the solubility of the active pharmaceutical ingredient, maintenance of the compositional effect of the composition. pharmaceutical, etc.), allow the use of the active pharmaceutical ingredients in the pharmaceutical compositions. The pharmaceutically acceptable excipients are described extensively; see, for example, Handbook of Phraumaceutical Excipients, Ainley Wade and Paul J. Weller, American Pharmaceutical Association, 1994. Therapeutically active proteins have also been described as active pharmaceutical ingredients in the compositions Pharmaceutical These pharmaceutical compositions also comprise various pharmaceutical excipients which, with their properties, allow the preparation of stable pharmaceutical compositions comprising therapeutically active proteins. Such pharmaceutical compositions are extensively described; see, for example, Yu-Chang John Wang and Musetta A. Hanson (1988), J of Parenteral Science & Technology, 42: S4-S26; Wong D. and Parasrampuria J. (1997), Biopharm: November 52-61. Stable pharmaceutical compositions comprising therapeutic protein granulocyte colony stimulating factor (G-CSF) are described in EP 373679 and are also described as to mainly stabilize G-CSF in solution with low conductivity and acidic pH between 2.75 and 4.0. To improve the stability, sticks sugars, amino acids, polymers and detergents were added. Particularly it has been emphasized that the pH of the G-CSF comprising the composition should be less than 4 in order to reduce the formation of aggregates and increase the stability of this form. The formation of aggregates and reduced stability with a pH exceeding 4.0 are in accordance with the data from the literature of the state of the art (Kuzníar et al. (2001), Pharm Dev Technol 6 (3): 441-7; Bartkowski et al. (2002), J Protein Chem 21 (3): 137-43, Narhi et al. (1991), J Protein Chem 10 (4): 359-367, Wang W (1999), Int J Pharmaceut 185: 129-188. The stability of G-CSF, described in other pharmaceutical compositions of patent and scientific literature, was achieved with the addition of several stabilizers, such as, for example, sulfate ions (EP 1129720), mixture of various preservatives, amino acids and surfactants (EP 607156), various pH regulating systems (phosphate, citrate, arginine, acetate) in the presence of a surfactant (EP 674525), molecular compounds high, such as hydroxypropylcellulose, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone and others (GB 2193621), a surfactant (EP 1060746), various pH regulating systems (TRIS, HEPES, TRICINE) (EP 0988861) sugars, such as cellobiose, gentiobiose, isomaltose, raffinose, trehalose and others (EP 0674524), and one or more amino acids (EP 1197221, WO51629, EP 1260230 and EP 1329224, EP 0975335). Although low ionic strength in the G-CSF comprising pharmaceutical compositions is preferred, various surfactants and other stabilizers are used for G-CSF stabilization in most cases described for use for stabilization of G-CSF. In addition, several pH regulator systems have been used in addition in most cases for the maintenance of pH. In the literature, the use of non-detergent sulfobetaines (NDSBs) as solubilizers (used in high concentrations of around 1 M of the solution) was described by protein renaturizations (Chong Y and chen H. (2000), Biotechniques 29 (6) : 1166-7; Vuillard L and others (1995), Biochem J 305: 337-43; Vuillard L and others (1995), Electrophoresis 16 (3): 295-7; Vuillard L and others (1998), Eur J Biochem 256: 128-135; Goldberg ME et al. (1995), Folding &Design 1: 21-27).

There is a current need to provide stabilized pharmaceutical compositions. A description of NDSBs is pharmaceutical compositions that have not been found in scientific or patent literature.

DESCRIPTION OF THE DRAWINGS Figure 1: SE-HPLC of samples of the invention and of reference sample, stored at 40 ° C (± 2 ° C) for 1 month (40). Figure 2: SE-HPLC of samples of the invention, stored at 40 ° C (± 2 ° C) for 1 month (40).

DESCRIPTION OF THE INVENTION In the context of the present invention it has been discovered that an NDSB can be used as an excipient in a pharmaceutical composition. By using an NDSB, pharmaceutical compositions can be provided which are stabilized. Accordingly, the present invention relates to a pharmaceutical composition comprising an NDSB. In a first aspect of the present invention there is provided a pharmaceutical composition comprising an active pharmaceutical ingredient and a non-detergent sulfobetaine (NDSB). The active pharmaceutical ingredient of the present invention is selected from the group consisting of a synthetic molecule u Therapeutically effective natural organic (for example, natural synthetic and organic molecules poorly soluble in water), and a therapeutically effective protein (e.g., poorly water soluble and / or hydrophobic proteins) and / or other active pharmaceutical ingredients that have a therapeutic effect . The active pharmaceutical ingredient is preferably comprised in a therapeutically effective amount. The term "therapeutically effective amount of active pharmaceutical ingredient" as used herein, refers to the active pharmaceutical ingredient in the amount that has a therapeutic effect. The pharmaceutical composition of the present invention comprises a non-detergent sulfobetaine (NDSB). The term "non-detergent sulfobetaine" as used herein, refers to a sulfobetaine that does not form micelles in water solution. In a preferred embodiment of the pharmaceutical composition of the present invention, the NDSB is a quaternary ammonium salt wherein the groups R1, R2, R3 and R4-SO "3 are linked to the central nitrogen atom, and wherein: R1 is methyl, ethyl, propyl, butyl, pentyl, hexyl or their derivatives, R2 is methyl, ethyl, propyl, butyl, pentyl, hexyl or their derivatives, R3 is methyl, ethyl, propyl, butyl, pentyl, hexyl or their derivatives; all combinations of R1, R2 and R3, R4 is (CH2) n, where n is between 1 and 6; n most preferred is 3.

The quaternary nitrogen atom can be a part of an aliphatic or aromatic ring structure as well. Accordingly, in a preferred embodiment of the pharmaceutical composition of the present invention, the NDSB is a quaternary ammonium salt of Formula 1, Formula 1 wherein R1, R2 and R3 may be the same and / or different and are selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, hexyl or their derivatives, and R4 is (CH2) n, wherein n is between 1 and 6; more preferably n is 3. Preferably, the NDSB which is selected from the group consisting of dlmethylethyl- (3-sulfopropyl) -ammonium salt (SB195, Vuillard et al. (1994) FEBS Letters is used in the pharmaceutical composition of the present invention. , 353, 394-296; Goldberg et al. (1995/1996) Folding &Design, 1, 21-27), 3- (1-pyridine) - 1 -propanesulfonate (SB201), dimethylbenzylammonium propansulfonate (SB256) ), dimethyl-t-butyl- (3-sulfopropyl) ammonium salt (SB222t), 3- (1-methylpiperidine) -1-propanesulfonate (SB221) and dimethyl- (2-hydroxyethyl). (sulfopropyl) -ammonium salt (SB211; Vuillard et al. (1995) Anal Biochem, 230, 290-294) Two or more indicated NDSBs can also be used in all possible combinations.

Preference is given to using dimethyl-t-butyl- (3-sulfopropyl) ammonium salt (SB222t), dimethyl- (3-sulfopropyl) -ammonium salt (SB195) and 3- (1-methylpiperidine) -1-propansulfonate ( SB221). More preferably, dimethyl-t-butyl- (3-sulfopropyl) ammonium salt (SB222t) is used. The concentration of NDSB used depends on the pH intended to be adjusted and / or maintained. It is selected from the range of 1 to 1000 mM, preferably from 5 to 100 mM. The pH of the pharmaceutical composition of the present invention may be within the range of 2 to 9, preferably between 3 and 8, and more preferably 3.5 to 7.5. In a second aspect of the present invention there is provided the pharmaceutical composition comprising a therapeutically effective protein and a non-detergent sulfobetaine (NDSB). The term "therapeutically effective protein" as used herein, refers to a protein with therapeutic effect. A therapeutically effective protein used in the pharmaceutical composition of the present invention is selected from the group consisting of granulocyte colony stimulating factor (G-CSF), interferons (IFNs); such as IFN-alpha2a, INF-alpha 2b, IFN-beta, IFN-gamma 1b; interleukins (lls), such as IL-1, IL-2, IL-3, IL-4, IL-5 up to IL-10; granulocyte-macrophage colony stimulating factor (GM-CSF); macrophage colony stimulating factor (M-CSF); epidermal growth factor (EGF); erythropoietin (EPO); follicle stimulating hormone (FSH); human serum albumin (HSA); deoxyribonulease (DNase); fibroblast growth factor (aFGF or bFGF); tumor necrosis factor alpha (TNF-alpha) and tumor necrosis factor beta (TNF-beta); calcitonin; hematoprotein, plasminogenic activators and their precursors (t-PA, urokinase, pro-urokinase, streptokinase, protein C); cytosines; family of TNF ligands (TRAIL, FasL, osteoprotegerin); soluble receptors (p55, p75), growth hormone, for example, human growth corone, bovine growth hormone and parathyroid hormone; Lipoproteins; alpha-1-antit-ripsin; insulin, proinsulin, insulin A subunit, B subunit of insulin; glucagons; blood coagulant factors, such as, for example, Factor HIV, Factor IX, tissue factor, von Willebrand factor; bombina; thrombin; enkephalinase; macrophage inflammatory protein (MIP-1-alpha); A subunit of relaxin, B subunit of relaxin, prorelaxin; inhibin; activin; vascular endothelial growth factor (VEGF); hormone receptors or growth factor receptors; integrins; protein A, protein D; rheumatoid factors; bone-derived neurotrophic factor (BDNF), neurotropin-3, -4, -5, or -6; nerve growth factor (NGF); platelet-derived growth factor (PDGF); fibroblast growth factor (aFGF and bFGF); transforming growth factor (TGF-alpha and TGF-beta); insulin-like growth factor (IGF1 and IGF2); thrombopoietin (TPO); bone morphogenetic protein (BMP); superoxide dismutase; biologically active fragments of the aforementioned proteins, and other therapeutically effective proteins.

The therapeutically effective proteins of the present invention are used in therapeutically effective amounts. The term "therapeutically effective amount of a protein" as used herein, refers to the amount of a protein that has a therapeutic effect. The most preferred active pharmaceutical ingredient is G-CSF preferably used in therapeutically effective amounts. In the context of the present invention, the pharmaceutical composition comprising G-CSF and a non-detergent sulfobetaine (NDSB) is provided. The term "G-CSF" as used herein refers to the protein that regulates the differentiation and proliferation of hematopoietic cells in mammals and activation of mature cells of the hematopoietic system. It is selected from the group consisting of: human G-CSF and its derivatives and analogues defined below. Preferably, G-CSF refers to recombinant human G-CSF, produced by expression in the E. coli bacterium. The pharmaceutical composition of the present invention can be used for all types of G-CSFs; it can be used among others also in the case of isolation of forms derived from G-CSF, such as methionyl G-CSF (Met-G-CSF), glycolized G-CSF, from above and chemically modified (such as, for example : pegylated), G-CSF analogs and fusion proteins comprising G-CSF. The term "therapeutically effective amount of G-CSF" as is used here, refers to the amount of G-CSF that allows the therapeutic effect of G-CSF. The pharmaceutical composition of the present invention further optionally comprises a polyol. The term "polyol" refers to any polyhydric alcohol, that is, a chemical compound that contains one or more hydroxyl groups per molecule. Preferably, the polyol is selected from the group consisting of sorbitol, glycerol, inositol, trehalose, and mannitol. The preferred polyol concentration is in the range of 1% to 10% (m / v). The pharmaceutical composition of the present invention further optionally comprises one or more of the pharmaceutically acceptable excipients. The pharmaceutically acceptable excipient is selected from the group consisting of metal cation scavengers (e.g., EDTA and similar chelators), free radical scavengers and solvents (e.g., DMSO), various acids (e.g., acetic acid, citronic, methanesulfonic, phosphoric, hydrochloric and others), various bases (e.g., NaOH or N organic bases, e.g. Good's pH regulators, such as TRIS, TES, HEPES), various pH-regulating systems (e.g., acetic acid) / acetate, glutamic acid / glutamate, maleic acid / maleate, citric acid / citrate, phosphoric acid / phosphate, and others), various pharmaceutically acceptable excipients for the maintenance of isotonicity of the solution (for example, inorganic salts, such as CaCl2 and NaCl), protein stabilizers, selected from the group consisting of active surface substances, such as: glycol and glycerol esters, macrogol esters and ethers, sorbitan derivatives or polysorbates (polysorbate 20, polysorbate 80), amino acids, poloxamers (Pluronic F68), polyvinylpyrrolidone (PVP), and others. Preferably, the pharmaceutically acceptable excipient is selected from the group consisting of EDTA and DMSO. The NDSB in the pharmaceutical compositions of the present invention can be combined with one or more of the aforementioned pharmaceutically acceptable excipients. The term "stabilizer" as used herein, refers to a pharmaceutically acceptable excipient that can stabilize an active pharmaceutical ingredient (e.g., a protein, e.g., G-CSF). The term "protein stabilizer" as used herein, refers to a pharmaceutically acceptable excipient that can stabilize a protein (e.g., G-CSF). The term "protein stability" (e.g., G-CSF) as used herein refers to the maintenance of protein content (e.g., G-CSF), as well as to the maintenance of the biological activity of protein (e.g. G-CSF). The decrease in protein stability (for example, G-CSF) can be influenced, among others, by following the procedures: adsorption of protein to container walls, protein denaturation or degradation, as well as aggregate formation, for example, for the dimer of protein (e.g., G-CSF dimer) and / or protein multimer (e.g., multimer of G-CSF) and / or similar molecules with higher molecular mass. These procedures may be the result of several factors, for example increased temperature, inappropriate containers, use of inappropriate protein stabilizers, exposure to light, freezing / melting, improper manufacturing procedure and / or improper storage. The pharmaceutical composition of the present invention can stabilize the protein (e.g., G-CSF) at temperatures above the refrigerator temperature (2-8 ° C), and also at room temperature (ie, below 25 ° C) and even higher temperatures (for example, around 40 ° C). The pharmaceutical composition of the present invention may comprise only a pharmaceutically acceptable excipient, ie, NDSB for protein stabilization (e.g., G-CSF) and for maintaining adequate pH of the solution. Accordingly, in another aspect of the present invention there is provided a pharmaceutical composition comprising an active pharmaceutical ingredient (as presented above) and an NDSB as the sole other excipient. NDSB in the pharmaceutical composition represents a protective molecule that stabilizes the protein and therefore represents protein stabilizers that are used in other pharmaceutical compositions (eg, sugars, amino acids and others), as well as a molecule that adjusts an appropriate pH of the solution and of this form is used in place of acids (for example, acetic, citric, methanesulfonic, phosphoric, hydrochloric and others), which are used for adjusting the pH of the solution in other pharmaceutical compositions. NDSBs can also maintain an adequate pH and therefore replace several pH regulating systems and / or combinations thereof used in other pharmaceutical compositions (eg, acetic acid / acetate, glutamic acid / glutamate, maleic acid / maleate, citric acid / citrate , phosphoric acid / phosphate, and others.In comparison with the use of two or more molecules with different functions, the use of a molecule with several different functions with respect to preparation economy, lower costs, as well as easier preparation and of the pharmaceutical composition, and also for the same patient, as concerns the entry of less additional substances into the body.The characteristics of NDSB presents an additional advantage, because it is a simple molecule that is not sensitive to light, temperature, varis oxidizing agents (for example, air oxygen), hydrolysis, it is not the chemically reactive molecule, it has the nature of zwiterion in wide pH area, which means that the interaction mechanism in wide pH range does not change substantially. One aspect of the present invention is the use of an NDSB as a stabilizer in a pharmaceutical composition. One aspect of the present invention is the use of an NDSB as a protein stabilizer in a pharmaceutical composition.

One aspect of the present invention is the use of an NDSB as a pH adjusting agent in a pharmaceutical composition. One aspect of the present invention is the use of an NDSB as a pH regulating agent in a pharmaceutical composition. The preferred pharmaceutical composition of the present invention is a liquid pharmaceutical composition; however, this does not limit the use of NDSBs in compositions comprising pharmaceutical protein I iofized in the context of the present invention. The pharmaceutical composition of the present invention allows parenteral administration subcutaneously, intravenously or intramuscularly without further reconstitution, dilution or further preparation which would lead to a decrease in the activity of the active pharmaceutical ingredient, eg protein, for example G-CSF and for problems additional technicians at the time of administration. Preferably, the pharmaceutical composition of the present invention does not contain human serum proteins with which viral contamination is possible. In this way the probability of occurrence of several allergic reactions is reduced, which could be the result of administration of human serum albumins. It was prepared in isotonic solution which is pharmaceutically acceptable and does not cause undesirable effects. In the pharmaceutical composition of the present invention, the therapeutically effective amount of a protein corresponds to therapeutically effective amounts of protein that are present in the market. In the case of the use of G-CSF, the therapeutically effective amount of G-CSF is selected from the range between 0.3 mg / ml and 1.2 mg / ml, which, however, does not limit the present invention. The pharmaceutical composition of the present invention can be used for the preparation of medicaments (for treatment) and for treatment of diseases indicated for therapeutically effective proteins, enumerated above. The pharmaceutical composition of the present invention can also be used for the treatment of all diseases and for the preparation of medicaments for the treatment of all diseases for which G-CSF is indicated. The indicated diseases can be selected from the group consisting of: neutropenia and its clinical sequelae, reduced hospitalization in febrile neutropenia after chemotherapy, mobilization of hematopoietic germ cells, alternative infusion of donor leukocytes, chronic neutropenia, neutropenic and non-neutropenic infections, transplant recipients, chronic inflammatory diseases, septic or septic shock, reduced risk, reduced morbidity, reduced mortality and reduced number of days of hospitalization in neutropenic and non-neutropenic infections, prevention of infections and complications of infections in neutropenic and non-neutropenic patients, prevention of nosocomial infections and reduced mortality and frequency of nosocomial infections, enteric application to newborn babies born, imposition of the neonatal immune system, improvement of the clinical outcome in patients in the intensive care unit and in critically ill patients, vaccination and management of burns, ulcers and skin lesions, intensification of chemotherapy and / or radiotherapy, pancytopenia, increase in anti-inflammatory cytokines, reduction of high-dose chemotherapy intervals with prophylactic use of G-CSF, potentiation of antitumor effects of photodynamic therapy, prevention and management of diseases caused by various brain dysfunctions, treatment of thrombotic diseases and their complications and recovery postradiation of erythropoiesis. One aspect of the present invention is the use of an NDSB for the preparation of a pharmaceutical composition. The pharmaceutical composition of the present invention can be filled into pharmaceutical packaging selected from the group consisting of ampules, injection syringes and bottles. This pharmaceutical packaging allows the application in volumes in the range of 0.2 ml to 2 ml (per dose). Furthermore, the object of the invention is also the process for the preparation of the pharmaceutical composition of the present invention. The process for the preparation of the pharmaceutical composition of the present invention comprises the mixing of an NDSB with a therapeutically effective amount of an active pharmaceutical ingredient (e.g., of protein, for example G-CSF). The present invention is illustrated in detail through the following examples but is not restricted to them. In particular, the examples relate to preferred embodiments of the present invention.

EXAMPLES Analytical Methods The following methods are used for the analysis of the pharmaceutical composition of the present invention: high performance liquid chromatography with exclusion of size (SE-HPLC), reserved phase HPLC (RP-HPLC), polyacrylamide gel electrophoresis of sodium dodecylsulfate (SDS-PAGE), and measurement of in vitro biological activity. SE-HPLC SE-HPLC is used to determine concentrations of aggregates of G-CSF, particularly of dimers and higher aggregates. The detection limit for the determination of higher dimers and aggregates is 0.01%. High performance liquid chromatography (HPLC) consists of: UV detector, inline gas cleaner, binary pump module and auto-sampler with thermostat (for example HPLC from Wateers Alliance). The analysis is carried out under the following conditions: Chromatographic conditions: Column: TSK G3000 SW, 10 μm, 300 x 7.5 mm ID Column temperature: 30 ° C Mobile phase: pH 7.0 phosphate buffer (5 mM sodium phosphate, 50 mM NaCl) Flow rate: 0.8 ml / min, Socratic form Detection: UV detector, wavelength 215 nm. Injection volume: 20 μ \ (amount of protein injected: 6-12 μg) Auto-sampler temperature: +2 to + 8 ° C Operating Time: 20 minutes. RP-HPLC RP-HPLC is used to determine the content of G-CSF and for quantitative determination of impurities that vary according to the degree of hydrophobicity. The HPLC system consists of: a UV detector, in-line gas cleaner, a binary pump module and self-timer with thermostat and column department regulated with thermostat (for example, Waters Alliance HPLC systems). The analysis is carried out under the following conditions: Chromatographic conditions: Column: YMC-ODS-AQ package, 200 Á, spherical, 3 μm, 150 x 4.6 mm i.d. Column temperature: 65 ° C Mobile phase: Phase A: 0.1% trifluoroacetic acid (TFA) and 50% acetonitrile (ACN) in water Phase B: 0.1% TFA and 95% ACN in water for HPLC Flow Rate: 1.0 mL / min, gradient: Detection: UV detector, wavelength 215 nm. Injection volume: 10 μL (injected amount of protein 3-6 μg) Auto-sampler temperature: +2 to + 8 ° C Operating Time: 25 minutes.

SDS-PAGE SDS-PAGE is used for visual detection of protein dimers present and other aggregated forms (trimers and forms with high molecular mass). The charge samples are prepared in the pH regulator of free charge of reducing agent. The vertical SDS-PAGE, NuPAGE Bis-Tris 12% gel 8 x 8 cm, thickness of 1.0 mm, 15 steps (Invitrogen) in electrophoresis pH buffer of MOPS SDS (Invitrogen). The electrophoresis runs at 1 hour at a constant voltage of 200 V. The samples are colored with Commassie blue color (0.1% of Phást Gel Blue R 350 in 30% methanol). In vitro biological activity test of G-CSF The biological activity of G-CSF is determined by the method based on stimulation of cell proliferation (NFS-60 cells) using the known method (Hammerling, U. and others, in J Pharm Biomed Anal 13, 9-20 (1995)) and the use of recombinant human G-CSF of international standard (88/502, yeast cell derivative; NIBSC Potrees Bar, Hertfordshire, UK; see Mire-Sluis, AR and others v J Immunol Methods 179, 117-126 (1995).

Measurement of pH value The pH is measured using MA 5741 pH meter (Iskra) and Biotrode electrodes (Hamilton). The pH meter is calibrated to the pH range of 3.0 to 5.0 with suitable fresh calibration regulators. The pH is measured at a temperature of 25 ° C. The standard deviation of the pH measurement is 0.003 of the pH value (0.3%).

The conditions to test the stability of G-CSF in pharmaceutical compositions 4 ° C: stored in refrigerator at the refrigerator temperature (in the range of + 4 ° C to + 6 ° C) 40 ° C: stored at 40 ° C ± 2 ° C 25 ° C: stored at room temperature between 25 ° C and 30 ° C in 1-ml syringes filled during agitation at 75 rpm in the Vidromix 314EVT shaker.

Example 1: Stability tests The following liquid pharmaceutical compositions are prepared: FP1 0.3 mg / ml G-CSF, 39 mM NDSB, 5 mM NA EDTA, pH 4.4 FP2 0.3 mg / ml G-CSF, 39 mM NDSB, 5 mM NA EDTA, 5% DMSO pH 4.4 FP3 0.3 mg / ml G-CSF, 7 mM NDSB, 5% sorbitol, pH 4.4 FP4 0.6 mg / ml G-CSF, 6 mM NDSB, 8% sorbitol, pH 4.6 FP5 0.6 mg / ml G-CSF, 13 mM NDSB, 8% sorbitol, pH 4.3 FP6 0.6 mg / ml G-CSF, 10 mM NDSB, 8% sorbitol, pH 4.5 FP7 0.6 mg / ml of G-CSF, 10 mM of NDSB, 5% of sorbitol, pH 4.4 FP8 0.6 mg / ml of G-CSF, 10 mM of NDSB, 10% of sorbitol, pH 4.4 FP9 0.9 mg / ml of G-CSF, 10 mM NDSB, 8% sorbitol, pH 4.4 FP10 0.6 mg / ml G-CSF, 10 mM NDSB, 8% sorbitol, pH 4.4 FP11 0.6 mg / ml G-CSF, 50 mM NDSB, 8 Sorbitol%, pH 4.9 • Pharmaceutical reference composition A ((S16-10ACT): 0.3 mg / ml G.CSF, 10 mM acetic acid, 5% (m / v) sorbitol, 0.004% Tween 80, pH adjusted to 4.0 cpn NaC! (Identical to Neupogen.) Samples with a G-CSF concentration of 0.6 mg / ml were stored at 40 ° C for 1 month, analyzed using SE-HPLC, and 6 μg of G-CSF was loaded onto the column. respective results (AU = absorption unit) Legend of Figure 1: 1 FP1 2 FP2 R S16-10ACT Samples with a concentration of G-CSF of 0.6 mg / ml were stored at 40 ° C for 1 month. were analyzed using SE-HPLC, the application of G-CSF to the column was 6 μg The results are shown in Figure 2 (AU = absorption unit) Legend of Figure 2: 4 FP4 5 FP5 9 FP9 ESSABILITY TEST RESULTS The SE-HPLC analysis of samples FP1 and FP2, stored for 1 month at 40 ° C, showed that the samples are stable, since there is no visible increase in the content of aggregates and hydrophobic degradation products (Table 1, Figure 1). The stability is comparable with the reference sample (AS16-10ACT) which is identical to the pharmaceutical composition comprising G-CSF, and commercially available (Neupogen, G-CSF = 0.3 mg / ml). The stability is also confirmed with RP-HPLC analysis that shows no essential changes in impurities or protein content after the storage period. These results comply with the results of the SDS-PAGE analysis (the results are not indicated) and the in vitro measurement of the biological activity. The in vitro biological activity of G-CSF, which is used in the studies, is at the level of the international standard (Cat. No. 88/502; NIBSC, United Kingdom). The in vitro biological activity of the FP1 and FP2 samples does not change after storage under test conditions (the results are not shown).

Table 1 m: month; %: amount of dimer / higher aggregates with reference to the total amount of G-CSF; c = concentration of G-CSF. The results show that the stability of the pharmaceutical compositions comprising NDSBs are comparable to the reference sample (AS16-1 OACT). Higher NDSB concentrations reveal a more favorable effect on stability. The SE-HPLC analysis of samples from FP4 to FP11, stored at 25 ° C for 1 week and 1 month, shows that the samples are stable as long as there is no noticeable increase in the aggregate content and in the hydrophobic degradation products (Table 2, Figure 2). The stability is comparable to that of the reference sample (AS16-1 OACT), which is identical to the G-CSF comprising the pharmaceutical composition and which is present on the market (Neupogen, G-CSF = 0.3 mg / ml). Stability also confirms with the RP-HPLC analyzes that they do not show essential changes in the impurities or protein content after the storage period. These results comply with the results of the SDS-PAGE analysis (the results are not indicated) and the measurement of the in vitro biological activity. The in vitro biological activity of G-CSF, which is used in the studies, is at the international standard level (Cat. No. 88/502; NIBSC, United Kingdom). After storage under the study conditions, the in vitro biological activity of the samples from FP4 to FP11 is changed (the results are not shown).

Table 2 s: week, m: month; %: amount of dimer / higher aggregates with reference to the total amount of G-CSF; % polyol: m / v. The results in Table 2 show that the pharmaceutical compositions with the addition of NDSBs are stable. A slight reduction in stability with respect to the reference sample (Table 1) was noted in the samples that are exposed to more extreme conditions (1 month, 40 ° C). It is also possible that a slight reduction in stability results from the fact that the pH above 4.0 is not favorable for G-CSF, which is also evident from the state of the art. The reference sample is prepared using pH 4.0.

Example 2 Composition of inventive pharmaceutical compositions of G-CSF The compositions of the pharmaceutical compositions Inventives are presented in Table 3.

Table 3 Preparation of Bulk Concentrate The starting material of G-CSF for the preparation of bulk concentrate is produced by the expression in E. coli. The bulk concentrate is prepared in solution with pure acid (acetic acid or HCl) at a pH of 4.4 using the concentration of G-CSF 1.5 mg / ml. The SE-HPLC analysis of the bulk concentrate shows that the content of dimers and higher aggregates is below the limit of detection. The impurity test, determined with the RP-HPLC analysis, is in the range of 2-4%. (The RP-HPLC analysis of a new sample of Neupogen shows a comparable amount of impurities).

Quality of NDSB Substances: NDSB-195 (Calbiochem) for analysis, sorbitol: quality Ph. Eur .; glycerol: Merck; For analysis; inositol: myo-inositol (Fluka:> 99.5% HPLC), trhalosa (Fluka:> 99.5% HPLC), EDTA (Sigma: 99%), DMSO (Merck:> 99.5%), Tween 80 (Sigma , low level of peroxide, contains BHT as an antioxidant); water for injection: quality Ph. Eur .; Water for analysis: Milli-Q (Millipore).

Preparation of Reference Pharmaceutical Composition A (S16-10ACT): The fractions of the gel filtration comprising the G-CSF monomer are pooled and replaced with pH regulator containing 10 mM acetic acid and 5% sorbitol in water for injection. The pH of the pH regulator is adjusted with a NaOH solution to 3.96. The substitution is made in the TFF / Millipore system of Labscale ™, which uses three Ultracel-5 PLCCC membranes. Tween 80 was added until a concentration of 0.004% is obtained. The pH of the final solution after substitution is 4.0, with a concentration of 0.304 mg / ml.

Preparation of Inventive Pharmaceutical Compositions: General: The inventive pharmaceutical compositions are prepared with bulk concentrate dilution with a buffer solution adequate sterile, previously filtered through a 0.2 PES / Nalgene filter. The final G-CSF concentration is 0.3 mg / ml or 0.6 mg / ml, respectively. The pharmaceutical compositions FP1 and FP2 are filled in 2 ml bottles made of colorless glass, hydrolytic type I, washed, sterilized and closed with brombutyl rubber closures, equipped with aluminum caps. Other pharmaceutical compositions (FP3 to FP11) are filled (manually) into the injection syringes (volume 1.3-1.4 ml) so that there is the minimum amount of air around the closure. FP1, FP2, FP3: To 1 portion of the bulk concentrate add 4 portions of the appropriate pH buffer solution. The final concentrations indicated in Table 3 were obtained. The pH is no longer adjusted. FP4-FP11: To 4 portions of the concentrate, 6 portions of the solution were added with a suitable pH regulator. The final concentrations indicated in Table 3 were obtained. The pH is no longer adjusted.

Claims (16)

1. - A pharmaceutical composition comprising an active pharmaceutical ingredient and a non-detergent sulfobetaine (NDSB).

2. The pharmaceutical composition according to claim 1, wherein the active pharmaceutical ingredient is selected from the group consisting of a therapeutically effective natural synthetic or organic molecule and a therapeutically effective protein.

3. The pharmaceutical composition according to claim 2, wherein the therapeutically effective protein is selected from the group consisting of granulocyte colony stimulating factor, interferons, interleukins, granulocyte-macrophage colony stimulating factor, colony stimulating factor. of macrophage, epidermal growth factor; erythropoietin; follicle stimulating hormone, human serum albumin, deoxyribonulease, fibroblast calcitonin, hematoprotein, plasminogen activators and their precursors, cytosines; family of TNF ligands, soluble receptors, growth hormones, lipoproteins; alpha-1-antitrypsin; insulin, proinsulin, subunit A of insulin, subunit B of insulin; glucagons; blood coagulant factors, pumps; thrombin; enkephalinase; macrophage inflammatory protein (MIP-1-alpha); subunit A of relaxin, subunit B of relaxin, prorelaxin; inhibin; activin; growth factor vascular endothelial; hormone receptors or growth factor receptors; integrins; protein A, protein D; rheumatoid factors; neurotrophic factor derived from bone, neurotropin-3, -4, -5, or -6; nerve growth factor; platelet-derived growth factor; fibroblast growth factor, transformed growth factor, insulin-like growth factor, thrombopoietin, bone morphogenetic protein; superoxide dismutase.

4. The pharmaceutical composition according to claim 3, wherein the therapeutically effective protein is G-CSF.

5. The pharmaceutical composition according to any of the preceding claims, wherein the NDSB is a quaternary ammonium salt of the Formula I, Formula 1 wherein R1, R2 and R3 may be the same and / or different and are selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, hexyl or their derivatives, and R4 is (CH2) n, wherein n is between 1 and 6.

6. The pharmaceutical composition according to claim 5, wherein the NDSB is selected from the group that consists of dimethylethyl- (3-sulfopropyl) -ammonium salt, 3- (1-pyridino) -1-propanesulfonate propansulfonate, dimethylbenzylammonium propansulfonate, dimethyl-t-butyl- (3-sulfopropyl) ammonium salt, 3- ( 1-methylpiperidin) -1-propansulfonate and dimethyl- (2-hydroxyethyl) - (sulfopropyl) -ammonium salt.

7 '.- The pharmaceutical composition according to claim 6, wherein the NDSB is dimethyl-t-butyl- (3-suifopropyl) ammonium salt.

8. The pharmaceutical composition according to claims 1 to 7, wherein said composition optionally further comprises a polyol.

9. The pharmaceutical composition according to claim 8, wherein the polyol is selected from the group consisting of sorbitol, glycerol, inositol, trehalose and mannitol.

10. The pharmaceutical composition according to claims 1 to 9, wherein said composition optionally further comprises one or more pharmaceutically acceptable excipients.

11. The pharmaceutical composition according to claim 10, wherein a pharmaceutically acceptable excipient is selected from the group consisting of EDTA and DMSO.

12. A process for the preparation of a pharmaceutical composition, wherein said pharmaceutical composition is prepared by mixing an NDSB with the therapeutically effective amount of an active pharmaceutical ingredient.

13. - The use of an NDSB for the preparation of a pharmaceutical composition.

14. The use of an NDSB as a stabilizer in a pharmaceutical composition.

15. The use of an NDSB as a pH regulating agent in a pharmaceutical composition.

16. The use of an NDSB as a pH adjusting agent in a pharmaceutical composition.