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    • 专利摘要:
    The present invention relates to an aqueous parenteral pharmaceutical composition of multiple doses of insulinotropic peptide and use thereof. a long term storage formulation of the insulinotropic peptide can be obtained by the method of the present invention. the pharmaceutical composition of the present invention comprises: the insulinotropic peptide, the insulinotropic peptide analogue or analogue; pharmaceutically acceptable tonicity modifier (stavilizer); pharmaceutically acceptable preservative. the pharmaceutical composition of insulinotropic peptide is used in the preparation of drugs to treat diabetes and adipose.
    公开号:BR112016002939A2
    申请号:R112016002939-9
    申请日:2014-07-31
    公开日:2020-06-23
    发明作者:Xiong Chunlin；Chunlin XIONG；He Yunxia；Yunxia HE；Zuo Yajun；Yajun Zuo；Yu Gang；Gang Yu
    申请人:Shanghai Benemae Pharmaceutical Corporation；
    IPC主号:
    专利说明:

    [0001] [0001] The present invention relates to an aqueous parenteral pharmaceutical composition of multiple doses of insulinotropic peptide and uses thereof. BACKGROUND OF THE TECHNIQUE
    [0002] [0002] The glucagon-like peptide 1 (also called GLP-1) and Exendin are both insulinotropic peptides, and have 53% identity in their amino acid sequences. Pharmacology has proven that both GLP-1 and Exendin-4 act on the GLP-1 receptors of insulin-secreting B TC1 cells. These types of hormones can promote insulin secretion and have a concentration-dependent hypoglycemic effect.
    [0003] [0003] Similar to insulin, GLP-1 and Exendin are effective only when injected before meals. However, due to the fact that a protein or polypeptide molecule is unstable, it cannot be developed into an oral pharmaceutical composition and must be used by injection. Even if a drug being developed is in an injectable form, it tends to be a lyophilized powder for injection that is inconvenient to use.
    [0004] [0004] The instability of proteins and polypeptides includes two general aspects, that is, physical and chemical aspects. Physical instability includes, for example, denaturation, surface adsorption, aggregation, precipitation, gelatinization and the like, and chemical instability includes, for example, hydrolysis, deamination, oxidation, racemization, isomerization, B-elimination, disulfide bond permutation and the like. Such series of destabilizing factors will all be altered with changes in the structures of proteins or polypeptides and, therefore, many protein or polypeptide drugs are produced using a freeze-drying method, so that the use of the formulation can meet the requirements for service life.
    [0005] [0005] However, the production of lyophilized injection powders using the freeze-drying method has many disadvantages: for example, the cost of production is high, it is inconvenient for patients (the powder for injection is in single doses, and before the
    [0006] [0006] Insulinotropic peptides, in particular GLP-1, have the properties of these polypeptides, particularly physical instability, such as gel formation and, therefore, in order to successfully develop aqueous parenteral pharmaceutical compositions of multiple doses, these physical and chemical instabilities described above need to be resolved to allow the compositions to reach the pharmaceutically available shelf life.
    [0007] [0007] In the development of aqueous multiple dose parenteral pharmaceutical compositions, adding a preservative to the pharmaceutical composition needs to be taken into account, in order to ensure that there is no microbial contamination during the duration of storage and the period of use. However, most preservatives are harmful to proteins or polypeptides and interact with proteins to make them unstable, leading to aggregation. For example, phenol preservatives, such as metacresol and phenol, cause human growth hormones to aggregate (Kirsch et al, 1993), phenol allows for an increase in B times in insulin-like drugs, and benzyl alcohol allows that recombinant human interferon-y aggregates. Therefore, when screening pharmaceutical compositions, the relationship between the antimicrobial effect of the preservative and the stability of the protein or polypeptide must be balanced. The most complicated difficulty in developing parenteral solutions is to allow the formula to be stored for 2 years or more at 4ºC after adding a preservative. Many drugs of crude material or protein or polypeptide stock solutions do not present problems in storage for 2 years or more at 4ºC, but have difficulty in meeting the requirements for shelf life after adding the preservative, just because the addition of the preservative will drastically influence the stability of the drug.
    [0008] [0008] The dissolution enhancers selected for most proteins or polypeptides are surfactants and PEG. Surfactants are mainly Tween, Span, Poloxamer, Pluronic, Brij and the like. In addition to these substances, the surfactants selected in the present invention additionally include propylene glycol and dextran. Propylene glycol and dextran have very beneficial effects when used as GLP-1 dissolution enhancers.
    [0009] [0009] Patent document WO00 / 37098, filed by Brader, Mark, L., also mentions the instability of GLP-1: physical stability is insufficient between pH 6.8 and 7.5, the formulation will become cloudy at a pH value less than 8.0 after adding a preservative, and chemical stability is reduced when the pH value is greater than 8.8, so the appropriate pH range is narrow, pH 8.2 to 8.8. The claimed range within it is 0.3 mg / ml to 0.65 mg / ml and a particularly stable concentration is 0.5 mg / ml. They carried out the work using synthetic GLP-1, which also indicates that GLP-1 is, in fact, unstable.
    [0010] [0010] While the present invention provides an aqueous parenteral pharmaceutical composition that allows GLP-1 to have greater stability and the aqueous parenteral pharmaceutical composition has a much higher concentration of GLP-1 than the drug concentration that can be achieved by the prior art. SUMMARY OF THE INVENTION
    [0011] [0011] In one aspect, the present invention provides an aqueous parenteral pharmaceutical composition of multiple doses of insulinotropic peptide that can be stored for a long time and has greater stability. The formulated aqueous parenteral pharmaceutical composition can meet the requirement for shelf life. The aqueous parenteral pharmaceutical composition comprises an insulinotropic peptide, a pharmaceutically acceptable isotonic agent, a pharmaceutically acceptable dissolution enhancer, a pharmaceutically acceptable preservative and a pharmaceutically acceptable buffer salt, wherein the aqueous parenteral pharmaceutical composition has a pH value of 3 to 5 .
    [0012] [0012] In the aqueous parenteral pharmaceutical composition of the present invention, the insulinotropic peptide is GLP-1, Exendin-4, a GLP-1 analogue, an Exendin-4 analogue, a GLP-1 derivative or an Exendin derivative 4.
    [0013] [0013] In the present application, the term "analog" serves to indicate such a peptide in which one or more amino acid residues of the parent peptide have been replaced by other amino acid residues and / or in which one or more amino acid residues of the parental peptide were excluded and / or where one or more amino acid residues were added to the parental peptide. This addition can occur at the N-terminal or C-terminal, or both, of the parental peptide. In general, an "analog" is such a peptide in which six or less amino acids of the parent peptide have been substituted and / or added and / or deleted, more preferably, it is such a peptide in which three or less amino acids of the parent peptide have been replaced and / or added and / or deleted and, most preferably, is such a peptide in which an amino acid of the parent peptide has been substituted and / or added and / or deleted.
    [0014] [0014] In the present application, the term "derivative" serves to indicate such a peptide in which one or more amino acid residues of the parent peptide have a substituent introduced therein, and typical variants of the substituent include starch, sugars, aquyl, acyl, ester, pegylation and the like.
    [0015] [0015] The insulinotropic peptide can be GLP-1, a GLP-1 analog and a GLP-1 derivative.
    [0016] [0016] Preferably, GLP-1 and GLP-1 analogs have a sequence of: 6 10 20 30 37
    [0017] [0017] The insulinotropic peptide can alternatively be Exendin-4, an analogue of Exendin-4 and a derivative of Exendin-4.
    [0018] [0018] Preferably, Exendin-4 and the Exendin-4 analogue have a sequence of:
    [0019] [0019] For example, Exendin-4 and the Exendin-4 analogue can have a sequence of: HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS.
    [0020] [0020] As is well known, the insulinotropic peptide has low stability and it is very difficult for the aqueous solution to be maintained for the entire duration of storage (2 years at 2 to 8ºC). However, the present invention has developed a pharmaceutical composition with the ability to allow the insulinotropic peptide, the analog and the derivative to be stable and to allow them to meet expectations for shelf life. For example, in the formulation of the present invention, the concentration of GLP-1 is up to 2 mg / ml, and in the case that a preservative is added, it can be stored for 2 years at 4ºC.
    [0021] [0021] Concentration is a factor that influences stability. If a drug molecule has good stability, its fixed concentration is high and, on the contrary, its fixed concentration is low. For a particular drug molecule in particular, the drug has greater stability at a low concentration than at a higher concentration.
    [0022] [0022] In the pharmaceutical composition of the present invention, the insulinotropic peptide has a concentration of about 0.1 to 20 mg / ml, more preferably, about 0.2 to 10 mg / ml, more preferably about 0.05 to 0.5 mg / ml, more preferably about 1 to 5 mg / ml and, more preferably, about 2 to 4 mg / ml. In this application, "about" refers to the difference of a numerical value established in a range of 10%.
    [0023] [0023] Another factor that has an important function in stability is the maintenance of the pH value of the pharmaceutical composition and, in particular, it was found, in the present invention, that the maintenance of the pH value at about 3.5 to 4 , 0 is very good, and GLP-1 can maintain good stability within this range. In addition, it was found that the pH value at which stability is maintained has a very narrow range. The drug is very unstable at a pH value between about 4.5 and 6.5, and turbidity or precipitation will occur as long as the GLP-1 drug molecules are shifted to that pH range. When the pH value is less than about 3.5, acid hydrolysis will occur, which is likewise also unstable. In addition, the formulation for injection requires that the pH value is not less than about 3.0, with maximum preference, which is not less than about 4.0, and a pH value less than about 3, 5 or less is unfavorable for animal and human bodies.
    [0024] [0024] The pharmaceutical composition of the present invention has a pH value of about 3.5 to 5.0, more preferably, about 3.5 to 4.5, more preferably, about 3.6 to 4.2 , more preferably, about 3.6 to 4.0 and, more preferably, about
    [0025] [0025] In the process of formulating the pharmaceutical composition formulation of GLP-1, it is generally necessary to add a buffer salt to maintain the pH of the pharmaceutical composition. In addition, the type of buffer will also influence the stability of GLP-1. Phosphate has low stability, and the buffer salt must be capable of providing a buffer salt within this pH range, that is, histidine - HCl, sodium acetate - acetic acid, glycine - HCl, disodium hydrogen phosphate - citric acid, hydroxide sodium - citric acid, sodium citrate - citric acid, succinate - succinic acid, lactate - lactic acid, glutaminate - glutamic acid, malate - malic acid, benzoate - benzoic acid, tartrate - tartaric acid and the like can be used. This buffer salt must be a pharmaceutically acceptable buffer salt, that is, it has no adverse effect on GLP-1 and has pharmacology and toxicology that meets the requirements. The buffer salt is preferably histidine and sodium acetate - acetic acid and, most preferably, sodium acetate - acetic acid.
    [0026] [0026] The concentration of the buffer salt has a very large influence on the GLP-1 polypeptide. GLP-1 is highly sensitive to salt and is extremely unstable to salt at a high concentration. The concentration of the selected buffer salt in the present invention is about 2 to 200 mmol / l, more preferably about 5 to 200 mmol / l, more preferably about 5 to 50 mmol / l, most preferably about 5 to 20 mmol / l, most preferably, about 7.5 to 10 mmol / l.
    [0027] [0027] For a multiple dose parenteral solution, the preservative is an essential ingredient of the pharmaceutical composition formulation. The preservative refers to a natural or synthetic chemical ingredient, to add food, drugs, pigments, biological specimens and the like, to delay decomposition caused by microbial growth or chemical changes and, therefore, to prolong the life of the food, drugs, pigments, biological specimens and the like. If no preservatives are added to the polypeptide drug, it is extremely difficult to meet microbial quality control requirements for multiple administrations. The use of the preservative has a lot to do with the type of preservative, the pH value of the pharmaceutical composition, the packaging material and the sealing material. Preservatives of types such as Nipagin and benzoic acid have high preservative efficacy in an acidic condition, and reduced efficacy in an alkalinity condition. All of the various preservatives have effective antimicrobial concentrations and the concentration in use should not be less than these concentrations. In addition, the preservative must be used in an amount that is not too high, to prevent it from causing damage to the bodies of humans. Preservatives that can be used in drugs can influence the stability of the polypeptide. In general, phenols are used in the pharmaceutical composition as preservatives, but phenol preservatives have serious influences on the stability of polypeptides, as well as GLP-1. Therefore, it is a difficult problem that needs to be solved when selecting a preservative for a GLP-1 drug solution that not only has a preservative effect, but will also not significantly influence the stability of GLP-1, that is, after the use, it allows the pharmaceutical composition to be able to meet the requirements for storage duration. The present invention has successfully solved this difficult problem.
    [0028] [0028] When the insulinotropic peptide is GLP-1, a GLP-1 analog or a GLP-1 derivative, the preservative can be phenol, benzyl alcohol, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, p- propyl hydroxybenzoate, butyl p-hydroxybenzoate, chlorobutanol, 2-phenoxyethanol, 2-phenethyl alcohol, benzalkonium chloride (bromide), mertiolate or any combination thereof. When the insulinotropic peptide is Exendin-4, an analogue of Exendin-4 or a derivative of Exendin-4, the preservative can be phenol, metacresol, benzyl alcohol, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, p-hydroxybenzoate propyl, butyl p-hydroxybenzoate, chlorobutanol, 2-phenoxyethanol, 2-phenethyl alcohol, benzalkonium chloride (bromide), mertiolate or any combinations thereof. For a pharmaceutical composition of GLP-1, preferably benzyl alcohol, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate and phenol, more preferably benzyl alcohol, phenol, or two of the above are used in combination. For a pharmaceutical composition of Exendin-4, preferably metacresol, benzyl alcohol, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate and phenol, more preferably, metacresol, benzyl alcohol, phenol, or two of those above are used in combination.
    [0029] [0029] The concentration of the preservative is also a factor to be taken into account. Different types of preservatives may have different antimicrobial concentrations in use. If metacresol or phenol is selected, the concentration in use is about 1 mg / ml to hand / ml, more preferably about 1 mg / ml to 5 mg / ml and, most preferably, about 1.5 mg / ml at 3 ma / ml. If benzyl alcohol is selected, the concentration in use is about 5 mg / ml to 20 mg / ml, more preferably about 5 mg / ml to 10 mg / ml and, most preferably, about 7.5 mg / ml at 10 mg / ml.
    [0030] [0030] In the formulation of the pharmaceutical composition, an isotonic agent must be carefully selected to allow the pharmaceutical composition to have a tonicity that is close to human tonicity. In addition, many isotonic agents act as stabilizers at the same time. Not only an intrinsic tonicity of the isotonic agent, but also the influences of other ingredients in the pharmaceutical composition on the general tonicity of the composition must be taken into account when selecting the concentration of the isotonic agent. Isotonic agents used in the present invention include polyols, for example, mannitol, sorbitol, inositol, xylitol, glycerin, propylene glycol and the like; sodium chloride; sugar, sucrose, trehalose, lactose, fructose and glucose and the like, and mannitol, glycerin and sorbitol are preferably used, most preferably, mannitol.
    [0031] [0031] When polyol or sugar are used as the isotonic agent, they are used in a concentration of about 10 mg / ml to 100 mg / ml and, more preferably, about 30 mg / ml to 50 mg / ml. When sodium chloride is used as the isotonic agent, it is used in a concentration of about 1 to 30 mg / ml, more preferably about 1 to 15 mg / ml, more preferably about 5 mg / ml to 15 mg / ml and, more preferably, about 7 mg / ml to 9 mg / ml.
    [0032] [0032] Due to the fact that GLP-1 has a strong hydrophobicity and is susceptible to self-association in macromolecular aggregates or the generation of gels, a dissolution enhancer has a very beneficial effect on the dissolution of GLP-1. The dissolution enhancers used in the present invention include Tween 20, Tween 40, Tween 80, Span 20, Span 40, Span 80, Poloxamer 188, Pluronic F68, Brij 35, dextran, PEG 400, PEG 1000, PEG 1500, PEG 2000, propylene glycol, and the like, and propylene glycol and PEG 400 are preferably used, most preferably, propylene glycol. The dissolution enhancer is used in a concentration of about 0.1 mg / ml to 10 mg / ml and, preferably, about 0.2 mg / ml to 5 mg / ml.
    [0033] [0033] In another aspect, the present invention further provides the use of the aqueous parenteral pharmaceutical composition formulated in the present invention, in particular, its use in the preparation of drugs to treat diabetes and adipose.
    [0034] [0034] The aqueous parenteral pharmaceutical composition of the present invention overcomes the problem with a parenteral solution in the prior art as to the difficulty in meeting the requirements for shelf life after adding a preservative and still preserving the ability to be stored for 2 years at 4ºC in case a preservative is added to it at a concentration of up to 2 mg / ml.
    [0035] [0035] The present invention is further presented below together with the specific modalities. However, the present invention is not limited to those specific embodiments or examples.
    [0036] [0036] The influence of pH value and ionic strength on the stability of GLP-1.
    [0037] [0037] The freeze-dried powders of GLP-1 were taken, dissolved with a buffer of 0.01 M sodium acetate - acetic acid to 10 mg / ml, and then the GLP-1 was replaced in buffers at various values of pH through dialysis or through a G25 chromatography method. Each buffer is also designed to have 4 concentrations of salt. The collected samples were quantified by an HPLC method and then the concentration of GLP-1 was adjusted to 4 mg / ml, followed by the addition of an adjuvant to a required concentration. The final concentration of GLP-1 was 2 mg / ml. The types of buffers and pharmaceutical composition designs are shown in Table 1. TABLE 1. OBSERVATION OF INFLUENCES OF PH AND SALT CONCENTRATION ON PHYSICAL STABILITY OF GLP-1
    [0038] [0038] The samples were placed at two temperatures, that is, 25ºC and 35ºC, for the treatment, and removed for detection in 8 days.
    [0039] [0039] A research method for physical stability: before determination, the samples were observed, in relation to appearance, with the naked eye. If there was evident precipitation or turbidity, no further determination was made. If the samples did not have any abnormal phenomenon observable with the naked eye, the samples were taken and subjected to the determination of absorption values at 360 nm and detection by HPLC, in which the former investigates physical stability and the latter investigates chemical stability.
    [0040] [0040] The absorption values determined at a wavelength of 360 nm were compared, in order to compare the differences in physical stability among the samples. A higher absorption value indicated less satisfactory physical stability.
    [0041] [0041] An investigation method for chemical stability: a test sample was taken and the sample was analyzed on a C18 column (3.5 µm, 300 Å, & 4.6 x 50 mm). An analytical method: mobile phase A was 0.1% trifluoroacetic acid, B was phase A with 80% acetonitrile added to it, the wavelength of detection was 280 nm, 60% A and 40% B were equilibrated for 3 minutes, and the sample amount was 2a5 µl. Elution was performed for 8 minutes with 40% to 53% of phase B and 60% to 47% of phase A. The peak area was calculated using the normalization method. A standard sample was determined using the same method. The concentration of peptide in the sample was calculated by comparison with the standard sample, and compared to the determination result on day 0, to calculate the retention of the peptide content in the sample. Higher retention of the peptide content indicated better chemical stability.
    [0042] [0042] The results on the stability of GLP-1 in solutions at various pH values are as follows. TABLE 2. INFLUENCES OF PH AND SALT CONCENTRATION ON GLP-1 STABILITY | air [NaCl EE EDER
    [0043] [0043] Note: an absorption value of the control background color was from 0 to 01, and the opalescence or turbidity could be seen with the naked eye when it was greater than 0.12.
    [0044] [0044] Conclusions: when the GLP-1 was at pH 5.0 to 6.5, turbidity or precipitation occurred while the GLP-1 sample was prepared, which indicates that GLP-1 is unstable at these pH values ; whereas, when the pH was 4.5, the prepared sample was clear and transparent, but turbidity occurred in the case of high salt concentration when formulated as a pharmaceutical composition and then subjected to the heating treatment. At pH 3.5 and pH 4.0 turbidity occurred in the sample in the presence of 0.5 M NaCl, and no turbidity or precipitation was seen at pH 3.0. As can be seen from the absorption values determined at 360 nm, the absorption value was increased with an increase in the pH value between pH 3.0 and 4.5, which indicates that physical stability has been decreased. With the same pH value, the absorption value was also increased with the increase in the concentration of NaCl salt, which indicates that the salt decreased physical stability. However, the analysis by HPLC was the reverse, the retention of the peptide content was increased with the increase of the pH value, which indicates that a superacid condition is unfavorable for the chemical stability of GLP-1. While, at the same pH value, the retention of the peptide content was increased with increasing salt concentration, possibly due to a certain inhibitory effect of salt on the adsorption of GLP-1. At pH 7.0 and 7.5, physical stability was completely normal, but the retention of the peptide content was significantly less compared to samples with pH 3.0 to 4.5 and therefore chemical stability was lower at pH 7.0 to 7.5.
    [0045] [0045] The following table shows the physical stability and chemical stability of the pharmaceutical composition when the pH of the GLP-1 pharmaceutical composition was in the range of 3.6 to 4.2 and the sodium chloride salt concentration was below 20 mmol / I (1.17 mg / ml). A monitoring method for the physical stability of the pH value was the fluorescence value, that is, tioflavin T was added to the sample at a final concentration of 5 umol / L, then the fluorescence absorption value was determined (in a excitation wavelength of 435 nm, and an emission wavelength of 485 nm). The higher the absorption value, the more rigorous the gelatinization phenomenon of the sample and the less satisfactory the physical stability. TABLE 3. INFLUENCE OF PH VALUE ON GLP-1 STABILITY pH of% GE peptide content o [e] se es) rj a ess re xe |
    [0046] [0046] As can be seen from the results in Table 3, between a pH range 3.6 to 4.2, the lower the pH value, the better the physical stability and the less satisfactory the chemical stability and on the contrary, the higher the pH value, the better the chemical stability and the less satisfactory the physical stability. Therefore, the pH range in which GLP-1 is stable must be the result of a comprehensive consideration of both physical and chemical stability. EXAMPLE 2
    [0047] [0047] Preparation of the pharmaceutical composition of the formulation.
    [0048] [0048] 20 ml of GLP-1 peptide at 4 mg / ml (in a 20 mmol / l buffer, pH 3.5 to 4.5) was mixed with 20 ml of 80 mg / ml of mannitol - 5, 2 mg / ml phenol. The mixture was adjusted to a pH of 3.5 to 4.5 with NaOH or acetic acid, filtered through a 0.22 µm filter membrane and dispensed in 2 ml penicillin bottles. Each component was: GLP-1 2 mg / ml Mannitol 40 mg / ml Phenol 2.6 mg / ml NaAC-HAC 10 mmol / l pH 3.5a4.5
    [0049] [0049] The dispensed samples were placed at 25ºC and 35ºC, respectively. The samples were placed at different times for inspection and analysis, to investigate physical and chemical stability. EXAMPLE 3
    [0050] [0050] Influences of buffer systems and microbial agents on the physical stability of GLP-1.
    [0051] [0051] A solution of GLP-1 (referred to as a stock solution) that was replaced in different buffer systems (the buffers had a concentration 2 times that of the final pharmaceutical composition) was diluted with a buffer [for 4 mg / ml , and an equal volume of a concentrated stock adjuvant solution with a final concentration of 2 times was added to it. The solutions were mixed uniformly, filtered through a 0.22 µm filter membrane, dispensed in 2 ml penicillin bottles and placed at different temperatures for investigation. A series of sampling time points was arranged. After sampling, the samples were first observed with the naked eye for appearance. If evident turbidity or precipitation occurred, the sample was considered to be disqualified in relation to physical stability and was not submitted to the next HPLC analysis step.
    [0052] [0052] The projects and results are as shown in Table 4: TABLE 4. INFLUENCE OF THE COMPOSITION OF THE PHARMACEUTICAL COMPOSITION ON PHYSICAL STABILITY OF GLP-1 pharmaceutical nº. 2 mg / ml) preparation '
    [0053] [0053] As can be seen from the results in Table 4, metacresol drastically influenced the stability of GLP-1, which became cloudy immediately after formulation, while phenol and benzyl alcohol were better, since the solutions of pharmaceutical composition of GLP-1 maintained a clear and transparent state. EXAMPLE 4
    [0054] [0054] Influence of the adjuvant on the physical stability of GLP-1. TABLE 5. INFLUENCE OF THE ADJUVANT ON PHYSICAL STABILITY OF GLP-1
    [0055] [0055] The results in Table 5, show that carboxymethylcellulose and sodium heparin are not suitable as adjuvants to GLP-1. EXAMPLE 5
    [0056] [0056] Influence of the adjuvant (additive) on the stability of GLP-1.
    [0057] [0057] Formulation of the pharmaceutical composition:
    [0058] [0058] A solution of GLP-1 (referred to as a stock solution) that was replaced in different buffer systems (the buffers had a concentration of 2 times that of the final pharmaceutical composition) was diluted with a buffer to 4 mg / ml , and an equal volume of a 2-fold concentrated stock adjuvant solution was added to it. The solutions were adjusted to pH 3.5 to 4.0, mixed uniformly, filtered through a 0.22 µm filter membrane, dispensed in 2 ml penicillin bottles and placed at different temperatures for investigation. A series of sampling time points was arranged. The samples were first observed with the naked eye for appearance. If no turbidity or gelatinization occurred, the sample was considered to be qualified in relation to physical stability and then subjected to HPLC detection to analyze chemical stability.
    [0059] [0059] The HPLC detection method was performed according to Example 1.
    [0060] [0060] All 10 mmol / l histidine pharmaceutical compositions, pH 4.0, were placed at 4 ° C overnight and then a lactescence occurred. When the pharmaceutical compositions were moved to room temperature (above 25ºC), they became clear. EXAMPLE 6
    [0061] [0061] The formulation method and the HPLC detection method of the pharmaceutical compositions were performed according to Example 5. TABLE 7. PHARMACEUTICAL COMPOSITION COMPOSITION INFLUENCE ON GLP-1 STABILITY Results of HPLC analysis (0 days) after 25ºC, 42 days preparation Composition | Composition (final pharmaceutical concentration | GLP-1 of 2 mg / ml, 10 mmol / l of No. NaAC-HAC, pH 3.5) Concentration Retention Purity of Dept and Purity | of the content of o, o, í (%) (mg / ml) (%)
    [0062] [0062] The formulation method and the HPLC detection method of the pharmaceutical compositions were carried out according to Example 5.
    [0063] [0063] As can be seen from the results in Table 8, two pharmaceutical compositions, that is, 3.5% mannitol + 0.2% propylene glycol and 4% mannitol + 0.01% propylene glycol had the highest retention of the peptide content at 25 degrees in 42 days. EXAMPLE 8
    [0064] [0064] An analog of Em from Exendin-4 had a sequence as follows:
    [0065] [0065] HGEGTFTSDL SKQLEEEAVK LFIEWLKNGG PSSGAPPPR
    [0066] [0066] (1) preparation of the Em
    [0067] [0067] The preparation was carried out using a solid phase polypeptide synthesis method and then purification using a reverse phase C18 column and freeze-drying were performed in order to obtain the Em.
    [0068] [0068] (2) method of formulating the pharmaceutical composition.
    [0069] [0069] The lyophilized Em powder was weighed and dissolved 2 times with a pH 3.5 NaAC-HAC buffer. In addition, crystalline powder of mannitol and metacresol were weighed according to an amount 2 times that in the formulation and dissolved in water. Then, the two solutions above were mixed, stirred uniformly, filtered through a 0.22 µm membrane, and dispensed in penicillin bottles or Carlsberg bottles.
    [0070] [0070] (3) detection by HPLC method
    [0071] [0071] An investigation method for chemical stability: a test sample was taken and the sample was analyzed on a C18 column (5.0 um, 300 À, 0 4.6 x 150 mm). An analytical method: mobile phase A was 0.1% trifluoroacetic acid, B was phase A with 80% acetonitrile added to it, the wavelength of detection was 214 nm, 68% A and 32% B were equilibrated for 4 minutes, and the sample amount was at 40 µl. Elution was carried out for 15 minutes with 32% to 45% of phase B and 68% to 55% of phase A. The peak area was calculated using the normalization method. A standard sample was determined using the same method. The peptide concentration in the sample was calculated by comparison with the standard sample and compared to the determination result on day 0, to calculate the retention of the peptide content in the sample. Higher retention of the peptide content indicated better chemical stability. TABLE 9. COMPOSITION INFLUENCE OF COMPOSITION ON EM STABILITY [E - by HPLC (0 days) 25ºC, 42 days Composition | Composition (final concentration of | POST EM pharmaceutical preparation of 0.3 mam, 10 mmol / | of - (mg / ml) (%) [9 EESTI | om to EDTA - Na + 2.2 mg / ml of 97 , 6 0.296 92.78 | 93.40 metacresol
    [0072] [0072] As can be seen from the results in Table 9, the 50 mg / ml mannitol + 2.2 mg / ml metacresol formula had the highest peptide content retention, as well as the best purity at 25 degrees in 30 days. That is, the formulation without the addition of EDTA or Tween 80 had good stability, and the formulation with the addition of EDTA or Tween 80 had low stability.
    权利要求:
    Claims (10)
    [1]
    1. Aqueous parenteral pharmaceutical composition of a stable insulinotropic peptide that can be stored for a long time, characterized by the fact that the aqueous parenteral pharmaceutical composition comprises an insulinotropic peptide, a pharmaceutically acceptable osmotic agent, a pharmaceutically acceptable preservative, a pharmaceutically dissolving enhancer acceptable and a pharmaceutically acceptable buffer salt solution; wherein the aqueous parenteral pharmaceutical composition has a pH of 3.0 to 5.0 and, preferably, 3.5 to 4.5; the insulinotropic peptide has a concentration of 0.1 mg / ml to 20 mg / ml and, preferably, 0.5 mg / ml to 5 mg / ml; the insulinotropic peptide is a GLP-1, an Exendin-4, a GLP-1 analogue, an Exendin-4 analogue, a GLP-1 derivative or an Exendin-4 derivative; where GLP-1 and GLP-1 analogs have a sequence as follows: 610 20 30 37
    XHXEGTFTSD VSSYLEXQAA XEFIAWLVXG XX where position 6 is R or a deletion; position 8 is A, G or V; position 22 is G or E; position 26 is K, R, Q or N; position 34 is K, R, Q or N; position 36 is R, R-NH>, K or K-NH>; position 37 is G or a deletion; where Exendin-4 and the Exendin-4 analog have a sequence as follows:
    XXXGTXXXXX SKQXEEEAVX LXXXXLKNGG XXXXXXXXX where, position 1 is H, Rou Y; position 2 is S, G, A or T; position 3 is D or E; position 6 is Fou Y;
    position 7 is T You S; position 8 is S or Y; position 9 is D or E; position 10 is Lou 1; position 14 is L, 1, Vou M; position 20 is R or K; position 22 is Fou Y; position 23 is 1, V Lou M; position 24 is E or D; position 25 is W, Fou Y; position 31 is P or a deletion; position 32 is S or a deletion; position 33 is S or a deletion; position 34 is G or a deletion; position 35 is A or a deletion; position 36 is P or a deletion; position 37 is P or a deletion; position 38 is P or a deletion; and position 39 is S, R or a deletion.
    [2]
    Aqueous parenteral pharmaceutical composition according to claim 1, characterized in that the GLP-1 derivative or the Exendin-4 derivative refers to GLP-1, Exendin-4, a GLP-1 analogue or an Exendin-4 analogue that is subjected to modification by pegylation or modification by acyl grease in a side or C-terminal chain of the same through or without a spacer arm.
    [3]
    Aqueous parenteral pharmaceutical composition according to claim 1, characterized in that the insulinotropic peptide has a concentration of 1 mg / ml to 5 mg / ml and, preferably, 2 mg / ml to 4 mg / ml.
    [4]
    4. Aqueous parenteral pharmaceutical composition, according to claim 1, characterized by the fact that the isotonic agent is polyol, sodium chloride, sugar or any combination thereof; where the polyol is mannitol, sorbitol, inositol, xylitol,
    glycerin, propylene glycol or any combination thereof; and sugar is sucrose, trehalose, lactose, fructose, glucose or any combination thereof.
    [5]
    Aqueous parenteral pharmaceutical composition according to claim 4, characterized in that the polyol has a concentration of 10 mg / ml to 100 mg / ml and, preferably, 30 mg / ml to 50 mg / ml; wherein sodium chloride has a concentration of 1 mg / ml to 30 mg / ml, preferably 1 mg / ml to 15 mg / ml, more preferably 5 mg / ml to 15 mg / ml and, most preferably, 7 mg / ml to 9 mg / ml; and wherein the sugar has a concentration of 10 mg / ml to 100 mg / ml and, preferably, 30 mg / ml to 50 mg / ml.
    [6]
    6. Aqueous parenteral pharmaceutical composition according to claim 1, characterized in that the dissolution enhancer is Tween 20, Tween 40, Tween 80, Span 20, Span 40, Span 80, Poloxamer 188, Pluronic F68, Brij 35 , dextran-20, PEG 400, PEG 1000, PEG 1500, PEG 2000, propylene glycol or any combinations thereof; and the dissolution enhancer has a concentration of 0.01 mg / ml to 10 mg / ml, preferably 0.1 mg / ml to 10 mg / ml, more preferably 0.2 mg / ml to 2 mg / ml and , most preferably, 0.5 mg / ml to 1 mg / ml.
    [7]
    7. Aqueous parenteral pharmaceutical composition according to claim 1, characterized by the fact that when the insulinotropic peptide is GLP-1, the GLP-1 analogue or the GLP-1 derivative, the preservative is phenol, benzyl alcohol , methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate, chlorobutanol, 2-phenoxyethanol, 2-phenethyl alcohol, benzalkonium chloride (bromide), mertiolate or any combination thereof; and, when the insulinotropic peptide is Exendin-4, the Exendin-4 analogue or the derivative of Exendin-4, the preservative is phenol, metacresol, benzyl alcohol, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, p-hydroxybenzoate propyl, butyl p-hydroxybenzoate, chlorobutanol, 2-phenoxyethanol, 2-phenethyl alcohol, benzalkonium chloride (bromide), mertiolate or any combination thereof; and the preservative has a concentration of 1 mg / ml to 20 mg / ml.
    [8]
    8. Aqueous parenteral pharmaceutical composition according to claim 1, characterized by the fact that the buffer salt is histidine - hydrochloric acid (histidine-HCI), sodium citrate - citric acid, disodium hydrogen phosphate - citric acid, NaOH -
    citric acid, sodium acetate - acetic acid (NaAC-HAC), succinate - succinic acid, lactate - lactic acid, glutaminate - glutamic acid, malate - malic acid, benzoate - benzoic acid, tartrate - tartaric acid or glycine - hydrochloric acid ( GIy-HCI) or any combination thereof; and the buffer salt has a concentration of 2 to 200 mmol / l, preferably 5 to 50 mmol / l, more preferably 5 to 20 mmol / l and, most preferably, 7.5 to 10 mmol / l.
    [9]
    9. Use of the aqueous parenteral pharmaceutical composition, as defined in any of claims 1 to 8, characterized by the fact that it is for the manufacture of a medicament to treat diabetes.
    [10]
    10. Use of the aqueous parenteral pharmaceutical composition, as defined in any of claims 1 to 8, characterized by the fact that it is for the manufacture of a medicament to treat adipose.
    summary
    STABLE AQUEOUS PHARMACEUTICAL PHARMACEUTICAL COMPOSITIONS OF PEPTIDES
    INSULINOTROPICS The present invention relates to an aqueous parenteral pharmaceutical composition of multiple doses of insulinotropic peptide and the use thereof. A long-term storage formulation of the insulinotropic peptide can be obtained using the method of the present invention. The pharmaceutical composition of the present invention comprises: the insulinotropic peptide, the insulinotropic peptide analog or analog; pharmaceutically acceptable tonicity modifier (stabilizer); pharmaceutically acceptable preservative; and pharmaceutically acceptable dissolution enhancer and pharmaceutically acceptable buffer solution. The pharmaceutical composition of the insulinotropic peptide is used in the preparation of drugs to treat diabetes and adipose.
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    同族专利:
    公开号 | 公开日
    RU2016104653A|2017-09-19|
    CA2921250A1|2015-02-19|
    CN103405753A|2013-11-27|
    JP2016532698A|2016-10-20|
    KR102350489B1|2022-01-11|
    CN103405753B|2016-05-11|
    HK1226302A1|2017-09-29|
    AU2014308320B2|2020-01-16|
    CA2921250C|2021-10-05|
    EP3034090A1|2016-06-22|
    US20160235855A1|2016-08-18|
    KR20160042120A|2016-04-18|
    WO2015021861A1|2015-02-19|
    EP3034090A4|2017-08-09|
    JP6987500B2|2022-01-05|
    AU2014308320A1|2016-03-10|
    MX2016001971A|2016-10-26|
    JP2020011975A|2020-01-23|
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    法律状态:
    2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|
    2018-02-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2019-05-21| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |
    2019-10-01| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2021-11-03| B350| Update of information on the portal [chapter 15.35 patent gazette]|
    优先权:
    申请号 | 申请日 | 专利标题
    CN201310351740.5A|CN103405753B|2013-08-13|2013-08-13|Stable insulin secretion accelerating peptide liquid drugs injection pharmaceutical composition|
    CN201310351740.5|2013-08-13|
    PCT/CN2014/083370|WO2015021861A1|2013-08-13|2014-07-31|Stable insulin secretagogue peptide hydro-injection pharmaceutical composition|
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