Patent Publication Number: US-2016235855-A1

Title: Stable aqueous parenteral pharmaceutical compositions of insulinotropic peptides

Description:
PRIORITY CLAIM 
     This application is a continuation of International Application No. PCT/CN2014/083370, filed Jul. 31, 2014, which claims priority to Chinese Patent Application No. 201310351740.5, filed Aug. 13, 2013, both of which are incorporated herein in their entirety. 
    
    
     TECHNICAL FIELD 
     The present disclosure relates to an insulinotropic peptide multi-dose aqueous parenteral pharmaceutical composition and use thereof. 
     BACKGROUND ART 
     Glucagon-like peptide 1 (also designated GLP-1) and Exendin-4 are both insulinotropic peptides, and have 53% identity in amino acid sequences thereof. Pharmacology has proven that both GLP-1 and Exendin-4 act on GLP-1 receptors of insulin-secreting β TC1 cells. This type of hormones can promote insulin secretion, and exert a glucose concentration-dependent hypoglycemic effect. 
     Similar to insulin, GLP-1 and Exendin are effective only when injected before meals. However, because a protein or polypeptide molecule is unstable, it cannot be developed to an oral pharmaceutical composition and must be used by injection. Even if a drug under development is in an injectable form, it tends to be a lyophilized injection powder that is inconvenient to use. 
     Instability of proteins and polypeptides includes two general aspects, i.e., physical and chemical aspects. Physical instability includes, for example, denaturation, surface adsorption, aggregation, precipitation, gelatination, and the like, and chemical instability includes, for example, hydrolysis, deamination, oxidization, racemization, isomerization, β-elimination, disulfide bond exchange, and the like. Such a series of unstabilizing factors will all change with the alteration in structures of the proteins or polypeptides, and therefore many protein or polypeptide drugs are produced by employing a freezing-drying method, such that the use of the formulation can meet the requirements for shelf life. 
     However, the production of lyophilized injection powders by employing the freezing-drying method suffers from many disadvantages: for example, high production cost, inconvenient for patients (the injection powder is in single doses, and prior to use each time, the patient needs to dissolve the injection powder with water, draw the mixture from a Penicillin bottle, and then injected the mixture), i.e., poor conformability, and therefore the market competitiveness is poor. Therefore, the development of multi-dose parenteral solutions not only provides convenience to patients, but also reduces the production cost, and thus has rather important significance for the improvement of market competitiveness. 
     Insulinotropic peptides, in particular GLP-1, have properties of these polypeptides, particularly physical instability, such as formation of gel, and therefore, in order to develop successfully multi-dose aqueous parenteral pharmaceutical compositions, these physical and chemical instability described above must be solved, to allow the compositions to achieve the pharmaceutically available period of validity. 
     In the development of multi-dose aqueous parenteral pharmaceutical compositions, adding a preservative into the pharmaceutical composition must be taken into consideration, so as to ensure that there is no microbial contamination during the storage duration and the usage period. However, most of preservatives are harmful to proteins or polypeptides, and interact with the 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 β-folds in insulin-type drugs to increase, and benzyl alcohol allows recombinant human interferon-γ to aggregate. Therefore, in the screening of pharmaceutical compositions, the relationship between the antimicrobial effect of the preservative and stability of the protein or polypeptide should be balanced. The trickiest difficulty in the development of parenteral solutions is to allow the formula to be able to be stored for 2 years or more at 4° C. after addition of a preservative. Many raw material drugs or stock solutions of proteins or polypeptides have no problem in storage for 2 years or more at 4° C., but have difficulty in meeting the requirements for shelf life after the addition of the preservative, just because the addition of the preservative will severely influence stability of the drug. 
     Dissolution enhancers selected for most proteins or polypeptides are surfactants and PEG. Surfactants are mostly Tween, Span, Poloxamer, Pluronic, Brij and the like. In addition to these substances, surfactants selected in the present disclosure further include propylene glycol and dextran. Propylene glycol and dextran have very good effects when used as GLP-1 dissolution enhancers. 
     Patent WO00/37098 filed by Brader, Mark, L. also mentions GLP-1 instability: physical stability is poor between pH 6.8 and 7.5, the formulation will become turbid at a pH value less than 8.0 after the addition of an preservative, and chemical stability is reduced when the pH value is greater than 8.8, therefore the suitable pH range is narrow, pH 8.2 to 8.8. A range claimed therein 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 employing synthetic GLP-1, which also indicates that GLP-1 is unstable indeed. 
     Whereas the present disclosure provides an aqueous parenteral pharmaceutical composition that allows GLP-1 to have greater stability, and the aqueous parenteral pharmaceutical composition has a GLP-1 concentration far higher than the drug concentration achievable by the prior art. 
     SUMMARY OF THE INVENTION 
     In one aspect, the present disclosure provides an insulinotropic peptide multi-dose aqueous parenteral pharmaceutical composition that can be stored for a long term and have greater stability. The formulated aqueous parenteral pharmaceutical composition can meet the requirement for storage duration. 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. 
     In the aqueous parenteral pharmaceutical composition of the present disclosure, the insulinotropic peptide is GLP-1, Exendin-4, a GLP-1 analogue, an Exendin-4 analogue, a GLP-1 derivative or an Exendin-4 derivative. 
     In the present application, the term “analogue” serves to denote such a peptide wherein one or more amino acid residues of the parent peptide have been substituted with other amino acid residues and/or wherein one or more amino acid residues of the parent peptide have been deleted and/or wherein one or more amino acid residues have been added into the parent peptide. This addition may be occurred at N-terminus or C-terminal or both, of the parent peptide. In general, an “analogue” is such a peptide wherein six or less amino acids of the parent peptide have been substituted and/or added and/or deleted, more preferably is such a peptide wherein three or less amino acids of the parent peptide have been substituted and/or added and/or deleted, and most preferably is such a peptide wherein one amino acid of the parent peptide have been substituted and/or added and/or deleted. 
     In the present application, the term “derivative” serves to denote such a peptide wherein one or more amino acid residues of the parent peptide have a substituent introduced therein, and typical variants of the substituent include amide, sugars, alkyl, acyl, ester, PEGylation and the like. 
     The insulinotropic peptide may be GLP-1, a GLP-1 analogue and a GLP-1 derivative. 
     Preferably, GLP-1 and the GLP-1 analogues have a sequence of: 
     
       
         
           
               
            
               
                 6  10    20      30  37 
               
               
                 X 6 HX 8 EGTFTSD VSSYLEX 22 QAA X 26 EFIAWLVX 34 G X 36 X 37   
               
            
           
         
       
     
     wherein X 6  is: R or a deletion; 
     wherein X 8  is: A, G or V; 
     X 22  is: G or E; 
     X 26  is: K, R, Q or N; 
     X 34  is: K, R, Q or N; 
     X 36  is: R, R—NH 2 , K or K—NH 2 ; and 
     X 37  is: G or a deletion. 
     The insulinotropic peptide may alternatively be Exendin-4, an Exendin-4 analogue and an Exendin-4 derivative. 
     Preferably, Exendin-4 and the Exendin-4 analogue have a sequence of: 
     
       
         
           
               
            
               
                 X 1 X 2 X 3 GTX 6 X 7 X 8 X 9 X 10  SKQX 14 EEEAVX 20  LX 22 X 23 X 24 X 25 LKNGG 
               
               
                 X 31 X 32 X 33 X 34 X 35 X 36 X 37 X 38 X 39   
               
            
           
         
       
     
     wherein, X 1  is H, R or Y; 
     X 2  is S, G, A or T; 
     X 3  is D or E; 
     X 6  is F or Y; 
     X 7  is T, Y or S; 
     X 8  is S or Y; 
     X 9  is D or E; 
     X 10  is L or I; 
     X 14  is L, I, V or M; 
     X 20  is R or K; 
     X 22  is F or Y; 
     X 23  is I, V, L or M; 
     X 24  is E or D; 
     X 25  is W, For Y; 
     X 31  is P or a deletion; 
     X 32  is S or a deletion; 
     X 33  is S or a deletion; 
     X 34  is G or a deletion; 
     X 35  is A or a deletion; 
     X 36  is P or a deletion; 
     X 37  is P or a deletion; 
     X 38  is P or a deletion; and 
     X 39  is S, R or a deletion. 
     For example, Exendin-4 and the Exendin-4 analogue may have a sequence of: 
     HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS 
     As is well known, the insulinotropic peptide has poor stability, and it is very difficult for the aqueous solution thereof to get through its storage duration (2 years at 2 to 8° C.). However, the present disclosure has developed a pharmaceutical composition capable of allowing the insulinotropic peptide, the analogue and the derivative to be stable, and allow them to meet the requirement for the storage duration. For example, in the formulation of the present disclosure, the GLP-1 concentration is up to 2 mg/ml, and in the event that an preservative is added, it can be stored for 2 years at 4° C. 
     Concentration is a factor that influences stability. If a drug molecule has good stability, its steady concentration is high, and on the contrary, its steady concentration is low. For a certain particular drug molecule, the drug has greater stability at a low concentration than that at a higher concentration. 
     In the pharmaceutical composition of the present disclosure, 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 0.5 to 5 mg/mL, more preferably about 1 to 5 mg/mL, and more preferably about 2 to 4 mg/mL. In the present application, “about” refers to the difference from a stated numerical value in a range of ±10%. 
     Another factor that that plays an important role in stability is maintenance of the pH value of the pharmaceutical composition, and in particular, it is found in the present disclosure that 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. Also, it is found that the pH value at which stability is kept 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 be occurred so long as the GLP-1 drug molecules are shifted into this pH range. When the pH value is lower than about 3.5, acid hydrolysis will be occurred, which is unstable likewise. Also, the injection formulation requires that the pH value should not be lower than about 3.0, most preferably not be lower than about 4.0, and a pH value lower than about 3.5 or lower is unfavorable for animal and human bodies. 
     The pharmaceutical composition of the present disclosure 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 3.6 to 3.9. At this pH, stability of GLP-1 comes up to what is expected, and can be stored for 2 years or more at 2 to 8° C. 
     In the process of formulating the GLP-1 pharmaceutical composition formulation, it is generally required to add a buffer salt to maintain pH of the pharmaceutical composition. In addition, the kind of the buffer will also influence the stability of GLP-1. Phosphate has poor stability, and the buffer salt should be able to provide a buffer salt within this pH range, hence histidine-HCl, sodium acetate-acetic acid, glycine-HCl, disodium hydrogen phosphate-citric acid, sodium hydroxide-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 may be employed. This buffer salt must be a pharmaceutically acceptable buffer salt, i.e., it has no adverse effect on GLP-1, and has pharmacology and toxicology that meet the requirements. The buffer salt is preferably histidine and sodium acetate-acetic acid, and most preferably sodium acetate-acetic acid. 
     Concentration of the buffer salt has a very great influence on the GLP-1 polypeptide. GLP-1 is highly sensible to the salt and is extremely unstable to the salt at a high concentration. Concentration of the buffer salt selected in the present disclosure is about 2 to 200 mmol/L, more preferably about 5 to 200 mmol/L, more preferably about 5 to 50 mmol/L, more preferably about 5 to 20 mmol/L, and most preferably about 7.5 to 10 mmol/L. 
     For a multi-dose parenteral solution, the preservative is an essential ingredient of the pharmaceutical composition formulation. Preservative refers to a natural or synthetic chemical ingredient, for adding into food, drugs, pigments, biological specimens and the like, to delay decomposition caused by microbial growth or chemical changes, and thereby to prolong shelf life of the food, drugs, pigments, biological specimens and the like. If no preservative is added into the polypeptide drug, it is extremely difficult to meet the quality control requirements of microbes for multiple administrations. Usage of the preservative has much to do with the kind of the preservative, the pH value of the pharmaceutical composition, the packaging material and the sealing material. Preservatives of the types such as Nipagin and benzoic acid have high preservative efficacy at an acidic condition, and reduced efficacy at an alkaline condition. Various preservatives all have effective antimicrobial concentrations thereof, and the concentration in use should not be lower than these concentrations. Also, the preservative should be used in an amount that is not too high, to prevent from doing harm to human bodies. Preservatives that may be used in drugs may influence stability of the polypeptide. Generally, phenols are used in the pharmaceutical composition as preservatives, but phenol preservatives have severe influences on the stability of polypeptides, and GLP-1 as well. Therefore, it is a difficult problem that must be solved to carefully select an preservative for a GLP-1 drug solution, which not only has an preservative effect, but also will not notably influence the stability of GLP-1, i.e., after employment, it allows the pharmaceutical composition to be able to meet the requirements for storage duration. The present disclosure has successfully solved this difficult problem. 
     When the insulinotropic peptide is GLP-1, a GLP-1 analogue or a GLP-1 derivative, the preservative may be phenol, benzyl alcohol, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate, chlorobutanol, 2-phenoxyethanol, 2-phenethyl alcohol, benzalkonium chloride (bromide), merthiolate or any combinations thereof. When the insulinotropic peptide is Exendin-4, an Exendin-4 analogue or an Exendin-4 derivative, the preservative may be phenol, metacresol, benzyl alcohol, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate, chlorobutanol, 2-phenoxyethanol, 2-phenethyl alcohol, benzalkonium chloride (bromide), merthiolate or any combinations thereof. For a GLP-1 pharmaceutical composition, preferably benzyl alcohol, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate and phenol are used, more preferably benzyl alcohol, phenol, or two of the above are used in combination. For an Exendin-4 pharmaceutical composition, preferably metacresol, benzyl alcohol, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate and phenol are used, more preferably metacresol, benzyl alcohol, phenol, or two of the above are used in combination. 
     Concentration of the preservative is also a factor to be taken into consideration. Different kinds 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 10 mg/mL, more preferably about 1 mg/mL to 5 mg/mL, and most preferably about 1.5 mg/mL to 3 mg/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 to 10 mg/mL. 
     In the formulation of the pharmaceutical composition, an isotonic agent should be selected carefully to allow the pharmaceutical composition to have an tonicity that is close to the human tonicity. In addition, many isotonic agents function as stabilizers at the same time. Not only an intrinsic tonicity of the isotonic agent, but also influences of other ingredients in the pharmaceutical composition on the overall tonicity of the composition should be taken into consideration in the selection of concentration of the isotonic agent. Isotonic agents employed in the present disclosure 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 used preferentially, most preferably mannitol. 
     When polyol or sugar is used as the isotonic agent, it is used at 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 at 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. 
     Because GLP-1 has strong hydrophobicity and is susceptible to self-association into macromolecular aggregates or generation of gels, a dissolution enhancer has a very good effect on the dissolution of GLP-1. Dissolution enhancers employed in the present disclosure 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 used preferentially, most preferably propylene glycol. The dissolution enhancer is used at a concentration of about 0.1 mg/mL to 10 mg/mL, and preferably about 0.2 mg/mL to 5 mg/mL. 
     In another aspect, the present disclosure further provides use of the aqueous parenteral pharmaceutical composition formulated in the present disclosure, in particular, use thereof in the preparation of drugs for treating diabetes and adiposis. 
     The aqueous parenteral pharmaceutical composition of the present disclosure overcomes the problem with a parenteral solution in the prior art of difficulty in meeting the requirements for shelf life after the addition of an preservative, and is still able to be stored for 2 years at 4° C. in the event that an preservative is added therein at a concentration up to 2 mg/ml. 
     The present disclosure is further set forth below in conjunction with particular embodiments. However, the present disclosure is not limited to these particular embodiments or examples. 
    
    
     EXAMPLE 1 
     Influence of pH Value and Ionic Strength on Stability of GLP-1 
     GLP-1 lyophilized powders were taken, dissolved with a 0.01 M sodium acetate-acetic acid buffer to 10 mg/mL, and then GLP-1 was replaced into buffers at various pH values through dialysis or through a G25 chromatographic method. Each buffer was also designed to have 4 salt concentrations. Samples collected were quantified by a HPLC method, and then GLP-1 concentration was adjusted to 4 mg/mL, followed by addition of an adjuvant to a required concentration. The final concentration of GLP-1 was 2 mg/mL. The kinds of the buffers and designs of pharmaceutical compositions are as shown in Table 1. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Observation of influences of pH and salt concentration 
               
               
                 on physical stability of GLP-1 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Replacement 
                 NaCl con- 
                   
               
               
                 Type of the 
                   
                 methods of the 
                 centration 
                 Situations of samples 
               
               
                 buffers 
                 pH 
                 buffers 
                 (mmol/L) 
                 after preparation 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 20 mmol/L 
                 3.0 
                 Sephadex G25 
                 0 
                 Clear and transparent 
               
               
                 NaAC-HAC 
                   
                   
                 20 
                 Clear and transparent 
               
               
                   
                   
                   
                 150 
                 Clear and transparent 
               
               
                   
                   
                   
                 500 
                 Clear and transparent 
               
               
                   
                 3.5 
                 Sephadex G25 
                 0 
                 Clear and transparent 
               
               
                   
                   
                   
                 20 
                 Clear and transparent 
               
               
                   
                   
                   
                 150 
                 Clear and transparent 
               
               
                   
                   
                   
                 500 
                 Turbid 
               
               
                   
                 4.0 
                 Sephadex G25 
                 0 
                 Clear and transparent 
               
               
                   
                   
                   
                 20 
                 Clear and transparent 
               
               
                   
                   
                   
                 150 
                 Clear and transparent 
               
               
                   
                   
                   
                 500 
                 Turbid 
               
               
                   
                 4.5 
                 Dialysis 
                 0 
                 Difficult to filter 
               
               
                   
                   
                   
                 20 
                 Difficult to filter 
               
               
                   
                   
                   
                 150 
                 Difficult to filter 
               
               
                   
                   
                   
                 500 
                 Difficult to filter, 
               
               
                   
                   
                   
                   
                 turbid 
               
               
                   
                 5.0 
                 Dialysis 
                 0 
                 Turbid or precipitated 
               
               
                   
                   
                   
                 20 
               
               
                   
                   
                   
                 150 
               
               
                   
                   
                   
                 500 
               
               
                 20 mmol/L 
                 5.5 
                 Dialysis 
                 0 
                 Turbid or precipitated 
               
               
                 citrate 
                   
                   
                 20 
               
               
                 sodium- 
                   
                   
                 150 
               
               
                 citric acid 
                   
                   
                 500 
               
               
                   
                 6.0 
                 Sephadex G25 
                 0 
                 Precipitate sticking 
               
               
                   
                   
                   
                   
                 to the wall 
               
               
                   
                   
                   
                 20 
                 Precipitate sticking 
               
               
                   
                   
                   
                   
                 to the wall 
               
               
                   
                   
                   
                 150 
                 Precipitate sticking 
               
               
                   
                   
                   
                   
                 to the wall 
               
               
                   
                   
                   
                 500 
                 Precipitate sticking 
               
               
                   
                   
                   
                   
                 to the wall 
               
               
                   
                 6.5 
                 Sephadex G25 
                 0 
                 Turbid 
               
               
                   
                   
                   
                 20 
                 Turbid 
               
               
                   
                   
                   
                 150 
                 Turbid 
               
               
                   
                   
                   
                 500 
                 Turbid 
               
               
                 20 mmol/L 
                 7.0 
                 Sephadex G25 
                 0 
                 Clear and transparent 
               
               
                 Na 2 HPO 4−   
                   
                   
                 20 
                 Clear and transparent 
               
               
                   
                   
                   
                 150 
                 Clear and transparent 
               
               
                   
                   
                   
                 500 
                 Clear and transparent 
               
               
                 NaH 2 PO 4   
                 7.5 
                 Sephadex G25 
                 0 
                 Clear and transparent 
               
               
                   
                   
                   
                 20 
                 Clear and transparent 
               
               
                   
                   
                   
                 150 
                 Clear and transparent 
               
               
                   
                   
                   
                 500 
                 Clear and transparent 
               
               
                   
               
            
           
         
       
     
     Samples were placed at two temperatures, i.e., 25° C. and 35° C., for treatment, and taken out for detection at 8 days. 
     An investigation method for physical stability: before determination, the samples were observed for appearance with the naked eye. If there was evident precipitation or turbidity, no further determination would be carried out. If the samples had no abnormal phenomena observable with the naked eye, the samples were taken and subjected to determination of absorption values at 360 nm and HPLC detection, wherein the former investigated the physical stability, and the latter investigated the chemical stability. 
     Absorption values determined at a wavelength of 360 nm were compared, so as to compare differences in physical stability among the samples. A higher absorption value indicated poorer physical stability. 
     An investigation method for chemical stability: a test sample was taken, and analyzed on a C18 column (3.5 μm, 300 Å, φ 4.6×50 mm). An analytical method: mobile phase A was 0.1% trifluoroacetic acid, B was the A phase with 80% acetonitrile added therein, the detection wavelength was 280 nm, 60% A and 40% B were balanced for 3 min, and the sample amount was 2 to 5 μL. Elution was performed with linear gradient for 8 min with 40% to 53% of the B phase and 60% to 47% of the A phase. Peak area was calculated using the normalization method. A standard sample was determined with the same method. The peptide concentration in the sample was calculated by comparison with the standard sample, and compared with the determination result at day 0, to calculate the retention of the peptide content in the sample. A higher retention of the peptide content indicated better chemical stability. 
     Stability results of GLP-1 in solutions at various pH values are as follows. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Influences of pH and salt concentration on stability of GLP-1 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Results of HPLC analysis, 
               
               
                   
                 NaCl 
                 Absorption values 
                 retention of the peptide 
               
               
                   
                 concentration 
                 at 360 nm 
                 content (%) 
               
            
           
           
               
               
               
               
               
               
            
               
                 pH 
                 (mg/mL) 
                 35° C., 8 days 
                 45° C., 8 days 
                 35° C., 8 days 
                 45° C., 8 days 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 3.0 
                 0 
                 0.0706 
                 0.0844 
                 95.46 
                 87.19 
               
               
                   
                 1.17 
                 0.0716 
                 0.0724 
                 93.69 
                 87.93 
               
               
                   
                 8.77 
                 0.1697 
                 0.1812 
                 95.03 
                 87.86 
               
               
                   
                 29.22 
                 0.2052 
                 0.2417 
                 96.42 
                 91.16 
               
               
                 3.5 
                 0 
                 0.0684 
                 0.0689 
                 94.73 
                 88.66 
               
               
                   
                 1.17 
                 0.0694 
                 0.0823 
                 96.92 
                 92.54 
               
               
                   
                 8.77 
                 0.1882 
                 0.1780 
                 98.06 
                 92.90 
               
            
           
           
               
               
               
            
               
                   
                 29.22 
                 Samples became turbid by placement for a period of 
               
               
                   
                   
                 time after formulation. 
               
            
           
           
               
               
               
               
               
               
            
               
                 4.0 
                 0 
                 0.0686 
                 0.0843 
                 96.07 
                 92.77 
               
               
                   
                 1.17 
                 0.1354 
                 0.1448 
                 97.43 
                 93.76 
               
               
                   
                 8.77 
                 0.1907 
                 0.1974 
                 98.73 
                 Turbid 
               
            
           
           
               
               
               
            
               
                   
                 29.22 
                 Samples became turbid after formulation. 
               
            
           
           
               
               
               
               
               
               
            
               
                 4.5 
                 0 
                 0.1326 
                 0.1370 
                 97.23 
                 92.45 
               
               
                   
                 1.17 
                 0.1655 
                 0.1814 
                 95.29 
                 Turbid 
               
               
                   
                 8.77 
                 1.4096 
                 1.3635 
                 Turbid 
                 Turbid 
               
            
           
           
               
               
               
            
               
                   
                 29.22 
                 Samples became turbid after formulation. 
               
               
                 5.0 
                 0 
                 Precipitate was generated while GLP-1 was 
               
               
                   
                 1.17 
                 prepared. 
               
            
           
           
               
               
               
            
               
                   
                 8.77 
                   
               
               
                   
                 29.22 
               
            
           
           
               
               
               
            
               
                 5.5 
                 0 
                 Precipitate was generated while GLP-1 was 
               
               
                   
                 1.17 
                 prepared. 
               
               
                   
                 8.77 
               
               
                   
                 29.22 
               
               
                 6.0 
                 0 
                 Precipitate was generated after formulation and 
               
               
                   
                 1.17 
                 placement of the sample. 
               
               
                   
                 8.77 
               
               
                   
                 29.22 
               
               
                 6.5 
                 0 
                 Turbidity was generated after formulation and 
               
               
                   
                 1.17 
                 placement of the sample. 
               
            
           
           
               
               
               
            
               
                   
                 8.77 
                   
               
               
                   
                 29.22 
               
            
           
           
               
               
               
               
               
               
            
               
                 7.0 
                 0 
                 0.0813 
                 0.0828 
                 94.30 
                 88.83 
               
               
                   
                 1.17 
                 0.0864 
                 0.0865 
                 93.35 
                 90.43 
               
               
                   
                 8.77 
                 0.0824 
                 0.0841 
                 94.34 
                 92.79 
               
               
                   
                 29.22 
                 0.0850 
                 0.0862 
                 95.83 
                 95.37 
               
               
                 7.5 
                 0 
                 0.0807 
                 0.0859 
                 90.89 
               
               
                   
                 1.17 
                 0.0799 
                 0.0937 
                 91.88 
                 85.88 
               
               
                   
                 8.77 
                 0.0846 
                 0.0902 
                 92.15 
                 87.48 
               
               
                   
                 29.22 
                 0.0861 
                 0.0942 
                 93.74 
                 88.04 
               
               
                   
               
               
                 Note: 
               
               
                 an absorption value of the control background color was 0 to 0.1, and opalescence or turbidity could be seen with the naked eye when it was greater than 0.12. 
               
            
           
         
       
     
     Conclusions: when GLP-1 was at pH 5.0 to 6.5, turbidity or precipitation was occurred while the GLP-1 sample was prepared, indicating that GLP-1 is unstable at these pH values; whereas when pH was 4.5, the sample prepared was clear and transparent, but turbidity was occurred in case of high salt concentration when formulated as a pharmaceutical composition and then subjected to heating treatment. At pH 3.5 and pH 4.0, turbidity was occurred in the sample in the presence of 0.5 M NaCl, and no turbidity or precipitation was seen only at pH 3.0. As can also be seen from the absorption values determined at 360 nm, the absorption value was increased with the increasing pH value between pH 3.0 and 4.5, indicating that the physical stability was decreased. At the same pH value, the absorption value was also increased with the increasing NaCl salt concentration, indicating that the salt decreased the physical stability. However, the HPLC analysis was then contrary, the retention of the peptide content was increased with the increasing pH value, indicating that a superacidic condition is unfavorable for the chemical stability of GLP-1. Whereas at the same pH value, the retention of the peptide content was increased with the increasing salt concentration, possibly due to a certain inhibitory effect of the salt on GLP-1 adsorption. At pH 7.0 and 7.5, the physical stability was all normal, but the retention of the peptide content was significantly lower as compared with the samples at pH 3.0 to 4.5, and therefore the chemical stability was poor at pH 7.0 to 7.5. 
     The following table shows the physical stability and chemical stability of the pharmaceutical composition when pH of the GLP-1 pharmaceutical composition ranged from 3.6 to 4.2 and the sodium chloride salt concentration was below 20 mmol/L (1.17 mg/mL). A monitoring method for physical stability of the pH value was the fluorescence value, that is, thioflavine T was added into the sample at a final concentration of 5 μmol/L, then the fluorescence absorption value was determined (at an excitation wavelength of 435 nm, and an emission wavelength of 485 nm). The higher the absorption value is, the severer the gelatination phenomena of the sample is, and the poorer the physical stability is. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Influence of the pH value on stability of GLP-1 
               
            
           
           
               
               
               
            
               
                   
                 Results of HPLC analysis, 
                   
               
               
                   
                 retention of the peptide content % 
                 Fluorescence value 
               
            
           
           
               
               
               
               
               
            
               
                 pH 
                 25° C., 30 days 
                 35° C., 30 days 
                 25° C., 35 days 
                 35° C., 35 days 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 3.6 
                 93.04 
                 86.25 
                 8.49 
                 9.95 
               
               
                 3.7 
                 95.15 
                 86.11 
                 9.11 
                 14.55 
               
               
                 3.8 
                 97.10 
                 89.48 
                 11.01 
                 31.58 
               
               
                 3.9 
                 98.08 
                 90.36 
                 26.46 
                 78.27 
               
               
                 4.0 
                 97.85 
                 91.80 
                 59.32 
                 113.18 
               
               
                 4.1 
                 98.90 
                 91.35 
                 139.48 
                 157.00 
               
               
                 4.2 
                 98.24 
                 93.96 
                 N.D 
                 N.D 
               
               
                   
               
            
           
         
       
     
     As can be seen from the results in Table 3, between a range from pH 3.6 to 4.2, the lower the pH value is, the better the physical stability is, and the poorer the chemical stability is, and on the contrary, the higher the pH value is, the better the chemical stability is, and the poorer the physical stability is. Therefore, the pH range at which GLP-1 is stable is supposed to be a result of comprehensive consideration of both physical and chemical stability. 
     EXAMPLE 2 
     Preparation of the Formulation Pharmaceutical Composition 
     20 mL of 4 mg/mL GLP-1 peptide (in a 20 mmol/L buffer, pH 3.5 to 4.5) was mixed with 20 mL of 80 mg/mL mannitol-5.2 mg/mL phenol. The mixture was adjusted to pH 3.5-4.5 with NaOH or acetic acid, filtered through a 0.22 μm filter membrane, and dispensed into 2 mL Penicillin bottles. Each of the components was: 
     
       
         
           
               
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 GLP-1 
                 2 
                 mg/mL 
               
               
                   
                 Mannitol 
                 40 
                 mg/mL 
               
               
                   
                 Phenol 
                 2.6 
                 mg/mL 
               
               
                   
                 NaAC-HAC 
                 10 
                 mmol/L 
               
            
           
           
               
               
               
            
               
                   
                 pH 
                 3.5 to 4.5 
               
               
                   
                   
               
            
           
         
       
     
     The samples dispensed were placed at 25° C. and 35° C. respectively. Samples were taken at different times for inspection and analysis, to investigate physical and chemical stability. 
     EXAMPLE 3 
     Influences of Buffer Systems and Antimicrobial Agents on the Physical Stability of GLP-1 
     The GLP-1 solution (referred to as a stock solution) that had been replaced into different buffer systems (the buffers had a concentration 2 times that of the final pharmaceutical composition) was diluted with a buffer to 4 mg/mL, and an equal volume of a concentrated stock adjuvant solution with a 2-time final concentration was added therein. The solutions were mixed uniformly, filtered through a 0.22 μm filter membrane, dispensed into 2 mL Penicillin bottles, and placed at different temperatures for investigation. A series of sampling time points were arranged. After sampling, the samples were firstly observed with the naked eye for appearance. If evident turbidity or precipitation was occurred, the sample was considered as disqualified as for physical stability, and would not be subjected to the next step of HPLC analysis. 
     Designs and results are as shown in Table 4: 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Influence of composition of the pharmaceutical composition on the physical stability of GLP-1 
               
            
           
           
               
               
               
               
            
               
                 Pharmaceutical 
                 Composition 
                 Appearance 
                   
               
               
                 composition No. 
                 (GLP-1 final concentration 2 mg/mL) 
                 after preparatio   
                 25° C., 14 days 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 10 mmol/L NaAC-HAC pH 3.5, 40 mg/mL 
                 Turbid 
                 Turbid 
               
               
                   
                 mannitol, 3 mg/mL metacresol 
               
               
                 2 
                 10 mmol/L NaAC-HAC pH 3.5, 45 mg/mL 
                 Turbid 
                 Turbid 
               
               
                   
                 mannitol, 3 mg/mL metacresol 
               
               
                 3 
                 10 mmol/L NaAC-HAC pH 3.5, 50 mg/mL 
                 Turbid 
                 Turbid 
               
               
                   
                 mannitol, 3 mg/mL metacresol 
               
               
                 4 
                 10 mmol/L NaAC-HAC pH 3.5, 55 mg/mL 
                 Turbid 
                 Turbid 
               
               
                   
                 mannitol, 3 mg/mL metacresol 
               
               
                 5 
                 10 mmol/L NaAC-HAC pH 3.5, 40 mg/mL 
                 Transparent and 
                 Transparent and 
               
               
                   
                 mannitol, 2 mg/mL phenol 
                 clear 
                 clear 
               
               
                 6 
                 10 mmol/L NaAC-HAC pH 3.5, 45 mg/mL 
                 Transparent and 
                 Transparent and 
               
               
                   
                 mannitol, 2 mg/mL phenol 
                 clear 
                 clear 
               
               
                 7 
                 10 mmol/L NaAC-HAC pH 3.5, 50 mg/mL 
                 Transparent and 
                 Transparent and 
               
               
                   
                 mannitol, 2 mg/mL phenol 
                 clear 
                 clear 
               
               
                 8 
                 10 mmol/L NaAC-HAC pH 3.5, 55 mg/mL 
                 Transparent and 
                 Transparent and 
               
               
                   
                 mannitol, 2 mg/mL phenol 
                 clear 
                 clear 
               
               
                 9 
                 10 mmol/L NaAC-HAC pH 3.5, 10 mg/mL 
                 Turbid 
                 Turbid 
               
               
                   
                 glycerin, 3 mg/mL metacresol 
               
               
                 10 
                 10 mmol/L NaAC-HAC pH 3.5, 1.50 
                 Turbid 
                 Turbid 
               
               
                   
                 mg/mL glycerin, 3 mg/mL metacresol 
               
               
                 11 
                 10 mmol/L NaAC-HAC pH 3.5, 20 mg/mL 
                 Turbid 
                 Turbid 
               
               
                   
                 glycerin, 3 mg/mL metacresol 
               
               
                 12 
                 10 mmol/L NaAC-HAC pH 3.5, 25 mg/mL 
                 Turbid 
                 Turbid 
               
               
                   
                 glycerin, 3 mg/mL metacresol 
               
               
                 13 
                 10 mmol/L histidine pH 4.0, 40 mg/mL 
                 Transparent and 
                 Transparent and 
               
               
                   
                 mannitol, 2 mg/mL phenol 
                 clear 
                 clear 
               
               
                 14 
                 10 mmol/L histidine pH 4.0, 45 mg/mL 
                 Transparent and 
                 Transparent and 
               
               
                   
                 mannitol, 2 mg/mL phenol 
                 clear 
                 clear 
               
               
                 15 
                 10 mmol/L histidine pH 4.0, 50 mg/mL 
                 Transparent and 
                 Transparent and 
               
               
                   
                 mannitol, 2 mg/mL phenol 
                 clear 
                 clear 
               
               
                 16 
                 10 mmol/L histidine pH 4.0, 55 mg/mL 
                 Transparent and 
                 Transparent and 
               
               
                   
                 mannitol, 2 mg/mL phenol 
                 clear 
                 clear 
               
               
                 17 
                 10 mmol/L histidine pH 4.0, 15 mg/mL 
                 Transparent and 
                 Transparent and 
               
               
                   
                 glycerin, 2 mg/mL phenol 
                 clear 
                 clear 
               
               
                 18 
                 10 mmol/L histidine pH 4.0, 20 mg/mL 
                 Transparent and 
                 Transparent and 
               
               
                   
                 glycerin, 2 mg/mL phenol 
                 clear 
                 clear 
               
               
                 19 
                 10 mmol/L histidine pH 4.0, 25 mg/mL 
                 Transparent and 
                 Transparent and 
               
               
                   
                 glycerin, 2 mg/mL phenol 
                 clear 
                 clear 
               
               
                 20 
                 10 mmol/L NaAC-HAC pH 3.5, 40 mg/mL 
                 Transparent and 
                 Transparent and 
               
               
                   
                 mannitol, 9 mg/mL benzyl alcohol 
                 clear 
                 clear 
               
               
                 21 
                 10 mmol/L NaAC-HAC pH 3.5, 45 mg/mL 
                 Transparent and 
                 Transparent and 
               
               
                   
                 mannitol, 9 mg/mL benzyl alcohol 
                 clear 
                 clear 
               
               
                 22 
                 10 mmol/L NaAC-HAC pH 3.5, 50 mg/mL 
                 Transparent and 
                 Transparent and 
               
               
                   
                 mannitol, 9 mg/mL benzyl alcohol 
                 clear 
                 clear 
               
               
                   
               
               
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     As can be seen from the results in Table 4, metacresol severely influenced the stability of GLP-1, which became turbid immediately after the formulation, whereas phenol and benzyl alcohol were better, as the pharmaceutical composition solutions of GLP-1 maintained in a clear and transparent state. 
     EXAMPLE 4 
     Influence of the Adjuvant on Physical Stability of GLP-1 
       
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Influence of the adjuvant on physical stability of GLP-1 
               
            
           
           
               
               
               
               
            
               
                 Pharmaceutical 
                 Composition (GLP-1 final concentration 2 
                 Appearance 
                   
               
               
                 composition No. 
                 mg/mL, 10 mmol/L NaAC-HAC pH 3.5) 
                 after preparation 
                 25° C., 14 days 
               
               
                   
               
               
                 23 
                 40 mg/mL sorbitol, 2 mg/mL phenol 
                 Transparent and 
                 Transparent and 
               
               
                   
                   
                 
                   
                 
                 
                   
                 
               
               
                 24 
                 45 mg/mL sorbitol, 2 mg/mL phenol 
                 Transparent and 
                 Transparent and 
               
               
                   
                   
                 
                   
                 
                 
                   
                 
               
               
                 25 
                 50 mg/mL sorbitol, 2 mg/mL phenol 
                 Transparent and 
                 Transparent and 
               
               
                   
                   
                 
                   
                 
                 
                   
                 
               
               
                 26 
                 5 mg/mL hydroxypropyl-beta-cyclodextrin, 
                 Transparent and 
                 Transparent and 
               
               
                   
                 2 mg/mL phenol 
                 
                   
                 
                 
                   
                 
               
               
                 27 
                 20 mg/mL hydroxypropyl-beta- 
                 Transparent and 
                 Transparent and 
               
               
                   
                 cyclodextrin, 2 mg/mL phenol 
                 
                   
                 
                 
                   
                 
               
               
                 28 
                 5 mg/mL hydroxypropyl-beta-cyclodextrin 
                 Turbid 
                 Turbid 
               
               
                   
                 (HP), 3 mg/mL metacresol 
               
               
                 29 
                 20 mg/mL hydroxypropyl-beta- 
                 Turbid 
                 Turbid 
               
               
                   
                 cyclodextrin, 3 mg/mL metacresol 
               
               
                 30 
                 5 mg/mL hydroxypropyl-beta-cyclodextrin, 
                 Transparent and 
                 Transparent and 
               
               
                   
                 9 mg/mL benzyl alcohol 
                 
                   
                 
                 
                   
                 
               
               
                 31 
                 20 mg/mL hydroxypropyl-beta- 
                 Transparent and 
                 Transparent and 
               
               
                   
                 cyclodextrin, 9 mg/mL benzyl alcohol 
                 
                   
                 
                 
                   
                 
               
               
                 32 
                 0.5 mg/mL carboxyl methyl cellulose 
                 Turbid 
                 Turbid 
               
               
                   
                 (CMC), 2 mg/mL phenol 
               
               
                 33 
                 0.5 mg/mL carboxyl methyl cellulose 
                 Turbid 
                 Turbid 
               
               
                   
                 (CMC), 3 mg/mL metacresol 
               
               
                 34 
                 0.5 mg/mL carboxyl methyl cellulose 
                 Turbid 
                 Turbid 
               
               
                   
                 (CMC), 9 mg/mL benzyl alcohol 
               
               
                 35 
                 2 mg/mL carboxyl methyl cellulose (CMC), 
                 Turbid 
                 Turbid 
               
               
                   
                 2 mg/mL phenol 
               
               
                 36 
                 2 mg/mL carboxyl methyl cellulose (CMC), 
                 Turbid 
                 Turbid 
               
               
                   
                 3 mg/mL metacresol 
               
               
                 37 
                 2 mg/mL carboxyl methyl cellulose (CMC), 
                 Turbid 
                 Turbid 
               
               
                   
                 9 mg/mL benzyl alcohol 
               
               
                 38 
                 0.05 mg/mL heparin sodium, 2 mg/mL 
                 Turbid 
                 Turbid 
               
               
                   
                 
                   
                 
               
               
                 39 
                 0.05 mg/mL heparin sodium, 3 mg/mL 
                 Turbid 
                 Turbid 
               
               
                   
                 metacresol 
               
               
                 40 
                 0.05 mg/mL heparin sodium, 9 mg/mL 
                 Turbid 
                 Turbid 
               
               
                   
                 benzyl alcohol 
               
               
                 41 
                 0.3 mg/mL heparin sodium, 2 mg/mL 
                 Turbid 
                 Turbid 
               
               
                   
                 phenol 
               
               
                 42 
                 0.3 mg/mL heparin sodium, 3 mg/mL 
                 Turbid 
                 Turbid 
               
               
                   
                 metacresol 
               
               
                 43 
                 0.3 mg/mL heparin sodium, 9 mg/mL 
                 Turbid 
                 Turbid 
               
               
                   
                 benzyl alcohol 
               
               
                   
               
               
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     Results in Table 5 show that carboxy methylcellulose and heparin sodium are not suitable as adjuvants of GLP-1. 
     EXAMPLE 5 
     Influence of the Adjuvant (Additive) on the Stability of GLP-1 
     Formulation of the Pharmaceutical Composition: 
     The GLP-1 solution (referred to as a stock solution) that had been replaced into different buffer systems (the buffers had a concentration 2 times that of the final pharmaceutical composition) was diluted with a buffer to 4 mg/mL, and an equal volume of a concentrated stock adjuvant solution with a 2-time concentration was added therein. The solutions were adjusted to pH 3.5-4.0, mixed uniformly, filtered through a 0.22 μm filter membrane, dispensed into 2 mL Penicillin bottles, and placed at different temperatures for investigation. A series of sampling time points were arranged. The samples were firstly observed with the naked eye for appearance. If no evident turbidity or gelatination was occurred, the sample was considered as qualified as for physical stability, and then subjected to HPLC detection to analyze the chemical stability. 
     The HPLC detection method was performed according to Example 1. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Influence of composition of the pharmaceutical composition on the stability of GLP-1 
               
            
           
           
               
               
               
            
               
                   
                 Results of HPLC 
                   
               
               
                   
                 analysis 0 day 
                 25° C., 14 days 
               
            
           
           
               
               
               
               
            
               
                   
                 after the 
                   
                 Retention 
               
               
                   
                 preparation 
                   
                 of the 
               
            
           
           
               
               
               
               
               
               
            
               
                 Pharmaceutical 
                   
                   
                 Peptide 
                   
                 peptide 
               
               
                 composition 
                 Composition (GLP-1 final 
                 Purity 
                 concentration 
                 Purity 
                 content 
               
               
                 No. 
                 concentration 2 mg/mL) 
                 (%) 
                 (mg/ml) 
                 (%) 
                 (%) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 44 
                 10 mmol/L 
                 45 mg/ml mannitol, 2 mg/ml 
                 97.97 
                 1.96 
                 Gelatination 
               
               
                   
                 histidine- 
                 phenol, 0.1 mg/ml 
               
               
                   
                 HCl pH 
                 Tween 80 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 45 
                 4.0 
                 45 mg/ml mannitol, 2 mg/ml 
                 97.78 
                 2.00 
                 97.12 
                 91.51 
               
               
                   
                   
                 phenol, 0.5 mg/ml 
               
               
                   
                   
                 Tween 80 
               
               
                 46 
                   
                 45 mg/ml mannitol, 2% 
                 97.62 
                 2.00 
                 97.72 
                 86.55 
               
               
                   
                   
                 phenol 2 mg/ml Tween 
               
               
                   
                   
                 80 
               
               
                 47 
                 10 mmol/L 
                 45 mg/ml mannitol, 2 mg/ml 
                 98.24 
                 1.97 
                 97.99 
                 92.39 
               
               
                   
                 NaAC- 
                 phenol 0.1 mg/ml 
               
               
                   
                 HAC, pH 
                 Tween 80 
               
               
                 48 
                 3.5 
                 45 mg/ml mannitol, 2 mg/ml 
                 98.35 
                 1.96 
                 98.42 
                 92.21 
               
               
                   
                   
                 phenol, 0.5 mg/ml 
               
               
                   
                   
                 Tween 80 
               
               
                 49 
                   
                 45 mg/ml mannitol, 2 mg/ml 
                 98.37 
                 2.01 
                 97.57 
                 91.86 
               
               
                   
                   
                 phenol, 2 mg/ml 
               
               
                   
                   
                 Tween 80 
               
               
                 50 
                 10 mmol/L 
                 45 mg/ml mannitol, 2 mg/ml 
                 97.88 
                 2.01 
                 97.72 
                 80.37 
               
               
                   
                 NaA-HAC, 
                 phenol, 0.2 mg/ml 
               
               
                   
                 pH 4.0 
                 PEG 400 
               
               
                 51 
                   
                 45 mg/ml mannitol, 2 mg/ml 
                 97.78 
                 2.03 
                 97.79 
                 93.55 
               
               
                   
                   
                 phenol, 0.5 mg/ml 
               
               
                   
                   
                 PEG 400 
               
               
                 52 
                   
                 45 mg/ml mannitol, 2 mg/ml 
                 97.59 
                 2.00 
                 97.74 
                 89.60 
               
               
                   
                   
                 phenol, 1 mg/ml 
               
               
                   
                   
                 PEG 400 
               
               
                 53 
                 10 mmol/L 
                 45 mg/ml mannitol, 2 mg/ml 
                 98.28 
                 2.06 
                 97.19 
                 90.07 
               
               
                   
                 histidine- 
                 phenol 0.2, mg/ml 
               
               
                   
                 HCl pH 
                 PEG 400 
               
               
                 54 
                 4.0 
                 45 mg/ml mannitol, 2 mg/ml 
                 98.48 
                 2.06 
                 97.74 
                 90.30 
               
               
                   
                   
                 phenol, 0.5% 
               
               
                   
                   
                 PEG 400 
               
               
                 55 
                   
                 45 mg/ml mannitol, 2 mg/ml 
                 98.42 
                 1.85 
                 97.39 
                 87.39 
               
               
                   
                   
                 phenol, 1 mg/ml 
               
               
                   
               
            
           
         
       
     
     All pharmaceutical compositions of 10 mmol/L histidine pH 4.0 were placed at 4° C. overnight, and then milkiness appeared. When the pharmaceutical compositions were shifted to room temperature (above 25° C.), they became clear. 
     EXAMPLE 6 
     The formulation method and HPLC detection method of the pharmaceutical compositions were performed according to Example 5. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Influence of composition of the pharmaceutical composition on the stability of GLP-1 
               
            
           
           
               
               
               
            
               
                   
                 Results of HPLC 
                   
               
               
                   
                 analysis (0 day) 
               
               
                   
                 after the preparation 
                 25° C., 42 days 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 Peptide 
                   
                 Retention of the 
               
               
                 Pharmaceutical 
                 Composition (GLP-1 final concentration 2 
                 Purity 
                 concentration 
                 Purity 
                 peptide content 
               
               
                 composition No. 
                 mg/mL, 10 mmol/L NaAC-HAC pH 3.5) 
                 (%) 
                 (mg/mL) 
                 (%) 
                 (%) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 56 
                 50 mg/mL mannitol + 2 mg/mL 
                 99.5 
                 2.04 
                 Gelatination 
               
               
                   
                 phenol + 0.5 mg/mL dextran 20 
               
               
                 57 
                 50 mg/mL mannitol + 2 mg/mL 
                 100 
                 2.01 
                 Gelatination 
               
               
                   
                 phenol + 1 mg/mL dextran 20 
               
               
                 58 
                 50 mg/mL mannitol + 2 mg/mL 
                 99.5 
                 2.14 
                 Gelatination 
               
               
                   
                 phenol + 2 mg/mL dextran 20 
               
               
                 59 
                 50 mg/mL mannitol + 2 mg/mL + 
                 99.6 
                 2.11 
                 Gelatination 
               
               
                   
                 3 mg/mL dextran 20 
               
            
           
           
               
               
               
               
               
               
            
               
                 60 
                 50 mg/mL mannitol + 2 mg/mL 
                 99.4 
                 1.95 
                 97.8 
                 98.5 
               
               
                   
                 phenol + 0.1 mg/mL propylene 
               
               
                 61 
                 50 mg/mL mannitol + 2 mg/mL 
                 99.7 
                 1.95 
                 97.8 
                 98.37 
               
               
                   
                 phenol + 0.4 mg/mL propylene 
               
               
                 62 
                 50 mg/mL mannitol + 2 mg/mL 
                 100 
                 2.00 
                 97.9 
                 97.69 
               
               
                   
                 phenol + 1 mg/mL propylene 
               
               
                   
               
            
           
         
       
     
     EXAMPLE 7 
     The formulation method and HPLC detection method of the pharmaceutical compositions were performed according to Example 5. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Influence of the ratio of mannitol to propylene glycol on the stability of GLP-1 
               
            
           
           
               
               
               
            
               
                   
                 Results of HPLC 
                   
               
               
                   
                 analysis (0 day) 
               
               
                   
                 after the preparation 
                 25° C., 42 days 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 Peptide 
                   
                 Retention of the 
               
               
                 Pharmaceutical 
                 Composition (GLP-1 final concentration 2 
                 Purity 
                 concentration 
                 Purity 
                 peptide content 
               
               
                 composition No. 
                 mg/mL, 10 mmol/L NaAC-HAC pH 3.5) 
                 (%) 
                 (mg/mL) 
                 (%) 
                 (%) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 63 
                 40 mg/mL mannitol + 2.2 mg/mL 
                 99.7 
                 2.01 
                 97.0 
                 96.65 
               
               
                   
                 phenol 
               
               
                 64 
                 40 mg/mL mannitol + 2.2 mg/mL 
                 99.4 
                 1.99 
                 97.8 
                 97.62 
               
               
                   
                 phenol + 0.1 mg/mL propylene 
               
               
                   
                 glycol 
               
               
                 65 
                 38 mg/mL mannitol + 2.2 mg/mL 
                 99.4 
                 1.99 
                 97.8 
                 96.66 
               
               
                   
                 phenol + 1 mg/mL propylene 
               
               
                   
                 glycol 
               
               
                 66 
                 35 mg/mL mannitol + 2.2 mg/mL 
                 99.5 
                 2.00 
                 97.5 
                 97.93 
               
               
                   
                 phenol + 2 mg/mL propylene 
               
               
                   
                 glycol 
               
               
                 67 
                 30 mg/mL mannitol + 2.2 mg/mL 
                 99.5 
                 2.00 
                 96.7 
                 97.19 
               
               
                   
                 phenol + 5 mg/mL propylene 
               
               
                   
                 glycol 
               
               
                 68 
                 22 mg/mL mannitol + 2.2 mg/mL 
                 99.5 
                 2.00 
                 97.7 
                 97.07 
               
               
                   
                 phenol + 7.5 mg/mL propylene 
               
               
                   
                 glycol 
               
               
                   
               
            
           
         
       
     
     As can be seen from results in Table 8, two pharmaceutical compositions, i.e., 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 at 42 days. 
     EXAMPLE 8 
     An analogue Em of Exendin-4 had a sequence as follows: 
     
       
         
           
               
               
            
               
                   
                 HGEGTFTSDL SKQLEEEAVK LFIEWLKNGG PSSGAPPPR 
               
            
           
         
       
     
     (1) Preparation of Em 
     The preparation was performed using a solid phase polypeptide synthesis method, and then purification using a reversed phase C18 column and lyophilization were performed, so as to obtain Em. 
     (2) Formulation Method of the Pharmaceutical Composition 
     Lyophilized Em powder was weighed, and dissolved with 2 times of a pH 3.5 NaAC-HAC buffer. In addition, mannitol crystalline powder and metacresol were weighed according to an amount 2 times that in recipe, and dissolved in water. Then, the above two solutions were mixed, stirred uniformly, filtered through a 0.22 μm membrane, and dispensed into Penicillin bottles or carlsberg&#39;s flasks. 
     (3) HPLC Detection Method 
     An investigation method for chemical stability: a test sample was taken, and analyzed on a C18 column (5.0 μm, 300 Å, φ 4.6×150 mm). An analytical method: mobile phase A was 0.1% trifluoroacetic acid, B was the A phase with 80% acetonitrile added therein, the detection wavelength was 214 nm, 68% A and 32% B were balanced for 4 min, and the sample amount was 20 to 40 μL. Elution was performed for 15 min with 32% to 45% of the B phase and 68% to 55% of the A phase. Peak area was calculated using the normalization method. A standard sample was determined with the same method. The peptide concentration in the sample was calculated by comparison with the standard sample, and compared with the determination result at day 0, to calculate the retention of the peptide content in the sample. A higher retention of the peptide content indicated better chemical stability. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Influence of composition of the composition on the stability of EM 
               
            
           
           
               
               
               
            
               
                   
                 Results of HPLC 
                   
               
               
                   
                 analysis (0 day) 
               
               
                   
                 after the preparation 
                 25° C., 42 days 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 Peptide 
                   
                 Retention of the 
               
               
                 Pharmaceutical 
                 Composition (EM final concentration 0.3 
                 Purity 
                 concentration 
                 Purity 
                 peptide content 
               
               
                 composition No. 
                 mg/mL, 10 mmol/L NaAC-HAC pH 3.5) 
                 (%) 
                 (mg/mL) 
                 (%) 
                 (%) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 69 
                 50 mg/mL mannitol + 0.3 
                 97.6 
                 0.296 
                 92.78 
                 93.40 
               
               
                   
                 mg/mLEDTA-Na 2  + 2.2 mg/mL 
               
               
                   
                 metacresol 
               
               
                 70 
                 50 mg/mL mannitol + 0.2 mg/mL 
                 97.80 
                 0.281 
                 93.64 
                 96.3 
               
               
                   
                 Tween 80 + 2.2 mg/mL metacresol 
               
               
                 71 
                 50 mg/mL mannitol + 2.2 mg/mL 
                 96.33 
                 0.289 
                 96.05 
                 97.37 
               
               
                   
                 metacresol 
               
               
                 72 
                 50 mg/mL mannitol + 0.3 mg/mL 
                 96.86 
                 0.288 
                 93.75 
                 95.14 
               
               
                   
                 EDTA + 2.2 mg/mL metacresol + 
               
               
                   
                 0.2 mg/mL Tween 80 
               
               
                   
               
            
           
         
       
     
     As can be seen from results in Table 9, the 50 mg/mL mannitol+2.2 mg/mL metacresol formula had the highest retention of the peptide content as well as the best purity at 25 degrees at 30 days. That is to say, the recipe without the addition of EDTA or Tween 80 had good stability, and the recipe with the addition of EDTA or Tween 80 had poor stability. 
     EXAMPLE 9 
     Influence of Adjuvants (Additives) on the Long-Term Stabilities of GLP-1 
     GLP-1 formulations (pH 3.5-4.0) comprising 0.2% phenol, 20 mmol/L NaAC-HAC, and the adjuvants listed in Table 10 were prepared as described in Example 5. 
     The stabilities of GLP-1 were determined by HPLC using a C18 column. The GLP-1 concentration in the sample was determined by comparison with a standard sample as disclosed in Example 1. The GLP-1 purity was determined by comparing the GLP-1 peak area with the total peak area. 
     The GLP-1 stability parameter (also referred herein as the retention of the peptide content) was determined by comparing the GLP-1 concentration with the GLP-1 concentration on day 0 after preparation (the initial GLP-1 concentration) using the following formula: 
       GLP-1 Stability Parameter (%)=GLP-1 Concentration/Initial GLP-1 Concentration×100%
 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Long-term stability of GLP-1 in GLP-1 formulations 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 0 day 
                   
                   
               
               
                   
                   
                 after the 
                 2-8° C., 
                 2-8° C., 
               
               
                 Pharmaceutical 
                 Composition (main 
                 preparation 
                 12 months 
                 24 months 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 composition No. 
                 adjuvants) 
                 A* 
                 B** 
                 A* 
                 B** 
                 C*** 
                 A* 
                 B** 
                 C*** 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 73 
                 4.5% mannitol + 
                 99.1 
                 1.96 
                 97.55 
                 1.90 
                 96.94 
                 95.0 
                 1.89 
                 96.43 
               
               
                   
                 0.5% PEG400 
               
               
                 74 
                 5% mannitol + 
                 99.4 
                 1.96 
                 98.4 
                 1.96 
                 100.00 
                 96.2 
                 1.94 
                 98.98 
               
               
                   
                 0.05% dextran 
               
               
                 75 
                 5% mannitol + 0.2% 
                 99.8 
                 1.96 
                 98.45 
                 1.96 
                 100.00 
                 96.2 
                 1.95 
                 99.49 
               
               
                   
                 dextran 
               
               
                 76 
                 5% mannitol + 0.3% 
                 99.1 
                 1.97 
                 98.5 
                 1.95 
                 98.98 
                 96.4 
                 1.93 
                 97.97 
               
               
                   
                 dextran 
               
               
                 77 
                 5% mannitol + 
                 99.1 
                 1.98 
                 98.45 
                 1.96 
                 98.99 
                 96.2 
                 1.96 
                 98.99 
               
               
                   
                 0.01% propylene 
               
               
                   
                 glycol 
               
               
                 78 
                 5% mannitol + 0.1% 
                 98.7 
                 2.00 
                 98.45 
                 1.98 
                 99.00 
                 96.45 
                 1.96 
                 98.00 
               
               
                   
                 propylene glycol 
               
               
                 79 
                 5% mannitol + 
                 99.2 
                 1.97 
                 98.05 
                 1.97 
                 100.00 
                 96.45 
                 1.95 
                 98.99 
               
               
                   
                 0.25% propylene 
               
               
                   
                 glycol 
               
               
                 80 
                 4.5% mannitol + 
                 99.2 
                 1.97 
                 98.05 
                 1.96 
                 99.49 
                 96.7 
                 1.96 
                 99.49 
               
               
                   
                 0.1% propylene 
               
               
                   
                 glycol 
               
               
                 81 
                 4.5% mannitol + 
                 99.2 
                 1.98 
                 98.65 
                 1.97 
                 99.49 
                 96.3 
                 1.96 
                 98.99 
               
               
                   
                 0.45% propylene 
               
               
                   
                 glycol 
               
               
                   
               
               
                 *A is GLP-1 Purity, also referred to as purity in this disclosure (%) 
               
               
                 **B is GLP-1 concentration, also referred to as peptide concentration in this disclosure (mg/mL) 
               
               
                 ***C is GLP-1 Stability Parameter, also referred to as the retention of the peptide content in this disclosure (%) 
               
            
           
         
       
     
     As shown in Table 10, GLP-1 showed good stabilities in all GLP-1 formulations tested when kept at 4° C. for 2 years. The GLP-1 purities remained at 95% or higher. Among the tested formulations, GLP-1 formulation No. 73 showed the highest degradation rate of GLP-1: 3.57%; and GLP-1 formulation Nos. 75 and 80 showed the best stability parameter of GLP-1: 99.49%.