Source: https://patents.google.com/patent/US8748376B2/en
Timestamp: 2019-09-23 09:29:45
Document Index: 114974918

Matched Legal Cases: ['Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 05104050', 'Application No. 05104172', 'art 0', 'art 0']

US8748376B2 - Stable formulations of peptides - Google Patents
Stable formulations of peptides Download PDF
US8748376B2
US8748376B2 US12/643,330 US64333009A US8748376B2 US 8748376 B2 US8748376 B2 US 8748376B2 US 64333009 A US64333009 A US 64333009A US 8748376 B2 US8748376 B2 US 8748376B2
US12/643,330
US20100173844A1 (en
Anne-Mette Lilleøre
2004-11-12 Priority to DKPA200401753 priority Critical
2004-11-12 Priority to DKPA200401753 priority
2004-11-12 Priority to DK200401753 priority
2004-11-18 Priority to US62911504P priority
2004-12-08 Priority to DK200401906 priority
2004-12-08 Priority to DKPA200401906 priority
2004-12-10 Priority to US63531104P priority
2005-05-13 Priority to EP05104050 priority
2005-05-13 Priority to EP05104050.9 priority
2005-05-18 Priority to EP05104172.1 priority
2005-05-18 Priority to EP05104172 priority
2005-05-19 Priority to US68272405P priority
2005-11-11 Priority to PCT/EP2005/055916 priority patent/WO2006051103A2/en
2005-11-11 Priority to WOPCT/EP2005/055916 priority
2005-11-11 Priority to EPPCT/EP2005/055916 priority
2005-11-14 Priority to PCT/EP2005/055946 priority patent/WO2006051110A2/en
2007-11-06 Priority to US66704007A priority
2009-12-21 Priority to US12/643,330 priority patent/US8748376B2/en
2009-12-21 Application filed by Novo Nordisk AS filed Critical Novo Nordisk AS
2010-07-08 Publication of US20100173844A1 publication Critical patent/US20100173844A1/en
2014-06-10 Publication of US8748376B2 publication Critical patent/US8748376B2/en
2014-11-11 First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=39560969&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US8748376(B2) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Stable pharmaceutical composition comprising insulinotropic peptide.
This application is a divisional of U.S. application Ser. No. 11/667,040 filed Nov. 6, 2007, which is a National Stage filing of International Application No. PCT/EP2005/055946, filed Nov. 14, 2005, which claims priority from U.S. Application No. 60/629,115, filed Nov. 18, 2004, and U.S. Application No. 60/635,311, filed Dec. 10, 2004, and U.S. Application No. 60/682,724, fled May 19, 2005, and which also claims priority from Danish Patent Application No. PA 2004 01753, filed Nov. 12, 2004, and Danish Application No. PA 2004 01906, filed Dec. 8, 2004, and European Application No. 05104050.9, filed May 13, 2005, and European Application No. 05104172.1, filed May 18, 2005, and European Application No. PCT/EP2005/055916, filed Nov. 11, 2005.
The present invention relates to the field of pharmaceutical formulations. More specifically the invention pertains to shelf-stable pharmaceutical formulations comprising an insulinotropic peptide.
Therapeutic peptides are widely used in medical practise. Pharmaceutical compositions of such therapeutic peptides are required to have a shelf life of several years in order to be suitable for common use. However, peptide compositions are inherently unstable due to sensitivity towards chemical and physical degradation. Chemical degradation involves change of covalent bonds, such as oxidation, hydrolysis, racemization or crosslinking. Physical degradation involves conformational changes relative to the native structure of the peptide, which may lead to aggregation, precipitation or adsorption to surfaces.
Glucagon has been used for decades in medical practise within diabetes and several glucagon-like peptides are being developed for various therapeutic indications. The preproglucagon gene encodes glucagon as well as glucagon-like peptide 1 (GLP-1) and glucagon-like peptide 2 (GLP-2). GLP-1 analogs and derivatives as well as the homologous lizard peptide, exendin-4, are being developed for the treatment of hyperglycemia within type 2 diabetes. GLP-2 are potentially useful in the treatment of gastrointestinal diseases. However, all these peptides encompassing 29-39 amino acids have a high degree of homology and they share a number of properties, notably their tendency to aggregate and formation of insoluble fibrils. This property seems to encompass a transition from a predominant alpha-helix conformation to beta-sheets (Blundell T. L. (1983) The conformation of glucagon. In: Lefébvre P. J. (Ed) Glucagon I. Springer Verlag, pp 37-55, Senderoff R. I. et al., J. Pharm. Sci. 87 (1998)183-189, WO 01/55213). Aggregation of the glucagon-like peptides are mainly seen when solutions of the peptides are stirred or shaken, at the interface between solution and gas phase (air), and at contact with hydrophobic surfaces such as Teflon®.
WO 01/77141 discloses heat treatment of Arg34-GLP-1(7-37) at elevated temperatures for less than 30 seconds. WO 04/55213 discloses microfiltration of Arg34-GLP-1(7-37) at pH 9.5. WO 01/55213 discloses treatment of Val8-GLP-1(7-37) at pH 12.3 for 10 minutes at room temperature. WO 03/35099 discloses the preparation of zinc crystals of GLP-1 at alkaline pH.
FIG. 1. Both samples contain a formulation of 1.2 mM Liraglutide, 14 mg/ml propylene glycol, 40 mM phenol, 10 mM NaCl, pH 7.7. Poloxamer-188 is added to a final concentration of 200 ppm in one sample.
FIG. 2. All samples contain 1.67 mM Liraglutide, 58 mM phenol, 14 mg/ml propylene glycol, 8 mM sodium phosphate pH 7.7. Poloxamer 188 is added to two samples.
FIG. 3. Both samples contain 1.2 mM Liraglutide, 40 mM phenol, 14 mg/ml propylene glycol, 10 mM NaCl, pH 7.7. Polysorbate 20 added to one sample
FIG. 4. Measurement of NTU versus time during a rotation test of liraglutide compositions without surfactant (F1) and with surfactant (F2 and F3).
FIG. 5. Measurement of ThT fluorescence versus time during a rotation test of liraglutide compositions without surfactant (F1) and with surfactant (F2). The lower curve is the trace of F2.
FIG. 6. Time course for fibril formation.
FIG. 7. Physical stability of liraglutide prepared by heat treatment at 60° C.
FIG. 8. Purity of liraglutide after heat treatment at 60° C.
FIG. 9. Physical stability of liraglutide prepared by heat treatment at 80° C.
FIG. 10. Purity of liraglutide after heat treatment at 80° C.
FIG. 11. Physical stability of liraglutide prepared by 15 min. of heat treatment at 22, 40, 60, and 80° C.
FIG. 12. Physical stability of liraglutide prepared by heat treatment at 50 and 80° C. at pH 10.
FIG. 13. Purity of liraglutide after heat treatment at 50 and 80° C. at pH 10.
FIG. 14. Physical stability of liraglutide prepared by heat treatment at 60 and 80° C. at pH 9 and 10.
FIG. 15. This figure shows 5 different formulations. 4 different formulations containing various amounts of Solutol HS-15 in either phosphate or tricine buffer. One formulation (Ref. formulation) is liraglutide in phosphate buffer without surfactant.
FIG. 16. This figure shows 5 different formulations. 4 different formulations containing various amounts of Pluronic F-127 in either phosphate or tricine buffer. One formulation (Ref. formulation) is liraglutide in phosphate buffer without surfactant.
FIG. 17. Physical stability of liraglutide after heat treatment at 50-70° C. for 60-120 minutes.
FIG. 18. Penfill® heat treated at different times and temperatures which were subsequently subjected to rotation.
The term “pharmaceutical composition” as used herein means a product comprising an active compound or a salt thereof together with pharmaceutical excipients such as buffer, preservative and tonicity modifier, said pharmaceutical composition being useful for treating, preventing or reducing the severity of a disease or disorder by administration of said pharmaceutical composition to a person. Thus a pharmaceutical composition is also known in the art as a pharmaceutical formulation. It is to be understood that pH of a pharmaceutical composition which is to be reconstituted is the pH value which is measured on the reconstituted composition produced by reconstitution in the prescribed reconstitution liquid at room temperature.
The term “shelf-stable pharmaceutical composition” as used herein means a pharmaceutical composition which is stable for at least the period which is required by regulatory agencies in connection with therapeutic proteins. Preferably, a shelf-stable pharmaceutical composition is stable for at least one year at 5° C. Stability includes chemical stability as well as physical stability.
The term “analogue” as used herein referring to a peptide means a modified peptide wherein one or more amino acid residues of the peptide have been substituted by other amino acid residues and/or wherein one or more amino acid residues have been deleted from the peptide and/or wherein one or more amino acid residues have been deleted from the peptide and or wherein one or more amino acid residues have been added to the peptide. Such addition or deletion of amino acid residues can take place at the N-terminal of the peptide and/or at the C-terminal of the peptide. In one embodiment an analogue comprises less than 6 modifications (substitutions, deletions, additions) relative to the native peptide. In another embodiment an analogue comprises less than 5 modifications (substitutions, deletions, additions) relative to the native peptide. In another embodiment an analogue comprises less than 4 modifications (substitutions, deletions, additions) relative to the native peptide. In another embodiment an analogue comprises less than 3 modifications (substitutions, deletions, additions) relative to the native peptide. In another embodiment an analogue comprises less than 2 modifications (substitutions, deletions, additions) relative to the native peptide. In another embodiment an analogue comprises only a single modification (substitutions, deletions, additions) relative to the native peptide.
The term “GLP-1 compound” as used herein means GLP-1(7-37) (SEQ ID NO. 1), insulinotropic analogue thereof and insulinotropic derivatives thereof. Non-limiting examples of GLP-1 analogues are GLP-1(7-36) amide, Arg34-GLP-1(7-37), Gly8-GLP-1(7-37), Val8-GLP-1(7-36)-amide and Val8Asp22-GLP-1(7-37). Non-limiting examples of GLP-1 derivatives are desamino-His7, Arg26, Lys34(Nε-(γ-Glu(Nα-hexadecanoyl)))-GLP-1(7-37), desamino-His7, Arg26, Lys34(Nε-octanoyl)-GLP-1(7-37), Arg26,34, Lys38(Nε-(ω-carboxypentadecanoyl))-GLP-1(7-38), Arg26,34, Lys36(Nε-(γ-Glu(Nα-hexadecanoyl)))-GLP-1(7-36) and Arg34, Lys26(Nε-(γ-Glu(Nα-hexadecanoyl)))-GLP-1(7-37).
The functional receptor assay is carried out by measuring cAMP as a response to stimulation by the insulinotropic peptide or insulinotropic compound. Incubations are carried out in 96-well microtiter plates in a total volume of 140 mL and with the following final concentrations: 50 mM Tris-HCl, 1 mM EGTA, 1.5 mM MgSO4, 1.7 mM ATP, 20 mM GTP, 2 mM 3-isobutyl-1-methylxanthine (IBMX), 0.01% w/v Tween-20, pH 7.4. Compounds are dissolved and diluted in buffer. GTP is freshly prepared for each experiment: 2.5 pg of membrane is added to each well and the mixture is incubated for 90 min at room temperature in the dark with shaking. The reaction is stopped by the addition of 25 mL 0.5 M HCl. Formed cAMP is measured by a scintillation proximity assay (RPA 542, Amersham, UK). A dose-response curves is plotted for the compound and the EC50 value is calculated using GraphPad Prism software.
The term “exendin-4 compound” as used herein is defined as exendin-4(1-39) (SEQ ID NO. 2), insulinotropic fragments thereof, insulinotropic analogs thereof and insulinotropic derivatives thereof. Insulinotropic fragments of exendin-4 are insulinotropic peptides for which the entire sequence can be found in the sequence of exendin-4 (SEQ ID NO. 2) and where at least one terminal amino acid has been deleted. Examples of insulinotropic fragments of exendin-4(1-39) are exendin-4(1-38) and exendin-4(1-31). The insulinotropic property of a compound may be determined by in vivo or in vitro assays well known in the art. For instance, the compound may be administered to an animal and monitoring the insulin concentration over time. Insulinotropic analogs of exendin-4(1-39) refer to the respective molecules wherein one or more of the amino acids residues have been exchanged with other amino acid residues and/or from which one or more amino acid residues have been deleted and/or from which one or more amino acid residues have been added with the proviso that said analogue either is insulinotropic or is a prodrug of an insulinotropic compound . An example of an insulinotropic analog of exendin-4(1-39) is Ser2Asp3-exendin-4(1-39) wherein the amino acid residues in position 2 and 3 have been replaced with serine and aspartic acid, respectively (this particular analog also being known in the art as exendin-3). Insulinotropic derivatives of exendin-4(1-39) and analogs thereof are what the person skilled in the art considers to be derivatives of these peptides, i.e. having at least one substituent which is not present in the parent peptide molecule with the proviso that said derivative either is insulinotropic or is a prodrug of an insulinotropic compound. Examples of substituents are amides, carbohydrates, alkyl groups, esters and lipophilic substituents. An example of an insulinotropic derivatives of exendin-4(1-39) and analogs thereof is Tyr31-exendin-4(1-31)-amide.
In a first aspect the present invention relates to a shelf-stable pharmaceutical composition comprising an insulinotropic peptide, a pharmaceutically acceptable preservative, a poloxamer or polysorbate 20 surfactant at a concentration of from about 10 mg/L to about 400 mg/L, and optionally a pharmaceutically acceptable tonicity modifier, where said composition has a pH that is in the range from about 7.0 to about 8.5.
In another embodiment the concentration of surfactant is from about 50 mg/L to about 200 mg/L.
In another embodiment the concentration of surfactant is from about 10 mg/L to about 200 mg/L.
In another embodiment of the invention the pharmaceutical composition comprises two different surfactants wherein at least one surfactant is a non-ionic surfactant.
In another embodiment of the invention the pharmaceutical composition comprises two different surfactants wherein the two different surfactants are both non-ionic surfactants.
In another embodiment of the invention the pharmaceutical composition comprises two different surfactants wherein all surfactants are non-ionic surfactants.
In another embodiment of the invention the pharmaceutical composition comprises poloxamer 188 and polysorbate 20.
In another embodiment of the invention the pharmaceutical composition comprises a tonicity modifier selected from the group consisting of glycerol, propylene glycol and mannitol.
In another embodiment of the invention the pharmaceutical composition the preservative is selected from the group consisting of phenol, m-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, thiomerosal and mixtures thereof.
In another embodiment of the invention the pharmaceutical composition comprises an insulinotropic peptide which is a DPP-IV protected peptide.
In another embodiment of the invention the pharmaceutical composition the insulinotropic peptide comprises a lipophilic substituent selected from the group consisting of CH3(CH2)nCO— wherein n is 4 to 38, and HOOC(CH2)mCO— wherein m is from 4 to 38.
In another embodiment of the invention the pharmaceutical composition the insulinotropic peptide is acylated GLP-1 or an acylated GLP-1 analogue.
In another embodiment of the invention the pharmaceutical composition comprises an insulinotropic peptide which is an acylated GLP-1 analogue wherein said GLP-1 analogue is selected from the group consisting of Arg34-GLP-1(7-37), Gly8-GLP-1(7-36)-amide, Gly8-GLP-1(7-37), Val8-GLP-1(7-36)-amide, Val8-GLP-1(7-37), Aib8-GLP-1(7-36)-amide, Aib8-GLP-1(7-37), Val8Asp22-GLP-1(7-36)-amide, Val8Asp22-GLP-1(7-37), Val8Glu22-GLP-1(7-36)-amide , Val8Glu22-GLP-1(7-37), Val8Lys22-GLP-1(7-36)-amide, Val8Lys22-GLP-1(7-37), Val8Arg22-GLP-1(7-36)-amide, Val8Arg22-GLP-1(7-37), Val8His22-GLP-1(7-36)-amide, Val8His22-GLP-1(7-37), Val8Trp19Glu22-GLP-1(7-37), Val8Glu22Val25-GLP-1(7-37), Val8Tyr16Glu22-GLP-1(7-37), Val8Trp16Glu22-GLP-1(7-37), Val8Leu16Glu22-GLP-1(7-37), Val8Tyr18Glu22-GLP-1(7-37), Val8Glu22His37-GLP-1(7-37), Val8Glu22Ile33-GLP-1(7-37), Val8Trp16Glu22Val25Ile33-GLP-1(7-37), Val8Trp16Glu22Ile33-GLP-1(7-37), Val8Glu22Val25Ile33-GLP-1(7-37), Val8Trp16Glu22Val25-GLP-1(7-37), and analogues thereof.
In another embodiment of the invention the pharmaceutical composition the insulinotropic peptide is Arg34, Lys26(Nε-(γ-Glu(Nα-hexadecanoyl)))-GLP-1(7-37).
In another embodiment of the invention the concentration of said insulinotropic peptide is in the range from about 0.1 mg/ml to about 25 mg/ml, in the range from about 1 mg/ml to about 25 mg/ml, in the range from about 2 mg/ml to about 15 mg/ml, in the range from about 3 mg/ml to about 10 mg/ml, or in the range from about 5 mg/ml to about 8 mg/ml.
In another embodiment of the invention the insulinotropic peptide is exendin-4 or ZP-10, i.e. HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-NH2.
In another embodiment of the invention the pharmaceutical composition the insulinotropic peptide is acylated exendin-4 or an acylated exendin-4 analogue.
In another embodiment of the invention the pharmaceutical composition the insulinotropic peptide is [N-epsilon(17-carboxyheptadecanoic acid)20 exendin-4(1-39)-amide
In another embodiment of the invention the pharmaceutical composition the concentration of the insulinotropic peptide in the pharmaceutical composition is from about 5 μg/mL to about 10 mg/mL, from about 5 μg/mL to about 5 mg/mL, from about 5 μg/mL to about 5 mg/mL, from about 0.1 mg/mL to about 3 mg/mL, or from about 0.2 mg/mL to about 1 mg/mL.
The present invention also relates to a method for preparation of a stable solution of a GLP-1 compound, which method comprises heating a solution of said GLP-1 compound at alkaline pH to a temperature above 40° C. for at least 5 minutes. Concentrations of the GLP-1 compound during the heat treatment is generally preferred to be in the range from 10 g/L to 100 g/L. The GLP-1 compound may be dissolved in an aqueous solution having the final temperature, or it may be dissolved in aqueous solution having room temperature followed by heating to the appropriate temperature for the specified time.
It has been shown that the physical stability of liraglutide was significantly improved as the temperature of heat treatment was increased (22 to 80° C.). For temperatures of 60 and 80° C., time of heat treatment was shown to have a strong influence on the physical stability of liraglutide, as 120 minutes of heat treatment showed to improve the physical stability significantly in comparison to 1 minute of heat treatment. It has also been shown that the physical stability of liraglutide was significantly improved by increasing the temperature from 22 to 50-80° C. at pH 9-10 (cn.f. examples). For all temperatures, time of heat treatment was shown to have an influence on the physical stability of liraglutide, as 15 to 20 minutes of heat treatment showed to improve the physical stability significantly compared to 1 minute of heat treatment. Optimal conditions for heat treatment to dissolve fibril germs appear to be 3-20 minutes at pH 9-10.5 and 70-85° C. In production scale, this could be performed using common methods for fast heating and cooling of large volumes by heat exchangers.
In another aspect the present invention relates to a method for preparation of a stable solution of a GLP-1 compound, which method comprises heating a solution of said GLP-1 compound having a pH between pH 8.0 to pH 10.5 to a temperature between 50° C. and 80° C. for a period of time which is between 3 minutes and 180 minutes.
In one embodiment the present invention relates to a method for preparation of a stable solution of a GLP-1 compound, which method comprises heating a solution of said GLP-1 compound having a pH between pH 8.0 to pH 10.0 to a temperature between 50° C. and 80° C. for a period of time which is between 3 minutes and 180 minutes.
In another embodiment the present invention relates to a method for preparation of a stable solution of a GLP-1 compound, which method comprises heating a solution of said GLP-1 compound having a pH between pH 8.0 to pH 10.0 to a temperature between 50° C. and 80° C. for a period of time which is between 3 minutes and 120 minutes.
In another embodiment the temperature is between 60° C. and 80° C. for a period of time which is between 5 minutes and 15 minutes.
In another embodiment the temperature is between 60° C. and 80° C. for a period of time which is between 1 minute and 15 minutes.
In another embodiment the temperature is between 60° C. and 80° C. for a period of time which is between 3 minutes and 30 minutes.
In another embodiment the temperature is between 60° C. and 80° C. for a period of time which is between 5 minutes and 30 minutes.
In another embodiment the present invention relates to a method for preparation of a stable solution of exendin-4, which method comprises heating a solution of exendin-4 having a pH between pH 8.0 to pH 10.0 to a temperature between 50° C. and 80° C. for a period of time which is between 3 minutes and 120 minutes.
In another embodiment the present invention relates to a method for preparation of a stable solution of Aib8,35-GLP-1(7-36)-amide, which method comprises heating a solution of Aib8,35-GLP-1(7-36)-amide having a pH between pH 8.0 to pH 10.0 to a temperature between 50° C. and 80° C. for a period of time which is between 3 minutes and 120 minutes.
In another embodiment the GLP-1 compound is Arg34, Lys26(Nε-(γ-Glu(Nα-hexadecanoyl)))-GLP-1(7-37).
In another aspect the present invention relates to a method for preparation of a shelf-stable pharmaceutical composition of a GLP-1 compound, which method comprises heating a solution of said GLP-1 compound having a pH between pH 8.0 to pH 10.0 to a temperature between 50° C. and 80° C. for a period of time which is between 3 minutes and 180 minutes.
In one embodiment the present invention relates to a method for preparation of a shelf-stable pharmaceutical composition of a GLP-1 compound, which method comprises heating a solution of said GLP-1 compound having a pH between pH 8.0 to pH 10.0 to a temperature between 50° C. and 80° C. for a period of time which is between 3 minutes and 120 minutes.
In another aspect the present invention relates to a method for the treatment of hyperglycemia comprising parenteral administration of an effective amount of the pharmaceutical composition according to the invention to a mammal in need of such treatment.
In another aspect the present invention relates to a method for the treatment of obesity, beta-cell deficiency, IGT or dyslipidemia comprising parenteral administration of an effective amount of the pharmaceutical composition according to the invention to a mammal in need of such treatment.
F = f i + m i ⁢ t + f f + m f ⁢ t 1 + ⅇ - [ ( t - t 0 ) / τ ] Eq . ⁢ ( 1 )
The ThT fibrillation assay of a pharmaceutical composition of the acylated GLP-1 analogue liraglutide is shown in FIG. 1 (experimental performed along procedures described in “General procedure”). After approximately 10 hours the ThT fluorescence emission increases indicating the on-set of fibrillation. This signal increases steadily and reaches a plateau before the assay is terminated. In the presence of 200 ppm Poloxamer 188, however, the ThT fluorescence signal remains at the background level. This indicates that no fibrillation occurs and, hence, the pharmaceutical composition is physical stable under these conditions. The pharmaceutical compositions used in example 1 (FIG. 1) is not added a buffer.
The effect of Poloxamer 188 in a pharmaceutical composition of liraglutide containing sodium phosphate as a buffer is shown in FIG. 2 (experimental performed along procedures described in “General procedure”). Here, the presence of 50 ppm Poloxamer 188 prolongs the lag time before on-set of fibrillation, whereas 100 ppm Poloxamer 188 completely inhibits fibrillation during the assay time.
Polysorbate 20 does also stabilise formulations of liraglutide. One such example is shown in FIG. 3 (experimental performed along procedures described in “General procedure”). The presence of 200 ppm Polysorbate 20 attenuates the fibrillation, which is observed as a slower growth rate of the ThT fluorescence signal. Hence, a significantly smaller ThT fluorescence signal is observed in the Polysorbate 20 sample than in the reference after 40 hours of incubation.
F1. 1.2 mM liraglutide, 14 mg/ml propylene glycol, 40 mM phenol, 3 Zn/hexamer, aspart 0.6 mM, 8 mM bicine, 50 ppm poloxamer 188, pH 7.7.
F2. 1.2 mM liraglutide, 14 mg/ml propylene glycol, 40 mM phenol, 3 Zn/hexamer, aspart 0.6 mM, 8 mM bicine, pH 7.7.
Physical stability of the pharmaceutical compositions are evaluated by means of an accelerated stressed test. The stressed test is performed as a rotation test. 50 μL air is added to 5 cartridges (glass vials) of each formulation. The cartridges are rotated with a frequency of 30 rotations per minute for 4 hours daily. The test is stopped after 22 days of rotation. The inspection of the cartridges is followed daily or as required. The turbidity of the pharmaceutical compositions is characterized by nephelometric measurement of the turbidity on a HACH Turbidimeter 2100AN. The turbidity measurement of a liquid is specified in “Nephelometric Turbidity Unit” (NTU). Physical instability of the protein is characterised by high turbidity measurements.
Three pharmaceutical compositions were prepared:
F1. 1.6 mM liraglutide, 14 mg/ml propylene glycol, 40 mM phenol, 8 mM sodium phosphate, pH 7.7.
F2. 1.6 mM liraglutide, 14 mg/ml propylene glycol, 40 mM phenol, 8 mM sodium phosphate, 100 μg/ml poloxamer 188, pH 7.7.
F3. 1.6 mM liraglutide, 14 mg/ml propylene glycol, 40 mM phenol, 8 mM sodium phosphate, 200 μg/ml poloxamer 188, pH 7.7.
The pharmaceutical compositions F1-F3 were subjected to the rotation test as described in example 4. The resulting NTU measurements versus time are shown in FIG. 4.
Two pharmaceutical compositions were prepared:
F1. 1.6 mM liraglutide, 14 mg/ml propylene glycol, 40 mM phenol, 8 mM sodium phosphate, 0 μg/ml poloxamer 407 (Pluronic F-127), pH 7.7.
F2. 1.6 mM liraglutide, 14 mg/ml propylene glycol, 40 mM phenol, 8 mM sodium phosphate, 200 μg/ml poloxamer 407 (Pluronic F-127), pH 7.7.
The formulations were tested with respect to physical stability using the Thioflavin T assay. The formulations are placed in 96-well plates (Black NUNC) and incubated at 37° C. for up to 72h at the BMG FLUOstar microtiterplate fluorimeter using the following program: [300 rpm 15 min, 5 min rest]n=72. The resulting measurements are shown in FIG. 5 (lower curve being F2)
Solution 1 was prepared by dissolving preservative, isotonic agent, and buffer in water, pH was adjusted to 7.3. In another container solution 2 was prepared: liraglutide was dissolved in 60° C. hot water and kept on a water bath at 60° C. for 1, 20, and 120 minutes. The heat treatment of liraglutide was carried out in solution having pHs of about 8 and 10. After heat treatment solution 2 was cooled to 22° C. where after the two solutions were mixed and pH adjusted to 7.7 using sodium hydroxide and/or hydrochloric acid. Finally, the formulation was filtered through a 0.22 μm filter.
The physical stability of the liraglutide preparations was evaluated by the use of a florescence method; the Thioflavine T-test where the histological thiazole dye Thioflavine T (ThT) was used as an indicator of fibril formation. By the use of Thioflavine T-test it was possible to determine the presence of fibrils in the different formulations. The method was based on the fluorescent characteristics of ThT. In the presence of fibrils, the fluorescence of ThT exhibited an excitation maximum at 450 nm and enhanced emission at 482 nm. The ThT fluorescence intensity has been shown to be linear with an increase in fibril concentration.
ThT was used in a stress test applying the different formulations in microtiter plates with ThT at 35° C. and shaken with 350 rpm until the formulations were fibrillated. Graphs of the fluorescence intensity (FI) as a function of time (sec) were obtained. The response variable was; time (seconds) to achieve a fluorescence intensity of 400, e.g. the longer time to reach FI=400, the more stable a formulation.
The purity of the liraglutide preparations was measured by RP-HPLC.
Results from the experiments are depicted in FIGS. 7 and 8.
Solution 1 was prepared by dissolving preservative, isotonic agent, and buffer in water, pH was adjusted to 7.3. In another container solution 2 was prepared: liraglutide was dissolved in 80° C. hot water and kept on a water bath at 80° C. for 1, 30, and 120 minutes. The heat treatment of liraglutide was carried out in solution having pHs of about 8 and 10. After heat treatment solution 2 was cooled to 22° C. where after the two solutions were mixed and pH adjusted to 7.7 using sodium hydroxide and/or hydrochloric acid. Finally, the formulation was filtered through a 0.22 μm filter.
Physical stability and purity of the preparations were measured as described in example 7. Results from the experiments are depicted in FIGS. 9 and 10.
Solution 1 was prepared by dissolving preservative, isotonic agent, and buffer in water, pH was adjusted to 7.3. In another container solution 2 was prepared: liraglutide was dissolved in water of various temperatures: 22, 40, 60, and 80° C. and kept on a water bath for 15 minutes for all the investigated temperatures. The heat treatments of liraglutide were carried out in solution having a pH of about 10. After heat treatment solution 2 was cooled to 22° C. where after the two solutions were mixed and pH adjusted to 7.7 using sodium hydroxide and/or hydrochloric acid. Finally, the formulation was filtered through a 0.22 μm filter.
Physical stability of the preparations was measured as described in example 7.
Results from the experiments are depicted in FIG. 11.
Heat treatment during preparation (30, 60, and 120 min. at 60° C. compared to 60 min. at 22° C.) followed by formulation of drug product at pH of 8.15 is performed. Furthermore, the investigation of an additive effect of heat treatment (1 and 3 min. at 80° C.) of liraglutide drug substance prior to freeze drying followed by a second heat treatment (8 min. at 75° C.) of liraglutide during preparation is performed.
liraglutide was dissolved in water at room temperature and pH was adjusted to pH 10. The solution was heated on a water bath at 50 and 80° C. for 1, 3, 5 and 20 minutes. After heat treatment, the solution was cooled to 22° C. on a water batch. The solution was then filtered through a 0.22 μm filter and freeze dried. The powder was dissolved in a solution containing preservative, isotonic agent and buffer components and pH was adjusted to pH 7.7.
The results are depicted in FIGS. 12 and 13.
liraglutide was dissolved in water at room temperature and pH was adjusted to pH 9 and 10. The solution was heated on a water bath at 60 and 80° C. for 1 and 15 minutes. After heat treatment, the solution was cooled to 22° C. on a water batch. The solution was then filtered through a 0.22 μm filter and freeze dried. The powder was dissolved in a solution containing preservative, isotonic agent and buffer components and pH was adjusted to pH 7.7.
The results are depicted in FIG. 14.
Excipients held constant
Liraglutide 6.25 mg/ml
Propylene glycol 14.0 mg/ml
Phenol 5.50 mg/ml
Thioflavin T 1 mM
Specific excipients.
Excipients Concentration
Solutol HS-15 100 or 200 μg/ml
Pluronic F-127 (Poloxamer 407) 100 or 200 μg/ml
Di-sodium hydrogen phosphate, di-hydrate 8 mM
8×250 μl of each formulation (8 repeats) was pipetted into a 96-well plate (Black NUNC). Subsequently, the plates were sealed using “Sealing tape for plates, NUNC”.
The plate was inserted into a BMG FLUOstar microtiter plate fluorimeter. Excitation was measured at 440±10 mm and emission at 480±10 mm. Data were sampled for 72 h (approx. 260.000 sec). The BMG FLUOstar microtiter plate fluorimeter was programmed as indicated here: [600 rpm for 300 sec, rest 100 sec]n=72 using double orbital rotation.
From what can be seen in FIGS. 1 and 2, the formulations containing Solutol HS-15 in phosphate buffer are only slightly more stable than the reference formulation. The formulations containing either 100 or 200 μg/ml Pluronic F-127 in phosphate buffer are more stable. Interestingly, formulations containing either Solutol HS-15 or Pluronic F-127 in tricine buffer are exceptionally stable, especially the latter.
Solution 1 was prepared by dissolving preservative, isotonic agent, and buffer in water, pH was adjusted to 7.9. In another container solution 2 was prepared: liraglutide was dissolved in 60-70° C. hot water and kept on a water bath at 50, 60, and 70° C. for 60, 90, and 20 minutes. The heat treatment of liraglutide was carried out in solution having pHs of about 8 and 10. After heat treatment solution 2 was cooled to 22° C. where after the two solutions were mixed and pH adjusted to 8.15 using sodium hydroxide and/or hydrochloric acid. Finally, the formulation was filtered through a 0.22 μm filter.
ThT was used in a stress test applying the different formulations in microtiter plates with ThT at 35° C. and shaken with 350 rpm until the formulations were fibrillated. Graphs of the fluorescence intensity (FI) as a function of time (sec) were obtained. The response variable was; time (sec) to achieve a fluorescence intensity of 400, e.g. the longer time to reach FI=400, the more stable a formulation.
The results are depicted in FIG. 17.
Solution 1 was prepared by dissolving preservative, isotonic agent, and buffer in water, pH was adjusted to 7.9. In another container solution 2 was prepared: liraglutide was dissolved in 60-70° C. hot water and kept on a water bath at 60, 65, and 70° C. for 30, 45, 150, and 180 minutes. The heat treatment of liraglutide was carried out in solution having pHs of about 8 and 10. After heat treatment solution 2 was cooled to 22° C. where after the two solutions were mixed and pH adjusted to 8.15 using sodium hydroxide and/or hydrochloric acid. Finally, the formulation was filtered through a 0.22 μm filter.
Heat treatment of liraglutide drug product in Penfill®.
Penfill ® containing fibrillated liraglutide
were heat treated for 30 min at 85° C.
Penfill before heat Penfill after heat
treatment (NTU) treatment (NTU)
Approx. 50 0.382
(average of 10 0.182
penfill containing 0.275
fibrillated 0.174
liraglutide DP) 0.284
Freshly produced liraglutide drug product has a turbidity of approx. 0.2-1.0 NTU. Thus, heat treatment of highly fibrillated liraglutide drug product can dissolve the otherwise very stable fibril structures.
FIG. 18 shows Penfill® heat treated at different times and temperatures which were subsequently subjected to rotation.
1. A method for the preparation of a shelf-stable pharmaceutical composition of a GLP-1 compound comprising: dissolving said GLP-1 compound in aqueous medium, adjusting the pH of the solution to between about 8.0 and about 10.5, and heating the solution to a temperature between about 50° C. and about 95° C. for a period of time from about 3 minutes to about 180 minutes to result in a shelf-stable pharmaceutical solution.
2. The method according to claim 1, wherein the temperature is between 60° C. and 95° C.
3. The method according to claim 1, wherein the temperature is between 50° C. and 80° C.
4. The method according to claim 1, wherein the temperature is between 70° C. and 80° C.
5. The method according to claim 1, wherein the temperature is between 60° C. and 80° C.
6. The method according to claim 1, wherein the pH is between about 8.0 to 10.0.
7. The method according to claim 1, wherein the pH is about 8.15.
8. The method according to claim 1 wherein the heating is continued for a period of time which is between 15 minutes and 120 minutes.
9. The method according to claim 1 wherein the heating is continued for a period of time which is between 10 minutes and 90 minutes.
10. The method according to claim 1 wherein the heating is continued for a period of time which is between 3 minutes and 30 minutes.
11. The method according to claim 1 wherein the heating is continued for a period of time which is between 5 minutes and 15 minutes.
12. The method according to claim 1, further comprising freeze drying the solution after the heating step; wherein the resulting freeze-dried composition is shelf-stable.
13. The method according to claim 1, further comprising the addition of a pharmaceutically acceptable excipient after the heating step; wherein the resulting pharmaceutical composition is shelf-stable.
14. The method according to claim 1, wherein said GLP-1 compound is Arg34, Lys26(Nε-(γ-Glu(Nα-hexadecanoyl)))-GLP-1(7-37).
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EP2498801B1 (en) 2018-01-24 PHARMACEUTICAL COMPOSITION COMPRISING desPro36Exendin-4(1-39)-Lys6-NH2 AND METHIONINE