JP7371208B2 - von Willebrand factor-containing preparations - Google Patents

Description

Related Applications This application claims the priority benefit of Application No. 2022-006389 filed with the Japan Patent Office on January 19, 2020. The priority application is hereby incorporated by reference in its entirety.

The present invention relates to a pharmaceutical preparation of von Willebrand factor (VWF) or a complex of blood coagulation factor VIII (FVIII) and von Willebrand factor (VWF), which suppresses the generation of floating substances after dissolution. Concerning composition.

Von Willebrand factor (VWF) is a plasma protein produced by vascular endothelial cells and bone marrow megakaryocytes. VWF is involved in the adhesion and aggregation of platelets to bleeding sites, and binds to blood coagulation factor VIII (FVIII) in circulating plasma, functioning as its carrier protein. Von Willebrand disease (VWD) is a disease in which a bleeding tendency occurs due to congenital quantitative reduction, deficiency, or qualitative abnormality of VWF. The clinical symptoms of VWD are characterized by bleeding symptoms in the skin and mucous membranes, and are often noticed as nosebleeds from childhood or difficulty in stopping bleeding during tooth extraction, and women with VWD are prone to excessive menstruation. Unlike hemophilia, VWD rarely causes intra-articular or intramuscular bleeding. A definitive diagnosis of VWD is made based on VWF antigen amount (VWF:Ag), ristocetin cofactor activity (VWF:RCo), as well as FVIII activity, ristocetin agglutination ability, and multimer, and is classified into Types 1, 2, and 3. Type 2 is further divided into subclasses A, B, M, and N. For hemostatic therapy of VWD, VWF preparations and VWF preparations containing FVIII (FVIII/VWF preparations) are used as VWF replacement therapy (Non-Patent Document 1).

Since VWF is a carrier protein for FVIII, VWF functions as a stabilizer for FVIII, and in hemophilia A treatment where FVIII is deficient or reduced, FVIII/VWF formulations can reduce the half-life of the administered FVIII formulation. It is believed that it may have a number of advantages, including prolongation and protective effects against anti-FVIII antibodies.

Human plasma-derived VWF preparations and FVIII/VWF preparations currently sold overseas are summarized in Table 2 of Non-Patent Document 2. FVIII/VWF preparations include Alphanate (Grifols), Factor BY (Bio Products Laboratory), Fanhdi (Grifols), Haemate P (CSL Behring), Immunate (Baxter), and Wilfate (Octapharma). Wilfactin (LFB) is a VWF preparation, which is a single VWF preparation with the FVIII content reduced as much as possible. On the other hand, as a formulation other than Non-Patent Document 2, the FVIII/VWF formulation Voncento (CSL Behring) is also sold in Europe (Non-Patent Document 3), and in Japan, Confact F (KM Biologics Co., Ltd.: Non-Patent Document 3) is also sold in Europe. Patent Document 4) and Conchoate-HT (Japan Blood Products Organization) are commercially available (Non-Patent Document 5). Furthermore, as a recombinant VWF preparation, Bonbendy 1300 for intravenous injection (Takeda Pharmaceutical Company Limited: Non-Patent Document 6) is on the market. This is a lyophilized preparation in which VWF is expressed and purified using Chinese hamster ovary (CHO) cell line, and the concentration of VWF is 130 IU/mL. Both formulations are lyophilized to enhance long-term storage stability.

Information on the above VWF formulation and FVIII/VWF formulation is summarized in Table 1.
#: Quoted from non-patent document 2

As described in Table 2 of Non-Patent Document 2, in FVIII/VWF preparations, the ratio of VWF:RCo to FVIII activity (VWF:RCo/FVIII) is indicated (non-patented VWF single preparation Wilfactin). The values in Table 2 of Reference 2 are academic reference values). Currently, among FVIII/VWF formulations, the formulations with the highest VWF:RCo/FVIII ratios in the world are Haemate P and Voncento (CSL Behring) and Confact F (KM Biologics Co., Ltd.).

When focusing on the concentration of VWF derived from human plasma, the concentration of VWF in Voncento (CSL Behring) is 240 IU/mL, which is the highest concentration in the world. However, although the types of additives in Voncento have been disclosed, the concentrations of those additives have not been made clear (Non-Patent Document 7).
In addition, Bonbendy Intravenous Injection 1300 of Non-Patent Document 7, which is a recombinant VWF (rVWF) preparation, has a VWF concentration of 130 IU/mL, and contains 1 mg/mL glycine, 20 mg/mL mannitol, and It is stated that it contains 10 mg/mL trehalose hydrate and 0.1 mg/mL polysorbate 80.

When a lyophilized preparation containing VWF is dissolved with a solvent, proteinaceous aggregates may be generated and observed as floating substances (hereinafter referred to as VWF floating substances). This is described in the package inserts of freeze-dried preparations containing VWF as precautions during dissolution, and is described as "floating matter", "particles", etc. (Non-patent Documents 4, 6). The amount of VWF suspended matter increases as the VWF concentration at the time of dissolution increases. It is desirable to reduce VWF floaters as much as possible because the generation of VWF floaters causes problems such as, for example, causing a substantial decrease in VWF concentration, clogging of the injection needle, or undesirable appearance.

Patent Document 1 discloses that in rVWF preparations with VWF concentrations of 70 to 130 IU/mL, additives for lyophilized preparations were screened using the degree of rVWF aggregation as an index, and additives such as 15 mM Na citrate, 15 mM glycine, and 10 mg It is described that a formulation composition containing trehalose/mL, 20 mg/mL mannitol, and 0.1 mg/mL polysorbate 80 was found.

The activity of VWF in the present invention is based on the role of VWF as a cofactor for the glycopeptide antibiotic ristocetin (VWF:RCo) in its ability to induce platelet aggregation (Non-Patent Documents 8, 9). This activity is expressed in international units (IU VWF: RCo) and its concentration in IU/milliliter (IU VWF: RCo/mL).

The activity of FVIII in the present invention relates to the coagulation activity of FVIII (FVIII:C) based on the role of FVIII as a cofactor in the activation of FX in the presence of FIXa, calcium ions and phospholipids (non-patent References 10, 11). It is quantified for the chromogenic substrate and expressed in international units (IU FVIII), and its concentration is expressed in IU FVIII/ml (IU FVIII/mL).

Patent application 2011-532347

Journal of Thrombosis and Hemostasis 20 (1): 3-5, 2009.WEB site: https://www.jsth.org/publications/pdf/tokusyu/20_1.003.2009.pdf (confirmed December 15, 2021) Ther Clin Risk Manag. 2016 Jun 30;12:1029-37. Website: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4936816/ (confirmed December 15, 2021) CSL Behring website: https://labeling.csl.com/SDS/CORE/Voncento/EN/Voncento-Safety-Data-Sheet.pdf (verified December 15, 2021) KM Biologics website: https://www.kmbiologics.com/medical/plasma/pdf/confact_pi_2109_01.pdf (confirmed December 15, 2021) Japan Blood Products Organization website: https://www.jbpo.or.jp/med/di/file/cae4b0ab30064f33990edc885e401bbe/ (confirmed on December 15, 2021) Takeda Pharmaceutical Co., Ltd. website: https://www.takedamed.com/mcm/medicine/download.jsp?id=1234&type=ATTACHMENT_DOCUMENT (confirmed on December 15, 2021) European Medicines Agency website: https://www.ema.europa.eu/en/documents/product-information/voncento-epar-product-information_en.pdf (confirmed December 15, 2021) European Pharmacopoeia No. 07/2006:20721 Clinical guidelines for von Willebrand disease 2021 edition Journal of Thrombosis and Hemostasis 32 (4): 413-481, 2021.Website: https://www.jsth.org/wordpress/wp-content/uploads/2015/04/von-Willebrand %E7%97%85%E3%81%AE%E8%A8%BA%E7%99%82%E3%82%AC%E3%82%A4%E3%83%89%E3%83%A9%E3 %82%A4%E3%83%B32021%E5%B9%B4%E7%89%88.pdf (confirmed December 28, 2021) European Pharmacopoeia No. 07/2006:20704 Biological product standards (Ministry of Health, Labor and Welfare Notification No. 410: December 23, 2021) Dry concentrated human blood coagulation factor VIII p.228 Website: https://www.niid.go.jp/niid /images/qa/seibutuki/seibutsuki_japanese/20211223.pdf (confirmed December 28, 2021) Vox Sang. 2008 Nov;95(4):298-307. Website: https://pubmed.ncbi.nlm.nih.gov/19138259/ (confirmed December 15, 2021) J Lab Clin Med. 1981 Jun;97(6):864-80. Website: https://pubmed.ncbi.nlm.nih.gov/6971912/ (confirmed December 15, 2021) J Chromatogr B Biomed Appl. 1994 Dec 9;662(2):181-90. Website: https://pubmed.ncbi.nlm.nih.gov/7719474/ (confirmed December 15, 2021) Ann Clin Lab Sci. Mar-Apr 1989;19(2):84-91. Website: http://www.annclinlabsci.org/content/19/2/84.full.pdf (December 15, 2021 confirmation) Haemophilia. 2009 May;15(3):788-96. Website: https://onlinelibrary.wiley.com/doi/10.1111/j.1365-2516.2009.01995.x (verified December 15, 2021)

When a preparation containing VWF is dissolved with a solvent, proteinaceous aggregates may be generated and observed as floating substances (hereinafter referred to as VWF floating substances). It is desirable to reduce VWF floaters as much as possible because the generation of VWF floaters causes problems such as, for example, causing a substantial decrease in VWF concentration, clogging of the injection needle, or undesirable appearance.
It is an object of the present invention to provide a pharmaceutical formulation in which VWF or a complex of FVIII and VWF is stable.

In order to solve the above problems, the present inventors have found a composition in which VWF or a complex of FVIII and VWF is stable after dissolution of a pharmaceutical preparation containing VWF, and have completed the present invention.

Accordingly, the invention includes;
[Section 1]
A pharmaceutical formulation comprising von Willebrand factor (VWF) or a complex of blood coagulation factor VIII (FVIII) and VWF, the pharmaceutical formulation comprising polysorbates and/or macrogols, wherein VWF or FVIII is present in the pharmaceutical formulation. and VWF, wherein the complex is stable,
[Section 2]
The formulation according to [Item 1], containing polysorbates and macrogols,
[Section 3]
The formulation according to [Item 1], wherein the concentration of polysorbates is 0.15 mg/mL or more,
[Section 4]
The formulation according to [Item 1], wherein the polysorbate is polysorbate 80,
[Section 5]
The formulation according to [Item 1], wherein the concentration of macrogol is 0.25 mg/mL or more,
[Section 6]
The formulation according to [Item 1], wherein the macrogol is macrogol 4000,
[Section 7]
The formulation according to [Item 1], wherein the concentration of VWF is 120 IU/mL or more,
[Section 8]
The formulation according to [Item 1], further comprising human serum albumin, glycine, sodium chloride, sodium citrate hydrate, or L-histidine,
[Section 9]
The formulation according to [Item 8], which has a pH in the range of about 6.5 to about 8.0,
[Section 10]
The formulation according to [Item 8], wherein the pharmaceutical formulation is a lyophilized formulation,
[Section 11]
A method for stabilizing VWF or a complex of FVIII and VWF by containing polysorbates and/or macrogols at a predetermined concentration in a pharmaceutical preparation containing VWF or a complex of FVIII and VWF,
[Section 12]
A method for producing a pharmaceutical preparation containing VWF or a complex of FVIII and VWF, the method comprising a step of containing polysorbates and/or macrogols at a predetermined concentration. Method of manufacturing the formulation.

Additionally, the invention includes;
[Section 1]
A pharmaceutical formulation comprising von Willebrand factor (VWF) or a complex of blood coagulation factor VIII (FVIII) and VWF, the pharmaceutical formulation comprising polysorbates and/or macrogols, wherein VWF or FVIII is present in the pharmaceutical formulation. and VWF, wherein the complex is stable,
[Section 2]
The formulation according to [Item 1], containing polysorbates and macrogols,
[Section 3]
The formulation according to [Item 1], in which the concentration of polysorbates exceeds 0.1 mg/mL,
[Section 4]
The formulation according to [Item 1], in which the concentration of polysorbates exceeds 0.2 mg/mL,
[Section 5]
The formulation according to [Item 1], wherein the polysorbate is polysorbate 80,
[Section 6]
The formulation according to [Section 1], in which the concentration of macrogol exceeds 0.35 mg/mL,
[Section 7]
The formulation according to [Section 1], in which the concentration of macrogol exceeds 0.5 mg/mL,
[Section 8]
The formulation according to [Item 1], wherein the macrogol is macrogol 4000,
[Section 9]
The formulation according to [Item 1], wherein the concentration of VWF is more than 60 IU/mL,
[Section 10]
The formulation according to [Item 1], wherein the concentration of VWF is more than 240 IU/mL,
[Section 11]
The preparation according to [Item 1], wherein the concentration of VWF is more than 500 IU/mL,
[Section 12]
The formulation according to [Item 1], further comprising human serum albumin, glycine, sodium chloride, sodium citrate hydrate, or L-histidine,
[Section 13]
The formulation according to [Item 12], which has a pH in the range of about 6.5 to about 8.0,
[Section 14]
The formulation according to [Item 12], wherein the pharmaceutical formulation is a lyophilized formulation,
[Section 15]
A method for stabilizing VWF or a complex of FVIII and VWF by containing polysorbates and/or macrogols at a predetermined concentration in a pharmaceutical preparation containing VWF or a complex of FVIII and VWF,
[Section 16]
A method for producing a pharmaceutical preparation containing VWF or a complex of FVIII and VWF, the method comprising a step of containing polysorbates and/or macrogols at a predetermined concentration. Method of manufacturing the formulation.

According to the present invention, in a pharmaceutical formulation containing VWF or a complex of FVIII and VWF, the generation of floating VWF in the pharmaceutical formulation can be suppressed because the VWF or the complex of FVIII and VWF is "stable". Pharmaceutical formulations containing VWF that can be stored for long periods of time can be provided, or pharmaceutical formulations containing high concentrations of VWF can be provided.

FIG. 1 is a graph showing float scores and dissolution times of formulations containing different concentrations of P80 or PEG. FIG. 2 is a graph showing float scores for formulations containing combinations of P80 and/or PEG at different concentrations.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

The present invention provides a pharmaceutical formulation containing VWF that suppresses the generation of VWF floating substances.

In one embodiment, the present invention provides a pharmaceutical formulation comprising von Willebrand factor (VWF) or a complex of blood coagulation factor VIII (FVIII) and VWF, wherein said pharmaceutical formulation contains polysorbates and/or macrogols. and in which the VWF or the complex of FVIII and VWF is stable. In another aspect, the present invention provides for stabilizing VWF or the complex of FVIII and VWF by including a predetermined concentration of polysorbates and/or macrogols in a pharmaceutical formulation containing VWF or a complex of FVIII and VWF. Concerning how to make this happen. In yet another aspect, the present invention provides a method for producing a pharmaceutical preparation comprising VWF or a complex of FVIII and VWF, the method comprising a step of containing polysorbates and/or macrogols at a predetermined concentration. Alternatively, the present invention relates to a method for producing a pharmaceutical preparation containing a complex of FVIII and VWF.

VWF is produced as a 2,813-residue amino acid, and after the 22-residue signal peptide is cleaved to become proVWF, the 741-residue large propeptide is removed and matured with 2,050 amino acids. Becomes VWF. VWF is a glycoprotein with a size of about 250 kD and circulates in plasma as a multimer of about 500 to about 20,000 kD. In the present disclosure, the VWF may form a monomer or have an average size of about 500, 750, 1000, 1250, 1500, 1750, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000. , 10,000, 12,500, 15,000, 17,500, 20,000 kDa or any number therebetween. The multimeric form of VWF is composed of approximately 250 kD polypeptide subunits linked by disulfide bonds. VWF can be prepared by ion exchange chromatography or immunoaffinity chromatography (Non-patent Documents 12, 13). VWF may be purified from blood or recovered as a recombinant protein.

FVIII is a single-chain glycoprotein consisting of 2332 amino acids and has a domain structure of A1-A2-B-A3-C1-C2. In plasma, it exists as a heterodimer of a heavy chain consisting of A1-A2-B domains and a light chain consisting of A3-C1-C2 domains. FVIII can be prepared by ion exchange chromatography or immunoaffinity chromatography (Non-patent Documents 12, 14, 15). FVIII may be purified from blood or recovered as a recombinant protein.

FVIII non-covalently binds to VWF to form a complex. The complex can be detected by a method using ELISA (Non-Patent Document 16).

As used herein, "VWF suspended matter" refers to a visually visible suspended matter in a solution containing a preparation containing VWF. The visible size is not particularly limited, but when measuring the longest diameter of floating objects, it is approximately 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8mm, 0.9mm, 1.0mm, 1.1mm, 1.2mm, 1.3mm, 1.4mm, 1.5mm, 1.6mm, 1.7mm, 1.8mm, 1.9mm, 2. 0 mm, 3.0 mm, 4.0 mm, 5.0 mm, 6.0 mm, 7.0 mm, 8.0 mm, 9.0 mm, 10.0 mm or more, or any number in between. A size factor can be assigned based on the size of the floating object; for example, if the size of the floating object is less than 1 mm, it is 5, if it is 1 mm or more and less than 2 mm, it is 10, and if it is 2 mm or more and less than 5 mm, it is 20. , it can be set as 40 if it is 5 mm or more and less than 10 mm, and 80 if it is 10 mm or more. The degree of VWF floating matter in a formulation solution containing VWF may be calculated as a total score of floating matter by multiplying the number of floating matter by a size coefficient. A "VWF suspension" is a proteinaceous aggregate and may contain VWF or a complex of FVIII and VWF.

In this specification, unless otherwise specified, when referring to "concentration", particularly "concentration" of an ingredient in a pharmaceutical formulation, it refers to the final concentration when the pharmaceutical formulation is administered to a patient. For example, if a composition contains 10 mg of a particular component and is administered by dissolving the composition in 10 mL of a solvent, the concentration of that component in the composition is 1 mg/mL.

As used herein, the term "stable" for VWF or the complex of FVIII and VWF means that the complex of VWF or FVIII and VWF does not precipitate or form floating substances in solution. Since VWF or the complex of FVIII and VWF is "stable", it is possible to suppress the occurrence of floating VWF in pharmaceutical formulations, provide a pharmaceutical formulation containing VWF that can be stored for a long time, or containing a high concentration of VWF. Pharmaceutical formulations can be provided. In certain embodiments, when VWF or a complex of FVIII and VWF is "stable," the number, size, or The total score, or total float score, is lower for pharmaceutical formulations of the compositions of the present disclosure. In one embodiment, if the VWF or the complex of FVIII and VWF is "stable", no VWF floatation of the pharmaceutical formulation is visible. In another embodiment, when the VWF or the complex of FVIII and VWF is "stable", for example, the size of the VWF floaters is about 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0. 5mm, 0.6mm, 0.7mm, 0.8mm, 0.9mm, 1.0mm, 1.1mm, 1.2mm, 1.3mm, 1.4mm, 1.5mm, 1.6mm, 1.7mm, 1.8mm, 1.9mm, 2.0mm, 3.0mm, 4.0mm, 5.0mm, 6.0mm, 7.0mm, 8.0mm, 9.0mm, 10.0mm or less or any number in between It is. In yet another embodiment, when the VWF or FVIII and VWF complex is "stable", for example, the number of VWF floaters is 1, 2, 3, 4, 5 or less. In other embodiments, when the VWF or the complex of FVIII and VWF is "stable", for example, the floater total score is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, less than 10, 15, 20, 25, 30, 35, 40, 45, 50. In yet other embodiments, if the VWF or FVIII and VWF complex is "stable," the composition of the present disclosure may be The total float score of a pharmaceutical formulation of, for example, 90%, 85%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20% of a pharmaceutical formulation that differs from the composition of the present disclosure. %, less than 10%.

In one embodiment, the pharmaceutical formulation has a concentration of VWF of 60 IU/mL, 120 IU/mL, 180 IU/mL, 240 IU/mL, 300 IU/mL, 360 IU/mL, 420 IU/mL, 480 IU/mL, 500 IU/mL, 540 IU /mL, 600IU/mL, 700IU/mL, 800IU/mL, 900IU/mL, 1000IU/mL or more. In another embodiment, the pharmaceutical formulation has a concentration of VWF of 60 IU/mL, 120 IU/mL, 180 IU/mL, 240 IU/mL, 300 IU/mL, 360 IU/mL, 420 IU/mL, 480 IU/mL, 500 IU/mL, More than 540 IU/mL, 600 IU/mL, 700 IU/mL, 800 IU/mL, 900 IU/mL, 1000 IU/mL. In one embodiment, the pharmaceutical formulation has a concentration of FVIII greater than or equal to 25 IU/mL, 50 IU/mL, 75 IU/mL, 100 IU/mL, 125 IU/mL, 150 IU/mL. In another embodiment, the pharmaceutical formulation has a concentration of FVIII greater than 25 IU/mL, 50 IU/mL, 75 IU/mL, 100 IU/mL, 125 IU/mL, 150 IU/mL.

In one embodiment, the pharmaceutical formulation may include a surfactant and/or polyethylene glycol. The surfactant is, for example, an ionic surfactant or a nonionic surfactant, preferably a nonionic surfactant. Nonionic surfactants are, for example, polysorbates. Polysorbates include polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, and polysorbate 80. The polyethylene glycol is preferably for medical use, such as macrogols. The macro goals include macro goal 200, macro goal 400, macro goal 1500, macro goal 4000, macro goal 6000, and macro goal 20000.

In one embodiment, the concentration of surfactant is 0.1 mg/mL or more, 0.15 mg/mL or more, 0.20 mg/mL or more, 0.25 mg/mL or more, 0.30 mg/mL or more, 0.35 mg /mL or more, 0.40 mg/mL or more, 0.45 mg/mL or more, or 0.50 mg/mL or more. In another embodiment, the concentration of surfactant is greater than 0.1 mg/mL, greater than 0.2 mg/mL, greater than 0.3 mg/mL, greater than 0.4 mg/mL, or 0.5 mg/mL. More than mL. In other embodiments, the concentration of polyethylene glycol is 0.1 mg/mL or more, 0.15 mg/mL or more, 0.20 mg/mL or more, 0.25 mg/mL or more, 0.30 mg/mL or more, 0.35 mg /mL or more, 0.40mg/mL or more, 0.45mg/mL or more, 0.50mg/mL or more, 0.55mg/mL or more, 0.60mg/mL or more, 0.65mg/mL or more, or 0.70mg /mL or more. In another embodiment, the concentration of polyethylene glycol is greater than 0.1 mg/mL, greater than 0.15 mg/mL, greater than 0.20 mg/mL, greater than 0.25 mg/mL, greater than 0.30 mg/mL. exceeds, exceeds 0.35 mg/mL, exceeds 0.40 mg/mL, exceeds 0.45 mg/mL, exceeds 0.50 mg/mL, exceeds 0.55 mg/mL, exceeds 0.60 mg/mL, More than 0.65 mg/mL, or more than 0.70 mg/mL.

In one embodiment, the pharmaceutical formulation may include excipients. Additives include, but are not limited to, albumin, amino acids, salts, surfactants, polymer compounds, or pH regulators. The albumin is preferably serum albumin, more preferably human serum albumin. Amino acids are, for example, glycine or L-histidine. Salts are inorganic or organic salts. Inorganic salts are, for example, sodium chloride, potassium chloride, sodium chloride hydrate, and potassium chloride hydrate. Surfactants are, for example, polysorbates. The polymer compound is, for example, polyethylene glycol. Examples of pH regulators include hydrochloric acid and sodium hydroxide. The pharmaceutical formulation is adjusted to a pH of 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 or any number between them. The pharmaceutical formulation preferably has a pH in the range of about 6.0 to about 8.5, more preferably in the range of about 6.5 to about 8.0. Those skilled in the art can appropriately select the above-mentioned stabilizers and set their concentrations.

In one embodiment, the pharmaceutical formulation is, but is not limited to, a biological product, preferably a blood product. Blood products are classified into "whole blood products," "blood component products," and "plasma fraction products," and those skilled in the art can obtain the products by methods known to those skilled in the art.

In another embodiment, the pharmaceutical formulation is a lyophilized formulation to which the physician or patient will add solvents and/or diluents before use.

The pharmaceutical formulations herein may be used to treat diseases caused by a deficiency or reduction in VWF or FVIII, such as von Willebrand disease and hemophilia A.

As used herein, "about" means a range of ±10%, preferably ±5% of the indicated numerical value.

EXAMPLES Hereinafter, the present invention will be explained in detail with reference to Examples, but the present invention is not limited to these Examples in any way.

Setting the floating object score and calculating the total floating object score:
It is an object of the present invention to provide a pharmaceutical formulation containing stable VWF or a complex of FVIII and VWF. To this end, we considered it essential to establish a floating matter score evaluation method. Therefore, in order to more accurately evaluate the VWF suspended matter after dissolution, a coefficient was set for each size of the VWF suspended matter. After dissolving the VWF preparation, the formulation solution was visually observed, and the sum of the numbers of each floating substance multiplied by the size coefficient was calculated as the total score of floating substances.

Preparation of FVIII/VWF original drawing:
(1) Separation of cryoprecipitate
Raw plasma (fresh frozen plasma (obtained from the Japanese Red Cross Society)) stored at -20°C or lower was chilled and thawed at 6°C or lower. Centrifugation was performed at approximately 8,300 x g (cryocentrifugation) to obtain cryoprecipitate.

(2) Purification of cryoprecipitate The cryoprecipitate obtained in (1) above was mixed with 3.5 times the amount of buffer A (NaCl: 10.55 g/L, glycine: 15 g/L, calcium chloride dihydrate). :0.088 g/L, pH 7.3) (NaCl, glycine, and calcium chloride hydrate were obtained from Tomita Pharmaceutical Co., Ajinomoto Co., and Tomita Pharmaceutical Co., respectively), and 5 times more than cryoprecipitate. I increased it to the amount. Aluminum hydroxide gel (Croda's "Alhydrogel" was obtained) was added at a concentration of 0.3% and centrifuged at about 12,000 x g to obtain a supernatant. This was developed on a cation exchange chromatography column (SP Toyopearl 550C manufactured by Tosoh Corporation, functional group: sulfopropyl group).
Afterwards, the column was washed with buffer A containing 1w/v% polysorbate 80, and buffer B (NaCl: 35 g/L, glycine: 15 g/L, calcium chloride dihydrate: 0.088 g/L, FVIII/VWF was eluted with trisaminomethane (2.4 g/L, pH 7.5) (trisaminomethane was obtained from Fujifilm Wako Pure Chemical's "2-amino-2-hydroxymethyl-1,3-propanediol"). did.). The obtained elution fraction was filtered through a membrane to remove viruses, and then concentrated to obtain an original FVIII/VWF image.

Confirmation of total float score using Confact F composition:
Confact F is a FVIII/VWF formulation manufactured and sold in Japan by KM Biologics Co., Ltd., and is obtained by adding additives to the above FVIII/VWF original image, freeze-drying it, and subjecting it to dry heat treatment at 65°C for 96 hours. It is a formulation. When this drug is dissolved in the attached solvent (Japanese Pharmacopoeia Water for Injection), the composition in one vial is as shown in the table below. (VWF concentration is 60 IU/mL).

Regarding the current Confact F, the total score of suspended matter when the lyophilized preparation was dissolved in water for injection was confirmed (Table 4). When dissolved to a VWF concentration of approximately 60 IU/mL, all floaters generated were less than 5 mm, and the total float score ranged from 36.7 to 55.6.
Furthermore, when 1000 unit preparations were dissolved to a VWF concentration of 485.6 IU/mL, large floating particles of about 2 cm were generated, and the floating particle score was 83.3. Floating particles larger than 1 cm have an unfavorable appearance and are difficult to provide as pharmaceuticals. Therefore, it has been found that it is necessary to investigate a composition that further reduces suspended matter in FVIII/VWF formulations.


*1: Shows the average value ±SD when data was obtained from 3 lots x 3 times (9 times in total).
*2: Shows the average value ±SD when data was obtained for 1 lot x 3 times (3 times in total).

Screening study for high concentration composition based on Confact F composition:
Among the components of Confact F, sodium citrate hydrate (Na citrate) (we obtained Fujifilm Wako Pure Chemical Industries, Ltd.'s ``Sodium Citrate Hydrate ``Manufacturing Only''''), L-Histidine (L- His) (obtained from Merck & Co.), macrogol 4000 (PEG) (obtained from Sanyo Chemical Co., Ltd.), and polysorbate 80 (P80) (obtained from NOF Corporation "Polysorbate 80 (SS)"). We investigated the effectiveness of the ingredients in suppressing suspended matter after dissolution. For Confact F shown in Table 3, a study was conducted assuming a formulation with twice the FVIII and VWF concentrations (50 IU/mL and 120 IU/mL, respectively).
Each component was used as a factor, and three levels were set for each concentration as shown in Table 5, and assigned using an L9 orthogonal array. The composition of No. 5 was set to be the same composition and concentration as the current composition of Confact F, except that the FVIII and VWF concentrations were twice as high.
A 250-unit lyophilized preparation was prepared with these 9 compositions, and the sample was dissolved so that the VWF concentration was approximately 120 IU/mL (the solution concentration before lyophilization and the concentration after dissolution were the same). The total floating matter score was calculated by the method of Example 1 (n=6: mean value ± SD).
As a result, compositions Nos. 4 and 8 had particularly few suspended substances.

*Concentrations in the table are expressed in mg/mL. The concentrations of each component are shown when dissolved to a VWF concentration of approximately 120 IU/mL.

Using the measurement results of prepared samples No. 1 to No. 9, a significant difference test (α = 0.05) between levels was performed by one-way analysis of variance for each factor.
As a result, only P80 of each factor showed a significant difference between levels, and the suspended matter score decreased in a P80 concentration-dependent manner. Although no significant differences were observed for any of the other factors, there was a tendency for the suspended matter score to decrease in a somewhat concentration-dependent manner for PEG.
Furthermore, regarding the time required for dissolution (dissolution time), a significant difference between levels was observed only for P80 among each factor, and the dissolution time was shortened depending on the concentration of P80. Although no significant difference was observed with PEG, there was a tendency for the dissolution time to shorten in a concentration-dependent manner. From these results, it was found that P80 has the effect of improving solubility. (Figure 1)

Confirmation of total floating matter score using highly concentrated composition - 1:
Among the compositions of Example 4, three lots of freeze-dried preparations were prepared using composition No. 4 (hereinafter referred to as highly concentrated composition), which contained fewer suspended substances, in the same manner as in Example 4 but using different raw materials.

When these preparations were dissolved in 5, 10, and 20 mL of water for injection, respectively, so that the VWF concentration was about 120 IU/mL, the total score of suspended matter was confirmed. When dissolved to a VWF concentration of approximately 120 IU/mL, no floaters were generated and the total floater score was 0.


*1: For each standard, the average value ± SD is shown when data was obtained from 3 lots x 3 times (9 times in total).

Confirmation of total floating matter score using highly concentrated composition - 2:
Regarding the formulation composition prepared in Example 5, the total score of suspended matter was confirmed when one representative lot of 1000 units of formulation was dissolved in water for injection so that the VWF concentration was 500.8 IU/mL. As a result, the total score of floating particles was 1.7±2.9, and the size of all floating particles was 1 mm or less. This is because the total score of suspended matter is lower than that of Confact F (VWF concentration: 60 IU/mL), which is currently provided as a pharmaceutical product, as shown in Example 3, so the VWF concentration was set to 500 IU/mL or higher. It was found that even if dissolved, the generation of suspended matter was suppressed.

*1: Indicates the average value ±SD when data was obtained for 1 lot x 3 times (3 times in total).

Long-term storage stability of highly concentrated formulations:
Regarding the formulation prepared in Example 5, the total score of floating matter after long-term storage under the conditions shown in the table below was confirmed.

After storage under these conditions, one representative lot of the 1000-unit preparation was dissolved in 5 mL of water for injection to give a VWF concentration of 520.4 IU/mL, and the total score of suspended matter was confirmed. As a result, the total score of floating particles was 5.0±5.0, and the size of all floating particles was 1 mm or less. This had a lower total floating matter score than Confact F (VWF concentration: 60 IU/mL), which is currently provided as a pharmaceutical product, as shown in Example 3. Therefore, it was found that even after storage at 10° C. for 36 months, the formulation prepared with the composition of Example 5 suppressed the generation of suspended matter when dissolved at a VWF concentration of 500 IU/mL or higher.

*1: Indicates the measured value when data was obtained for 1 lot x 3 times (3 times in total).
*2: Indicates the measured value when data was obtained for 1 lot x 1 time (total 1 time).

Confirmation of the effects of macrogol 4000 and polysorbate 80:
From Example 4, it was considered that the addition of macrogol 4000 (PEG) and polysorbate 80 (P80) was effective in suppressing the generation of suspended matter, so the mutual influence of these components was evaluated. Therefore, freeze-dried preparations having nine types of compositions No. 1 to No. 9 shown in Tables 11 and 12 were prepared in the same manner as in Example 4. In addition, in order to confirm differences between batches of FVIII/VWF original drawings, original drawing materials that tend to generate floating substances were used.
For these nine types of lyophilized preparations, the suspended matter was collected using the method of Example 1 for samples dissolved so that the VWF concentration was approximately 120 IU/mL (the solution concentration before lyophilization and the concentration after dissolution were the same). A score was calculated (n=3: average value).
The results are shown in Table 12 and Figure 2. When the concentration of PEG and P80 was higher than 0.25 mg/mL and 0.15 mg/mL, respectively, the floating matter score became smaller, and it was confirmed that both contributed to the suppression of floating matter generation.

INDUSTRIAL APPLICABILITY The present invention can be used as a method for producing a stable lyophilized preparation containing VWF in which the generation of floating substances is suppressed.

Claims (11)

A pharmaceutical formulation comprising von Willebrand factor (VWF) or a complex of blood coagulation factor VIII (FVIII) and VWF, wherein said pharmaceutical formulation comprises polysorbates and macrogols, and wherein said pharmaceutical formulation is in a solvent . A pharmaceutical preparation containing the polysorbate and the macrogol dissolved in a solvent such that the polysorbate and the macrogol are 0.15 mg/mL or more and 0.25 mg/mL or more when dissolved. A pharmaceutical preparation in which VWF or a complex of FVIII and VWF is stable and the generation of VWF floating matter is suppressed. Claim 1: The pharmaceutical preparation contains the polysorbates and the macrogol so that when dissolved in a solvent, the polysorbate and/or the macrogol are 0.30 mg/mL or more and/or 0.50 mg/mL or more . The formulation described in. 3. The formulation according to claim 1 or 2 , wherein the polysorbate is polysorbate 80. 3. The formulation according to claim 1 or 2 , wherein the macrogol is macrogol 4000. 3. The formulation according to claim 1 or 2 , wherein the pharmaceutical formulation contains VWF such that when dissolved in a solvent, the concentration of VWF is 120 IU/mL or more. 3. The formulation according to claim 1 or 2 , wherein the polysorbate is polysorbate 80 and the macrogol is macrogol 4000. 2. The formulation of claim 1, further comprising human serum albumin, glycine, sodium chloride, sodium citrate hydrate, or L-histidine. 8. The formulation of claim 7 , wherein the pharmaceutical formulation has a pH in the range of about 6.5 to about 8.0 when dissolved in a solvent . 8. The formulation according to claim 7 , wherein the pharmaceutical formulation is a lyophilized formulation. A pharmaceutical formulation containing VWF or a complex of FVIII and VWF , the pharmaceutical formulation containing polysorbates and macrogol , wherein the polysorbate is 0.15 mg/mL or more and the macrogol is 0.25 mg/mL or more . A method for stabilizing VWF or a complex of FVIII and VWF by containing galls at a predetermined concentration, the method comprising suppressing the generation of VWF floating substances. A method for producing a pharmaceutical formulation comprising VWF or a complex of FVIII and VWF , the pharmaceutical formulation comprising polysorbates and macrogols , the method comprising: 0.15 mg/mL or more of the polysorbates and 0.25 mg/mL A method for producing a pharmaceutical preparation containing stable VWF or a complex of FVIII and VWF, the method comprising the step of containing mL or more of the macrogols at a predetermined concentration, the production method suppressing the generation of VWF suspended matter. .