Liquid, aqueous, pharmaceutical compositions of factor vii polypeptides

(54) Title: LIQUID LIQUID PHARMACEUTICAL COMPOSITION, METHOD FOR PREPARING AND USING THE SAME, METHOD FOR TREATING A SYNDROME RESPONSIVE TO FACTOR VII, E, HERMETIC CONTAINER (30) Unionist Priority: 03/18/2003 dk pa 2003 00413: 23 / 05/2003 DK PA 2003 00788; 06/25/2003 DK PA 2003 00959;

07/07/2003 DK PA 2003 00955; 08/14/2003 DK PA 2003 01161 (71) Depositor (s): Novo Nordisk Health Care AG (CH) (72) Inventor (s): Michael Bech Jensen, Birthe Lykkegaard Hansen, Troeis Kornfelt, Kirsten Kramer Jakobsen, Janus Krarup , Egon Persson, Anders Klarskov Petersen, Andrew Neil Bowler (74) Attorney: Momsen, Leonardos & Cia (86) International Request: pctDK2004 / 00018I dei8 / 03/2004 (87) International Publication: W0 2004/082708 of 30/09 / 2004 (57) Summary: LIQUID LIQUID PHARMACEUTICAL COMPOSITION, METHOD FOR PREPARING AND USING THE SAME, METHOD FOR TREATING A SYNDROME RESPONSIVE TO FACTOR VII, E, HERMETIC CONTAINER. The invention relates to an aqueous liquid pharmaceutical composition comprising a Factor VII polypeptide (for example, human Vila Factor) and buffering agent; where the molar ratio of non-complexed calcium ions (Ca²⁺) for the Factor VII polypeptide is less than 0.5. The composition may further comprise a stabilizing agent (for example, copper or magnesium ions, benzamidine or guanidine), a nonionic surfactant, a tonicity modifying agent, an antioxidant and a preservative. The composition is useful for treating a factor VII-responsive syndrome, such as bleeding disorders, including those caused by coagulation factor deficiencies (for example, hemophilia A, hemophilia B, coagulation factor XI deficiency, factor VII deficiency of coagulation): by thrombocytopenia or von Willebrand's disease or by inhibitors of the coagulation factor and intracerebral hemorrhage or excessive bleeding from any cause. The preparations can also be administered to patients in association with surgery or other trauma or to patients receiving anticoagulant therapy.

. V »t 4 ϋ« · «« · *> ·· ♦ ····. ··· · 9 9 * ·· 9 9 9 9 9 9 9 9 * * · · · «> ·“ WATER LIQUID PHARMACEUTICAL COMPOSITION, METHOD FOR PREPARING AND USING THE SAME, METHOD FOR TREATING A FACTOR VII RESPONSIVE SYNDROME, AND, HERMETIC CONTAINER ”

FIELD OF THE INVENTION

The present invention is directed to liquid, aqueous pharmaceutical compositions containing factor VII polypeptides and methods for preparing and using such compositions, as well as containers containing such compositions and the use of such compositions in the treatment of a factor VII responsive syndrome. More particularly, the invention relates to liquid compositions stabilized against chemical and / or physical degradation.

BACKGROUND OF THE INVENTION

A variety of factors involved in the blood clotting process have been identified, including Factor VII (FVII), a plasma glycoprotein. Coagulation is initiated by the formation of a complex between the Tissue Factor (TF) that is exposed to the circulating blood following damage to the vessel wall, and the FVIIa that is present in the circulation in an amount corresponding to about 1% of the total FVII protein mass. FVII exists in plasma primarily as a single-chain zymogen that is cleaved by FXa in its activated, double-stranded form, FVIIa. Recombinant activated Vila Factor (rFVIIa) was developed as a pro hemostatic agent. Administration of rFVIIa provides a rapid and highly effective prohemostatic response in haemophiliac patients with bleeding, who cannot be treated with other coagulation factor products due to antibody formation. Also bleeding in patients with Factor VII deficiency or patients having a normal clotting system but suffering from excessive bleeding can be successfully treated with FVIIa.

It is desirable to have forms of administration of Factor Vila suitable for both storage and release. Ideally, the medicated product is stored and administered as a liquid. Alternatively, the medicated product is lyophilized, that is, freeze-dried and then reconstituted by adding a suitable diluent before use by the patient. Ideally, the medicated product has sufficient stability to be kept in long-term storage, that is, more than six months.

The decision to keep the finished drug product as a liquid or to freeze dry it is usually based on the stability of the protein drug in these forms. Protein stability can be affected inter alia by factors such as ionic concentration, pH, temperature, repeated freeze / thaw cycles and exposure to shear forces. The active protein can be lost as a result of physical instabilities, including denaturation and aggregation (the formation of both soluble and insoluble aggregates), as well as chemical instabilities, including, for example, hydrolysis, deamidation, isomerization and oxidation, to name just a few. few. For a general review of the stability of pharmaceutical protein products, see, for example, Manning, et al., Pharmaceutical Research 6: 903 to 918 (1989).

Although the possible occurrence of protein instabilities is widely assessed, it is impossible to predict particular instability problems for a particular protein. Any of these instabilities can result in the formation of a by-product or protein derivative, with decreased activity, increased toxicity and / or increased immunogenicity. In fact, precipitation of the protein can lead to thrombosis, inhomogeneity of form and dosage amount, as well as clogged syringes. In addition, post translational modifications such as, for example, the carboxylation range of certain N-terminus glutamic acid residues and the addition of carbohydrate side chains provide potential sites that may be susceptible to storage modification. Also, specific to Fator Vila, which is a serine protease, fragmentation due to autocatalysis can occur (enzymatic degradation). Thus, the safety and effectiveness of any protein composition is directly related to its stability. Maintaining stability in a liquid form is generally different from maintaining stability in a lyophilized form because of the highly increased potential for molecular movement and thereby increased likelihood of molecular interactions. Maintaining stability in a concentrated form is also different from the above, because of the propensity for aggregate formation at increased protein concentrations.

When developing a liquid composition, many factors are taken into account. Short duration, that is, less than six months, the stability of liquid in general depends on avoiding major structural changes, such as denaturation and aggregation. These processes are described in the literature for various proteins and many examples of stabilizing agents exist. It is well known that an agent effective in stabilizing one protein truly acts to destabilize others. Once the protein has been stabilized against major structural changes, the development of a liquid composition for long-term stability (for example, more than six months) depends on further stabilizing the protein from specific degradation types for that protein. More specific types of degradation may include, for example, mixing disulfide bond, oxidation of certain residues, deamidation, cyclization. Although it is not always possible to accurately identify the species of individual degradation, assays are developed to monitor subtle changes in order to monitor the ability of specific excipients to uniquely stabilize the protein of interest.

It is desirable that the pH of the composition is in a physiologically suitable range for injection / infusion, otherwise pain and discomfort for the patient may result.

For a general review of protein compositions, see, for example, Cleland et al., The development of stable protein compositions: A closer look at protein aggregation, deamidation and oxidation, Critical Reviews in Therapeutic Drug Carrier Systems 1993, 10 (4) : 307 to 377; and Wang et al., Parenteral compositions of proteins and peptides: Stability and stabilizers, Journal of Parenteral Science and Technology 1988 (Supplement), 42 (2S).

Fator Vila degrades through several pathways, especially aggregation (dimerization), oxidation and autolytic cleavage (clipping of the main peptide chain). In addition, precipitation can occur. Many of these reactions can be significantly slowed by removing water from the protein. However, the development of an aqueous composition of Fator Vila has the advantages of eliminating reconstitution errors, thereby increasing the dosage accuracy, as well as simplifying the use of the product clinically, thereby increasing patient compliance. Ideally, Fator Vila compositions should be stable for more than 6 months over a wide range of protein concentrations. This allows for flexibility in administration methods. In general, more highly concentrated forms allow the administration of lower volumes, which is highly desirable from the patient's point of view. Liquid compositions can have many advantages over freeze-dried products with respect to ease of administration and use.

Currently, the only recombinant manufactured, commercially available FVII polypeptide composition is a product of

Freeze-dried FVIIa factor that is reconstituted before use; it contains a relatively low concentration of Vila Factor, for example, about 0.6 mg / ml. One vial (1.2 mg) of NovoSeven® (Novo Nordisk A / S, Denmark) contains 1.2 mg of recombinant human Vila Factor, 5.84 mg of NaCI, 2.94 mg CaCl₂.2H₂O, 2.64 mg of GlyGly, 0.14 mg of polysorbate 80 and 60.0 mg of mannitol; it is reconstituted at pH 5.5 by 2.0 ml of water for injection (WFI). When reconstituted, the protein solution is stable for use for 24 hours. Thus, no ready-to-use Factor VII products or liquid concentrates are currently commercially available.

WO 03/055512 discloses an aqueous liquid pharmaceutical composition comprising a Factor VII polypeptide, a buffer and an agent selected from a calcium salt, a magnesium salt and a mixture thereof, in particular a calcium salt, in a concentration at least 15 mM. The calcium / magnesium salt provides stability to the liquid, aqueous composition.

In view of the above, it is an object of this invention to provide further liquid, aqueous Factor VII polypeptide compositions that provide acceptable control of chemical and / or physical degradation products such as enzymatic degradation products or autocatalysis.

SUMMARY OF THE INVENTION

The present inventors have now found that although several references in the prior art recommend the use of a relatively high concentration of calcium ions in the purification steps and in aqueous liquids for the storage of Factor polypeptides. VII, it is also possible to obtain excellent storage stability for liquid, aqueous pharmaceutical compositions of Factor VII polypeptides by ensuring that the relative ratio between calcium (Ca) ions and the Factor VII polypeptide is very low.

Thus, an aspect of the present invention concerns an aqueous liquid pharmaceutical composition comprising a polypeptide of the

Factor VII (i) and a buffering agent (ii) suitable for maintaining the pH in the range of about 5.0 to about 9.0; where the molar ratio of calcium ions (Ca²⁺) not complexed for the Factor VII polypeptide is less than 0.5.

A second aspect of the present invention concerns a method for preparing an aqueous liquid pharmaceutical composition of a Factor VII polypeptide that comprises the step of providing the Factor VII polypeptide (i) in a solution that comprises a buffering agent (ii) suitable for maintaining the pH in the range of about 5.0 to about 9.0; while ensuring that, in the final composition, the molar ratio of uncomplexed calcium (Ca) ions to the Factor VII polypeptide is less than 0.5.

A third aspect of the present invention concerns an aqueous liquid pharmaceutical composition as defined above for use as a medicament.

A fourth aspect of the present invention concerns the use of an aqueous liquid pharmaceutical composition as defined above for the preparation of a medicament to treat a VIL Factor responsive syndrome.

A fifth aspect of the present invention concerns a method for treating a Factor VII-responsive syndrome, the method comprising administering to a patient in need thereof an effective amount of an aqueous liquid pharmaceutical composition as defined above.

A sixth aspect of the present invention concerns an airtight container, at least partially filled containing an aqueous liquid pharmaceutical composition as defined above and optionally an inert gas, said container comprising (i) a wall portion and (ii) one or more closing means which do not form part of said wall portion.

DETAILED DESCRIPTION OF THE INVENTION

As mentioned above, the present invention resides in the development of a new aqueous liquid stabilized pharmaceutical composition comprising a Factor VII polypeptide. More specifically, the aqueous liquid pharmaceutical composition comprises a Factor VII polypeptide (i) and a buffering agent (ii) suitable for maintaining the pH in the range of about 5.0 to about 9.0; in which reason

Π molar I of calcium (Ca) ions not complexed for the Factor VII polypeptide is less than 0.5.

When used here, the term "the concentration of uncomplexed calcium ions" is intended to mean the difference between the total concentration of calcium ions and the concentration of calcium bound to calcium chelators. In this regard, the Factor VII polypeptide is not considered to be a "calcium chelator" although calcium is expected to bind or become associated with the Factor VII polypeptide under certain conditions.

Preferably, the molar ratio of calcium ions (Ca²⁺) not complexed for the Factor VII polypeptide is less than 0.5, for example, in the range of 0.001 to 0.499, such as from 0.005 to 0.050 or in the range of 0.000 to 0.499, such as in the range of 0.000 to 0.050 or about of 0.000. In order to obtain the low relative ratio between calcium ions (Ca²⁺) and the Factor VII polypeptide, it may be necessary or desirable to add a calcium chelator in order to bind (complex) excess calcium ions. This is particularly relevant where the relationship between calcium ions and the Factor VII polypeptide in a solution of a process step that precedes the formulation step exceeds the limit established above. Examples of "calcium chelators" include EDTA, citric acid, NTA, DTPA, tartaric acid, lactic acid, malic acid, succinic acid, HIMDA, ADA and similar compound.

Factor VII (i) Polypeptide

The biological effect of the pharmaceutical composition is mainly attributed to the presence of the Factor VII polypeptide, although other active ingredients can be included in combination with the Factor VII polypeptide.

As used herein, the term "Factor VII polypeptide" encompasses wild type Factor VII (i.e. a polypeptide having the amino acid sequence disclosed in US Patent 4,784,950), as well as Factor VII variants that exhibit substantially the same activity biological or improved over Factor VII wild type. The term "Factor VII" is intended to cover Factor VII polypeptides in their non-cleaved form (zymogen), as well as those that have been proteolytically processed to produce their respective bioactive forms, which can be called Vila Factor. Typically, Factor VII is cleaved between residues 152 and 153 to produce Fator Vila. The term "Factor VII polypeptide" also encompasses polypeptides, including variants, in which the biological activity of Factor Vila has been substantially modified or slightly reduced in relation to wild type Factor Vila activity. These polypeptides include, without limitation, Factor VII or Vila Factor in which specific amino acid sequence changes have been introduced that modify or disrupt the polypeptide's bioactivity.

The biological activity of Factor Vila in the blood clot derives from its ability to (i) bind to the Tissue Factor (TF) and (ii) catalyze the proteolytic dividing of Factor IX or Factor X to produce activated Factor IX or X (Factor IXa or Xa, respectively).

For the purposes of the invention, the biological activity of Factor VII polypeptides ("Factor VII biological activity") can be quantified by measuring the ability of a preparation to promote blood clotting, as per Assay 4 described herein. In this assay, biological activity is expressed as the reduction in clotting time compared to a control sample and is converted to “units of Factor VII” by comparison with a standard pooled human serum containing 1 unit / ml of Factor VII activity . Alternatively, the biological activity of Factor Vila can be quantified (i) by measuring the ability of Factor VHa or a polypeptide related to Factor VII to produce activated Factor X (Factor Xa) in a system comprising TF embedded in a lipid membrane and Factor X. (Persson et al., J. Biol. Chem. 272: 19919 to 19924, 1997); (ii) measuring Factor X hydrolysis in an aqueous system (“In Vitro Proteolysis Assay, see Assay 2 below); (iii) measuring the physical binding of Factor VHa or a Factor VII-related polypeptide to TF using an instrument based on surface plasma resonance (Persson, FEBS Letts. 413: 359 to 363, 1997); (iv) measure the hydrolysis of a synthetic substrate by Factor Vila and / or a polypeptide related to Factor VII (“In Vitro Hydrolysis Assay, see Assay 1 below); or (v) measure thrombin generation in an in vitro TF-independent system (see Test 3 below).

Variants of Factor VII having substantially the same biological or improved activity over Factor Vila wild type include those that exhibit at least about 25%, such as, for example, at least about 50%, at least about 75 % or at least about 90% of the specific activity of Vila Factor that was produced in the same type of cell, when tested in one or more of a coagulation assay (Assay 4), proteolysis assay (Assay 2) or TF binding assay as described above. Variants of Factor VII having substantially reduced biological activity compared to the wild-type Factor Vila are those that exhibit less than about 25%, such as, for example, less than about 10% or less than about 5 % of the specific activity of the wild type Vila Factor that was produced in the same cell type when tested in one or more of a coagulation assay (Assay

4), proteolysis assay (Assay 2) or TF binding assay as described above. Variants of Factor VII having a substantially modified biological activity in relation to wild-type Factor VII include, without limitation, Factor VII variants that exhibit TF-independent Factor X proteolytic activity and those that bind TF but do not cleave Factor X .

Variants of Factor VII, whether exhibiting substantially the same or better bioactivity than Factor VII of the wild type or, alternatively, exhibiting substantially modified or reduced bioactivity in relation to Factor VII of the wild type, include, without limitation, polypeptides having a sequence amino acid that differs from the Factor VII sequence of the wild type by the insertion, deletion or substitution of one or more amino acids.

Non-limiting examples of Factor VII variants having substantially the same biological activity as wild type Factor VII include S52A-FVIIa, S60A-FVIIa (Lino et al., Arch. Biochem. Biophys. 352: 182 to 192, 1998) ; variants of FVIIa that exhibit increased proteolytic stability as disclosed in US Patent 5,580,560; Vila factor that was proteolytically cleaved between residues 290 and 291 or between residues 315 and 316 (Mollerup et al., Biotechnol. Bioeng. 48: 501 to 505, 1995); oxidized forms of the Vila Factor (Komfelt et al., Arch. Biochem. Biophys. 363: 43 to 54, 1999); FVII variants as disclosed in PCT / DK02 / 00189; and FVII variants that exhibit increased proteolytic stability as disclosed in WO 02/38162 (Scripps Research Institute); FVII variants having a modified Gla domain and exhibiting enhanced membrane binding as disclosed in WO 99/20767 (University of Minnesota); and FVII variants as disclosed in WO 01/58935 (Maxygen ApS).

Non-limiting examples of Factor VII variants having increased biological activity compared to wild-type FVIIa include FVII variants as disclosed in WO 01/83725, WO 02/22776, WO 02/077218, WO 03/27147, WO 03/37932 ; WO 02/38162 (Scripps Research Institute); and FVIIa variants with enhanced activity as disclosed in JP 2001061479 (Chemo-Sero-Therapeutic Res Inst.).

Non-limiting examples of Factor VII variants having substantially reduced or modified biological activity in relation to wild type Factor VII include R152E-FVIIa (Wildgoose et al., Biochem 29: 3413 to 3420, 1990), S344A-FVIIa (Kazama et al., J. Biol. Chem. 270: 66 to 72, 1995), FFR-FVIIa (Holst et al., Eur. J. Vasc. Endovasc. Surg. 15: 515 to 520, 1998) and Factor Vila that lacks from the Gla domain, (Nicolaisen et al., FEBS Letts. 317: 245 to 249, 1993).

Examples of Factor VII polypeptides include, without limitation, Wild Type Factor VII, L305V-FVII, L305V / M306D / D309SFVII, L305I-FVII, L305T-FVII, F374P-FVII, V158T / M298Q-FVII, V158D / E296 M298Q-FVII, K337A-FVII, M298Q-FVII, V158D / M298QFVII, L305V / K337A-FVII, V158D / E296V / M298Q / L305V-FVII, V158D / E296V / M298Q / L337V / M298Q / L335 K337A-FVII,

K157A-FVII, E296V-FVII, E296V / M298Q-FVII, V158D / E296V-FVII, V158D / M298K-FVII, and S336G-FVII, L305V / K337A-FVII, L305V / V158DFV, L158DFVI, L158DFVI, - FVII, L305V / V158T-FVII, L305V / K337A / V158T-FVII, L305V / K337A / M298Q-FVII, L305V /

K337A / E296V-FVII, S314E / L305V / V158D-FVII, S314E / L305V / E296VFVII, S314E / L305V / M298Q-FVII, S314E / L305V / Vl 5 8T-FVII, S314E / L33, V3, K3 L305V / K337A / M298Q-FVII, S314E / L305V / K337A / E296V-FVII, S314E / L305V / K337A / V158D-FVII, S314E / L305V / Vl 58D / M298Q-FVII, S314E / L6 S314E / L305V / V158T / M298Q-FVII, S314E / L305V / V158T / E296V-FVII, S314E /

L305V / E296V / M298Q-FVII, S314E / L305V / V158D / E296V / M298Q-FVII,

S314E / L305V / V158T / E296V / M298Q-FVII, S314E / L305V / V158T /

K337A / M298Q-FVII, S314E / L305V / V158T / E296V / K337A-FVII, S314E /

L305V / V158D / K337A / M298Q-FVII, S314E / L305V / V158D / E296V / Κ337ΑFVII, S314E / L305V / V158D / E296V / M298Q / K337A-FVII, S314E /

L305V / V158T / E296V / M298Q / K337A-FVII, K316H / L305V / K337A-FVII,

K316H / L305V / V158D-FVII, K316H / L305V / E296V-FVII, K316H / L305V / M298Q-FVII, K316H / L305V / V158T-FVII, K316H / L305V / K337A / LQ KI7I / V38TFVI, K316H K316H / L305V / K337A7E296VFVII, K316H / L305V / K337A / V158D-FVII, K316H / L305V / V158D / M298QFVII, K316H / L305V / V158D / E296V-FVI / K5V / V6 / V6 / V6 / V6 FVII, K316H /

L305V / V158D / E296V / M298Q-FVII, K316H / L305V / V158T / E296V /

M298Q-FVII, K316H / L305V / V158T / K337A / M298Q-FVII, K316H /

L305V / V158T / E296V / K337A-FVII, K316H / L305V / V158D / K337A / V298QFVII, K316H / L305V / V158D / E296V / K337A-FVII, K316H / L305V /

V158D / E296V / M298Q / K337A-FVII, K316H / L305V / V158T / E296V /

M298Q / K337A-FVII, K316Q / L305V / V158D-FVII, K316Q / L305V / E296VFVII, K316Q / L305V / V158T-FVII, K316Q / L305V / K337A / V158T-FVII,

K316Q / L305V / K337A / M298Q-FVII, K316Q / L305V / K337A / E296V-FVII, K316Q / L305V / K337A / V158D-FVII, K316Q / L305V / V158D / E296VQ FVII, K516V-FVII, K30 K316Q / L305V / V158T / E296V-FVII, K316Q / L305V / E296V / M298Q-FVII, K316Q / L305V /

V158D / E296V / M298Q-FVII, K316Q / L305V / V158T / E296V / M298Q-FVII,

K316Q / L305V / V158T / K337A / M298Q-FVII, K316Q / L305V / V158T /

E296V / K337A-FVII, K316Q / L305V / V158D / K337A / M298Q-FVII,

K316Q / L305V / V158D / E296V / K337A-FVII, K316Q / L305V / V158D /

E296V / M298Q / K337A-FVII, K316Q / L305V / V158T / E296V / M298Q /

K337A-FVII, F374Y / K337A-FVII, F374Y / V158D-FVII, F374Y / E296VFVII, F374Y / M298Q-FVII, F374Y / V158T-FVII, F374Y / S314E-FVII,

F374Y / L305V-FVII, F374Y / L305V / K337A-FVII, F374Y / L305V / V158DFVII, F374Y / L305V / E296V-FVII, F374Y / L305V / M298Q-FVII, F374Y /

L305V / V158T-FVII, F374Y / L305V / S314E-FVII, F374Y / K337A / S314EFVII, F374Y / K337A / V158T-FVII, F374Y / K337A / M298Q-FVII, F374Y /

K337A / E296V-FVII, F374Y / K337A / V158D-FVII, F374Y / V158D / S314EFVII, F374Y / V158D / M298Q-FVII, F374Y / V158D / E296V-FVII, F374Y / V158D / F154Y / V158 FVII, F374Y / V158T / E296VFVII, F374Y / E296V / S314E-FVII, F374Y / S314E / M298Q-FVII, F374Y / E296V / M298Q-FVII, F374Y / L305V / K337A / V158 / F334

K337A / E296V-FVII, F374Y / L305V / K337A / M298Q-FVII, F374Y / L305V /

K337A / V158T-FVII, F374Y / L305V / K337A / S314E-FVII, F374Y / L305V /

V158D / E296V-FVII, F374Y / L305V / V158D / M298Q-FVII, F374Y / L305V /

V158D / S314E-FVII, F374Y / L305V / E296V / M298Q-FVII, F374Y / L305V /

E296V / V158T-FVII, F374Y / L305V / E296V / S314E-FVII, F374Y / L305V /

M298Q / V158T-FVII, F374Y / L305V / M298Q / S314E-FVII, F374Y / L305V /

V158T / S314E-FVII, F374Y / K337A / S314E / V158T-FVII, F374Y / K337A /

S314E / M298Q-FVII, F374Y / K337A / S314E / E296V-FVII, F374Y / K337A /

5314E / V158D-FVII, F374Y / K337A / V158T / M298Q-FVII, F374Y / K337A /

V158T / E296V-FVII, F374Y / K337A / M298Q / E296V-FVII, F374Y / K337A /

M298Q / V158D-FVII, F374Y / K337A / E296V / V158D-FVII, F374Y / V158D /

S314E / M298Q-FVII, F374Y / V158D / S314E / E296V-FVII, F374Y / V158T /

S314E / EZ96V-FVII, F374Y / V158T / M298Q / E296V-FVII, F374Y / E296V /

S314E / M298Q-FVII, F374Y / L305V / M298Q / K337A / S314E-FVII, F374Y / L305V / E296V / K337A / S314E-FVII, F374Y / E296V / M298Q / K337A / SQ14V / M298 / -FVII, F374Y / L305V /

E296V / M298Q / S314E-FVII, F374Y / V158D / E296V / M298Q / K337A-FVII,

F374Y / VI58D / E296V / M298Q / S314E-FVII, F374Y / L305V / V158D /

K337A / S314E-FVII, F374Y / V158D / M298Q / K337A / S314E-FVII, F374Y / V158D / E296V / K337A / S314E-FVII, F374Y / L305V / V158D / E296V / M298Q /

FVII, F374Y / L305V / V158D / M298Q / K337A-FVII, F374Y /

L305V / V158D / E296V / K337A-FVII, F374Y / L305V / V158D / M298Q / S314EFVH, F374Y / L305V / V158D / E296V / S314E-FVII, F374Y / V158T /

E296V / M298Q / K337A-FVII, F374Y / V158T / E296V / M298Q / S314E-FVII,

F374Y / V158T / M298Q / K337A / S314E-FVII, F374Y / V158T / E296V /

K337A / S314E-FVII, F374Y / L305V / V158T / E296V / M298Q-FVII, F374Y / L305V / V158T / M298Q / K337A-FVII, F374Y / L305V / V158T / E296V / K337VV / K337VI / VT FVII, F374Y / L305V /

V158T / E296V / S314E-FVII, F374Y / E296V / M298Q / K337A / V158T / S314EFVH, F374Y / V158D / E296V / M298Q / K337A / S314E-FVII, F374Y /

L305V / V158D / E296V / M298Q / S314E-FVII, F374Y / L305V / E296V /

M298Q / V158T / S314E-FVII, F374Y / L305V / E296V / M298Q / K337A / V158TFVII, F374Y / L305V / E296V / K337A / V158T / S314E-FVII,

K316H / L305V / E296V / M298Q-FVII, F374Y / L305V / M298Q / K337A /

V158T / S314E-FVII, F374Y / L305V / V158D / E296V / M298Q / K337A-FVII,

F374Y / L305V / V158D / E296V / K337A / S314E-FVII, F374Y / L305V /

VI58D / M298Q / K337A / S314E-FVII, F374Y / L305V / E296V / M298Q /

K337A / V158T / S314E-FVII, F374Y / L305V / V158D / E296V / M298Q /

K337A / S314E-FVII, S52A-Factor VII, S60A-Factor VII; R152E-Factor VII, S344A-Factor VII, Vila Factor that lacks the Gla domain; and Pl 1Q / K33E-FVII, T106N-FVII, K143N / N145T-FVII, V253N-FVII, R290N / A292T-FVII, G291N-FVII, R315N / V317T-FVII, K143N / N145T / R315N / VVI; and FVII having substitutions, additions or deletions in the amino acid sequence from 233Thr to 240Asn, FVII having substitutions, additions or deletions in the amino acid sequence from 304Arg to 329Cys and FVII having substitutions, deletions or additions in the amino acid sequence Ilel53-Arg223.

In some embodiments, the Factor VII polypeptide is human Vila Factor (hFVIIa), preferably recombinantly manufactured human Vila Factor (rhVIIa).

In other embodiments, the Factor VII polypeptide is a sequence variant of Factor VII.

In some embodiments, the Factor VII polypeptide has a different glycosylation than human wild type Factor VII.

In various embodiments, for example, those where the Factor VII polypeptide is a Factor VII related polypeptide or a Factor VII sequence variant, the ratio between the activity of the Factor VII polypeptide and the activity of the native Factor Vila human (wild type FVIIa) is at least about 1.25, preferably at least about 2.0 or 4.0, most preferred at least about 8.0, when tested in the “In Vitro Proteolysis Assay” (Assay 2) as described in this specification.

In some embodiments, the Factor VII polypeptides are polypeptides related to Factor VII, in particular variants, in which the ratio between the activity of said Factor VII polypeptide and the activity of the native human Factor Vila (wild type FVIIa) it is at least about 1.25 when tested in the “In Vitro Hydrolysis Assay” (see Assay 1 below); in other embodiments, the ratio is at least about 2.0; in other embodiments, the ratio is at least about 4.0.

In a pharmaceutical composition, it is often desirable for the concentration of the active ingredient to be such that the application of a unit dose does not cause unnecessary discomfort to the patient. Thus, a unit dose of more than about 2 to 10 ml is often undesirable. For the purpose of the present invention, the concentration of the Factor VII polypeptide is therefore at least 0.01 mg / ml. In different embodiments, the Factor VII polypeptide is present in a concentration of 0.01 to 20 mg / ml; 0.1 to 10 mg / ml; 0.5 to 5.0 mg / ml; 0.6 to 4.0 mg / ml; 1.0 to 4.0 mg / ml; 0.1 to 5 mg / ml; 0.1 to 4.0 mg / ml; 0.1 to 2 mg / ml; or 0.1 to 1.5 mg / ml.

The concentration of Vila Factor is conveniently expressed as mg / ml or as IU / ml, with 1 mg usually representing 43,000 a

56,000 IU or more.

Buffering agent (ii)

In order to make the aqueous liquid pharmaceutical composition useful for direct parenteral administration to a mammal such as a human, it is normally required that the pH value of the composition be kept within reasonable limits, such as from about 5.0 to about 9.0. To ensure an adequate pH value under the given conditions, the pharmaceutical composition also comprises a buffering agent (ii) suitable for maintaining the pH in the range of about 5.0 to about 9.0.

The term "buffering agent" encompasses those agents or combinations of agents that maintain the pH of the solution in an acceptable range of about 5.0 to about 9.0.

In one embodiment, the buffering agent (ii) is at least one component selected from the groups consisting of acids and salts of MES, PIPES, ACES, BES, TES, HEPES, TRIS, histidine, imidazole, glycine, glycylglycine, glycinamide, phosphoric acid, acetic acid (eg sodium acetate), lactic acid, glutaric acid, citric acid, tartaric acid, malic acid, maleic acid and succinic acid. It is to be understood that the buffering agent can comprise a mixture of two or more components, wherein the mixture is capable of providing a pH value in the specified range. As examples can be mentioned acetic acid and sodium acetate, etc.

Due to the fact that the composition comprises very small amounts of calcium, it is possible to use a buffer system based on phosphoric acid, that is, a phosphate buffer, without the undesirable precipitation of calcium phosphates. Thus, in an interesting embodiment, the buffer is a phosphate buffer.

The concentration of the buffering agent is chosen in order to maintain the preferred pH of the solution. In various embodiments, the concentration of the buffering agent is 1 to 100 mM; 1 to 50 mM; 1 to 25 mM; or 2 to 20 mM.

In one embodiment, the pH of the composition is maintained from about 5.0 to about 8.0; such as from about 5.0 to about 7.5; about 5.0 and about 7.0; from about 5.0 to about 6.5, from about 5.0 to about 6.0, from about 5.5 to about 7.0; about 5.5 to about 6.5, about 6.0 to about 7.0, about 6.4 to about 6.6 or about 5.2 to about 5, 7.

As used herein, the pH values ​​specified as "about" are understood to be ± 0.1, for example, about pH 8.0 includes pH 8.0 ± 0.1.

Stabilizing agent (iii)

In a currently preferred embodiment, the composition further comprises a stabilizing agent (iii).

The stabilizing agent (iii), when included, is typically present in a concentration of at least 5 μΜ, at least 25 μΜ, at least 50 μΜ, at least 100 μΜ, at least 200 μΜ, at least 400 μΜ, at least 500 μΜ, at least 800 μΜ, at least 900 μΜ, at least 1000 μΜ, at least 5 mM, such as 20 to 2000 μΜ, 50 to 5000 μΜ, 0.1 to 10 mM, 0.2 to 20 mM or 0 , 5 to 50 mM.

Bivalent metal-type stabilizing agent (iiia)

In one embodiment, the stabilizing agent (iii) includes at least one metal-containing agent (iiia), wherein said metal is selected from the group consisting of metals from the +11 oxidation state first transition series.

The present inventors have the impression that the metals in the +11 oxidation state first transition series have not previously been used as stabilizing agents in connection with ready-to-use pharmaceutical compositions.

When used here, the term “oxidation state transition metals series + ΙΙ” is intended to cover the metals titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper and zinc.

Although titanium and vanadium can exist in the +11 oxidation state in aqueous environments, it is more typical to select the metal (s) among chromium, manganese, iron, cobalt, nickel, copper and zinc. Illustrative Examples of agents containing metal (iiia) corresponding to these metals are chromium (II) chloride, manganese (II) chloride, iron (II) chloride, cobalt (II) chloride, nickel (II) chloride and copper (II) chloride. It is to be understood that the metal-containing agent (iiia) can comprise two or more metals, for example, two or more metals from the first transition series. So in some cases, two or more of the agents mentioned above can be used in combination.

So far, the most promising metals are copper and manganese. Illustrative examples of corresponding metal-containing agents (iiia) are copper (II) chloride and manganese (II) chloride.

The concentration of the metal-containing agent (or agents) (iiia) is typically at least 1 μΜ. The desirable (or necessary) concentration typically depends on the selected metal-containing agent (or agents), more specifically on the binding affinity of the selected metal from oxidation state +11 to the Factor VII polypeptide.

In different embodiments, the metal-containing agent (iiia) is present in a concentration of at least 5 μΜ, at least 25 μΜ, at least 50 μΜ, at least 100 μΜ, at least 200 μΜ, at least 400 μΜ, at least 500 μΜ, at least 800 μΜ, at least 900 μΜ, at least 1000 μΜ, at least 5 mM, at least 25 mM, at least 50 mM, at least 100 mM, at least 200 mM, at least 400 mM, at least 800 mM, at least 900 mM or at least 1000 mM.

In a particular embodiment, the metal of the metal-containing agent (iiia) is copper and the concentration of said agent is at least 5 μΜ, such as at least 10 μΜ or at least 15 μΜ.

In another particular embodiment, the metal of the metal-containing agent (iiia) is manganese and the concentration of said agent is at least 100 μΜ, such as at least 500 μΜ or at least 1 mM. Benzamidine / arginine (iiib) stabilizing agent

In another embodiment, the stabilizing agent includes at least one agent (iiib) that comprises a -C ^ NZ ^ R motif¹) NH-Z²-R², on what

Z¹ and Z² independently are selected from the group consisting of -O-, -S-, -NR^H- and a simple link, where R^H is selected from the group consisting of hydrogen, Cm alkyl, aryl and arylmethyl and R and R are independently selected from the group consisting of hydrogen, Ci alkyl.₆ optionally substituted, alkenyl C₂.₆ optionally substituted, optionally substituted aryl, optionally substituted heterocyclyl or

Z² and R² are as defined above and -C ^ NZ ^ R¹ forms part of a heterocyclic ring or

Z and R are as defined above and -C-NH-Z -R forms part of a heterocyclic ring or

-C (= NZ¹-R¹) -NH-Z²-R² forms a heterocyclic ring in which z'-R'-R²-Z²-is a bi-radical.

The term "Cm alkyl" is intended to cover saturated acyclic and cyclic hydrocarbon residues that have 1 to 6 carbon atoms and that can be linear or branched. Particular examples are methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, secbutyl, tert-butyl, cyclopropylmethyl, n-pentyl, isopentyl, n-hexyl, etc. Similarly, the term "Cm alkyl" encompasses saturated acyclic and cyclic hydrocarbon residues that have 1 to 4 carbon atoms and that can be linear or branched.

Similarly, the term “C alkenyl₂-6 ”is intended to cover acyclic and cyclic hydrocarbon residues that have 2 to 6 carbon atoms and comprise an unsaturated bond, which can be linear or branched. Examples of alkenyl groups C₂.₆ are vinyl, allyl, but-l-en-l-yl, but-2-en-l-yl, pent-l-en-l-yl and hex-l-en-l-yl.

The term "optionally substituted" in connection with C 1 alkyl groups.₆ and alkenyl C₂.₆ is intended to denote that the group in question may be substituted once or several times, preferably 1 to 3 times, with group (s) selected from the group consisting of hydroxy, C1-6 alkoxy (i.e. C1-6 alkyloxy), alkenyloxy C₂.6, oxo (forming a keto or aldehyde functionality), aryl, aryloxy, arylcarbonyl, heterocyclyl, heterocyclyloxy, heterocyclylcarbonyl, amino, mono- and di- (Ομ alkyl₆) amino, halogen, where any aryl and heterocyclyl can be substituted as specifically described below by optionally substituted aryl and heterocyclyl.

"Halogen" includes fluorine, chlorine, bromine and iodine.

When used herein, the term "aryl" is intended to denote a carbocyclic ring or ring system wholly or partially aromatic, such as phenyl, naphthyl, 1,2,3,4-tetrahydronaftila, anthracyl, phenanthracyl, pyrenyl, benzopyrenyl, fluorenyl and xanthenyl, among which phenyl is a preferred example.

The term "heterocyclyl" is intended to denote a saturated, partially unsaturated, partially aromatic or totally aromatic carbocyclic ring or ring system where one or more of the carbon atoms have been replaced with hetero atoms, for example, nitrogen atoms (= N- or -NH), sulfur (-S-) and / or oxygen (-O-). Examples of such heterocyclyl groups are oxazolyl, oxazolinyl, oxazolidinyl, isoxazolyl, isoxazolinyl, isoxazolidinyl, oxadiazolyl, oxadiazolinyl, oxadiazolidinyl, thiazolyl, isothiazolyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrrolidinyl, pyrrolidinyl, pyrrolidinyl, pyrrolidinyl, pyrrolidinyl, pyrrolidinyl, pyrrolidinyl, pyrrolidinyl, pyrrolidinyl, pyrrolidinyl, pyrrolidinyl, pyrrolidinyl, pyrrolidinyl, pyrrolidinyl, imidazol , coumarila, fiirila, quinolila, benzothiazolila, benzotriazolila, benzodiazolila, benzoxozolila, diazolila, diazolinila, diazolidinila, triazolila, triazolinyl, triazolidinyl, tetrazole, etc. preferred heterocyclyl groups are 5, 6 or 7 membered monocyclic groups such as isoxazolyl, isoxazolinyl, oxadiazolyl, oxadiazolinyl, pyrrolyl, pyrrolinyl, diazolyl, diazolinyl, triazolyl, triazolinyl, imidazolyl, etc.

The term "heterocyclic ring" is intended to mean a ring corresponding to those defined under "heterocyclyl".

In connection with the terms "aryl", "heterocyclyl" and "heterocyclic ring", the term "optionally substituted" is intended to denote that the group in question can be substituted once or several times, preferably 1 to 3 times, with the (s) selected hydroxy group (s) (which when present in an enol system can be represented in tautomeric keto form), C1-6 alkyl, C alkenyl₂-6, phenyl, benzyl, C1-6 alkoxy, oxo (which can be represented in tautomeric enol form), carboxy, Cj.6 alkoxycarbonyl, Cj.6 alkylcarbonyl, amino, mono- and di- (Cj-o alkyl) amino , dihalo-C1.4 alkyl, trihalo-C1 alkyl.₄ and halogen. The most typical examples of substituents are hydroxyl, C1 alkyl.₄, phenyl, benzyl, Cm alkoxy, oxo, amino, mono- and di-methylamino and halogen.

In addition to the fact that R¹ and R² independently can be selected from the group consisting of hydrogen, optionally substituted Cm alkyl, C alkenyl₂.₆ optionally substituted, optionally substituted aryl, optionally substituted heterocyclyl, it is also possible that a part of the -C (= NZ¹-R¹) -NH-Z²-R² may be part of a heterocyclic ring, while the other parts of the motif have the defined meaning for Z¹, Z², R¹ and R², respectively. In some interesting embodiments, -C = NZ¹-R¹ may form part of a heterocyclic ring selected from the group consisting of a 1,2-diazole ring, an isoxazole ring, a 1,2,4-triazole ring and a 1,2,4-oxadiazole ring or - C-NH-Z R² may form part of a heterocyclic ring selected from the group consisting of a 1,2-diazoline ring, an isoxazoline ring, an

1,2,4-triazoline and a 1,2,4-oxadiazoline ring. Such heterocyclic rings can be replaced as described above.

In some embodiments, at least one of R and R is hydrogen, for example, both are hydrogen. Furthermore, in some embodiment, which can be combined with the previously mentioned embodiments, at least one of Z and Z is a single bond, for example, both are a single bond. In special embodiments, R¹ and R² are both hydrogen and Z¹ and Z² they are both a simple link.

The reason -C (= NZ¹-R¹) -NH-Z²-R² is particularly important for the stabilizing effect of the stabilizing agent (iiib). In particular, the reason -C (= NZ¹-R¹) -NH-Z²-R² mimic an arginine portion of a substrate for the Factor VII polypeptide.

In more specific embodiments, the stabilizing agent (iiib) is at least one selected from the group consisting of amidine compounds that comprise a -CC (= NZ -R) -NH-Z 2 11 R motif and guanidine compounds that comprise a reason> NC (= NZ -R) NH-Z²-R².

In some embodiments, the stabilizing agent (iiib) is at least one amidine compound selected from the group consisting of benzamidines comprising the -C motif₆H₄-C (= NZ -R) -NH-Z -R, where C₆H₄ denotes an optionally substituted benzene ring, of which benzamidine (R¹ and R² are hydrogen and Z¹ and Z² are a simple link) is a particular embodiment.

In other of its particular embodiments, benzamidines comprise the motif> N-C6H4-C (= NZ¹-R¹) -NH-Z²-R², where C₆H₄ denotes an optionally substituted benzene ring, i.e. an oamino-benzamidine, a m-amino-benzamidine or a p-amino-benzamidine, of which p-amino-benzamidines, such as p-amino-benzamidine, are the most currently promising.

Other illustrative examples of p-amino-benzamidines are those disclosed by Aventis in EP 1 162 194 Al, in particular those defined in claims 1 to 6 and sections [0009] to [0052] and EP 1 270 551 Al, in particular in accordance with claims 1 and 2 and sections [0010] to [0032].

In another embodiment, the stabilizing agent (iiib) is at least one guanidine compound selected from the group consisting of guanidine compounds comprising a -CH2-NH-C motif (= NZ¹R¹) -NH-Z²-R². Examples of guanidine compounds are those selected from the group consisting of arginine, arginine derivatives and peptides of 2 to 5 amino acid residues that comprise at least one arginine residue. Arginine is a particular embodiment.

The term "arginine derivatives" is intended to encompass arginine counterparts, arginines functionalized at the N-terminus (eg, N-methylated and N-acylated derivatives (eg, acetylated)), arginines functionalized at the C-terminus (eg, derivatives C-amidates, C-alkylamidates and C-alkylates) and combinations thereof.

As mentioned above, the only crucial reason for stabilizing agents is -C (= NZ¹-R¹) -NH-Z²-R². Other parts of the stabilizing agent may also be important, in particular with regard to the optimization of the stabilizing effect and patient tolerance. Typically, the stabilizing agent has the formula YC (= NZ¹-R¹) -NH-Z²-R², where Y is an organic radical. The Y radical is typically selected to improve the efficiency of the stabilizing effect. Also, the Y radical can comprise an r 112 2 or more additional motifs of the formula -C (= NZ -R) -NH-Z -R.

The molecular weight of the stabilizing agent is typically a maximum of 1000 Da, as well as a maximum of 500 Da.

The concentration of the stabilizing agent (or agents) (iiib) is typically at least 1 pM. The desirable (or necessary) concentration typically depends on the selected stabilizing agent (or agents), more specifically on the binding affinity of the selected stabilizing agent to the Factor VII polypeptide.

In different embodiments, the stabilizing agent (iiib) is present in a concentration of at least 5 pM, at least 10 pM, at least 20 pM, at least 50 pM, at least 100 pM, at least 150 pM, at least 250 pM, at least 500 pM, at least 1 mM, at least 2 mM, at least 4 mM, at least 5 mM, at least 8 mM, at least 9 mM, at least 10 mM, at least 15 mM, at least 20 mM, such as 20 to 2000 pM, 50 to 5000 pM, 0.1 to 10 mM, 0.2 to 20 mM or 0.5 to 50 mM.

In one embodiment, the stabilizing agent (iiib) is benzamidine and the concentration of said agent is at least 0.5 mM, such as at least 2 mM, although it is considered that the substituted benzamidines may be more potent, which is why they can be added in lower concentrations.

In one embodiment, the stabilizing agent (iiib) is arginine and the concentration of said agent is at least 2 mM, such as at least 10 mM.

It is to be understood that one or more agents containing metal (iiia) and one or more stabilizing agents (iiib) can be used in combination.

Nonionic surfactant (iv)

In another embodiment that can be combined with the above embodiments that relate to the presence of a stabilizing agent (iii) (for example, a metal-containing agent (iiia) or an agent (iiib)), the composition comprising a non-ionic surfactant (iv). Surfactants (also known as detergents) in general include those agents that protect the protein from stresses induced by the air / solution interface and stresses induced by the solution / surface interface (for example, which result in protein aggregation).

Typically, the nonionic surfactant (iv) is at least one selected from polysorbates, poloxamers, polyoxyethylene alkyl ethers, polyethylene / polypropylene block copolymers, polyethylene glycol (PEG), polyoxyethylene stearates and polyoxyethylene castor oils.

Illustrative examples of non-ionic surfactants are Tween®, polysorbate 20, polysorbate 80, Brij-35 (polyoxyethylene dodecyl ether), poloxamer 188, poloxamer 407, PEG8000, polyuria Pluronic, polyoxy-23-lauryl ether, Myrj 49, Solutol HS15 and Cremophor A.

In one embodiment, the non-ionic surfactant is present in an amount of 0.005 to 2.0% by weight.

Tonicity modifying agent - High ionic concentration

In another embodiment that can be combined with the above embodiments that relate to the presence of a stabilizing agent (iii) (for example, a metal containing agent (iiia) or an agent (iiib)) and / or a nonionic surfactant (iv), the composition may comprise a tonicity modifying agent (v).

As used herein, the term "tonicity modifying agent" includes agents that contribute to the osmolality of the solution. The tonicity modifying agent (v) includes at least one agent selected from the group consisting of neutral salts, amino acids, peptides from 2 to 5 amino acid residues, monosaccharides, disaccharides, polysaccharides and sugar alcohols. In some embodiments, the composition comprises two or more of such agents in combination.

By "neutral salt" is meant a salt that is neither an acid nor a base when dissolved in an aqueous solution.

In one embodiment, at least one tonicity modifying agent (v) is a neutral salt selected from the groups consisting of sodium salts, potassium salts and magnesium salts, such as sodium chloride, potassium chloride, chloride magnesium, magnesium acetate, magnesium gluconate and magnesium levulate.

In another embodiment, the tonicity modifying agent (v) includes sodium chloride in combination with at least one selected from the group consisting of magnesium chloride and magnesium acetate.

In yet another embodiment, the tonicity modifying agent (v) is at least one selected from the group consisting of sodium chloride, sucrose, glucose and mannitol.

In different embodiments, the tonicity modifying agent (v) is present in a concentration of at least 1 mM, at least 5 mM, at least 10 mM, at least 20 mM, at least 50 mM, at least 100 mM, at least 200 mM, at least 400 mM, at least 800 mM, at least 1000 mM, at least 1200 mM, at least 1500 mM, at least 1800 mM, at least 2000 mM or at least 2200 mM.

In a number of embodiments, the tonicity modifying agent (v) is present in a concentration of 5 to 2200 mM, such as 25 to 2200 mM, 50 to 2200 mM, 100 to 2200 mM, 200 to 2200 mM, 400 at 2200 mM, 600 to 2200 mM, 800 to 2200 mM, 1000 to 2200 mM, 1200 to 2200 mM, 1400 to 2200 mM, 1600 to 2200 mM, 1800 to 2200 mM or 2000 to 2200 mM; 5 1800 mM, 25 to 1800 mM, 50 to 1800 mM, 100 to 1800 mM, 200 to 1800 mM, 400 to 1800 mM, 600 to 1800 mM, 800 to 1800 mM, 1000 to 1800 mM, 1200 to 1800 mM, 1400 1800 mM, 1600 to 1800 mM; 5 to 1500 mM, 25 to 1400 mM, 50 to 1500 mM, 100 to 1500 mM, 200 to 1500 mM, 400 to 1500 mM, 600 to 1500 mM, 800 to 1500 mM, 1000 to 1500 mM, 1200 to 1500 mM; 5 to 1200 mM, 25 to 1200 mM, 50 to 1200 mM, 100 to 1200 mM, 200 to 1200 mM,

400 1200 mM, 600 to 1200 mM or 800 to 1200 mM.

In a preferred embodiment of the invention, at least one tonicity modifying agent (v) is an ionic concentration modifying agent (v / a).

As used herein, the term "ionic concentration modifying agent" includes agents that contribute to the ionic concentration of the solution. The agents include, but are not limited to, neutral salts, amino acids, peptides of 2 to 5 amino acid residues. In some embodiments, the composition comprises two or more of such agents in combination.

Preferred examples of ionic concentration modifying agents (v / a) are neutral salts such as sodium chloride, potassium chloride and magnesium chloride. A preferred agent (v / a) is sodium chloride.

The term "ionic concentration" is the ionic concentration of the solution (μ) which is defined by the equation: μ =% Σ ([i] (Z;²)), where μ is the ionic concentration, [i] is the millimolar concentration of an ion, and Z_; it is the charge (+ or -) of that ion (see, for example, Solomon, Journal of Chemical Education, 78 (12): 1691 to 1692, 2001; James Fritz and George Schenk: Quantitative Analytical Chemistry, 1979).

In different embodiments of the invention, the ionic concentration of the composition is at least 50, such as at least 75, at least 100, at least 150, at least 200, at least 250, at least 400, at least 500, at least 650, at least 800, at least 1000, at least 1200, at least 1600, at least 2000, at least 2400, at least 2800 or at least 3200.

In some specific embodiments, the total concentration of the tonicity modifying agent (v) and the ionic concentration modifying agent (v / a) is in the range of 1 to 500 mM, such as 1 to 300 mM or 10 to 200 mM or 20 to 150 mM, depending on the effect that any other ingredients may have on ionic tonicity and concentration.

In one embodiment, the composition is isotonic; in another, it is hypertonic. The term "isotonic" means "isotonic with the serum", that is to say about 300 ± 50 milliosmol / kg. Tonicity is intended to be a measure of the solution's osmolality prior to administration. The term “hypertonic” is intended to mean levels of osmolality above the physiological level of serum, such as levels above 300 ± 50 milliosmol / kg.

Also, a particular embodiment of the present invention concerns the combination of the stabilizing agent (iii) with a reasonably high concentration of an ionic concentration modifying agent (v / a) selected from the group consisting of sodium salts and magnesium salts . In this embodiment, the ionic concentration modifying agent (v / a), i.e. the sodium salt and / or magnesium salt, is present in a concentration of 15 to 1000 mM, such as 25 to 1000 mM, 50 to 1000 mM, 100 to 1000 mM, 200 to 1000 mM, 300 to 1000 mM, 400 to 1000 mM, 500 to 1000 mM, 600 to 1000 mM, 700 to 1000 mM; 15 to 800 mM, 25 to 800 mM, 50 to 800 mM, 100 to 800 mM, 200 to 800 mM, 300 to 800 mM, 400 to 800 mM, 500 to 800 mM; 15 at 600 mM, 25 to 600 mM, 50 to 600 mM, 100 to 600 mM, 200 to 600 mM, 300 to 600 mM; 15 to 400 mM, 25 to 400 mM, 50 to 400 mM or 100 to 400 mM.

Within these embodiments, the sodium salt can be sodium chloride and the magnesium salt can be selected from the group consisting of magnesium chloride, magnesium acetate, magnesium gluconate, magnesium levulate and strong acid magnesium salts . In a more specific embodiment, a magnesium salt is used in combination with sodium chloride.

In a currently preferred embodiment, the composition comprises one or more ion concentration modifying agents selected from magnesium salts (Mg³⁺), for example, one or more salts selected from the group consisting of magnesium chloride, magnesium acetate, magnesium sulfate, magnesium gluconate, magnesium levulate, magnesium salts of strong acids. In one of its forms of realization, the

IQ concentration of the magnesium (Mg) salt (s) is at least 2 mM, such as at least 5 mM or about 10 mM.

Other ingredients

In addition to the above-mentioned components, the aqueous, liquid pharmaceutical composition may comprise additional components beneficial to the preparation, formulation, stability, or administration of the composition.

Thus, the composition may further comprise an antioxidant (vi). In different embodiments, the antioxidant is selected from the group consisting of L-methionine, D-methionine, methionine analogs, methionine-containing peptides, ascorbic acid, cysteine, homocysteine, glutathione, cystine, and cystathionine. In a preferred embodiment, the antioxidant is L-methionine.

The concentration of the antioxidant is typically 0.1 to 5.0 mg / ml, such as 0.1 to 4.0 mg / ml, 0.1 to 3.0 mg / ml, 0.1 to 2.0 mg / ml, or 0.5 to 2.0 mg / ml.

In particular embodiments, the composition does not include an antioxidant; instead, the susceptibility of the Factor VII polypeptide to oxidation is controlled by excluding atmospheric air. The use of an antioxidant naturally can also be combined with the exclusion of atmospheric air.

Thus, the present invention also provides an airtight container (for example, a vial or a cartridge (such as a cartridge for a pen-shaped applicator)) containing an aqueous, liquid pharmaceutical composition as defined herein, and optionally an inert gas. This aspect is discussed below.

In addition to the mandatory components, the stabilizing agent (iii), the nonionic surfactant (iv), the tonicity modifying agent (v) and the optional antioxidant (vi), the pharmaceutical composition can also comprise a preservative (vii).

A preservative can be included in the composition to slow microbial development and thereby allow the "multiple use" packaging of Factor VII polypeptides. Examples of preservatives include phenol, benzyl alcohol, ortho-cresol, meta-cresol, para-cresol, methyl paraben, propyl paraben, benzalkonium chloride, and benzethonium chloride. The preservative is usually included in a concentration of 0.1 to 20 mg / ml depending on the pH range and the type of preservative.

Furthermore, the composition can also include an agent capable of inhibiting deamidation and isomerization.

Particular embodiments

The present inventors have currently identified the following embodiments as particularly advantageous: An aqueous, liquid pharmaceutical composition comprising:

0.1 to 10 mg / ml of a Factor VII (i) polypeptide; a buffering agent (ii) suitable for maintaining the pH in the range of about 5.0 to about 9.0; and a tonicity modifying agent (v) in a concentration of at least 5 mM, • a.

wherein the molar ratio of non-complex calcium (Ca) ions to the Factor VII polypeptide is less than 0.5.

An aqueous, liquid pharmaceutical composition comprising:

0.1 to 10 mg / ml of a Factor VII (i) polypeptide; a buffering agent (ii) suitable for maintaining the pH in the range of about 5.0 to about 9.0;

a nonionic surfactant (iv); and a tonicity modifying agent (v) at a concentration of at least 5 mM, where the molar ratio of non-complex calcium ions (Ca²⁺) 10 for the Factor VII polypeptide is less than 0.5.

An aqueous, liquid pharmaceutical composition comprising:

0.1 to 10 mg / ml of a Factor VII (i) polypeptide; a buffering agent (ii) suitable for maintaining the pH in the range of about 5.0 to about 9.0;

a stabilizing agent (iii); a nonionic surfactant (iv); and a tonicity modifying agent (v) at a concentration of at least 5 mM, where the molar ratio of non-complex calcium ions (Ca) to the Factor VII polypeptide is less than 0.5.

An aqueous, liquid pharmaceutical composition comprising:

0.1 to 10 mg / ml of a Factor VII (i) polypeptide;

a buffering agent (ii) suitable for maintaining the pH in the range of about 5.0 to about 9.0;

a copper-containing agent (iiia) at a concentration of at least 5 μΜ and / or a manganese-containing agent (iiia) at a concentration of at least 100 μΜ;

a nonionic surfactant (iv); and a tonicity modifying agent (v) at a concentration of at least 5 mM, where the molar ratio of non-complex calcium ions (Ca²⁺) for the Factor VII polypeptide is less than 0.5.

An aqueous, liquid pharmaceutical composition comprising:

0.1 to 10 mg / ml of a Factor VII (i) polypeptide; a buffering agent (ii) suitable for maintaining the pH in the range of about 5.0 to about 9.0;

at least one stabilizing agent (iiib) comprising the -CôH4-C motif (= NZ¹-Rⁱ) -NH-Z²-R² at a concentration of at least 5 μΜ and / or at least one stabilizing agent (iiib) comprising the CH motif₂-NH-C (= NZ¹-R¹) -NH-Z²-R² at a concentration of at least 500 μΜ;

a nonionic surfactant (iv); and a tonicity modifying agent (v) at a concentration of at least 5 mM, where the molar ratio of non-complex calcium ions (Ca²⁺) for the Factor VII polypeptide is less than 0.5.

In the above embodiments, the buffering agent preferably comprises phosphoric acid.

Properties of the compositions of the present invention

The compositions according to the present invention are useful as stable compositions and preferably ready for use with Factor VII polypeptides. In addition, it is believed that the specific principles, guidelines and embodiments provided here are also applicable for the bulky storage of Factor VII polypeptides, mutatis mutandis.

The compositions are typically stable for at least six months, and preferably up to 36 months; when stored at temperatures ranging from 2 ° C to 8 ° C. The compositions are chemically and / or physically stable, in particular chemically stable, when stored for at least 6 months at 2 ° C to 8 ° C.

The term "stable" is intended to denote that (i) after storage for 6 months at 2 ° C to 8 ° C the composition maintains at least 50% of its initial biological activity as measured by a single stage clot test ( Test 4), or (ii) after storage for 6 months at 2 ° C to 8 ° C, the content of heavy chain degradation products is a maximum of 40% (w / w) in the event that the initial sample does not comprise no heavy chain degradation products (ie, only the Factor VII polypeptide is introduced in the percentage calculation).

For the purpose of determining biological activity as measured by a single stage clot assay (Assay 4), the sample to be tested is diluted in 50 mM Tris (pH 7.5), 0.1% BSA and 100 μΐ is incubated with 100 μΐ of Factor VII deficient plasma and 200 μί of

Thromboplastin C is 10 mM Ca. Clotting times are measured and compared to a standard curve using a reference standard or a reservoir of normal human plasma citrated in the serial dilution.

Preferably, the stable composition maintains at least 70%, such as at least 80%, or at least 85%, or at least 90%, or at least 95%, of its initial activity after storage for 6 months at 2 to 8 ° C.

For the purpose of determining the content of the heavy chain degradation products, a reverse phase HPLC was conducted on a 4.5 x 250 mm butyl bonded silica column registered with a 5 pm particle size and pore size of 300 À. Column temperature: 70 ° C. Buffer A: 0.1% v / v trifluoroacetic acid. Buffer B: 0.09% v / v trifluoroacetic acid, 80% v / v acetonitrile. The column was eluted with a linear gradient from X to (X + 13)% B over 30 minutes. X was adjusted so that FVIIa elutes with a retention time of approximately 26 minutes. Flow rate: 1.0 ml / min. Detection: 214 nm.

Load: 25 pg of FVIIa.

The term "physically stable" is intended to mean a composition that remains visually clear. The physical stability of the compositions is assessed by means of visual inspection and turbidity after storing the composition at different temperatures for various periods of time. Visual inspection of the compositions is carried out in sharp focused light with a dark background. A composition is classified as physically unstable when it has visual turbidity.

The term "physical stability" of Factor VII polypeptides refers to the formation of insoluble and / or soluble aggregates in the form of dimeric, oligomeric and polymeric forms of Factor VII polypeptides as well as any structural deformation and denaturation of the molecule.

The term "chemically stable" is intended to refer to a composition that maintains at least 50% of its initial biological activity after storage for 6 months at 2 to 8 ° C, as measured by a single stage clot test (Test 4) .

The term "chemical stability" is intended to refer to the formation of any chemical changes in Factor VII polypeptides in solution storage under accelerated conditions. Examples are hydrolysis, deamidation, isomerization and oxidation as well as enzymatic degradation resulting in the formation of Factor VII polypeptide fragments. In particular, sulfur-containing amino acids are prone to oxidation with the formation of the corresponding sulfoxides.

Preparation of the compositions of the present invention

In another aspect, the invention also provides a method for preparing the aqueous, liquid pharmaceutical compositions of the invention.

Thus in one embodiment, the method for preparing the aqueous, liquid pharmaceutical composition of a Factor VII polypeptide comprises the step of providing the Factor VII polypeptide (i) in a solution comprising a buffering agent (ii) suitable for maintaining the pH in the range of about 5.0 to about 9.0; while ensuring that, in the final composition, the molar ratio of non-complex calcium ions (Ca²⁺) for the Factor VII polypeptide is less than 0.5.

Thus in another embodiment, the method for preparing an aqueous, liquid pharmaceutical composition of a Factor VII polypeptide comprises the step of providing the Factor VII polypeptide (i) in a solution comprising a buffering agent (ii) suitable for maintaining the pH in the range of about 5.0 to about 9.0; at least one metal-containing agent (iii), wherein said metal is selected from the group consisting of metals from the +11 oxidation state first transition series; and a non-ionic surfactant (iv); while ensuring that, in the final composition, the molar ratio of non-complex calcium ions (Ca) to the Factor VII polypeptide is less than 0.5.

Thus, in yet another embodiment, the method for preparing the liquid, aqueous pharmaceutical composition of a Factor VII polypeptide comprises the step of providing the Factor VII polypeptide in a concentration of at least 0.01 mg / ml (i ) in a solution comprising a buffering agent (ii) suitable for maintaining the pH in the range of about 5.0 to about 9.0; and at least one stabilizing agent (iiib) comprising a -C (= NZ¹-R¹) -NH-Z²-R², on what

Z and Z are independently selected from the group consisting of -O-, -S-, -NR^H- and a simple link, where R^H is selected from the group consisting of hydrogen, C1- alkyl₄, aryl and arylmethyl, and R and R are independently selected from the group consisting of hydrogen, alkyl

Optionally substituted C_6, C alkenyl₂_₆ optionally substituted, optionally substituted aryl, optionally substituted heterocyclyl, or

Z² and R² are as defined above and form part of a heterocyclic ring, or

Z¹ and R¹ are as defined above and -C-NH-Z²-R² forms part of a heterocyclic ring, or

-C (= NZ¹-R¹) -NH-Z²-R² forms a heterocyclic ring in which Z¹-R¹-R²-Z²-is a bi-radical;

while ensuring that, in the final composition, the molar ratio of non-complex calcium ions (Ca²⁺) for the Factor VII polypeptide is less than 0.5.

It should be understood that maintaining the molar ratio of ions

Não i non-complex calcium (Ca) for the Factor VII polypeptide less than 0.5 can be carried out by selecting the appropriate starting materials where the concentration of “free” (ie, non-complex) calcium ions is very low, or adding a calcium chelator to bind calcium ions. In the last example, the calcium chelator is typically added in an amount that roughly corresponds to the concentration of the "free" calcium ions.

Usage methods

The aqueous, liquid pharmaceutical compositions defined herein can be used in the field of medicine as ready-to-use compositions or a bulky solution for the preparation of ready-to-use compositions. Thus, the present invention in particular provides the aqueous, liquid pharmaceutical compositions defined herein for use as a medicament, more particularly for use as a medicament to treat a factor VII responsive syndrome.

Consequently, the present invention also provides the use of the aqueous liquid pharmaceutical composition as defined herein for the preparation of a medicament to treat a Factor VII-responsive syndrome, as well as a method for treating a Factor VII-responsive syndrome, the method comprising administering to a patient in need thereof an effective amount of the aqueous liquid pharmaceutical composition as defined herein. The preparations of the present invention can be used to treat any Factor VII-responsive syndrome, such as, for example, bleeding disorders, including those caused by coagulation factor deficiencies (e.g., hemophilia A, hemophilia B, factor XI deficiency coagulation, deficiency of Coagulation Factor VII); by thrombocytopenia or von Willebrand's disease, or by coagulation factor inhibitors, and intra-cerebral hemorrhage, or excessive bleeding from any cause. The preparations can also be administered to patients in association with surgery or other trauma or to patients receiving anticoagulant therapy.

The term "effective amount" is the effective dose to be determined by a qualified physician, who can titrate the dosages to obtain the desired response. Factors for dose consideration will include potency, bioavailability, desired pharmacokinetic / pharmacodynamic profiles, condition of treatment, factors related to the patient (eg weight, health, age, etc.), presence of co-administered medications (eg , anticoagulants), time of administration, or other factors known to a physician.

The term "treatment" is defined as the handling and care of a patient, for example, a mammal, in particular a human being, for the purpose of combating the disease, condition, or disorder and includes the administration of a Factor VII polypeptide to prevent the onset of symptoms or complications, or to relieve symptoms or complications, or to eliminate the disease, condition, or disorder. The pharmaceutical compositions according to the present invention containing a Factor VII polypeptide can be administered parenterally to patients in need of such treatment. Parenteral administration can be performed by subcutaneous, intramuscular or intravenous injection using a syringe, optionally a pen-type syringe. Alternatively, parenteral administration can be performed by means of an infusion pump.

In important embodiments, the pharmaceutical composition is adapted for subcutaneous, intramuscular or intravenous injection according to methods known in the art. The possibly high concentration of metal ions (in particular the divalent metal ions of the metal-containing agent (iiia)) in the pharmaceutical compositions defined herein can be disadvantageous for certain groups of patients. The present invention therefore also provides a method before use to decrease the concentration of metal ion in a liquid, aqueous pharmaceutical composition, wherein said method comprises the step of contacting the liquid, aqueous pharmaceutical composition defined herein with a cation exchange.

An example of a cation exchange material is Chelex-100 (Fluka-Riedel / Sigma-Aldrich). The cation exchange material, for example, the Chelex-100, is preferably contained in a sterile container, for example, in a glass or plastic cartridge.

The aqueous, liquid pharmaceutical composition is considered to be contacted with the cation exchange material, for example, by passing through a cartridge containing the cation exchange material, immediately before use. In a particular embodiment, the cartridge is considered to be an integral part of a syringe assembly.

Suitable container for the pharmaceutical composition

As mentioned above, the present invention also provides an airtight container (for example, a vial or a cartridge (such as a cartridge for a pen-shaped applicator)) containing an aqueous, liquid pharmaceutical composition as defined herein, and optionally a gas inert. The inert gas can be selected from the group consisting of nitrogen, argon, etc. The container (e.g., bottle or cartridge) is typically made of glass or plastic, in particular glass, optionally closed by a rubber septum or other closure means taking into account penetration with preservation of the integrity of the pharmaceutical composition. In a particular embodiment of this, the composition does not comprise a preservative (vii). In another embodiment, the container is a bottle or cartridge enclosed in a sealed bag, for example, a sealed plastic bag, such as a laminate (for example, metal-laminated plastic bag (such as aluminum)).

More particularly, the airtight container, at least partially filled, contains an aqueous, liquid pharmaceutical composition as defined herein, and optionally an inert gas, said container comprising (i) a wall portion and (ii) one or more closure means which they do not form part of said wall portion. Preferably, the pharmaceutical composition does not comprise a preservative (vii).

In particular, the inner wall material of the container is a material selected from the group consisting of silica coated glass, silicon coated glass, non-cyclic olefin polymers, cycloolefin polymers, and linear cycloolefin / olefin copolymers.

In a variant, the inner wall of a container includes varying degrees / types of glass to which a coating of silica (silicon dioxide, SiO₂) was applied; one such material that is very well suited is so-called "Type I" glass (as defined in European Pharmacopeia, Ph. Eur.) coated with silica. For the definition of, and characterization tests for, Type I glass and other types of pharmaceutically applicable glass (Types II, III and IV), see, for example, section 3.2.1 of Ph. Eur. At the World Wide Web following: http: // online .pheur.org / 404TER / ep404. dll? f = templates & fn = main-h.htm & 2.0.

Type I glass containers are described in section 3.2.1 of Ph. Eur. (4^The Edition, online) as follows: “They are made of neutral glass and have a high hydrolytic resistance due to the chemical composition of the glass itself.”, The neutral glass being defined as follows: “The neutral glass is a borosilicate glass containing significant amounts of boric oxide, aluminum or alkaline earth oxides. Due to its composition, neutral glass has a high thermal shock resistance and a very high hydrolytic resistance. ”

The silica coating on the inner wall of such a container will preferably have a substantially uniform thickness of at least about 0.05 pm, although a substantially uniform thickness is believed to be in the range of about 0.1 pm to about 0 , 2 pm in general is more desirable. Chemical Vapor Deposition (CVD) appears to be a technique that is very well suited for applying such a substantial and uniformly thick coating of silica to glass surfaces, and Type I glass containers (eg vials) in which a coating silica which has been deposited by a CVD technique on the inner surface of the container, and which are very suitable for use in the context of the invention, are commercially available, for example, Schott Type I plus® containers from Schott Glaskontor, Muliheim / Baden, Germany. Reference can be made, for example, to the following article on WorldWideWeb for a description of CVD techniques: http://www.azom.com/details.asp?ArticleID=1552.

As also indicated above, other preferred materials for the inner wall of a container include varying degrees / types of glass that normally after washing or initial infusion in water or another aqueous medium to remove water-leachable substances or species have been coated with a silicone. As before, a preferred type of glass in this context is Type I (Ph. Eur.) Glass.

The term "silicone" is used widely here to denote not only silicones per se, which are typically dialkylated, diarylated or monoalkylated + polymeric monoarylated siloxanes, but also copolymers, typically block and graft copolymers comprising segments of silicone and segments of other materials polymeric materials such as polystyrene, polyolefins, polyamides or polyurethane.

The coating material may suitably be an oil (poly (dialkyl siloxane)) or copolymer, and suitable types of poly (dialkyl siloxane) which in this context include poly (dimethyl siloxane) (PDMS), poly (dipropyl siloxane) and poly ( diexyl-siloxane).

The viscosity of the oil when applied to the component can be of importance, especially for the elimination of the slip-stick phenomenon that can arise, for example, when the container in question is a cartridge or the like comprising a displaceable diver used to expel the liquid ( protein formulation) of the container. The more viscous, the lower the risk of a slip-stick phenomenon with which the diver's smooth movement is prevented. In one embodiment, the coating comprises a linear or branched hydrophilized poly (dialkyl siloxane) oil. The viscosity of the oil is preferably above 200,000 centistokes, as well as above 500,000 centistokes when applied to the component.

The silicone coating can also comprise a cross-linked or gelled silicone oil, such as a hydrophilized poly (dialkylsiloxane) oil, or a mixture of a cross-linked and a non-cross-linked oil. Using a crosslinked or gelled oil, the oil's migration capacity is significantly reduced, and the coating can be considered as a solid material.

A crosslinked, or cured silicone oil is typically obtained by applying a linear, or branched, silicone oil with reactive functionality that is used to crosslink the coating in a subsequent step. There are several different crosslinking methods available, for example, curing by irradiation with UV light, curing by heating at elevated temperature, and curing in the presence of water. A cross-linked silicone oil can also be obtained by first applying a straight or branched chain silicone oil, and then by irradiating the oil with a high energy irradiation source, for example, an electron source or light source X. The crosslinkable silicone oil may suitably be a medical grade, for example, MDX® supplied by Dow Corning (Fluid MDX44159); other suitable types include Wacker E2 silicone oil, supplied as an aqueous emulsion of approx. 35 %.

In another embodiment, the silicone coating comprises a block copolymer and a hydrophilized poly (dialkyl siloxane) graft. The copolymer can be any block and graft copolymer that comprises polymeric poly (dialkyl siloxane) segments, such as PDMS. The polymeric segments, for example, can be combined with polymeric segments of polystyrene, polyolefins, polyamides or polyurethane to form the desired copolymer. The copolymer can be prepared by any suitable known method, for example by sequential anionic polymerization, or various grafting procedures.

The hydrophilicity of a silicone coating can be achieved by any appropriate method, for example, by subjecting the coating to an oxidative treatment, such as plasma treatment or corona treatment, after it has been applied to the glass surface. Hydrophilicity can also be achieved by capping a copolymer with hydrophilic group or chain segments at the end. The hydrophilic group, for example, can be a negatively charged chemical group or phosphorylcholine (PC) groups, and the chain segment, for example, can be poly (ethylene oxide) (PEO) or poly (243 hydroxyethyl methacrylate) (pHEMA).

Plasma-treated surfaces can be modified to decrease protein absorption by linking hydrophilic polymeric segments or functional groups. These polymeric segments or functional groups can be of the same type as those described above, and can be further linked to the functional groups generated during plasma treatment.

The thickness of the silicone coating depends on the specific coating, and is preferably from 0.005 to 10 pm, more preferably from 0.01 to 1 pm. The optimum thickness depends on the dimensions, shape and type of the container, and can easily be determined by a person skilled in the art. In the case, for example, of a cartridge with a displaceable diver or part of the plunger, if the coating is too thin it can be broken in use, thereby increasing the friction between the diver and the wall part. When the coating thickness has reached a certain threshold value, the frictional forces are approximately constant, even when the thickness is more increased. For any coating composition the coating should preferably be as thin as possible to reduce costs. Such a thin coating may suitably have a thickness of 0.005 to 0.4 pm, such as 0.015 to 0.25 pm, more preferably about 0.2 pm.

Depending on the migration capacity of the silicone coating, the hydrophilic groups in the coating will tend to go into the coating leaving the surface hydrophobic due to the hydrophobicity of the surrounding air. In the case of a container that must be filled with the aqueous pharmaceutical composition, liquid of a Factor VII polypeptide, it is therefore desirable - in order to minimize any tendency of the protein in an aqueous liquid formulation to absorb onto the surface of the internal container - which the coating remains hydrophilic during storage until the liquid protein formulation is introduced into the container. This is most simply achieved by filling the container with the protein formulation abruptly after the coating process has taken place.

As indicated above, other preferred materials for the inner wall of a container in the context of the present invention include polymers of non-cyclic olefins (i.e., straight or branched chain), i.e., polyalkenes. Among such materials, useful polymers derived from a single monomer include polyethylenes and polypropylenes, numerous grades of which are partially crystalline in structure. Non-cyclic olefin copolymers [for example, copolymers of ethylene (ethylene) and propylene (propene)] are thus of interest as the inner wall materials in the context of the invention.

As also indicated above, other preferred materials for the inner wall of a container include cycloolefin polymers, and suitable types of these include those consisting of substantially 100% 5- to 7-membered aliphatic cyclic hydrocarbon rings. Suitable commercially available containers made of cycloolefin polymeric material include containers made of CZ® resin, available from Daikyo Seiko Ltd., Tokyo, Japan. Other relevant polymeric materials of this type include Zeonor® and Zeonex®, both from Nippon Zeon Co. Ltd. Tokyo, Japan.

Suitable types of cycloolefin / linear olefin copolymers include materials with an amorphous structure, such as highly transparent copolymers of the Topas® type (obtainable from Ticona GmbH, Frankfurt am Main, Germany), which are available in a variety of grades ( for example, Topas® 8007, Topas® 5013, Topas® 6013, Topas® 6015 and Topas® 6017).

In a variant, the container having as a material of the inner wall of the container a solid phase material which, when incubated for at least 24 months at a temperature not exceeding 40 ° C in contact with water or an aqueous solution having a pH of about 3 to about 8 releases at most about 3 μΜ of a trivalent metal ion in the solution; the container comprising (i) a portion of the wall and (ii) one or more closing means that are not part of the portion of the wall.

Although it is believed (as indicated above) that an acceptable upper limit for the level / concentration released of trivalent metal ions is about 3 μΜ (ie, level released <about 3 μΜ), a level released at most, about 2.5 μΜ (i.e., <about 2.5 μΜ), most desirably at most, about 1 μΜ (i.e., <about 1 μΜ), as at most, about 0.5 μΜ (ie, <about 0.5 μΜ), appears to be advantageous.

With respect to trivalent metal ions in the context of the two later aspects of the present invention, the release of Al appears to be particularly undesirable; Fe is another example of a trivalent metal ion whose release into the solution must be avoided. In addition to preventing the release of trivalent metal ions into the solution, it is still believed that it is desirable to prevent the release into the solution of certain divalent metal ions, particularly Zn. In this context, the released levels should probably not exceed about 3 μΜ (ie, released level <about 3 μΜ), more preferably about 1 μΜ (ie released level <about 1 μΜ), as at most about 0.5 μΜ (that is, <about 0.5 μΜ).

It can be mentioned at this point that although coated glass materials, notably silica coated glass (notably silica coated Type I glass) and silicon coated glass (notably silicone coated Type I glass), are among the preferred inner wall materials in the context of various aspects of the invention, they can in order to satisfy the criteria demonstrated above with respect to the release of trivalent or bivalent ions in the solution - in some embodiments they are sufficient to use a glass, particularly a Type I glass (Ph. Eur.), which has been subjected to a washing or extraction treatment that reduces the level of extractable trivalent and divalent metal ions present on the glass surface. Such treatments include infusion in (extraction with) hot water (preferably at least 90 ° C) or another aqueous medium, for example, ammonium sulfate solution.

In another embodiment, said container is a vial or cartridge comprising a closure means comprising a needle-penetrable, self-sealing elastomeric septum. In particular, the container is a cartridge further comprising a displaceable plunger means with which the liquid present in said container can be expelled from said container.

EXPERIMENTS

General methods

The percentages are (weight / weight) both when referring to solids dissolved in solution and to liquids mixed in solutions. For example, for Tween, they are the weight of 100% stock / weight of the solution. Adequate assays to determine the biological activity of Factor VII polypeptides

Factor VII polypeptides useful in accordance with the present invention can be selected by suitable assays that can be performed as simple preliminary in vitro tests. Thus, the present specification discloses a simple test (titled “In Vitro Hydrolysis Assay”) for the activity of Factor VII polypeptides.

In vitro hydrolysis assay (Assay 1)

Polypeptides of Factor Vila and Factor VII natural (wild type) (both hereinafter referred to as “Factor Vila”) can be tested for specific activities. They can also be tested in parallel to directly compare your specific activities. The assay is performed on a microtiter plate (MaxiSorp, Nunc, Denmark). The chromogenic substrate D-Ile-Pro-Arg-p-nitroanilide (S-2288, Chromogenix, Sweden), final concentration of 1 mM, is added to Factor Vila (final concentration of 100 nM) in 50 mM HEPES, pH 7 , 4, containing 0.1 M NaCl, 5 mM CaC12 and 1 mg / ml of bovine serum albumin. The absorbance at 405 nm is measured continuously on a SpectraMax® 340 plate reader (Molecular Devices, USA). The absorbance developed during a 20-minute incubation, after subtracting the absorbance in an empty reservoir containing no enzyme, is used to calculate the ratio between the activities of the Factor VII and Factor Vila polypeptides of the wild type:

Ratio = (A405 nm of Factor VII polypeptide) / (A405 nm of Factor Vila wild type).

Based on this, Factor VII polypeptides with an activity less than, comparable to, or greater than the natural Factor Vila can be identified, such as, for example, Factor VII polypeptides where the ratio between the activity of the polypeptide of Factor VII and the activity of natural Factor VII (wild type FVII) is about 1.0 versus over 1.0.

The activity of Factor VII polypeptides can also be measured using a physiological substrate such as Factor X (“In Vitro Proteolysis Assay”), appropriately at a concentration of 100 to 1000 nM, where the generated Factor Xa is measured after adding a suitable chromogenic substrate (for example, S-2765). In addition, the activity test can be conducted at physiological temperature.

In Vitro Proteolysis Assay (Assay 2)

The natural Factor Vila and Factor VII polypeptides (wild type) (both hereinafter referred to as “Factor Vila”) are tested in parallel to directly compare their specific activities. The assay is performed on a microtiter plate (MaxiSorp, Nunc, Denmark). Vila Factor (10 nM) and X Factor (0.8 microM) in 100 μΐ of 50 mM

HEPES, pH 7.4, containing 0.1 M NaCl, 5 mM CaCl₂ and 1 mg / ml of bovine serum albumin, are incubated for 15 min. Factor X divination is then interrupted by the addition of 50 μί of 50 mM HEPES, pH 7.4, containing 0.1 M NaCl, 20 mM EDTA and 1 mg / ml of bovine serum albumin. The amount of Factor Xa generated is measured by the addition of the chromogenic substrate ZD-Arg-Gly-Arg-p-nitroanilide (S-2765, Chromogenix, Sweden), final concentration of 0.5 mM. The absorbance at 405 nm is measured continuously on a SpectraMax® 340 plate reader (Molecular Devices, USA). The absorbance developed during 10 minutes, after subtracting the absorbance in an empty reservoir containing no FVIIa, is used to calculate the ratio between the proteolytic activities of the Factor VII and Factor Vila polypeptides of the wild type:

Ratio = (A405 nm of Factor VII polypeptide) / (A405 nm of Factor Vila wild type).

Based on this, the Factor VII polypeptide with an activity less than, comparable to, or greater than the natural Vila Factor can be identified, such as, for example, Factor VII polypeptides where the ratio between the activity of the polypeptide of Factor VII and the activity of natural Factor VII (wild type FVII) is about 1.0 versus above 1.0. Thrombin generation assay (Assay 3)

The ability of Factor Vila or Factor VII polypeptides to generate thrombin can also be measured in an assay (Assay 3) comprising all relevant factors and coagulation inhibitors in physiological concentrations (negative Factor VIII when mimicking hemophilia A conditions) and platelets activated (as described on page 543 in Monroe et al. (1997) Brit. J. Haematol. 99, 542 to 547, which is hereby incorporated by reference).

Single stage coagulation test (Test 4)

The biological activity of Factor VII polypeptides can also be measured using a single stage coagulation assay (Assay 4). For this purpose, the sample to be tested is diluted in 50 mM PIPES buffer (pH 7.5), 0.1% BSA and 40 μΐ are incubated with 40 μΐ of Factor VII deficient plasma and 80 μΐ of factor of recombinant human tissue containing 10 mM Ca²⁺ and synthetic phospholipids. Clotting times are measured and compared to a standard curve using a reference standard in a parallel line assay.

Preparation and purification of Factor VII polypeptides

The purified human Vila Factor suitable for use in the present invention is preferably manufactured by recombinant DNA technology, for example, as described by Hagen et al., Proc. Natl. Acad. Know. USA 83: 2412 to 2416, 1986, or as described in European Patent N² 0 200 421 (ZymoGenetics, Inc.).

Factor VII can also be produced by the methods described by Broze and Majerus, J. Biol. Chem. 255 (4): 1242 to 1247, 1980 and Hedner and Kisiel, J. Clin. Invest. 71: 1836 to 1841, 1983. These methods produced Factor VII without detectable amounts of the other blood coagulation factors. Yet another preparation of the purified Factor VII can be obtained by including additional gel filtration as the final purification step. Factor VII is then converted to Factor Vila activated by known means, for example, by several different plasma proteins, such as Factor Xlla, IXa or Xa. Alternatively, as described by Bjoem et al. (Research Disclosure, 269 September 1986, pages 564 to 565), Factor VII can be activated by passing it through an ion exchange chromatography column, such as MonoQ® (Pharmacia fine Chemicals) or the like, or by self-activation in solution.

Factor VII-related polypeptides can be produced by modification of wild-type Factor VII or by recombinant technology. Polypeptides related to Factor VII with altered amino acid sequence when compared to Factor VII of the wild type can be produced by modifying the nucleic acid sequence that encodes Factor VII of the wild type by changing the amino acid codons or by removing some of the amino acid codons in the nucleic acid encoding natural Factor VII by known means, for example, by site-specific mutagenesis.

It will be evident to those skilled in the art that substitutions can be made outside regions critical to the function of the Factor Vila molecule and still result in an active polypeptide. Amino acid residues essential for the activity of the Factor VII polypeptide, and therefore preferably not subject to substitution, can be identified according to procedures known in the art, such as site-directed mutagenesis or Alanine scan mutagenesis (see, for example, Cunningham and Wells, 1989, Science 244: 1081 to 1085). In the subsequent technique, mutations are introduced into each positively charged residue on the molecule, and the resulting mutant molecules are tested for coagulant, respectively crosslinking activity to identify amino acid residues that are critical to the molecule's activity. The substrate-enzyme interaction sites can also be determined by analyzing the three-dimensional structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photo-affinity labeling (see, for example, by Vos et al., 1992, Science 255: 306 to 312; Smith et al., 1992, Journal of Molecular Biology 224: 899 to 904; Wlodaver et al., 1992, FEBS Letters 309: 59 to 64).

The introduction of a mutation in the nucleic acid sequence to exchange a nucleotide for another nucleotide can be effected by site-directed mutagenesis using any of the methods known in the art. Particularly useful is the procedure that uses a double-stranded, super-spiral DNA vector with an insert of interest and two synthetic primers containing the desired mutation. The oligonucleotide primers, each complementary to the opposite strands of the vector, extend during temperature cycling by means of Pfu DNA polymerase. Upon incorporation of the primers, a mutated plasmid containing staggered incisions is generated. Following temperature cycling, the product is treated with Dpnl which is specific for methylated and semi-methylated DNA to digest the precursor DNA pattern and select the synthesized DNA containing the mutation. Other procedures known in the art to create, identify and isolate variants can also be used, such as, for example, gene scrambling or phage display techniques.

The separation of polypeptides from their original cell can be achieved by any method known in the art, including, without limitation, removing the cell culture medium containing the desired product from an adherent cell culture; centrifugation or filtration to remove non-adherent cells; and others.

Optionally, Factor VII polypeptides can be further purified. Purification can be achieved using any method known in the art, including, without limitation, affinity chromatography, such as, for example, on an anti-Factor VII antibody column (see, for example, Wakabayashi et al., J. Biol Chem. 261: 11097, 1986, and Thim et al., Biochem. 27: 7785, 1988); hydrophobic interaction chromatography; ion exchange chromatography; size exclusion chromatography; electrophoretic procedures (eg, preparative isoelectric focusing (IEF), differential solubility (eg, ammonium sulfate precipitation), or extraction and others. See, in general, Scopes, Protein Purification, SpringerVerlag, New York, 1982; and Protein Purification, JC Janson and Lars Ryden, editors, VCH Publishers, New York, 1989. Following purification, the preparation preferably contains less than 10% by weight, more preferably less than 5% and most preferably less than 1%, of non-Factor VII polypeptides derived from the host cell.

Factor VII polypeptides can be activated by proteolytic divination, using Factor Xlla or other proteases having trypsin-like specificity, such as, for example, Factor IXa, kallikrein, Factor Xa, and thrombin. See, for example, Osterud et al., Biochem. 11: 2853 (1972); Thomas, US Patent 4,456,591; and Hedner et al., J. Clin. Invest. 71: 1836 (1983). Alternatively, Factor VII polypeptides can be activated by passing them through an ion exchange chromatography column, such as MonoQ® (Pharmacia) or the like, or by autoactivation in solution. The resulting activated Factor VII polypeptide can then be formulated and administered as described in the present application.

The following examples illustrate the practice of the invention. These examples are included for illustrative purposes only and are not intended in any way to limit the scope of the claimed invention. Work Examples

Example 1 - Effect of calcium content in aqueous rFVIIa solutions on heavy chain degradation (autocatalytic divage)

In order to investigate the effect of calcium ions on rFVIIa, the following procedure was followed:

RFVIIa (M_w approx. 50,000) was transferred to the following solutions by desalination on a PD-10 column (Amersham Biosciences):

Formulation 1-1:

rFVIIa

PIPES-di-Na

1 M NaOH or 1 M HCl

Ca ratio²⁺/ FVII

Formulation 1-2:

1.0 mg / ml

17.32 mg / ml (50 mM) added to pH 6.5 0 rFVIIa

Calcium chloride 2 H₂Sodium chloride Glycylglycine Sodium acetate Histidine

1 M NaOH or 1 M HCI

1.0 mg / ml

1.47 mg / ml (10 mM) 2.92 mg / ml (50 mM) 1.32 mg / ml (10 mM) 0.82 mg / ml (10 mM) 1.55 mg / ml (10 mM) added to pH 6.5

Ca ratio²⁺/ FVII 500

The formulations were stored at a temperature of

5 ° C or 25 ° C, respectively, and the analyzes were performed at the times indicated in Table 1.

Table 1 - Content of heavy chain degradation products (%) in rFVIIa formulations

<td></td><td>T = 0</td><td>T = 1 month</td><td>T = 2 months</td><td>T = 3 months</td>

<td>Formulation 1-1, 5 ° C</td><td> 9,6</td><td> 9,8</td><td> 9,8</td><td> 10,6</td>

<td>Formulation 1-2, 5 ° C</td><td> 10,2</td><td> 17,0</td><td> 23,8</td><td> 30,4</td>

<td>Formulation 1-1, 25 ° C</td><td> 9,6</td><td> 9,7</td><td> 9,7</td><td> 10,3</td>

<td>Formulation 1-2, 30 ° C</td><td> 10,2</td><td> 18,5</td><td> 23,5</td><td>na</td>

As can be seen from Table 1, the increase in the content of heavy chain degradation products in Formulation 1-2 was much greater than the increase for Formulation 1-1.

The content of the heavy chain degradation products was determined by RP-HPLC as described in the following:

Reverse phase HPLC was conducted on a 4.5 x 250 mm butyl bonded silica column registered with a particle size of 5 pm and pore size of 300 µm. Column temperature: 70 ° C. Buffer A: 0.1% v / v trifluoroacetic acid. Buffer B: 0.09% v / v trifluoroacetic acid, 80% v / v acetonitrile. The column was eluted with a linear gradient from X to (X + 13)% B over 30 minutes. X was adjusted so that the FVIIa elutes with a retention time of approximately 26 minutes. Flow rate: 1.0 ml / min. Detection: 214 nm. Load: 25 pg of FVIIa.

Example 2 - Effect of the content of calcium and divalent metal ions in aqueous rFVIIa solutions on heavy chain degradation (autocatalytic cleavage)

In order to investigate the effect of calcium ions and divalent metal ions on rFVIIa, the following procedure was followed:

The rFVIIa was transferred to the following solutions by desalination on a PD-10 column (Amersham Biosciences):

All formulations (2-1 to 2-8) included rFVIIa

Calcium chloride 2 H₂Sodium Chloride Glycylglycine Histidine

1 M NaOH or 1 M HCl

1.0 mg / ml

1.47 mg / ml (10 mM) 2.92 mg / ml (50 mM) 1.32 mg / ml (10 mM) 1.55 mg / ml (10 mM) added to pH 6.5 and still included benzamidine and EDTA as shown in Table 2

Table 2

<td>Formulation N²</td><td>Benzamidine (mM)</td><td>EDTA (mM)</td><td>non-complex calcium ions (mM)</td><td>Ca ratio²⁺/ rFVIIa</td>

<td> 2-1</td><td> 10</td><td> 0</td><td>about 10</td><td> 500</td>

<td> 2-2</td><td> 10</td><td> 9,9</td><td>about 0.1</td><td> 5</td>

<td> 2-3</td><td> 10</td><td> 15</td><td>about 0.0</td><td> 0,0</td>

<td> 2-4</td><td> 1</td><td> 0</td><td>about 10</td><td> 500</td>

<td> 2-5</td><td> 1</td><td> 9,9</td><td>about 0.1</td><td> 5</td>

<td> 2-6</td><td> 1</td><td> 15</td><td>about 0.0</td><td> 0,0</td>

<td> 2-7</td><td> 0</td><td> 0</td><td>about 10</td><td> 500</td>

The formulations were stored at a temperature of

5 ° C and the analyzes were performed at the times indicated in Table 3.

Table 3 - Content of heavy chain degradation products (%) in rFVIIa formulations

<td></td><td>T = 0</td><td>T = 2 weeks</td><td>T = 3 weeks</td><td>T = 4 weeks</td><td>T = 8 weeks</td>

<td>Formulation 2-1</td><td> 7,2</td><td> 8,0</td><td> 8,9</td><td> -</td><td> 9,9</td>

<td>Formulation 2-2</td><td> 7,3</td><td> 7,5</td><td> 7,8</td><td> -</td><td> 8,2</td>

<td>Formulation 2-3</td><td> 7,3</td><td> 7,3</td><td> 7,6</td><td> -</td><td> 7,9</td>

<td>Formulation 2-4</td><td> 7,5</td><td> 10,7</td><td> 12,8</td><td> -</td><td> 19,3</td>

<td>Formulation 2-5</td><td> 7,4</td><td> 8,2</td><td> 8,7</td><td> -</td><td> 10,3</td>

<td>Formulation 2-6</td><td> 7,2</td><td> 7,7</td><td> 8,1</td><td> -</td><td> 8,7</td>

<td>Formulation 2-7</td><td> 8,1</td><td> -</td><td> -</td><td> 16,3</td><td> -</td>

As can be seen from Table 2, the content of non-complex calcium ions had a significant influence on rFVH heavy chain degradation. The results also show that the stabilizing agent (benzamidine) was a more efficient stabilizer when the concentration of non-complex calcium ions was decreased. Thus, it was estimated that about the same stability could be obtained by concurrently reducing the concentration of benzamidine and the concentration of non-complex calcium ions (compare Formulations 2-3 and 2-6).

The content of the heavy chain degradation products was determined by RP-HPLC as described in Example 1.

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