HK1090284A - Stabilized aqueous compositions comprising tissue factor pathway inhibitor (tfpi) or tissue factor pathway inhibitor variant - Google Patents

Description

Stable liquid composition containing tissue factor pathway inhibitor or tissue factor pathway inhibitor variant

The present application claims copending provisional application filed on 8/1/2003: serial No.60/438,519, filed 13/8/2003, serial No.60/494,577, filed 8/19/2003, serial No.60/509,260, filed 20/10/2003, priority of the patent serial No.60/512,090, the contents of which are incorporated herein by reference.

Technical Field

The present invention relates to stable compositions comprising tissue factor pathway inhibitor protein (TFPI). More particularly, the present invention relates to compositions comprising TFPI or a TFPI variant, a cosolvent, and an antioxidant.

Background

The Tissue Factor Pathway Inhibitor (TFPI) protein is 276 amino acids in length and functions as an inhibitor of tissue factor-mediated blood clotting. The amino acid sequence is shown in SEQ ID NO: 1 is shown. The amino terminus of TFPI is negatively charged and the carboxy terminus is positively charged. The TFPI protein contains 3 Kunitz-type enzyme inhibitory domains. TFPI contains 18 cysteine residues, which when properly folded form 9 disulfide bonds. Its primary sequence contains 3N-linked consensus glycosylation sites (Asn-X-Ser/Thr). Asparagine residues at the glycosylation sites are located at positions 145, 195 and 256. TFPI is also known as lipoprotein-associated procoagulant inhibitor (LACI), Tissue Factor Inhibitor (TFI) and Exogenous Pathway Inhibitor (EPI).

TFPI has been suggested to be useful in the treatment of a variety of diseases, including sepsis (u.s.6,063,764 and WO93/24143), deep vein thrombosis (u.s.5,563,123, u.s.5,589,359 and WO 96/04378), ischemia (u.s.5,885,781, u.s.6,242,414, and WO 96/40224), restenosis (u.s.5,824,644 and WO 96/01649), and cancer (u.s.5,902,582 and WO 97/09063). TFPI variants differ from TFPI by the addition of an alanine residue at the amino terminus ("ala-TFPI"). Carr et al have reported (Circ Shock 1994 NOV; 44 (3): 126-37) that TFPI variants are effective in treating sepsis in animal models.

Following preparation, the TFPI or TFPI variant compositions may be packaged for storage in liquid form or in a frozen state. However, TFPI or TFPI variants form polymers when stored in liquid dosage forms. Polymerization resulting from interactions between TFPI or TFPI variant molecules may result in oligomer formation. These oligomers may remain soluble during storage or form large, visible polymer precipitates from solution. TFPI or TFPI variants may play an adverse role in the biological activity of the polymer formed during storage of the liquid composition, resulting in its loss of efficacy as an anti-coagulant in the treatment of various diseases, including sepsis. In addition, polymer formation may lead to other problems, such as clogging of tubing, membranes or pumps when compositions containing TFPI or TFPI variants are administered using infusion systems. To minimize these problems, there is a need in the art to improve the stability of TFPI and TFPI variant compositions.

Summary of The Invention

The present invention is based on the recognition that TFPI or a TFPI variant liquid composition comprising a co-solvent and an antioxidant has significantly improved stability. The antioxidant may be in the form of an oxygen displacing gas, an oxygen or radical scavenger or a chelating agent.

The present invention provides at least the following embodiments.

One embodiment of the invention is a liquid composition comprising about 0.05 to 15mg/ml TFPI or a TFPI variant; about 50-600mM of a cosolvent selected from the group consisting of (i) arginine or an analog thereof, (ii) lysine or an analog thereof, (iii) a mixture of (i) and (ii); and an antioxidant selected from the group consisting of (i) an oxygen-substituted gas, (ii) an oxygen or free radical scavenger, (iii) a chelating agent, and (iv) mixtures thereof; the liquid composition has (a) a percent polymerization stability of about 45% or greater; (b) the percent oxidation stability is about 45% or greater; (c) the pH is between about 4 and 8.

Another embodiment of the invention is a method of making a liquid TFPI or TFPI variant composition comprising adding to a liquid composition comprising 0.05 to 15mg/ml of TFPI or TFPI variant about 50 to 600mM of a cosolvent selected from the group consisting of (i) arginine or an analog thereof, (ii) lysine or an analog thereof, (iii) a mixture of (i) and (ii); and an antioxidant selected from the group consisting of (i) an oxygen-substituted gas, (ii) an oxygen or free radical scavenger, (iii) a chelating agent, and (iv) a mixture of (i), (ii), and (iii); the liquid composition has (a) a percent polymerization stability of about 45% or greater; (b) the percent oxidation stability is about 45% or greater; (c) the pH is about 4 to 8.

Yet another embodiment of the present invention is a pharmaceutical composition comprising the liquid composition and a pharmaceutically acceptable excipient. The liquid composition comprises about 0.05-15mg/ml TFPI or TFPI variant; and about 50 to 600mM of a cosolvent selected from the group consisting of (i) arginine or an analog thereof, (ii) lysine or an analog thereof, (iii) a mixture of (i) and (ii); and an antioxidant selected from the group consisting of (i) an oxygen-substituted gas, (ii) an oxygen or free radical scavenger, (iii) a chelating agent, and (iv) a mixture of (i), (ii), and (iii); the liquid composition has (a) a percent polymerization stability of about 45% or greater; (b) the percent oxidation stability is about 45% or greater; (c) the pH is about 4-8.

Brief description of the drawings

FIG. 1 shows the half-lives (t) during storage of 4 standard ala-TFPI compositions analyzed by ion exchange high pressure liquid chromatography (IEX-HPLC) at 50 deg.C_1/2By day) is a function of arginine concentration. All formulations contained 0.15mg/ml ala-TFPI buffered with L-arginine-base and citric acid buffer or L-arginine hydrochloride and 10mM citric acid/sodium citrateThe pH of the solution was adjusted to 5.5. The specific ala-TFPI formulations comprise: (a)20-150mM of L-arginine hydrochloride and 10mM of citric acid/sodium citrate as a buffer solution; (b)20-150mM L-arginine acid-base, titrating with citric acid; (c)100-300mM L-arginine hydrochloride, 10mM citric acid/sodium citrate as buffer; (d) 100-300 mML-arginine acid-base titrated by citric acid;

figure 2 shows the stability of a standard ala-TFPI composition as a function of dissolved oxygen concentration expressed as a percentage of complete saturation of air. The percentage of soluble ala-TFPI in stable samples stored at 30 ℃ was analyzed by Reverse Phase (RP) HPLC. The standard ala-TFPI composition contains 0.15mg/ml ala-TFPI, 20mM citric acid/sodium citrate and 300mM L-arginine. The pH value is 5.5.

FIG. 3 shows the half-life (t) of standard ala-TFPI compositions during storage_1/2In weeks) is a function of dissolved oxygen concentration expressed as a percentage of complete saturation of the air. The percentage of soluble ala-TFPI in stable samples stored at 30 ℃ was analyzed by Reverse Phase (RP) HPLC. The standard ala-TFPI composition contains 0.15mg/ml TFPI, 20mM citric acid/sodium citrate and 300mM L-arginine. The pH value is 5.5.

FIG. 4 shows the stability of a standard ala-TFPI composition containing the chelating agents EDTA and DTPA, added at 0, 1 or 4 mM. The percentage of soluble ala-TFPI in stable samples stored at 30 ℃ was analyzed by Reverse Phase (RP) HPLC. The standard ala-TFPI composition contains 0.15mg/ml ala-TFPI, 20mM citric acid/sodium citrate and 300mM L-arginine. The pH value is 5.5.

FIG. 5 shows the stability of standard ala-TFPI compositions containing the oxygen scavenger methionine added at 0, 2,5 or 10 mM. The percentage of soluble ala-TFPI in stable samples stored at 30 ℃ was analyzed by Reverse Phase (RP) HPLC. The standard ala-TFPI composition contains 0.15mg/ml ala-TFPI, 20mM citric acid/sodium citrate and 300mM L-arginine. The pH value is 5.5.

FIG. 6 is an RP-HPLC profile of ala-TFPI samples. Peaks A-F are described in example 1.

FIG. 7 is a Kaplan-Neier survival plot. The X-axis is the survival number; the Y-axis is time (hours).

Detailed Description

The liquid composition of the present invention is based on the following findings: adding to a liquid TFPI or TFPI variant composition i) an amino acid cosolvent (e.g., arginine, lysine, or analogs thereof) and ii) an antioxidant (wherein the liquid composition has a pH of about 4 to 8). Results in a substantial increase in the stability of a composition comprising TFPI or a TFPI variant during storage compared to a composition comprising TFPI or a TFPI variant which has not been combined with the two additional components during the production process. This overall improvement in the stability of the composition is achieved by the co-solvent in combination with the antioxidant, so that the composition is resistant not only to polymerization during storage, but also to deleterious oxidation, especially to the methionine residue of TFPI. The liquid compositions of the present invention are also resistant to other deleterious effects (e.g., unfolding, refolding and denaturation) that can result in their loss of biological activity or other undesirable properties.

Because the co-solvent and antioxidant primarily affect separate mechanisms of degradation of TFPI or TFPI variants (polymerization and methionine oxidation, respectively), the use of the co-solvent and antioxidant in combination, in combination with the non-combined use, even if one of the two ingredients is used, results in a more stable TFPI or TFPI variant composition. For example, oxidation of TFPI or TFPI variant methionine is undesirable even when TFPI or TFPI variant is biologically active.

Stability of liquid compositions

The liquid compositions of the present invention comprising TFPI or a TFPI variant generally have increased stability during storage with respect to one or more degradative effects (e.g., polymerization and methionine oxidation) as compared to compositions prepared without the use of co-solvents and antioxidants as described herein. This is because the percentage of polymerization stability and the percentage of oxidation stability of the TFPI or TFPI variant-containing compositions of the invention are increased, so that the half-life of unpolymerized, unoxidized TFPI or TFPI variant is also increased. The percent polymerization stability and percent oxidation stability of the TFPI or TFPI variant samples can be varied independently. More preferably, the TFPI or TFPI variant of the liquid compositions of the present invention are biologically active, as measured by the prothrombin time assay described below.

The liquid compositions of the present invention have a polymerization stability of at least 45%. "percent polymerization stability" refers to the ratio of soluble fractions measured in an accelerated stability assay at 40 ℃ for a sample of TFPI or a TFPI variant. In the accelerated stability assay at 40 ℃, TFPI or TFPI variant samples were incubated at 40 ℃ for 8 weeks. After incubation, TFPI or TFPI variant samples are filtered through a 0.2 μm filter and subjected to an ion exchange high performance liquid chromatography (IEX-HPLC) assay to determine the amount of soluble TFPI or TFPI variant in solution. IEX-HPLC tests below 45% are described below. Thus, for example, a TFPI or TFPI variant composition having 60% polymerization stability refers to a composition that has 60% soluble TFPI or TFPI variant as tested in an accelerated stability assay at 40 ℃. A TFPI or TFPI variant composition having 80% polymerization stability means that the composition has 80% soluble TFPI or TFPI variant as tested in an accelerated stability assay at 40 ℃. The percent polymerization stability of the TFPI or TFPI variant compositions of the present invention is preferably about 45, 50, 60, 70, or 75% or more, more preferably about 80, 82, 84, 85, 90, 92, 94, 95, 96, 97, 98, or 99% or more, as measured in an accelerated stability test at 40 ℃, and can range, for example, from about 45% or more to about 99% or more, from about 45% or more to about 70% or more, from about 60% or more to about 80% or more, from about 70% or more to about 90% or more, from about 80% or more to about 90% or more, from about 45% or more to about 70% or more.

The liquid composition of the present invention also has an oxidation stability of 45% or more. "percent oxidative stability" refers to the ratio of TFPI or TFPI variant samples that do not contain an oxidized methionine moiety as measured in an accelerated stability test at 30 ℃. In the accelerated stability assay at 30 ℃, TFPI or TFPI variant samples are incubated at 30 ℃ for 8 weeks. After incubation, TFPI or TFPI variant samples are assayed for the amount of methionine oxidized TFPI or TFPI variant in solution using a reverse phase high performance liquid chromatography (RP-HPLC) assay. The RP-HPLC test is described below. Thus, for example, a TFPI or TFPI variant composition having 60% oxidative stability means that the composition does not contain oxidized methionine for 60% of the TFPI or TFPI variant as measured in the accelerated stability test at 30 ℃. A TFPI or TFPI variant composition having 80% oxidative stability means that the composition does not contain oxidized methionine in 80% of the TFPI or TFPI variant as measured in an accelerated stability test at 30 ℃. The percent oxidative stability of the TFPI or TFPI variant compositions of the present invention is preferably about 45, 50, 60, 70, or 75% or more, more preferably about 80, 82, 84, 85, 89, 90, 91, 92, 94, 95, 96, 97, 98, or 99% or more, as measured in an accelerated stability test at 30 ℃, and can range, for example, from about 45% or more to about 99% or more, from about 45% or more to about 70% or more, from about 60% or more to about 80% or more, from about 70% or more to about 90% or more, from about 80% or more to about 90% or more.

The storage half-life of TFPI or TFPI variant in a liquid composition of the invention typically ranges from about 1 month to about 36 months (e.g., 1, 2, 3, 4,5, 10, 15, 20, 25, 30, 35, or 36 months), depending on the storage temperature. In view of polymerization and/or oxidative stability, the liquid compositions of the present invention comprising TFPI or a TFPI variant, a co-solvent, and an antioxidant typically have a half-life in storage of greater than 8 weeks at 15 ℃. These liquid compositions have a pH of about 4 to about 8. For example, TFPI or a TFPI variant has a storage half-life of from about 1 month to about 24 months (e.g., about 1, 2,4, 6,8, 10, 12, 14, 16, 18, 20, 22, or 24 months) at a temperature of about 15 ℃ or 30 ℃.

Storage temperature

The liquid composition of the present invention has improved storage stability whether stored as a liquid for later use or frozen for thawing before use. Storage temperatures can range from about-70 ℃ to about 25 ℃ (e.g., about-70, -60, -50, -40, -30, -20, -10,0, 1, 2, 3, 4,5, 6, 7, 8,9, 10, 12, 15, 18, 20, 21, 22, 23, 24, or 25 ℃). Preferably, the liquid compositions of the present invention are stored in a liquid form which increases storage stability, can be conveniently administered without reconstitution, and can be provided as a pre-filled, ready-to-use formulation in a syringe or as a multi-dose formulation if the formulation is compatible with the bacteriostatic agent. The preferred storage temperature for liquid formulations is about 2 ℃ to 8 ℃ (e.g., about 2, 3, 4,5, 6, 7, or 8 ℃).

TFPI and TFPI variants

TFPI is a polypeptide whose amino acid sequence is set forth in SEQ ID NO: 1. preferably, the TFPI is a recombinant human protein produced by a microbial host. WO 01/24814 further characterizes and describes the biological activity of TFPI.

TFPI variants include analogs and derivatives of TFPI, as well as fragments of TFPI, TFPI analogs, and TFPI derivatives. TFPI variants may be obtained by human or other mammalian sources, synthetic or recombinant techniques. Analogs thereof are TFPI molecules having one or more amino acid substitutions, insertions, deletions and/or additions. Conservative substitutions, i.e. the substitution of one amino acid by another of similar nature, are preferred. Examples of conservative substitutions include, but are not limited to: gly * Ala, Val * Ile * Leu, Lys * Arg, Asn * Gln and Phe * Trp * Tyr. They are typically in the range of 1 to 5 amino acids (e.g., 1, 2, 3, 4, or 5 amino acids). The additional amino acids may be added at any position of the molecule, especially at the amino or carboxy terminus. For example, N-L-alanyl-TFPI, is a TFPI analog with an alanine added to its amino terminus. The added amino acids may be 1, 2,5, 10, 25, 100 or more. Fusion proteins are included in this definition.

Fragments refer to a portion of TFPI, a TFPI analog, or a TFPI derivative. Examples of fragments include Kunitz domain 1, 2 or 3, Kunitz domain 1 and 2 or 2 and 3, or N-terminal or C-terminal deletions, or both. Practical guidelines for making variants are found in U.S.5,106,833. Fragments of TFPI contain seq id NO: 1, at least 20 conserved amino acids. For example, a fragment may be 20, 25, 30, 50, 100, 150, 200, 250, or 275 conserved amino acids in length. Biologically inactive fragments of TFPI are described in u.s.5,106,833. These fragments are also used in the present invention.

Derivatives are defined as TFPI, TFPI analogs, or TFPI fragments with an added moiety. Examples of such additional moieties include glycosylation, phosphorylation, acylation or amidation.

TFPI variants and SEQ ID NO: the percent homology between 1 can be determined using the Blast2 alignment program (Blosum62, Expect10, standard genetic code, open gap 11, extension gap 1, gapx _ dropoff 50, low complexity filtering). The amino acid sequence of the TFPI variant is identical to SEQ ID NO: 1 is generally 70% or greater, preferably about 80% or greater, more preferably about 90% to 95% (e.g., 90, 91, 92, 93, 94, or 95%) or greater, and most preferably 98% or 99% identical.

Preparation of amino acid sequence variants of TFPI can be performed by altering the DNA sequence encoding TFPI. Methods for altering nucleotide sequences are well known in the art. For example, see Walker and Gaastra editors (1983) molecular biology techniques (MacMillan press, New York), Kunkel (1985) proc.natl.acad.sci.usa 82: 488-492, Kunkel et al (1987) methods in enzymology (methods enzymol) 154: 367, 382, Sambrook et al (1989) molecular cloning: a laboratory manual (cold spring Harbor, New York), U.S.4,873,192, and references cited therein.

The TFPI variants preferably have a certain amount of biological activity, such as 10%, 30%, 50%, 60%, 80%, 90% or more of the biological activity of TFPI as measured by the Prothrombin (PT) assay described below. It is clear that any change in the DNA encoding the TFPI variant must not place its sequence out of frame and preferably does not produce a complementary region that can form the structure of the secondary mRNA. Computer programs well known in the art may be used to find technical guidelines for determining which amino acid residues may be substituted, inserted or deleted without causing TFPI or a TFPI variant to lose biological or immunological activity: for example, DNASTAR software, or Atlas of protein sequences and Structure (Natl. biomed. Res. found, Washington, D.C.) by Dayhoff et al (1978). Stable biologically inactive variants of TFPI are also contemplated.

TFPI or TFPI variants can be produced by recombinant methods, see u.s.4,966,852. For example, the cDNA for the desired protein can be incorporated into a plasmid and expressed in prokaryotes or eukaryotes. Those skilled in the art will know of many references that may provide details for the expression of proteins by microorganisms. See U.S.4,847,201 and Maniatas et al, 1982, molecular cloning: a laboratory Manual (Cold spring Harbor, New York).

Various techniques have been available for transforming microorganisms and expressing TFPI or TFPI variants using the transformed microorganisms. The following are only examples of some of the possible methods. The DNA sequence of TFPI or TFPI variants may be linked to appropriate control sequences. The DNA sequence of TFPI or a variant of TFPI may be incorporated into plasmids such as pUC13 or pBR322, which are available from Boehringer-Mannhein et al. Once the DNA sequence of TFPI or a TFPI variant is inserted into a vector, the vector may be cloned into an appropriate host. The DNA was amplified using techniques as described in U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,683,195. mRNA is produced by inducing cells (e.g., HepG2 and SKHep hepatoma cells) to obtain cDNA, and then the mRNA is identified and isolated, and reverse transcribed to obtain cDNA. After the expression vector is transformed into a host cell (e.g., E.coli), the bacterium is cultured to express the protein. Prokaryotic microorganisms are preferred, and E.coli is particularly preferred. A preferred microorganism for use in the present invention is Escherichia coli K-12, strain MM294, deposited at the American type culture Collection on 9/14 of 1984 under the terms of the Budapest treaty. The institute is now located at university road 10801, Manassas, virginia (accession number 39607).

TFPI or TFPI variants can be produced using bacteria or yeast and then purified. The procedures used are generally as described in U.S.5,212,091, U.S.6,063,764 and U.S.6,103,500 or in WO 96/40784. TFPI or a TFPI variant can be purified, solubilized and refolded as described in WO96/40784 and Gustafson et al (prot.express.Pur.5: 233). For example, when ala-TFPI is prepared as described in example 9 of WO96/40784, the resulting ala-TFPI preparation contains biologically active ala-TFPI in an amount of 85% to 90% by weight of total protein.

The amount of TFPI or TFPI variant added to a liquid composition of the present invention is typically from about 0.05mg/ml to 15mg/ml (e.g., 0.05, 0.15, 0.5, 1, 2.5, 5, 7.5, 10, 12.5, or 15 mg/ml).

Amino acid cosolvent

The amino acid co-solvent added to the TFPI or TFPI variant-containing compositions of the present invention primarily serves to protect the TFPI or TFPI variant from polymerization, thereby improving its stability during storage. The reduction in polymer formation is in a concentration dependent manner with the addition of an amino acid co-solvent. Increasing the concentration of amino acid co-solvent results in increased stability of the TFPI or TFPI variant compositions. Since the formation of polymer during storage is correspondingly reduced.

Preferred amino acid co-solvents are arginine, lysine or analogs of arginine, lysine. Arginine or lysine may be present in the form of the free base or in the form of a salt, for example in the form of a hydrochloride. Analogs of arginine or lysine may also exist in free base form or salt form. Arginine analogues include aminoguanidino arginine ethyl ester, arginine hydroxamate and arginine p-nitroanilide. Lysine analogs include lysinamide, lysine ethyl ester, lysine hydroxamate, and lysine p-nitroanilide. The co-solvent is preferably arginine in the form of its free base or hydrochloride salt. Natural arginine or lysine L-stereoisomers are also preferred as co-solvents, however, D-stereoisomers or mixtures of L-and D-stereoisomers may be added to the stabilizing compositions of the present invention.

Arginine or lysine co-solvents or analogs thereof are added to the liquid compositions of the invention in amounts that provide the desired stabilization of TFPI or TFPI variants during storage, and thus the formulations exhibit increased resistance to degradation as compared to similar compositions without added co-solvent. The total amount of co-solvent in the composition is preferably from about 50 to 600mM (e.g., 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600mMO), more preferably from about 100mM to 400mM, and most preferably about 300 mM.

The amount of a particular amino acid base added to a TFPI-or TFPI variant-containing liquid composition to reduce polymer formation, increase polypeptide stability, and storage stability of the composition is readily determined using methods well known to those skilled in the art, e.g., as described in example 6 below. For example, determining the effect of arginine or lysine co-solvents on the storage stability of TFPI or a TFPI variant in a liquid composition can be conveniently measured by detecting changes in one or more possible properties of the composition, such as the concentration of soluble polypeptide over time. The amount of soluble polypeptide in solution can be quantitatively determined using ion-exclusion (IEC) -HPLC. If the primary pathway for degradation of TFPI or TFPI variant is polymerization, the effective amount of co-solvent added to the TFPI or TFPI variant composition to achieve increased stability is an amount that reduces polymer formation during this time, thereby allowing more soluble polypeptide to remain in solution in the form of non-polymerized (e.g., monomeric) molecules.

Antioxidant agent

The liquid TFPI or TFPI variant compositions of the present invention also comprise an antioxidant. An "antioxidant" is a component that reduces oxidation of TFPI or TFPI variants, particularly oxidation of methionine residues in the molecule. Oxidation of methionine residues in the TFPI or TFPI variant molecules is one of the major degradation pathways during storage of TFPI or TFPI variant compositions. The oxidation is related to contaminants present in the composition which may either react directly with methionine residues or may catalyze the oxidation reaction. Thus, the addition of certain antioxidants to combat the effects of these contaminants can greatly improve the stability of TFPI or TFPI variant compositions even if the co-solvents of the present invention have been added to the compositions. Preferred antioxidants are pharmaceutically acceptable at a concentration of about 0.01 to 50mM (e.g., 0.01, 0.1, 1, 2,5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mM). The term "pharmaceutically acceptable" means that the formulation is not significantly associated with biological side effects when administered to a patient. The term "patient" includes both humans and animals.

Three general types of antioxidants are effective in TFPI or TFPI variant compositions of the present invention: namely an oxygen-displacing gas, an oxygen or radical scavenger and a chelating agent.

Oxygen substituted gas

Dissolved oxygen in liquid TFPI or TFPI variant compositions can eventually lead to methionine oxidation, resulting in loss of the therapeutic efficacy of TFPI or incorporation of oxidized amino acids (e.g., methionine sulfoxide) into TFPI or TFPI variant polypeptides, possibly with unknown or undesirable physiological effects. An oxygen-displacing gas is a gas that is effective in scavenging or displacing dissolved oxygen. Oxygen substitution of the gas can significantly reduce the dissolved oxygen concentration compared to the dissolved oxygen concentration of a composition equilibrated with ambient air. The preferred oxygen-substituted gas reduces the dissolved oxygen concentration to less than about 10% as compared to the dissolved oxygen concentration of a TFPI or TFPI variant liquid composition that does not contain oxygen-substituted gas. This greatly improves stability.

Preferred oxygen-substituted gases are substantially inert to the TFPI or TFPI variant compositions, i.e., do not undergo significant chemical reactions upon exposure of the TFPI composition to the oxygen-substituted gas, thereby retaining the biological activity of the TFPI. Suitable oxygen-displacing gases include nitrogen, nitrogen-enriched air, nitrogen-enriched oxygen, noble gases (e.g., helium or argon), methane, ethane, propane, carbon dioxide, or mixtures of these gases. "Nitrogen-enriched air" and "nitrogen-enriched oxygen" are each mixtures of nitrogen and air or oxygen. They contain nitrogen at higher concentrations than in the atmosphere (e.g., greater than 79 vol-%). Nitrogen is the preferred oxygen displacing gas.

The oxygen-displacing gas in the composition may be in any concentration up to and including its solubility limit. The TFPI or TFPI variant composition may be stored in a pressurized environment to increase the dissolved oxygen-displacing gas therein, such as by having the displacing gas above the liquid level of the composition in a closed container. Alternatively, a pressure below atmospheric pressure is maintained above the liquid surface to reduce the dissolution of the oxygen displacement gas.

Oxygen-substituted gas can be introduced into the TFPI or TFPI variant composition by any conventional means. For example, the replacement gas is blown into the headspace of a vial or other container containing TFPI or TFPI variant composition, the replacement gas is sparged or bubbled through the TFPI or TFPI variant composition, the replacement gas is cyclically pressurized/depressurized, the replacement gas is re-pressurized and then evacuated, and the like.

After the above-described oxygen substitution occurs, oxygen re-dissolution into the TFPI or TFPI variant composition is prevented because the air is sequestered by the oxygen-substituted gas.

Oxygen or free radical scavengers

Another useful class of antioxidants in the present invention are oxygen scavengers or free radical scavengers. In general, such capture agents are more reactive with oxygen and/or free radicals than TFPI or TFPI variants. They act as "sacrificial" molecules to react with available oxygen, thereby preventing deleterious oxygen-TFPI or oxygen-TFPI variant interactions, particularly oxidation of methionine residues. In a preferred embodiment, the concentration of oxygen or free radical scavenger is about 0.1 mM to about 10 mM.

Suitable oxygen or free radical scavengers are stable in the TFPI or TFPI variant compositions of the invention. Pharmaceutically acceptable, preferred oxygen or free radical scavengers include: methionine, ascorbic acid or sodium ascorbate, L-, DL-or D-alpha tocopherol and L-, DL-or D-alpha tocopheryl acetate, beta-carotene, selenium, pyritinol, propyl gallate, Butylated Hydroxyanisole (BHA) and butylated hydroxytoluene. The appropriate form of oxygen or free radical trap naturally depends on its compatibility with the TFPI or TFPI variant composition. In general, hydrophilic antioxidants such as ascorbic acid or alpha tocopherol acetate (e.g., alpha tocopheryl acetate) may be suitably added to the compositions of the present invention.

Any stereoisomer of methionine (L-, D-or DL-) or mixtures of these isomers may be used. A particularly preferred antioxidant is methionine, especially L-methionine. Generally, better results are obtained when methionine is added in an amount at least equivalent to methionine in TFPI or TFPI variants on a molar basis. Native TFPI contains 5 methionine residues per protein molecule. Methionine that is part of the TFPI or TFPI variant protein is referred to as "TFPI or TFPI variant methionine" to distinguish it from methionine that is added to the composition as an antioxidant and is not part of the TFPI or TFPI variant protein. Of course, methionine in the polypeptide that is not TFPI or a TFPI variant methionine may also be used as an oxygen scavenger in the present invention. For example, a polypeptide containing methionine may reduce the oxidation rate of TFPI or TFPI variant methionine, similar to the case where free methionine is added to the composition. It is therefore important to distinguish between the above-defined "TFPI or TFPI variant methionine" and the "methionine not present in TFPI or TFPI variant", the latter including any methionine added to the composition in free form or bound in a polypeptide other than TFPI or TFPI variant.

In terms of the molar ratio of non-TFPI or non-TFPI variant methionine to TFPI or TFPI variant methionine, it is preferred that methionine be added in an amount of about 1: 1 to about 10,000: 1, more preferably about 1: 1 to about 5,000: 1, more preferably about 100: 1 to about 1,000: 1, still more preferably about 300: 1 to about 1,000: 1, and yet more preferably about 500: 1 to about 1,000: 1. With respect to the absolute concentration of methionine, it is preferably present in the composition at a concentration of about 1-10mM (e.g., about 1, 2, 3, 4,5, 6, 7, 8,9, or 10 mM). Although the concentration of methionine may vary depending on the concentration of TFPI or TFPI variant in the compositions of the invention. An important role of methionine or other oxygen scavengers is to prevent the formation of methionine sulfoxide residues of TFPI or TFPI variants that can lead to undesirable or unknown effects under physiological conditions even if the TFPI or TFPI variant has biological activity. Thus, the antioxidant should be added in an amount sufficient to inhibit oxidation of the methionine residue. The amount of methionine oxysulfide produced by subjecting the added methionine to oxidation is acceptable to the regulatory authorities. Generally, this means that no more than about 10% -30% of the methionine residues are present in the composition as methionine sulfoxide.

Chelating agents

Another antioxidant useful in the present invention is a chelating agent, also known as a sequestering agent, which effectively binds transition metal ions (e.g., Fe)³⁺). Transition metal ions can be present in the composition and can catalyze detrimental oxidation reactions, leading to protein degradation and polymerization. The chelating agent(s) selected should have little or no chemical reactivity with the other ingredients of the composition, and should be generally compatible with the base(s) for maintaining the desired physiological properties of the composition (e.g., pH and isotonicity). Thus, if transition metal cations in the composition are not intentionally added to maintain a pH or an osmolality equivalent, it is preferred to use a chelating agent in the composition.

Preferably a pharmaceutically acceptable chelating agent. Preferred, pharmaceutically acceptable chelating agents include various aminocarboxylate compounds that are capable of forming metal ligand complexes with one or more transition metal ions in solution. These aminocarboxylates include ethylenediaminetetraacetic acid (EDTA) and diethyltriaminepentaacetic acid (DTPA), 1, 2-bis (2-aminophenoxy) ethane-N, N, N ', N' -tetraacetic acid (BAPTA), ethylene glycol bis (2-aminoethyl) -N, N, N ', N' -tetraacetic acid ethyl Ester (EGTA) and other aminocarboxylate compounds containing one or more carboxylate groups. Derivative salt forms (e.g., disodium salt forms) of these aminocarboxylate chelants may also be employed so long as they have some ability to complex free transition metal ions in the TFPI or TFPI variant compositions. Other forms of these chelating agents than salt forms are also effective, including the various ester, anhydride and halogenated forms of these compounds.

Buffer solution

The pH of a TFPI or TFPI variant composition affects the solubility of the protein and thus also its stability. See Chen et al (1999) j.pham.science 88: 881-888. The preferred pH range for the compositions of the present invention is from about 4 to about 8 (e.g., 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8), more preferably from about 5 to about 6.5. Because pH is a major factor in TFPI solubility, the use of buffers to maintain a suitable pH may additionally improve the stability of such formulations. The liquid compositions of the present invention may therefore also comprise a buffer to maintain the pH of the solution. The buffer is preferably substantially free of the buffer salt form of the acid, the salt form of the acid, or a combination of the acid and its salt forms.

The pH of the compositions of the present invention is preferably maintained with a cosolvent of arginine or lysine in base form and an acid in substantially non-salt form. This combination allows for a lower osmolality of the solution relative to the use of acids and their salt forms as buffers and in combination with amino acid bases. The advantage of this combination is that higher concentrations of arginine or lysine co-solvent and/or antioxidant (e.g., methionine) can be added to the liquid composition without exceeding the isotonicity of the solution. By "substantially non-salt form of the acid" is meant an acid that is less than 2% of the salt form thereof that is used as a buffering agent in the liquid composition.

When buffers containing an acid are used in liquid compositions, the buffer is typically prepared using the salt form of the acid or a combination of the salt form of the acid and its conjugate base. Thus, for example, such buffers may be prepared using sodium, potassium, ammonium, calcium and/or magnesium salts of acids and their conjugate bases. When the buffer selected comprises arginine or lysine as a co-solvent in base form and an acid in substantially non-salt form, the buffer is preferably selected from citric acid, succinic acid, phosphoric acid, glutamic acid, maleic acid, malic acid, acetic acid, tartaric acid and aspartic acid. Particularly preferred is the use of citric acid and succinic acid in combination with arginine in free base form as buffering agents. In addition, as previously described, a salt form of arginine (e.g., a hydrochloride salt form of arginine) may also be used. In this case, the buffer generally comprises a combination of the acids described above and their conjugate base salt forms. Other buffers that may be used include histidine and imidazole. Generally, preferred buffer concentrations are about 0 to about 50mM (e.g., 0, 1, 2,5, 10, 15, 20, 25, 30, 35, 40, 45, or 50mM), with a concentration of about 5 to about 30mM being more preferred.

If the buffering agent used is an amino acid base and an acid in a substantially non-salt form, the prepared composition comprising TFPI or a TFPI variant is substantially isotonic, without the inclusion of an added isotonicity agent (e.g., sodium chloride). Substantially isotonic compositions result in little or no water flow through the membranes of the surrounding cells after in vivo administration. In general, it is desirable that the liquid composition be isotonic in order to reduce the pain of administration and to minimize the potential hemolytic effects associated with hypertonic or hypotonic compositions. Isotonic conditions corresponding to osmolality of the solution are about 240-340 mOsmol/L (e.g., 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, or 340mOsmol/L), which is also suitable for the present invention. More preferably, isotonic conditions are achieved with an osmolality of about 290 mOsmol/L.

In some cases, however, the acid used as the buffer may be a salt of the acid or a mixture of the acid and its salt, depending on the desired properties to be maintained (e.g., pH and osmolality) of the TFPI or TFPI variant composition. In this case, the preferred buffer is a mixture of an acid and a salt thereof. These acids may be citric acid, succinic acid, phosphoric acid, glutamic acid, maleic acid, malic acid, acetic acid, tartaric acid or aspartic acid. The salt form of the selected acid may be the sodium, potassium, calcium or magnesium salt of its conjugate base. Particularly preferred salts of those conjugate bases are sodium salts of buffers. These buffers include citric acid/sodium citrate, succinic acid/sodium succinate, phosphoric acid/sodium phosphate, glutamic acid/sodium glutamate, maleic acid/sodium maleate, malic acid/sodium malate, acetic acid/sodium acetate, tartaric acid/sodium tartrate and aspartic acid/sodium aspartate. When arginine, or even its free base form, is used as a co-solvent, the preferred buffer is citric acid/sodium citrate or succinic acid/sodium succinate. In this case, the preferred buffer concentration is about 5mM to 30mM (e.g., 5,10, 15, 20, 25 or 30mM), more preferably about 20 mM.

When the amino acid base is buffered with the acid in a substantially non-salt form, the nearly isotonic formulation may have a higher concentration of the stabilizing amino acid than when a mixture of the acid and its salt is used as a buffer system. In such cases, the high concentration of co-solvent associated with the substantially isotonic composition may also improve the stability of TFPI or TFPI variants, thereby extending their shelf life.

For example, citric acid is used to buffer arginine base added to a composition containing TFPI or TFPI variant and at a pH of 5.5, the concentration of arginine can be increased to 300mM while still maintaining isotonicity of the composition. This results in a 30% increase in the storage life of TFPI or TFPI variants at 50 ℃. Although similar shelf-life can be achieved with TFPI or TFPI variants using the same arginine concentration and citric acid/sodium citrate as the buffer, arginine must be added in its acid form to achieve a similar pH and the resulting composition is hypertonic. The ability to utilize high concentrations of amino acids as primary stabilizers eliminates the need for more traditional co-solvents (e.g., bovine serum albumin or human serum albumin) of TFPI or TFPI variants, which are less than ideal because of the potential for viral contamination.

Other stabilizers

Other compounds that increase the action or improve the quality of TFPI or TFPI variants may be included in TFPI or TFPI variant compositions of the invention, provided they do not adversely affect the basic stabilization achieved by amino acid co-solvents in combination with antioxidants. For example, degradation of TFPI or TFPI variant polypeptides may result from freeze-thawing or mechanical shearing during processing of the TFPI or TFPI variant compositions of the present invention, which degradation may be inhibited by the addition of surfactants to the composition to reduce the surface tension at the liquid-air interface. Suitable surfactants are nonionic surfactants including polyoxyethylene sorbitol esters (such as polysorbate 80 (tween 80) and polysorbate 20 (tween 20)); polyoxypropylene-polyoxyethylene esters (e.g., Pluronic (Pluronic) F68); polyoxyethylene alcohols (e.g., Brij 35); dimethyl silicone oil; polyethylene glycol (e.g., PEG 400); lysophosphatidylcholine and polyoxyethylene-p-t-octylphenol (e.g., Triton X-100). Classical drug stabilization by surfactants or emulsifiers is described by Levine et al (1991) j. 160-165. A preferred surfactant for use in the practice of the present invention is polysorbate 80.

Other stabilizers (e.g., albumin) may optionally be added to further increase the stability of the TFPI or TFPI variant compositions. The albumin may be added in an amount of about 1% w/v or less. Sugars or sugar alcohols may also be included in TFPI or TFPI variant compositions of the invention. Any saccharide such as a monosaccharide, disaccharide or polysaccharide or a water-soluble polysaccharide (e.g., fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch and carboxymethyl cellulose-sodium) may be used. Sucrose is the most suitable carbohydrate additive. Sugar alcohols (i.e. C containing hydroxy groups)₄-C₈Hydrocarbons) may also be used, for example mannitol, sorbitol, inositol, galactitol (galactitol), dulcitol, xylitol or arabitol. Mannitol is the most preferred sugar alcohol additive. The above sugars or sugar alcohols may be used alone or in combination. The amount thereof is not fixedly limited as long as the sugar or sugar alcohol is soluble in the liquid formulation and does not adversely affect the stabilization effect achieved by the method of the present invention. The sugar or sugar alcohol is preferably present at a concentration of about 1% w/v to about 15% w/v, more preferably about 2% w/v to about 10% w/v.

Preparation of Stable compositions

The compositions of the present invention are preferably prepared by pre-mixing the co-solvent, antioxidant, optional buffering agent and other excipients that are added prior to TFPI or TFPI variants. After addition of suitable amounts of co-solvent and antioxidant to increase the stability of TFPI or TFPI variants, the pH of the composition may be adjusted to be within the optimal range for TFPI or TFPI variants as described herein. Although the pH may be adjusted after addition of TFPI or TFPI variants, it is preferably adjusted before addition, so that the risk of denaturation is reduced. The components can then be suitably mixed by suitable mechanical means.

Pharmaceutical compositions

Preferably, the liquid composition of the present invention may be in a form for administration to a subject or may be in a form for preparing a formulation for administration to a subject. The liquid composition comprising TFPI or a TFPI variant may be formulated as a single dosage form or may be formulated as an injectable or infusible dosage form (e.g., a solution, suspension or emulsion). The liquid composition of the present invention is preferably stored as a liquid formulation, which has the advantage that the storage stability achieved by the present invention and as described below can be improved. The pharmaceutical compositions of TFPI or TFPI variants are preferably sterilized by membrane filtration and stored in single-dose or multi-dose containers (e.g., sealed vials or ampoules). These compositions may also be stored cold.

Other methods of formulating compositions are generally known in the art, so long as they do not adversely affect the beneficial effects of the co-solvents, antioxidants, and buffers described herein, and can be used to further improve the storage stability of liquid TFPI or TFPI variant compositions. For a discussion of formulating and selecting pharmaceutically acceptable carriers, co-solvents, etc., see Remirgton' Pharmaceutical Science (1990) (18 th ed., MackPub. CO., Eaton, Pennsylvania).

The following examples are provided for the purpose of illustration and not limitation. The contents of all patents, patent applications, and references cited herein are incorporated by reference.

Experiment of

The following protocols in examples 1-6 were employed to determine the degradation and stability effects of particular co-solvents and/or antioxidants on liquid TFPI or TFPI variant compositions during storage.

Reverse Phase (RP) HPLC

RP-HPLC was performed on a Waters 626 LC system equipped with a 717 autosampler (Waters Inc., Milford, Maine) using a Vydac214BTP 54C₄Columns and Vydac214GCCP54 pre-column (Separation Group, Hesparia, California). The column was first equilibrated with mobile phase a (10% acetonitrile, 0.1% TFA). The RP-HPLC method uses the detected TFPI or TFPI variant monomer as the main peak. Other compounds containing single or multiple oxideA peak for a protein of a methionine residue and peaks representing acetylated and carbamylated forms of TFPI or TFPI variants.

Ion exchange HPLC (IEX-HPLC)

Ion Exchange (IEX) -HPLC was performed on a Pharmacia Mono-s HR5/5 glass column using a Waters 626 LC system and 717 heating/cooling autosampler as described by Chen et al, supra. The column was first equilibrated with 80% mobile phase A (70: 30 v/v, 20mM sodium acetate: acetonitrile, pH5.4) and 20% mobile phase B (70: 30 v/v, 20mM sodium acetate and 1M ammonium chloride: acetonitrile, pH 5.4). After injection, TFPI and TFPI variants were eluted with mobile phase B increasing to 85% at a flow rate of 0.7ml/min for 21 minutes. TFPI and TFPI variants were washed as a single peak at approximately 16.5 minutes and the UV absorbance at 280nm was measured using a Waters absorbance detector. Data were obtained and processed using a Perkin-Elmer Turbochrom system. Protein concentration was estimated by comparing the integrated area of the peak to a standard curve for samples of known concentration.

Determination of the pH and osmolality

The pH of the solutions of the various formulations was measured using an Orion pH meter (type 611, Orion Research incorporated laboratory Products Group, Boston, Mass.). The PH meter was checked according to the manufacturer's recommended double buffer calibration method. The two buffers were a pH4 standard (Fisher Scientific, Cat. NO. SB101-500) and a pH7 standard (Fisher Scientific, Cat. NO. SB107-500).

The osmolality of the solutions of these formulations was measured using a Wescor vapor pressure osmometer (model 5500, Wescor Inc, Logan, Utah). The osmometer was checked against two standards provided by the manufacturer, 290mmol/kg standard (Wescor, Reorder No. OA-010) and 1,000mmol/kg standard (Wescor, Reorder No. OA-029), respectively.

Other materials and methods

A buffer solution of the formulation was prepared with a Chiron Tech Service. 10-cc of I-shaped tubular glass vials and Daikyo Gummi layered non-siliconized stoppers were obtained for the following study.

Dissolved oxygen levels in TFPI and TFPI variant vials were determined using Nova BioProfile 200. The apparent first order rate constant of TFPI oxidation was estimated using the KaleidaGraph * Software (Stnergy Software, Reading pennsylvania) program.

Example 1

Prothrombin time assay

Suitable prothrombin time assays are described in U.S. Pat. No. 5,888,968 and WO 96/40784. Briefly, prothrombin time can be measured using a coagulometer (e.g., Coag-A-Mate MTXII from Organon Teknika). A suitable assay buffer is 100mM sodium chloride, 50mM Tris, adjusted to pH 7.5, containing 1mg/ml bovine serum albumin. Other reagents that are desirable are normal human plasma (e.g., "Verifyl" from Organon Teknika), thromboplastin reagent (e.g., "Simplatin Excel" from Organon Teknika), and a TFPI standard solution (e.g., 20 μ g of ala-TFPI at 100% purity (or equivalent) per ml of assay buffer).

A standard curve is obtained by analyzing the clotting time of a series of dilutions (e.g., 1-5 μ g/ml final concentration) of a TFPI standard solution. To determine clotting time, the sample or TFPI standard is first diluted with assay buffer and added to normal human plasma. A thromboplastin reagent was added to initiate the clotting reaction. The instrument will record the clotting time. A linear TFPI standard curve was obtained by plotting the logarithm of the clotting time versus the logarithm of the TFPI concentration. The standard curve is calibrated to a concentration of TFPI corresponding to an equivalent of 100% pure standard based on the purity of the TFPI standard. For example, if the standard is 97% biochemically pure ala-TFPI preparation (i.e., containing 3% by molecular weight of a substance that is not biologically active in TFPI), the concentration of each dilution of the standard is multiplied by 0.97 to obtain the actual TFPI concentration. Then, at the actual concentration of 97% pure formulation, 1.0. mu.g/ml of TFPI standard corresponds to a concentration of 1.0X 0.97 or 0.97. mu.g/ml and is treated at this actual concentration. The efficacy of TFPI in the treatment of sepsis and a range of other conditions can also be determined, including a reduction in the mortality rate for various causes and improvement in some Multiple Organ Dysfunction (MOD) over placebo on a 28-day basis.

Example 2

Effect of L-arginine concentration on ala-TFPI stability in various compositions

An ala-TFPI composition having a pH of 5.5 and a final ala-TFPI concentration of 0.15mg/ml may be prepared using a 0.6mg/ml storage solution. The buffer of the stock solution was dialyzed, the concentration of the resulting ala-TFPI was analyzed by UV/Vis photometry and then diluted to the initial target concentration of 0.15mg/ml with citrate buffer, which may or may not contain sodium citrate. Sodium citrate was added to the samples only when the L-arginine co-solvent was L-arginine hydrochloride, whereas the composition containing L-arginine base used only citric acid as a buffer.

These solutions were aliquoted into 3-cc vials (1 ml each) and stored stably. Enough vials were left for starting the concentration measurements at the time points. The other vials were placed in a 50 ℃ incubator for accelerated stability studies. The composition of the four samples contained 0.15mg/mlala-TFPI, pH5.5, with co-solvents and buffer concentrations listed below:

1)20-150mM L-arginine hydrochloric acid cosolvent and 10mM citric acid/sodium citrate buffer solution;

2)20-150mM L-arginine acid-base cosolvent, titrating to pH5.5 with citric acid;

3) 100-;

4)100 plus 300mM L-arginine acid-base cosolvent, titrating to pH5.5 by using citric acid;

on days 3,7, 14 and 30, the contents of each vial were transferred to a 1.7ml microcentrifuge tube and then centrifuged at 10,000rpm for about 2 minutes. After centrifugation, the soluble proteins in the sample are separated from the polymerized/precipitated proteins. The content of soluble protein was determined by IEX-HPLC (Chen et al (1999) J.Pharm.Sci.88 (9): 881-888). Will be used as a storage time boxNumerical concentration data applied to a first order exponential decay model (Y ═ T)₀e^-ks) And the half-life of the remaining soluble protein during storage was calculated using KaleidaGraph * graphic software.

Half-life (t) of ala-TFPI preparation during storage_1/2) Values were plotted as a function of arginine concentration (see figure 1). The data show that the storage half-life of ala-TFPI is extended with increasing L-arginine concentration. The use of L-arginine as a co-solvent alone in the composition results in a significantly longer storage half-life than compositions with very small amounts of no co-solvent.

Example 3

Degradation kinetics of ala-TFPI formulations

One of the major degradation pathways of ala-TFPI when stored at 2-8 ℃ is the oxidation of methionine residues. The oxidized methionine fraction was resolved by reverse phase-HPLC (RP-HPLC) and eluted earlier than the main peak. FIG. 6 is an RP-HPLC chromatogram of an ala-TFPI sample showing resolvable oxidized methionine. Peak a contains multiple MetSO, peak C contains one MetSO, peak D is norvaline (norvaline) -substituted ala-TFPI, and peaks E and F are acylated and/or carbamylated ala-TFPI. The A and C peaks were integrated separately. All remaining fractions, including the main peak, the D peak, the E peak and the F peak, were combined together as the main peak integral.

To understand the degradation kinetics at 30 ℃, 2mlala-TFPI samples were prepared (as described in example 2). Each sample contained 0.15mg/ml TFPI, 20mM citrate/sodium citrate buffer and 300mM L-arginine. These samples were injected into 10-cc glass vials (2 ml samples per vial) and incubated at 30 ℃. Loss of soluble protein due to polymerization/precipitation was first detected as this phenomenon resulted in a reduction in the total HPLC peak area. Both IEX-HPLC and RP-HPLC showed a 2% -5% decrease in the total peak area of the stable samples after 8 weeks of storage at 30 ℃, indicating that only a relatively small amount of ala-TFPI polymerizes/precipitates with the above formulation. Degradation by methionine oxidation was evaluated by plotting the main peak, peak A and peak C of RP-HPLC as a function of storage time at 30 ℃ and this was done. The A and C peaks increased with a decrease in the main peak. After 8 weeks of storage, approximately 11% and 9% of the oxidized methionine was single MetSO and multiple MetSO methionine. This suggests that methionine oxidation is an important degradation pathway under standard storage conditions, based on currently available assays. The results in table 1 reveal that the formation of MetSO increases with temperature.

TABLE 1 Effect of temperature on ala-TFPI Oxidation

Temperature of	Area of ala-TFPIETSO Peak (C Peak) (%) determined by RP-HPLC
			Starting material	After 4 weeks
	40℃	6.8			23.7
30℃			6.8	10.4
	2-8℃	6.8			7.0

Example 4

Effect of dissolved oxygen on ala-TFPI stability

Samples of the composition were prepared as described in example 3. The dissolved oxygen level was varied by blowing nitrogen/air over the vial surface space through the fermenter unit instead of the gas mixture. The buffer for each sample was adjusted to ph 5.5. To facilitate equilibration of displaced gas between the head space and the liquid, the vial was shaken at 200rpm for 1 hour while sparging. The vial was then maintained at 30 ℃ and ala-TFPI samples were taken at the indicated time points for stability analysis. The dissolved oxygen level in each vial was again determined at each time point for stability analysis.

In an initial pilot study, ala-TFPI vials were prepared to contain different dissolved oxygen levels, e.g., 0%, 20%, 100%, and 200% air saturation (assuming air contains 21% oxygen at 100% saturation). FIG. 2 shows the results of stability evaluation at 30 ℃. The results show that oxidation of ala-TFPI is substantially suppressed when the oxygen level is reduced to near 0% air saturation. 0% air saturation means that the atmosphere above the liquid level is essentially pure nitrogen substitute gas. In comparison, the increase in stability resulting from the decrease in dissolved oxygen from 200% to 20% air saturation is relatively small.

A second study was then conducted to more specifically evaluate the stability performance of ala-TFPI samples containing dissolved oxygen from 0% to 12% air saturation. The essential effect on stability is found in this range. The relationship between storage half-life of ala-TFPI and dissolved oxygen level at 30 ℃ is shown in FIG. 3. The ala-TFPI stability is greatly improved when the dissolved oxygen level of the sample falls below 5% air saturation (about 1% oxygen content). Dissolved oxygen levels were also determined in individual sample vials at the ala-TFPI concentration analysis time point, and no significant change in dissolved oxygen levels in the vials was observed. These results show that replacement of a sufficient amount of oxygen with a replacement gas such as nitrogen greatly improves the storage stability of ala-TFPI if the dissolved oxygen concentration can be reduced to a sufficiently low level. Nitrogen, a displacing gas, inhibits the oxidation of ala-TFPI and therefore may be considered an antioxidant.

Example 5

Effect of Metal chelators on ala-TFPI Oxidation

10mg/ml of the main ala-TFPI liquid is diluted to 0.15mg/ml with a buffer containing the metal chelator EDTA or DTPA at a concentration of 1mM or 4 mM. These compositions also contained 20mM citric acid/sodium citrate and 300mM L-arginine as a co-solvent. The diluted ala-TFPI solution was injected into 10-cc glass vials (2 ml sample per vial) and stored at 2-8 deg.C or 30 deg.C for stability analysis.

The stability curve of the main peak area maintained at the storage temperature of 30 ℃ was analyzed by RP-HPLC and is shown in FIG. 4. Table 2 below sets forth the half-life data obtained in this study at 2-8 ℃ and 30 ℃. The metal chelator stabilizes ala-TFPI in a concentration-dependent manner, suggesting that ala-TFPI methionine residue oxidation is catalyzed by metal ions in solution. Regardless of its actual mechanism of action, the metal chelating agent prevents ala-TFPI from oxidizing and is therefore a potent antioxidant.

Example 6

Effect of free methionine on ala-TFPI Oxidation

10mg/ml of the ala-TFPI master was diluted to 0.15mg/ml with methionine containing buffer. These compositions also contained 20mM citric acid/sodium citrate and 300mM L-arginine as a co-solvent. The diluted ala-TFPI solution was injected into 10-cc glass vials (2 ml sample per vial) and stored at 2-8 deg.C or 30 deg.C for stability analysis.

The stability curve of the main peak area maintained at the storage temperature of 30 ℃ was analyzed by RP-HPLC and is shown in FIG. 5. Table 2 below sets forth the half-life data obtained in this study at 2-8 ℃ and 30 ℃. These data indicate that compositions containing 2-10mM methionine are effective in inhibiting the oxidation of ala-TFPI methionine residues. In fact, no oxidative degradation of ala-TFPI was detected in the presence of 2-10mM methionine even after 6 months of storage at 2-8 ℃. The stability of ala-TFPI compositions containing L-arginine as a co-solvent is again enhanced by the use of an antioxidant, in this case the oxygen scavenger methionine. Without being bound by any particular theory, it is believed that the inhibition of ala-TFPI oxidation by free methionine is performed by providing "sacrificial" methionine, such that methionine on the protein is less likely to be affected.

Methionine oxidation can be caused by a variety of factors, including metal ions, dissolved oxygen, and peroxides. Several antioxidants have been identified to prevent the oxidation of methionine in proteins. Such as chelating agents, oxygen scavengers, reducing agents and displacing gases. The chelating agent may complex metal ions that catalyze oxidation reactions. The oxygen scavenger is capable of reacting with oxygen to preferentially oxidize, thereby removing the oxidation source and protecting the protein. The reducing agent can reduce the oxidation of the protein by the oxidizing agent. The displacing gas reduces the partial pressure of oxygen in the space above the container to lower the dissolved oxygen concentration.

Table 2 compares the effect of a metal chelator (as tested in example 4) and the oxygen scavenger methionine on the reduction of ala-TFPI oxidation. All antioxidants increased the storage half-life of ala-TFPI compared to a control sample containing 0.15mg/mlala-TFPI, 20mM citrate/sodium citrate buffer and 300mM L-arginine (prepared as in example 3). All conditions were evaluated, in which the addition of 10mM methionine to the ala-TFPI formulation protected the ala-TFPI protein from oxidative degradation particularly effectively.

TABLE 2 comparison of antioxidant effect on ala-TFPI stability

Antioxidant agent	Half life at 2-8 deg.C (moon)	Half life storage at 30 ℃ (moon)
			Metal chelating agent (example 4)
1mMEDTA	63	25
			4mMEDTA	157	28
1mMDTPA	52	11
			4mMDTPA	160	23
Oxygen scavenger (example 5)
			2mM methionine	No degradation was detected until 6 months of storage	23
5mM methionine	24
		10mM methionine	39
Control combination
			20mM citrate, 300mM arginine, pH5.5	36	5.3

Example 7

Effect of ala-TFPI protein concentration on ala-TFPI oxidation

Detecting the effect of ala-TFPI on the oxidation of ala-TFPI when the concentration of ala-TFPI is 0.15mg/ml-10 mg/ml. Stability test samples were prepared by diluting the 10mg/ml host solution with 20mM citric acid/sodium citrate buffer (as in example 3) to 3,1, 0.6, 0.3 and 0.15 mg/ml. These samples also contained 300mM L-arginine. The undiluted and diluted primary samples were then injected into 10-cc glass vials (2 ml samples per vial) and stoppered for stability evaluation at 2-8 ℃ or 30 ℃.

The main peak stability curves maintained at 30 ℃ accelerated temperature and 2-8 ℃ actual storage conditions were analyzed by RP-HPLC, indicating that the storage half-life of ala-TFPI strongly depends on its protein concentration in an inverse relationship. The half-life values of these stability curves are listed in table 3. The oxidation rate increases at lower protein concentrations. Without being bound by any particular theory, it is believed that an increase in the ratio of oxidant to protein molecules in solution may result in an increase in the rate of oxidation.

TABLE 3 major peak storage half-life of phase 3 TFPI stored at different concentrations at 30 ℃ or 2-8 ℃ as determined by RP-HPLC

Storage temperature	Storage of T1/2 (month) at different protein concentrations
							10(mg/ml)	3(mg/ml)	1(mg/ml)	0.6(mg/ml)	0.3(mg/ml)	0.15(mg/ml)
	30℃	22	28	9.3	8.5	6.4							5.6
2-8℃							195	157	98	85	59	44

Example 8

Survival study

To compare freshly prepared clinical grade recombinant ala-TFPI (rtpi) (TFPI92) to clinical grade, partially deamidated and oxidized TFPI (TFPI78), mouse cecal ligation and puncture studies were performed. This model induces intraperitoneal and systemic infection by a variety of microorganisms through direct fecal contamination and caecum necrosis, closely mimicking human intraabdominal sepsis (Opal et al, clinical Care Medicine 29, 13-18, 2001).

Two TFPI formulations were prepared as described in the following patent documents: serial No.60/494,546, submitted on 8/13/2003; serial No.60/509,227, submitted on 8/10/2003 and serial No.60/512,199, submitted on 20/10/2003. The contents of these applications are incorporated by reference. rtpi 78, rtpi 92 or diluted controls were administered blindly over a 48 hour period (SQ q12 hours x 4 dose). Blood was collected before and 48 hours after surgery to determine the quantitative levels of bacteremia, endotoxin and cytokines (α -tumor necrosis factor and interleukin-6). Animals were observed daily and recorded when death occurred. All animals were necropsied, evaluated for histological evidence of organ damage at the end of the experiment and subjected to quantitative bacteriological tests.

The Kaplan-Meier survival plot is shown in FIG. 7. It can be seen that mice receiving freshly formulated rtpi have a significant survival advantage over mice receiving partially oxidized, deamidated forms of rtpi. Both rtpi groups performed better than the control mice that received the dilutions. As expected, sham (surgical intervention to identify the cecum without ligation and puncture) mice survived for 7 days during the study. There was no significant difference in the secondary endpoints of bacteremia, endotoxemia, or cytokine production between the two groups treated with rTFPI.

This study demonstrates that TFPI appears to confer a survival advantage, although the mechanism of action cannot be explained by plasma levels of bacteria, endotoxin or cytokines. Deamidated, oxidized TFPI is less protective than freshly prepared TFPI.

Sequence listing

<110> Selong (CHIRON CORPORATION)

<120> Stable liquid composition comprising Tissue Factor Pathway Inhibitor (TFPI) or tissue factor pathway inhibitor variant

<130>12441.00055

<150>US 60/438,519

<151>2003-01-08

<150>US 60/474,577

<151>2003-08-13

<150>US 60/509,260

<151>2003-10-08

<150>US 60/512,090

<151>2003-10-20

<160>1

<170>PatentIn version 3.1

<210>1

<211>276

<212>PRT

<213> human (Homo sapiens)

<400>1

Asp Ser Glu Glu Asp Glu Glu His Thr Ile Ile Thr Asp Thr Glu Leu

1 5 10 15

Pro Pro Leu Lys Leu Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp

20 25 30

Gly Pro Cys Lys Ala Ile Met Lys Arg Phe Phe Phe Asn Ile Phe Thr

35 40 45

Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn

50 55 60

Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp Asn

65 70 75 80

Ala Asn Arg Ile Ile Lys Thr Thr Leu Gln Gln Glu Lys Pro Asp Phe

85 90 95

Cys Phe Leu Glu Glu Asp Pro Gly Ile Cys Arg Gly Tyr Ile Thr Arg

100 105 110

Tyr Phe Tyr Asn Asn Gln Thr Lys Gln Cys Glu Arg Phe Lys Tyr Gly

115 120 125

Gly Cys Leu Gly Asn Met Asn Asn Phe Glu Thr Leu Glu Glu Cys Lys

130 135 140

Asn Ile Cys Glu Asp Gly Pro Asn Gly Phe Gln Val Asp Asn Tyr Gly

145 150 155 160

Thr Gln Leu Asn Ala Val Asn Asn Ser Leu Thr Pro Gln Ser Thr Lys

165 170 175

Val Pro Ser Leu Phe Glu Phe His Gly Pro Ser Trp Cys Leu Thr Pro

180 185 190

Ala Asp Arg Gly Leu Cys Arg Ala Asn Glu Asn Arg Phe Tyr Tyr Asn

195 200 205

Ser Val Ile Gly Lys Cys Arg Pro Phe Lys Tyr Ser Gly Cys Gly Gly

210 215 220

Asn Glu Asn Asn Phe Thr Ser Lys Gln Glu Cys Leu Arg Ala Cys Lys

225 230 235 240

Lys Gly Phe Ile Gln Arg Ile Ser Lys Gly Gly Leu Ile Lys Thr Lys

245 250 255

Arg Lys Arg Lys Lys Gln Arg Val Lys Ile Ala Tyr Glu Glu Ile Phe

260 265 270

Val Lys Asn Met

275

Claims (32)

1. A liquid composition, comprising:

about 0.05-15mg/ml of TFPI or a TFPI variant;

about 50-600mM of a co-solvent selected from the group consisting of: (i) arginine or an analog thereof, (ii) lysine or an analog thereof, (iii) a mixture of (i) and (ii);

and an antioxidant selected from the group consisting of: (i) an oxygen-displacing gas, (ii) an oxygen or radical scavenger, (iii) a chelating agent, and (iv) mixtures thereof;

wherein the liquid composition has:

a percent polymerization stability of about 45% or greater;

a percent oxidative stability of about 45% or greater;

the pH value is about 4-8.

2. The composition of claim 1 comprising a TFPI variant, wherein the TFPI variant has about 70% or greater homology to TFPI (SEQ ID NO: 1).

3. The composition of claim 2 wherein the TFPI variant is ala-TFPI.

4. The composition of claim 1, wherein the co-solvent is arginine in a form selected from the group consisting of hydrochloride salt, L-arginine, and free base.

5. The composition of claim 1, wherein the composition comprises about 300mM co-solvent.

6. The composition of claim 1, wherein the antioxidant is an oxygen displacing gas.

7. The composition of claim 6 wherein said dissolved oxygen concentration is about 10% less than a liquid composition of TFPI or a TFPI variant that does not contain oxygen-substituted gas.

8. The composition of claim 6, wherein the oxygen-displacing gas is selected from the group consisting of: nitrogen-enriched air, nitrogen-enriched oxygen, nitrogen, noble gases, methane, ethane, propane, carbon dioxide, and mixtures thereof.

9. The composition of claim 8, wherein the oxygen displacing gas is nitrogen.

10. The composition of claim 1, wherein the antioxidant is an oxygen or free radical scavenger or chelating agent and the concentration of the antioxidant is about 0.01 mM to about 20 mM.

11. The composition of claim 10, wherein the antioxidant is at a concentration of about 1mM to about 10 mM.

12. The composition of claim 1, wherein the antioxidant is an oxygen or free radical scavenger and is present at a concentration of about 0.1 mM to about 10 mM.

13. The composition of claim 1, wherein the antioxidant is selected from the group consisting of oxygen or free radical scavengers: methionine, ascorbic acid, sodium ascorbate, L-alpha-tocopherol, DL-alpha-tocopherol, D-alpha-tocopherol, L-alpha-tocopheryl acetate, DL-alpha-tocopheryl acetate, D-alpha-tocopheryl acetate, beta-carotene, selenium, arsenthiol, propyl gallate, butylated hydroxyanisole, butylated hydroxytoluene methionine and mixtures thereof.

14. The composition of claim 13, wherein the antioxidant is methionine and the methionine is L-methionine.

15. The composition of claim 13 wherein said antioxidant is methionine and methionine is added in an amount such that said composition contains a molar ratio of non-TFPI methionine to TFPI methionine of from about 1: 1 to about 1000: 1.

16. The composition of claim 1, wherein the antioxidant is a chelating agent selected from the group consisting of: (i) an aminocarboxylate compound or a derivative thereof; (ii) EDTA or derivatives thereof; (iii) DTPA or a derivative thereof; (iv) BAPTA or derivatives thereof; (v) EGTA or a derivative thereof; and (vi) mixtures of (ii), (iii) (iv) and (v).

17. The composition of claim 1, wherein the pH is about 5 to about 6.5.

18. The composition of claim 1, wherein the osmolality of the composition is from about 240mOsmol/L to about 600 mOsmol/L.

19. The composition of claim 18, wherein the osmolality of the composition is about 290 mOsmol/L.

20. The composition of claim 1 having a storage half-life of about 1 to about 24 months at about 30 ℃.

21. The composition of claim 1, further comprising a buffering agent selected from the group consisting of: (i) an acid substantially in the form of a non-salt, (ii) an acid in the form of a salt, (iii) a mixture of an acid and its salt form.

22. The composition of claim 21, wherein the buffering agent is an acid in substantially non-salt form and is selected from the group consisting of: citric acid, succinic acid, phosphoric acid, glutamic acid, maleic acid, malic acid, acetic acid, tartaric acid, and aspartic acid.

23. The composition of claim 21, wherein the buffer comprises a mixture of an acid and a salt thereof, and the acid is selected from the group consisting of: citric acid, succinic acid, phosphoric acid, glutamic acid, maleic acid, malic acid, acetic acid, tartaric acid, and aspartic acid; and the salt form of the acid is selected from: sodium, potassium, calcium and magnesium salts of the conjugate base of the acid.

24. The composition of claim 23, wherein the buffering agent is selected from the group consisting of: citric acid/sodium citrate, succinic acid/sodium succinate, phosphoric acid/sodium phosphate, glutamic acid/sodium glutamate, maleic acid/sodium maleate, malic acid/sodium malate, acetic acid/sodium acetate, tartaric acid/sodium tartrate and aspartic acid/sodium aspartate.

25. The composition of claim 21, wherein the buffer is at a concentration of about 5mM to about 50 mM.

26. The composition of claim 1, wherein the percent polymerization stability is from about 45% or greater to about 50% or greater.

27. The composition of claim 1, wherein the percent polymerization stability is from about 45% or greater to about 99% or greater.

28. The composition of claim 1, wherein the percent oxidation stability is about 89% or greater.

29. The composition of claim 1, wherein the percent oxidation stability is from about 45% or greater to about 99% or greater.

30. A pharmaceutical composition comprising the liquid composition of claim 1 and a pharmaceutically acceptable excipient.

31. The pharmaceutical composition of claim 30, wherein the percent polymerization stability is from about 45% or greater to about 99% or greater.

32. The pharmaceutical composition of claim 30, wherein the percent oxidative stability is from about 45% or greater to about 99% or greater.