WO2007103425A2

WO2007103425A2 - Kits and methods for preparing pharmaceutical compositions comprising tissue factor pathway inhibitor (tfpi)

Info

Publication number: WO2007103425A2
Application number: PCT/US2007/005814
Authority: WO
Inventors: Augustus Okhamafe; Chin-Yi Huang
Original assignee: Novartis Ag
Priority date: 2006-03-06
Filing date: 2007-03-06
Publication date: 2007-09-13
Also published as: WO2007103425A3

Abstract

The polypeptide Tissue Factor Pathway Inhibitor (TFPI) and variants thereof can be advantageously stored in a form (e.g., a concentrated aqueous solution) that exhibits high stability and thereafter converted (e.g., by mixing with a diluent) into a form that is therapeutically effective (e.g., in the treatment of severe Community Acquired Pneumonia) when administered parenterally (e.g., intravenously) to patients. The separate storage of components of pharmaceutical compositions comprising TFPI and TFPI variants provides several important advantages. The components are generally a concentrated polypeptide solution and a diluent.

Description

KITS AND METHODS FOR PREPARING PHARMACEUTICAL COMPOSITIONS COMPRISING TISSUE FACTOR PATHWAY INHIBITOR (TFPI)

FIELD OF THE INVENTION

[01] The present invention relates to the packaging and storage of components used to prepare pharmaceutical compositions comprising Tissue Factor Pathway Inhibitor (TFPI) or variants thereof. The pharmaceutical compositions are therapeutically beneficial (e.g., for the treatment of severe Community Acquired Pneumonia).

BACKGROUND OF THE INVENTION

[02] Drug products are in many cases packaged and/or stored in forms that differ significantly from those employed when the drug is ultimately administered to a patient. Drug storage forms may vary in terms of physical state, composition (e.g., concentration), or conditions (e.g., temperature) relative to their corresponding administration forms. For example, a drug may be administered in a solution form that is relatively unstable and therefore not suitable for extended storage periods. Often in such cases the drug can be stored for an extended period (which may include packaging and shipping time) in a powdered or lyophilized form that is considerably more stable than its administration form. Other drugs, although they may be effectively stored in solution form, require dilution prior to administration. In either case, the storage form of the drug must be diluted or reconstituted with a diluent (e.g., an aqueous solution containing any of a number of solubilizers, stabilizers such as antioxidants, buffers, or other agents compatible with the drug) to obtain the administration form.

[03] Regardless of the stored form of a drug and/or diluent, an overriding consideration, especially in the case of drugs administered parenterally (e.g. , intravenously) is the maintenance of these components in a sterile environment. This applies to the storage form of the drug during the packaging, storage, and shipping periods, as well as to the administration form of the drug during reconstitution and administration. Unfortunately, maintaining sterility and stability often present conflicting challenges. For example, the "terminal sterilization" (i.e., after packaging and sealing) of some drug products using the common practice of heating to about 120⁰C (248°F) can destabilize them to the point where their biological activity is wholly or partly lost. Otherwise, overcoming stability problems (e.g., aggregation and/or precipitation) may require manufacturing a drug in high volumes and at a low concentration, whereby the correspondingly large manufacturing scale poses a significant barrier to maintaining aseptic conditions. Manufacturing and shipping costs, which are often related to the ability of a particular drug formulation to be made in relatively low volumes and without significant modifications to existing,, standard facilities, represent other major considerations. For all of these reasons, appropriate storage and administration forms are highly drug-specific and require extensive investigation into the properties and requirements of the particular drug at issue.

[04] The polypeptide tissue factor pathway inhibitor (TFPI), also known as lipoprotein associated coagulation inhibitor (LACI), tissue factor inhibitor (TFI), and extrinsic pathway inhibitor (EPI), is 276 amino acid residues in length and functions as an inhibitor of tissue factor-mediated blood coagulation. TFPI was first purified from a human hepatoma cell, Hep G2 (Broze and Miletich (1987) PROC. NATL. ACAD. SCI. USA 84:1886-1890) and subsequently from human plasma (Novotny et al. (1989) JL BlOL. CHEM. 264:18832-18837); and Chang liver and SK hepatoma cells (Wun et al. (1990) J. BlOL. CHEM. 265:16096-16101). TFPI cDNA have been isolated from placental and endothelial cDNA libraries (Wun et al. (1988) J. BIOL. CHEM. 263:6001-6004); Girard et al. (1989) THROMB. RES. 55:37-50). For reviews, see Rapaport (1989) BLOOD 73:359-365 (1989) and Broze et al. (1990) BIOCHEMISTRY 29:7539-7546. The cloning of the TFPI cDNA. which encodes TFPI, is further described in U.S. Patent No. 4,966,852; see also U.S. Patent Nos. 5,773,251 and 5,849,875.

[05] Variants of TFPI are known in the art. See, for example, U.S. Patent No. 5,212,091, where a non-glycosylated form of recombinant TFPI has been produced and isolated from E. coli; U.S. Patent No. 5,106,833, where analogs and fragments are disclosed; and U.S. Patent No. 5,378,614, where production of TFPI analogs in yeast is described. A TFPI variant, which differs from TFPI by the addition of an alanine residue at the amino terminus ("ala-TFPI"), has been shown to be efficacious in animal models for the treatment of sepsis. Carr et al., ClRC SHOCK 44(3): 126-37 (November 1994). Procedures for the purification of TFPI and TFPI analogs are described in U.S. Published Application No. 2005/0037475.

[06] Use of TFPI has been proposed for the treatment of various indications, including severe pneumonia (U.S. Published Application No. 2003/0139339), sepsis (U.S. Patent No. 6,063,764 and WO 93/24143), deep vein thrombosis (U.S. Patent Nos. 5,563,123 and 5,589,359, and WO 96/04378), ischemia (U.S. Patent No. 5,885,781 and U.S. Patent No- 6,242,414, and WO 96/40224), restenosis (U.S. Patent No. 5,824,644 and WO 96/01649), and cancer (U.S. Patent No. 5,902,582 and WO 97/09063).

[07] The effect of various agents and conditions (e.g., pH and temperature) on TFPI solubility and stability are described in U.S. Patent No. 6,525,102 and U.S. Published Application Nos. 2004/0224886 and 2002/0137884. From these disclosures, it is apparent that TFPI, while promising for the treatment of a number of conditions, is nevertheless a hydrophobic protein with limited solubility in aqueous solutions. Moreover, TFPI is subject to losing its biological activity or undergoing chemical changes through several possible mechanisms. Many factors, including those described above, must therefore be considered in determining how TFPI can be stored in a stabilized form and thereafter converted into a form appropriate for parenteral administration to patients. This determination is further complicated by the need to maintain sterility over the entire period from the manufacturing of solutions containing TFPI until and including the administration of the TFPI drug composition to patients.

BRIEF SUMMARY OF THE INVENTION

[08] Despite the above-noted characteristics of TFPI, methods and kits for effectively storing TFPI and thereafter using it in pharmaceutical compositions, suitable for parenteral administration to patients, have been now been discovered. These methods and kits effectively take advantage of a combination of properties of TFPI, thereby allowing the cost effective provision of sterile TFPI pharmaceutical compositions with essentially complete maintenance of biological activity. In particular, these properties of TFPI include its ability to be concentrated in solutions comprising some of the agents used in a TFPI pharmaceutical composition, the ability of other agents to undergo sterilization by heating and/or irradiation, and the directionally increasing stability of TFPI against oxidation at increasing concentrations.

[09] In one embodiment, therefore, the present invention is a method for preparing a pharmaceutical composition for parenteral administration to a patient. The method comprises aseptically packaging an aqueous solution comprising from about 1 mg/ml , to about 20 mg/ml of TFPI or a TFPI variant having about 70% or greater amino acid sequence identity to SEQ ID NO:1. The method further comprises combining the aqueous solution with a diluent, packaged in a second container, to provide the pharmaceutical composition. The first container and second container are selected from the group consisting of a vial, a flexible bag, an ampule, and a cartridge. In another embodiment, the TFPI variant is selected from the group consisting of (i) Tissue Factor Pathway Inhibitor (TFPI), (ii) ala-TFPI, and (iii) analogs of (i) or (ii) having from 1 to 5 amino acid substitutions. In another embodiment, the TFPI variant is ala-TFPI. In another embodiment, the pharmaceutical composition has a pH from about 4 to about 8 and comprises from about 0.10 mg/ml to about 0.50 mg/ml of ala- TFPI, from about 100 mM to about 400 mM of arginine, from about 2 mM to about 10 mM methionine, and from about 10 mM to about 50 mM of citric acid/sodium citrate. In another embodiment, the method further comprises, prior to combining the aqueous solution with the diluent, terminally sterilizing the diluent. In another embodiment, the aqueous solution is maintained at a temperature from about 2°C (36°F) to about 8°C (46°F) prior to combining the aqueous solution with the diluent. In another embodiment, the aqueous solution has a volume of less than about 100 ml.

[10] In another embodiment, the present invention is a method for preparing a pharmaceutical composition for parenteral administration to a patient. The method comprises providing a first container comprising an aseptically packaged aqueous solution comprising from about 1 mg/ml to about 20 mg/ml of TFPI or a TFPI variant having about 70% or greater amino acid sequence identity to SEQ ID NO:1. The method further comprises providing a second container comprising a terminally sterilized diluent. The method further comprises exposing the aseptically packaged aqueous solution to the diluent under aseptic conditions to provide the pharmaceutical composition. [11] In another embodiment, the present invention is a method for stabilizing a an aqueous solution prior to its use in the preparation of a pharmaceutical composition. The method comprises aseptically packaging an aqueous solution comprising TFPI or a TFPI variant having about 70% or greater amino acid sequence identity to SEQ ID NO:1. The method further comprises storing the aqueous solution under conditions whereby the aqueous solution has half-life with respect to both aggregation and oxidation from about 25 months to about 200 months and thereafter exposing the aseptically packaged aqueous solution to the diluent to provide the pharmaceutical composition. In another embodiment, the conditions include a concentration of the TFPI or TFPI variant in the aqueous solution from about 1 mg/ml to about 20 mg/ml and a temperature of the aqueous solution from about 2°C (36°F) to about 8°C (46°F). In another embodiment, the aqueous solution is stored for a period from about 6 months to about 24 months.

[12] In another embodiment, the present invention is a method for providing a pharmaceutical composition to a remote location. The method comprises aseptically packaging, at a first location, an aqueous solution comprising from about 1 mg/ml to about 20 mg/ml of TFPI or a TFPI variant having about 70% or greater amino acid sequence identity to SEQ ID NO:1. The method further comprises diluting, at a second location, the aqueous solution with a diluent to provide the pharmaceutical composition.

[13] In other embodiments, the present invention is a pharmaceutical composition prepared according to any of the above methods.

[14] In another embodiment, the present invention is a kit for preparing a pharmaceutical composition for parenteral administration to a patient. The kit comprises an aqueous solution, packaged under aseptic conditions and comprising from about 1 mg/ml to about 20 mg/ml of TFPI or a TFPI variant having about 70% or greater amino acid sequence identity to SEQ ED NO:1. The kit further comprises a diluent, wherein the aqueous solution and the diluent are packaged in separate containers. In another embodiment, the separate containers are connected to allow the aqueous solution and the diluent to be aseptically combined upon establishing flow communication between the separate containers. In another embodiment, the aqueous solution is packaged in a vial container and the diluent is packaged in a flexible bag container. In another embodiment, the diluent comprises a buffer. In another embodiment, the aqueous solution further comprises a solubilizer and an antioxidant. In another embodiment, when the aqueous solution and the diluent are combined, the resulting pharmaceutical composition has a pH from about 4 to about 8 and comprises from about 0.10 mg/ml to about 0.50 mg/ml of ala-TFPI, from about 100 mM to about 400 mM of arginine, from about 2 mM to about 10 mM methionine, and from about 10 mM to about 50 mM of citric acid/sodium citrate.

[15] These and other embodiments are apparent from the following Detailed Description.

DETAILED DESCRIPTION OF THE INVENTION

[16] The present invention is based on the finding that TFPI and TFPI variants can be advantageously stored in a form (e.g., a concentrated aqueous solution) that exhibits high stability and thereafter converted (e.g., by mixing with a diluent) into a form that is therapeutically effective when administered parenterally (e.g., intravenously) to patients. The components (i.e., the aqueous solution and the diluent) of the TFPI pharmaceutical composition can be prepared in a sterile environment or otherwise sterilized and thereafter aseptically combined prior to use.

[17] Tissue Factor Pathway Inhibitor (TFPI) is a polypeptide having the amino acid sequence shown in SEQ DD NO: 1. TFPI may be recombinant human protein generated in a microbial host. TFPI is further characterized and described with respect to its biological activity in WO 01/24814.

[18] TFPI variants and the production of TFPI variants are described in detail in U.S. Patent Application Publication No. 2004/0224886 and include polypeptide analogs, fragments, and derivatives of TFPI. Analogs are TFPI molecules with one or more amino acid substitutions, insertions, deletions, and/or additions. Typical variants which are analogs include those having from 1 to 5 amino acid substitutions. In the case of TFPI analogs having one or more amino acid additions, these additional amino acids can be added at any position in the molecule, for example at the amino or carboxy terminus. One TFPI analog, N-L-alanyl-TFPI ("ala-TFPI") has an additional alanine residue at the amino terminal end. Ala-TFPI may be produced recombinantly (r- ala-TFPI) according to procedures described in U.S. Patent Application Publication No. 2004/0224886.

[19] Fragments are portions of TFPI, TFPI analogs, or TFPI derivatives and include Kunitz domains 1 ; 2; 3; 1 and 2; or 2 and 3, or deletions of the N-terminus, C-terminus, or both. Fragments of TFPI comprise at least 20 consecutive amino acids of SEQ ID NO: 1. Derivatives are defined as TFPI, TFPI analogs, or TFPI fragments having additional moieties. Examples of such additions include those generated by glycosylation, phosphorylation, acetylation, or amidation.

[20] Percent homology or identity between a TFPI variant and SEQ ID NO: 1 is determined using the Blast2 alignment program (Blosum62, Expect 10, standard genetic codes, open gap 11, extension gap 1, gap x_dropoff 50, and low complexity filter off). TFPI variants will generally have about 70% or greater, typically about 80% or greater, usually about 90% to about 95% {e.g., 90, 91, 92, 93, 94, or 95%) or greater, and often about 98% or 99% amino acid sequence identity to SEQ ID NO: 1.

[21] The TFPI or TFPI variant in pharmaceutical compositions of the present invention for parenteral administration is present in concentrations generally ranging from about 0.05 to about 5 mg/ml, more typically from about 0.05 to about 1 mg/ml, often from about 0.1 to about 1 mg/ml, and usually from about 0.10 to about 0.50 mg/ml. For example, concentrations of ala-TFPI ranging from about 0.15 to about 0.45 mg/ml have been found to be therapeutically effective in the treatment of indications such as severe Community Acquired Pneumonia (CAP). In general, the TFPI or TFPI variant pharmaceutical compositions are administered parenterally. Parenteral administration refers to administration routes other than through the gastrointestinal tract, including intravenous, intramuscular, intradermal, subcutaneous, intrathecal, intraarterial, intracardiac, transdermal, transmucosal, etc. Intravenous administration is often employed. For example, representative parenteral administrations to patients include continuous intravenous infusions of about 10 μg/hr/kg to about 100 μg/hr/kg for a period from about 24 hours to about 168 hours. [22] Pharmaceutical compositions comprising TFPI or a TFPI variant may generally comprise any of a number of solubilizers, antioxidants, and/or buffers. Suitable solubilizers include amino acids such as arginine or lysine, which may be present in either a free base or a salt form, for example the hydrochloric acid salt form. Arginine and lysine analogs and the L-stereoisomers of arginine and lysine, any of which may also be in their free base or salt forms, are described in U.S. Patent Application Publication No. 2004/0224886 and may also be used as solubilizers. Arginine is beneficial not only for increasing the solubility of the TFPI or TFPI variant in aqueous solutions, but also for increasing, in a concentration dependent manner, the stability of the polypeptide against aggregation/precipitation (i.e., aggregation stability). The concentration of solubilizer in the pharmaceutical composition generally ranges from about 50 to about 600 mM, typically from about 100 to about 400 raM, and is often about 300 mM.

[23] Suitable antioxidants for use in TFPI or TFPI variant compositions of the present invention are described in U.S. Patent Application Publication No. 2004/0224886 and include oxygen displacement gases (e.g., nitrogen), scavengers of oxygen or free radicals (e.g., methionine), and chelating agents (e.g., EDTA). Methionine is particularly effective, and is believed to function as a "sacrificial" scavenger of oxygen molecules that could otherwise oxidize the methionine residues which are part of the TFPI or TFPI variant polypeptide. Native TFPI, for example, has 5 methionine residues per polypeptide molecule.

[24] When methionine is used in the TFPI or TFPI variant compositions of the present invention, it is present in an amount such that the molar ratio of non-TFPI or non- TFPI variant methionine to TFPI or TFPI variant methionine is generally at least about 1:1, typically at least about 10:1, usually at least about 25:1, and often at least about 100:1. The upper bound of this molar ratio may generally be up to 10,000:1, typically up to 5,000:1, and usually up to 1,000:1. The phrase "TFPI or TFPI variant methionine" is used to indicate methionine that is part of the TFPI or TFPI variant polypeptide, in contrast to "non-TFPI or non-TFPI variant methionine," which is methionine added to the composition as an antioxidant and which is not part of the TFPI or TFPI variant polypeptide. It is possible that the "non-TFPI or non-TFPI variant methionine" may itself be bound methionine, such as when methionine is added in the form of a polypeptide. For example, a polypeptide comprising poly(methionine) may be added to the composition as an antioxidant and function in a manner similar to free methionine. In terms of absolute concentration, when an antioxidant such as methionine is used, it is present in the pharmaceutical composition in a concentration generally from about 1 mM to about 10 mM and typically from about 2 mM to about 10 mM. Often, free methionine is introduced at a concentration of about 5 mM.

[25] Buffers which may be used in the TFPI or TFPI variant compositions of the present invention are described in U.S. Patent Application Publication No. 2004/0224886. Typically, when a buffer comprising an acid is used in an aqueous composition, it is prepared using a salt form of the acid or a combination of the acid and a salt form of the conjugate base of the acid. Thus, for example, the buffer may be prepared using an acid in combination with the sodium, potassium, ammonium, calcium, and/or magnesium salt of its conjugate base. Typical acids employed in such combinations include citric acid, succinic acid, phosphoric acid, glutamic acid, maleic acid, malic acid, acetic acid, tartaric acid, and aspartic acid. Citric acid or succinic acid, for example, may be used in the composition together with their respective conjugate bases. The combination citric acid/sodium citrate represents one such buffer system. The buffer may be present in the composition in a concentration from about 5 mM to about 50 mM, and is often present in a concentration from about 10 mM to about 30 mM. A typical buffer concentration is about 20 mM.

[26] For any buffer system, the amounts of acid and salt form of its conjugate base to buffer a solution at a given buffer strength (i.e., concentration) and pH may be readily determined. The pH of TFPI or TFPI variant compositions of the present invention significantly affects the polypeptide solubility and hence its stability. Normally, the TFPI or TFPI variant composition will therefore be buffered at a pH from about 4 to about 8, and often from about 5 to about 6.5. A pH of about 5.5, for example, can provide good stability of the TFPI or TFPI variant. In view of the above considerations, a suitable TFPI pharmaceutical composition therefore comprises, in addition to TFPI or a TFPI variant, from about 100 mM to about 400 mM of arginine, from about 2 to about 10 mM methionine, and from about 10 to about 50 mM of citric acid/sodium citrate, at a pH from about 4 to about 8. [27] The above pharmaceutical compositions comprising TFPI or a TFPI variant are generally prepared from an aqueous solution having a higher concentration of polypeptide, which is stored and later diluted, prior to use of the pharmaceutical composition. The "solution" or "aqueous solution" of TFPI or TFPI variant therefore refers to the generally more concentrated form used to store the polypeptide (i.e., the storage form), which is diluted with a diluent, prior to use, to provide the pharmaceutical composition containing this polypeptide (i.e., the administration form). The term "storage" refers to all time periods during which the polypeptide is maintained in its storage form, including packaging (e.g., under aseptic conditions), labeling, shelving, shipping, etc. Typical storage periods for the TFPI or TFPI variant containing aqueous solution generally range from about 1 to about 60 months, typically from about 5 to about 36 months, and often from about 6 to about 24 months.

[28] The TFPI or variant concentration in the aqueous solution, therefore, is typically in the range from about 1 mg/ml to about 20 mg/ml, and often from about 5 mg/ml to about 10 mg/ml. These concentrations of the polypeptide in solution are achieved using a suitable solubilizer described above. Normally, solutions having relatively high TFPI or TFPI variant concentrations in the above ranges are susceptible to loss of soluble protein due to aggregation/precipitation. This phenomenon represents a major degradation pathway for TFPI and it can adversely impact biological activity and therapeutic efficacy of the polypeptide.

[29] The solubilizer arginine can significantly improve the aggregation stability of TFPI and TFPI variants. Arginine is therefore generally present in the TFPI or TFPI variant solution in an amount that brings about the desired effect of stabilizing the polypeptide during storage, such that, relative to a similar solution but without added arginine, the solution exhibits improved resistance to aggregation (i.e., aggregation stability) during storage. In general, at least a portion of the solubilizer (e.g., arginine) that is present in the pharmaceutical compositions described above is stored with the TFPI or TFPI variant solution to allow the polypeptide to be effectively concentrated and stabilized against aggregation/precipitation. A significant portion, typically at least about 70% and usually at least about 80%, of solubilizer is also normally present in the diluent. This allows the solubilizer concentrations in the solution, diluent, and pharmaceutical composition to be maintained essentially constant (e.g., the solubilizer concentration in solution maintained within about +/- 20% of the solubilizer concentration in the pharmaceutical composition, after combining the diluent and solution to provide the pharmaceutical composition).

[30] Aggregation stability is expressed in terms of the polypeptide (i.e., TFPI or TFPI variant) solution half-life with respect to aggregation. The loss of soluble polypeptide due to aggregation/precipitation is monitored over time, by maintaining one or more stability samples and, at various time intervals, separating soluble protein from aggregated/precipitated protein in a given volume (e.g., through centrifugation in a microcentrifuge tube). The concentration of soluble protein is determined based on the decrease in total polypeptide peak area, as measured using ion-exchange high pressure liquid chromatography (EEX-HPLC) (Chen et al., J. PHARM. SCI. 88:881-888 (1999)) or reverse-phase high pressure liquid chromatography (RP-HPLC). The loss in the concentration of soluble polypeptide (Ysoi), from its initial concentration (Y_o,_S0i) over storage time (t) can then be fitted to a first-order exponential decay model

to determine the deactivation rate constant with respect to aggregation (k_agg). The estimated half-life (t^gg) with respect to aggregation is 0.693/kagg. The solution half-life with respect to aggregation is highly dependent on solution temperature, requiring the stability sample(s) to be maintained at essentially constant temperature. For purposes of the present invention, the solution comprising TFPI or a TFPI variant has a half-life with respect to aggregation generally of at least about 15 days, and typically from about 20 days to about 70 days, at 50⁰C.

[31] Advantageously, increasing the TFPI or TFPI variant concentration in the aqueous solution in which the polypeptide is stored (relative to its concentration in the pharmaceutical composition that is ultimately administered to a patient) directionally improves oxidation stability. Consequently, TFPI or TFPI variant solutions stored at a sufficiently high polypeptide concentration and/or at a sufficiently low temperature may have adequate oxidation stability for commercial use, even without the need for (i.e._t in the absence of) an antioxidant such as the oxygen free radical scavenger methionine, discussed above. If an antioxidant is used, at least a portion of the antioxidant (e.g., methionine) present in the pharmaceutical compositions described above is stored with the TFPI or TFPI variant solution to improve the polypeptide stability against oxidation during storage. As with the solubilizer described above, a significant portion, typically at least about 70% and usually at least about 80%, of the antioxidant is also normally present in the diluent. This allows the antioxidant concentrations in the solution, diluent, and pharmaceutical composition to be maintained essentially constant (e.g., the antioxidant concentration in solution maintained within about +/- 20% of the antioxidant concentration in the pharmaceutical composition, after combining the diluent and aqueous solution to provide the pharmaceutical composition). It is possible to store significant portions of both the solubilizer and antioxidant with the diluent, as they can generally undergo terminal sterilization by heat without suffering from a loss of functionality (i.e., the ability to soiubilize the polypeptide or hinder its oxidation) in the pharmaceutical composition. Storage of significant portions of the solubilizer and antioxidant with the diluent reduces the amount of material that must be prepared and introduced into a storage container with the TFPI or TFPI variant solution under aseptic conditions.

[32] Oxidation stability is expressed in terms of the polypeptide (i.e., TFPI or TFPI variant) solution half-life with respect to oxidation. Degradation by the oxidation of TFPI or TFPI variant methionine can be measured in stability samples over time, based on the decrease in the polypeptide peak area(s) corresponding to non-oxidized species (i.e., species not having one or more oxidized methionine residues, such as methionine sulfoxide), as measured using RP-EDPLC. The loss in the concentration of non-oxidized polypeptide (Y_nOn-ox) from its initial concentration (Y₀,non-ox) over storage time (t) can then be fitted to a first-order exponential decay model (Y_non-

to determine the deactivation rate constant (IC_0X) and half-life (ti/2_)Oχ) with respect to oxidation. The estimated half-life with respect to oxidation is 0.693/koχ- The solution half-life with respect to oxidation is dependent on solution temperature, requiring the stability sample(s) to be maintained at constant temperature. For purposes of the present invention, the solution comprising TFPI or a TFPI variant has a half-life with respect to oxidation generally of at least about 5 months, typically from about 10 months to about 100 months, and often from about 25 months to about 50 months, at 30⁰C. [33] In cases where TFPI or TFPI variant solutions having very high stability with respect to both aggregation and oxidation are desired, the solution may be stored under refrigerated conditions. For example, the solution may be maintained at a temperature from about 2°C (36°F) to about 8⁰C (46°F) at polypeptide concentrations given above, prior to dilution to provide the pharmaceutical composition. Under such conditions, the solution half-life with respect to both aggregation and oxidation is generally at least about 10 months, typically at least about 20 months, and often in the range from about 25 months to about 200 months. A half-life from about 75 months to about 200 months with respect to both aggregation and oxidation is representative of the polypeptide solution stability according to the present invention. In many cases, depending on the solution composition (e.g., the amounts of TFPI or TFPI variant, solubilizer, and/or antioxidant) and solution temperature, essentially no degradation of the polypeptide with respect to either aggregation or oxidation may be detectible for at three months of storage, and often for at least six months of storage. As such, TFPI or TFPI variant solutions may generally be stored for periods of up to about 36 months without suffering a significant loss of biological activity. Typical storage periods can range from about 6 months to about 24 months.

[34] As explained above, as an alternative (or in addition) to the use of an antioxidant, the oxidation stability of TFPI or variant may also be directionally improved as the solution polypeptide concentration is increased. This characteristic provides important advantages in the kits and methods of the present invention where a TFPI or a TFPI variant solution is stored in a relatively concentrated aqueous solution (i.e., in a state less susceptible to degradation by oxidation) and then diluted to provide drug concentrations for parenteral administration at practical therapeutic administration rates. For example, a 100 ml vial of concentrated TFPI or TFPI variant may be stored with sufficient polypeptide for polypeptide administration at a representative rate from about 10 to about 100 μg/hr/kg body weight, and usually from about 25 to about 15 μg/hr/kg body weight, for a time from about 96 to about 120 hours. Typical concentrated TFPI or TFPI variant solution vials, however, will contain from about 5 mg to about 450 mg, and often from about 20 to about 150 mg, of polypeptide. These amounts of TFPI or TFPI variant will generally necessitate the use of multiple vials over the course of administration (e.g., by intravenous infusion), which typically requires a total dosage from about 48 mg to about 2160 mg, and often from about 48 mg to about 1080 mg.

[35] The ability to effectively store TFPI or a TFPI variant in concentrated aqueous solutions, for example those comprising from about 1 mg/ml to about 20 mg/ml of polypeptide, is also important in terms of providing manufacturing flexibility. In particular, the availability of manufacturing facilities having the capacity to produce a commercial batch of TFPI or TFPI variant at the relatively low concentrations used for administration (e.g., from about 0.15 mg/ml to about 0.45 mg/ml) is very limited. For example, a typical production run at these concentrations would likely involve the manufacture of approximately 25,000 to 50,000 type I glass vials. This scale of production typically requires the use of tanks having a capacity on the order of 5,000 liters as well as other large-scale equipment. Moreover, the storage and distribution (e.g., shipping) of large quantities of product vials or containers, especially under refrigerated conditions, would greatly add to the overall costs associated with the production of TFPI or TFPI variant compositions in the form in which they are normally administered to patients. Thus, while small-scale studies, including clinical trials, were conducted with ala-TFPI produced in the same form as administered to patients, this is not a desired practice for commercial scale manufacturing operations. Production costs and processing times can be significantly reduced by manufacturing the polypeptide in a more concentrated form.

[36] If it is desired to buffer the pharmaceutical composition of the present invention using any of the buffer systems as described above, at least a portion, typically at least about 70%, and usually at least about 80%, of the buffer (e.g., citric acid/sodium citrate) is stored with the diluent, as the buffer can generally be terminally stabilized by heat without suffering from a loss of functionality (i.e., the ability to maintain pH) in the pharmaceutical composition. Storage of a significant portion of the buffer with the diluent also reduces the amount of material that must be prepared and introduced into a storage container with the TFPI or TFPI variant under aseptic conditions. Storing at least some portion of the buffer with the TFPI or TFPI variant solution, however, allows for the maintenance of a desired solution pH and can therefore improve TFPI or TFPI variant solubility and stability in the solution. As with the solubilizer and stabilizer described above, storing a significant portion of the buffer with the diluent allows the buffer concentrations in the solution, diluent, and pharmaceutical composition to be maintained essentially constant (e.g., the buffer concentration in solution maintained within about +/- 20% of the antioxidant concentration in the pharmaceutical composition, after combining the diluent and solution to provide the pharmaceutical composition). In one embodiment of the invention, therefore, both the TFPI or TFPI variant solution and the diluent have a pH from about 4 to about 8 and comprise from about 100 mM to about 400 mM arginine, from about 2 to about 10 mM methionine, and from about 10 mM to about 50 mM of citric acid/sodium citrate buffer. The amount of diluent used will generally be from about 50 ml to about 1000 ml, typically from about 50 ml to about 500 ml, and often from about 100 ml to about 250 ml:

[37] In any event, regardless of the disposition of the solubilizer, antioxidant, and/or buffer (i.e., whether stored in the TFPI or TFPI variant solution, diluent, or both), the pharmaceutical composition resulting from the combination of these components (i.e., the aqueous solution and the diluent) will generally comprise from about 0.10 mg/ml to about 10 mg/ml of the polypeptide, from about 50 mM to about 600 mM solubilizer, from about 1 mM to about 10 mM antioxidant, and from about 5 mM to about 50 mM buffer. It is also within the scope of the invention to combine two or more components (e.g., two or more diluents) with the TFPI or ala-TFPI solution to provide pharmaceutical compositions described herein, with the realization of equivalent benefits and advantages. Such embodiments of the invention, which similarly take advantage of the characteristics of TFPI and TFPI variants described above, would be apparent to those having skill in the art and having regard for the present disclosure.

[38] TFPI or TFPI variant solutions of the present invention are aseptically packaged and maintained in a sterile environment prior to (as well as after) their subsequent dilution to provide the pharmaceutical compositions described above. The presence of harmful bacteria in the polypeptide solution is therefore avoided. TFPI and TFPI variants, however, are heat-sensitive and cannot be exposed to the elevated temperatures and/or radiation doses commonly used for sterilization, without suffering from a loss in biological activity. For these reasons, the TFPI or TFPI variant solutions must be manufactured and introduced into a container in a sterile mode. A number of medical containers such as molded plastic containers may be used for aseptically packaging and containing TFPI or TFPI variant solutions for parenteral administration. For example, containers having a closure system that provides a sterile barrier or seal are described in U.S. Patent No. 6,371,319. Flexible bags that may be filled and maintained under aseptic conditions are described, for example, in U.S. Patent No. 4,840,017. Other methods for aseptically packaging solutions into containers are described, for example, in U.S. Patent No. 6,769,231. Medical containers used for aseptic packaging are normally sterilized (prior to the aseptic introduction of the TFPI or TFPI variant solution into this container), by a sterilization process such as autoclaving. Autoclaving subjects the container to temperatures typically in the range of about 115°C (240⁰F) to about 125°C (260⁰F).

[39] So-called "form, fill, and seal" machines can be used for packaging or introducing the TFPI or TFPI variant solution into a container under aseptic conditions. These machines typically form a bag from a web of flexible material and pass the bag directly to a filling station where a solution may be fed by gravity or otherwise introduced into the bag through an opening in the bag. The same machine then seals the bag opening to enclose the product. Thus, the formation of the container, its filling with solution, and the subsequent formation and application of a seal for the container are achieved aseptically in an uninterrupted sequence of operations, without exposure to nonsterile environments between operations.

[40] In contrast to the TFPI or TFPI variant solution, the diluent (which is later combined with this solution to provide the pharmaceutical composition) may advantageously be sterilized (e.g., in a second container that is separate from a first container into which the TFPI or TFPI variant solution is introduced) by heating and/or irradiation, without adverse effects on the ability of the diluent to solubilize, stabilize, and/or buffer the pharmaceutical composition. As an alternative to aseptic packaging of the diluent, therefore, terminal sterilization may be employed subsequent to packaging. Terminal sterilization refers to the process of sterilizing the packaged material (e.g., the diluent, packaged in a flexible, collapsible "bag" container) and often involves heating the diluent to a temperature from about 80⁰C (175°F) to about 150⁰C (300°F), and typically from about 105°C (220⁰F) to about 130⁰C (265°F), for a time from about 5 minutes to about 1 hour, and typically from about 10 minutes to about 30 minutes. [41] The ability to terminally sterilize a component of pharmaceutical composition (i.e., the diluent) provides additional, important commercial advantages in the methods and kits of the present invention. In particular, aseptic packaging and storage of the component of the pharmaceutical comprising the TFPI or TFPI variant, combined with terminal sterilization of another component of the pharmaceutical composition prior to dilution, reduces overall production costs, relative to the situation where the entire pharmaceutical composition requires aseptic packaging and storage. Moreover, terminal sterilization of the diluent prior to dilution (i.e., combining the TFPI or TFPI variant solution with the diluent to provide the pharmaceutical composition) provides a high assurance of its sterility and can compensate for any non-aseptic conditions encountered in prior steps, such as packaging of the diluent.

[42] Sterile filtration may be employed in either the aseptic packaging of the TFPI or TFPI variant solution or in the terminal sterilization of the diluent. Likewise, the subsequent mixing of these components, to reduce the polypeptide concentration of the solution and to provide the pharmaceutical composition, may be accompanied by sterile filtration. When converting the storage form (i.e., the TFPI or TFPI variant solution) to the administration form (i.e., the TFPI or TFPI variant pharmaceutical composition) sterility concerns arise especially in cases where the components to be mixed, although stored in a sterile environment, are susceptible to contact with bacteria during or after mixing (e.g., into a third, separate container). In order to ensure that either or both of the TFPI or TFPI variant solution and the diluent are sterile upon mixing, a process employing a sterilizing-grade filter or other known sterilization processes may be used to maintain a sterile mixing environment. In certain packaging systems, however, the TFPI or TFPI variant solution may be diluted with the diluent in a completely enclosed system, including those described below, such that aseptic conditions can be maintained without additional sterilization steps.

[43] Overall, the methods described herein allow pharmaceutical compositions comprising TFPI or a TFPI variant to be conveniently prepared in remote locations (i.e., away from the manufacturing site, such as in a hospital), whereby the components of such compositions are maintained under both sterile and stable conditions. A concentrated TFPI or TFPI variant solution, as a first component, may be manufactured in commercially practical volumes per manufacturing batch. Advantageously, concentrating the TFPI or TFPI variant directionally improves its stability with respect to oxidation. The avoidance of large manufacturing capacity requirements provides flexibility in the choice of manufacturing site and/or allows for minimal modification of existing commercial drug manufacturing facilities (e.g., to provide sterile manufacturing in large-scale operations). Moreover, limiting the number of vials or other containers to be stored, especially under refrigerated conditions, reduces overall costs.

[44] A diluent, as a second component, may contain one or more solubilizers, antioxidants, and/or buffers which are capable of undergoing sterilization by heat and/or irradiation without a loss of functionality. Thus, the TFPI or TFPI variant solution and diluent may be provided to the remote location in a ready-to-use format (e.g., in a kit) which does not necessarily require special handling, for example in a hospital pharmacy, to generate the pharmaceutical composition. The kits of the present invention, for preparing the pharmaceutical compositions described herein, may therefore comprise separate containers in which the aqueous solution comprising TFPI or a TFPI variant and the diluent are packaged, respectively. In one embodiment, the aqueous solution is packaged under aseptic conditions and the diluent is terminally sterilized as described above or otherwise aseptically packaged as well. The aseptically packaged aqueous solution is then exposed to the diluent (e.g., by opening vials, breaking seals, etc.) under aseptic conditions to provide the pharmaceutical composition.

[45] The term "separate containers" as used to describe the packaging of the TFPI or TFPI solution and the diluent means that these components are not in fluid communication. This term, however, does not preclude the use, for example, of systems whereby the separate containers for these components are themselves physically connected to allow the TFPI or TFPI variant solution and diluent to be aseptically combined upon establishing flow communication between the separate containers. In one such system, the aqueous solution comprising TFPI or a TFPI variant may be packaged in a glass vial container and the diluent packaged in a flexible bag container that is attached to the glass vial via an umbilical transfer tube. The combination of the vial and diluent bag therefore represents a kit of the present invention, where the components are provided in a format that allows for their mixing (and consequently the provision of the pharmaceutical composition) under aseptic conditions. Packaging the diluent in a flexible bag allows for its terminal sterilization by heating or irradiation. In another embodiment, the TFPI or TFPI variant solution vial may be screwed or "docked" onto a flexible bag containing the diluent. A threaded slot can provide an air-tight connection, allowing the components to mix aseptically when a stopper or cap on the solution vial is removed. The ADD- VANTAGE^® system (Hospira, Inc., Lake Forest, IL, USA) exemplifies such as system.

[46] The containers used for the TFPI or TFPI variant solution and the diluent may be made from conventional materials such as glass or plastics which are used to package pharmaceuticals in a sterile environment. Kits of the present invention may therefore utilize vials, ampules, cartridges, bags or other large-volume containers, etc. The container used for the diluent is preferably capable of undergoing terminal sterilization, without adverse effects (e.g., thermal degradation of the container). Representative containers for the diluent include vials and flexible plastic bags. For example, diluent in an amount generally ranging from about 10 ml to about 500 ml, typically from about 50 ml to about 200 ml, may be contained in flexible bags which allow terminal sterilization of the bag contents. Examples of such bags include those which are made from polyolefin (e.g., polypropylene or polyethylene), polyvinyl chloride (PVC), ethylene vinyl acetate (EVA), and other thermally stable materials.

[47] In a representative kit of the present invention, the aqueous solution component comprises TFPI or TFPI variant at a relatively high concentration, a solubilizer, an antioxidant, and a buffer. The diluent component comprises additional amounts of the solubilizer, antioxidant, and buffer. In one embodiment, the TFPI or TFPI variant solution and the diluent, when combined (e.g., by establishing flow communication between these components) results in a pharmaceutical composition having a pH from about 4 to about 8 and comprising from about 0.10 mg/ml to about 0.50 ala-TFPI, from about 100 mM to about 400 mM arginine, from about 2 to about 10 mM methionine, and from about 10 mM to about 50 mM of citric acid/sodium citrate buffer.

[48] All references cited in this specification, including without limitation, all U.S., international, and foreign patents and patent applications, as well as all abstracts and papers {e.g., journal articles, periodicals, etc.), are hereby incorporated by reference into this specification in their entireties.

[49] As various changes could be made in the above methods and compositions without departing from the scope of the invention, it is intended that all matter contained in this application, including all theoretical mechanisms and/or modes of interaction described above, shall be interpreted as illustrative only and not limiting in any way the scope of the appended claims.

[50] The following examples are set forth as representative of the present invention. These examples are not to be construed as limiting the scope of the invention as these and other equivalent embodiments will be apparent in view of the present disclosure and appended claims.

EXAMPLE l

[51] An aqueous solution of the TFPI variant ala-TFPI, as described above, is prepared and purified in a manufacturing facility according to procedures outlined in U.S. Published Application No. 2005/0037475. A 5 ml quantity of solution containing 10 mg/ml ala-TFPI, 300 mM L-arginine, 5 mM methionine, and 20 mM of a citric acid/sodium citrate buffer to maintain a pH of 5.5, is aseptically transferred to a glass vial for use in the ADD- V ANT AGE^® system. A flexible diluent bag, also part of this system, is filled with 328 ml of aqueous diluent containing arginine, methionine, and buffer in the same concentrations as in the ala-TFPI solution. The contents of this bag are terminally sterilized by subjecting the filled bag to a temperature of 120⁰C (248°F) for about 15 minutes. The contents of the vial are stored at about 5°C (41⁰F)₅ allowing the aqueous solution to be stored for at least a 24 months.

[52] The sterile ala-TFPI solution and diluent described above are combined aseptically at the hospital where the pharmaceutical composition resulting from this combination is ultimately administered intravenously to a patient. Protective covers on both the ala- TFPI solution vial and the vial port of the diluent bag are removed, according to instructions supplied with the vial/bag kit. The vial is screwed onto to the vial port of the diluent bag until an air-tight seal is achieved. The inner cap of the vial is then pulled into the interior of the diluent container, allowing the ala-TFPI solution and diluent to mix aseptically within the diluent bag. These components are mixed thoroughly in the bag to provide a 0.15 mg/ml ala-TFPI pharmaceutical composition in 300 mM arginine, 5 mM methionine, and 20 mM of citric acid/sodium citrate buffer at a pH of 5.5. This resulting pharmaceutical composition is administered to a patient by continuous intravenous infusion of the polypeptide at a rate of 25 μg/kg/hr. The administration period begins within 48 hours of mixing the concentrated ala-TFPI aqueous solution and diluent.

EXAMPLE 2

[53] A 10 ml portion of the concentrated ala-TFPI aqueous solution (10 mg/ml) and 212 ml of the aqueous diluent described in Example 1 are aseptically packaged in a vial portion and a bag portion, respectively, of a pre-attached vial and bag assembly (or kit). The vial and bag are physically attached through a tubing connection, but fluid communication between the vial and bag is prevented by a partition. Following a storage period and within 48 hours prior to intravenous administration of the ala-TFPI pharmaceutical composition to a patient, removal of this partition allows the aqueous solution and diluent to mix aseptically and provide an ala-TFPI pharmaceutical composition comprising 0.45 mg/ml ala-TFPI pharmaceutical composition in 300 mM arginine, 5 mM methionine, and 20 mM of citric acid/sodium citrate buffer at a pH of 5.5.

EXAMPLE 3

[54] After manufacture of the ala-TFPI solution described in Example 1 , it is packaged aseptically in a container that has been sterilized by autoclaving. The aqueous diluent described in Example 1 is packaged in a flexible bag which is then terminally sterilized at 120⁰C (248°F) for about 15 minutes. A kit comprising the separate containers is prepared (with instructions for combining the contents of the containers prior to administration) and transferred to a hospital where the kit is stored for up to 24 months under refrigerated conditions. The ala-TFPI solution and aqueous diluent are ultimately mixed under aseptic conditions to provide a pharmaceutical composition that is administered intravenously to a patient.

Claims

WHAT IS CLAIMED IS:

L. A method for preparing a pharmaceutical composition for parenteral administration to a patient, the method comprising:

aseptically packaging, in a first container, an aqueous solution comprising from about 1 mg/ml to about 20 mg/ml of TFPI or a TFPI variant having about 70% or greater amino acid sequence identity to SEQ ID NO:1;

combining said aqueous solution with a diluent, packaged in a second container, to provide said pharmaceutical composition,

wherein said first container and said second container are selected from the group consisting of a vial, a flexible bag, an ampule, and a cartridge.

2. The method of claim 1, wherein said TFPI variant selected from the group consisting of (i) Tissue Factor Pathway Inhibitor (TFPI), (ii) ala-TFPI, and (iii) analogs of (i) or (ii) having from 1 to 5 amino acid substitutions.

3. The method of claim 1, wherein said TFPI variant is ala-TFPI.

4. The method of claim 1, wherein said diluent comprises a buffer.

5. The method of claim 4, wherein said buffer comprises citric acid/sodium citrate.

6. The method of claim 5, wherein said pharmaceutical composition has a pH from, about 4 to about 7 and comprises from about 0.10 mg/ml to about 0.50 mg/ml of ala-TFPI, from about 100 mM to about 400 mM of arginine, from about 2 raM to about 10 mM methionine, and from about 10 mM to about 50 mM of citric acid/sodium citrate.

7. The method of claim I₅ further comprising, prior to combining said aqueous solution with said diluent, terminally sterilizing said diluent.

8. The method of claim 7, wherein said sterilizing comprises heating or irradiating said diluent.

. The method of claim 8, wherein said sterilizing comprises heating said diluent to a temperature from about 80⁰C (175°F) to about 150°C (300⁰F) for a time from about 5 minutes to about 1 hour.

10. The method of claim 1, wherein said aqueous solution is maintained at a temperature from about 2°C (36°F) to about 8⁰C (46°F) prior to combining said aqueous solution with said diluent.

11. The method of claim 1, wherein said aqueous solution has a volume of less than about 100 ml.

12. A pharmaceutical composition prepared according to the method of claim 1.

13. A method for preparing a pharmaceutical composition for parenteral administration to a patient, the method comprising:

providing a first container comprising an aseptically packaged aqueous solution comprising from about 1 mg/ml to about 20 mg/ml of TFPI or a TFPI variant having about 70% or greater amino acid sequence identity to SEQ ID NO:1;

providing a second container comprising a terminally sterilized diluent; and

exposing said aseptically packaged aqueous solution to said diluent under aseptic conditions to provide said pharmaceutical composition.

14. A method for stabilizing an aqueous solution prior to its use in the preparation of a pharmaceutical composition, the method comprising:

aseptically packaging an aqueous solution comprising TFPI or a TFPI variant having about 70% or greater amino acid sequence identity to SEQ DD NO:1 to provide an aseptically packaged aqueous solution; storing said aqueous solution under conditions whereby said aqueous solution has half- life with respect to both aggregation and oxidation from about 25 months to about 200 months; and thereafter

exposing said aseptically packaged aqueous solution to said diluent to provide said pharmaceutical composition.

15. The method of claim 14, wherein said conditions include a concentration of said TFPI or TFPI variant in said aqueous solution from about 1 mg/ml to about 20 mg/ml and a temperature of said aqueous solution from about 2°C (36°F) to about 8°C (46°F).

16. The method of claim 14, wherein said aqueous solution is stored for a period from about 6 months to about 24 months.

17. A method for providing a pharmaceutical composition at a remote location, the method comprising:

aseptically packaging, at a first location, an aqueous solution comprising from about 1 mg/ml to about 20 mg/ml of TFPI or a TFPI variant having about 70% or greater amino acid sequence identity to SEQ ID NO:1;

diluting, at a second location, said aqueous solution with a diluent to provide said pharmaceutical composition.

18. A kit for preparing pharmaceutical composition for parenteral administration to a patient, the kit comprising:

an aqueous solution, packaged under aseptic conditions and comprising from about 1 mg/ml to about 20 mg/ml of TFPI or a TFPI variant having about 70% or greater amino acid sequence identity to SEQ ID NO: 1 ; and

a diluent,

wherein said aqueous solution and said diluent are packaged in separate containers.

19. The kit of claim 18, wherein said separate containers are connected to allow said aqueous solution and said diluent to be aseptically combined upon establishing flow communication between said separate containers.

20. The kit of claim 19, wherein said aqueous solution is packaged in a vial container and said diluent is packaged in a flexible bag container.

21. The kit of claim 18, wherein said diluent is terminally sterilized by heating or irradiation.

22. The kit of claim 21, wherein said diluent is terminally sterilized by heating to a temperature from about 80⁰C (175°F) to about 150⁰C (300⁰F) for a time from about 5 minutes to about 1 hour.

23. The kit of claim 18, wherein said TFPI variant is ala-TFPI.

24. The kit of claim 18, wherein said diluent comprises a buffer.

25. The kit of claim 24, wherein said aqueous solution further comprises a solubilizer and an antioxidant.

26. The kit of claim 25, wherein said solubilizer is arginine, said antioxidant is methionine, and said buffer is citric acid/sodium citrate.

27. The kit of claim 26, wherein, when said aqueous solution and said diluent are combined, a resulting pharmaceutical composition, having a pH from about 4 to about 8, comprises from about 0.10 mg/ml to about 0.50 mg/ml of ala-TFPI, from about 100 mM to about 400 mM of arginine, from about 2 mM to about 10 mM methionine, and from about 10 mM to about 50 mM of citric acid/sodium citrate.