WO2008022151A1 - Prophylactic treatment of hemophilia - Google Patents

Abstract

A method for the prophylactic treatment of Hemophilia A or B is disclosed. The method involves administration of Factor VIII (Hemophilia A) or Factor IX (Hemophilia B) to a patient at regular intervals to maintain a predetermined minimum level of Factors VIII or IX in the blood stream for an extended period. The method eliminates the need for on demand Factor treatment except for traumatic injury and surgery.

Description

PROPHYLACTIC TREATMENT OF HEMOPHILIA

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 60/837,784, filed August 15, 2006, entitled "Prophylactic Treatment of Hemophilia," which is hereby incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

[0002] The invention relates to methods of treating Hemophilia prophylactically by administering Factor VIII (Hemophilia A) and Factor IX (Hemophilia B) at regular intervals to provide a constant level of Factor in the blood. Description of the Related Art

[0003] Bleeding disorders can result from a deficiency in the functional levels of one or more of the blood proteins, collectively known as blood coagulation factors, that are required for normal hemostasis, i.e. blood coagulation. The severity of a given bleeding disorder is dependent on the blood level of functional coagulation factors. Mild bleeding disorders are generally observed when the functional level of a given coagulation factor reaches about 5% of normal, but if the functional level falls below 1%, severe bleeding is likely to occur with any injury to the vasculature.

[0004] Medical experience has shown that essentially normal hemostasis can be temporarily restored by intravenous infusion of biological preparations containing one or more of the blood coagulation factors. So-called replacement therapy, whereby a biological preparation containing the deficient blood coagulation factor is infused when bleeding occurs (on demand) or to prevent bleeding (prophylactically), has been shown to be effective in managing patients with a wide variety of bleeding disorders. In general, for replacement therapy to be effective, intravenous infusions of the missing coagulation factor are targeted to achieve levels that are well above 5% of normal over a two- to three-day period. Historically, patients who suffer from hemophilia, a genetically acquired bleeding disorder that results from a deficiency in either blood coagulation Factor VIII (hemophilia A) or Factor IX (hemophilia B), were successfully treated by periodic infusion of whole blood or blood plasma fractions of varying degrees of purity.

[0005] Hemophilia A, the most common, results from a mutation in the gene for Factor VIII; Hemophilia B, also known as Christmas Disease, results from a mutation in the gene for Factor IX. Hemophilia B, like Hemophilia A, is X-linked and accounts for approximately 12% of hemophilia cases. The symptoms are identical to those of Hemophilia A: excessive bleeding upon injury; and spontaneous bleeding, especially into weight-bearing joints, soft tissues, and mucous membranes. Repeated bleeding into joints results in hemarthroses, causing painful crippling arthropathy that often necessitates joint replacement. Hematomas in soft tissues can result in pseudo tumors composed of necrotic coagulated blood; they can obstruct, compress, or rupture into adjacent organs and can lead to infection. Once formed the hematomas are difficult to treat, even with surgery. Recovery of nerves after compression is poor, resulting in palsy. Those bleeding episodes that involve the gastrointestinal tract, central nervous system, or airway/retroperitoneal space can lead to death if not detected. Intracranial bleeding is a major cause of death in hemophiliacs.

[0006] Current treatment of these symptoms consists of intravenous replacement therapy with Factor VIII or Factor IX concentrates. Treatment of major bleeding episodes is by bolus injection of concentrate. As described above, however, tissue damage remains even after prompt detection and treatment. Prophylactic treatment by intravenous administration is recommended to prevent this pain and debilitation. Upon injection, 50% of Factor IX is immediately bound to vascular endothelial cells and/or diffuses into the extravascular space. The remaining 50% has a half life in circulation of approximately 24 hours. These infusion kinetics result in the need for injections twice per week or more to maintain minimal therapeutic levels in the plasma. While this regimen is inconvenient and stressful for the patient, it is also not totally effective. Progressive, cumulative tissue damage continues with each bleeding episode when hemostatic levels of coagulation factor are not present in the circulation.

[0007] Prophylactic treatment in severe hemophilic dogs by daily injection of relatively high concentrations (50 IU/kg) Factor IX resulted in a continuous therapeutic level of Factor IX and was accompanied by a two-fold increase in recovery levels by day 5 compared to administration with a single bolus. Plasma levels of Factor IX were maintained above 10% of normal at all times. This corresponds to a prophylactic regimen for prevention of spontaneous or trauma-induced hemorrhage in surgery (Brinkhous, et al. (October 1, 1996) Blood, vol. 88, no. 7: pp 2603-2610).

[0008] U.S. Patent No. 6,320,029 discloses continuous administration of Factor using a pump device to provide continuous injection or infusion. However, this has the obvious disadvantage that the implantable device or external pump requires frequent servicing and has to be attached to the patient. In addition, this patent does not teach the prophylactic level of coagulation factor required to prevent tissue damage.

[0009] Both a pulse replacement regiment and a continuous replacement regimen have been used to administer recombinant Factor IX intravenously to patients undergoing surgery. An initial bolus was administered pre-operatively and subsequent doses were administered at 7.25-25.5 hour intervals pre-, peri-, and post-operatively. Peri- and postoperative blood loss was similar to non-hemophilic individuals and hemostasis was rated as excellent or good in 34 out of 35 procedures. Dosage was determined on the basis of type of procedure, subject's experience, protocol guidelines and pharmokinetic estimates (Ragni, et al. (2002) Haemophilia 8: 91-97). No data was provided for long range, permanent hemostasis for hemophilic patients.

[0010] Factor IX has also been administered to dogs intratracheally by inhalation. However, bioavailability was lower compared to IV administration (Russell, et al.(2001) Thromb. Haemost. vol 85: 445-449) and the level needed to maintain normal hemostasis was not disclosed.

[0011] Besides the technical difficulty associated with delivery of a relatively labile protein to the bloodstream of a hemophiliac in an effective amount, another issue is the economic feasibility of administering frequent doses of blood clotting factors such as Factor VIII and IX. Prophylactic care has been recommended by the Medical and Scientific Advisory Committee for the National Hemophilia Foundation. However, the cost of such care could drive the annual cost to well over $250,000 per year for an adult hemophiliac. Given that life-time insurance caps of about $1 million are generally associated with most policies in the United States, hemophiliacs are severely constrained in terms of the amount of commercial product that they can afford for care which, at the least, affects their quality of life during adulthood and, at the worst, raises the risk of life-threatening bleeding and death.

[0012] Commercial production of properly processed blood clotting factors is another technical barrier preventing a "cure" for hemophilia by prophylactic treatment. Subcutaneous administration has been limited by the low concentration of plasma derived Factor IX in commercial concentrates (McCarthy, et al. (2002) Thromb Haemost 87:824- 830). While higher protein concentrations of Factor VIII and Factor IX have been produced using recombinant methods, much of the recombinantly produced material is not functional due to insufficient processing making the final product available for human use both scarce and expensive.

SUMMARY OF THE INVENTION

[0013] Embodiments of the invention are directed to the prophylactic treatment of hemophilia by administering a therapeutic agent to a hemophiliac at regular intervals for an extended period for a therapeutic effect. In preferred embodiments, the therapeutic agent is administered at a level sufficient to prevent spontaneous bleeding episodes. In preferred embodiments, the therapeutic agent is Factor VIII, Factor IX, mutants thereof and/or derivatives thereof. In preferred embodiments, the therapeutic effect is either maintaining a predetermined minimum level of the therapeutic agent in the blood stream and/or maintaining a level of the therapeutic agent sufficient for a predetermined maximal Activated Thromboplastin Time (APTT).

[0014] In preferred embodiments, a therapeutic effect is maintaining a predetermined minimum level of the therapeutic agent in the blood stream and the predetermined minimum level of the therapeutic agent is at least 3% of the level present in normal individuals, more preferably at least 5%, yet more preferably at least 7.5%, yet more preferably at least 17%, and most preferably at least 50% of the level present in normal individuals.

[0015] In preferred embodiments, a therapeutic effect is maintaining a level of therapeutic agent sufficient for a predetermined maximal Activated Thromboplastin Time (APTT) and the APTT is no more than 2 minutes, more preferably the APTT is no more than 1 minute, most preferably the APTT is corrected into the normal range for blood clotting. [0016] In preferred embodiments, the therapeutic agent is administered as a single dose, once per day. In alternate preferred embodiments, the therapeutic agent is administered incrementally throughout the day. Preferably, the therapeutic agent is administered 2-4 times daily. Preferably, administration is oral.

[0017] ^~ In preferred embodiments, the therapeutic agent is Factor IX and a dosage of 10-200 IU of Factor IX/kg body weight/day is administered, more preferably, a dosage of 20-150 IU of Factor IX/kg body weight/day is administered, more preferably, a dosage of 40- 100 IU of Factor IX/kg body weight/day is administered.

[0018] In some preferred embodiments, administration of the therapeutic agent is intravenous. In alternate preferred embodiments, administration of the therapeutic agent is non-intravenous. Preferably, the non-intravenous administration is oral, subcutaneous, transdermal or by inhalation. Preferably, the subcutaneous administration is by injection or catheter. Preferably, the oral administration is by hard gel capsule, soft gel capsule, tablet or liquid form. In some preferred embodiments, oral administration is with a sustained release formulation.

[0019] In some preferred embodiments, administration is oral and the therapeutic agent is mixed with a therapeutically effective amount of a molecule which facilitates the oral absorption of the therapeutic agent.

[0020] In some preferred embodiments, administration is by inhalation and the therapeutic agent is mixed with a therapeutically effective amount of a molecule which facilitates the absorption of the therapeutic agent by inhalation.

[0021] In preferred embodiments, administration is for an extended period such as 1-12 months, more preferably, 1- 5 years and most preferably administration is greater than 5 years and up to the lifetime of the treated hemophiliac.

[0022] Embodiments of the invention are directed to a pharmaceutical composition which includes a therapeutic agent for prophylactically treating a patient with hemophilia which is in a form for oral administration including but not limited to hard gel capsules, soft gel capsules, tablets and sustained relief formulations.

[0023] Embodiments of the invention are directed to methods for the prophylactic treatment of hemophilia B by administering a recombinant Factor IX to a hemophiliac at regular intervals for an extended period for a therapeutic effect, where preferably the recombinant Factor IX is prepared by a process which includes the steps of transfecting a mammalian cell with a gene encoding Factor IX operably linked to a promoter and at least one gene encoding a processing factor(s) operably linked to at least one promoter, either simultaneously or sequentially; and harvesting the Factor IX product.

[0024] In preferred embodiments, the processing factor is a nucleic acid such as paired basic amino acid converting enzyme (PACE), vitamin K dependent epoxide reductase (VKOR), vitamin K dependent γ-glutamyl carboxylase (VKGC) and combinations thereof operably linked to one or more promoter(s).

[0025] Preferably, at least one gene is overexpressed. In some preferred embodiments, the overexpressed gene is operably linked to a Chinese hamster elongation factor 1-α (CHEFl) promoter.

[0026] In preferred embodiments, the mammalian cell is a Chinese Hamster Ovary (CHO) cell.

[0027] Preferably, transfecting a mammalian cell with a gene encoding Factor IX operably linked to a promoter and at least one gene encoding a processing factor(s) operably linked to at least one promoter, is sequential. More preferably, the method of transfecting the mammalian cell also includes selecting for cells which express high levels of the Factor IX protein product or the processing factor(s); cloning the selected cells; and amplifying the cloned cells.

[0028] Further aspects, features and advantages of this invention will become apparent from the detailed description of the preferred embodiments which follow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure IA compares predicted peak/trough values for 50 units /kg body wt Factor IX administered intravenously every 72 hours to the same amount administered orally, twice daily. Figure 1 B compares predicted peak/trough values for 50 units /kg body wt Factor IX administered intravenously every 72 hours to 20 units /kg body wt. administered orally, twice per day.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT [0030] While the described embodiment represents the preferred embodiment of the present invention, it is to be understood that modifications will occur to those skilled in the art without departing from the spirit of the invention. The scope of the invention is therefore to be determined solely by the appended claims. Definitions

[0031] "Factor" in the context of the present invention refers to either Factor VIII or Factor IX. Variants, analogs and homologs of Factor VIII and IX proteins are encompassed by the terms "Factor VIII", "Factor IX" and "Factor" as well as the native protein. Precursors of Factor VIII and IX are encompassed as well as modified and unmodified forms. In particular, modified forms include forms of Factor IX which have been γ-glutamylated.

[0032] "Trough" refers to a minimal level of a blood clotting factor in the bloodstream.

[0033] The term "prophylactic" as used herein has its usual and customary meaning. More specifically, in the context of the invention, prophylactic treatment of hemophilia means a treatment of an individual having hemophilia in order to prevent spontaneous bleeding episodes. Prophylactic treatment according to embodiments of the invention means that the appropriate blood clotting factor is administered to the individual in sufficient amounts and sufficient intervals such that the need for "on demand" treatment with biological preparations containing one or more blood clotting factors is reduced or avoided altogether. By prophylactic treatment according to embodiments of the invention, the hemophiliac approximates the response of a normal individual who does not have hemophilia.

[0034] The term "spontaneous bleeding" refers to bleeding with little or no trauma or injury.

[0035] Hemophilia is generally classified in three different categories based upon the level of Factor in the patient's bloodstream. Mildly effected individuals have >5% of Factor VIII and IX in their bloodstream. Intervention is only required for traumatic events. Moderate bleeders have 2-5% levels of Factors VIII/IX. Severely effected individuals are those with a level of <1% Factor VIII or IX in their bloodstream. Spontaneous hemorrhage generally occurs only in patients with severe hemophilia. Patients in the severe category will have joint bleeds with subsequent joint destruction as a lifelong problem. These individuals are preferred candidates for prophylactic therapy. Currently, the Medical and Scientific Advisory Board of the National Hemophilia Foundation recommends prophylactic therapy for these individuals by maintaining a minimum trough level of at least 1% which can usually be accomplished by administration of 25-50 Factor VIII units/kg three times per week or every other day, or 40-100 Factor IX units/kg two to three times weekly (adopted by National Hemophilia Foundation Board of Directors June 3, 2006). Administration currently is intravenous. One unit equals the activity of clotting factor in 1 ml of pooled, normal plasma.

[0036] Prophylactic administration is usually intravenous because of the volume required to provide an effective dose. Subcutaneous injection is not a practical option because multiple injections at several sites are required to deliver the large volumes necessary for effective levels of Factor. Prophylactic administration by injection or infusion using a pump device is inconvenient for the patient and also not completely effective as described above. More frequent administration is desirable in order to prevent the high peaks and low troughs in the level of Factor VIII or Factor IX, resulting from administration which is typically 2-3 times per week. While daily administration is desirable, this is not practical when administration relies upon intravenous administration for delivery of large volumes.

[0037] One object of preferred embodiments is to provide a convenient prophylactic treatment that encourages patient compliance and moves a severe patient up into the moderate range that avoids the high peaks and low troughs that are the result of administration 2-3 times per week. Normally, Factor VIII or IX is administered as an intravenous bolus injection that may raise Factor levels to 50-100% of normal. This high initial level of Factor is needed because it is not practical to administer coagulation factors intravenously at more frequent intervals. Consequently, a larger dose than what is actually needed is administered so that levels will be maintained above 1% until it is time for another administration. Naturally, administration of such unnecessarily large doses increases the likelihood that the patient will produce antibodies against the blood factors being administered. [0038] Antibodies generated against coagulation factors are called inhibitor antibodies and are produced in about 15% of patients with severe hemophilia. A distinct advantage of preferred embodiments are that lower doses of coagulation factors may be administered at more frequent intervals so that likelihood that the patient will develop inhibitors is decreased. Also, a maximal range of Factor VIII or IX can be maintained in the bloodstream, avoiding the high peaks and low troughs associated with less frequent administration. This is because the Factor produced as described contains a higher proportion of active material. Therefore, the amount of material necessary to provide the recommended dose of 40-100 units/kg when administered two to three times weekly (as recommended by the National Hemophilia Foundation) is much less so the chance of an immune response is reduced.

[0039] The prophylactic treatment according to embodiments of the invention means a treatment that is for an extended period of time which means treatment on a continuous and ongoing basis. This treatment is continuous in that it is performed without major interruptions. The treatment may be modified for certain events and still be considered continuous as this term is used herein. For example, the administration of blood clotting factor(s) may be modified as the result of a surgery or a traumatic event such as a car accident. The dosage of blood clotting factor(s) may be adjusted in light of the aging of the patient or in light of other health conditions which may arise during the lifetime of the patient. Such administration is considered "continuous" in the context of the invention. This treatment is "ongoing" because it is for an extended period which typically is the remaining lifetime of the patient, although shorter extended periods such as months or years are also beneficial to the patient. Any period of time in which the patient is protected from spontaneous bleeding events is beneficial.

[0040] The formulation described herein provides for both intravenous and non- intravenous administration. The formulation described herein is highly concentrated and bioavailable thus avoiding high dose bolus administration. More frequent doses are possible which allows a lower amount of material per dose and a more constant level of Factor in the bloodstream of the patient and consequently less total amount (units) of factor being administered again reducing the probability of inhibitor antibody formation. In preferred embodiments, administration is non-intravenous. Non-intravenous administration includes but is not limited to subcutaneous, transdermal, oral and inhalation routes.

[0041] Preferred embodiments of the present invention relate to administration of an oral form of Factor VIII or Factor IX. Oral administration has the advantage that the patient can easily take more frequent doses, preferably daily. By taking daily doses, each individual dose can be much smaller. This way, loss due to the administration of a bolus injection can be avoided leading to safer and more efficient use of coagulation factor. Also, troughs where the level of Factors VIII or IX drops to levels too low to be effective are also avoided. While the patient needs to continue to take Factors VIII or IX in order to maintain an optimal level of Factor in the bloodstream, the individual doses are smaller, safer and more convenient.

[0042] Administration may be as often as necessary to maintain a predetermined level of Factor VIII or IX, effective to prevent spontaneous bleeding. In preferred embodiments, administration may be once, twice, three times or even four times per day. As a result of prophylactic treatment, the "on demand" requirement for Factor VIII or IX is eliminated except for traumatic injury and scheduled surgery. That is, the hemophiliac is effectively cured as occasions necessitating blood transfusion are the same as for the non- hemophiliac (normal) individual. In preferred embodiments, as a consequence of being maintained on the prophylactic regimen a person suffering from Hemophilia A or B will have an activated thromboplastin time (APTT) which is within or close to the normal range. Generally, 25 to 39 seconds is considered a normal clotting time for a standard test. In preferred embodiments, APTT for a patient undergoing prophylactic treatment is preferably under 2 minutes, more preferably under 1 minute, yet more preferably under 45 seconds. In a most preferred embodiment, clotting time is within the normal range of 25 to 39 seconds.

[0043] In preferred embodiments the level of Factor VIII or IX in the blood is maintained at a level of at least 3%, preferably at least 5%, more preferably at least 7.5%, yet more preferably at least 17%, most preferably at least 50% of that of a normal (non- hemophiliac) individual.

[0044] In preferred embodiments, the amount of Factor administered is 10-200 Units/ kg body wt./day, more preferably 20-150 Units/ kg body wt./day, yet more preferably 40-100 Units/ kg body wt./day, and most preferably 60-80 Units/ kg body wt./day. Levels less than 10 Units/ kg body wt./day may be appropriate for individuals with mild or moderate forms of Hemophilia A or B.

[0045] A method for creating a genetically engineered cell that produces a high percentage of biologically active Factor VIII or IX in quantities suitable for prophylactic administration of Factor VIII or Factor IX by intravenous and non-intravenous means is described. While embodiments of the invention are described with respect to production of Factor IX, similar principles also pertain to Factor VIII.

[0046] Among the first synthetic blood coagulation protein to become commercially available was Factor IX . Although recombinant Factor IX can be produced by recombinant methods, it is not optimal as a treatment for Hemophilia B because its posttranslational modification in tissue culture cells is not identical to plasma derived material and consequently its bioavailability to patients is variable. While reasonable levels of recombinant Factor IX protein can be expressed by genetically engineered CHO cells, e.g. up to 188 mg/L, the levels of fully functional Factor IX that are produced are on the order of only 0.5 mg/L due to the limited ability of the CHO cells to fully gamma-carboxylate the first 12 glutamic acid residues in the amino terminal region of the protein referred to as the gla- domain. In addition to this deficiency in the post-translational modification of Factor IX, subsequent work demonstrated that pro-Factor IX, a form of Factor IX that contains a propeptide domain that is required for the efficient intracellular gamma-carboxylation of the protein, is not processed completely prior to secretion from the CHO cell. As a consequence, it was found that well over half of the Factor IX secreted from genetically engineered CHO cells still contains the propeptide region and is non-functional.

[0047] The present application describes a method for the prophylactic treatment of hemophilia. The method avoids the problems discussed above by using a recombinant Factor VIII or Factor IX that is made in extraordinarily high concentration in CHO cells and is completely biologically active. The resulting product is properly processed and in high concentration. The delivery of Factor VIII or IX is by intravenous or non-intravenous administration. In preferred embodiments the coagulation Factor is delivered by a non- intravenous route such as inhalation, subcutaneous or transdermal. In a most preferred embodiment the coagulation Factor is delivered by the oral route. Oral administration is made possible as a consequence of the described production system which provides high yields of recombinant coagulation factor in active form.

[0048] In preferred embodiments, Factor VIII or Factor IX useful for prophylactic treatment of hemophilia is produced from tissue culture cells, which is at least 10% biologically active and is capable of production at a level of at least about 30 mg/L.

[0049] As used herein, "biologically activity" for Factor IX is determined with reference to a Factor IX standard derived from human plasma, such as MONONINE® (ZLB Behring). The biological activity of the Factor IX standard is taken to be 100%. Preferably, the Factor IX according to embodiments of the invention has at least 5% of the activity of the Factor IX standard, more preferably at least 10% of the activity of the Factor IX standard, more preferably at least 15% of the activity of the Factor IX standard, more preferably at least 20% of the activity of the Factor IX standard, more preferably at least 25% of the activity of the Factor IX standard, more preferably at least 30% of the activity of the Factor IX standard, more preferably at least 35% of the activity of the Factor IX standard, more preferably at least 40% of the activity of the Factor IX standard, more preferably at least 45% of the activity of the Factor IX standard, more preferably at least 50% of the activity of the Factor IX standard, more preferably at least 55% of the activity of the Factor IX standard, more preferably at least 60% of the activity of the Factor IX standard, more preferably at least 65% of the activity of the Factor IX standard, more preferably at least 70% of the activity of the Factor IX standard, more preferably at least 75% of the activity of the Factor IX standard, more preferably at least 80% of the activity of the Factor IX standard, more preferably at least 85% of the activity of the Factor IX standard, more preferably at least 90% of the activity of the Factor IX standard.

[0050] Cultured cells producing Factor VIII or Factor IX according to the invention are capable of production at a level of at least about 20 mg/L, preferably at least about 30 mg/L, more preferably at least about 40 mg/L, more preferably at least about 50 mg/L, yet more preferably at least about 60 mg/L, yet more preferably at least about 70 mg/L, yet more preferably at least about 80 mg/L, yet more preferably at least about 90 mg/L, yet more preferably at least about 100 mg/L, yet more preferably at least about 110 mg/L, yet more preferably at least about 120 mg/L, yet more preferably at least about 130 mg/L, yet more preferably at least about 140 mg/L, yet more preferably at least about 150 mg/L, yet more preferably at least about 160 mg/L, yet more preferably at least about 170 mg/L, yet more preferably at least about 180 mg/L, yet more preferably at least about 190 mg/L, yet more preferably at least about 200 mg/L, yet more preferably at least about 210 mg/L of biologically active Factor VIII or Factor IX protein.

[0051] The term "processing factor" is a broad term which includes any protein, peptide, non-peptide cofactor, substrate or nucleic acid which promotes the formation of a Factor IX. Examples of such processing factors include, but are not limited to PACE, VKOR and VKGC.

[0052] Many transfection methods to create genetically engineered cells that express large quantities of recombinant proteins are well known. Monoclonal antibodies, for example, are routinely manufactured from genetically engineered cells that express protein levels in excess of 1000 mg/L. Preferred embodiments are not dependent on any specific transfection method that might be used to create a genetically engineered cell.

[0053] Many expression vectors can be used to create genetically engineered cells. Some expression vectors are designed to express large quantities of recombinant proteins after amplification of transfected cells under a variety of conditions that favor selected, high expressing, cells. Some expression vectors are designed to express large quantities of recombinant proteins without the need for amplification under selective pressure. Preferred embodiments are not dependent on the use of any specific expression vector.

[0054] To create a genetically engineered cell to produce large quantities of Factor VIII or Factor IX, cells are transfected with an expression vector that contains the cDNA encoding the protein. In preferred embodiments, a transfected cell is created that is capable, under optimized growth conditions, of producing a minimum of 20 mg/L of Factor VIII or Factor IX. Higher levels of production of Factor VIII or Factor IX protein may be achieved and could be useful in some preferred embodiments of the invention. However, the optimum level of production of Factor VIII or Factor IX protein is a level at or above 20 mg/L that can be obtained in a significantly increased functional form when the Factor VIII or Factor IX protein is expressed with selected co-transfected enzymes that cause proper post- translational modification to occur in a given cell system.

[0055] The cell may be selected from a variety of sources, but is otherwise a cell that may be transfected with an expression vector containing a nucleic acid, preferably a cDNA of a Factor VIII or Factor IX protein.

[0056] From a pool of transfected cells, clones are selected that produce quantities of the Factor VIII or Factor IX protein over a range (Target Range) that extends from the highest level to the lowest level that is minimally acceptable for the production of a commercial product. Cell clones that produce quantities of the Factor VIII or Factor IX protein within the Target Range may be combined to obtain a single pool or multiple sub- pools that divide the clones into populations of clones that produce high, medium or low levels of the Factor VIII or Factor IX protein within the Target Range.

[0057] Transfected cells that produce a Factor VIII or Factor IX protein within the Target Range may be analyzed to determine the extent to which fully functional protein is produced. Such analysis will provide insight into the specific enzyme deficiencies that limit the production of fully functional protein. Further, it is anticipated that analysis of sub-pools consisting of cell clones that produce high, medium, or low levels of the Factor VIII or Factor IX protein within the Target Range will provide insight into the specific enzyme deficiencies that limit the production of fully functional protein at varying levels of production of the Factor VIII or Factor IX protein.

[0058] Transfection of the pool of cells with an expression vector containing a nucleic acid, preferably a cDNA for a protein that, when expressed by a cell clone, to eliminate enzyme deficiencies within a pool of transfected clones that limits the production of fully functional Factor IX protein within the Target Range, mitigates the enzyme deficiency in whole or in part. In some embodiments, more than one enzyme deficiency must be mitigated or mitigation of a deficiency in post-translational modification of the Factor IX protein may require the presence of the activities of more than one enzyme or protein or other processing factor that may be provided in the method of preferred embodiments of the present invention by the simultaneous or subsequent (sequential) transfection of the cell clones with additional expression vectors containing cDNA for given proteins.

[0059] One such protein would have the enzymatic activity of vitamin K epoxide reductase (VKOR). Another such enzyme would have the enzymatic activity of vitamin Independent gamma-glutamyl carboxylase (VKGC). Another such enzyme would have the enzymatic activity of paired amino acid cleaving enzyme, i.e. PACE or furin.

[0060] Pools of cell clones that produce a Factor IX protein within the Target Range are subsequently transfected to provide a specific protein or multiple proteins in various combinations. Transfected pools of cell clones are then analyzed to determine the relative percentages of fully functional Factor IX protein that are now produced by transfectant pools that co-express the various proteins. The transfectant pool that produces the highest percentage of fully functional Factor IX protein with the minimum number of co- expressed proteins, is selected for subsequent cloning.

[0061] The selected transfectant pool is cloned to determine the optimal level of production of fully functional Factor IX protein that is attained by co-expression of additional protein(s). Higher percentages of fully functional Factor IX protein will be produced by cell clones that produce lower total amounts of the Factor IX protein within the Target Range. On the other hand, some cell clones may be superproducers of Factor IX protein without significant improvements in post translational processing. Nevertheless, such superproducer lines produce usable amounts of functional protein as the overall production level is high. The optimal level of production will be the highest level of functional Factor IX protein.

[0062] Preferred embodiments of the invention employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al., "Molecular Cloning; A Laboratory Manual", 2nd ed (1989); "DNA Cloning", VoIs. I and II (D. N Glover ed. 1985); "Oligonucleotide Synthesis" (M. J. Gait ed. 1984); "Nucleic Acid Hybridization" (B. D. Hames & S. J. Higgins eds. 1984); "Transcription and Translation" (B. D. Hames & S. J. Higgins eds. 1984); "Animal Cell Culture" (R. I. Freshney ed. 1986); "Immobilized Cells and Enzymes" (IRL Press, 1986); B. Perbal, "A Practical Guide to Molecular Cloning" (1984); the series, Methods in Enzymology (Academic Press, Inc.), particularly VoIs. 154 and 155 (Wu and Grossman, and Wu, eds., respectively); "Gene Transfer Vectors for Mammalian Cells" (J. H. Miller and M. P. Calos eds. 1987, Cold Spring Harbor Laboratory); " Immunochemical Methods in Cell and Molecular Biology", Mayer and Walker, eds. (Academic Press, London, 1987); Scopes, "Protein Purification: Principles and Practice", 2nd ed. 1987 (Springer-Verlag, N. Y.); and "Handbook of Experimental Immunology" VoIs I-IV (D. M. Weir and C. C. Blackwell eds 1986). All patents, patent applications, and publications cited in the background and specification are incorporated herein by reference. Modification of the propeptide

[0063] One strategy to increase γ-carboxylation is to replace the native propeptide sequence with a propeptide sequence that has a lower affinity for the gamma carboxylase as discussed in U.S. Application No. 2003/0220247, which is incorporated herein by reference. Useful propeptide sequences include altered forms of wild type sequences or propeptide sequences, or combinations of the same, for heterologous vitamin K dependent proteins. The propeptide sequence in Factor IX protein is the recognition element for the enzyme which directs gamma carboxylation of the protein. Factor IX proteins are not fully functional unless they comprise a high percentage of gamma carboxylated moieties. Thus, it is important when generating recombinant versions of these proteins that mechanisms be put in place to ensure full gamma carboxylation of the same.

[0064] The sequence alignment of several propeptide sequences is shown in FIG. 3 of US. 2003/0220247. Thus, propeptides which are useful in preferred embodiments of the invention are those which have the sequences shown in FIG. 3 wherein an 18 amino acid sequence of several useful propeptides is shown along with the relative affinities of these propeptides for gamma carboxylase. A low affinity propeptide may be generated by modifying any one of amino acids -9 or -13 on either prothrombin or protein C. Preferred modifications include the substitution of an Arg or a His residue at position -9 and the substitution of a Pro or a Ser residue at position -13. Other preferred chimeric proteins include a propeptide selected from the group consisting of altered Factor IX, Factor X, Factor VII, Protein S, Protein C and prothrombin, or an unaltered propeptide in combination with the mature Factor IX protein which is not native to the chosen propeptide sequence. [0065] The term "fully gamma carboxylated protein" is used herein to refer to a protein wherein at least about 80% of the amino acids which should be gamma carboxylated are carboxylated. Preferably, at least about 85%, more preferably, at least about 90%, more preferably at least about 95% and even more preferably, at least about 99% of the amino acids which should be gamma carboxylated are gamma carboxylated. Paired basic amino acid converting enzyme (PACE)

[0066] As used herein, the term "PACE" is an acronym for paired basic amino acid converting (or cleaving) enzyme. PACE, originally isolated from a human liver cell line, is a subtilisin-like endopeptidase, i.e., a propeptide-cleaving enzyme which exhibits specificity for cleavage at basic residues of a polypeptide, e.g., -Lys-Arg-, -Arg-Arg, or -Lys- Lys-. PACE is stimulated by calcium ions; and inhibited by phenylmethyl sulfonyl fluoride (PMSF). A DNA sequence encoding PACE (or furin) appears in FIG. 1 [SEQ ID NO: 1] of U.S. Patent No. 5,460,950, which is incorporated herein by reference. The co-expression of PACE and a proprotein which requires processing for production of the mature protein results in high level expression of the mature protein. Additionally, co-expression of PACE with proteins requiring γ-carboxylation for biological activity permits the expression of increased yields of functional, biologically active mature proteins in eukaryotic, preferably mammalian, cells. Vitamin K dependent epoxide reductase

[0067] Vitamin K dependent epoxide reductase (VKOR) is important for vitamin K dependent proteins because vitamin K is converted to vitamin K epoxide during reactions in which it is a cofactor. The amount of vitamin K in the human diet is limited. Therefore, vitamin K epoxide must be converted back to vitamin K by VKOR to prevent depletion. Consequently, co-transfection with VKOR provides sufficient vitamin K for proper functioning of the vitamin K dependent enzymes such as the vitamin K dependent γ-glutamyl carboxylase (VKCG). Proper functioning of vitamin K dependent VKCG is essential for proper γ-carboxylation of the gla-domain of vitamin K dependent coagulation factors. Vitamin K dependent gamma carboxylase

[0068] Vitamin K dependent γ-glutamyl carboxylase (VKGC) is an ER enzyme involved in the post-translation modification of vitamin K dependent proteins. VKGC incorporates CO₂ into glutamic acid to modify multiple residues within the vitamin K dependent protein within about 40 residues of the propeptide. The loss of three carboxylations markedly decreases the activity of vitamin K-dependent proteins such as vitamin K dependent coagulation factors. The cDNA sequence for human vitamin K dependent γ-glutamyl carboxylase is described by U.S. Patent No. 5,268,275, which is incorporated herein by reference. The sequence is provided in SEQ ID NO: 15 of U.S. Patent No. 5,268,275 Genetic Engineering Techniques

[0069] The production of cloned genes, recombinant DNA, vectors, transformed host cells, proteins and protein fragments by genetic engineering is well known. See, e.g., U.S. Pat. No. 4,761,371 to Bell et al. at Col. 6 line 3 to Col. 9 line 65; U.S. Pat. No. 4,877,729 to Clark et al. at Col. 4 line 38 to Col. 7 line 6; U.S. Pat. No. 4,912,038 to Schilling at Col. 3 line 26 to Col. 14 line 12; and U.S. Pat. No. 4,879,224 to Wallner at Col. 6 line 8 to Col. 8 line 59.

[0070] A vector is a replicable DNA construct. Vectors are used herein either to amplify DNA encoding Vitamin K Dependent Proteins and/or to express DNA which encodes Vitamin K Dependent Proteins. An expression vector is a replicable DNA construct in which a DNA sequence encoding a Vitamin K dependent protein is operably linked to suitable control sequences capable of effecting the expression of a Vitamin K dependent protein in a suitable host. The need for such control sequences will vary depending upon the host selected and the transformation method chosen. Generally, control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation.

[0071] Amplification vectors do not require expression control domains. All that is needed is the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants.

[0072] Vectors comprise plasmids, viruses (e.g., adenovirus, cytomegalovirus), phage, and integratable DNA fragments (i.e., fragments integratable into the host genome by recombination). The vector replicates and functions independently of the host genome, or may, in some instances, integrate into the genome itself. Expression vectors should contain a promoter and RNA binding sites which are operably linked to the gene to be expressed and are operable in the host organism.

[0073] DNA regions are operably linked or operably associated when they are functionally related to each other. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.

[0074] Transformed host cells are cells which have been transformed or transfected with one or more vector(s) constructed using recombinant DNA techniques. Expression of multiple proteins

[0075] By providing the cell with the necessary enzymes and cofactors to process Factor IX, higher yields of biologically active Factor IX proteins are achieved. When adequate levels of fully functional Factor IX protein is produced by a recombinant cell, lengthy purification steps designed to remove the useless, partially modified, or unmodified Factor IX protein from the desired product are avoided. This lowers the production cost and eliminates inactive material that may have undesirable side effects for the patient.

[0076] Methods for producing Factor IX proteins by co-expression with PACE, VKGC and/or VKOR can include the following techniques. First, a single vector containing coding sequences for more than one protein such as PACE and Factor IX can be inserted into a selected host cell. Alternatively, two or more separate vectors encoding Factor IX plus one or more other proteins, can be inserted into a host. Upon culturing under suitable conditions for the selected host cell, the two or more polypeptides are produced and interact to provide cleavage and modification of the proprotein into the mature protein.

[0077] Another alternative is the use of two transformed host cells wherein one host cell expresses the Factor IX protein and the other host cell expresses one or more of PACE, VKGC and/or VKOR which will be secreted into the medium. These host cells can be co-cultured under conditions which allow expression and secretion or release of the recombinant Factor IX protein and the co-expressed recombinant polypeptides, including cleavage into the mature form by the extracellular PACE and gamma carboxylation of N- terminal glutamates. In this method, it is preferred that the PACE polypeptide lacks the transmembrane domain so that it secretes into the medium.

[0078] In some instances, it may be desirable to have a plurality of copies, two or more, of the gene expressing the Factor IX protein in relation to the other genes, or vice versa. This can be achieved in a variety of ways. For example, one may use separate vectors or plasmids, where the vector containing the Factor IX protein encoding polynucleotide has a higher copy number than the vector containing the other polynucleotide sequences, or vice versa. In this situation, it would be desirable to have different selectable markers on the two plasmids, so as to ensure the continued maintenance of the plasmids in the host. Alternatively, one or both genes could be integrated into the host genome, and one of the genes could be associated with an amplifying gene, (e.g., dhfr or one of the metallothionein genes).

[0079] Alternatively, one could employ two transcriptional regulatory regions having different rates of transcriptional initiation, providing for the enhanced expression of either Factor IX protein or the expression of any of the other processing factor polypeptides, relative to Factor IX protein. As another alternative, one can use different promoters, where one promoter provides for a low level of constitutive expression of Factor IX protein, while the second promoter provides for a high level of induced expression of the other products. A wide variety of promoters are known for the selected host cells, and can be readily selected and employed in the invention by one of skill in the art such as CMV, MMTV, SV 40 or SRa promoters which are well known mammalian promoters.

[0080] In a preferred embodiment, a promoter for the elongation factor -lα from Chinese hamster is used (CHEFl) to provide high level expression of a Factor VIII, Factor IX coagulation factor and/or processing factor(s). The CHEFl vector is used as described in Deer, et al.(2004) "High-level expression of proteins in mammalian cells using transcription regulatory sequences from the Chinese Hamster EF-lα gene" Biotechnol. Prog. 20: 880-889 and in U.S. Patent No. 5,888,809 which is incorporated herein by reference. The CHEFl vector utilizes the 5' and 3' flanking sequences from the Chinese hamster EF-lα. The CHEFl promoter sequence includes approximately 3.7 kb DNA extending from a Spel restriction site to the initiating methionine (ATG) codon of the EF- lα protein. The DNA sequence is set forth in SEQ ID NO: 1 of U.S. Patent No. 5,888,809.

[0081] Production of biologically active vitamin K dependent proteins such as Factor IX, are maximized by overexpression of one or more of PACE, VKOR, and/or VKGC and/or by modification of the gla region to maximize γ-carboxylation. That is, rate limiting components are expressed in sufficient quantity so that the entire system operates to produce a commercially viable quantity of Factor IX protein. Host cells

[0082] Suitable host cells include prokaryote, yeast or higher eukaryotic cells such as mammalian cells and insect cells. Cells derived from multicellular organisms are a particularly suitable host for recombinant Factor VIII or Factor IX protein synthesis, and mammalian cells are particularly preferred. Propagation of such cells in cell culture has become a routine procedure (Tissue Culture, Academic Press, Kruse and Patterson, editors (1973)). Examples of useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and WI138, HEK 293, BHK, COS-7, CV, and MDCK cell lines. Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located upstream from the DNA encoding Factor VIII or Factor IX protein to be expressed and operatively associated therewith, along with a ribosome binding site, an RNA splice site (if intron-containing genomic DNA is used), a polyadenylation site, and a transcriptional termination sequence. In a preferred embodiment, expression is carried out in Chinese Hamster Ovary (CHO) cells using the expression system of U.S. Patent No. 5,888,809, which is incorporated herein by reference.

[0083] The transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells are often provided by viral sources. For example, commonly used promoters are derived from polyoma, Adenovirus 2, and Simian Virus 40 (SV40). See. e.g.. U.S. Pat. No. 4,599,308.

[0084] An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV 40 or other viral (e.g. Polyoma, Adenovirus, VSV, or BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.

[0085] Rather than using vectors which contain viral origins of replication, one can transform mammalian cells by the method of cotransformation with a selectable marker and the DNA for the Factor VIII or Factor IX protein. Examples of suitable selectable markers are dihydrofolate reductase (DHFR) or thymidine kinase. This method is further described in U.S. Pat. No. 4,399,216 which is incorporated by reference.

[0086] Other methods suitable for adaptation to the synthesis of Factor VIII or Factor IX protein in recombinant vertebrate cell culture include those described in M-J. Gething et al., Nature 293, 620 (1981); N. Mantei et al, Nature 281, 40; A. Levinson et al., EPO Application Nos. 117,06OA and 117,058 A.

[0087] Host cells such as insect cells (e.g., cultured Spodoptera frugiperda cells) and expression vectors such as the baculovirus expression vector (e.g., vectors derived from Autographa californica MNPV, Trichoplusia ni MNPV, Rachiplusia ou MNPV, or Galleria ou MNPV) may be employed in carrying out preferred embodiments of the invention, as described in U.S. Pat. Nos. 4,745,051 and 4,879,236 to Smith et al. In general, a baculovirus expression vector comprises a baculovirus genome containing the gene to be expressed inserted into the polyhedrin gene at a position ranging from the polyhedrin transcriptional start signal to the ATG start site and under the transcriptional control of a baculovirus polyhedrin promoter.

[0088] Prokaryotic host cells include gram negative or gram positive organisms, for example Escherichia coli (E. coli) or Bacilli. Higher eukaryotic cells include established cell lines of mammalian origin as described below. Exemplary host cells are E. coli W3110 (ATCC 27,325), E. coli B, E. coli Xl 776 (ATCC 31,537), E. coli 294 (ATCC 31,446). A broad variety of suitable prokaryotic and microbial vectors are available. E. coli is typically transformed using pBR322. Promoters most commonly used in recombinant microbial expression vectors include the betalactamase (penicillinase) and lactose promoter systems (Chang et al., Nature 275, 615 (1978); and Goeddel et al., Nature 281, 544 (1979)), a tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res. 8, 4057 (1980) and EPO App. Publ. No. 36,776) and the tac promoter (H. De Boer et al., Proc. Natl. Acad. Sci. USA 80, 21 (1983)). The promoter and Shine-Dalgarno sequence (for prokaryotic host expression) are operably linked to the DNA encoding the Factor VIII or Factor IX protein(s), i.e., they are positioned so as to promote transcription of Factor VIII or Factor IX Protein(s) messenger RNA from the DNA.

[0089] Eukaryotic microbes such as yeast cultures may also be transformed with Factor VIII or Factor IX Protein-encoding vectors, see, e.g., U.S. Pat. No. 4,745,057. Saccharomyces cerevisiae is the most commonly used among lower eukaryotic host microorganisms, although a number of other strains are commonly available. Yeast vectors may contain an origin of replication from the 2 micron yeast plasmid or an autonomously replicating sequence (ARS), a promoter, DNA encoding one or more Factor VIII or Factor IX proteins, sequences for polyadenylation and transcription termination, and a selection gene. An exemplary plasmid is YRp7, (Stinchcomb et al., Nature 282, 39 (1979); Kingsman et al., Gene 7, 141 (1979); Tschemper et al., Gene 10, 157 (1980)). Suitable promoting sequences in yeast vectors include the promoters for metallothionein, 3 -phosphogly cerate kinase (Hitzeman et al., J. Biol. Chem. 255, 2073 (1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7, 149 (1968); and Holland et al., Biochemistry 17, 4900 (1978)). Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman et al., EPO Publn. No. 73,657.

[0090] Cloned genes of preferred embodiments of the invention may code for any species of origin, including mouse, rat, rabbit, cat, porcine, and human, but more preferably code for Factor VIII or Factor IX proteins of human origin. DNA encoding Factor VIII or Factor IX that is hybridizable with DNA encoding for proteins disclosed herein is also encompassed. Hybridization of such sequences may be carried out under conditions of reduced stringency or even stringent conditions (e.g., conditions represented by a wash stringency of 0.3M NaCl, 0.03M sodium citrate, 0.1% SDS at 60° C or even 70° C to DNA encoding the Factor VIII or Factor IX protein disclosed herein in a standard in situ hybridization assay. See J. Sambrook et al., Molecular Cloning, A Laboratory Manual (2d Ed. 1989)(Cold Spring Harbor Laboratory)).

[0091] As noted above, a functional Factor IX protein may be provided by a method which includes carboxylation of the N-terminal glu residues. The strategy may include co-expressing Factor IX protein along with VKOR, VKGC and/or PACE in a single host cell. In general, the method comprises culturing a host cell which expresses a Factor IX protein and supporting proteins; and then harvesting the proteins from the culture. While some host cells may provide some Factor IX protein, VKOR, VKGC and/or PACE at basal levels, the vector DNA encoding PACE, VKGC and/or VKOR is included to enhance carboxylation. The culture can be carried out in any suitable fermentation vessel, with a growth media and under conditions appropriate for the expression of the Factor IX protein(s) by the particular host cell chosen. The Factor IX protein harvested from the culture is found to be carboxylated due to the expression of the supporting proteins in the host cell. Factor IX protein can be collected directly from the culture media, or the host cells lysed and the Factor IX protein collected therefrom. Factor IX protein can then be further purified in accordance with known techniques.

[0092] As a general proposition, the purity of the recombinant protein will preferably be an appropriate purity known to the skilled art worker to lead to the optimal activity and stability of the protein. For example, when the recombinant protein is Factor VIII or Factor IX , the Factor VIII or Factor IX is preferably of ultrahigh purity. Preferably, the recombinant protein has been subjected to multiple chromatographic purification steps, such as affinity chromatography, ion-exchange chromatography and preferably immunoaffinity chromatography to remove substances which cause fragmentation, activation and/or degradation of the recombinant protein during manufacture, storage and/or use. Illustrative examples of such substances that are preferably removed by purification include thrombin and Factor IXa; other protein contaminants, such as modification enzymes like PACE/furin, VKOR, and VKGC; proteins, such as hamster proteins, which are released into the tissue culture media from the production cells during recombinant protein production; non-protein contaminants, such as lipids; and mixtures of protein and non-protein contaminants, such as lipoproteins. Purification procedures for vitamin K dependent proteins are known in the art. For example, see U.S. Patent No. 5,714,583, which is incorporated herein by reference.

[0093] Methods of preparing Factor VIII as well as variants and homologs of Factor VIII are known as taught, for example, in the following US patent applications which are incorporated herein by reference: U.S. Patent Application No. 10/122,264; U.S. Patent Application No. 10/283,648; U.S. Patent Application No. 10/383,206; U.S. Patent Application No. 10/974,534; U.S. Patent Application No. 10/782,362; and US Provisional Application No. 60/818,177.

[0094] Factor IX DNA coding sequences, along with vectors and host cells for the expression thereof, are disclosed in European Patent App. 373012, European Patent App. 251874, PCT Patent Appl. 8505376, PCT Patent Appln. 8505125, European Patent Appln. 162782, and PCT Patent Appln. 8400560. Genes for other coagulation factors are also known and available, for example, Factor II (Accession No. NM 000506), Factor VII (Accession No. NM_019616, and Factor X (Accession No. NM_000504). The above disclosures are incorporated herein by reference. Formulations

[0095] The blood clotting Factor formulations may be formed by methods well known in the art. Factor VIII or Factor IX compositions may be blended with conventional excipients such as binders, including gelatin, pre-gelatinized starch, and the like; lubricants, such as hydrogenated vegetable oil, stearic acid and the like; diluents, such as lactose, mannose, and sucrose; disintegants, such as carboxymethyl cellulose and sodium starch glycolate; suspending agents, such as povidone, polyvinyl alcohol, and the like; absorbents, such as silicon dioxide; preservative, such as methylparaben, propylparaben, and sodium benzoate; surfactants, such as sodium lauryl sulfate, polysorbate 80, and the like; and colorants, such as F.D & C. dyes and the like.

[0096] Inert, pharmaceutically acceptable carriers may be used which are either solid or liquid form. Solid form preparations include powders, tablets, dispersible granules, capsules, and cachets. A solid carrier is suitably one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders or tablet disintegrating agents. The solid carrier material also includes encapsulating material. In powders, the carrier is finely divided active compounds. In the tablet, the active compound is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired. Suitable solid carriers include, but are not limited, to magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. Delivery may use a sustained release form as described in U.S. Patent No. 6,855,334, which is incorporated herein by reference.

[0097] Liquid form preparations include solutions, suspensions, and emulsions. Aqueous solutions suitable for oral use are prepared by dissolving the active component in water or other suitable liquid and adding suitable colorants, flavors, stabilizing agents, and thickening agents as desired. Aqueous solutions suitable for oral use may also be made by dispersing the finely divided active component in water or other suitable liquid with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other suspending agents known in the art.

[0098] Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for either oral or parental administration. Such liquid forms include solutions, suspensions, and emulsions. These particular solid form preparations are provided in unit dose form and as such are used to provide a single liquid dosage unit. Alternatively, sufficient solid preparation may be provided so that the after conversion to liquid form, multiple individual liquid doses may be obtained by measuring predetermined volumes of the liquid form preparation as with a syringe, teaspoon, or other volumetric measuring device.

[0099] Pharmaceutical compositions of Factor VIII or Factor IX for injection comprise therapeutically effective amounts of Factor VIII or Factor IX and an appropriate physiologically acceptable carrier. A variety of aqueous carriers may be used, e.g., buffered water, saline, 0.3% glycine and the like. Stabilizers such as plant-derived glycoproteins, albumin, lipoprotein, fibronectin and/or globulin may also be added. Other components of the pharmaceutical compositions of the invention can include pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.

[0100] The solid and liquid forms may contain, in addition to the active material, flavorants, colorants, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like. The liquid utilized for preparing the liquid form preparation is suitably water, isotonic water, ethanol, glycerin, propylene glycol, and the like, as well as combinations thereof. The liquid utilized will be chosen with regard to the route of administration.

[0101] Preferably, the preparations are unit dosage form. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active components. The unit dosage form can be a packaged preparation, such as packaged tablets or capsules. The unit dosage can be a capsule, cachet, or tablet itself or it can be the appropriate number of any of these in packaged form.

[0102] The quantity of active material in a unit dose of preparation is varied according to the particular application and potency of the active ingredients.

EXAMPLE Example 1

[0103] Figure 1 compares bolus administration of Factor IX once every 72 hours by intravenous injection with oral administration of Factor IX twice daily at 50 units/kg body wt. (Figure IA) and 20 units/kg body wt. (Figure IB). At 50 units/kg body wt, the initial plasma recovery of recombinant Factor IX administered intravenously is about 0.35 - 0.40 units/ml, whereas about 1.0 unit/ml would be predicted. If oral administration of Factor IX results in a 10% recovery, the initial plasma level would be 0.1 units/mL, that is 10% of normal. Given a plasma half-life of about 18 hours, oral administration of Factor IX (50 units/kg body wt.) twice a day would result in a "peak/trough" steady state of 27% to 17% of normal (Figure IA). At a dosage of 20 units/kg body wt. (twice daily), a "peak/trough" range of about 11% to 7% is predicted (Figure IB).

[0104] It will be understood by those of skill in the art that numerous and various modifications can be made without departing from the spirit of the present invention. Therefore, it should be clearly understood that the forms of the present invention are illustrative only and are not intended to limit the scope of the present invention.

Claims

WHAT IS CLAIMED IS:

1. A method for the prophylactic treatment of hemophilia comprising administering a therapeutic agent to a hemophiliac at regular intervals for an extended period for a therapeutic effect at a level sufficient to prevent spontaneous bleeding episodes.

2. The method of claim 1, wherein the therapeutic agent is selected from the group consisting of Factor VIII, Factor IX, mutants thereof and derivatives thereof.

3. The method of claim 1, wherein the therapeutic effect is selected from the group consisting of maintaining a predetermined minimum level of the therapeutic agent in the blood stream, maintaining a level of the therapeutic agent sufficient for a predetermined maximal Activated Thromboplastin Time (APTT), and combinations thereof.

4. The method of claim 3, wherein the therapeutic effect is maintaining a predetermined minimum level of the therapeutic agent in the blood stream and wherein the predetermined minimum level of the therapeutic agent is at least 3% of the level present in normal individuals.

5. The method of claim 4, wherein the predetermined minimum level of the therapeutic agent is at least 5% of the level present in normal individuals.

6. The method of claim 5, wherein the predetermined minimum level of the therapeutic agent is at least 7.5% of the level present in normal individuals.

7. The method of claim 6, wherein the predetermined minimum level of the therapeutic agent is at least 17% of the level present in normal individuals.

8. The method of claim 7, wherein the predetermined minimum level of the therapeutic agent is at least 50% of the level present in normal individuals.

9. The method of claim 3, wherein the therapeutic effect is maintaining a level of therapeutic agent sufficient for a predetermined maximal Activated Thromboplastin Time (APTT) and wherein the APTT is no more than 2 minutes.

10. The method of claim 9, wherein the APTT is no more than 1 minute.

11. The method of claim 10, wherein the APTT is corrected into the normal range for blood clotting.

12. The method of claim 1, wherein the therapeutic agent is administered as a single dose, once per day.

13. The method of claim 1, wherein the therapeutic agent is administered incrementally throughout the day.

14. The method of claim 13, wherein the therapeutic agent is administered 2-4 times daily.

15. The method of claim 14, wherein administration is oral.

16. The method of claim 2, wherein the therapeutic agent is Factor IX and wherein a dosage of 10-200 IU of Factor IX/kg body weight/day is administered.

17. The method of claim 16, wherein a dosage of 20-150 IU of Factor IX/kg body weight/day is administered.

18. The method of claim 17, wherein a dosage of 40-100 IU of Factor IX/kg body weight/day is administered.

19. The method of claim 1, wherein administration of the therapeutic agent is intravenous.

20. The method of claim 1 , wherein administration of the therapeutic agent is non- intravenous.

21. The method of claim 20, wherein non-intravenous administration is oral, subcutaneous, transdermal or by inhalation.

22. The method of claim 21, wherein the subcutaneous administration is by injection or catheter.

23. The method of claim 21, wherein the oral administration is by hard gel capsule, soft gel capsule, tablet or liquid form.

24. The method of claim 21, wherein the oral administration comprises an oral sustained release formulation.

25. The method of claim 21, wherein administration is oral and wherein the therapeutic agent is mixed with a therapeutically effective amount of a molecule which facilitates the oral absorption of the therapeutic agent.

26. The method of claim 21, wherein administration is by inhalation and wherein the therapeutic agent is mixed with a therapeutically effective amount of a molecule which facilitates the absorption of the therapeutic agent by inhalation.

27. The method of claim 1, wherein the extended period is 1-12 months.

28. The method of claim 1, wherein the extended period is 1- 5 years.

29. The method of claim 1, wherein the extended period is more than 5 years and up to the lifetime of the treated hemophiliac.

30. A pharmaceutical composition comprising a therapeutic agent for prophylactically treating a patient with Hemophilia which is in a form for oral administration selected from the group consisting of hard gel capsules, soft gel capsules, tablets and sustained release formulations.

31. A method for the prophylactic treatment of hemophilia B comprising administering a recombinant Factor IX to a hemophiliac at regular intervals for an extended period for a therapeutic effect, wherein recombinant Factor IX is prepared by a process comprising the steps of: transfecting a mammalian cell with a gene encoding Factor IX operably linked to a promoter and at least one gene encoding a processing factor(s) operably linked to at least one promoter, either simultaneously or sequentially; and harvesting the Factor IX product.

32. The method of claim 31, wherein the processing factor is a nucleic acid selected from the group consisting of paired basic amino acid converting enzyme (PACE), vitamin K dependent epoxide reductase (VKOR), vitamin K dependent γ-glutamyl carboxylase (VKGC) and combinations thereof operably linked to one or more promoter(s).

33. The method of claim 31, wherein at least one gene is overexpressed and wherein the overexpressed gene is operably linked to a Chinese hamster elongation factor 1-α (CHEFl) promoter.

34. The method of claim 31, wherein the mammalian cell is a Chinese Hamster Ovary (CHO) cell.

35. The method of claim 31, wherein transfection is sequential and wherein transfecting the mammalian cell further comprises: selecting for cells which express high levels of the Factor IX protein product or the processing factor(s); cloning the selected cells; and amplifying the cloned cells.