MXPA01005124A - Method of treating sickle cell disease and thalassemia - Google Patents

Abstract

The present invention provides a method of treatment of sickle cell disease (SCD) or thalassemia with protein C. The claimed invention provides a needed therapy for potentially serious and debilitating disorders while avoiding complications such as bleeding tendency, toxicityand general side effects of currently available anti-coagulant agents.

Description

METHOD FOR TREATING FALCIFORM CELL DISEASE AND TALASEMIA Description of the Invention This application claims priority over Provisional Application Serial No. 60 / 109,474 sent on November 23, 1998. This invention relates to medical science, particularly the treatment of sickle cell disease or thalassemia using the protein C.

Protein C is a serine protease dependent on vitamin K and a naturally occurring anticoagulant that plays a role in the regulation of hemostasis by inactivating the Va and Villa Factors in the coagulation cascade. Human protein C circulates in the manner of a 2-chain zymogen, but functions on the endothelial surface and platelets after conversion to activated protein C (aPC) by limited proteolysis with thrombin forming complexes with the protein of the membrane of the cell surface, thrombomodulin.

Ref: 12B487 Together with other proteins, the aPC functions perhaps as the most important regulator of blood coagulation, resulting in protection against thrombosis. In addition to its anticoagulation functions, aPC has anti-inflammatory effects by inhibiting the generation of cytokines (for example, TNF and IL-1) and also exerts profibrinolytic properties that facilitate the lysis of clots. Therefore, the protein C enzyme system represents a major physiological mechanism of anticoagulation, anti-inflammation, and fibrinolysis. Sickle cell disease (SCD) and thalassemia are inherited hemoglobulinopathies characterized by a structural defect in hemoglobin. SCD includes diseases that cause the falciformation of erythrocytes, which includes sickle cell anemia (which results from two hemoglobulin S genes), sickle cell r thalassemia (a hemoglobin S gene, and a thalassemia gene). r), and the SC disease of hemoglobin (one of hemoglobin S and one of hemoglobin C), and the rarer disease, Harlem hemoglobin C. Thalassemia includes β-thalassemia and T-thalassemia. These hereditary diseases have significant morbidity and significant mortality, and affect individuals of African-American heritage, as well as those of Mediterranean, Middle Eastern and Southeast Asian descent. This disease commonly causes severe pain in those who suffer from it, in part due to ischemia caused by damage to erythrocytes that block free flow through the circulatory system. SCD is considered a pre-thrombotic state, since certain characteristics of sickle cells, such as abnormal adhesion and absence of asymmetry of the phospholipid membrane, are involved in the thrombotic process [Marfaing-Koka, et al. , Nouv Rev Fr Hamatol 35: 425-430, 1993]. The majority of SCD morbidity appears to be related to the appearance of occlusion of the microvasculature, resulting in generalized ischemia and irreversible organ damage. In addition, pulmonary microthromboemboli have been described in 441 of the autopsies of patients with thalassemia [Chuansumrit et al. , J Med Assoc Thai, 76 (2): 80-84, 1993]. Researchers who use sensitive titers for coagulation have documented signs of a hypercoagulable state, resulting in occlusion of blood vessels in adults with SCD. The occlusion of blood vessels is a complex process that involves vascular, cellular and humoral factors and, possibly, thrombotic events. The occurrence of stroke is probably the most devastating complication of SCD in children [Peters, et al. , Thrombosis and Haemostasis 71 (2): 69-172, 1994; Tam D., Journal of Child Neurology 12 (1): 19-21, 1997]. Deficiencies of protein C and increased generation of thrombin have been reported in patients with SCD or thalassemia [Karayalcin, et al. , The American Journal of Pediatric Hematology / Oncology 11 (3): 320-323, 1989; Hazmi, et al. , Acta Haematol 90: 114-119, 1993; Peters, 1994]; Shirahata et al. , Southeast Asian J Trop Med Pub Health 23 (2): 65-73]. The lower levels of protein C in SCD or thalassemia are due, either to decreased production or increased consumption.

Therefore, there is an imbalance in the coagulation in patients with SCD or thalassemia which, in turn, may be responsible for the adverse clinical effects observed in these patients. Currently, there is no effective therapy to prevent pain associated with SCD or thalassemia, or to correct the genes that cause the disease. The current treatment methodology includes intravenous glucose and electrolyte solutions, narcotic analgesics and anti-inflammatory agents [Green et al. , Ame.rican Journal of Hematology 23: 317-321, 1986]. Recently, the chemotherapeutic agent hydroxyurea has been used in an increased number of patients with sickle cell anemia. In the most severe cases or after an ischemic stroke, exchange transfusions and bone marrow transplants have been used [Ferrera et al. , American Journal of Emergency Medicine 15 (7): 671-679, 1997]. Prophylactic transfusion is the only therapy accepted for patients with SCD who have had a stroke. Therefore, there is a need for safe and effective therapy in patients with SCD or thalassemia.

The present invention is the first to describe the treatment of SCD or thalassemia using protein C. Protein C, with its anticoagulant, anti-inflammatory and profibrinolytic activities, is useful for the treatment of hypercoagulable state or protein C deficiency that occurs in SCD or in thalassemic patients. The present invention provides a method for treating a patient suffering from sickle cell disease (SCD) or thalassemia, which comprises administering to this patient a pharmaceutically effective amount of protein C. The present invention further provides a method for treating the disease of sickle cell or thalassemia in a patient in need thereof, which comprises administering to the patient a pharmaceutically effective amount of activated protein C, such that an activated protein C plasma level of about 2 ng / ml to about 300 ng / ml is achieved.

For the purposes of the present invention, as described and claimed herein, the following terms are as defined below.

The term "protein C" refers to a serine protease dependent on vitamin K with anticoagulant, anti-inflammatory and profibrinolytic properties including, but not limited to, protein C derived from plasma or produced by genetic recombination methods. Protein C includes and preferably is human protein C, although protein C can also include other species or derivatives having proteolytic, amidolytic, sterolitic and biological activities of protein C (anticoagulant, profibrinolytic and anti-inflammatory). Examples of protein C derivatives are as described by Gerlitz, et al. , Patent of E. U. A. No. 5,453,373, and Foster, et al. , U.S. Patent No. 5,516,650, the complete teachings are incorporated herein by reference. The term "Zimógeno" is an enzymatically inactive precursor of a proteolytic enzyme. The protein C zymogen, as used herein, it refers to the secreted and inactive forms, either single-stranded or double-stranded, of protein C. The term "activated protein C" or "aPC" refers to the zymogen of protein C that has been converted, through limited proteolysis, to its activated form. The aPC includes and is preferably human protein C, although the aPC may also include other species or derivatives that have proteolytic, amidolytic, sterolitic and biological activities of protein C (anticoagulant or profibrinolytic). Examples of protein C derivatives are mentioned above in the description of protein C. HPC - human protein C zymogen. r-hPC - recombinant human protein C zymogen. r-aPC - Recombinant activated human protein C that is produced by activating r-hPC in vi tro or by direct secretion of the activated form of protein C from prokaryotic cells, eukaryotic cells, and transgenic animals or plants, which include , for example, secretion from human kidney 293 cells in the manner of a zymogen and then purified and activated by techniques that are well known to the patient. technically skilled and are demonstrated in Yan, Patent of E. U.

A. No. 4,981,952, and Cottingham, WO97 / 20043, and the complete teachings of which are incorporated herein by reference. Activated protein C derived from plasma - is activated protein C that is produced by activating plasma HPC as described in Eibl, Patent of E. U. A.

No. 5,478,558, of which the complete teachings are incorporated herein for reference. The term "continuous infusion" is to continue in a substantially uninterrupted manner, the introduction of a solution into a vein for a specified period of time. The term "bolus injection" is the injection of a drug in a defined amount (called a bolus) for a period of time up to approximately 120 minutes. The term "suitable for administration" is a lyophilized formulation or a solution that is suitable for administration as a therapeutic agent. The term unit dose form - refers to physically discrete units suitable in the manner of unit dose for human subjects, each unit contains a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a pharmaceutically suitable excipient. The term "pharmaceutically effective amount" means and represents an amount of a compound of the invention that is capable of inhibiting sepsis in humans. The particular dose of the compound administered according to this invention is, of course, determined by the attending physician and assesses the particular circumstances surrounding the case. The present invention provides a treatment for sickle cell disease (SCD) or thalassemia using protein C. Protein C, with its anticoagulant, anti-inflammatory, and profibrinolytic activities, is useful for the treatment of the hypercoagulable state of the deficiency of protein C that occurs in SCD or thalassemic patients. The protein C that is administered according to this invention can be generated and / or isolated by means known in the art or as described in the patent. from U.A. No. 4,981,952, and in U.S. Patent No. 5,550,036, which are incorporated herein by reference. For example, protein C can be produced by secreting a full-length soluble protein C, or variants of the biologically active polypeptide of protein C from a cell comprising (a) structuring a vector comprising the DNA encoding protein C; (b) transfecting the cell with the vector; and (c) culturing the cell that is transfected in this way in a culture medium under conditions such that the full-length soluble protein C or the biologically active variants of the protein C polypeptide are secreted. In addition, the cell is a eukaryotic cell, for example the cell of a mammal such as the Syrian hamster AV12 cell, the human embryo 293 cell, or the neonate hamster kidney cell. The protein C that is used in the treatment of SCD or thalassemia can be formulated according to known methods to prepare pharmaceutically useful compositions. For example, a desired formulation is one that is a stable and lyophilized product of high purity which comprises a filler, such as sucrose, a salt such as sodium chloride, a buffer such as sodium citrate and protein C or aPC. Protein C is administered parenterally to ensure its administration into the bloodstream in an effective manner by injecting the appropriate dose in the form of a continuous infusion for approximately 1 hour to approximately 240 hours. Those skilled in the art can easily optimize pharmaceutically effective doses and administration regimens for the therapeutic compositions comprising protein C, as determined by good medical practice and the clinical condition of the individual patient. Generally, the amount of protein C administered is from about 5.0 μg / kg / hr to about 250 μg / kg / hr. Preferably, the C protein used in the treatment of SCD is activated protein C (aPC). The amount of aPC that is administered is from about 1.0 μg / kg / hr to about 96 μg / kg / hr. More preferably the amount of aPC that is administered is from about 1.0 μg / kg / hr to approximately 50 μg / kg / hr. Although more preferably, the amount of aPC that is administered is from about 1.0 μg / kg / hr to about 35 μg / kg / hr. Even more preferably, the amount of aPC that is administered is from about 5.0 μg / kg / hr to about 30 μg / kg / hr. Even more preferably, the amount of aPC that is administered is from about 15 μg / kg / hr to 30 μg / kg / hr. Even more preferably, the amount of aPC that is administered is from about 20 μg / kg / hr to 30 μg / kg / hr. The preferable amount of aPC that is administered is from about 24 μg / kg / hr. The most preferable amount of aPC aPC that is administered is from about 48 μg / kg / hr. The appropriate dose of aPC that is administered results in a reduction in thrombotic complications associated with SCD. The values of the plasma ranges that are obtained from the amount of aPC that is administered are from about 2 ng / ml to about 300 ng / ml. The preferred plasma ranges are from about 2 ng / ml to 200 ng / ml. More preferably, the plasma ranges are from about 30 ng / ml to about 150 ng / ml and even more preferably at about 100 ng / ml. Alternatively, the aPC is administered by injecting one third of the appropriate dose per hour in the manner of a bolus injection followed by the remaining two thirds of the dose per hour as a continuous infusion for one hour followed by a continuous infusion of the appropriate dose for 23 hours which results in an appropriate dose that is administered over a period of 24 hours. In addition, the bolus injection is administered via an intravenous pump drop by drop or a syringe pump at two times the normal speed for 15 minutes followed by 1.5 times the normal speed for 45 minutes. The normal speed, that is, the rate at which it has been determined to administer the appropriate level of the therapeutic agent dose over a period of time, then continues for up to a period of 240 hours. The use of protein C in the treatment of SCD or thalassemia as presented in the present invention provides a therapy required for a debilitating and potentially serious disorder. The use of protein C is effective and avoids complications such as the tendency to bleeding, toxicity, and other general side effects of currently available anticoagulants. The following examples are merely provided to further illustrate the present invention. The scope of the invention should not be construed as merely consisting of the following examples.

Preparation 1 Preparation of Human Protein C A recombinant human protein C (r-hPC) is produced in human kidney 293 cells by techniques that are well known to the skilled artisan, such as those set forth in Yan, US Pat. No. 4,981,952, the complete teachings of which are incorporated herein for reference. The gene coding for human protein C is described and claimed in Bang, et al. , Patent of E. U. A. No. 4,775,624, the complete teachings of which are incorporated herein for reference. The plasmid that is used to express human protein C in 293 cells is the pLPC plasmid described in Bang, et al. , U.S. Patent No. 4,992,373, the complete teachings of which are incorporated herein by reference. The construction of the pLPC plasmid is also described in European Patent Publication No. 0 445 939, and in Grinnell, et al. , 1987, Bio / Technology 5: 1189-1192, the teachings of which are incorporated into the present for reference. In brief, the plasmid is transfected into 293 cells, then stable transformants are identified, and are subcultured and cultured in a serum-free medium. After fermentation, the cell-free medium is obtained by microfiltration. Human protein C is separated from the culture fluid by an adaptation of the techniques of Yan, U.A. Patent No. 4,981,952. The purified medium is made 4 mM in EDTA before it is absorbed in an anion exchange resin (Fast-Flow Q, Pharmacia). After washing with 4 column volumes of 20 mM Tris, 200 mM NaCl, pH 7.4 and 2 column volumes of 20 mM Tris, 150 mM NaCl, pH 7.4, the agglutinated zymogen of recombinant human protein C is eluted with 20 mM Tris, 150 mM NaCl, 10 mM CaCl 2, pH 7.4. The eluted protein is greater than 951 pure after elution as judged by SDS-polyacrylamide electrophoresis. Further purification of the protein is achieved by making the 3 M protein in NaCl after adsorption in a hydrophobic interaction resin (Toyopearl Phenyl 650 M, TosoHaas) equilibrated in 20 mM Tris, 3 M NaCl, 10 mM CaCl 2, pH 7.4 . After washing with 2 column volumes of the equilibrium buffer without CaCl 2, the recombinant human protein C is eluted with 20 mM Tris, pH 7.4. The eluted protein is prepared for activation by removing residual calcium. The recombinant human protein C is passed over an affinity metal column (Chelex-100, Bio-Rad) to remove the calcium and again agglutinate in an anionic permutator (Fast Flow Q, Pharmacia). Both of these columns are serially configured and equilibrated in 20 mM Tris, 150 mM NaCl, 5 M EDTA, pH 7.4. After loading the protein, the Chelex-100 column is washed with a volume of calcium from the same buffer before disconnecting from the series. The anion exchange column is washed with 3 column volumes of the equilibrium buffer before eluting the protein with 0.4 M NaCl, 20 mM Tris-acetate, pH 6.5. The protein concentrations of the solutions of recombinant human protein C and recombinant activated protein C are quantified by UV depreciation at 280 nm ED-1% = 1.81 or 1.85, respectively.

Preparation 2 Activation of Recombinant Human Protein C Bovine thrombin is coupled to activated CH-Sepharose 4B (Pharmacia) in the presence of 50 mM HEPES, pH 7.5 at 4 ° C. The coupling reaction which is carried out on resin already packed in a column using about 5000 units of thrombin / mL resin. The thrombin solution is circulated through the column for approximately 3 hours before adding 2-amino-ethanol (MEA) at a concentration of 0.6 mL / L of the solution in circulation. The solution containing MEA is circulated for an additional 10-12 hours to ensure complete blockage of the unreacted amines on the resin. After blocking, the resin coupled with thrombin is washed with 10 column volumes of 1 M NaCl, 20 mM Tris, pH 6.5 to remove all non-specifically bound protein, and is used in the activation reactions after equilibration in a buffer of activation. The purified r-hPC is made 5 mM in EDTA (to chelate any residual calcium) and diluted to a concentration of 2 mg / mL with 20 mM Tris, pH 7.4 or 20 mM Tris-acetate, pH 6.5. This material is passed through a thrombin column equilibrated at 37 ° C with 50 mM NaCl either 20 mM Tris pH 7.4 or 20 mM Tris-acetate pH 6.5. The flow rate is adjusted to allow for about 20 minutes of contact time between r-hPC and the thrombin resin to flow. The effluent is collected and immediately evaluated for its amidolytic activity. If the material does not have a specific activity (amidolytic) comparable to a standard established pattern of protein C, it recycle in the thrombin column to activate the r-hPC until its term. This is followed by a 1: 1 dilution of the material with 20 mM buffer as above, with a pH of either 7.4 or 6.5 to keep protein C at lower concentrations while waiting for the next processing step.

Removal of leached thrombin from protein C material is achieved by binding protein C to an anion exchange resin (Fast Flow Q, Pharmacia) equilibrated in an activation buffer (either 20 mM Tris, pH 7.4 or 20 mM Tris-acetate, pH 6.5) with 150 mM NaCl. Thrombin does not interact with the anion exchange resin under these conditions, but passes through the column in the effluent of the application sample. Once protein C is loaded onto the column, a wash of 2-6 column volumes with 20 mM equilibrium buffer is made before eluting agglutinated protein C with an elution step using 0.4 M NaCl- either in 5 mM Tris-acetate, pH 6.5 or 20 mM Tris, pH 7.4. Higher volume washes of the column facilitate a more complete removal of the dodecapeptide. The material that is eluted from this column is stored either in a frozen solution (-20 ° C) or in the form of a lyophilized powder. The anticoagulant activity of activated protein C is determined by quantifying the prolongation of the clotting time in the assessment of the coagulation time of activated partial thromboplastin (APTT). A standard curve is prepared in the dilution buffer (1 mg / mL quality bovine serum albumin for radioimmunoassay [BSA], 20 • mM Tris, pH 7.4, 150 mM NaCl, 0.02% NaN3) which covers the concentration of C protein from 125-1000 ng / mL, while samples are prepared in several dilutions at this concentration range. To each tube of the sample, 50 μL cold equine plasma and 50 μL of a reconstituted activated partial thromboplastin time reagent (APTT Reagent, Sigma) is added and incubated at 37 ° C for 5 minutes. After the incubation period, 50 μL of the appropriate samples or prototypes are added to each tube. The dilution buffer is used in place of the sample or prototype to determine the baseline coagulation time. The timer of the fibrometer (Analyzer with CoA Haemostasis Detector, American Labor) begins immediately after the addition of 50 μL 37 ° C 30 mM CaCl2 to each sample or prototype. The concentration of activated protein C in the samples is calculated from the linear regression equation or the standard curve. The types of coagulation that are reported are the average for a minimum of three replicates, including the samples of the standard curve. The above descriptions allow someone with the skill in the art to prepare protein C for use in the treatment of sickle cell disease. Preparation 3 Activated Protein C Formulation A lyophilized stable formulation of activated protein C is prepared by a process comprising lyophilizing a solution comprising approximately 2.5 mg / mL activated protein C, approximately 15 mg / mL sucrose, approximately 20 mg / mL of NaCl, and a sodium citrate buffer having a pH greater than 5.5 but less than 6.5. In addition, the lyophilized stable formulation of activated protein C comprises lyophilizing a solution comprising approximately 5 mg / mL of activated protein C, approximately 30 mg / mL of sucrose, approximately 38 mg / mL of NaCl, and a citrate buffer having a pH greater than 5.5 but less than 6.5. The proportion of protein C: salt: filling agent (w: w: w) is an important factor in the proper accumulation for the lyophilization process. The proportion varies depending on the concentration of protein C, the selection and concentration of the salt and the selection and concentration of the filler. Particularly, a proportion of about one part of activated protein C in about 7.6 parts of salt in about 6 parts of filler is preferred. A unit dose formulation of activated protein C suitable for administration by continuous infusion is prepared by mixing activated protein C, NaCl, sucrose, and a sodium citrate buffer. After mixing, 4 mL of the solution is transferred to a unit dosage receptacle and lyophilized. The receptacle for the dosage unit containing about 5 mg to about 20 mg of activated protein C, suitable for administering a dose of about 0.01 g / kg / hr to about 0.05 mg / kg / hr to patients in need of this, is sealed and stored until its utilization.

Example 1 Double-blind, placebo-controlled trial of human and recombinant activated protein C (r-aPC) in patients with sickle cell disease (SCD) Studies of patients with sickle cell disease (SCD) in pain crisis have demonstrated abnormalities of platelet aggregation and survival as well as alterations in coagulation factors. Vascular occlusion in the microcirculation is a key factor in the pathogenesis of homozygous SCD. The vaso-occlusion process is multifactorial and coagulation disturbances probably contribute.

The current treatment methodology for patients with SCD includes intravenous glucose and electrolyte solutions, narcotic analgesics, and / or anti-inflammatory agents. In the most severe cases or after an ischemic stroke, exchange transfusions and bone marrow transplants are used. This trial aims to show that the infusion of r-aPC results in a statistically significant reduction in the combined endpoint of reduced pain crises and incidences of ischemic stroke in patients with homozygous SCD. Inclusion criteria include patients with homozygous sickle cell disease from a population-based study of children and young adults. These patients are presented in a trial after 48 hours of admission to the hospital and typically in a pain crisis. Patients who meet the inclusion criteria for SCD are given intravenous glucose and electrolyte solutions and narcotic analgesics. In addition, patients receive either a placebo or r-aPC during 96 hours The r-aPC is administered in a dose of 24 μg / kg / hr. The main end point of the study is the reduction / elimination of pain crises. The safety and efficacy of r-aPC is compared to placebo. The secondary endpoint of the trial is the reduction / elimination of ischemic stroke associated with SCD.

It is noted that in relation to this date, the best known method for the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects or products to which it refers.

Claims (10)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A method for treating a patient suffering from sickle cell disease (SCD) or thalassemia characterized in that it comprises administering to the patient a pharmaceutically effective amount of protein C.

2. The method according to claim 1, characterized in that protein C is the zymogen of human protein C.

3. The method according to claim 1, characterized in that protein C is activated protein C of human.

4. The method according to claim 3, characterized in that the amount of activated protein C of human is from about 1 μg / kg / hr to about 96 μg / kg / hr.

5. The method according to claim 4, characterized in that the activated protein C of human is administered by a continuous infusion for about 1 to about 240 hours.

6. A method for treating sickle cell disease or thalassemia in a patient in need thereof, characterized in that it comprises administering to the patient a pharmaceutically effective amount of activated protein C such that the plasma level of activated protein C is approximately 2. ng / ml up to approximately 300 ng / ml is achieved.

7. The method according to claim 6, characterized in that the activated protein C is administered in a bolus injection.

8. The method according to claim 6, characterized in that the activated protein C is administered by a continuous infusion for about 1 to about 240 hours.

9. The method according to claim 6, characterized in that the activated protein C is first administered as a bolus and then as a continuous infusion.

10. The method according to claim 9, characterized in that a third of the activated protein C that is required to achieve the plasma levels of activated protein C in the range of about 2 ng / ml to about 300 ng / ml is administered in the form of bolus injection and then a continuous infusion of the remaining two thirds of the activated protein C.