Title:  Methods for treating vascular disorders

United States Patent:  6,426,071

Inventors:  Grinnell; Brian W. (Indianapolis, IN); Howey; Daniel C (Indianapolis, IN); Jackson; Charles V (Indianapolis, IN)

Assignee:  Eli Lilly and Company (Indianapolis, IN)

Appl. No.:  877759

Filed:  June 8, 2001

A method of treatment for patients with vascular occlusion and thromboembolic disorders including the acquired disease state of thrombotic stroke, by administering activated protein C. The administration of aPC provides a highly selective therapeutic agent with a low potential for causing bleeding complications. The administration of aPC is beneficial in preventing the local extension of the microvascular and macrovascular occluding arterial thrombus, thereby reducing the neurological deficit resulting from the stroke.

DETAILED DESCRIPTION OF THE INVENTION

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

Activated protein C (aPC) refers to recombinant activated protein C. aPC includes and is preferably human protein C although aPC may also include other species or derivatives having full protein C proteolytic, amidolytic, esterolytic, and biological (anticoagulant or profibrinolytic) activities. Examples of protein C derivatives are described by Gerlitz, et al., U.S. Pat. No. 5,453,373, and Foster, et al., U.S. Pat. No. 5,516,650, the entire teachings of which are hereby included by reference. Recombinant activated protein C may be produced by activating recombinant human protein C zymogen in vitro or by direct secretion of the activated form of protein C. Protein C may be produced in procaryotic cells, eukaryotic cells, transgenic animals, transgenic plants, or gene therapy, including, for example, secretion from human kidney 293 cells as a zymogen then purified and activated by techniques known to the skilled artisan.

Continuous infusion--continuing substantially uninterrupted the introduction of a solution into a blood vessel for a specified period of time.

Bolus injection--the injection of a drug in a defined quantity (called a bolus) over a period of time up to about 120 minutes.

Suitable for administration--A formulation or solution preferably prepared from lyophilized aPC that is appropriate to be given as a therapeutic agent.

Zymogen--refers to secreted, inactive forms, whether one chain or two chains, of protein C.

The term "vial" refers broadly to a reservoir suitable for retaining the lyophilized activated protein C and diluent in a contained sterile state. Vials suitable for packaging products for parental administration are well-known and recognized in the art.

The present invention provides a method of treatment for human patients with vascular occlusive and arterial thromboembolic disorders which comprises administering to said patient a dosage of about 0.01 mg/kg/hr to about 0.05 mg/kg/hr of activated protein C by continuous infusion for about 4 to about 96 hours.

Applicants have found that pre-clinical toxicology studies in non-human primates indicate the safety of r-aPC for a 96 hour infusion is limited at a top dose of around 0.05 mg/kg/hr. These data are unexpected when compared to the prior art. In fact, the dose levels of r-aPC for humans that have been based on previous pre-clinical and clinical studies are above the toxicological range established in the above toxicological studies.

The present invention also demonstrates the effect of intravenous administration of r-aPC on reperfusion of totally occluded coronary arteries in a canine model of occlusive coronary artery thrombosis (Example 2). surprisingly, five of six animals treated with r-aPC demonstrated vessel reperfusion compared to vessel reperfusion in none of the six control animals.

aPC administered in accordance with the present invention is useful in treating vascular occlusive or arterial thromboembolic disorders, including thrombotic stroke, peripheral arterial thrombosis, emboli originating from the heart or peripheral arteries, acute myocardial infarction, and coronary arterial disease without the concomitant bleeding problems that may be associated with high dose levels.

Furthermore, the present invention provides an article of manufacture comprising packaging material and a vial comprising a lyophilized formulation of activated protein C, wherein said packaging material comprises a label which indicates that said lyophilized formulation be stored at refrigerated temperature; that said lyophilized formulation be reconstituted with normal saline, sterile water or comparable diluent; that said reconstituted formulation may be stored at refrigerated temperature to about 22^oC.; and that said reconstituted formulation be administered within 48 hours.

The present claimed articles of manufacture are useful for administration of aPC. Applicants have discovered that the reconstituted formulation of activated protein C may be administered at a dosage of about 0.01 mg/kg/hr to about 0.05 mg/kg/hr by continuous infusion for about 4 to about 96 hours.

The aPC can be formulated according to known methods to prepare pharmaceutically useful compositions. The aPC is preferably administered parenterally to ensure its delivery into the bloodstream in an effective form by injecting the appropriate dose as continuous infusion for about 4 to about 96 hours. Preferably, the appropriate dose of aPC will be administered by continuous infusion for about 4 to about 72 hours. More preferably, the appropriate dose of aPC will be administered by continuous infusion for about 4 to about 48 hours. More preferably, the appropriate dose of aPC will be administered by continuous infusion for about 12 to about 48 hours. More preferably, the appropriate dose of aPC will be administered by continuous infusion for about 12 to about 36 hours. More preferably, the appropriate dose of aPC will be administered by continuous infusion for about 4 to about 36 hours. More preferably, the appropriate dose of aPC will be administered by continuous infusion for about 12 to about 24 hours. Most preferably, the appropriate dose of aPC will be administered by continuous infusion for about 24 hours. The administration of aPC will begin as soon as possible following diagnosis of the vascular occlusive or arterial thromboembolic disorder.

The amount of aPC administered is from about 0.01 mg/kg/hr to about 0.05 mg/kg/hr which is equivalent to about 20 mg/70 kg/24 hours to about 84 mg/70 kg/24 hours. While the dose level is identified as a specific amount per 24 hours, one skilled in the art would recognize that this is a designation of the dose level and is not necessarily limited to a 24 hour infusion but may include continuous infusion for various times, for example, from about four hours to about ninety-six hours. More preferably the amount of aPC administered is about 0.01 mg/kg/hr to about 0.04 mg/kg/hr (about 20 mg/70 kg/24 hours to about 67 mg/70 kg/24 hours). While more preferably the amount of aPC administered will be about 0.01 mg/kg/hr to about 0.03 mg/kg/hr (about 20 mg/70 kg/24 hours to about 50 mg/70 kg/24 hours). Furthermore, the amount of aPC administered is from about 0.02 mg/kg/hr to about 0.05 mg/kg/hr which is equivalent to about 34 mg/70 kg/24 hours to about 84 mg/70 kg/24 hours. More preferably the amount of aPC administered is about 0.024 mg/kg/hr to about 0.048 mg/kg/hr (about 40 mg/70 kg/24 hours to about 80 mg/70 kg/24 hours). While more preferably the amount of aPC administered will be about 0.027 mg/kg/hr to about 0.045 mg/kg/hr (about 45 mg/70 kg/24 hours to about 75 mg/70 kg/24 hours). While more preferably the amount of aPC administered will be about 0.030 mg/kg/hr to about 0.042 mg/kg/hr (about 50 mg/70 kg/24 hours to about 70 mg/70 kg/24 hours). While more preferably the amount of aPC administered will be about 0.033 mg/kg/hr to about 0.039 mg/kg/hr (about 55 mg/70 kg/24 hours to about 65 mg/70 kg/24 hours). Preferable amounts of aPC administered are about 0.024 mg/kg/hr (about 40 mg/70 kg/24 hours), about 0.027 mg/kg/hr (about 45 mg/70 kg/24 hours) or, about 0.030 mg/kg/hr to about 0.042 mg/kg/hr (about 50 mg/70 kg/24 hours).

Alternatively, the aPC will be administered by injecting a portion of the appropriate dose per hour as a bolus injection over a time from about 5 minutes to about 120 minutes, followed by continuous infusion of the appropriate dose for about twenty three hours to about 96 hours which results in the appropriate dose administered over 24 hours to 96 hours.

As noted previously, the dosage levels of aPC presented above are in contrast to those presented by Griffin, et al. Griffin claimed dose levels in the range of 0.07 mg/kg/hr to 1.1 mg/kg/hr for the treatment of thrombotic occlusion. In contrast, the dose levels claimed herein are equivalent to a tenth of this dose or a range of about 0.01 mg/kg/hr to about 0.05 mg/kg/hr. The most preferable dose level of aPC to be administered for thrombolitic occlusion as described herein will be about 0.024 mg/kg/hr. It is significant to note that the most preferable dose level of 0.024 mg/kg/hr as indicated herein is 3 fold less than the lowest dose level claimed by Griffin and 44 fold less than the highest dose level claimed by Griffin.

Preparation 1

Preparation of Human Protein C

Recombinant human protein C (rHPC) was produced in Human Kidney 293 cells by techniques well known to the skilled artisan such as those set forth in Yan, U.S. Pat. No. 4,981,952, the entire teaching of which is herein incorporated by reference. The gene encoding human protein C is disclosed and claimed in Bang, et al., U.S. Pat. No. 4,775,624, the entire teaching of which is incorporated herein by reference. The plasmid used to express human protein C in 293 cells was plasmid pLPC which is disclosed in Bang, et al., U.S. Pat. No. 4,992,373, the entire teaching of which is incorporated herein by reference. The construction of plasmid pLPC 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 also incorporated herein by reference. Briefly, the plasmid was transfected into 293 cells, then stable transformants were identified, subcultured and grown in serum-free media. After fermentation, cell-free medium was obtained by microfiltration.

The human protein C was separated from the culture fluid by an adaptation of the techniques of Yan, U.S. Pat. No. 4,981,952, the entire teaching of which is herein incorporated by reference. The clarified medium was made 4 mM in EDTA before it was absorbed to 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 bound recombinant human protein C zymogen was eluted with 20 mM Tris, 150 mM NaCl, 10 mM CaCl₂, pH 7.4. The eluted protein was greater than 95% pure after elution as judged by SDS-polyacrylamide gel electrophoresis.

Further purification of the protein was accomplished by making the protein 3 M in NaCl followed by adsorption to a hydrophobic interaction resin (Toyopearl Phenyl 650M, TosoHaas) equilibrated in 20 mM Tris, 3 M NaCl, 10 mM CaCl₂, pH 7.4. After washing with 2 column volumes of equilibration buffer without CaCl₂, the recombinant human protein C was eluted with 20 mM Tris, pH 7.4.

The eluted protein was prepared for activation by removal of residual calcium. The recombinant human protein C was passed over a metal affinity column (Chelex-100, Bio-Rad) to remove calcium and again bound to an anion exchanger (Fast Flow Q, Pharmacia). Both of these columns were arranged in series and equilibrated in 20 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 6.5. Following loading of the protein, the Chelex-100 column was washed with one column volume of the same buffer before disconnecting it from the series. The anion exchange column was washed with 3 column volumes of equilibration buffer before eluting the protein with 0.4 M NaCl, 20 mM Tris-acetate, pH 6.5. Protein concentrations of recombinant human protein C and recombinant activated protein C solutions were measured by UV 280 nm extinction E^0.1% =1.85 or 1.95, respectively.

Preparation 2

Activation of Recombinant Human Protein C

Bovine thrombin was coupled to Activated CH-Sepharose 4B (Pharmacia) in the presence of 50 mM HEPES, pH 7.5 at 4^oC. The coupling reaction was done on resin already packed into a column using approximately 5000 units thrombin/ml resin. The thrombin solution was circulated through the column for approximately 3 hours before adding MEA to a concentration of 0.6 ml/l of circulating solution. The MEA-containing solution was circulated for an additional 10-12 hours to assure complete blockage of the unreacted amines on the resin. Following blocking, the thrombin-coupled resin was washed with 10 column volumes of 1 M NaCl, 20 mM Tris, pH 6.5 to remove all non-specifically bound protein, and was used in activation reactions after equilibrating in activation buffer.

Purified rHPC was 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 was passed through a thrombin column equilibrated at 37^oC. with 50 mM NaCl and either 20 mM Tris pH 7.4 or 20 mM Tris-acetate pH 6.5. The flow rate was adjusted to allow for approximately 20 min. of contact time between the rHPC and thrombin resin. The effluent was collected and immediately assayed for amidolytic activity. If the material did not have a specific activity (amidolytic) comparable to an established standard of aPC, it was recycled over the thrombin column to activate the rHPC to completion. This was followed by 1:1 dilution of the material with 20 mM buffer as above, with a pH of anywhere between 7.4 or 6.0 (lower pH being preferable to prevent autodegradation) to keep the aPC at lower concentrations while it awaited the next processing step.

Removal of leached thrombin from the aPC material was accomplished by binding the aPC to an anion exchange resin (Fast Flow Q, Pharmacia) equilibrated in activation buffer (either 20 mM Tris, pH 7.4 or preferably 20 mM Tris-acetate, pH 6.5) with 150 mM NaCl. Thrombin passes through the column and elutes during a 2-6 column volume wash with 20 mM equilibration buffer. Bound aPC is eluted with a step gradient using 0.4 M NaCl in either 5 mM Tris-acetate, pH 6.5 or 20 mM Tris, pH 7.4. Higher volume washes of the column facilitated more complete removal of the dodecapeptide. The material eluted from this column was stored either in a frozen solution (-20^oC.) or as a lyophilized powder.

The amidolytic activity (AU) of aPC was determined by release of p-nitroanaline from the synthetic substrate H-D-Phe-pip-Arg-p-nitroanilide (S-2238) purchased from Kabi Vitrum using a Beckman DU-7400 diode array spectrophotometer. One unit of activated protein C was defined as the amount of enzyme required for the release of 1 .mu.mol of p-nitroaniline in 1 min. at 25^oC., pH 7.4, using an extinction coefficient for p-nitroaniline at 405 nm of 9620 M^-1 cm^-1.

The anticoagulant activity of activated protein C was determined by measuring the prolongation of the clotting time in the activated partial thromboplastin time (APTT) clotting assay. A standard curve was prepared in dilution buffer (1 mg/ml radioimmunoassay grade BSA, 20 mM Tris, pH 7.4, 150 mM NaCl, 0.02% NaN₃) ranging in protein C concentration from 125-1000 ng/ml, while samples were prepared at several dilutions in this concentration range. To each sample cuvette, 50 .mu.l of cold horse plasma and 50 .mu.l of reconstituted activated partial thromboplastin time reagent (APTT Reagent, Sigma) were added and incubated at 37^oC. for 5 min. After incubation, 50 .mu.l of the appropriate samples or standards were added to each cuvette. Dilution buffer was used in place of sample or standard to determine basal clotting time. The timer of the fibrometer (CoA Screener Hemostasis Analyzer, American Labor) was started upon the addition of 50 .mu.l 37^oC. 30 mM CaCl₂ to each sample or standard. Activated protein C concentration in samples are calculated from the linear regression equation of the standard curve. Clotting times reported here are the average of a minimum of three replicates, including standard curve samples.

Claim 1 of 17 Claims

What is claimed is:

1. A method of treating a human patient with vascular occlusive and/or arterial thromboembolic disorders, which comprises administering a continuous infusion of recombinant human activated protein C for about 4 to about 96 hours, and wherein an activated protein C plasma concentration in said patient ranges from 20 ng/ml to 80 ng/ml when measured at 24 hours after starting the continuous infusion.
 

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