MXPA99008727A - Methods for treating vascular disorders - Google Patents

Abstract

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.

Description

METHODS TO TREAT VASCULAR DISORDERS FIELD OF THE INVENTION This invention relates to medical science, particularly to the treatment of vascular disorders with activated protein C.

BACKGROUND OF THE INVENTION Protein C is a serine protease and a naturally occurring anticoagulant that has a role in the regulation of homeostasis by deactivating the Va and VIIIa factors in the coagulation cascade. Human protein C is produced in vivo mainly in the liver as a single polypeptide of 461 amino acids. This precursor molecule undergoes multiple post-translational modifications including: 1) separation of a signal sequence of 42 amino acids; 2) proteolytic removal of a zymogen chain from the lysine residue at position 155 and the arginine residue at position 156 to produce the two-chain form of the molecule (ie, a light chain of 155 amino acid residues linked through a disulfide bridge to a heavy chain of 262 amino acid residues containing serine protease); 3) carboxylation dependent on vitamin K REF .: 31161 of nine glutamic acid residues grouped in the first 42 amino acids of the light chain, resulting in 9 gamma-carboxyglutamic residues; and 4) carbohydrate binding at four sites (one in the light chain and three in the heavy chain). The heavy chain contains a well-established serine protease triad of Asp 257, His 211 and Ser 360. Finally, the circulating two-chain zymogen is activated in vivo by thrombin on a phospholipid surface in the presence of calcium ion. The activation results from the removal of a dodecapeptide in the N-terminal part of the heavy chain, which produces activated protein C (aPC) which possesses enzymatic activity. Along with other proteins, protein C functions perhaps as the most important downstream regulator of blood coagulation. In other words, the protein C enzyme system represents a major physiological mechanism of anticoagulation. The coagulation system is best observed as a chain reaction involving the sequential activation of zymogens in active serines proteases that ultimately produce the enzyme, thrombin, which through limited proteolysis converts plasma fibrinogen into insoluble gel, fibrin. Two key phenomena in the coagulation cascade are the conversion of the coagulation factor X to Xa by the coagulation factor IXa and the conversion of prothrombin into thrombin by the coagulation factor Xa. Both reactions occur on cell surfaces, most notably on the platelet surface. Both reactions require cofactors. The main cofactors, factors V and VIII in the system circulate as relatively inactive precursors, but when some thrombin molecules are formed for the first time, thrombin forms a return cycle and activates the cofactors through limited proteolysis. Activated cofactors, Va and Villa, accelerate both the conversion of prothrombin into thrombin and the conversion of factor X to factor Xa by approximately five orders of magnitude. Activated protein C overwhelmingly prefers two plasma protein substrates which hydrolyze and irreversibly destroy. These plasmatic protein substrates are the activated forms of the Va and Villa coagulation cofactors. Activated protein C minimally degrades inactive precursors, coagulation factors V and VIII. It has been shown that activated protein C in dogs markedly increases the circulating concentrations of the main physiological fibrinolytic enzyme, tissue plasminogen activator (tPA). It has been shown in vitro that activated protein C improves lysis of fibrin in whole human blood. Therefore, activated protein C represents an important adjuvant for fibrinolysis in vivo in man.

Currently, there are few effective treatments available for vascular occlusions, which include thrombotic attack. Treatment with tPA, if administered within three hours after the onset of the attack, has recently been approved by the FDA. The treatment of attacks with either heparin or oral anticoagulants, although occasionally beneficial, presents an elevated risk of bleeding in the infarcted brain area. The use of recombinant aPC (r-aPC) in the treatment of thrombotic occlusion or thromboembolism in a balloon model has been reported by Griffin et al., In U.S. Patent No. 5,084,274. Griffin states that the dose levels in the range of 0.2 mg / kg / h to 1.1 mg / kg / h for the treatment of thrombotic occlusion. However, applicants have found that these dose levels are in a range significantly above the toxicological concentration of r-aPC. For example, preclinical toxicology studies in non-human primates indicate the safety of r-aPC for a 96-hour infusion is limited to a maximum dose of approximately 0.05 mg / kg / h. Therefore, the lowest dose level presented by Griffin et al., Ie, 0.2 mg / kg / h is at a level four times higher than the toxic dose established by the applicants for humans. Therefore, even at the lower dose level mentioned by Griffin may carry a high risk of hemorrhage in the infarcted brain area, thus aggravating the neurological deficit that accompanies the attack. Consequently, even in view of the teachings of Griffin et al., The need remains to identify an effective thrombus arterial formation therapy in humans with aPC. Contrary to the teachings of previous investigators, the applicants have discovered that only a low dose therapy with r-aPC is useful in the treatment of thrombotic attacks. The administration of aPC is also beneficial to avoid local extension of the microvascular and macrovascular occlusion arterial thrombus, which reduces the neurological deficit that results after the attack.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides a method of treatment for human patients with arterial and vascular occlusive thromboembolic disorders, which comprises administering to the patient a dosage of about 0.01 mg / kg / h to about 0.05 mg / kg / h of activated protein C. This invention also provides a unit dosage form suitable for continuous infusion administration which comprises a unit dosage receptacle containing about 5 mg to about 20 mg of activated protein C suitable to deliver a dosage of about 0.01 mg / kg / hr. approximately 0.05 mg / kg / h.

DETAILED DESCRIPTION OF THE INVENTION For purposes of the present invention, as described and claimed herein, the following terms are defined below. The term aPC or activated protein C refers to protein C either recombinant or derived from plasma. The term aPC includes and preferably is human protein C, although aPC may also include other species or derivatives that have protein C with proteolytic, amidolytic, sterolytic and biological activity (anticoagulant or profibrinolytic). Examples of protein C derivatives are described by Gerlitz et al., U.S. Patent Number 5,453,373 and Foster, et al., U.S. Patent Number 5,516,650, the entire disclosure of which is hereby incorporated by reference. . APTT - activated partial thromboplastin time AU - amidolytic units. HPC - human protein C zymogen MEA-2-aminoethanol tPA - plasminogen tissue activator r-HPC - recombinant human protein C zymogen. r-aPC - recombinant activated protein C produced by activating the zymogen of protein C in vi tro or in vivo or by direct secretion of the activated form of protein C from prokaryotic cells, eukaryotic cells or transgenic animals including, for example, secretion from human kidney 293 cells as a zymogen which is then purified and activated by techniques well known to those familiar in the art and demonstrated in Yan, U.S. Patent No. 4,981,952 and Cottingham, WO document 97/20043, the full description of which is incorporated herein by reference. Infusion continues - substantially continuous and uninterrupted introduction of a solution into a blood vessel for a specified period of time. Bolus injection - the injection of a medication in a defined amount (called a bolus) for a period of time up to approximately 120 minutes. Suitable for administration - a formulation or solution preferably prepared from lyophilized aPC that is suitable for delivery as a therapeutic agent. Zymogen - an enzymatically inactive precursor of a proteolytic enzyme. "Protein C" zylogen, as used herein, refers to inactive secreted forms, either a chain or two chain, protein C. Applicants have found that preclinical toxicological studies in non-human primates indicate that the Safety of r-aPC for an infusion of ^ 96 hours is limited to a maximum dose of approximately 0.05 mg / kg / h. 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 preclinical and clinical studies are above the toxicological range established in the previous toxicological studies. The present invention provides a method of treatment for human patients with arterial and vascular occlusive thromboembolic disorders which comprises administering to the patient a dosage of about 0.01 mg / kg / h to about 0.05 mg / kg / h of activated protein C. Activated protein C administered at low dose levels is useful for the treatment of thrombotic attack without the concomitant bleeding problems that may be associated with high dose concentrations. The present invention also demonstrates the use of protein Recombinant human C (r-aPC) in a human clinical trial to determine the plasma blood concentration of r-aPC (Example 1) .

The present invention also demonstrates the effect of intravenous administration of r-aPC on reperfusion of totally occluded coronary arteries in a dog model of occlusive coronary artery thrombosis (Example 2). Surprisingly, five of six animals - treated with r-aPC showed reperfusion of the vessels compared to vessel reperfusion in none of the six control animals. The present invention relates to the treatment with activated protein C of vascular or arterial occlusive thromboembolic disorders, including thrombotic attack, peripheral arterial thrombosis, embolisms originating from the cardiac or peripheral arteries, acute myocardial infarction and coronary artery disease. 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 manner by injecting the appropriate dose as a continuous infusion for about 1 to about 48 hours. Most preferably, the appropriate dose of aPC will be administered by continuous infusion for about 4 to about 36 hours. Even 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 approximately 24 hours. The administration of aPC will begin as soon as possible after the diagnosis of the attack. The amount of aPC administered is from about 0.01 mg / kg / h to about 0.05 mg / kg / h, which is equivalent to about 20 mg / 70 kg / 24 h to about 84 mg / 70 kg / 24 h. Although the dose level is identified as the specific amount per 24 hours, a person familiar with the art will 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 by various times, for example, from about one hour to about forty-eight hours. More preferably, the amount of aPC administered is from about 0.01 mg / kg / h to about 0.04 mg / kg / h (from about 20 mg / 70 kg / 24 h to about 67 mg / 70 kg / 24 h). Although more preferably, the amount of aPC administered will be from about 0.01 mg / kg / h to about 0.03 mg / kg / h (from about 20 mg / 70 kg / 24 h to about 50 mg / 70 kg / 24 h) . The most preferable amount of aPC administered is about 0.024 mg / kg / h (approximately 40 mg / 70 kg / 24 h).

Alternatively, the aPC will be administered by injecting a portion of the appropriate dose per hour as a bolus injection for a period from about 5 minutes to about 30 minutes, followed by continuous infusion of the appropriate dose for about 23 hours to about 47 hours, which results in an appropriate dose administered for 24 hours to 48 hours. As previously indicated, the aPC dosage levels presented above contrast with those presented by Griffin et al. Griffin presents dose levels in the range of 0.2 mg / kg / h to 1.1 mg / kg / h for the treatment of thrombotic occlusion. In contrast, the dose levels claimed herein are equivalent to one tenth of this dose or a range of about 0.01 mg / kg / h to about 0.05 mg / kg / h. The most preferable dose level of aPC that is administered for thrombotic occlusion as described herein will be about 0.024 mg / kg / h. It is important to note that the most preferable dosage level of 0.024 mg / kg / h as indicated herein is 8 times lower than the lowest dose level claimed by Griffin and 44 times lower than the highest dose level claimed by Griffin.

Preparation 1 Preparation of Human Protein C Recombinant human protein C (rHPC) in cells 293 of human kidney by techniques well known to those familiar in the art in the manner set forth in Yan, U.S. Patent No. 4,981,952, the full disclosure of which is incorporated herein by reference. The gene encoding human protein C is described and presented in Bang et al. , U.S. Patent No. 4,775,624, the entire disclosure of which is incorporated herein by reference. The plasmid used to express human protein C in 293 cells is plasmid pLPC which is described in Bang et al., U.S. Patent No. 4,992,373, the full disclosure of which is incorporated herein by reference. The construction of plasmid pLPC is also described in European Patent Publication Number 0 445 939, and in Grinnel et al., 1987, Bio / Technoloqy 5: 1189-1192, the descriptions of which are incorporated herein by reference. Briefly, the plasmid is transfected into 293 cells, then stable transformants are identified, subcultured and grown in 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.S. Patent No. 4,981,952, the full disclosure of which is incorporated herein by reference. The clarified medium is made in 4 mM EDTA before it is absorbed by anion exchange resin (Fast-Flow A, 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, bound recombinant human protein zymogen is eluted with 20 mM Tris , 150 mM NaCl, 10 mM CaCl2, pH 7.4. The eluted protein is less than 95% pure after elution, considered by electrophoresis in SDS-polyacrylamide gel. Additional purification of the protein is carried out when preparing the 3 M protein n NaCl followed by absorption to a hydrophobic interaction resin (Toyopearl Phenyl 650M, TosoHaas) equilibrated in 20 mM Tris, 3 M NaCl, 10 mM CaCl 2, pH 7.4 . After washing with 2 column volumes of equilibrium buffer without CaCl2, recombinant human protein C is eluted with 20 mM Tris, pH 7.4. The eluted protein is prepared for activation by removal of residual calcium. The recombinant human protein C is passed over a metal affinity column (Chelex-100, Bio-Rad) to remove the calcium and binds again to an anion exchanger (Fast Flow Q, Pharmacia). Both columns are placed in series and equilibrated in 20 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 6.5. After loading the protein, the Chelex-100 column is washed with a column volume of the same buffer before disconnecting it from the series. The anion exchange column is washed with 3 column volumes of equilibrium 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 are measured by UV extinction 280 nM E ° -1% "1.85 or 1.95, 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 is performed on resin already packed in a column using approximately 5000 thrombin units / ml resin. The thrombin solution is circulated through a column for about 3 hours before adding MEA at a concentration of 0.6 ml / 1 of circulating solution. The solution containing MEA is circulated for an additional 10-12 hours to ensure complete blockage of amines that have not reacted in the resin. After the blockade, the resin coupled with thrombin is washed with 10 column volumes of 1 M NaCl, 20 mM Tris, pH 6.5 to remove any bound protein in a non-specific manner, and is used in activation reactions after equilibration in the activation buffer. 5mM purified rHPC is made 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 and either 20 mM Tris, pH 7.4 or 20 mM Tris-acetate, pH 6.5. The flow rate is adjusted to allow for approximately 20 min of contact time between rHPC and the thrombin resin. The effluent is collected and tested immediately to determine amidolytic activity. If the material does not have specific (amidolytic) activity comparable to an established aPC standard, it is recycled onto the thrombin column to activate the rHPC until complete. This is done by a 1: 1 dilution of the 20 mM buffer material as above, with a pH anywhere between 7.4 or 6.0 (a lower pH is preferable to avoid autodegradation) to keep aPC at lower concentrations while wait for the next stage of processing.

The removal of the leached thrombin from the aPC material is carried out by joining aPC to the 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 wash with 2-6 column volumes with 20 mM equilibration buffer. APC bound with a step or step gradient is eluted using 0.4 M NaCl in either 5 mM Tris-acetate, pH 6.5 or 20 mM Tris, pH 7.4. Larger volumes of column wash facilitate a more complete removal of dodecapeptide. The material eluted from this column is stored either in a frozen solution (-20 ° C) or as a lyophilized powder. The amidolytic activity (AU) of aPC is determined by release of p-nitroaniline from the synthetic substrate HD-Phe-Pip-Arg-p-nitroanilide (S-2238) purchased from Kabi Vitrum using a diode array spectrophotometer Beckman DU -7400. One activated protein C unit is defined as the amount of enzyme required for the release of 1 μmol of p-nitroaniline in 1 min, at 25 ° C pH 7.4, using an extension coefficient for p-nitroaniline at 405 nm of 9620 M "1 was" 1. Protein anticoagulant activity was determined C activated by measuring the prolongation of the clotting time in the activated partial thromboplastin coagulation assay (APTT). A standard curve is prepared in dilution buffer (1 mg / ml BSA grade radioimmunoassay, 20 mM Tris, pH 7.4, 150 mM NaCl, 0.02% NaN3) which varies in protein C concentration from 125-1000 ng / ml, while that the samples are prepared at various dilutions in this concentration range. To each sample cuvette, 50 μl of cold horse plasma and 50 μl of activated partial thromboplastin reconstituted reagent (APTT reagent, Sigma) are added and incubated at 37 ° C for 5 minutes. After incubation, 50 μl of the appropriate samples or standards are added to each cuvette. The dilution buffer is used in place of the sample or standard to determine the basal coagulation time. The chronometer of the fibrometer (CoA Screener Hemostasis Analyzer, American Labor) is initiated by the addition of 50 μl of 30 mM CaCl2 at 37 ° C to each sample or standard. The concentration of activated protein C in the samples is calculated from the linear regression equation of the standard curve. The coagulation times presented here are the average of a minimum of three reproductions, which include standard curve samples. The above descriptions allow a person with the appropriate skill in the art to prepare aPC for use in the treatment of thrombotic attack.

Example 1 Plasmatic Human Concentrations of aPC Six human patients received an i.v. of aPC at 1 mg / m2 / h or approximately 0.024 mg / kg / h during a period of 24 hours. The aPC administered is a lyophilized formulation containing 10 mg of aPC, buffer 5 mM Tris acetate and 100 mM sodium chloride reconstituted with 2 ml of water and adjusted to pH 6.5. Plasma concentrations of aPC were measured using an immunocapture-amidolytic assay. Blood is collected in the presence of anticoagulant citrate and benzamidine, a reversible inhibitor of aPC. The enzyme is captured from the plasma by a mouse monoclonal antibody specific for aPC, C3, removed by washing and the amidolytic activity of aPC is measured using an oligopeptide chromogenic substrate. After incubation for 16-20 h at 37 ° C, the absorbance at 405 nm is measured and the data analyzed by a weighted linear curve fitting algorithm. The aPC concentrations are estimated from the standard curve with variable concentrations of 0-100 ng / ml. The limit of the assay quantification is 1.0 ng / ml. The aPC dose concentrations and plasma concentrations are measured at approximately 24 hours. The plasma ranges are from 2 ng / ml to less than 100 ng / ml.

Preferred plasma ranges are from about 20 ng / ml to 80 ng / ml. More preferably, the plasma ranges are from about 30 ng / ml to about 60 ng / ml, and even more preferably about 50 ng / ml. Therefore, the dose of 0.024 mg / kg / h provides the most preferable plasma concentration of 50 ng / ml at 24 hours for the treatment of thrombotic attack without the concomitant bleeding problems of the higher dose concentrations.

Example 2 Reperfusion Induced in a Canine Model of Occlusive Coronary Artery Thrombosis Twelve dogs (17-22 kg, of both sexes, Butler Farms) were anesthetized with sodium pentobarbital (30 mg / kg, i.v.) and ventilated with ambient air. Cannulae were placed for blood pressure measurement, drug administration and blood samples in the carotid artery, femoral vein and jugular vein; respectively. Left thoracotomy was performed, the heart was suspended in a basket for the pericardium and a 2 cm segment of the left circumflex coronary artery (LCCA) proximal to the first main diagonal branch was isolated. The LCCA is instrumented with an electromagnetic flow probe, a stimulation electrode and an external occluder to measure coronary blood flow, cause vessel damage and provide critical stenosis; respectively. Damage to the vessel is calculated by placing the stimulation electrode (anode) in contact with the intimal side of the vessel and stimulating the anode with a current of 100 μA d.c. (direct current) (the circuit is completed by placing the cathode in a subcutaneous site). The damage current is continued for 60 minutes and then stops whether the vessel is occluded or not. The vessels reach total occlusion in approximately 60 minutes from the onset of vessel damage.

Thirty minutes after the total occlusion of the vessel (established as a coronary blood flow of 0 for 30 minutes), a continuous intravenous infusion of 2.0 mg / kg / h of aPC or Tris buffer is delivered by infusion for 2 h. ml, pH 7.4 (vehicle group). The preparations are followed • for 4 h starting from the moment of damage initiation to LCCA. The arterial blood pressure, heart rate and coronary blood flow are determined and analyzed. At different points throughout the experiment, blood samples are drawn to determine total coagulation times (Hemochron 801) and gingival template bleeding times are determined using a bleeding time device Simplate II. A second set of blood samples (citrated) was collected during the experiment for determination of plasma plasminogen activator inhibitor-1 (PAI-1). Plasma PAI-1 levels were determined using a plasma IMUBIND ^ ELISA PAI-1 kit (American Diagnostica). All data (presented as mean + standard error mean) are analyzed for statistical differences using simple ANOVA followed by Student-Neuman-Keuls analysis for significance at a level of p < 0.05 The incidence of reperfusion and patency are analyzed using Fisher's exact test at a level of p < 0.05. A continuous infusion of 2.0 mg / kg / h of aPC produces a 6-fold increase in total blood coagulation time in APTT at the end of the 2-hour infusion of medication (Table 1). APTT begins to return to normal values at the end of the experiment. There is no observable effect on the coagulation time of thrombin or the bleeding time of the template. Table 1 shows the results. Table 1 Effect of aPC on Coagulation and Template Bleeding Times in Anesthetized Dogs Treatment Parameter Premedicamento 60 m in of 120 min of End infusion infusion Vehicle Time of 36 + 1 38 + 4 33 + 1 34 + I Thrombin (sec) (n-6) APTT (sec) 100 + 6 95 + 5 89 + 10 91 + 10 Bleeding time 132 + 15 182 + 14 152 + 15 159 13 are templates (sec) aPC Time of 33 + 1 34 + 1 34 + 1 34 ± 1 Thrombin (sec) (n = 6) APTT (sec) 96 ± 6 573 + 237 670 + 209 138 + 13 Bleeding time 199 + 41 272 + 84 204 + 20 193 ± 39 se templates (sec) The dosing regimen used for the vehicle treated group is 20 ml of saline buffered with Tris delivered by infusion for 2 h and aPC (2.0 mg / kg / hx 2 h) administration started 30 minutes after the total occlusion of glass. * Denotes statistical difference at a level of p < 0.05 compared to the group treated by vehicle. Each value represents the mean + SEM.

Table 2 illustrates the effects of intravenous administration of aPC in reperfusion of completely occluded coronary arteries. The time for total thrombotic occlusion of the coronary arteries was similar between the two groups; 66 + 7 and 62 + 6 minutes, treated with vehicle and treated with aPC, respectively. Five of the six recipients in the aPC group demonstrated reperfusion compared to none of the 6 recipients who received the vehicle; the time for reperfusion in the group treated with aPC was 128 ± 17 min. Coronary blood flow in the group treated with aPC is significantly greater than the group treated with corresponding vehicle; the group treated with aPC reaches 13.7 + 2.7 ml / min during the reperfusion period and a flow volume of 1069 + 623 ml (this represents a restoration of approximately 60-70% of the coronary blood flow prior to thrombosis, in this group ). Three of the 5 receptors exposed to aPC were still patent at the end of the 4 h experiment. Therefore, the data demonstrate that aPC is effective in the treatment of occlusive coronary artery thrombosis in the canine model.

Table 2 Effects of aPC in the Restoration of Coronary Blood Flow in the Canine Coronary Artery Thrombosis Model Parameter Vehicle aPC (n-6) (n = 6) Time for occlusion (min) 66 + 7 62 +6 Thrombus mass (g) 10.8 2.1 8.2 + 1.2 Incidence of reperfusion 0 5 of 6 * Time for reperfusion (m in) 128 ± 17 * Patency of the vessels 0 out of 6 3 out of 5 End of CBF experiment during reperfusion 13.7+ "2.7 * (ml / min) Reperfusion Volume (mi) 1069 + 623 * Indicates a statistical difference at a level of p < 0.05 versus the vehicle group. Each value represents the mean +. SEM.

The blood samples taken during each experiment show that there is a significant correlation with the venous infusion of aPC and the circulating concentrations of plasminogen activator inhibitor-1 (PAI-1). At the end of the intravenous infusion of aPC, the plasma concentrations of PAI-1 decreased by 80%. Upon cessation of aPC infusion, plasma concentrations of PAI-1 began to return to pre-infusion concentrations. Although these dosage levels in this canine model appear to be greater than the claimed dosage levels for humans, Applicants have found that the dog is especially insensitive to human activated protein C, and therefore the claimed dosage levels are appropriate for humans . It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (15)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. The use of an activated protein C for the manufacture of a medicament for human patients with arterial and thromboembolic arterial occlusive disorders, characterized in that it comprises administering to the patient a dosage of approximately 0.01 mg / kg / h to approximately 0.05 mg / kg / h of activated protein C.
2. 2. The use according to claim 1, characterized in that the vascular or thromboembolic occlusive disorder is thrombotic attack.
3. 3. The use according to claim 2, characterized in that it comprises administering to the patient from approximately 0.01 mg / kg / h to approximately 0.03 mg / kg / h of activated protein C.
4. 4. The use according to claim 3, characterized in that it comprises administering to the patient approximately 0.024 mg / kg / h of activated protein C.
5. 5. The use according to claim 4, characterized in that the activated protein C is human activated protein C.
6. 6. The use according to claim 1, characterized in that the activated protein C is administered in a continuous infusion for about 1 hour to about 48 hours.
7. The use according to claim 6, characterized in that the activated protein C is administered in a continuous infusion for approximately 12 hours to approximately 36 hours.
8. 8. The use according to claim 7, characterized in that the activated protein C is administered in a continuous infusion for approximately 24 hours.
9. 9. The use according to claim 8, characterized in that the activated protein C is human activated protein C.
10. 10. The use according to claim 9, characterized in that it comprises administering approximately 0.024 mg / kg / h of activated protein C.
11. 11. The use according to claim 1, characterized in that the activated protein C is administered in a bolus injection.
12. 12. The use according to claim 11, characterized in that the bolus injection of activated protein C is followed by a continuous infusion.
13. 13. A unit dosage form comprising a unit dosage receptacle which contains from 5 to 20 mg of activated C protein suitable for administration at a dosage of 0.01 mg / kg. / h up to 0.05 mg / kg. / h.
14. 14. A unit dosage according to claim 13, characterized in that the administration is continuous infusion from approximately 1 to 48 hours.
15. 15. A unit dosage according to claim 14, characterized in that the activated protein C is lyophilized.