WO2002024215A2 - Method for using activated protein c for the treatment of coagulation-associated disorders - Google Patents

Method for using activated protein c for the treatment of coagulation-associated disorders Download PDF

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WO2002024215A2
WO2002024215A2 PCT/US2001/026028 US0126028W WO0224215A2 WO 2002024215 A2 WO2002024215 A2 WO 2002024215A2 US 0126028 W US0126028 W US 0126028W WO 0224215 A2 WO0224215 A2 WO 0224215A2
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protein
activated protein
apc
administered
plasma
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PCT/US2001/026028
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French (fr)
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WO2002024215A3 (en
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Sau-Chi Betty Yan
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Eli Lilly And Company
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4866—Protein C (3.4.21.69)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid

Definitions

  • FIELD OP THE INVENTION This invention relates to methods of decreasing the duration of time a human patient with a hypercoagulable state and/or protein C deficiency remains in an intensive care unit, a hospital and/or on mechanical ventilation which comprises administering to said patient activated Protein C (aPC) .
  • aPC patient activated Protein C
  • Protein C is a serine protease and naturally occurring anti-coagulant produced as an inactive precursor or zymogen by the liver. Human protein C is made in vivo as a single polypeptide of 461 amino acids. This polypeptide undergoes multiple post-translational modifications including: 1) cleavage of a 42 amino acid signal sequence; 2) cleavage of lysine and arginine residues (positions 156 and 157) to make a 2-chain inactive zymogen (a 155 amino acid residue light chain attached via a disulfide bridge to a 262 amino acid residue heavy chain); 3) vitamin K-dependent carboxylation of nine glutamic acid residues located within the amino- terminal 45 residues (gla-domain) ; and, 4) carbohydrate attachment at four sites (one in the light chain and three in the heavy chain) .
  • the protein C zymogen circulates in the plasma and, upon removal of a dodecapeptide at the N- terminus of the heavy chain, results in aPC possessing enzymatic activity.
  • aPC plays a key role in regulating hemostasis by inactivating Factors V a and Villa in the coagulation cascade.
  • Blood coagulation is a highly complex process regulated by the balance between pro-coagulant, anti-coagulant, and fibrinolytic mechanisms. This balance determines a condition of either normal hemostasis or abnormal pathological thrombus generation and the progression of hypercoagulable states. Two major factors control this balance; the generation of fibrin and the activation and subsequent aggregation of platelets, both processes controlled by the generation of the enzyme thrombin, which occurs following activation of the clotting cascade. Thrombin, in complex with thrombomodulin, also functions as a potent anti-coagulant since it activates protein C zymogen to the active enzyme, aPC .
  • thrombin-thrombomodulin complex In large blood vessels, the activation of protein C zymogen to aPC by thrombin- thrombomodulin complex is further augmented by an endothelial transmembrane protein, endothelial-protein receptor (EPCR) [Stearn-Kurosawa, et al . Proc. Natl. Acad. Sci. USA 93:10212-10216, 1996].
  • EPCR endothelial transmembrane protein
  • APC inhibits the generation of thrombin.
  • aPC functions as perhaps the most important down-regulator of blood coagulation resulting in protection against thrombosis.
  • aPC exerts profibrinolytic properties that facilitate clot lysis and exerts anti-inflammatory effects via inhibiting the release of inflammatory mediators, such as, tumor necrosis factor and various interleukins .
  • inflammatory mediators such as, tumor necrosis factor and various interleukins .
  • the present invention is the first to describe administering aPC to decrease the duration a human patient with a hypercoagulable state and/or a protein C deficient state remains in a hospital, an intensive care unit and/or undergoing ventilation therapy.
  • Sepsis is defined clinically as a systemic response to infection or suspected infection complicated by one or more organ failures. Sepsis is associated with and mediated by the activation of a number of host defense mechanisms including the cytokine network, leukocytes, and the complement and coagulation/fibrinolysis systems. [Mesters, et al . , Blood 88:881-886, 1996]. Disseminated intravascular coagulation [DIC] , with widespread deposition of fibrin in the microvasculature of various organs, is an early manifestation of sepsis/septic shock. DIC is an important mediator in the development of the multiple organ failure syndrome and contributes to the poor prognosis of patients with severe sepsis.
  • DIC Disseminated intravascular coagulation
  • Purpura fulminans ecchymotic skin lesions, fever, hypotension associated with bacterial sepsis, viral, bacterial or protozoan infections
  • DIC DIC
  • numerous bacterial, viral, or protozoan infections include but are not limited to infections caused by Rickettsia (Rocky Mountain Spotted fever, tick bite fever, typhus, etc.) [Graybill, et al . , Southern Medical Journal, 66 (4) : 410-413 , 1973; Loubser, et al .
  • Staphylococci [Levin, M. , Pediatric Nephrology, 8:223-229]; Haemophilus aegyptius (Brazilian purpuric fever) ; Neisseria (gonococcemia, meningococcemia) ; and mycojacter-ium tuberculosis (miliary tuberculosis) .
  • VOP hepatic venocclusive disease
  • Organ dysfunction after BMT including pulmonary, central nervous system, hepatic or renal, is a complication that occurs in a high percentage of transplant patients [Haire, et al . , JAMA 274:1289-1295, (1995)].
  • a single organ dysfunction in BMT is a strong predictor of multiple organ dysfunction syndrome (MODS) which is the leading cause of death in BMT patients.
  • MODS multiple organ dysfunction syndrome
  • DIC Disseminated intravascular coagulation
  • DIC due to a massive activation of the coagulation system and widespread deposition of fibrin in the microvasculature of various organs is an important mediator in the development of MODS [Fourrier, et al .
  • Burned patients have supranormal in vi tro clotting activity and frequently develop DIC which is characterized by the sudden onset of diffuse hemorrhage; the consumption of fibrinogen, platelets, and Factor VIII activity; intravascular hemolysis; secondary fibrinolysis; and biopsy evidence of microthrombi [McManis, et al . , J. of Trauma 13:416-422, (1973)].
  • PREGNANCY causes multiple changes in the coagulation system which may lead to a hypercoagulable state .
  • the risk of venous thrombosis is almost fivefold higher than in the non- pregnant state.
  • clotting factors increase, natural inhibitors of coagulation decrease, changes occur in the fibrinolytic system, venous stasis increases, as well as increases in vascular injury at delivery from placental separation, cesarean section, or infection [Barbour, et al . , Obstet Gynecol 86:621-633, 1995].
  • preeclampsia and eclampsia and other obstetrical complications such as amniotic fluid embolism and placenta abruption in pregnant women appear to be- a state of increased coagulopathy and disseminated intravascular coagulation as indicated by an increase in fibrin formation, activation of the fibrinolytic system, platelet activation and a decrease in platelet count [Clin Obstet Gynecol 35:338-350, 1992].
  • Preeclampsia is thought to be the result of uteroplacental ischemia due to an anomaly of the "vascular insertion" of the placenta.
  • preeclampsia Consequences of preeclampsia include hypertension as well as DIC which leads to the release of numerous microthrombi which cause placental, renal, hepatic and cerebral lesions [Rev Fr Gynecol Obstet 86:158-163, 1991]. Furthermore, preeclampsia can lead to a severe and life threatening condition known as the HELLP syndrome which is defined as preeclampsia complicated by thrombocytopenia, hemolysis and disturbed liver function [Rathgeber, et al . , Anasth
  • CABG coronary artery bypass grafting
  • anti-coagulant therapy is important as a prophylactic therapy to prevent venous thromboembolic events in major surgery or trauma patients [Thomas, et al . , 1989; LeClerc, 1997]. For example, many patients who succumb from pulmonary embolism have no clinical evidence of preceding thromboembolic events and die before the diagnosis is made and the treatment is instituted [LeClerc, 1997].
  • Existing prophylactic methods e.g., warfarin, low molecular weight heparins, have limitations such as residual proximal thrombosis or the need for frequent dose adjustments.
  • ARDS Adult respiratory distress syndrome
  • lung edema characterized by lung edema, microthro bi, inflammatory cell infiltration, and late fibrosis . Pivotal to these multiple cellular and inflammatory responses is the activation of coagulation resulting in a hypercoagulable state.
  • Common ARDS-associated coagulation disorders include intravascular coagulation and inhibition of fibrinolysis . Fibrin formed by the activation of the coagulation system and inhibition of fibrinolysis presumably contributes to the pathogenesis of acute lung injury. Sepsis, trauma and other critical diseases are important risk factors that lead to ARDS [Hasegawa, et al . , Chest 105 (1) : 268-277 , 1994].
  • ARDS is associated with an activation of coagulation and inhibition of fibrinolysis .
  • the present invention provides a method of reducing the duration of time a human patient with a hypercoagulable state and/or protein C deficiency remains in a hospital; remains in an intensive care unit; and/or requires ventilation therapy, which comprises administering to said patient activated Protein C (aPC) .
  • the invention further provides a method of reducing the duration a human patient with a hypercoagulable state and/or protein C deficiency remains in a hospital; an intensive care unit; and/or requires ventilation therapy, which comprises administering said patient activated Protein C (aPC) to achieve activated Protein C plasma levels in the range of 25 ng/ l to 100 ng/ml.
  • Another aspect of this invention provides methods for reducing the duration of time a human patient remains in a hospital; an intensive care unit; and/or requires ventilation therapy wherein the patient has a condition selected from one or more of: sepsis (including, severe sepsis and septic shock) , disseminated intravascular coagulation, purpura fulminans, major trauma, major surgery, burns, adult respiratory distress syndrome (ARDS) , melioidosis, preeclampsia, eclampsia, amniotic fluid embolism, placenta abruption, transplantations, deep vein thrombosis, heparin-induced thrombocytopenia, sickle cell disease, thalassemia, viral hemorrhagic fever, thrombotic thrombocytopenic purpura, he olytic uremic syndrome, acute coronary syndromes (ACS; e.g., unstable angina, myocardial infarction) and acquired and congenital protein C deficiency
  • an article of manufacture comprising packaging material and activated Protein C contained within said packaging material, wherein the packaging material comprises a label which indicates that activated Protein C can be used to decrease the duration of a patient's stay in a hospital; an intensive care unit; and/or requires ventilation therapy.
  • APC activated human protein C molecule and/or derivatives thereof, whether plasma derived or produced by recombinant or transgenic means.
  • Recombinant and transgenic activated Protein C may be produced by activating the human protein C zymogen in vi tro or by direct secretion or production of the activated form of protein C.
  • Protein C may be produced in cells, eukaryotic cells, transgenic animals, or transgenic plants, including, for example, secretion from human kidney 293 cells as a zymogen then purified and activated by techniques known to the skilled artisan.
  • Derivatives included within the scope of this invention include truncated light chain derivatives wherein the N-terminus comprises amino acids: 1-150, 1-151, or 1-152 of activated Protein C and/or having a truncated heavy chain wherein the C-terminus comprises amino acids 170-415.
  • Treating describes the management and care of a patient for the purpose of combating a disease, condition, or disorder. Treating may also include prophylaxis, preventing or prophylactic administration to prevent the onset of the symptoms or complications of the disease, condition, or disorder.
  • Intensive Care Unit or ICU There are different degrees of comprehensiveness of ICUs. Detailed recommendation for minimum requirements of staffing, quality of staff, quality and structure of the facility have been published. ICUs meeting minimum requirements are suitable for patients with one organ failure. Patients with multiple organ failures need to be cared for in ICUs with more comprehensive staffing and facilities. Specialty ICUs, such as ICU for burns and for children tend to have more intensive staffing and are more expensive.
  • Mechanical ventilation - includes controlled ventilation with or without positive end-expiratory pressure; controlled ventilation with muscle relaxation; intermittent mandatory or assisted ventilation; and/or continuous positive airway pressure. See Miranda et al . (1996) Crit. Care Med. 24:64-73; table 2, # 5.
  • Bolus injection the injection of a drug in a defined quantity (called a bolus) over a period of time, for example, for about 1-120 minutes.
  • Suitable for administration a formulation or solution that is appropriate to be given as a therapeutic agent.
  • Receptacle - a container such as a vial or bottle that is used to receive the designated material, i.e., aPC .
  • Unit dosage form - refers to physically discrete units suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
  • Hypercoagulable states - excessive coagulability associated with disseminated intravascular coagulation, pre- thrombotic conditions, activation of coagulation, or congenital or acquired deficiency of clotting factors such as Protein C .
  • Zymogen - Protein C zymogen refers to secreted, inactive forms, whether one chain or two chains, of protein C.
  • Effective amount - a therapeutically efficacious amount of a pharmaceutical compound or compounds, particularly aPC.
  • Sepsis - refers to a systemic response to infection or suspected infection complicated by one or more organ failures.
  • the term "sepsis” also includes severe sepsis (sepsis with evidence of one of more organ failure.
  • Organs can be cardiovascular, metabolic, mental status/central nervous system, hematologic/coagulation, renal, respiratory, hepatic) and septic shock (defined as hypotension or hypoperfusion to end organs) .
  • Protein C deficiency can be determined and defined by two primary means depending upon the availability of patient data. For a patient whose normal plasma protein C level is known, or if serial plasma protein C level is performed on a patient (for example, every 6-12 hours), acquired protein C deficiency can be defined as a 10% or greater decrease from either the patient's own known normal level or from a recent protein C level value that was within a normal range.
  • the acquired protein C deficiency is generally defined as below the lower limit of the normal range of protein C as established or used by the laboratory that performs the protein C assay.
  • the aPC used in the present invention may be made by techniques well known in the art utilizing eukaryotic cell lines, transgenic animals, or transgenic plants. Skilled artisans will readily understand that appropriate host eukaryotic cell lines include but are not limited to HEPG-2, LLC-MK , CHO-Kl, 293, or AV12 cells, examples of which are described by Grinnell in US Patent No. 5,681,932, herein incorporated by reference. Furthermore, examples of transgenic production of recombinant proteins are described by Drohan, et al., in U.S. Patent No. 5,589,604 and Archibald, et al . , U.S. Patent No. 5,650,503, herein incorporated by reference. U.S. Patent No.
  • the aPC made by any of these methods must undergo post-translational modifications such as the carboxylation of the side-chain of nine glutamate residues to gamma-carboxy-glutamates (gamma-carboxylation, i.e., Gla content), the hydroxylation of the side chain of one aspartate residue to erythro-beta-hydroxy-Asp (beta- hydroxylation) , the glycosylation of the side chain of four asparagine residues to Asn-linked oligosaccharides (glycosylation) , the removal of the leader sequence (42 amino acid residues) and removal of the dipeptide Lys 156-
  • gamma-carboxylation i.e., Gla content
  • the hydroxylation of the side chain of one aspartate residue to erythro-beta-hydroxy-Asp beta- hydroxylation
  • aPC is not fully functional or is non-functional .
  • Protein C levels can be determined in patient citrated plasma samples using appropriately approved diagnostic kits by appropriately certified laboratories and trained laboratory technicians. There are generally three types of diagnostic kits for measuring protein C levels from various commercial companies. One is to measure the antigenic level of protein C in plasma by an ELISA type methodology. The other two methods are to measure the protein C activity level. Protein C is first converted to activated Protein C, generally using a protease extracted from snake venom, and then the activity is measured either by its amidolytic activity (amidolytic activity kit) or by its anticoagulant activity (clotting activity kit) . Any of the three diagnostic kits can be used to determine protein C deficiency in patients. For acquired protein C deficiency or an acquired hypercoagulable state where liver dysfunction may be involved, the preferred or more clinically relevant method for determining the protein C level in a patient is the clotting activity diagnostic kit.
  • Protein C levels are usually measured in patient citrated plasma.
  • a patient's blood sample is usually collected into either a 2.8 ml (pediatric size) or 4.5 ml vacutainer containing either 3.2% or 3.8% citrate.
  • the blood sample can be obtained either via veni-puncture or via a central line. If heparin contamination cannot be avoided when collecting the blood sample, for example, via central line, then only the antigenic method can be used to measure accurately the protein C levels in that sample.
  • the citrated blood sample is centrifuged at about 2000xg for 10 to 20 minutes. The citrated plasma, which is the supernatant can be removed and used for the protein C level measurement .
  • the measurement of plasma protein C levels using any one of the three kinds of diagnostic kits can be carried out using manual, semi-automated or automated equipment. Appropriately certified laboratories and technicians usually have detailed standard operating procedures for performing the protein C assays. The standard operating procedures should include appropriate validation of the assays and the equipment used prior to assaying patient samples. In general, human plasma standard samples with known levels of protein C are used to calibrate and validate the assay and equipment. The intra- and inter-day variation of the assay results using these known standards should be less than 10% CV. Determination of Normal (100%) Level of Human Plasma
  • Protein C Normal (100%) of human plasma protein C level is defined as the amount of protein C in a pooled normal plasma sample.
  • This pooled normal human plasma sample can be the established WHO international standard (1 ml of pooled citrated plasma) . This can also be supplied as part of the commercially available protein C diagnostic kit. This is usually prepared by combining citrated plasma from 20 to more than a hundred normal human donors . The pooled plasma is then aliquoted and generally stored as a 1 ml lyophilized or frozen liquid in vials with specified expiration date.
  • human plasma protein C is generally determined by each laboratory as part of the validation for determining human plasma protein C level for the purpose of providing clinical diagnosis of the patient by the clinical staff.
  • the normal range will vary slightly from laboratory to laboratory depending upon the diagnostic kit/method and
  • the normal range is determined by measuring the concentration of protein C in a citrated plasma sample from about 30 - 120 normal healthy individual donors who are not on any medications that can affect their blood clotting chemistry.
  • the lower and upper limit of the normal range are determined by taking two standard deviations from the mean (if the range is of normal distribution) or the median (if the range is not of normal distribution) .
  • the lower limit of normal range for adult human is usually around 60-80% of pooled normal plasma.
  • the upper limit of normal range for adult human (> 18 years of age) is usually around 140 - 180%.
  • a normal new born usually has a plasma protein C level of about 30-40% of an normal adult.
  • the plasma protein C level in a normal child will reach to about the lower limit of a normal adult.
  • the normal range in children is different from that of adult and needs to be determined separately.
  • PCZ Human Protein C Zymogen Recombinant human PCZ was produced in Human Kidney 293 cells by techniques well known to the skilled artisan such as those set forth in Yan, U.S. Patent 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. Patent 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. Patent No. 4,992,373, the entire teaching of which is incorporated herein by reference.
  • plasmid pLPC 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. Patent 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) .
  • an anion exchange resin Frazier-Flow Q, Pharmacia
  • the eluted protein was greater than 95% pure after elution as judged by SDS-polyacrylamide gel electrophoresis.
  • 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 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/1 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.
  • 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 PCZ was made 5mM 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°C 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 PCZ and thrombin resin. The effluent was collected and immediately assayed for amidolytic activity.
  • 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 PCZ 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
  • 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 aPC was defined as the amount of enzyme required for the release of 1 ⁇ mol of p- nitroaniline in 1 min. at 25°C, pH 7.4, using an extinction coefficient for p-nitroaniline at 405 nm of 9620 M -:1 -cm-l.
  • the anticoagulant activity of aPC 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.
  • 50 ⁇ l of cold horse plasma and 50 ⁇ l of reconstituted activated partial thromboplastin time reagent APTT Reagent, Sigma
  • Example 1 Human Plasma Levels of aPC Six human patients received an intravenous infusion of aPC at 1 mg/m ⁇ /hr or about 0.024 mg/kg/hr over a 24 hour period.
  • the aPC administered was a lyophilized formulation containing 10 mg aPC, 5 mM Tris acetate buffer and 100 mM sodium chloride reconstituted with two ml of water and adjusted to pH 6.5. Plasma concentrations of aPC were measured using an
  • Immunocapture-Amidolytic Assay Blood was collected .in the presence of citrate anticoagulant and benzamidine, a reversible inhibitor of aPC .
  • the enzyme was captured from plasma by an aPC specific murine monoclonal antibody, C3 , immobilized on a microtiter plate. The inhibitor was removed by washing and the amidolytic activity of aPC was measured using an oligopeptide chromogenic substrate.
  • aPC concentrations were estimated from a standard curve ranging in concentrations from 0-100 ng/ml. The limit of quantitation of the assay was 1.0 ng/ml.
  • the aPC dose levels and plasma concentrations were measured at about 24 hours. The dose of 0.024 mg/kg/hr yields a plasma concentration of about 50 ng/ml at 24 hours.
  • rhAPC or placebo was administered as a continuous intravenous infusion over a fixed interval of 48 hours; in Stage 2, rhAPC or placebo was administered over a fixed interval of 96 hours.
  • the initial dose of rhAPC was 12 ⁇ g/kg/hr; subsequent increases to 18, 24, and 30 ⁇ g/kg/hr in Stage 1 and to 18 and 24 ⁇ g/kg/hr in Stage 2 were determined by a Data Monitoring Board, which reviewed safety, pharmacokinetic, and pharmacodynamic data.
  • Statistical Methods Approximately 18 patients in Stage 1 and 20 patients in Stage 2 were enrolled at each dose level (12, 18, 24, and 30 ⁇ g/kg/hr). After study completion, data for qualitative variables were presented as incidence rates (number and percent) , and data for continuous variables were summarized using measures of central tendency and dispersion. Variables were compared between the placebo group and the rhAPC dose and dose duration using appropriate methods. Statistical tests were performed using analysis of variance (ANOVA) based on rank and unranked data. Frequency analyses were compared using contingency table techniques and confidence intervals for relative risks and odds ratios were used to determine 28-day all-cause mortality rates. Two-sided 5% significance levels and 95% confidence intervals were used for all primary and secondary efficacy and safety analyses. All primary pharmacokinetics, outcomes, and safety analyses were performed on the intent-to-treat population, which were defined as the set of all enrolled patients who received an infusion of either rhAPC or placebo.
  • Results A total of 135 patients were randomized to study drug (rhAPC or placebo) . Four of these patients did not receive study drug, and of the 131 remaining patients, 41 received placebo and 90 received rhAPC. Patients were randomly assigned to rhAPC or placebo in a 2:1 ratio in Stage 1 and a 3:1 ratio in Stage 2. Data were summarized by combining treatment groups into high-dose rhAPC-treated (24 ⁇ g/kg/hr and 30 ⁇ g/kg/hr doses) , low-dose rhAPC-treated (12 ⁇ g/kg/hr and 18 ⁇ g/kg/hr doses) , all rhAPC-treated, and all placebo patients.
  • GI gastrointestinal
  • CAD coronary artery disease
  • an effective prophylactic treatment for sepsis may prevent the development of organ failures and thus may keep the treated patients out of an ICU, hospital and off of a ventilator.
  • the administration of aPC in order to practice the present methods of therapy is carried out by administering an effective amount of aPC, preferably rhAPC, to the patient in need thereof.
  • the effective amount, and the appropriate dosing regimen is determined, in _the final analysis, by the physician in charge of the case, but depends on factors such as the exact disease or diseases to be treated, the severity of the disease and other diseases or conditions from which the patient suffers, the specific route of administration, other drugs and treatments which the patient may concomitantly require, and other factors in the physician's judgment .
  • the aPC is administered by continuous infusion for up to about 144 hours at a dosage of about 1 ⁇ g/kg/hr to about 50 ⁇ g/kg/hr. More preferably, the amount of aPC administered will be about 4 ⁇ g/kg/hr to about 48 ⁇ g/kg/hr.
  • the amount of aPC administered will be: about 6 ⁇ g/kg/hr to about 44 ⁇ g/kg/hr; about 8 ⁇ g/kg/hr to about 40 ⁇ g/kg/hr; about 10 ⁇ g/kg/hr to about 36 ⁇ g/kg/hr; about 12 ⁇ g/kg/hr to about 34 ⁇ g/kg/hr; about 24 ⁇ g/kg/hr to about 30 ⁇ g/kg/hr; about 16 ⁇ g/kg/hr to about 24 ⁇ g/kg/hr; about 18 ⁇ g/kg/hr to about 20 ⁇ g/kg/hr; about 6 ⁇ g/kg/hr to about 22 ⁇ g/kg/hr; or about 10 ⁇ g/kg/hr to about 20 ⁇ g/kg/hr; or about 5 ⁇ g/kg/hr to about 25 ⁇ g/kg/hr; or about 5 ⁇ g/kg/hr to about 30 ⁇ g/kg
  • a bolus may be administered at various intervals before during or after discontinuation of the infusion.
  • the bolus is preferably in the range of about 25 to 100 ⁇ g/kg/hr (bolus followed by infusion) .
  • a physician may also dose the aPC to achieve preferred aPC plasma levels. Should the physician desire rapid aPC plasma levels, aPC will be administered in a bolus or in an increased amount .
  • Examples of preferred protein C plasma level ranges include: about 10 ng/ml to about 180 ng/ml; about 25 ng/ml to about 160 ng/ml; about 25 ng/ml to about 100 ng/ml; about 30 ng/ml to about 140 ng/ml; about 40 ng/ml to about 120 ng/ml; about 40ng/ml to about 100 ng/ml; and about 40 to about 80 ng/ml.
  • various boluses of aPC may be used at various intervals, as is preferred in the judgement of the physician.
  • Table I sets forth normalized dose levels of several studies in humans or non- human primates. The human studies were done utilizing plasma derived PCZ while the non-human primate study utilized recombinant human aPC.
  • ** 1 U is defined as the amount which doubles the activated prothrombin time (APTT) in normal human plasma. This converts to approximately 5 Units/ug APC.
  • aPC formulations are prepared by known procedures using well-known and readily available ingredients.
  • the aPC will be administered parenterally to ensure delivery into the bloodstream in an effective form.
  • aPC is formulated according to the disclosure herein.
  • Example 3 Formulation of APC
  • a stable lyophilized formulation of aPC is prepared by a process which comprises lyophilizing a solution comprising about 2.5 mg/mL aPC, about 15 mg/mL sucrose, about 20 mg/mL NaCl, and a sodium citrate buffer having a pH greater than 5.5 but less than 6.5. Additionally, the stable lyophilized formulation of aPC comprises lyophilizing a solution comprising about 5 mg/mL aPC, about 30 mg/mL sucrose, about 38 mg/mL NaCl, and a citrate buffer having a pH greater than 5.5 and, preferably, less than 6.5.
  • the ratio of aPC : salt :bulking agent (w:w:w) is believed to be an important factor in a formulation suitable for the freeze drying process.
  • the ratio varies depending on the concentration of aPC, salt selection and concentration and bulking agent selection and concentration. Particularly, a ratio of about 1 part aPC to about 7.6 parts salt to about 6 parts bulking agent is believed to be preferred.
  • a unit dosage formulation of aPC suitable for parenteral administration, preferably subcutaneous administration or continuous intravenous infusion is prepared by mixing aPC, NaCl, sucrose, and sodium citrate buffer. After mixing, 4 mL of the solution is transferred to a unit dosage receptacle and lyophilized.
  • the ratio of aPC to sucrose to sodium chloride is believed to be an important formulation variable affecting the collapse and glass- transition temperatures.
  • the sodium chloride concentration must be high enough (preferably 325 mM for 2.5 mg/mL aPC and 650 mM for 5 mg/mL aPC formulations) to cause the sodium chloride to crystallize-out during the freezing part of the freeze- drying process.
  • Formulations of aPC can be processed in a conventional freeze dryer to produce lyophilized products consisting of 1 part aPC, 6 parts sucrose, and 7.6 parts sodium chloride by weight. The formulated aPC is placed in a container. A product label and labeling accompanies the aPC.

Abstract

A method of reducing the duration a human patient with a hypercoagulable state or protein C deficiency remains in a hospital, an intensive care unit, and/or remains on mechanical ventilation, which comprises administering to said patient activated Protein C (aPC).

Description

Methods for using Activated Protein C to Reduce Duration in an Intensive Care Unit and Associated Expenses
FIELD OP THE INVENTION This invention relates to methods of decreasing the duration of time a human patient with a hypercoagulable state and/or protein C deficiency remains in an intensive care unit, a hospital and/or on mechanical ventilation which comprises administering to said patient activated Protein C (aPC) .
BACKGROUND OF THE INVENTION
It is estimated that there are more than 500,000 cases of sepsis each year in the U.S. alone and more than 1.2 million cases of sepsis each year in the U.S., Canada and major European countries (Sands et al . 1997. JAMA 278:234- 240) . Sepsis-related healthcare costs for North America and the major European countries are estimated to be around $34 billion annually. Many of these costs are incurred from prolonged stays in hospitals, particularly in intensive care units where septic patients often undergo mechanical ventilation to remain alive. The average septic patient spends 29 days in the hospital and 18 days in an intensive care unit. Even with the medical technology and attention provided in ICUs, the sepsis mortality rate remains around 30-50%. Expensive and time-consuming research efforts have been underway to improve mortality outcome for septic patients. Fortunately, recombinant human activated protein C (rhAPC) has recently been shown to improve mortality outcome in patients with severe sepsis. Health economists may be led to predict that a drug capable of improving mortality outcome in septic patients may actually increase the overall healthcare costs. The logic that reducing mortality is more expensive than patient death comes from the fact that dead patients do not continue to incur expensive healthcare costs (hospital and ICU days, mechanical ventilation, etc.).
Surprisingly, recent evidence has demonstrated that when patients with severe sepsis were treated with rhAPC, these patients actually spent less time in a hospital, less time in an ICU, and less time undergoing mechanical ventilation. The average cost per ICU day, general ward hospital day and mechanical ventilation day is about $1830, $900 and $470 respectively. Therefore, treatment with rhAPC may provide an enormous reduction in healthcare costs ($8.3 billion annually, based on 2.7 less days of ICU stay, 3.1 less days of hospital days and 2.3 less days of mechanical ventilation for each patient and assuming 80% of the 1.2 million patients survived after treating with rhAPC) . One skilled in the art would expect similar cost savings when rhAPC is administered to treat other hypercoagulable states or protein C deficient states.
Protein C is a serine protease and naturally occurring anti-coagulant produced as an inactive precursor or zymogen by the liver. Human protein C is made in vivo as a single polypeptide of 461 amino acids. This polypeptide undergoes multiple post-translational modifications including: 1) cleavage of a 42 amino acid signal sequence; 2) cleavage of lysine and arginine residues (positions 156 and 157) to make a 2-chain inactive zymogen (a 155 amino acid residue light chain attached via a disulfide bridge to a 262 amino acid residue heavy chain); 3) vitamin K-dependent carboxylation of nine glutamic acid residues located within the amino- terminal 45 residues (gla-domain) ; and, 4) carbohydrate attachment at four sites (one in the light chain and three in the heavy chain) . The protein C zymogen circulates in the plasma and, upon removal of a dodecapeptide at the N- terminus of the heavy chain, results in aPC possessing enzymatic activity. aPC plays a key role in regulating hemostasis by inactivating Factors Va and Villa in the coagulation cascade.
Blood coagulation is a highly complex process regulated by the balance between pro-coagulant, anti-coagulant, and fibrinolytic mechanisms. This balance determines a condition of either normal hemostasis or abnormal pathological thrombus generation and the progression of hypercoagulable states. Two major factors control this balance; the generation of fibrin and the activation and subsequent aggregation of platelets, both processes controlled by the generation of the enzyme thrombin, which occurs following activation of the clotting cascade. Thrombin, in complex with thrombomodulin, also functions as a potent anti-coagulant since it activates protein C zymogen to the active enzyme, aPC . In large blood vessels, the activation of protein C zymogen to aPC by thrombin- thrombomodulin complex is further augmented by an endothelial transmembrane protein, endothelial-protein receptor (EPCR) [Stearn-Kurosawa, et al . Proc. Natl. Acad. Sci. USA 93:10212-10216, 1996]. APC, in turn inhibits the generation of thrombin. Thus, through the feedback regulation of thrombin generation via the inactivation of Factors Va and Villa, aPC functions as perhaps the most important down-regulator of blood coagulation resulting in protection against thrombosis. Additionally, aPC exerts profibrinolytic properties that facilitate clot lysis and exerts anti-inflammatory effects via inhibiting the release of inflammatory mediators, such as, tumor necrosis factor and various interleukins . A Phase III, clinical end point study of rhAPC vs. placebo in patients with severe sepsis demonstrated evidence of significant active treatment benefit.
The present invention is the first to describe administering aPC to decrease the duration a human patient with a hypercoagulable state and/or a protein C deficient state remains in a hospital, an intensive care unit and/or undergoing ventilation therapy.
Examples of certain hypercoagulable states contemplated within the scope of this invention are described below.
SEPSIS
Sepsis is defined clinically as a systemic response to infection or suspected infection complicated by one or more organ failures. Sepsis is associated with and mediated by the activation of a number of host defense mechanisms including the cytokine network, leukocytes, and the complement and coagulation/fibrinolysis systems. [Mesters, et al . , Blood 88:881-886, 1996]. Disseminated intravascular coagulation [DIC] , with widespread deposition of fibrin in the microvasculature of various organs, is an early manifestation of sepsis/septic shock. DIC is an important mediator in the development of the multiple organ failure syndrome and contributes to the poor prognosis of patients with severe sepsis. [Fourrier, et al . , Chest 101:816-823, 1992] . Purpura fulminans (ecchymotic skin lesions, fever, hypotension associated with bacterial sepsis, viral, bacterial or protozoan infections) and/or DIC have been associated with numerous bacterial, viral, or protozoan infections which include but are not limited to infections caused by Rickettsia (Rocky Mountain Spotted fever, tick bite fever, typhus, etc.) [Graybill, et al . , Southern Medical Journal, 66 (4) : 410-413 , 1973; Loubser, et al . , Annals of Tropical Pediatrics 13:277-280, 1993]; Salmonella (typhoid fever, rat bite fever) [Koul, et al . , Acta Haematol , 93:13-19,, 1995]; Pneumococci [Carpenter, et al . , Scand J Infect Pis, 29:479-483, 1997] Yersina pestis (Bubonic plague) [Butler, et al . , The Journal of Infectious Disease, 129:578-584, 1974]; Legionella pneumophila (Legionnaires Disease) ,- Plasmodium falciparum (cerebral malaria) [Lercari, et al . , Journal of Clinical Apheresis, 7:93-96, 1992]; Burkholderia pseudomallei (Melioidosis); Pseudomonas pseudomallei (Melioidosis) [Puthucheary, et al . , Transactions of the Royal Society of Tropical Medicine and Hygiene, 86:683-685, 1992]; Streptococci (Odontogenic infections) [Ota, Y. , J. Japanese Assoc. Infect. Pis.,
68:157-161]; zoster virus [Nguyen, et al . , Eur J Pediatr, 153:646-649, 1994]; Bacillus anthracis (Anthrax) [Franz, et al . , Journal of the American Medical Assoc . , 278 (5) : 399-411, 1997]; Leptospira interrogans (leptospirosis) [Hill, et al . , Seminars in Respiratory Infections, 12(l):44-49, 1997];
Staphylococci [Levin, M. , Pediatric Nephrology, 8:223-229]; Haemophilus aegyptius (Brazilian purpuric fever) ; Neisseria (gonococcemia, meningococcemia) ; and mycojacter-ium tuberculosis (miliary tuberculosis) .
TRANSPLANTATION A variety of transplantation associated thromboembolic complications may occur following bone marrow transplantation (BMT) , liver, kidney, or other organ transplantations [Haire, et al . , JAMA 274:1289-1295, (1995); Harper, et al . , Lancet 924-927 (1988); and Sorensen, et al . , J. Inter. Med 226:101-105 (1989); Gordon, et al . , Bone
Marrow Transplan. 11:61-65, (1993)]. Pecreased levels of circulating protein C have been reported after BMT [Bazarbachi, et al . , Nouv Rev Fr Hematol 35:135-140 (1993); Gordon, et al . , Bone Marrow Trans . 11:61-65 (1993)], renal transplantation [Sorensen, et al . , J. Inter. Med 226:101-105 (1989)], and liver transplantation [Harper, et al . , Lancet 924-927(1988)]. This deficiency in protein C contributes to a hypercoagulable state placing patients at risk for thromboembolic complications. For example, hepatic venocclusive disease (VOP) of the liver is the major dose-limiting complication of pre- transplantation regimens for BMT. VOD is presumably the result of small intrahepatic venule obliteration due to intravascular deposition of fibrin. [Faioni, et al . , Blood 81:3458-3462 (1993)]. In addition, VOD causes considerable morbidity and mortality following BMT [Collins, et al . , Throm. and Haemo. 72:28-33 (1994)]. A decreased level of protein C coincident with the peak incidence of VOD has been reported [Harper, et al . , Bone Marrow Trans . 5:39-42 (1990)] and is likely to be a contributing factor to the genesis of this condition. Organ dysfunction after BMT including pulmonary, central nervous system, hepatic or renal, is a complication that occurs in a high percentage of transplant patients [Haire, et al . , JAMA 274:1289-1295, (1995)]. A single organ dysfunction in BMT is a strong predictor of multiple organ dysfunction syndrome (MODS) which is the leading cause of death in BMT patients. Disseminated intravascular coagulation (DIC) due to a massive activation of the coagulation system and widespread deposition of fibrin in the microvasculature of various organs is an important mediator in the development of MODS [Fourrier, et al . , Chest 101:816-823 (1992)]. Thus, a deficiency in protein C levels in patients who have undergone bone marrow or other organ transplantations leads to a hypercoagulable state that predisposes the patients to venous thromboembolic complications and organ dysfunction.
BURNS
It has long been recognized that severely burned patients have complications associated with hypercoagulation [Curreri, et al . , Ann. Surg. 181:161-163 (1974)]. Burned patients have supranormal in vi tro clotting activity and frequently develop DIC which is characterized by the sudden onset of diffuse hemorrhage; the consumption of fibrinogen, platelets, and Factor VIII activity; intravascular hemolysis; secondary fibrinolysis; and biopsy evidence of microthrombi [McManis, et al . , J. of Trauma 13:416-422, (1973)]. Recently, it was reported that the levels of protein C were reduced drastically in severely burned patients and that this reduction of the natural anticoagulant may lead to an increase in the risk of DIC [Lo, et al . , Burns 20:186-187 (1994)]. In addition, Ueyama, et al . , in discussing the pathogenesis of DIC in the early stage of burn injury, concluded that massive thrombin generation and decrease of anticoagulant activity may occur in proportion to the severity of burns [Ueyama, et al . , Nippon Geka Gakkai Zasshi 92:907-12 (1991)]. DIC is one of the common complications in patients suffering from severe burn injuries.
PREGNANCY Pregnancy causes multiple changes in the coagulation system which may lead to a hypercoagulable state . For example, during pregnancy and postpartu , the risk of venous thrombosis is almost fivefold higher than in the non- pregnant state. In addition, clotting factors increase, natural inhibitors of coagulation decrease, changes occur in the fibrinolytic system, venous stasis increases, as well as increases in vascular injury at delivery from placental separation, cesarean section, or infection [Barbour, et al . , Obstet Gynecol 86:621-633, 1995]. Although the risk of a complication due to this hypercoagulable state in women without any risk factors is small, women with a history of thromboembolic events are at an increased risk for recurrence when they become pregnant. In addition, women with underlying hypercoagulable states, including the recent discovery of hereditary resistance to aPC, also have a higher recurrence risk [Dahlback, Blood 85:607-614, 1995] .
Therefore, it has been suggested" that women with a history of venous thromboembolic events who are found to have a deficiency in antithrombin-III, protein C, or protein S, are at an appreciable risk of recurrent thrombosis and should be considered for prophylactic anti-coagulant therapy [Conrad, et al . , Throm Haemost 63:319-320, 1990]. The conditions of preeclampsia and eclampsia and other obstetrical complications such as amniotic fluid embolism and placenta abruption in pregnant women appear to be- a state of increased coagulopathy and disseminated intravascular coagulation as indicated by an increase in fibrin formation, activation of the fibrinolytic system, platelet activation and a decrease in platelet count [Clin Obstet Gynecol 35:338-350, 1992]. Preeclampsia is thought to be the result of uteroplacental ischemia due to an anomaly of the "vascular insertion" of the placenta. Consequences of preeclampsia include hypertension as well as DIC which leads to the release of numerous microthrombi which cause placental, renal, hepatic and cerebral lesions [Rev Fr Gynecol Obstet 86:158-163, 1991]. Furthermore, preeclampsia can lead to a severe and life threatening condition known as the HELLP syndrome which is defined as preeclampsia complicated by thrombocytopenia, hemolysis and disturbed liver function [Rathgeber, et al . , Anasth
Intensivther Notfallmed 25:206-211, 1990]. Additionally, it has been documented that there is a reduction in protein C levels in pregnant women with severe preeclampsia when compared to normal pregnancies [De Stefano, et al . , Thromb Haemost 74:793-794, 1995].
Thus, the risk of venous thromboembolic complications occurring in pregnant women is a major concern, especially in women who have a" history of thromboembolic events. Although the possibility of severe complications such as preeclampsia or DIC is relatively low, it has been suggested that it is essential to start therapy of DIC as soon as it has been diagnosed by onset of inhibition of the activated coagulation system [Rathgeber, et al . , Anasth Intensivther Notfallmed 25:206-211, 1990]. The complications of preeclampsia or DIC is analogous to the situation that occurs in sepsis in that there is a hypercoagulable state and a decrease in the levels of protein C (Levi, et al . , N. Engl . J. Med. 341:586-592, 1999) .
MAJOR SURGERY/TRAUMA Patients recovering from major surgery or accident trauma frequently encounter blood coagulation complications as a result of an induced hypercoagulable state [Watkins, et al . , Klin Wochenschr 63:1019-1027, 1985]. Hypercoagulable states are increasingly recognized as causes of venous thromboembolism in surgical patients [Thomas, et al . , Am J Surg. 158:491-494, 1989; LeClerc, J.R., Clin Appl Thrombosis/Hemostasis 3 (3) : 153-156, 1997]. Furthermore, this hypercoagulable state can lead to complications with DIC-like symptoms, which is infrequently encountered but, nonetheless, is devastating and often fatal when it occurs. [Collins, et al . , Am J Surg. 124:375-380, 1977].
In addition, patients undergoing coronary artery bypass grafting (CABG) [Menges, et al . , J Cardiothor Vase An. 10:482-489, 1996], major spinal surgery [Mayer, et al . , Clin Orthop. 245:83-89, 1989], major abdominal surgery
[Blarney, et al . , Thromb Haemost . 54:622-625, 1985], major orthopedic surgery or arthroplastic surgery of the lower extremities [LeClerc, 1997] , or other types of surgery [Thomas, et al . , Am J Surg. 158:491-494, 1989], occasionally develop venous thromboembolic complications. Additionally, investigators in Japan have proposed treating microvascular thrombosis associated with spinal cord injury [patent application JP8325161A] with plasma derived PCZ at a dose of 1-10 mg/day for an adult, or preferably, 2-6 mg divided by 1-2 times to be administered as a bolus or by intravenous infusion. It has been suggested that anti-coagulant therapy is important as a prophylactic therapy to prevent venous thromboembolic events in major surgery or trauma patients [Thomas, et al . , 1989; LeClerc, 1997]. For example, many patients who succumb from pulmonary embolism have no clinical evidence of preceding thromboembolic events and die before the diagnosis is made and the treatment is instituted [LeClerc, 1997]. Existing prophylactic methods e.g., warfarin, low molecular weight heparins, have limitations such as residual proximal thrombosis or the need for frequent dose adjustments.
ARDS
Adult respiratory distress syndrome [ARDS] is characterized by lung edema, microthro bi, inflammatory cell infiltration, and late fibrosis . Pivotal to these multiple cellular and inflammatory responses is the activation of coagulation resulting in a hypercoagulable state. Common ARDS-associated coagulation disorders include intravascular coagulation and inhibition of fibrinolysis . Fibrin formed by the activation of the coagulation system and inhibition of fibrinolysis presumably contributes to the pathogenesis of acute lung injury. Sepsis, trauma and other critical diseases are important risk factors that lead to ARDS [Hasegawa, et al . , Chest 105 (1) : 268-277 , 1994]. ARDS is associated with an activation of coagulation and inhibition of fibrinolysis . Considerable clinical evidence exists for the presence of pulmonary vascular microemboli which is analogous to the hypercoagulation that is present in DIC. Therefore, a need currently exists for an effective treatment of this hypercoagulable state associated with ARDS.
SUMMARY OF THE INVENTION The present invention provides a method of reducing the duration of time a human patient with a hypercoagulable state and/or protein C deficiency remains in a hospital; remains in an intensive care unit; and/or requires ventilation therapy, which comprises administering to said patient activated Protein C (aPC) . The invention further provides a method of reducing the duration a human patient with a hypercoagulable state and/or protein C deficiency remains in a hospital; an intensive care unit; and/or requires ventilation therapy, which comprises administering said patient activated Protein C (aPC) to achieve activated Protein C plasma levels in the range of 25 ng/ l to 100 ng/ml. Another aspect of this invention provides methods for reducing the duration of time a human patient remains in a hospital; an intensive care unit; and/or requires ventilation therapy wherein the patient has a condition selected from one or more of: sepsis (including, severe sepsis and septic shock) , disseminated intravascular coagulation, purpura fulminans, major trauma, major surgery, burns, adult respiratory distress syndrome (ARDS) , melioidosis, preeclampsia, eclampsia, amniotic fluid embolism, placenta abruption, transplantations, deep vein thrombosis, heparin-induced thrombocytopenia, sickle cell disease, thalassemia, viral hemorrhagic fever, thrombotic thrombocytopenic purpura, he olytic uremic syndrome, acute coronary syndromes (ACS; e.g., unstable angina, myocardial infarction) and acquired and congenital protein C deficiency, which comprises administering aPC .
Also included is an article of manufacture, comprising packaging material and activated Protein C contained within said packaging material, wherein the packaging material comprises a label which indicates that activated Protein C can be used to decrease the duration of a patient's stay in a hospital; an intensive care unit; and/or requires ventilation therapy.
DETAILED DISCRIPTION OF THE INVENTION
For purposes of the present invention, as disclosed and claimed herein, the following terms are as defined below. "APC," "aPC," or "activated Protein C" refer to the activated human protein C molecule and/or derivatives thereof, whether plasma derived or produced by recombinant or transgenic means. Recombinant and transgenic activated Protein C may be produced by activating the human protein C zymogen in vi tro or by direct secretion or production of the activated form of protein C. Protein C may be produced in cells, eukaryotic cells, transgenic animals, or transgenic plants, including, for example, secretion from human kidney 293 cells as a zymogen then purified and activated by techniques known to the skilled artisan. Derivatives included within the scope of this invention include truncated light chain derivatives wherein the N-terminus comprises amino acids: 1-150, 1-151, or 1-152 of activated Protein C and/or having a truncated heavy chain wherein the C-terminus comprises amino acids 170-415.
Treating - describes the management and care of a patient for the purpose of combating a disease, condition, or disorder. Treating may also include prophylaxis, preventing or prophylactic administration to prevent the onset of the symptoms or complications of the disease, condition, or disorder. Intensive Care Unit or ICU - There are different degrees of comprehensiveness of ICUs. Detailed recommendation for minimum requirements of staffing, quality of staff, quality and structure of the facility have been published. ICUs meeting minimum requirements are suitable for patients with one organ failure. Patients with multiple organ failures need to be cared for in ICUs with more comprehensive staffing and facilities. Specialty ICUs, such as ICU for burns and for children tend to have more intensive staffing and are more expensive. The requirements for ICUs are published (Ferdinande et al . (1997) Intens . Care Med. 23:226-232; American College Critical Care Medicine of Society of Critical Care Medicine, (1999) Crit. Care Med. 27:422-426.). All such facilities are included within the scope of the invention. Mechanical ventilation - includes controlled ventilation with or without positive end-expiratory pressure; controlled ventilation with muscle relaxation; intermittent mandatory or assisted ventilation; and/or continuous positive airway pressure. See Miranda et al . (1996) Crit. Care Med. 24:64-73; table 2, # 5.
Continuous infusion - continuing substantially uninterrupted the introduction of a solution into a vein 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, for example, for about 1-120 minutes. Suitable for administration - a formulation or solution that is appropriate to be given as a therapeutic agent.
Receptacle - a container such as a vial or bottle that is used to receive the designated material, i.e., aPC . Unit dosage form - refers to physically discrete units suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Hypercoagulable states - excessive coagulability associated with disseminated intravascular coagulation, pre- thrombotic conditions, activation of coagulation, or congenital or acquired deficiency of clotting factors such as Protein C . Zymogen - Protein C zymogen, as used herein, refers to secreted, inactive forms, whether one chain or two chains, of protein C.
Effective amount - a therapeutically efficacious amount of a pharmaceutical compound or compounds, particularly aPC. Purpura fulminans - ecchymotic skin lesions, fever, hypotension associated with infection (bacterial sepsis, viral, bacterial or protozoan infections) or non-infectious causes. Disseminated intravascular coagulation is usually present . Sepsis - refers to a systemic response to infection or suspected infection complicated by one or more organ failures. For purposes of this application, the term "sepsis" also includes severe sepsis (sepsis with evidence of one of more organ failure. Organs can be cardiovascular, metabolic, mental status/central nervous system, hematologic/coagulation, renal, respiratory, hepatic) and septic shock (defined as hypotension or hypoperfusion to end organs) .
Protein C deficiency can be determined and defined by two primary means depending upon the availability of patient data. For a patient whose normal plasma protein C level is known, or if serial plasma protein C level is performed on a patient (for example, every 6-12 hours), acquired protein C deficiency can be defined as a 10% or greater decrease from either the patient's own known normal level or from a recent protein C level value that was within a normal range. When a patient's normal plasma protein C level is not known or cannot be obtained (for example, a patient upon admission to the hospital is already presenting with clinical symptoms of severe sepsis or laboratory tests indicate a hypercoagulable state) , then the acquired protein C deficiency is generally defined as below the lower limit of the normal range of protein C as established or used by the laboratory that performs the protein C assay.
The aPC used in the present invention may be made by techniques well known in the art utilizing eukaryotic cell lines, transgenic animals, or transgenic plants. Skilled artisans will readily understand that appropriate host eukaryotic cell lines include but are not limited to HEPG-2, LLC-MK , CHO-Kl, 293, or AV12 cells, examples of which are described by Grinnell in US Patent No. 5,681,932, herein incorporated by reference. Furthermore, examples of transgenic production of recombinant proteins are described by Drohan, et al., in U.S. Patent No. 5,589,604 and Archibald, et al . , U.S. Patent No. 5,650,503, herein incorporated by reference. U.S. Patent No. 5,009,889 (incorporated by reference herein) describes various procedures for isolating protein C from plasma. For additional examples of methods to prepare aPC, and formulations containing the same, See: U.S. Patent 5,580,962 (columns 3 and 4); U.S. Patent 5,831,025; European Patent Application 0662513A1; U.S. Patent 5,093,117; and U.S. Patent 5,084,273.
To be fully active, the aPC made by any of these methods must undergo post-translational modifications such as the carboxylation of the side-chain of nine glutamate residues to gamma-carboxy-glutamates (gamma-carboxylation, i.e., Gla content), the hydroxylation of the side chain of one aspartate residue to erythro-beta-hydroxy-Asp (beta- hydroxylation) , the glycosylation of the side chain of four asparagine residues to Asn-linked oligosaccharides (glycosylation) , the removal of the leader sequence (42 amino acid residues) and removal of the dipeptide Lys 156-
Arg 157. Without such post-translational modifications, aPC is not fully functional or is non-functional .
The following Methods, Preparations and Examples are illustrative and are not intended to limit the invention in any way.
Method for Determining Protein C Levels in Patient
Plasma Samples Protein C levels can be determined in patient citrated plasma samples using appropriately approved diagnostic kits by appropriately certified laboratories and trained laboratory technicians. There are generally three types of diagnostic kits for measuring protein C levels from various commercial companies. One is to measure the antigenic level of protein C in plasma by an ELISA type methodology. The other two methods are to measure the protein C activity level. Protein C is first converted to activated Protein C, generally using a protease extracted from snake venom, and then the activity is measured either by its amidolytic activity (amidolytic activity kit) or by its anticoagulant activity (clotting activity kit) . Any of the three diagnostic kits can be used to determine protein C deficiency in patients. For acquired protein C deficiency or an acquired hypercoagulable state where liver dysfunction may be involved, the preferred or more clinically relevant method for determining the protein C level in a patient is the clotting activity diagnostic kit.
Protein C levels are usually measured in patient citrated plasma. A patient's blood sample is usually collected into either a 2.8 ml (pediatric size) or 4.5 ml vacutainer containing either 3.2% or 3.8% citrate. The blood sample can be obtained either via veni-puncture or via a central line. If heparin contamination cannot be avoided when collecting the blood sample, for example, via central line, then only the antigenic method can be used to measure accurately the protein C levels in that sample. The citrated blood sample is centrifuged at about 2000xg for 10 to 20 minutes. The citrated plasma, which is the supernatant can be removed and used for the protein C level measurement .
The measurement of plasma protein C levels using any one of the three kinds of diagnostic kits can be carried out using manual, semi-automated or automated equipment. Appropriately certified laboratories and technicians usually have detailed standard operating procedures for performing the protein C assays. The standard operating procedures should include appropriate validation of the assays and the equipment used prior to assaying patient samples. In general, human plasma standard samples with known levels of protein C are used to calibrate and validate the assay and equipment. The intra- and inter-day variation of the assay results using these known standards should be less than 10% CV. Determination of Normal (100%) Level of Human Plasma
Protein C Normal (100%) of human plasma protein C level is defined as the amount of protein C in a pooled normal plasma sample. This pooled normal human plasma sample can be the established WHO international standard (1 ml of pooled citrated plasma) . This can also be supplied as part of the commercially available protein C diagnostic kit. This is usually prepared by combining citrated plasma from 20 to more than a hundred normal human donors . The pooled plasma is then aliquoted and generally stored as a 1 ml lyophilized or frozen liquid in vials with specified expiration date.
Determination of Normal Range of Human Plasma Protein C
The normal range of human plasma protein C is generally determined by each laboratory as part of the validation for determining human plasma protein C level for the purpose of providing clinical diagnosis of the patient by the clinical staff. The normal range will vary slightly from laboratory to laboratory depending upon the diagnostic kit/method and
> the equipment used to perform the protein C assay. A concentration standard curve is determined with the standards provided and the procedure accompanied by the diagnostic kit and the equipment. The normal range is determined by measuring the concentration of protein C in a citrated plasma sample from about 30 - 120 normal healthy individual donors who are not on any medications that can affect their blood clotting chemistry. The lower and upper limit of the normal range are determined by taking two standard deviations from the mean (if the range is of normal distribution) or the median (if the range is not of normal distribution) . The lower limit of normal range for adult human (> 18 years of age) is usually around 60-80% of pooled normal plasma. The upper limit of normal range for adult human (> 18 years of age) is usually around 140 - 180%. A normal new born usually has a plasma protein C level of about 30-40% of an normal adult. By about 1 year of age, the plasma protein C level in a normal child will reach to about the lower limit of a normal adult. Thus the normal range in children is different from that of adult and needs to be determined separately.
Preparation 1
Preparation of Human Protein C Zymogen (PCZ) Recombinant human PCZ was produced in Human Kidney 293 cells by techniques well known to the skilled artisan such as those set forth in Yan, U.S. Patent 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. Patent 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. Patent 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. Patent 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 PCZ 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 650 M, 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 400 mM NaCl, 20 mM Tris-acetate, pH 6.5. Protein concentrations of recombinant human PCZ and recombinant aPC solutions were measured by UV 280 nm extinction E® •1^=1.85 or 1.95, respectively.
Preparation 2 Activation of Recombinant Human PCZ Bovine thrombin was coupled to Activated CH-Sepharose 4B (Pharmacia) in the presence of 50 mM HEPES, pH 7.5 at
4°C. 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/1 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 PCZ was made 5mM 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°C 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 PCZ 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 PCZ 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 400 mM 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°C) 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 aPC was 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 extinction coefficient for p-nitroaniline at 405 nm of 9620 M-:1-cm-l. The anticoagulant activity of aPC 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 μl of cold horse plasma and 50 μl of reconstituted activated partial thromboplastin time reagent (APTT Reagent, Sigma) were added and incubated at 37°C for 5 min. After incubation, 50 μl 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 μl, 37°C, and 30 mM CaCl2 to each sample or standard. aPC concentration in samples is 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.
The above descriptions enable one with appropriate skill in the art to prepare aPC for use in the methods described herein.
Example 1 Human Plasma Levels of aPC Six human patients received an intravenous infusion of aPC at 1 mg/m^/hr or about 0.024 mg/kg/hr over a 24 hour period. The aPC administered was a lyophilized formulation containing 10 mg aPC, 5 mM Tris acetate buffer and 100 mM sodium chloride reconstituted with two ml of water and adjusted to pH 6.5. Plasma concentrations of aPC were measured using an
Immunocapture-Amidolytic Assay. Blood was collected .in the presence of citrate anticoagulant and benzamidine, a reversible inhibitor of aPC . The enzyme was captured from plasma by an aPC specific murine monoclonal antibody, C3 , immobilized on a microtiter plate. The inhibitor was removed by washing and the amidolytic activity of aPC was measured using an oligopeptide chromogenic substrate.
Following incubation for 16-20 hours at 37°C, the absorbance was measured at 405 nm and data are analyzed by a weighted linear curve-fitting algorithm. aPC concentrations were estimated from a standard curve ranging in concentrations from 0-100 ng/ml. The limit of quantitation of the assay was 1.0 ng/ml. The aPC dose levels and plasma concentrations were measured at about 24 hours. The dose of 0.024 mg/kg/hr yields a plasma concentration of about 50 ng/ml at 24 hours.
Example 2
Double-blinded Placebo-controlled
Trial in Human Patients With Sepsis, Stage 1
Study Design: A double-blind, randomized, placebo- controlled study was conducted in 40 medical centers. In Stage 1, rhAPC or placebo was administered as a continuous intravenous infusion over a fixed interval of 48 hours; in Stage 2, rhAPC or placebo was administered over a fixed interval of 96 hours. In both stages, the initial dose of rhAPC was 12 μg/kg/hr; subsequent increases to 18, 24, and 30 μg/kg/hr in Stage 1 and to 18 and 24 μg/kg/hr in Stage 2 were determined by a Data Monitoring Board, which reviewed safety, pharmacokinetic, and pharmacodynamic data. This study assessed, inter alia, the effect of rhAPC on reducing the duration of time a patient: 1-remains in an intensive care unit; 2-remains in a hospital; 3-remains free of shock; 4-requires mechanical ventilation and 5-manifests the symptoms of SIRS (systemic inflammatory response syndrome) .
Statistical Methods: Approximately 18 patients in Stage 1 and 20 patients in Stage 2 were enrolled at each dose level (12, 18, 24, and 30 μg/kg/hr). After study completion, data for qualitative variables were presented as incidence rates (number and percent) , and data for continuous variables were summarized using measures of central tendency and dispersion. Variables were compared between the placebo group and the rhAPC dose and dose duration using appropriate methods. Statistical tests were performed using analysis of variance (ANOVA) based on rank and unranked data. Frequency analyses were compared using contingency table techniques and confidence intervals for relative risks and odds ratios were used to determine 28-day all-cause mortality rates. Two-sided 5% significance levels and 95% confidence intervals were used for all primary and secondary efficacy and safety analyses. All primary pharmacokinetics, outcomes, and safety analyses were performed on the intent-to-treat population, which were defined as the set of all enrolled patients who received an infusion of either rhAPC or placebo.
Results: A total of 135 patients were randomized to study drug (rhAPC or placebo) . Four of these patients did not receive study drug, and of the 131 remaining patients, 41 received placebo and 90 received rhAPC. Patients were randomly assigned to rhAPC or placebo in a 2:1 ratio in Stage 1 and a 3:1 ratio in Stage 2. Data were summarized by combining treatment groups into high-dose rhAPC-treated (24 μg/kg/hr and 30 μg/kg/hr doses) , low-dose rhAPC-treated (12 μg/kg/hr and 18 μg/kg/hr doses) , all rhAPC-treated, and all placebo patients. In general, randomization resulted in well-balanced treatment groups with respect to demographic and other baseline parameters (Table A) . Baseline protein C functional activity data were available for 125 (95.4%) of the 131 patients in the study. Of these 125 patients, almost all (96.8%) were acquired protein C deficient at baseline (deficiency defined as protein C functional activity <80%) . More than half (50.4%) had protein C functional activity <40% at baseline.
Compared with placebo-treated patients, rhAPC-treated patients overall experienced on average 1.2 more ICU-free days (p = 0.54), 1.4 more shock-free days (p = 0.54), 2.0 more SIRS-free days (p = 0.33), 0.5 more invasive mechanical ventilation free days (p = 0.84), and 1.5 more hospital-free days (p = 0.38). High-dose rhAPC patients experienced on average 2.7 more ICU-free days (p = 0.26), 4.1 more shock- free days (p = 0.14), 4.8 more SIRS-free days (p = 0.049), 2.3 more invasive mechanical ventilation free days (p = 0.38), and 3.1 more hospital-free days (p = 0.11) (Table B) . No identifiable safety issues were noted through central laboratory parameters, vital signs, or trends in adverse events .
Table A. Baseline characteristics
Figure imgf000028_0001
Figure imgf000029_0001
GI = gastrointestinal; CAD = coronary artery disease
* Resolved before study entry; overall incidence >6%
* Overall incidence >10%
Table B. Difference in mean failure- and other-free days for high-dose rhAPC vs placebo
Figure imgf000029_0002
Figure imgf000030_0001
Table C. Difference in mean failure- and other-free days for high-dose rhAPC vs placebo in treated patients who survived 28 days.
Figure imgf000030_0002
In general, for a drug used to prevent the development of a hypercoaguable state, e.g., sepsis or severe sepsis, one would expect the treated patients to require less hospital resources. For example, an effective prophylactic treatment for sepsis may prevent the development of organ failures and thus may keep the treated patients out of an ICU, hospital and off of a ventilator.
When extending the lives of ill patients, however, health professionals and health economists, would be lead to predict that the use of healthcare resources would increase. The logic is that aPC treatment may allow the very severe septic patient to survive and these patients will require extensive intensive care and life-supporting systems. In terms of hospital resource utility, it may actually be cheaper to allow the patients to die. It is surprising that there is no increase in hospital, ICU and ventilation resource utilization.
In Table B and C, the utilization of hospital resources was calculated as "free days", days in which the patient was alive and not in hospital, alive and not in ICU, alive and not in shock, alive and not having SIRS, alive and not on mechanical ventilation for 28 days after initiating rhAPC or placebo treatment. A patient who died during the 28 day period may have the lowest free day score because the analysis was based on no "free days" after death. In table B, the data was calculated including all patients in the study. To analyze the data to remove any contribution of rhAPC ' s effect on improvement in mortality, table C only includes patients who survived the 28 day period. Data in table C unexpectedly suggest that rhAPC treatment for sepsis has a positive healthcare impact. At a minimum, there is no increased resource utilization and, the data further support a trend of using less healthcare resources . The administration of aPC in order to practice the present methods of therapy is carried out by administering an effective amount of aPC, preferably rhAPC, to the patient in need thereof. The effective amount, and the appropriate dosing regimen, is determined, in _the final analysis, by the physician in charge of the case, but depends on factors such as the exact disease or diseases to be treated, the severity of the disease and other diseases or conditions from which the patient suffers, the specific route of administration, other drugs and treatments which the patient may concomitantly require, and other factors in the physician's judgment . Preferably the aPC is administered by continuous infusion for up to about 144 hours at a dosage of about 1 μg/kg/hr to about 50 μg/kg/hr. More preferably, the amount of aPC administered will be about 4 μg/kg/hr to about 48 μg/kg/hr. Even more preferably the amount of aPC administered will be: about 6 μg/kg/hr to about 44 μg/kg/hr; about 8 μg/kg/hr to about 40 μg/kg/hr; about 10 μg/kg/hr to about 36 μg/kg/hr; about 12 μg/kg/hr to about 34 μg/kg/hr; about 24 μg/kg/hr to about 30 μg/kg/hr; about 16 μg/kg/hr to about 24 μg/kg/hr; about 18 μg/kg/hr to about 20 μg/kg/hr; about 6 μg/kg/hr to about 22 μg/kg/hr; or about 10 μg/kg/hr to about 20 μg/kg/hr; or about 5 μg/kg/hr to about 25 μg/kg/hr; or about 5 μg/kg/hr to about 30 μg/kg/hr.
Alternatively, a bolus may be administered at various intervals before during or after discontinuation of the infusion. The bolus is preferably in the range of about 25 to 100 μg/kg/hr (bolus followed by infusion) . A physician may also dose the aPC to achieve preferred aPC plasma levels. Should the physician desire rapid aPC plasma levels, aPC will be administered in a bolus or in an increased amount . Examples of preferred protein C plasma level ranges include: about 10 ng/ml to about 180 ng/ml; about 25 ng/ml to about 160 ng/ml; about 25 ng/ml to about 100 ng/ml; about 30 ng/ml to about 140 ng/ml; about 40 ng/ml to about 120 ng/ml; about 40ng/ml to about 100 ng/ml; and about 40 to about 80 ng/ml. Again, although the preferred doses and plasma ranges are stated herein, various boluses of aPC may be used at various intervals, as is preferred in the judgement of the physician. For examples of dosing regimens of aPC noted in literature and patent documents, Table I sets forth normalized dose levels of several studies in humans or non- human primates. The human studies were done utilizing plasma derived PCZ while the non-human primate study utilized recombinant human aPC.
TABLE I
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
* the normalized dose is a conversion of the reported dose to the equivalent ug/kg/hr designation.
• 1 III is equivalent to approximately 4 ug of PC
TABLE I Continued
Figure imgf000037_0002
paragraph]
Wada, et al . , Plasma-derived human APC 33 μg/hr for 6 Am. J. Hematol . was given at 4000 days 44:218-219, 1993 units** /day for 6 days, [p.219, column 1, 1st paragraph]
Wada, et al . , Plasma-derived human APC 0.8 to 2.5 Blood 94:28a, was given at 100 to 300 μg/kg/hr for 3 to 1999 units**/kg for 3 to 6 6 days days . This dose is not sufficient for treating purpura fulminans . [p.28a, column 2, #111]
Bang, et al . The dose of activated 1.8 to 18 ug/kg/hr U.S. Patent Protein C ranges from 1- An infusion time 4,775,624 10 mg as a loading dose was not given. followed by a continuous infusion in amounts ranging from 3-30 mg/day. [column 19, lines 55-59]
** 1 U is defined as the amount which doubles the activated prothrombin time (APTT) in normal human plasma. This converts to approximately 5 Units/ug APC.
aPC formulations are prepared by known procedures using well-known and readily available ingredients. Preferably, the aPC will be administered parenterally to ensure delivery into the bloodstream in an effective form. Preferably, aPC is formulated according to the disclosure herein.
Example 3 Formulation of APC A stable lyophilized formulation of aPC is prepared by a process which comprises lyophilizing a solution comprising about 2.5 mg/mL aPC, about 15 mg/mL sucrose, about 20 mg/mL NaCl, and a sodium citrate buffer having a pH greater than 5.5 but less than 6.5. Additionally, the stable lyophilized formulation of aPC comprises lyophilizing a solution comprising about 5 mg/mL aPC, about 30 mg/mL sucrose, about 38 mg/mL NaCl, and a citrate buffer having a pH greater than 5.5 and, preferably, less than 6.5.
The ratio of aPC : salt :bulking agent (w:w:w) is believed to be an important factor in a formulation suitable for the freeze drying process. The ratio varies depending on the concentration of aPC, salt selection and concentration and bulking agent selection and concentration. Particularly, a ratio of about 1 part aPC to about 7.6 parts salt to about 6 parts bulking agent is believed to be preferred.
A unit dosage formulation of aPC suitable for parenteral administration, preferably subcutaneous administration or continuous intravenous infusion is prepared by mixing aPC, NaCl, sucrose, and sodium citrate buffer. After mixing, 4 mL of the solution is transferred to a unit dosage receptacle and lyophilized. The unit dosage receptacle containing about 5 mg to about 20 mg of aPC, suitable for administering a dosage of about 0.02 mg/kg/hr to about 0.05 mg/kg/hr to patients in need thereof, is sealed and stored until use.
The ratio of aPC to sucrose to sodium chloride (in 10 or 20 mM citrate buffer) is believed to be an important formulation variable affecting the collapse and glass- transition temperatures. To be processed in a conventional freeze-dryer, the sodium chloride concentration must be high enough (preferably 325 mM for 2.5 mg/mL aPC and 650 mM for 5 mg/mL aPC formulations) to cause the sodium chloride to crystallize-out during the freezing part of the freeze- drying process. Formulations of aPC can be processed in a conventional freeze dryer to produce lyophilized products consisting of 1 part aPC, 6 parts sucrose, and 7.6 parts sodium chloride by weight. The formulated aPC is placed in a container. A product label and labeling accompanies the aPC.

Claims

We claim:
1. A method of reducing the duration of time a human patient with a hypercoagulable state and/or a protein C deficiency remains in a hospital, an intensive care unit, and/or on mechanical ventilation, which comprises administering to said patient activated Protein C (aPC) .
2. The method of Claim 1 wherein the duration the patient remains in a hospital is reduced.
3. The method of Claim 1 wherein the duration the patient remains in an ICU is reduced.
4. The method of Claim 1 wherein the duration of mechanical ventilation is reduced.
5. The method according to any of Claims 1, 2, 3 or 4, wherein said hypercoagulable state or protein C deficiency is associated with a disease or condition selected from: sepsis, severe sepsis, septic shock, disseminated intravascular coagulation, purpura fulminans, major trauma, undergoing or recovering from surgery, burns, adult respiratory distress syndrome, bone marrow and other organ transplantations, deep vein thrombosis, heparin- induced thrombocytopenia, sickle cell disease, thalassemia, viral hemorrhagic fever, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, unstable angina, myocardial infarction, meningococcemia, melioidosis, complications during pregnancy, preeclampsia, eclampsia, amniotic fluid embolism, placental abruption, and chemotherapy .
6. The method of Claim 5 wherein the hypercoagulable state or protein C deficiency is selected from sepsis, severe sepsis, and septic shock.
7. The method of any of Claim 1 wherein the activated Protein C is administered by continuous infusion at a dose of about 1 μg/kg/hr to about 50 μg/kg/hr.
8. The method of Claim 2, 3, 4 or 6 wherein the activated Protein C is administered by continuous infusion at a dose of about 1 μg/kg/hr to about 50 μg/kg/hr.
9. The method of Claim 5 wherein the activated Protein C is administered by continuous infusion at a dose of about 1 μg/kg/hr to about 50 μg/kg/hr.
10. The method of Claim 1 wherein the activated Protein C is administered by continuous infusion at a dose of about 24 μg/kg/hr to about 30 μg/kg/hr.
11. The method of Claim 2, 3, 4 or 6 wherein the activated Protein C is administered by continuous infusion at a dose of about 24 μg/kg/hr to about 30 μg/kg/hr.
12. The method of Claim 5 wherein the activated Protein C is administered by continuous infusion at a dose of about 24 μg/kg/hr to about 30 μg/kg/hr.
13. The method of Claim 1 wherein the activated
Protein C is administered to achieve an aPC plasma range of about 25 ng/ml to about 100 ng/ml.
14. The method of Claim 2 , 3 , 4 or 6 wherein the activated Protein C is administered to achieve an activated Protein C plasma range of about 25 ng/ml to about 100 ng/ml.
15. The method of Claim 5 wherein the activated Protein C is administered to achieve an activated Protein C plasma range of about 25 ng/ml to about 100 ng/ml.
16. The method of Claim 1 wherein the activated
Protein C plasma range is about 25 ng/ml to about 100 ng/ml,
17. The method of Claim 2 , 3 or 4 wherein the activated Protein C plasma range is about 25 ng/ml to about 100 ng/ml.
18. The method of Claim 12 wherein the activated Protein C plasma range is about 25 ng/ml to about 100 ng/ml.
19. The method of Claim 1, 2, 3, 4 or 6 wherein the aPC is produced recombinantly .
20. The method of Claim 1, 2, 3, 4 or 6 wherein the aPC is produced transgenically .
21. The method of Claim 1, 2, 3, 4 or 6 wherein the aPC is plasma derived.
22. The method of Claim 10, wherein the aPC is produced recombinantly.
23. The method of Claim 10, wherein the aPC is produced transgenically .
24. The method of Claim 10, wherein the aPC is plasma derived.
25. An article of manufacture comprising packaging material and activated Protein C contained within said packaging material, wherein the packaging material comprises a label or accompanying labeling which indicates that administration of activated Protein C can be used to reduce the duration a human patient with a hypercoagulable state or protein C deficiency remains in an intensive care unit.
26. The article of manufacture of Claim 25 wherein the activated Protein C is recombinant human activated Protein C and the disease or condition is selected from: sepsis, severe sepsis, septic shock, disseminated intravascular coagulation, purpura fulminans, major trauma, undergoing or recovering from surgery, burns, adult respiratory distress syndrome, bone marrow and other organ transplantations, deep vein thrombosis, heparin-induced thrombocytopenia, sickle cell disease, thalassemia, viral hemorrhagic fever, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, unstable angina, myocardial infarction, meningococcemia, melioidosis, complications during pregnancy, preeclampsia, eclampsia, amniotic fluid embolism, placental abruption, and chemotherapy.
PCT/US2001/026028 2000-09-18 2001-09-17 Method for using activated protein c for the treatment of coagulation-associated disorders WO2002024215A2 (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6008199A (en) * 1997-10-20 1999-12-28 Eli Lilly And Company Methods for treating hypercoagulable states or acquired protein C deficiency
WO2000030677A1 (en) * 1998-11-20 2000-06-02 Eli Lilly And Company Method of treating viral hemorrhagic fever
WO2000030676A1 (en) * 1998-11-23 2000-06-02 Eli Lilly And Company Method of treating sickle cell disease and thalassemia
WO2001089558A2 (en) * 2000-05-24 2001-11-29 Eli Lilly And Company Formulations and use of activated protein c and protein c zymogen for treating hypercoagulable states

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6008199A (en) * 1997-10-20 1999-12-28 Eli Lilly And Company Methods for treating hypercoagulable states or acquired protein C deficiency
WO2000030677A1 (en) * 1998-11-20 2000-06-02 Eli Lilly And Company Method of treating viral hemorrhagic fever
WO2000030676A1 (en) * 1998-11-23 2000-06-02 Eli Lilly And Company Method of treating sickle cell disease and thalassemia
WO2001089558A2 (en) * 2000-05-24 2001-11-29 Eli Lilly And Company Formulations and use of activated protein c and protein c zymogen for treating hypercoagulable states

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