Internet for Pharmaceutical and Biotech Communities
| Newsletter | Advertising |
 
 
 

  

Pharm/Biotech
Resources

Outsourcing Guide

Cont. Education

Software/Reports

Training Courses

Web Seminars

Jobs

Buyer's Guide

Home Page

Pharm Patents /
Licensing

Pharm News

Federal Register

Pharm Stocks

FDA Links

FDA Warning Letters

FDA Doc/cGMP

Pharm/Biotech Events

Consultants

Advertiser Info

Newsletter Subscription

Web Links

Suggestions

Site Map
 

 
   

 

  Pharmaceutical Patents  

 

Title:  Activated protein C variants with normal cytoprotective activity but reduced anticoagulant activity
United States Patent: 
7,498,305
Issued: 
March 3, 2009

Inventors:
 Griffin; John H. (Del Mar, CA), Mosnier; Laurent O. (San Diego, CA), Gale; Andrew J. (San Diego, CA)
Assignee: 
The Scripps Research Institute (La Jolla, CA)
Appl. No.: 
10/886,766
Filed: 
July 8, 2004


 

Training Courses -- Pharm/Biotech/etc.


Abstract

Variants (mutants) of recombinant activated protein C (APC) or recombinant protein C (prodrug, capable of being converted to APC) that have substantial reductions in anticoagulant activity but that retain normal levels of anti-apoptotic activity are provided. Two examples of such recombinant APC mutants are KKK191-193AAA-APC and RR229/230M-APC. APC variants and prodrugs of the invention have the desirable property of being cytoprotective (anti-apoptotic effects), while having significantly reduced risk of bleeding. The invention also provides a method of using the APC variants or prodrugs of the invention to treat subjects who will benefit from APC's cytoprotective activities that are independent of APC's anticoagulant activity. These subjects include patients at risk of damage to blood vessels or tissue in various organs caused, at least in part, by apoptosis. At risk patients include, for example, those suffering (severe) sepsis, ischemia/reperfusion injury, ischemic stroke, acute myocardial infarction, acute or chronic neurodegenerative diseases, or those undergoing organ transplantation or chemotherapy, among other conditions. Methods of screening for variants of recombinant protein C or APC that are useful in accordance with the invention are also provided.

Description of the Invention

FIELD OF THE INVENTION

The present invention relates to variants (mutants) of recombinant protein C and activated protein C, an enzyme that normally has anti-thrombotic, anti-inflammatory, and anti-apoptotic activities. The recombinant activated protein C mutants of the invention have markedly reduced anticoagulant activity, but retain near normal anti-apoptotic (cytoprotective) activity, so that the ratio of anti-apoptotic to anticoagulant activity is greater in the variants than it is in wild-type or endogenous activated protein C. This invention also relates to methods of using these variants. The activated protein C variants of the invention are useful as inhibitors of apoptosis or cell death and/or as cell survival factors, especially for cells or tissues of the nervous system, which are stressed or injured. The invention further relates to therapeutic use of the variants of this invention in subjects at risk for cell damage caused at least in part by apoptosis, and to therapeutic compositions comprising such mutant proteins, which compositions should provide the desired cytoprotective benefits while carrying a lower risk of bleeding, a side effect of activated protein C therapy.

BACKGROUND OF THE INVENTION

Protein C is a member of the class of vitamin K-dependent serine protease coagulation factors. Protein C was originally identified for its anticoagulant and profibrinolytic activities. Protein C circulating in the blood is an inactive zymogen that requires proteolytic activation to regulate blood coagulation through a complex natural feedback mechanism. Human protein C is primarily made in the liver as a single polypeptide of 461 amino acids. This precursor molecule is then post-translationally modified by (i) cleavage of a 42 amino acid signal sequence, (ii) proteolytic removal from the one-chain zymogen of the lysine residue at position 155 and the arginine residue at position 156 to produce the two-chain form (i.e., light chain of 155 amino acid residues attached by disulfide linkage to the serine protease-containing heavy chain of 262 amino acid residues), (iii) carboxylation of the glutamic acid residues clustered in the first 42 amino acids of the light chain resulting in nine gamma-carboxyglutamic acid (Gla) residues, and (iv) glycosylation at four sites (one in the light chain and three in the heavy chain). The heavy chain contains the serine protease triad of Asp257, His211 and Ser360.

Similar to most other zymogens of extracellular proteases and the coagulation factors, protein C has a core structure of the chymotrypsin family, having insertions and an N-terminus extension that enable regulation of the zymogen and the enzyme. Of interest are two domains with amino acid sequences similar to epidermal growth factor (EGF). At least a portion of the nucleotide and amino acid sequences for protein C from human, monkey, mouse, rat, hamster, rabbit, dog, cat, goat, pig, horse, and cow are known, as well as mutations and polymorphisms of human protein C (see GenBank accession P04070). Other variants of human protein C are known which affect different biological activities.

Activation of protein C is mediated by thromblin, acting at the site between the arginine residue at position number 15 of the heavy chain and the leucine residue at position 16 (chymotrypsin numbering) (See Kisiel, J. Clin. Invest., 64:761-769, 1976; Marlar et al., Blood, 59:1067-1072, 1982; Fisher et al. Protein Science, 3:588-599, 1994). Other proteins including Factor Xa (Haley et al., J. Biol. Chem., 264:16303-16310, 1989), Russell's viper venom, and trypsin (Esmon et al., J. Biol. Chem., 251:2770-2776, 1976) also have been shown to enzymatically cleave and convert inactive protein C to its activated form.

Thrombin binds to thrombomodulin, a membrane-bound thrombin receptor on the luminal surface of endothelial cells, thereby blocking the procoagulant activity of thrombin via its exosite I, and enhancing its anticoagulant properties, i.e., activating protein C. As an anticoagulant, activated protein C (APC), aided by its cofactor protein S, cleaves the activated cofactors factor Va and factor VIIa, which are required in the intrinsic coagulation pathway to sustain thrombin formation (Esmon et al., Biochim. Biophys. Acta., 1477:349-360, 2000a), to yield the inactivated cofactors factor Vi and factor VIIIi.

The thrombin/thrombomodulin complex mediated activation of protein C is facilitated when protein C binds to the endothelial protein C receptor (EPCR), which localizes protein C to the endothelial cell membrane surface. When complexed with EPCR, APC's anticoagulant activity is inhibited; APC expresses its anticoagulant activity when it dissociates from EPCR, especially when bound to negatively charged phospholipids on activated platelet or endothelial cell membranes.

Components of the protein C pathway contribute not only to anticoagulant activity, but also to anti-inflammatory functions (Griffin et al., Sem. Hematology, 39:197-205, 2002). The anti-inflammatory effects of thrombomodulin, recently attributed to its lectin-like domain, can protect mice against neutrophil-mediated tissue damage (Conway et al., J. Exp. Med. 196:565-577, 2002). The murine centrosomal protein CCD41 or centrocyclin, involved in cell-cycle regulation is identical to murine EPCR lacking the first N-terminal 31 amino acids (Rothbarth et al., FEBS Lett., 458:77-80, 1999; Fukodome and Esmon, J. Biol. Chem., 270:5571-5577,1995). EPCR is structurally homologous to the MHC class 1/CD1 family of proteins, most of which are involved in inflammatory processes. This homology suggests that the function of EPCR may not be limited to its ability to localize APC or protein C on the endothelial membrane (Oganesyan et al., J. Biol. Chem., 277:24851-24854, 2002). APC provides EPCR-dependent protection against the lethal effects of E.coli infusion in baboons (Taylor et al., Blood, 95:1680-1686, 2000) and can downregulate proinflammatory cytokine production and favorably alter tissue factor expression or blood pressure in various models (Shu et al., FEBS Lett. 477:208-212, 2000; Isobe et al., Circulation, 104:1171-1175, 2001; Esmon, Ann. Med., 34:598-605, 2002).

Inflammation is the body's reaction to injury and infection. Three major events are involved in inflammation: (1) increased blood supply to the injured or infected area; (2) increased capillary permeability enabled by retraction of endothelial cells; and (3) migration of leukocytes out of the capillaries and into the surrounding tissue (hereinafter referred to as cellular infiltration) (Roitt et al., Immunology, Grower Medical Publishing, New York, 1989).

Many serious clinical conditions involve underlying inflammatory processes in humans. For example, multiple sclerosis (MS) is an inflammatory disease of the central nervous system. In MS, circulating leukocytes infiltrate inflamed brain endothelium and damage myelin, with resultant impaired nerve conduction and paralysis (Yednock et al., Nature 366:63-66 (1992)). Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of tissue damage caused by self antigen directed antibodies. Auto-antibodies bound to antigens in various organs lead to complement-mediated and inflammatory cell mediated tissue damage (Theofilopoulos, A.N., Encyclopedia of Immunology, pp. 1414-1417 (1992)).

APC has not only anticoagulant and anti-inflammatory activities but also anti-apoptotic activity. EPCR has been found to be a required cofactor for the anti-apoptotic activity of APC in certain cells, as APC activation of protease activated receptor-1 (PAR-1) is EPCR-dependent (Riewald et al., Science, 2296:1880-1882, 2002; Cheng et al., Nat. Med., 9:338-342, 2003; Mosnier and Griffin, Biochem. J., 373:65-70, 2003). APC also has been shown potentially to inhibit staurosporine-induced apoptosis in endothelial cells in vitro by modulating the expression of NF.kappa.B subunits (Joyce et al., J. Biol. Chem., 276:11199-11203, 2001). Staurosporine-induced apoptosis in human umbilical vein endothelial cells (HUVEC) and tumor necrosis factor-.alpha.-mediated injury of HUVEC, based on transcriptional profiling, suggest that APC's inhibition of NF.kappa.B signaling causes down regulation of adhesion molecules (Joyce et al., supra, 2001). APC's induction of anti-apoptotic genes (e.g., Bcl2-related protein A1 or Bcl2A1, inhibitor of apoptosis 1 or clAP1, endothelial nitric oxide synthase or eNOS) has been interpreted as a possible mechanism linked to APC's anti-apoptotic effects in a staurosporine model of apoptosis.

APC has a remarkable ability to reduce all-cause 28-day mortality by 19% in patients with severe sepsis (Bernard et al., New Engl. J. Med. 344:699-709, 2001a), whereas, potent anticoagulant agents such as antithrombin III and recombinant TFPI have failed in similar phase III clinical trials (Warren et al., JAMA, 286:1869-1878, 2001; Abraham et al., Crit. Care Med., 29:2081-2089). The explanation for this difference may lie in the recently described anti-apoptotic activity of APC, as well as its anti-inflammatory activity. The clinical success of APC in treating sepsis may be related to its direct cellular effects that mediate its anti-apoptotic or anti-inflammatory activity.

In spite of the numerous in vivo studies documenting the beneficial effects of APC, there is limited information about the molecular mechanisms responsible for APC's direct anti-inflammatory and anti-apoptotic effects on cells. APC can directly modulate gene expression in human umbilical vein endothelial cells (HUVEC) with notable effects on anti-inflammatory and cell survival genes (Joyce et al., supra, 2001; Riewald et al., supra, 2002). Riewald et al. have shown this direct effect of APC on certain cells requires PAR-1 and EPCR (Riewald et al., supra, 2002), although they provided no data that related APC functional activity with PAR-1-signaling.

Recombinant activated protein C (rAPC), similar to Xigris (Eli Lilly & Co.), is approved for treating severe sepsis and it may eventually have other beneficial applications. However, clinical studies have shown APC treatment to be associated with increased risk of serious bleeding. This increased risk of bleeding presents a major limitation of APC therapy. If APC's effects in sepsis can be attributed to its anti-inflammatory and cell survival activities, a compound that retains the beneficial anti-apoptotic or cytoprotective activity but has a less anticoagulant activity is desirable.

SUMMARY OF THE INVENTION

It is an object of this invention to provide variants (mutants) of recombinant APC and prodrugs (e.g., variants of recombinant protein C) as therapeutics or research tools for use in alleviating or preventing cell damage associated at least in part with apoptosis. It is also an object of this invention to provide a method of alleviating or preventing cell damage associated at least in part with apoptosis, especially in subjects at risk for or suffering from such cell damage. Another object of this invention is to provide a means for screening candidate mutants for use in accordance with the invention.

The invention is directed to variants of recombinant APC and prodrugs (protein C variants) that provide reduced anticoagulant activity relative to anti-apoptotic activity compared to wild-type, and, therefore, have use as cytoprotective agents. Two examples of such recombinant APC mutants are KKK191-193AAA-APC (mutation of lysines 191, 192 and 193 to alanines) and RR229/230AA-APC (mutation of arginines 229 and 230 to alanines). As we demonstrate herein, these exemplary APC variants retain the desirable property of normal anti-apoptotic, cytoprotective activity but provide significantly reduced risk of bleeding, given their reduced anticoagulant activity. The APC and protein C variants of the invention provide a ratio of anti-apoptotic to anticoagulant activity greater than that of wild-type APC (i.e., >1.0).

In one embodiment of the invention, a method of preventing or alleviating damage associated at least in part with apoptosis is provided. In a related aspect of this embodiment, a method of treating subjects at risk for cell damage associated at least in part with apoptosis is provided. These subjects include patients at risk of damage to blood vessels or tissue in various organs caused, at least in part, by apoptosis. At risk patients include, for example, those suffering (severe) sepsis, ischemia/reperfusion injury, ischemic stroke, acute myocardial infarction, acute or chronic neurodegenerative diseases, or those undergoing organ transplantation or chemotherapy, among other conditions. The APC variants and prodrugs of the invention should be useful in treating subjects who will benefit from APC protective activities that are independent of APC's anticoagulant activity. Prodrug embodiments of this invention may involve recombinant protein C variants that, following conversion of protein C to APC, exhibit reduced anticoagulant activity while retaining normal or near-normal cell protective activities. For example, variants of protein C, when activated, will have the desired ratio of anti-apoptotic to anticoagulant activity of greater than 1.0.

In another embodiment of the invention, the APC mutants may be provided as therapeutics or in therapeutic compositions, to offer beneficial cytoprotective effects in cells, while carrying much less risk of bleeding. In yet another embodiment of the invention, methods of screening candidate recombinant APC variants having reduced anticoagulant activity, but retaining the beneficial cell protective and anti-inflammatory activities are provided.

Given the risk of bleeding associated with wild type activated protein C, the APC mutants of this invention offer advantages over currently available wild-type recombinant APC. Therefore, APC mutants of the invention are expected to provide superior therapy, either alone or adjunctive to other agents, whenever APC might be used for its anti-inflammatory or anti-apoptotic (cell survival) activities, rather than purely for its anticoagulant activity.

DETAILED DESCRIPTION OF THE INVENTION

Activated protein C (APC) has traditionally been regarded as an anticoagulant enzyme in the coagulation cascade, inhibiting thrombin formation and subsequent fibrin-clot formation by inactivating the cofactors factor Va and factor VIIIa (Esmon, supra, 2000a). However, APC also has the remarkable ability to reduce mortality in severe sepsis (Bernard et al., supra, 2001a; Bernard et al., Crit. Care Med., 29:2051-59, 2001b; Hinds, Brit. Med. J., 323:881-82, 2001; Kanji et al., Pharmacother., 21:1389-1402, 2001), while other anticoagulants such as antithrombin III and tissue factor pathway inhibitor have failed in this capacity (Warren et al., supra, 2001; Abraham et al., supra, 2001). This property of APC has peaked investigators' interest in the less extensively studied direct anti-inflammatory and anti-apoptotic activities attributed to APC (see, e.g., Cheng et al. Nat. Med., 9:338-42, 2003; Domotor et al., Blood, 101:4797-4801, 2003; Fernandez et al., Blood Cells Mol. Dis., 30:271-276, 2003; Esmon, J. Autoimmun., 15:113-116, 2000b). APC also has potential to protect the brain from damage caused by ischemic stroke (Cheng et al., supra, 2003; Esmon Thrombos Haemostas, 83:639-643, 2000c).

A major concern for the use of APC as a therapeutic is an increased risk of bleeding complications (Bernard et al., supra, 2001a; Bernard et al., supra, 2001b) due to APC anticoagulant activity. The APC variants of this invention solve this problem by having reduced anticoagulant activity over endogenous APC or wild-type recombinant APC, while retaining beneficial anti-apoptotic activity. Differentiating the anticoagulant activity from the anti-apoptotic activity was the first step in solving this problem. We have focused in part on the role of EPCR in regulation of these activities.

EPCR was originally discovered as a receptor capable of binding protein C and APC with equal affinities (Fukodome and Esmon, supra, 1995), and EPCR was shown to enhance the activation of protein C by the thrombin-thrombomodulin complex (Stearns-Kurosawa, et al., Proc. Nat'l Acad. Sci., USA, 93:10212-10216, 1996), apparently by optimizing the spatial localization of protein C for efficient activation by thrombomodulin-bound thrombin. Presumably EPCR binds APC to the endothelial surface and positions APC's active site proximate to the PAR-1 cleavage site at Arg41. Paradoxically, although EPCR function might be anticoagulant by stimulating protein C activation (Stearns-Kurosawa, et al., supra, 1996), APC anticoagulant activity is actually inhibited when APC is bound to EPCR (Regan et al., J. Biol. Chem., 271:17499-17503, 1996). Because binding of APC to EPCR is essential for APC's anti-apoptotic activity, we have concluded that the anti-apoptotic activity of APC is independent of its anticoagulant activity. We hypothesized that certain APC mutants could be generated which lack anticoagulant activity but retain anti-apoptotic activity. Such mutants could be therapeutically useful if they provided patients with direct cell survival activity without increased risks of bleeding.

We have determined the structural elements of APC required for its anti-apoptotic activity, by assaying different forms of APC for their anti-apoptotic activity. The staurosporine-induced apoptosis was blocked by pretreatment of APC with an anti-APC monoclonal antibody or heat denaturation of APC, thereby establishing the specificity of APC's anti-apoptotic activity (Mosnier and Griffin, supra, 2003). APC-mediated inhibition of staurosporine-induced apoptosis was found to require APC's active site, since the inactive protein C zymogen, as well as an inactive APC mutant, in which the active site Ser was replaced by Ala, S360A-APC (Gale et al., Protein Sci., 6:132-140, 1997), were devoid of anti-apoptotic activity (Mosnier and Griffin, supra, 2003). This implies that the anti-apoptotic activity of APC is mediated by proteolysis.

It was not known whether the APC-mediated inhibition of staurosporine-induced apoptosis (Joyce et al., supra, 2001) was dependent on PAR-1 and EPCR, until we demonstrated that inhibition of staurosporine-induced apoptosis by APC was dependent on PAR-1 and EPCR using a modified staurosporine-induced apoptosis model with EAhy926 endothelial cells (Mosnier and Griffin, supra, 2003). Inhibition of hypoxia-induced apoptosis in human brain endothelial cells also has been shown to require PAR-1 (Cheng et al., supra, 2003). Thus, consistent with the implication that APC's proteolytic active site is required for inhibition of apoptosis, preincubation of cells with blocking antibodies against PAR-1, but not against PAR-2, abolished APC-mediated inhibition of staurosporine-induced apoptosis (Mosnier and Griffin, supra, 2003). Furthermore, APC anti-apoptotic activity was abolished by an anti-EPCR antibody that blocks binding of APC to EPCR (Mosnier and Griffin, supra, 2003), and controls showed that this effect of the anti-EPCR antibody was neutralized by preincubation of the antibody with its peptide immunogen (Mosnier and Griffin, supra, 2003). Therefore, based on antibody blocking studies, PAR-1 and EPCR are required for APC to inhibit staurosporine-induced apoptosis of endothelial cells.

This requirement for PAR-1 and EPCR for inhibition of staurosporine-induced apoptosis of EAhy926 endothelial cells also is consistent with the finding that these receptors are important for APC's anti-apoptotic activity in the setting of hypoxic brain microvascular endothelial cells (Cheng et al., supra, 2003).

APC can cleave a synthetic extracellular N terminal PAR 1 polypeptide at Arg41, the thrombin cleavage site (Kuliopulos et al., Biochemistry, 38:4572-4585, 1999). Cleavage of this synthetic PAR 1 polypeptide by APC is 5,000-times slower than by thrombin (Kuliopulos et al., supra, 1999). When thrombin cleaves PAR 1 at Arg41, potent cell signaling pathways might be initiated. It is likely that APC cleavage of PAR 1 at Arg41initiates cell signals, including phosphorylation of MAP kinase (Riewald et al., supra, 2002). In brain endothelial cells subjected to hypoxia, an early result of APC signaling is the inhibition of increases in the levels of p53 (Cheng et al., supra, 2003). Previous studies suggest that APC directly alters the gene expression profiles of HUVEC so that several anti-apoptotic genes are upregulated (Joyce et al., supra, 2001; Riewald et al., supra, 2002) and that APC specifically downregulates levels of the pro-apoptotic factor, flax, while it upregulates levels of the anti-apoptotic factor, Bcl 2, in brain endothelial cells (Cheng et al., supra, 2003). The specific alteration of the critical ratio of Bax/Bcl 2 is likely of key importance for apoptosis. Other than these events, little can be stated about the mechanisms for PAR 1-dependent APC signaling. It is interesting to note that the PAR 1 agonist peptide, TFLLRNPNDK (SEO ID. 1), exhibited no protection from staurosporine-induced apoptosis of EAhy926 cells whereas this agonist provided partial rescue of brain endothelial cells from hypoxia-induced apoptosis, suggesting there are subtle, but significant, differences between APC's PAR 1-dependent anti-apoptotic activities in these two models.

In vivo data are consistent with an important distinction between the anticoagulant and cell protective activities of APC. APC-induced neuroprotective effects in a murine ischemia/reperfusion injury model were observed at low APC doses that had no effect on fibrin deposition or on restoration of blood flow, indicating that APC's neuroprotective effects, at least in part, were independent of APC's anticoagulant activity (Cheng et al., supra, 2003).

No inhibition of staurosporine-induced apoptosis of EAhy926 cells was observed with either PAR-1 or PAR-2 agonist peptides in the absence of APC. Moreover, thrombin, the archetype activator of PAR-1, did not inhibit staurosporine-induced apoptosis (Mosnier and Griffin, supra, 2003). The failure of these other activators of PAR-1 to provide cell survival activity indicates that the PAR-1-dependent anti-apoptotic effects of APC for staurosporine-induced apoptosis are specific for APC. Without being bound to a mechanism of action, we can speculate that when EPCR-bound APC cleaves and activates PAR-1, a significant modulation of PAR-1's intracellular signaling occurs, compared to signals triggered by thrombin or the PAR-1 agonist peptide. Another potential source of complexity may arise from the reported ability of EPCR to mediate nuclear translocation of APC (Esmon, supra, 2000c). The intracellular signals and pathways that cause inhibition of apoptosis by APC in various cell model systems remain to be elucidated.

The physiological relevance of APC EPCR-dependent signaling via PAR-1 is further demonstrated by the APC-induced neuroprotective effects in a murine ischemia/reperfusion injury model that requires PAR-1 and EPCR (Cheng et al., supra, 2003). APC may act via the EPCR and PAR-1 on stressed brain endothelial cells, or the PAR-1 and the protease activated receptor-3 (PAR-3) on stressed neurons, to activate anti-apoptotic pathways and/or pro-survival pathways in these stressed and/or injured brain cells. In human brain endothelium in vitro and in animals in vivo (ischemic stroke and NMDA models), APC can inhibit the p53-signaling pro-apoptotic pathway in stressed or injured brain cells (International Patent Application No. PCT/US03/38764).
 

Claim 1 of 21 Claims

1. A method of protecting cells against damage caused at least in part by apoptosis, comprising administering to a subject or to cells a therapeutic dose of a recombinant activated protein C mutant, wherein said recombinant activated protein C mutant is KKK191-193AAA-APC relative to SEQ ID NO:15.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]