The present invention provides methods of treating hemorrhagic fevers where selective inhibitors of fVIIa/TF are used as a treatment for hemorrhagic fevers and have therapeutic effects which include ameliorating and/or preventing coagulopathy and inflammatory responses. These inhibitors include certain proteins which are part of a family termed Nematode-Extracted Anticoagulant Proteins ("NAPs"). Other inhibitors include Tissue Factor Pathway Inhibitor ("TFPI") and TFPI analogs.

Description of the Invention

SUMMARY OF THE INVENTION

According to one aspect, the present invention is directed to treatment of viral hemorrhagic fever by administration of a selective inhibitor of the TF/VIIa complex which initiates the activation of coagulation.

In view of the published reports on the effects of inhibiting the TF/VIIa complex by different inhibitors, it is not predictable as to what effects a particular inhibitor of TF/VIIa will have on the associated inflammation responses induced by either bacterial or viral infection. Furthermore, little is known regarding the role of TF in mediating the coagulation and inflammatory responses to viral infection particularly hemorrhagic viral infection.

According to one aspect, the present invention is directed to a method for treating a mammal having a hemorrhagic fever which comprises administering to said mammal an amount of a selective inhibitor of Factor VIIa/tissue factor ("fVIIa/TF") effective to ameliorate symptoms of said fever.

According to an alternate aspect, the present invention is directed to a method of treating a mammal having a hemorrhagic fever which comprises administering to said mammal a therapeutically effective amount of a compound which specifically inhibits the catalytic activity of the fVIIa/TF complex in the presence of Factor Xa ("fXa") or catalytically active fXa derivative (including the zymogen Factor X ("fX")) and does not specifically inhibit the catalytic activity of the fVIIa/TF complex in the absence of fXa or catalytically inactive fXa derivative and does not specifically inhibit the activity of Factor VIIa ("fVIIa") in the absence of tissue factor ("TF") and does not specifically inhibit prothombinase.

In an additional aspect, the present invention is directed to a method of treating coagulopathy and/or an inflammatory response due to hemorrhagic fever in a mammal which comprises administering to said mammal an amount of a selective fVIIa/TF inhibitor effective to decrease or prevent said coagulopathy and/or inflammatory response in said mammal.

The hemorrhagic fever to be treated may be caused by a virus. Such hemorrhagic fevers may be caused by viruses such as the Filoviridae, Arenaviridae Bunyaviridae and Flaviridae. The methods of the present invention may be particularly suited to treat viral hemorrhagic fevers caused by the Filoviridae.

Suitable fVIIa/TF inhibitors and compounds which inhibit the catalytic activity of the fVIIa/TF complex include members of the family of Nematode-Extracted Anti-coagulant Proteins (NAPs") having fVIIa/TF inhibitory activity, tissue factor pathway inhibitor ("TFPI") and analogs of tissue factor pathway inhibitor.

Suitable NAPs for use in the methods of the present invention include an isolated protein having Factor VIIa/TF inhibitory activity and having one or more NAP domains, wherein each NAP domain includes the sequence: Cys-A1-Cys-A2-Cys-A3-Cys-A4-Cys-A5-Cys-A6-Cys-A7-Cys-A8-Cys-A9-Cys-A10, wherein

(a) A1 is an amino acid sequence of 7 to 8 amino acid residues;

(b) A2 is an amino acid sequence;

(c) A3 is an amino acid sequence of 3 amino acid residues;

(d) A4 is an amino acid sequence;

(e) A5 is an amino acid sequence of 3 to 4 amino acid residues;

(f) A6 is an amino acid sequence;

(g) A7 is an amino acid residue;

(h) A8 is an amino acid sequence of 11 to 12 amino acid residues;

(i) A9 is an amino acid sequence of 5 to 7 amino acid residues; and

A10 is an amino acid sequence; wherein each of A2, A4, A6 and A10 has an independently selected number of independently selected amino acid residues and each sequence is selected such that each NAP domain has in total less than about 120 amino acid residues.

Certain suitable NAPs for use according to the methods of the present invention include AcaNAPc2 (SEQ. ID. NO. 3) and AcaNAPc2/proline (SEQ. ID. NO. 4). Also suitable for use according to the methods of the present invention are TFPI and TFPI analogs.

DETAILED DESCRIPTION OF THE INVENTION

A. Inhibitors of fVIIa/TF

A number of selective inhibitors of fVIIa/TF have been reported. According to one aspect of the present invention, these inhibitors are used as a treatment for hemorrhagic fevers and have therapeutic effects which include ameliorating and/or preventing coagulopathy and inflammatory responses. These inhibitors include certain proteins which are part of a family termed Nematode-Extracted Anticoagulant Proteins ("NAPs"). Other inhibitors include Tissue Factor Pathway Inhibitor ("TFPI") and TFPI analogs.

1. NAP Proteins

U.S. Pat. No. 5,866,542 to Vlasuk et al. describes a family of proteins termed "Nematode-Extracted Anticoagulant Proteins ("NAPs"), one class of which is described as having Factor VIIa/TF inhibitory activity. This class of proteins include an isolated protein having Factor VIIa/TF inhibitory activity and having one or more NAP domains, wherein each NAP domain includes the sequence: Cys-A1-Cys-A2-Cys-A3-Cys-A4-Cys-A5-Cys-A6-Cys-A7-Cys-A8-Cys-A9-Cys-A10 ("FORMULA III"), wherein

(a) A1 is an amino acid sequence of 7 to 8 amino acid residues;

(b) A2 is an amino acid sequence;

(c) A3 is an amino acid sequence of 3 amino acid residues;

(d) A4 is an amino acid sequence;

(e) A5 is an amino acid sequence of 3 to 4 amino acid residues;

(f) A6 is an amino acid sequence;

(g) A7 is an amino acid residue;

(h) A8 is an amino acid sequence of 11 to 12 amino acid residues;

(i) A9 is an amino acid sequence of 5 to 7 amino acid residues; and

(j) A10 is an amino acid sequence; wherein each of A2, A4, A6 and A10 has an independently selected number of independently selected amino acid residues and each sequence is selected such that each NAP domain has in total less than about 120 amino acid residues.

Suitable NAP proteins within this aspect of the invention have at least one NAP domain. Preferred are NAPs having one or two NAP domains. Preferred are proteins having at least one NAP domain substantially the same as the NAP domain of AcaNAPc2 (SEQ. ID. No. 3). The NAP protein AcaNAPc2 (SEQ. ID. NO. 3) has one NAP domain and is a preferred NAP according to this aspect of the invention. Also preferred is AcaNAPc2/proline (SEQ. ID. NO. 4). AcaNAPc2/proline is a recombinant protein having the sequence of AcaNAPc2 and which has an additional proline residue at the C terminus.

Preferred NAP proteins include those in which A2 is an amino acid sequence of 3 to 5 amino acid residues, A4 is an amino acid sequence of 6 to 19 amino acid residues, A6 is an amino acid sequence of 3 to 5 amino acid residues, and A10 is an amino acid sequence of 5 to 25 amino acid residues.

In certain preferred NAPs, A3 has the sequence Asp-A3.sub.a-A3.sub.b, wherein A3.sub.a and A.sup.3.sub.b are independently selected amino acid residues. More preferably, A3 is Asp-Lys-Lys.

In certain preferred NAPs, A4 is an amino acid sequence having a net anionic charge.

Also preferred are NAPS where A5 has the sequence A5.sub.a-A5.sub.b-A5.sub.c-A5.sub.d wherein A5.sub.a through A5.sub.d are independently selected amino acid residues. Preferably, A5.sub.a is Leu and A5.sub.c is Arg.

In certain preferred NAPs, a preferred A7 amino acid residue is Val or Ile, more preferably Val.

Certain preferred NAPs include those in which A8 includes the amino acid sequence A8.sub.a-A8.sub.b-A8.sub.c-A8.sub.d-A8.sub.e-A8.sub.f-A8.sub.g

wherein

(a) A8.sub.a is the first amino acid residue in A8,

(b) at least one of A8.sub.a and A8.sub.b is selected from the group consisting of Glu or Asp, and

(c) A8.sub.c through A.sup.8.sub.g are independently selected amino acid residues.

Preferably, A8.sub.c is Gly, A8.sub.d is selected from the group consisting of Phe, Tyr, and Leu, A8.sub.e is Tyr, A8.sub.f is Arg, and A.sup.8.sub.g is selected from Asp and Asn. A preferred A8.sub.c-A8.sub.d-A8.sub.e-A8.sub.f-A8.sub.g sequence is Gly-Phe-Tyr-Arg-Asn (SEQ. ID. NO. 5).

In one embodiment, the present invention is directed to methods of using a suitable NAP molecule having factor VIIa/tissue factor inhibitory activity wherein

(a) A3 has the sequences Asp-A3.sub.a-A3.sub.b, wherein A3.sub.a and A3.sub.b are independently selected amino acid residues;

(b) A4 is an amino acid sequence having a net anionic charge;

(c) A5 has the sequence A5.sub.a-A5.sub.b-A5.sub.c-A5.sub.d, wherein A5.sub.a through A5.sub.d are independently selected amino acid residues, and

(d) A7 is selected from the group consisting of Val and Ile. Use of pharmaceutical compositions comprising one or more of these NAP proteins having factor VIIa/tissue factor inhibitory activity and a pharmaceutically acceptable carrier are contemplated by this embodiment. NAP proteins used according to the methods of the present invention have at least one NAP domain. Preferred are NAPs having one or two NAP domains. The NAP proteins AcaNAPc2 and AcaNAPc2/proline have one NAP domain and are preferred NAPs according to this embodiment of the invention.

In another preferred embodiment, a suitable NAP molecule for use in the methods of the present invention is one wherein

(a) A3 is Asp-Lys-Lys;

(b) A4 is an amino acid sequence having a net anionic charge;

(c) A5 has the sequence A5.sub.a-A5.sub.b-A5.sub.c-A5.sub.d wherein A5.sub.a through A5.sub.d are independently selected amino acid residues;

(d) A7 is Val;

(e) A8 includes an amino acid sequence A8.sub.a-A8.sub.b-Gly-Phe-Tyr-Arg-Asn (SEQ. ID. NO. 6), wherein at least one of A8.sub.a and A8.sub.b is Glu or Asp. Use of pharmaceutical compositions comprising such NAP proteins and a pharmaceutically acceptable carrier also are contemplated by this invention. These NAP proteins have at least one NAP domain. Preferred are NAPs having one or two NAP domains. The NAP proteins AcaNAPc2 (SEQ. ID. NO. 3) and AcaNAPc2/proline (SEQ. ID. NO. 4) each have one NAP domain and are preferred NAPs according to this embodiment of the invention.

Certain preferred NAP proteins having Factor VIIa/TF inhibitory activity as described above are derived from a nematode species. A preferred nematode species is selected from the group consisting of Ancylostoma caninum, Ancylostoma ceylanicum, Ancylostoma duodenale, Necator americanus, and Heligomosomoides polygyrus. These NAPs may conveniently be prepared by recombinant means. Particularly preferred are the NAP proteins AcaNAPc2 and AcaNAPc2/proline. AcaNAPc2 was derived from Ancylostoma caninum using recombinant methods.

U.S. Pat. No. 5,866,542 describes the preparation of recombinant NAP proteins, including AcaNAPc2 and AcaNAPc2/proline.

2. Tissue Factor Pathway Inhibitor

U.S. Pat. No. 6,300,100 describes Tissue Factor Pathway Inhibitor and process for its preparation by recombinant methods.

U.S. Pat. No. 5,378,614 describes the preparation of certain analogs of TFPI.

B. Selection of Compounds Having Factor VIIa/Tissue Factor Inhibitory Activity

The fVIIa/TF inhibitory activity of NAPs and other compounds used according to the methods of the present invention can be determined using protocols described herein. Example 3 describes fVIIa/TF assays. There, the fVIIa/TF-mediated cleavage and liberation of the tritiated activation peptide from radiolabeled human factor IX (.sup.3H-FIX) or the amidolytic hydrolysis of a chromogenic peptidyl substrate are measured. Interestingly, certain fVIIa/TF inhibitors require the presence of fXa or catalytically inactive fXa derivative in order to be active fVIIa/TF inhibitors. However, certain NAP and other fVIIa/TF inhibitors were equally effective in the presence of fXa in which the active site had been irreversibly occupied with the peptidyl chloromethyl ketone H-Glu-Gly-Arg-CMK (EGR), and thereby rendered catalytically inactive (EGR-fXa). While not wishing to be bound by any one explanation, it appears that these proteins having fVIIa/TF inhibitory activity form a binary complex with fXa by binding to a specific recognition site on the enzyme that is distinct from the primary recognition sites, P.sub.4-P-.sub.1, within the catalytic center of the enzyme. This is followed by the formation of a quaternary inhibitory complex with the fVIIa/TF complex. Consistent with this hypothesis is that EGR-fXa can fully support the inhibition of fVIIa/TF by these compounds which are inhibitory for fVIIa/TF despite covalent occupancy of the primary recognition sites (P.sub.4-P.sub.1) within the catalytic site of fXa by the tripeptidyl-chloromethyl ketone (EGR-CMK).

The fVIIa/TF inhibitory activity of these compounds also can be determined using the protocol in Example 7, as well as the fXa assays described above, and in Examples 5 and 6. There, the ability of a compound to inhibit the catalytic activity of a variety of enzymes is measured and compared to its inhibitory activity toward the fVIIa/TF complex.

Certain of these compounds specifically inhibit the catalytic activity of the fVIIa/TF complex in the presence of fXa or catalytically inactive fXa derivative (including the zymogen Factor X), but do not specifically inhibit prothrombinase. Preferred compounds according to this aspect of the invention have the characteristics described above for an isolated protein having Factor VIIa/TF inhibitory activity and having one or more NAP domains. A preferred protein according to this aspect of the invention is AcaNAPc2 or AcaNAPc2/Pro.

These compounds are identified by their fVIIa/TF inhibitory activity in the presence of fXa or a fXa derivative, whether the derivative is catalytically active or not. The protocol in Example 4 can detect a compound's inactivity toward free fXa or prothrombinase. Data generated using the protocols in Example 3 will identify compounds that require either catalytically active or inactive fXa to inhibit fVIIa/TF complex.

C. Administration and Formulation

According to one aspect of the present invention, the fVIIa/TF inhibitor is administered to a patient in need of treatment as soon as the hemorrhagic fever is detected. It may be preferred to treat the patient at as early a stage in the hemorrhagic fever as possible, preferably at or before onset of symptoms. Thus, with respect to hemorrhagic fever-causing viruses it may be advantageous to administer the fVIIa/TF prior to exposure to the virus or as soon after exposure as possible.

The compounds or pharmaceutical compositions thereof used according to the methods of the present invention are administered in vivo, ordinarily in a mammal, preferably in a human. In employing them in vivo, the compounds and pharmaceutical compositions can be administered to a mammal in a variety of ways, including orally, parenterally, intravenously, subcutaneously, intramuscularly, colonically, rectally, nasally or intraperitoneally, employing a variety of dosage forms. Administration is preferably parenteral, such as intravenous on a daily basis. Alternatively, for some compounds or pharmaceutical compositions administration is preferably oral, such as by tablets, capsules or elixirs taken on a daily basis.

In practicing the methods of the present invention, the compounds or pharmaceutical compositions used according to the methods of the present invention are administered alone or in combination with one another, or in combination with other therapeutic or in vivo diagnostic agents.

As is apparent to one skilled in the medical art, a therapeutically effective amount of the compounds or pharmaceutical compositions of the present invention will vary depending upon the age, weight and mammalian species treated, the particular compounds employed, the particular mode of administration and the desired effects and the therapeutic indication. Because these factors and their relationship to determining this amount are well known in the medical arts, the determination of therapeutically effective dosage levels, the amount necessary to achieve the desired result of ameliorating the effects caused by the hemorrhagic fever will be within the ambit of one skilled in these arts.

Typically, administration of the compounds or pharmaceutical composition of the present invention is commenced at lower dosage levels, with dosage levels being increased until the desired therapeutic effect is achieved which would define a therapeutically effective amount. For the compounds of the present invention, alone or as part of a pharmaceutical composition, such doses are between about 0.001 mg/kg and 100 mg/kg body weight, preferably between about 0.05 and 10 mg/kg, body weight.
 

Claim 1 of 15 Claims

1. A method of treating a mammal having a viral hemorrhagic fever which comprises administering to said mammal an effective amount of a Nematode-Extracted Anticoagulant Protein ("NAP") having Factor VIIa/tissue factor inhibitory activity ("fVIIa/TF"), wherein said NAP comprises one or more NAP domains, wherein each NAP domain includes the sequence: Cys-A1-Cys-A2-Cys-A3-Cys-A4-Cys-A5-Cys-A6-Cys-A7-Cys-A8-Cys-A9-- Cys-A10, wherein (a) A1 is an amino acid sequence of 7 to 8 amino acid residues; (b) A2 is an amino acid sequence of 3 to 5 amino acid residues; (c) A3 is an amino acid sequence of 3 amino acid residues; (d) A4 is an amino acid sequence of 6 to 19 amino acid residues; (e) A5 is an amino acid sequence of 3 to 4 amino acid residues; (f) A6 is an amino acid sequence of 3 to 5 amino acid residues; (g) A7 is an amino acid; (h) A8 is an amino acid sequence of 11 to 12 amino acid residues; (i) A9 is an amino acid sequence of 5 to 7 amino acid residues; and (j) A10 is an amino acid sequence of 5 to 25 amino acid residues.

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