Kallikrein-inhibitor therapies

This application is a continuation of U.S. patent application Ser. No. 11/466,979, filed Aug. 24, 2006, which is a continuation of U.S. patent application Ser. No. 10/953,902, filed on Sep. 27, 2004, which is a continuation-in-part of U.S. patent application Ser. No. 10/456,986, filed Jun. 6, 2003, issued as U.S. Pat. No. 7,064,107, which claims priority to U.S. provisional application No. 60/387,239, filed on Jun. 7, 2002, and 60/407,003, filed on Aug. 28, 2002, the contents of all of which are incorporated herein by reference.

Serine proteases such as, kallikrein (e.g., plasma kallikrein), are involved in pathways leading to excessive perioperative blood loss, the onset of systemic inflammatory response and nervous system pathophysiology. Inhibitors of kallikrein include proteinaceous and non-proteinaceous molecules. Exemplary plasma kallikrein inhibitors include those described in U.S. Pat. Nos. 6,333,402 and 6,057,287, the contents of which are incorporated herein by reference in their entirety.

Polypeptide and other inhibitors of plasma kallikrein can be used in therapeutic methods and compositions suitable for use in eliminating or reducing various ischemias, including but not limited to perioperative blood loss, cerebral ischemia, the onset of systemic inflammatory response, and/or reperfusion injury, e.g., reperfusion injury associated with cerebral ischemia or a focal brain ischemia. Perioperative blood loss results from invasive surgical procedures that lead to contact activation of complement components and the coagulation/fibrinolysis systems. The kallikrein inhibitors can be used to reduce or prevent perioperative blood loss and a systemic inflammatory response in patients subjected to invasive surgical procedures, especially cardiothoracic surgeries. The kallikrein inhibitors can also be used to reduce or prevent cerebral ischemia such as stroke, and/or reperfusion injury associated with cerebral ischemia. They can also prevent neurological and cognitive deficits associated with stroke, blood lose, and cerebral ischemia, e.g., events that are not associated with surgical intervention. The inhibitors can be administered prior to, during, or after an event, e.g., a cardiovascular event that can damage the central nervous system.

A variety of inhibitors of a kallikrein, e.g., a plasma kallikrein are described herein. Any one of these, or a combination of more than one of these, can be used in the following methods.

In one aspect, the disclosure features a method for preventing or reducing ischemia in a patient including administering to the patient a composition that includes an inhibitor of kallikrein, e.g., a plasma kallikrein. Typically the patient is a human patient. The composition can be administered in an amount effective to prevent or reduce ischemia in the patient. In a particular embodiment, the ischemia is at least partially due to blood loss, e.g., perioperative blood loss due to a surgical procedure performed on the patient. The surgical procedure can be, e.g., a cardiothoracic surgery, such as, for example, cardiopulmonary bypass or coronary artery bypass grafting. The inhibitor can be administered before, during, or after the procedure.

In another aspect, the disclosure features a method for preventing or reducing a systemic inflammatory response, e.g., a response associated with a surgical procedure in a patient or its onset. The method includes: administering to the patient a composition including an inhibitor of kallikrein, e.g., a plasma kallikrein. The composition can be administered before, during, or after surgery. In one embodiment, the surgical procedure is a cardiothoracic surgery, such as, for example, cardiopulmonary bypass or coronary artery bypass grafting.

In another aspect, the disclosure features a method for treating a brain or central nervous system (CNS) injury. The method can be used to prevent or reduce adverse effects of cerebral ischemia, e.g., stroke, and/or reperfusion injury, e.g., reperfusion injury associated with cerebral ischemia, in a patient including administering to the patient a composition including an inhibitor of kallikrein, e.g., a plasma kallikrein. In one embodiment, the cerebral ischemia is stroke, e.g., embolism-, thrombus- or hemorrhage-associated stroke. The method can include administering the inhibitor, before, during, or after the ischemia, e.g., at the time of reperfusion or at a time between 1-12 hours after an ischemic event, e.g., between 1-5 hours after such an event.

The inhibitor used in any disclosed method can have a Ki for kallikrein, e.g., plasma kallikrein of less than 50 nM, 5 nM, 1 nM, 500 pM, 100 pM, 50 pM, e.g., about 44 pM. The inhibitor can preferentially inhibit plasma kallikrein at least 100, 200, 500, or 1000 more than another kallikrein, e.g., human urine kallikrein, or another protease, e.g., plasmin or thrombin. For example, the inhibitor is other than aprotinin.

In one embodiment, the inhibitor is an agent that can cross the blood-brain barrier.

In one embodiment, the inhibitor includes a polypeptide that includes a Kunitz domain such as the amino acid sequence: Xaa1 Xaa2 Xaa3 Xaa4 Cys Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Gly Xaa13 Cys Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 Gly Gly Cys Xaa39 Xaa40 Xaa41 Xaa42 Xaa43 Xaa44 Xaa45 Xaa46 Xaa47 Xaa48 Xaa49 Xaa50 Cys Xaa52 Xaa53 Xaa54 Cys Xaa56 Xaa57 Xaa58 (SEQ ID NO:1).

The framework of the Kunitz domain can be human or can differ from a human Kunitz domain framework by fewer than six, five, four, three, or two amino acids. For example, the framework of the Kunitz domain can be the framework of one of the Kunitz domains of human lipoprotein-associated coagulation inhibitor (LACI) protein, e.g., the first second or third Kunitz domain. Typically the polypeptide differs from BPTI and/or one or more of the LACI Kunitz domains by at least one, two, three, or four amino acids, e.g., at least one, two or three amino acids in the binding loops and/or at least two, three, four, or six amino acids in the framework region. For example, the polypeptide can include a non-naturally occurring Kunitz domain that is derived from a naturally occurring Kunitz domain, e.g., a human Kunitz domain. In one embodiment, an inhibitor that includes a Kunitz domain binds to plasma kallikrein with an affinity that is at least 10, 100, or 500 fold better than BPTI and/or LACI.

In one embodiment, the polypeptide that inhibits kallikrein is not immunogenic on second use.

In one embodiment, the polypeptide can have one or more of the following features: Xaa1, Xaa2, Xaa3, Xaa4, Xaa56, Xaa57 or Xaa58 are each individually an amino acid or absent; Xaa10 is an amino acid selected from the group consisting of: Asp and Glu; Xaa11 is an amino acid selected from the group consisting of: Asp, Gly, Ser, Val, Asn, Ile, Ala and Thr; Xaa13 is an amino acid selected from the group consisting of: Arg, His, Pro, Asn, Ser, Thr, Ala, Gly, Lys and Gln; Xaa15 is an amino acid selected from the group consisting of: Arg, Lys, Ala, Ser, Gly, Met, Asn and Gln; Xaa16 is an amino acid selected from the group consisting of: Ala, Gly, Ser, Asp and Asn; Xaa17 is an amino acid selected from the group consisting of: Ala, Asn, Ser, Ile, Gly, Val, Gln and Thr; Xaa18 is an amino acid selected from the group consisting of: His, Leu, Gln and Ala; Xaa19 is an amino acid selected from the group consisting of: Pro, Gln, Leu, Asn and Ile; Xaa21 is an amino acid selected from the group consisting of: Trp, Phe, Tyr, His and Ile; Xaa22 is an amino acid selected from the group consisting of: Tyr and Phe; Xaa23 is an amino acid selected from the group consisting of: Tyr and Phe; Xaa31 is an amino acid selected from the group consisting of: Glu, Asp, Gln, Asn, Ser, Ala, Val, Leu, Ile and Thr; Xaa32 is an amino acid selected from the group consisting of: Glu, Gln, Asp Asn, Pro, Thr, Leu, Ser, Ala, Gly and Val; Xaa34 is an amino acid selected from the group consisting of: Thr, Ile, Ser, Val, Ala, Asn, Gly and Leu; Xaa35 is an amino acid selected from the group consisting of: Tyr, Trp and Phe; Xaa39 is an amino acid selected from the group consisting of: Glu, Gly, Ala, Ser and Asp; Xaa40 is an amino acid selected from the group consisting of: Gly and Ala; Xaa43 is an amino acid selected from the group consisting of: Asn and Gly; Xaa45 is an amino acid selected from the group consisting of: Phe and Tyr; and wherein the polypeptide inhibits kallikrein.

In a particular embodiment, individual amino acid positions of SEQ ID NO:1 can be one or more of the following: Xaa10 is Asp, Xaa11 is Asp, Xaa13 is Pro, Xaa15 is Arg, Xaa16 is Ala, Xaa17 is Ala, Xaa18 is His, Xaa19 is Pro, Xaa21 is Trp, Xaa31 is Glu, Xaa32 is Glu, Xaa34 is Ile, Xaa35 is Tyr, Xaa39 is Glu.

The polypeptide can include (or consist of) the following amino acid sequence: Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp (SEQ ID NO:2), or a fragment thereof, e.g., amino acids 3-60 of SEQ ID NO:2 or other fragment that binds and inhibits kallikrein. For example, the polypeptide can have fewer than 80, 70, 65, or 60 amino acids.

In one embodiment, the polypeptide is modified, e.g., to include one or more polymer moieties, e.g., a plurality of polymer moieties, e.g., as described in U.S.S.N. 10/931,153, filed Aug. 30, 2004, bearing attorney docket number 10280-119001. For example, the polypeptide can include a plurality of polyethylene glycol moieties, e.g., one on an N-terminal amine and one attached to each lysine of the polypeptide. The polyethylene glycol moieties can be less than 10, 8, 7, or 6 kDa in average molecular weight. Other exemplary modifications include a label, e.g., a radioactive or MRI-detectable label.

In one embodiment, the inhibitor does not include a peptide or polypeptide. For example, the inhibitor can be small molecule, e.g., a compound described in WO 04/062657, e.g., an acylated 4-amidino- or 4-guanidinobenzylamines. The compound can have the general formula (I) P4-P3-P2-P1 (I), where P4 is a mono- or poly-substituted or unsubstituted benzylsulphonyl group, P3 is a mono- or poly-substituted or unsubstituted, natural or unnatural alpha-amino or alpha-imino acid with the D-configuration, P2 is a mono- or poly-substituted or unsubstituted natural or unnatural alpha-amino or alpha-imino acid with the L-configuration and P1 is a mono- or poly-substituted or unsubstituted 4-amidino- or 4-guanidinobenzylamine group.

The methods described herein can include administering an effective amount of the inhibitor of kallikrein. Such an amount can be an amount sufficient to produce an improvement detectable to one skilled in the art, to ameliorate at least one symptom, or to modulate (e.g., improve) at least one physiological parameter, e.g., to a statistically significant degree.

All patents, patent applications, and publications cited herein are incorporated herein by reference. In the case of conflict, the present application controls.