Poly-Pegylated Protease Inhibitors

1 . A compound comprising:

(i) a Kunitz domain polypeptide that comprises a Kunitz domain that binds to and inhibits a protease; and

(ii) a plurality of polyethylene glycol moieties attached to the Kunitz domain polypeptide, wherein the average molecular weight of each of the moieties is less than 12 kDa, and each accessible primary amine of the Kunitz domain polypeptide is attached to one of the moieties.

2 - 22 . (canceled)

23 . A preparation that comprises Kunitz domain polypeptides that specifically bind and inhibit a protease, wherein at least 80% of the Kunitz domain polypeptides in the preparation

(i) bind and inhibit the protease, and

(ii) have a polyethylene glycol moiety attached at a first common site and a polyethylene glycol moiety attached at a second common site and wherein the average molecular weight of each of the attached polyethylene glycol moieties is less than 12 kDa or have a plurality of polyethylene glycol moieties attached to said Kunitz domain and wherein the average molecular weight of each of the attached polyethylene glycol moieties is less than 12 kDa.

24 - 51 . (canceled)

52 . A preparation that comprises Kunitz domain polypeptides that comprise

(i) the amino acid sequence of DX-890, wherein at least 80% of the DX-890-containing Kunitz domain polypeptides in the preparation have a polyethylene glycol moiety attached to each of four lysine residues and to the N-terminus of the polypeptide;

(ii) the amino acid sequence of DX-88, wherein at least 80% of the DX-88-containing Kunitz domain polypeptides in the preparation have a polyethylene glycol moiety attached to each of three lysine residues and to the N-terminus of the polypeptide; or

(iii) the amino acid sequence of DX-1000, wherein at least 80% of the DX-1000-containing Kunitz domain polypeptides in the preparation have a polyethylene glycol moiety attached to each of three lysine residues and to the N-terminus of the polypeptide.

53 - 54 . (canceled)

55 . A method of providing a pegylated Kunitz domain, the method comprising:

providing a polypeptide that comprises a Kunitz domain that has at least one lysine in the framework region of the Kunitz domain; and

contacting the polypeptide with activated polyethylene glycol, of average molecular weight less than 12 kDa, under conditions in which a plurality of polyethylene glycol moieties are attached to the polypeptide, at least one of which is attached to the lysine and at least one is attached to the N-terminal primary amine.

56 . A method of providing a PEGylated Kunitz domain, the method comprising:

providing a polypeptide that comprises a Kunitz domain that has at least two primary amine groups in the framework region of the Kunitz domain; and

contacting the polypeptide with activated polyethylene glycol, of average molecular weight less than 12 kDa, under conditions in which a plurality of polyethylene glycol moieties are attached to the polypeptide.

57 - 67 . (canceled)

68 . A method of treating a disorder characterized by excessive or undesired activity of a protease, the method comprising, administering to a subject having the disorder or suspected of having the disorder to pharmaceutical composition comprising the preparation of claim 1 , wherein the Kunitz domain polypeptide of the preparation inhibits the protease.

69 - 74 . (canceled)

75 . A preparation comprising molecules that comprise:

(i) a Kunitz domain polypeptide that comprises a Kunitz domain that binds to and inhibits a protease, and

(ii) a plurality of polyethylene glycol moieties attached to the Kunitz domain polypeptide, wherein the average molecular weight of each of the moieties is less than 12 kDa.

76 - 77 . (canceled)

78 . A method of treating a disorder characterized by excessive or undesired activity of a protease, the method comprising, administering to a subject having the disorder or suspected of having the disorder to pharmaceutical composition comprising the preparation of claim 75 , wherein the Kunitz domain polypeptide of the preparation inhibits the protease.