Poly-pegylated protease inhibitors

Abstract

Disclosed are compounds 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. Each accessible primary amine of the Kunitz domain polypeptide can be attached to one of the moieties. Also disclosed are related methods.

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 . The compound of  claim 1  wherein the average molecular weight of each of the moieties is less than 8 kDa.  
     
     
         3 . The compound of  claim 1  wherein each moiety has a molecular weight between 3-8 kDa.  
     
     
         4 . The compound of  claim 1  wherein the Kunitz domain polypeptide has a molecular weight less than 8. kDa, and the compound has a molecular weight greater than 16 kDa.  
     
     
         5 . The compound of  claim 1  wherein the plurality of polyethylene glycol moieties consists of four or five moieties, each attached to a different accessible primary amine.  
     
     
         6 . The compound of  claim 1  wherein each lysine is coupled to one of the moieties of the plurality.  
     
     
         7 . The compound of  claim 6  wherein the Kunitz domain polypeptide comprises an N-terminal primary amine, and each lysine and the N-terminal primary amine is coupled to one of the moieties.  
     
     
         8 . The compound of  claim 1  wherein the Kunitz domain polypeptide does not include a lysine in the Kunitz domain binding loops.  
     
     
         9 . The compound of  claim 1  wherein the Kunitz domain polypeptide includes at least two lysines in the framework region of the Kunitz domain.  
     
     
         10 . The compound of  claim 1  wherein the Kunitz domain polypeptide comprises three lysines in the framework region of the Kunitz domain.  
     
     
         11 . The compound of  claim 10  wherein the Kunitz domain polypeptide comprises four lysines in the framework region of the Kunitz domain.  
     
     
         12 . The compound of  claim 1  wherein the Kunitz domain polypeptide comprises a framework region that is identical to a corresponding region of a human Kunitz domain.  
     
     
         13 . The compound of  claim 12  wherein the Kunitz domain polypeptide comprises a framework region that is identical to corresponding residues in a LACI Kunitz domain or an ITI Kunitz domain.  
     
     
         14 . The compound of  claim 1  wherein the protease is elastase.  
     
     
         15 . The compound of  claim 14  wherein Kunitz domain polypeptide comprises the amino acid sequence of DX-890 or a sequence that differs by at least one, but fewer than six amino acid alterations from DX-890.  
     
     
         16 . The compound of  claim 14  wherein the Kunitz domain polypeptide comprises the amino acid sequence of the binding loops of DX-890.  
     
     
         17 . The compound of  claim 1  wherein the protease is kallikrein.  
     
     
         18 . The compound of  claim 17  wherein Kunitz domain polypeptide comprises the amino acid sequence of DX-88 or a sequence that differs by at least one, but fewer than six amino acid alterations from DX-88.  
     
     
         19 . The compound of  claim 17  wherein Kunitz domain polypeptide comprises the amino acid sequence of the binding loops of DX-88.  
     
     
         20 . The compound of  claim 1  wherein the protease is plasmin.  
     
     
         21 . The compound of  claim 20  wherein Kunitz domain polypeptide comprises the amino acid sequence of DX-1000 or a sequence that differs by at least one, but fewer than six amino acid alterations from DX-1000.  
     
     
         22 . The compound of  claim 20  wherein Kunitz domain polypeptide comprises the amino acid sequence of the binding loops of DX-1000.  
     
     
         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.  
     
     
         24 . The preparation of  claim 23  wherein the average molecular weight of each of the attached polyethylene glycol moieties is less than 10 kDa.  
     
     
         25 . The preparation of  claim 24  wherein the average molecular weight of each of the attached polyethylene glycol moieties is less than 8 kDa.  
     
     
         26 . The preparation of  claim 23  wherein at least 95% of the Kunitz domain polypeptides in the preparation have a polyethylene glycol moiety attached at the first common site and a polyethylene glycol moiety attached at the second common site.  
     
     
         27 . The preparation of  claim 23  wherein the at least 80% of the Kunitz domain polypeptides in the preparation further have a polyethylene glycol moiety attached at the third common site.  
     
     
         28 . The preparation of  claim 23  wherein the at least 80% of the Kunitz domain polypeptides in the preparation further have a polyethylene glycol moiety attached at the third common site and a polyethylene glycol moiety attached at the fourth common site.  
     
     
         29 . The preparation of  claim 23  wherein at least 80% of the Kunitz domain polypeptides in the preparation have a polyethylene glycol moiety attached at the third common site, a polyethylene glycol moiety attached at the fourth common site, and a polyethylene glycol moiety attached at the fifth common site.  
     
     
         30 . The preparation of  claim 23  wherein each of the Kunitz domain polypeptides in the preparation binds and inhibits the protease.  
     
     
         31 . The preparation of  claim 23  wherein 95% of the Kunitz domain polypeptides in the preparation binds and inhibits the protease.  
     
     
         32 . The preparation of  claim 23  wherein the at least 80% of the Kunitz domain polypeptides in the preparation have a polyethylene glycol moiety attached to each accessible primary amine.  
     
     
         33 . The preparation of  claim 32  wherein, with respect to the at least 80% of the Kunitz domain polypeptides in the preparation, each lysine is coupled to one of the moieties.  
     
     
         34 . The preparation of  claim 32  wherein, with respect to the at least 80% of the Kunitz domain polypeptides in the preparation, each lysine and the N-terminal primary amine is coupled to one of the moieties.  
     
     
         35 . The preparation of  claim 23  wherein the first common site is at an N-terminal primary amine and the second common site is at a lysine.  
     
     
         36 . The preparation of  claim 23  wherein the Kunitz domain polypeptides that specifically bind and inhibit the protease do not include a lysine in the Kunitz domain binding loops.  
     
     
         37 . The preparation of  claim 23  wherein the Kunitz domain polypeptides that specifically bind and inhibit the protease include at least two lysines in the framework region of the Kunitz domain.  
     
     
         38 . The preparation of  claim 23  wherein the Kunitz domain polypeptides that specifically bind and inhibit the protease include three lysines in the framework region of the Kunitz domain.  
     
     
         39 . The preparation of  claim 23  wherein the Kunitz domain polypeptides that specifically bind and inhibit the protease polypeptide include four lysines in the framework region of the Kunitz domain.  
     
     
         40 . The preparation of  claim 23  wherein the Kunitz domain polypeptides that specifically bind and inhibit the protease comprise a framework region that is identical to a corresponding region of a human Kunitz domain.  
     
     
         41 . The preparation of  claim 23  wherein the Kunitz domain polypeptides that specifically bind and inhibit the protease comprise a framework region that is identical to a LACI Kunitz domain or an ITI Kunitz domain.  
     
     
         42 . The preparation of  claim 23  wherein the protease is elastase.  
     
     
         43 . The preparation of  claim 42  wherein, with respect to the at least 80% of the Kunitz domain polypeptides in the preparation, the polypeptide comprises the amino acid sequence of DX-890 or a sequence that differs by at least one, but fewer than six amino acid alterations from DX-890.  
     
     
         44 . The preparation of  claim 42  wherein, with respect to the at least 80% of the Kunitz domain polypeptides in the preparation, the polypeptide comprises the amino acid sequence of the binding loops of DX-890.  
     
     
         45 . The preparation of  claim 23  wherein the protease is kallikrein.  
     
     
         46 . The preparation of  claim 45  wherein, with respect to the at least 80% of the Kunitz domain polypeptides in the preparation, the polypeptide comprises the amino acid sequence of DX-88 or a sequence that differs by at least one, but fewer than six amino acid alterations from DX-88.  
     
     
         47 . The preparation of  claim 45  wherein, with respect to the at least 80% of the Kunitz domain polypeptides in the preparation, the polypeptide comprises the amino acid sequence of the binding loops of DX-88.  
     
     
         48 . The preparation of  claim 23  wherein the protease is plasmin.  
     
     
         49 . The preparation of  claim 48  wherein, with respect to the at least 80% of the Kunitz domain polypeptides in the preparation, the polypeptide comprises the amino acid sequence of DX-1000 or a sequence that differs by at least one, but fewer than six amino acid alterations from DX-1000.  
     
     
         50 . The preparation of  claim 48  wherein, with respect to the at least 80% of the Kunitz domain polypeptides in the preparation, the polypeptide comprises the amino acid sequence of the binding loops of DX-1000.  
     
     
         51 . 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 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.  
     
     
         52 . A preparation that comprises Kunitz domain polypeptides that comprise 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.  
     
     
         53 . A preparation that comprises Kunitz domain polypeptides that comprise 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.  
     
     
         54 . A preparation that comprises Kunitz domain polypeptides that comprise 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.  
     
     
         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 . The method of  claim 56  wherein the framework comprises at least two lysines.  
     
     
         58 . The method of  claim 57  wherein the primary amine of each lysine of the polypeptide is attached to a polyethylene glycol moiety.  
     
     
         59 . The method of  claim 56  wherein the yield is greater than 40%.  
     
     
         60 . The method of  claim 56  wherein each accessible primary amine is attached to PEG moiety.  
     
     
         61 . The method of  claim 56  wherein the conditions for contacting are a pH greater than 7.5.  
     
     
         62 . The method of  claim 56  wherein a plurality of polypeptides are provided, and each polypeptide of the plurality becomes attached to a polyethylene glycol moiety at a first common site that is at a lysine and at a second common site that is the N-terminal primary amine.  
     
     
         63 . The method of  claim 56  wherein the conditions are such that at least 70% of the molecules are pegylated on each accessible primary amine.  
     
     
         64 . The method of  claim 56  wherein the conditions are such that at least 85% of the molecules are pegylated on each accessible primary amine.  
     
     
         65 . The method of  claim 56  wherein the conditions are such that at least 70% of the pegylated molecules have the same number of attached PEG moieties, the moieties being attached at the same positions.  
     
     
         66 . The method of  claim 65  wherein the conditions are such that at least 85% of the pegylated molecules have the same number of attached PEG moieties, the moieties being attached at the same positions.  
     
     
         67 . The method of  claim 56  further comprising formulating the pegylated polypeptide as a pharmaceutical composition.  
     
     
         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 . The method of  claim 68  wherein the protease is elastase and the Kunitz domain polypeptide comprises the amino acid sequence of DX-890 or a sequence that differs by at least one, but fewer than six amino acid alterations from DX-890.  
     
     
         70 . The method of  claim 69  wherein the disorder is cystic fibrosis, COPD, or an inflammatory disorder.  
     
     
         71 . The method of  claim 68  wherein the protease is kallikrein and the Kunitz domain polypeptide comprises the amino acid sequence of DX-88 or a sequence that differs by at least one, but fewer than six amino acid alterations from DX-88.  
     
     
         72 . The method of  claim 71  wherein the disorder is hemophilia, post-operative bleeding, peri-operative bleeding, or hereditary angioedema.  
     
     
         73 . The method of  claim 68  wherein the protease is plasmin and the Kunitz domain polypeptide comprises the amino acid sequence of DX-1000 or a sequence that differs by at least one, but fewer than six amino acid alterations from DX-1000.  
     
     
         74 . The method of  claim 73  wherein the disorder is fibrinolysis or fibrinogenolysis, excessive bleeding associated with thrombolytics, post-operative bleeding, peri-operative bleeding, and inappropriate androgenesis.  
     
     
         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 . The preparation of  claim 75  wherein at least 50% of the Kunitz domains have polyethylene glycol moieties attached to two or more sites and the average molecular weight of each of the polyethylene glycol moieties is less than 12 kDa.  
     
     
         77 . The preparation of  claim 76  wherein at least 50% of the Kunitz domains have polyethylene glycol moieties attached to three or more sites and the average molecular weight of each of the polyethylene glycol moieties is less than 12 kDa.  
     
     
         78 . The preparation of  claim 76  wherein at least 50% of the Kunitz domains have polyethylene glycol moieties attached to four or more sites and the average molecular weight of each of the polyethylene glycol moieties is less than 12 kDa.  
     
     
         79 . 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.