Modified ddah polypeptides comprising a pharmacokinetic enhancing moiety, improved pharmacology and their uses

Abstract

Modified DDAH polypeptides and their uses thereof are provided. Exemplary embodiments provide DDAH polypeptides which include one or more amino acid substitutions, additions, or deletions with natural or non-naturally encoded amino acids, and/or linkage to other biologically active molecules including other DDAH polypeptides, as well as PKEM. Additionally, use of said DDAH polypeptides for treatment of disease, such as heart failure or renal disease, is also provided.

What is claimed is: 
     
         1 . A DDAH polypeptide comprising
 an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 22 or fragment thereof; and   a pharmacokinetic enhancing moiety (PKEM) covalently linked to said DDAH polypeptide, wherein said DDAH polypeptide or fragment thereof has a molecular weight of at least about 150 kDa and exhibits asymmetric dimethylarginine (ADMA) metabolizing activity.   
     
     
         2 . The DDAH polypeptide of  claim 1  wherein said DDAH polypeptide comprises the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 17, or an amino acid sequence that differs from SEQ ID NO: 15 or SEQ ID NO: 17 by 1-10 amino acid modifications. 
     
     
         3 . The DDAH polypeptide of  claim 1  wherein said DDAH polypeptide comprises the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence that differs from SEQ ID NO: 17 by 1-10 amino acid modifications. 
     
     
         4 . The DDAH polypeptide of  claim 1  wherein said DDAH polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 15. 
     
     
         5 . The DDAH polypeptide of  claim 1  wherein said PKEM is selected from the group consisting of an acyl group, water soluble polymer, lipid, alkyl group, carbohydrate, polypeptide, polynucleotide, polysaccharide, antibody or antibody fragment, serum albumin, XTEN molecule, or adnectin. 
     
     
         6 . The DDAH polypeptide of  claim 5  wherein said DDAH polypeptide comprises two or more PKEMs covalently linked to said DAAH polypeptide wherein the two or more PKEMs are independently selected from polyethylene glycol (PEG), an acyl group, and an alkyl group. 
     
     
         7 . The DDAH polypeptide of  claim 5  wherein said PKEM is a DDAH polypeptide. 
     
     
         8 . The DDAH polypeptide of  claim 5  wherein said PKEM is an acyl group of the formula: CH 3 (CH 2 ) 12 C(═O)—, CH 3 (CH 2 ) 14 C(═O)—, CH 3 (CH 2 ) 16 C(═O)— or CH 3 (CH 2 ) 18 C(═O)—. 
     
     
         9 . The DDAH polypeptide of  claim 2  wherein said PKEM is polyethylene glycol. 
     
     
         10 . The DDAH polypeptide of  claim 9  wherein said DDAH polypeptide or fragment thereof has a molecular weight of about 250 kDa or greater. 
     
     
         11 . The DDAH polypeptide of  claim 1  wherein said PKEM is linked to the side chain of a lysine or cysteine residue of said DDAH polypeptide. 
     
     
         12 . A pharmaceutical composition comprising the DDAH polypeptide of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         13 . A method of reducing ADMA levels in a patient, said method comprising the steps of administering a modified DDAH polypeptide to a patient in need of ADMA reduction, said modified DDAH polypeptide comprising
 an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17, or an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 17; and   a PMET covalently linked to said amino acid sequence, wherein said modified DDAH has a molecular weight of at least about 150 kDa.   
     
     
         14 . The method of  claim 13  wherein the said DDAH polypeptide comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 15 or SEQ ID NO: 17, or an amino acid sequence that differs from SEQ ID NO: 15 or SEQ ID NO: 17 by 1-5 amino acid modifications. 
     
     
         15 . The method of  claim 14  wherein said DDAH polypeptide comprises two or more PKEMs covalently linked to said DDAH polypeptide, wherein the PKEMs are independently selected from the group consisting of polyethylene glycol (PEG), an acyl group, and an alkyl group. 
     
     
         16 . The method of  claim 15  wherein said PKEM is polyethylene glycol, and said DDAH polypeptide has a molecular weight of about 250 kDa or greater. 
     
     
         17 . A method of treating patients with elevated ADMA levels, said method comprising the steps of
 obtaining a biological sample from a patient;   measuring ADMA levels in the biological sample to identify patients with elevated levels of ADMA;   administering a modified DDAH polypeptide to said patients identified with elevated levels of ADMA in an amount effective to reduce ADMA levels in said patient.   
     
     
         18 . The method of  claim 17  wherein the biological sample is a plasma, urine or saliva sample. 
     
     
         19 . The method of  claim 17  wherein the patient with elevated ADMA levels is suffering from a disease or condition selected from the group consisting of cardiovascular disease, lung disease, fibrotic disease, kidney disease, hypertension, organ failure, sepsis, or COPD, and reduction of ADMA levels alleviates symptoms associated with said disease or condition. 
     
     
         20 . The method of  claim 17  further comprising the co-administration of
 one or more other active compounds selected from: antidiabetics, hypotensive agents, perfusion-enhancing agents, lipid metabolism modulators, antithrombotic agents, antioxidants, chemokine receptor antagonists, p38-kinase inhibitors, NPY agonists, orexin agonists, anorectics, PAF-AH inhibitors, antiphlogistics, COX inhibitors, LTB 4 -receptor antagonists, analgesics, prostacyclin analogs, endothelin receptor antagonist, PDE5 inhibitor, ACE inhibitor, angiotensin receptor antagonist, diuretics and aspirin.