Methods for preparing purified polypeptide compositions

Abstract

The present invention relates to purified peptidomimetic macrocycles. The invention additionally provides methods of preparing and using such macrocycles, for example in therapeutic applications.

1 .- 38 . (canceled) 
     
     
         39 . A method of treating a disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a pharmaceutically acceptable composition; wherein the pharmaceutically acceptable composition comprises:
 a peptidomimetic macrocycle or a pharmaceutically acceptable salt thereof comprising an alpha-helix and a cross-linker connecting a first amino acid and a second amino acid, the cross-linker spanning from 1 turn to 5 turns of the alpha-helix; and   a metal residue at a concentration of less than 75 ppm by weight.   
     
     
         40 . The method of  claim 39 , wherein the subject is a human. 
     
     
         41 . The method of  claim 39 , wherein the pharmaceutically acceptable composition is suitable for administration to a human subject. 
     
     
         42 . The method of  claim 39 , wherein the metal residue comprises ruthenium or osmium. 
     
     
         43 . The method of  claim 39 , wherein the metal residue comprises copper. 
     
     
         44 . The method of  claim 39 , wherein at least one of the first and second amino acids is an α,α-disubstituted amino acid. 
     
     
         45 . The method of  claim 39 , wherein the first amino acid and the second amino acid are separated by three amino acids. 
     
     
         46 . The method of  claim 39 , wherein the cross-linker comprises from 6 to 14 consecutive bonds. 
     
     
         47 . The method of  claim 39 , wherein the cross-linker comprises from 8 to 12 consecutive bonds. 
     
     
         48 . The method of  claim 39 , wherein the first amino acid and the second amino acid are separated by six amino acids. 
     
     
         49 . The method of  claim 39 , wherein the cross-linker comprises from 8 to 16 consecutive bonds. 
     
     
         50 . The method of  claim 39 , wherein the cross-linker comprises from 10 to 13 consecutive bonds. 
     
     
         51 . The method of  claim 39 , wherein the cross-linker spans 1 or 2 turns of the alpha helix. 
     
     
         52 . The method of  claim 39 , wherein the length of the cross-linker is about 5 Å to about 9 Å per turn of the alpha-helix. 
     
     
         53 . The method of  claim 39 , wherein the peptidomimetic macrocycle or the pharmaceutically acceptable salt thereof carries a net positive charge at pH 7.4. 
     
     
         54 . The method of  claim 39 , wherein the disease is mediated by p53 and/or MDM2. 
     
     
         55 . The method of  claim 39 , wherein the disease is a hyperproliferative disease or a cancer. 
     
     
         56 . The method of  claim 55 , wherein the cancer is a fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, uterine cancer, cancer of the head and neck, skin cancer, brain cancer, squamous cell carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular cancer, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposi sarcoma. 
     
     
         57 . The method of  claim 55 , wherein the cancer is breast cancer, lung cancer, liver cancer, colon cancer or ovarian cancer. 
     
     
         58 . The method of  claim 39 , wherein the disease is a disorder associated with an undesirable level of cell death or a disorder that lead to cell death associated with viral infection. 
     
     
         59 . The method of  claim 58 , wherein the disease is an infection associated with infection with human immunodeficiency virus (HIV). 
     
     
         60 . The method of  claim 39 , wherein the disease is selected from a group consisting of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), retinitis pigmentosa, spinal muscular atrophy, and various forms of cerebellar degeneration. 
     
     
         61 . The method of  claim 39 , wherein the disease is hematologic diseases associated with a decreased production of blood cells. 
     
     
         62 . The method of  claim 39 , wherein the disease is a BCL-2 family member mediated disease.