Peptide-mediated intravesical delivery of therapeutic and diagnostic agents

Abstract

The present invention relates to compositions comprising therapeutic and/or diagnostic anionic agents together with cationic peptides and their use in methods for delivering the anionic agents to bladder cells.

What is claimed: 
     
         1 . A method of treating a subject with an anionic therapeutic agent, comprising administering into the bladder of the subject an effective amount of an anionic therapeutic agent in combination with a cationic peptide(s) in an amount that promotes the uptake of the therapeutic agent into bladder cells. 
     
     
         2 . The method of  claim 1  which is a method of treating a subject with overactive bladder, comprising administering into the bladder of the subject a therapeutic amount of an antisense oligonucleotide toward nerve growth factor in combination with a cationic peptide(s) in an amount that promotes the uptake of the antisense oligonucleotide into bladder cells to reduce the level of nerve growth factor and reduce the frequency of urination and/or feeling of urgency to urinate. 
     
     
         3 . The method of  claim 1 , in which the cationic peptide(s) is a mixture of cationic peptides as comprised in a protamine solution. 
     
     
         4 . The method of  claim 1 , where the anionic therapeutic agent is an oligonucleotide. 
     
     
         5 . The method of  claim 4 , where the oligonucleotide inhibits expression of nerve growth factor. 
     
     
         6 . The method of  claim 1 , in which the cationic peptide(s) is a mixture of cationic peptides as comprised in a protamine solution and the anionic therapeutic agent is an antisense oligonucleotide directed against nerve growth factor. 
     
     
         7 . The method of any of the  claims 6 , where the cationic peptide(s) comprises one or more of the following sequences:
                     (SEQ ID NO: 1)         MARYRCCRSQ SRSRYYRQRQ RSRRRRRRSC QTRRRAMRCC         RPRYRPRCRR H;                   (SEQ ID NO: 2)         MVRYRVRSLS ERSHEVYRQQ LHGQEQGHHG QEEQGLSPEH         VEVYERTHGQ SHYRRRHCSR RRLHRIHRRQ HRSCRRRKRR         SCRHRRRHRR GCRTRKRTCR RH;                   (SEQ ID NO: 3)         MRRQASLPAR RRRRVRRTRV VRRRRRVGRR RH;                   (SEQ ID NO: 4)         PRRRRRSSSR PIRRRRRPRAS RRRRRGGRRR R;                   (SEQ ID NO: 5)         PRRRRSSRRP VRRRRRPRVSR RRRRGGRRRR;                   (SEQ ID NO: 6)         PRRRRSSSRP VRRRRRPRVSR RRRRGGRRRR;                   (SEQ ID NO: 7)         PRRRRASRRI RRRRRPRVSR RRRRGGRRRR;                                         and/or   a peptide comprising the sequence RRRX 1  X 2  RRR where X 1  or X 2  or both is/are G or V or A (SEQ ID NO:8).   
     
     
         8 . The method of  claim 6 , where the mass ratio of protamine to antisense oligonucleotide directed against nerve growth factor is between about 5:1 to about 15:1 respectively. 
     
     
         9 . The method of  claim 6 , where the mass ratio of protamine to antisense oligonucleotide directed against nerve growth factor is about 10:1 respectively. 
     
     
         10 . The method of  claim 6 , where the mass ratio of protamine to antisense oligonucleotide directed against nerve growth factor is about 5:1 respectively. 
     
     
         11 . The method of  claim 6 , comprising the antisense oligonucleotide directed against nerve growth factor in a concentration range between about 2 to about 20 μM. 
     
     
         12 . The method of  claim 6 , comprising protamine in a concentration range between about 20 to about 200 μM. 
     
     
         13 . The method of  claim 6 , comprising the antisense oligonucleotide directed against nerve growth factor in a concentration range between about 2 to about 20 μM and protamine in a concentration range between about 20 to about 200 μM. 
     
     
         14 . The method of  claim 6 , where the antisense oligonucleotide directed against nerve growth factor encoded by a mRNA having a corresponding cDNA sequence as follows: 
       
         
           
                 
                 
               
                   [SEQ ID NO: 9] 
                     
                 
                 
                 
                 
               
                      1 
                   tctgctttta ataagcttcc caatcagctc tcgagtgcaa agcgctctcc ctccctcgcc 
                     
                 
                     
                 
                     61 
                   cagccttcgt cctcctggcc cgctcctctc atccctccca ttctccattt cccttccgtt 
                 
                     
                 
                    121 
                   ccctccctgt cagggcgtaa ttgagtcaaa ggcaggatca ggttccccgc cttccagtcc 
                 
                     
                 
                    181 
                   aaaaatcccg ccaagagagc cccagagcag aggaaaatcc aaagtggaga gaggggaaga 
                 
                     
                 
                    241 
                   aagagaccag tgagtcatcc gtccagaagg cggggagagc agcagcggcc caagcaggag 
                 
                     
                 
                    301 
                   ctgcagcgag ccgggtacct ggactcagcg gtagcaacct cgccccctgc aacaaaggca 
                 
                     
                 
                    361 
                   gactgagcgc cagagaggac gtttccaact caaaaatgca ggctcaacag taccagcagc 
                 
                     
                 
                    421 
                   agcgtcgaaa atttgcagct gccttcttgg cattcatttt catactggca gctgtggata 
                 
                     
                 
                    481 
                   ctgctgaagc agggaagaaa gagaaaccag aaaaaaaagt gaagaagtct gactgtggag 
                 
                     
                 
                    541 
                   aatggcagtg gagtgtgtgt gtgcccacca gtggagactg tgggctgggc acacgggagg 
                 
                     
                 
                    601 
                   gcactcggac tggagctgag tgcaagcaaa ccatgaagac ccagagatgt aagatcccct 
                 
                     
                 
                    661 
                   gcaactggaa gaagcaattt ggcgcggagt gcaaatacca gttccaggcc tggggagaat 
                 
                     
                 
                    721 
                   gtgacctgaa cacagccctg aagaccagaa ctggaagtct gaagcgagcc ctgcacaatg 
                 
                     
                 
                    781 
                   ccgaatgcca gaagactgtc accatctcca agccctgtgg caaactgacc aagcccaaac 
                 
                     
                 
                    841 
                   ctcaagcaga atctaagaag aagaaaaagg aaggcaagaa acaggagaag atgctggatt 
                 
                     
                 
                    901 
                   aaaagatgtc acctgtggaa cataaaaagg acatcagcaa acaggatcag ttaactattg 
                 
                     
                 
                    961 
                   catttatatg taccgtaggc tttgtattca aaaattatct atagctaagt acacaataag 
                 
                     
                 
                   1021 
                   caaaaacaa  
                 
             
                
               
            
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         15 . The method of  claim 6 , where the antisense oligonucleotide directed against nerve growth factor comprises the following sequence: 
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 10) 
                 
                     
                   GCCCGAGACGCCTCCCGA.