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 overactive bladder, comprising administering into the bladder of the subject an effective amount of an antisense oligonucleotide directed against nerve growth factor in combination with protamine in an amount that promotes the uptake of the antisense oligonucleotide into bladder cells, wherein the antisense oligonucleotide and the protamine form a non-covalent attachment, and wherein the mass ratio of protamine to antisense oligonucleotide directed against nerve growth factor is 5:1 to 15:1 respectively. 
     
     
         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 the antisense oligonucleotide toward nerve growth factor in combination with protamine 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 , where the oligonucleotide inhibits expression of nerve growth factor. 
     
     
         4 . The method of any of the  claim 1 , where the protamine 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 RRRX1 X2 RRR where X1 or X2 or both is/are G or V or A (SEQ ID NO:8). 
 
     
     
         5 . The method of  claim 1 , where the mass ratio of protamine to antisense oligonucleotide directed against nerve growth factor is 10:1 respectively. 
     
     
         6 . The method of  claim 1 , where the mass ratio of protamine to antisense oligonucleotide directed against nerve growth factor is 5:1 respectively. 
     
     
         7 . The method of  claim 1 , comprising the antisense oligonucleotide directed against nerve growth factor in a concentration range between 2 to 20 μM. 
     
     
         8 . The method of  claim 1 , comprising protamine in a concentration range between 20 to 200 μM. 
     
     
         9 . The method of  claim 1 , comprising the antisense oligonucleotide directed against nerve growth factor in a concentration range between 2 to 20 μM and protamine in a concentration range between 20 to 200 μM. 
     
     
         10 . The method of  claim 1 , 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  
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         11 . The method of  claim 1 , where the antisense oligonucleotide directed against nerve growth factor comprises the following sequence: GCCCGAGACGCCTCCCGA (SEQ ID NO: 10).