Synthetic proteins for in vivo drug delivery and tissue augmentation

Abstract

Methods and compositions are provided which are useful for delivering a biologically active substance to a localized site in vivo and for altering the physical dimensions of a body tissue. These methods and compositions employ protein polymers having varying ratios of elastin-like, collagen-like, keratin-like repeating units and repeating units which promote protein crystallization such as silk-like repeating units. By varying the length of segments of the repeating units and/or the concentration of the protein polymers in the composition, the rate of delivery of a biologically active substance to a localized site can be greatly varied. Moreover, because the compositions are capable of acquiring a non-liquid form under normal physiological conditions, they find use as biocompatible tissue augmentation products.

What is claimed is:  
     
         1 . A method for delivering a biologically active substance to a localized site in vivo, said method comprising: 
 administering a composition to said localized site, said composition comprising (i) a protein polymer of at least 15 kD which comprises alternating blocks of at least 2 units each of (a) an amino acid sequence of from about 3 to 30 amino acids which promotes protein crystallization and (b) an amino acid sequence element selected from the group consisting of an elastin-like element, a collagen-like element or a keratin-like element, and (ii) a biologically active substance;    wherein said composition acquires a non-liquid form under physiological conditions and wherein said biologically active substance is delivered from said non-liquid to said localized site.    
     
     
         2 . The method according to  claim 1 , wherein said amino acid sequence which promotes protein crystallization is GAGAGS or SGAGAG.  
     
     
         3 . The method according to  claim 1 , wherein said amino acid sequence element (b) is the amino acid sequence VPGG, APGVGV, GXGVP or VPGXG, where X is valine, lysine, histidine, glutamic acid, arginine, aspartic acid, serine, tryptophan, tyrosine, phenylalanine, leucine, glutamine, asparagine, cysteine or methionine.  
     
     
         4 . The method according to  claim 3 , wherein amino acid X is valine or lysine.  
     
     
         5 . The method according to  claim 1 , wherein the delivery of said biologically active substance occurs over an extended period of time.  
     
     
         6 . The method according to  claim 1 , wherein the step of administering comprises injecting said composition in liquid form which acquires a non-liquid form subsequent to injection.  
     
     
         7 . The method according to  claim 6 , wherein the rate at which said liquid form acquires a non-liquid form is decreased by the addition to said liquid form of a compound which inhibits hydrogen bonding.  
     
     
         8 . The method according to  claim 7 , wherein said compound which inhibits hydrogen bonding is selected from the group consisting of urea, guanidine hydrochloride, dimethyl formamide, colloidal gold sol, aqueous lithium bromide and formic acid.  
     
     
         9 . The method according to  claim 6 , wherein the rate at which said liquid form acquires a non-liquid form is increased by the addition to said liquid form of a nucleating agent or accelerator.  
     
     
         10 . The method according to  claim 9 , wherein said nucleating agent is said protein polymer in precrystallized form.  
     
     
         11 . The method according to  claim 10 , wherein said protein polymer is SLP3 or SLP4.  
     
     
         12 . The method according to  claim 1 , wherein said protein polymer is about 10% (w/w) to about 50% (w/w) of said composition.  
     
     
         13 . The method according to  claim 1 , wherein said biologically active substance is selected from the group consisting of a protein or a nucleic acid.  
     
     
         14 . The method according to  claim 13 , wherein said protein has a molecular weight of from about 350 daltons to about 500,000 daltons.  
     
     
         15 . The method according to  claim 13 , wherein said nucleic acid is from about 60 to about 22,000 bases in length.  
     
     
         16 . The method according to  claim 1 , wherein said biologically active substance is selected from the group consisting of an anti-tumor agent, an analgesic, an antibiotic, an anti-inflammatory compound, a hormone or a vaccine.  
     
     
         17 . The method according to  claim 1 , wherein said biologically active substance is labeled.  
     
     
         18 . The method according to  claim 2 , wherein said amino acid sequence GAGAGS or SGAGAG is repeated from 2 to 16 times per alternating block.  
     
     
         19 . The method according to  claim 1 , wherein said protein polymer comprises an amino acid sequence selected from the group consisting of: 
 (a) [(VPGVG) 8 (GAGAGS) 8 ] 12 ;    (b) [(VPGVG) 12 (GAGAGS) 8 ] 9 ;    (c) [(VPGVG) 16 (GAGAGS) 8 ] 8 ;    (d) [(VPGVG) 32 (GAGAGS) 8 ] 5 ;    (e) [(VPGVG) 8 (GAGAGS) 6 ] 13 ;    (f) [(VPGVG) 8 (GAGAGS) 4 ] 13 ;    (g) [(GVGVP) 4  GKGVP (GVGVP) 3  (GAGAGS) 4 ] 12 ; or    (h) [GAGAGS (GVGVP) 4  GKGVP (GVGVP) 3  (GAGAGS) 2 ] 12 .    
     
     
         20 . A composition comprising (i) a protein polymer of at least 15 kD which comprises alternating blocks of at least 2 units each of (a) an amino acid sequence of from about 3 to 30 amino acids which promotes protein crystallization and (b) an amino acid sequence element selected from the group consisting of an elastin-like element, a collagen-like element or a keratin-like element, and (ii) a biologically active substance; 
 wherein said composition acquires a non-liquid form under physiological conditions.    
     
     
         21 . The composition according to  claim 20 , wherein said amino acid sequence which promotes protein crystallization is GAGAGS or SGAGAG.  
     
     
         22 . The composition according to  claim 20 , wherein said amino acid sequence element (b) is the amino acid sequence VPGG, APGVGV, GXGVP or VPGXG, where X is valine, lysine, histidine, glutamic acid, arginine, aspartic acid, serine, tryptophan, tyrosine, phenylalanine, leucine, glutamine, asparagine, cysteine or methionine.  
     
     
         23 . The composition according to  claim 22 , wherein amino acid X is valine or lysine.  
     
     
         24 . The composition according to  claim 20 , wherein said biologically active substance is selected from the group consisting of a protein or a nucleic acid.  
     
     
         25 . The composition according to  claim 24 , wherein said protein has a molecular weight of from about 350 daltons to about 500,000 daltons.  
     
     
         26 . The composition according to  claim 24 , wherein said nucleic acid is from about 60 to about 22,000 bases in length.  
     
     
         27 . The composition according to  claim 20 , wherein said biologically active substance is selected from the group consisting of an anti-tumor agent, an analgesic, an antibiotic, an anti-inflammatory compound, a hormone or a vaccine.  
     
     
         28 . The composition according to  claim 21 , wherein the amino acid sequence GAGAGS or SGAGAG is repeated from 2 to 16 times per alternating block.  
     
     
         29 . The composition according to  claim 20 , wherein said protein polymer comprises an amino acid sequence selected from the group consisting of: 
 (a) [(VPGVG) 8 (GAGAGS) 8 ] 12 ;    (b) [(VPGVG) 12 (GAGAGS) 8 ] 9 ;    (c) [(VPGVG) 16 (GAGAGS) 8 ] 8 ;    (d) [(VPGVG) 32 (GAGAGS) 8 ] 5 ;    (e) [(VPGVG) 8 (GAGAGS) 6 ] 13 ;    (f) [(VPGVG) 8 (GAGAGS) 4 ] 13 ;    (g) [(GVGVP) 4  GKGVP (GVGVP) 3  (GAGAGS) 4 ] 12 ; or    (h) [GAGAGS (GVGVP) 4  GKGVP (GVGVP) 3  (GAGAGS) 2 ] 12 .    
     
     
         30 . A method for altering the physical dimensions of a body tissue in a mammal, said method comprising: 
 introducing into or onto said body tissue a composition comprising a protein polymer of at least 15 kD which comprises alternating blocks of at least 2 units each of (a) an amino acid sequence of from about 3 to 30 amino acids which promotes protein crystallization and (b) an amino acid sequence element selected from the group consisting of an elastin-like element, a collagen-like element or a keratin-like element;    wherein said composition acquires a non-liquid form under physiological conditions.    
     
     
         31 . The method according to  claim 30 , wherein said amino acid sequence which promotes protein crystallization is GAGAGS or SGAGAG.  
     
     
         32 . The method according to  claim 30 , wherein said amino acid sequence element (b) is the amino acid sequence VPGG, APGVGV, GXGVP or VPGXG, where X is valine, lysine, histidine, glutamic acid, arginine, aspartic acid, serine, tryptophan, tyrosine, phenylalanine, leucine, glutamine, asparagine, cysteine or methionine.  
     
     
         33 . The method according to  claim 32 , wherein amino acid X is valine or lysine.  
     
     
         34 . The method according to  claim 30 , wherein the step of introducing comprises injecting said composition in liquid form which acquires a non-liquid form subsequent to injection.  
     
     
         35 . The method according to  claim 30 , wherein said protein polymer is about 10% (w/w) to about 50% (w/w) of said composition.  
     
     
         36 . The method according to  claim 31 , wherein said amino acid sequence GAGAGS or SGAGAG is repeated from 2 to 16 times per alternating block.  
     
     
         37 . The method according to  claim 30 , wherein said protein polymer comprises an amino acid sequence selected from the group consisting of: 
 (a) [(VPGVG) 8 (GAGAGS) 8 ] 12 ;    (b) [(VPGVG) 12 (GAGAGS) 8 ] 9 ;    (c) [(VPGVG) 16 (GAGAGS) 8 ] 8 ;    (d) [(VPGVG) 32 (GAGAGS) 8 ] 5 ;    (e) [(VPGVG) 8 (GAGAGS) 6 ] 13 ;    (f) [(VPGVG) 8 (GAGAGS) 4 ] 13 ;    (g) [(GVGVP) 4  GKGVP (GVGVP) 3  (GAGAGS) 4 ] 12 ; or    (h) [GAGAGS (GVGVP) 4  GKGVP (GVGVP) 3  (GAGAGS) 2 ] 12 .    
     
     
         38 . The method according to  claim 30 , wherein said composition further comprises a biologically active substance.