Polymer composition for delivering substances in living organisms

• US 6,030,941 A
• Filed: 04/30/1997
• Issued: 02/29/2000
• Est. Priority Date: 05/01/1996
• Status: Expired due to Term

• Current Assignee: AntiVirals, Inc. (Sarepta Therapeutics, Inc.)
• Original Assignee: Avi Biopharma, Inc.
• Inventors: Weller, Dwight D., Summerton, James E.
• Primary Examiner(s): Page, Thurman K.
• Assistant Examiner(s): CHANNAVAJJALA, LAKSHMI SARADA
• Application Number:
  US08/848,844
• Time in Patent Office: 1,035 Days
• Field of Search: 424/401, 514/2, 514/12, 514/21, 514/772, 514/773
• US Class Current: 514/1.2
• CPC Class Codes: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• CPC Class Codes: A61K 47/645 Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT

Abstract

Polymeric compositions effective for delivering compounds in living organisms are described. The compositions include polypeptides which exhibit solubility in both hydrophilic and lipophilic environments by undergoing a reversible pH-dependent transition from a low-pH, lipophilic form to a high-pH, hydrophilic form.

Claims

1. A composition for transporting a compound from a low-pH environment across a lipid layer to a higher-pH aqueous compartment, said composition comprising

(a) a polypeptide containing two or more pairs of side chain carboxyl groups, where

(i) the carboxyl groups of a pair are separated by zero, two or three amino acids,

(ii) the polypeptide contains, at at least one terminus, an initiator sequence consisting of 5-10 amino acid residues, wherein the ratio of non-acid side chain, lipophilic residues to acid side chain residues is greater than 1, to facilitate entry of said terminus into the lipid layer,

(iii) the sequence of the balance of the polypeptide is selected from (EXXXE)n, (XEXXE)n, and (EXEEXEXX)n, where E represents Glu or Asp, X represents a non-acid side chain amino acid, and n is an integer effective to give a total polypeptide length of up to 100 amino acid residues;

whereby the polypeptide exhibits solubility in both hydrophilic and lipophilic environments by undergoing a reversible transition between a lipophilic form, effective to partition from said low-pH environment into the lipid layer, and a hydrophilic form, effective to partition preferentially from the lipid layer into the higher-pH aqueous compartment, and is able to traverse the lipid layer from the low-pH environment to the higher-pH compartment, and

(b) the compound to be transported, covalently attached to the polypeptide.

2. The composition of claim 1, wherein each X represents an amino acid residue which is relatively non-polar and compatible with α

-helix formation.

3. The composition of claim 2, wherein each X represents an amino acid residue selected from the group consisting of phenylalanine, leucine, isoleucine, norleucine, methionine, valine, alanine, norvaline, and α

-amino butyric acid.

4. The composition of claim 3, wherein each X represents an amino acid residue selected from the group consisting of leucine, methionine, alanine, and α

-amino butyric acid.

5. The composition of claim 1, wherein the polypeptide has a defined length of between about 10 and 50 amino acid residues.

6. The composition of claim 1, wherein the composition contains a single attached compound, attached at or near a terminus of the polypeptide.

7. The composition of claim 1, wherein the compound is selected from the group consisting of taxol, cyclosporin, and amphotericin B, and a sequence-specific nucleic acid binding polymer.

8. The composition of claim 7, wherein the compound is a sequence-specific nucleic acid binding polymer.

9. The composition of claim 1, wherein non-acid side chain residues of the polypeptide which are not used for covalent attachment of the transported compound do not contain moieties which are cationic when the polypeptide is in a low-pH α

-helical conformation.

10. The composition of claim 1, wherein the polypeptide includes a group at its C-terminus or N-terminus which covalently bonds to, or includes, the terminal carboxyl or amino group, respectively, and which contains at least one remote hydroxyl or carbonyl group, which is effective to shield, by hydrogen bonding, one or more additional polar groups at or near said terminus.

11. A composition for transporting a compound from a low-pH environment across a lipid layer to a higher-pH aqueous compartment, said composition comprising

(a) a polypeptide containing two or more pairs of side chain carboxyl groups, where

(i) the carboxyl groups of a pair are separated by zero, two or three amino acids,

(ii) the polypeptide has a length of between about 8 and about 100 amino acid residues,

(iii) the polypeptide contains greater than 50% glutamic acid residues, and

(iv) the polypeptide contains, at at least one terminus, an initiator polypeptide sequence consisting of 5-10 amino acid residues, wherein the ratio of non-acid side chain, lipophilic residues to acid side chain residues is greater than 1, which sequence is effective to form an alpha helix at a pH higher than the pH at which a same-length polyglutamic acid forms an alpha helix, to facilitate entry of said terminus into the lipid layer,

whereby the polypeptide exhibits solubility in both hydrophilic and lipophilic environments by undergoing a reversible transition between a lipophilic form, effective to partition from said low-pH environment into the lipid layer, and a hydrophilic form, effective to partition preferentially from the lipid layer into the higher-pH aqueous compartment, and is able to traverse the lipid layer from the low-pH environment to the higher-pH compartment, and

(b) the compound to be transported, covalently attached to the polypeptide.

12. The composition of claim 11, wherein the polypeptide includes a group at its C-termiinus or N-terminus which covalently bonds to, or includes, the terminal carboxyl or amino group, respectively, and which contains at least one remote hydroxyl or carbonyl group, which is effective to shield, by hydrogen bonding, one or more additional polar groups at or near said terminus.

13. The composition of claim 12, wherein said group at the C- or N-terminus is selected from a β

-alanine moiety at the C-terminus, used to initiate the synthesis of the polypeptide, a dicarboxylic acid at the N-terminus, used to terminate the polypeptide, and a combination of the two.

14. The composition of claim 11, wherein the amino acid residues in said initiator sequence are selected from the group consisting of glutamic acid, leucine, methionine, alanine, 2-aminobutyric acid, norvaline, and β

-alanine, wherein the ratio of non-glutamic acid to glutamic acid residues is greater than 1.

15. A method of facilitating the transport of a compound from a low-pH environment across a lipid layer to a higher-pH aqueous compartment, comprising

(a) forming a polypeptide-compound conjugate by coupling the compound covalently to a polypeptide containing two or more pairs of side chain carboxyl groups, where

(i) the carboxyl groups of a pair are separated by zero, two or three amino acids,

(ii) the polypeptide contains, at at least one terminus, an initiator sequence consisting of 5-10 amino acid residues, wherein the ratio of non-acid side chain, lipophilic residues to acid side chain residues is greater than 1, to facilitate entry of said terminus into the lipid layer,

(iii) the sequence of the balance of the polypeptide is selected from (EXXXE)n, (XEXXE)n, and (EXEEXEXX)n, where E represents Glu or Asp, X represents a non-acid side chain amino acid, and n is an integer effective to give a total polypeptide length of up to 100 amino acid residues;

whereby the polypeptide exhibits solubility in both hydrophilic and lipophilic environments by undergoing a reversible transition between a lipophilic form, effective to partition from said low-pH environment into the lipid layer, and a hydrophilic form, effective to partition preferentially from the lipid layer into the higher-pH aqueous compartment; and

(b) introducing the polypeptide-compound conjugate to the low-pH environment,

whereby the polypeptide-compound conjugate is able to traverse the lipid layer from the low-pH environment to the higher-pH compartment.

16. The method of claim 15, wherein each X represents an amino acid residue which is relatively non-polar and compatible with α

-helix formation.

17. The method of claim 16, wherein each X represents an amino acid residue selected from the group consisting of phenylalanine, leucine, isoleucine, norleucine, methionine, valine, alanine, norvaline, and α

-amino butyric acid.

18. A method for facilitating the transport of a compound from a low-pH environment across a lipid layer to a higher-pH aqueous compartment, said composition comprising

(a) forming a polypeptide-compound conjugate by coupling the compound covalently to a polypeptide containing two or more pairs of side chain carboxyl groups, where

(i) the carboxyl groups of a pair are separated by zero, two or three amino acids,

(ii) the polypeptide has a length of between about 8 and about 100 amino acid residues,

(iii) the polypeptide contains greater than 50% glutamic acid residues, and

(iv) the polypeptide contains, at at least one terminus, an initiator polypeptide sequence consisting of 5-10 amino acid residues, wherein the ratio of non-acid side chain, lipophilic residues to acid side chain residues is greater than 1, which sequence is effective to form an alpha helix at a pH higher than the pH at which a same-length polyglutamic acid forms an alpha helix, to facilitate entry of said terminus into the lipid layer,

whereby the polypeptide exhibits solubility in both hydrophilic and lipophilic environments by undergoing a reversible transition between a lipophilic form, effective to partition from said low-pH environment into the lipid layer, and a hydrophilic form, effective to partition preferentially from the lipid layer into the higher-pH aqueous compartment, and is able to traverse the lipid layer from the low-pH environment to the higher-pH compartment;

and

(b) introducing the polypeptide-compound conjugate to the low-pH environment,

whereby the polypeptide-compound conjugate is able to traverse the lipid layer from the low-pH environment to the higher-pH compartment.

19. The method of claim 18, wherein the polypeptide includes a group at the C-terminus or the N-terminus which covalently bonds to, or includes, the terminal carboxyl or amino group, respectively, and which contains at least one remote hydroxyl or carbonyl group, which is effective to shield, by hydrogen bonding, one or more additional polar groups at or near the polypeptide terminus.

20. The method of claim 19, wherein said group at the C- or N-terminus is selected from a β

-alanine moiety at the C-terminus, used to initiate the synthesis of the polypeptide, a dicarboxylic acid at the N-terminus, used to terminate the polypeptide, and a combination of the two.