Composition comprising a polymeric reagent
The present invention provides conjugates having a degradable linkage and polymeric reagents useful in preparing such conjugates. The conjugates as well as the polymeric reagents used to form the conjugates include at least one of each the following: an aromatic moiety comprising an ionizable hydrogen atom, a spacer moiety, and a water-soluble polymer. Methods of making polymeric reagents and conjugates, as well as methods for administering conjugates and compositions, are also provided.
1. A method of preparing a polymeric reagent, said method comprising:
a. reacting an N,N-di-protected 2,7-diaminofluorene,
with a formylating agent to form 9-formyl-N,N-di-protected 2,7-diaminofluorene,
b. reducing the formyl group in 9-formyl-N,N-di-protected 2,7-diaminofluorene to form 9-hydroxymethyl-N,N-di-protected 2,7-diaminofluorene,
c. removing the N-protecting groups from 9-hydroxymethyl-N,N-di-protected 2,7-diaminofluorene to form 9-hydroxymethyl-2,7-diaminofluorene,
or a salt form thereof;
d. reacting 9-hydroxymethyl-2,7-diaminofluorene or a salt form thereof with carboxymethyl-methoxy-polyethylene glycol to form 9-hydroxymethyl-2,7-di(methoxypolyethylene glycol)-methylamide)fluorene,
and
e. reacting 9-hydroxymethyl-2,7-di(methoxypolyethylene glycol)-methylamide)fluorene with N-hydroxysuccinimide to form 9-hydroxymethyl-2,7-di(methoxypolyethylene glycol)-methylamide)fluorene-N-hydroxysuccinimide,
2. The method of claim 1 , wherein N,N-di-protected 2,7-diaminofluorene comprises Boc-protecting groups.
3. The method of claim 1 , wherein the formylating reagent of step (a) is ethyl formate.
4. The method of claim 1 , wherein a reducing agent used in reducing step (b) is sodium borohydride.
5. The method of claim 1 , where in step (c), the N-protecting groups are removed by addition of acid.
6. The method of claim 5 , wherein the acid is hydrochloric acid.
7. The method of claim 5 , wherein the product is a salt of 9-hydroxymethyl-2,7-diaminofluorene.
8. The method of claim 7 , wherein the salt of 9-hydroxymethyl-2,7-diaminofluorene is a dihydrochloride salt.
9. The method of claim 1 , wherein the carboxymethyl-methoxy-polyethylene glycol of step (d) is an N-succinimidyl ester.
10. The method of claim 1 , wherein the carboxymethyl-methoxy-polyethylene glycol of step (d) has a molecular weight in a range of about 100 daltons to about 150,000 daltons.
11. The method of claim 10 , wherein the carboxymethyl-methoxy-polyethylene glycol of step (d) has a molecular weight in a range of about greater than 5,000 daltons to about 100,000 daltons.
12. The method of claim 11 , wherein the carboxymethyl-methoxy-polyethylene glycol of step (d) has a molecular weight in a range of about 10,000 daltons to about 85,000 daltons.
13. The method of claim 1 , wherein step (d) results in a reaction mixture comprising 9-hydroxymethyl-2,7-di(methoxypolyethylene glycol)-methylamide)fluorene, and a tri-PEGylated species, 9-methyl-2-methoxypolyethylene glycol acetate-2,7-di(methoxypolyethylene glycol)-methylamide)fluorene.
14. The method of claim 13 , further comprising, prior to step (e), raising the pH of the reaction mixture to about 12, followed by ion-exchange chromatography to recover the 9-hydroxymethyl-2,7-di(methoxypolyethylene glycol)-methylamide)fluorene.
15. The method of claim 14 , wherein raising the pH comprises treating the reaction mixture with a base selected from lithium hydroxide, sodium hydroxide and potassium hydroxide.
16. The method of claim 1 , further comprising:
(f) reacting 9-hydroxymethyl-2,7-di(methoxypolyethylene glycol)-methylamide)fluorene-N-hydroxysuccinimide with an amine-containing biologically active agent to form a conjugate composition.
17. The method of claim 16 , wherein the biologically active agent is a small molecule.
18. The method of claim 16 , wherein the biologically active agent is a polypeptide.
19. The method of claim 18 , wherein the conjugate composition comprises a mixture of conjugates having different polyethylene glycol-to-polypeptide ratios.
20. The method of claim 18 , wherein the carboxymethyl-methoxy-polyethylene glycol of step (d) has a molecular weight of about 20,000 daltons.