CONJUGATES OF A CHOLINESTERASE MOIETY AND A POLYMER
Conjugates of a cholinesterase moiety and one or more nonpeptidic, water-soluble polymers are provided. Typically, the nonpeptidic, water-soluble polymer is poly(ethylene glycol) or a derivative thereof. Also provided, among other things, are compositions comprising conjugates, methods of making conjugates, and methods of administering compositions to a patient.
1 . A conjugate comprising a residue of a cholinesterase moiety covalently attached to a water-soluble polymer, wherein the residue of the cholinesterase moiety is covalently attached to the water-soluble polymer through a cysteine residue within the residue of the cholinesterase moiety.
2 . A conjugate comprising a residue of a cholinesterase moiety covalently attached to a water-soluble polymer, wherein the water-soluble polymer, prior to being covalently attached, is a polymeric reagent bearing a maleimide group.
3 . The conjugate of claim 1 , wherein the water-soluble polymer is a branched water-soluble polymer.
4 . The conjugate of claim 1 , wherein the cholinesterase moiety is acetylcholinesterase.
5 . The conjugate of claim 1 , wherein the cholinesterase moiety is butyrylcholinesterase.
6 . The conjugate of claim 1 , wherein the cholinesterase moiety is recombinantly prepared.
7 . The conjugate of claim 1 , wherein the water-soluble polymer is a polymer selected from the group consisting of poly(alkylene oxide), poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline, and poly(acryloylmorpholine).
8 . The conjugate of claim 7 , wherein the water-soluble polymer is a poly(alkylene oxide).
9 . The conjugate of claim 8 , wherein the poly(alkylene oxide) is a poly(ethylene glycol).
10 . The conjugate of claim 9 , wherein the poly(ethylene glycol) is terminally capped with an end-capping moiety selected from the group consisting of hydroxy, alkoxy, substituted alkoxy, alkenoxy, substituted alkenoxy, alkynoxy, substituted alkynoxy, aryloxy and substituted aryloxy.
11 . The conjugate of claim 1 , wherein the water-soluble polymer has a weight-average molecular weight in a range of from about 500 Daltons to about 100,000 Daltons.
12 . The conjugate of claim 1 , wherein the cysteine residue within the residue of the cholinesterase moiety corresponds to Cys66 of butyrylcholinesterase.
13 . The conjugate of claim 2 , wherein the polymeric reagent bearing a maleimide group has the following structure:
wherein:
X is a spacer moiety comprised of one or more atoms; and
each (n) is independently an integer having a value of from about 2 to about 4000.
14 . The conjugate of claim 13 , wherein the polymeric reagent bearing a maleimide group has the following structure:
wherein each (n) is independently an integer having a value of from about 225 to about 1930.
15 . The conjugate of claim 14 , wherein each (n) is defined so as to provide —(OCH 2 CH 2 )— as having a molecular weight of about 20 kDa.
16 . The conjugate of claim 3 , wherein the branched water-soluble polymer includes the following structure:
wherein each (n) is independently an integer having a value of from 2 to 4000.
17 . The conjugate of claim 1 , having the following structure:
wherein:
each (n) is independently an integer having a value of from 2 to 4000;
X is a spacer moiety comprised of one or more atoms; and
ChE is a residue of a cholinesterase moiety.
18 . The conjugate of claim 17 , having the following structure:
wherein each (n) is independently an integer having a value of from 2 to 4000.
19 . The conjugate of claim 1 , wherein the conjugate has from one to two water-soluble polymers attached to the residue of the cholinesterase moiety.
20 . The conjugate of claim 19 , wherein the conjugate has two water-soluble polymers attached to the residue of the cholinesterase moiety.
21 . The conjugate of claim 1 , wherein the residue of the cholinesterase moiety is in the form of a dimer derived from two separate cholinesterase moieties.
22 . The conjugate of claim 21 , wherein the conjugate has two water-soluble polymers attached to the residue of the cholinesterase moiety, one water-soluble polymer attached to each of the cholinesterase moieties forming the dimer.
23 . The conjugate of claim 2 , wherein the polymeric reagent bearing a maleimide group has a single maleimide group.
24 . The conjugate of claim 1 , wherein the cholinesterase moiety is glycosylated.
25 . The conjugate of claim 1 , wherein the conjugate is in an isolated and monoPEGylated form.
26 . (canceled)
27 . The conjugate of claim 1 , wherein the cholinesterase moiety is a precursor cholinesterase moiety.
28 . A pharmaceutical composition comprising a conjugate of claim 1 and a pharmaceutically acceptable excipient.
29 . A method for making a conjugate comprising contacting, under conjugation conditions, a cholinesterase moiety with a polymeric reagent bearing a thiol-reactive functional group.
30 . The method of claim 29 , wherein the contact step is carried out at a pH of greater than 8.0.
31 . A method for making a conjugate comprising:
(a) combining, under conjugation conditions, a reagent composition comprising a plurality of thiol-selective polymeric reagent molecules with a cholinesterase moiety composition comprising a plurality of cholinesterase moiety molecules, each molecule in the form of a dimer to form a conjugate mixture comprising monoconjugated dimers and diconjugated dimers;
(b) subjecting the conjugate mixture to reducing conditions to form a reduced mixture comprising reduced unconjugated monomers and reduced monoconjugated monomers;
(c) separating the reduced monoconjugated monomers from the reduced mixture to form a composition comprising reduced monoconjugated monomers; and
(d) removing the reducing conditions from the composition comprising reduced monoconjugated monomers to thereby form a composition of diconjugated dimers.
32 . The method of claim 31 , wherein the composition comprising reduced monoconjugated monomers is substantially free of reduced unconjugated monomers.