US 20160244741A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0244741 A1 Russell et al. (43) Pub. Date: Aug. 25, 2016 (54) POLYMERENGINEERED REGENERATING Publication Classification BOSCAVENGERS (51) Int. Cl. (71) Applicant: CARNEGE MELLON CI2N 9/96 (2006.01) UNIVERSITY, Pittsburg, PA (US) A638/48 (2006.01) A647/48 (2006.01) (72) Inventors: Alan J. Russell, Gibsonia, PA (US); Hironobu Murata, Pittsburgh, PA (US); CI2N 9/76 (2006.01) Chad Cummings, Pittsburgh, PA (US); CI2N 9/16 (2006.01) Richard R. Koepsel, Pittsburgh, PA A638/46 (2006.01) (52) U.S. Cl. (US) CPC. CI2N 9/96 (2013.01); C12N 9/16 (2013.01); (21) Appl. No.: 15/026,093 A61K 38/465 (2013.01); A61K47/48 176 (2013.01): CI2N 9/6427 (2013.01); A61 K (22) PCT Fled: Oct. 3, 2014 38/4826 (2013.01); C12Y-301/01 007 (2013.01); CI2Y 304/21001 (2013.01) (86) PCT NO.: PCT/US1.4/59171 S371 (c)(1), (57) ABSTRACT (2) Date: Mar. 30, 2016 Related U.S. Application Data Embodiments of the invention provide at least one polymer (60) Provisional application No. 61/961,097, filed on Oct. covalently conjugated to an esterase. The at least one polymer 3, 2013. includes a plurality of Oxime functional groups. Patent Application Publication Aug. 25, 2016 Sheet 1 of 19 US 2016/0244741 A1 E + 1 c > E + R — E + IR F.G. 1 Patent Application Publication Aug. 25, 2016 Sheet 2 of 19 US 2016/0244741 A1 R ar R Y a R w R R R 1- - n E0X - - + is R R - ? R R w . R Y R FG. 2 Patent Application Publication Aug. 25, 2016 Sheet 3 of 19 US 2016/0244741 A1 x stor $8 &888x Syss): 60 °C, 3 h SSX$88-88-8s x 8. FG. 3A Patent Application Publication Aug. 25, 2016 Sheet 4 of 19 US 2016/0244741 A1 i Nyir specisirs of sortine containing polyer I 3&iša-Šs a $2. assies by is 8:88: xy--------------xx-x-xy-y-ex-x-xx-xx-xx-x-x sists & & S.S. S.S. & 8 S& 8. 8, FG. 3B $x,Yess s^x,** iss & &-Saxxxxx. x^. s S. & 2-pyridine aidioxine oiuene, i20'c FG. 3C Patent Application Publication Aug. 25, 2016 Sheet 6 of 19 US 2016/0244741 A1 ED-NHS, wate: W FIG. 3E Patent Application Publication Aug. 25, 2016 Sheet 7 of 19 US 2016/0244741 A1 AChE, Buffer, SSYs S.S &S SS Patent Application Publication Aug. 25, 2016 Sheet 8 of 19 US 2016/0244741 A1 y & x --...--S 3. N Y. Y. - AChEee with ARR initiator bound to annine groups Šs Sulfonate monomer AR + Cu(1)Br, liganda-la. *S^ y o \ Š AS. so- K a toy-stiforate-AChE conjugate FG. 4A Patent Application Publication Aug. 25, 2016 Sheet 9 of 19 US 2016/0244741 A1 S .S X - s s w sessmewsweexsexyrgrks sys8* past fi 4.0 3.0 . O FG. 4B Patent Application Publication Aug. 25, 2016 Sheet 10 of 19 US 2016/0244741 A1 roy(2°AN-ACE & & xistiys six & 88 Š $8 is& is:s F.G. 5 Patent Application Publication Aug. 25, 2016 Sheet 11 of 19 US 2016/0244741 A1 Šs, s NH \- ti. - c. O*- - Y. Ö Y ACE-initiator y Airie N {{{titigate yer Cu()ci-Mes SREY water, C, 4 a AChE-PDMAAIAzide conjugate F.G. 6A Patent Application Publication Aug. 25, 2016 Sheet 12 of 19 US 2016/0244741 A1 N.R of AChE-Fi:AAiAxide corjigate www.www.rxexpyxswoxyxx-xxyyxx. Rys& 38 s& &. & & F.G. SB Patent Application Publication Aug. 25, 2016 Sheet 13 of 19 US 2016/0244741 A1 AChE-PDMAAJAzide conjugate & Alkyne-2-PAM Br CuSO4, HMTETA N - Ascoric acid N ^-a { W {{ 3 () is l i. s8 YssYSYS& RS '^s.Ns s Š s - s AChE-PAA:2-PA conjugate f --&ssr s SS- 88s F.G. 6C Patent Application Publication Aug. 25, 2016 Sheet 14 of 19 US 2016/0244741 A1 Nir of AChE-PSAAF2-PA conjugate i. :- aim k s ------------a^^^s. \\-yasa. F.G. 6) Patent Application Publication Aug. 25, 2016 Sheet 15 of 19 US 2016/0244741 A1 H NER of Aikyne-2-PAR d sta^\sssssssssssssssssssssssssssaara-ašai ill ^ --- ole & E && 8 visaw FIG. 6E Patent Application Publication Aug. 25, 2016 Sheet 16 of 19 US 2016/0244741 A1 Patent Application Publication Aug. 25, 2016 Sheet 17 of 19 US 2016/0244741 A1 i A. +W, y S. c\-e/ - e i ? \ . g f y i &s co-o & *ix. & r * , 8 c sk as f y s . Patent Application Publication Aug. 25, 2016 Sheet 18 of 19 US 2016/0244741 A1 is ; it is Patent Application Publication Aug. 25, 2016 Sheet 19 of 19 US 2016/0244741 A1 100 S 80 is 60 5 40 Š S} SS Š & & Š 20 Š S - POA CHT Š Native CHT 0 -------------------------------------------------------------------------------------------------------------- : O 5 O 15 2O 25 30 35 US 2016/0244741 A1 Aug. 25, 2016 POLYMERENGINEERED REGENERATING ketoxime. In various aspects, the invention may include a BOSCAVENGERS composition as provided in any of the aspects of the compo sition described herein, wherein the plurality of oxime func FIELD OF THE INVENTION tional groups may comprise at least one bis-pyridinium 0001. This invention describes the development of a broad Oxime. spectrum biocatalytic scavenger for use as an organophos 0008. In various aspects, the invention may include a com phate (OP) detoxifying agent. position as provided in any of the aspects of the composition described herein, wherein the at least one polymer covalently conjugated to the esterase may comprise a long lived covalent BACKGROUND OF THE INVENTION conjugate. 0002 The dramatic tragedy in the reemergence of chemi 0009. In various aspects, the invention may include a com cal weapons use in Syria highlights like never before the position as provided in any of the aspects of the composition urgent need to provide effective medical countermeasures for described herein, wherein the long lived covalent conjugate nerve agent poisoning. Less talked of, but even more urgent, may comprise a conjugate that is maintained in the body for a is the need for countermeasures against organophosphate time period ranging from at least one 24 hour day and pref (OP) pesticide poisoning that kills hundreds of thousands erably for more than about one day to more than about one world-wide every year. Although most OP pesticides were week. The long lived covalent conjugate may be maintained banned for use in the US, they still exist and are produced in in the body for a period of time of at least one week, or at least Kilo-tons in other countries and are easily accessible. Some two weeks, and preferably for a period of time sufficient to of them are highly toxic as witnessed by the fatal poisoning of eliminate or significantly reduce the inhibiting function of the school children in India in July 2013 caused by monocroto OP toxin. phos contamination of cooking oil. OP pesticides are a major 0010. In various aspects, of the invention, any of the worldwide health problem, even without accidental or delib aspects of the composition described herein may comprise at erate release with OP self-poisoning (suicide), responsible least one polymer which may have at least one environmen for 200,000 deaths a year worldwide. Eddleston M, Buckley tally responsive monomer. NA, Eyer P. Dawson A H. Management of acute organophos 0011. In various aspects of the invention, any of the phorus pesticide poisoning. Lancet, 371: 597-607 (2008). aspects of the composition described herein may comprise, at Further these poisonings are difficult to treat with the current least one polymer having a polymer length of two repeat standards of therapy. units, and preferably greater than two repeat units, and more preferably a polymer length ranging from a minimum of at SUMMARY OF THE INVENTION least 2 monomer repeats to about 1000 monomer repeats. In 0003. The present investigation represents a solution for certain aspects of the composition, there may be greater than the need for countermeasures against organophosphate pes 1000 monomer repeats. The only upper limit on the number ticide poisoning. A broad spectrum regenerating scavenger or length of monomers is that number or length that will avoid for use as an OP compound detoxifying agent following acute hindering contact of the oXime functional group or groups exposure is described herein. sufficient to interact with the active site of the enzyme for 0004. Accordingly, the present disclosure may include neutralization of the inhibiting moiety. various aspects of a composition and methods for making and 0012. In various aspects, the invention may include a com using the composition. For example, in various aspects, the position as provided in any of the aspects of the composition invention described herein provides a composition compris described herein, wherein the at least one polymer may be a ing at least one polymer covalently conjugated to an esterase. co-polymer that may comprise at least two different mono The at least one polymer may comprise a plurality of oxime mers, wherein at least one monomer may comprise a member functional groups. selected from the group consisting of aldoximes, ketoximes, 0005. In various aspects, the invention may include a com muco-adhesion monomers, polyethylene glycol, bis-pyri position wherein the esterase may comprise a member dinium oximes, N,N-dimethylacrylamide, N-isopropylacry selected from the group consisting of acetylcholinesterase lamide, (meth)acrylate, N,N-dimethylaminoethyl methacry and butyrylcholinesterase. In various aspects, the invention late, carboxyl acrylamide, 2-hydroxylethylmethacrylate, may include a composition wherein the esterase may not N-(2-hydroxypropyl)methacrylamide, quaternary ammo comprise acetylcholinesterase.