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Activated Polyoxazolines and Conjugates and Compositions Comprising the Same

a technology of activated polyoxazolines and conjugates, which is applied in the field of polyoxazoline polymers and polyoxazoline derivatives, can solve the problems of low work done on the activation of poz for coupling to target molecules, high polydispersity, and limitations of each of the previously described poz derivatives, so as to improve in-vivo stability, reduce renal clearance, and increase protein effective size

Inactive Publication Date: 2014-01-09
SERINA THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present patent provides monofunctional POZ derivatives that can be used to attach a wide range of target molecules under different reaction conditions. These monofunctional POZ derivatives have a variety of active groups that can react with different target molecules and groups, making them more versatile and effective than previous methods. This increases the likelihood of successful conjugation and can lead to the creation of stable target molecule-POZ conjugates. This results in a significant advantage over previous methods that could only work with a limited number of target molecules and groups.

Problems solved by technology

There has been little work done on activation of POZ for coupling to target molecules.
However, each of the previously described POZ derivatives suffers from limitations.
However, Jordan, Hoogenboom and others have shown that initiation of polymerization with different activating groups can require greatly differing reaction conditions requiring extensive studies to determine optimal reaction conditions.
Also, as discussed below, initiation of polymerization with an alkyl halide does not proceed by a living-cation mechanism and thus high polydispersities are found.
However, glutarate and succinate derivatives have: a hydrolytically unstable ester linkage connecting the target molecule to the POZ compound.
This derivative could in theory be coupled to a protein thiol group to give a disulfide linkage, although this was not done by Hsiue, but it is known that disulfides are unstable and subject to ready reduction in plasma.
An additional problem hindering use of known POZ polymers for modification of target molecules is that some POZ polymers do not possess a single active functional group; i.e., they are not “monofunctional”.
However, this technique was not designed to provide monofunctional POZ polymers but rather produces multifunctional compounds with pendent groups along the backbone.
Also there are instances when one would desire to have a single target molecule coupled to a polymer, and multi-functional POZ polymers will not permit this.
There has been a great deal of work showing that MWs and PDs in the above range cannot be achieved for POZ with conventional techniques.
Use of unusually low polymerization temperatures combined with reaction times of several weeks has been shown to give acceptable PDs, but such conditions are not practical for commercial-scale preparations (J. S. Park and K. Kataoka, Macromolecules, 39, 6622 (2006)).
Hoogenboom, Schubert and colleagues indicate that low-PD POZ can be prepared by using microwave irradiation, but again commercial-scale polymerizations are not available with this technique (R. M. Paulus, T. Erdmenger, C. R. Becer, R. Hoogenboom and U.S. Schubert, Macromol.
As a consequence of the generally found broad polydispersities, the functional POZ compounds described to date are seriously limited for use in polymer therapeutics.
Yet another problem hindering use of POZ derivatives in modification of target molecules is the unavailability of a range of appropriate activated POZ molecules capable of reaction with the target molecules under a range of conditions.
Furthermore, the POZ molecules presently available are multifunctional or contain hydrolytically unstable bonds when conjugated to target molecules, with the disadvantages associated therewith, or the active substituent is added during the initiation reaction, with the disadvantages associated therewith.
Furthermore, pendent functionality has been described, but these derivatives are multifunctional and not suitable for the current application.

Method used

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  • Activated Polyoxazolines and Conjugates and Compositions Comprising the Same
  • Activated Polyoxazolines and Conjugates and Compositions Comprising the Same
  • Activated Polyoxazolines and Conjugates and Compositions Comprising the Same

Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of 20 kDa H-PEOZ-Hydrazid

[0201]

[0202]The synthesis of 20 kDa polyoxazoline succimidyl thiopropionate has been described (H-PEOZ-T-SPA 20K, GFC shows 83% of —O-Su; GPC showed a Mn=18,243 Da, PDI=1.055, Mp=19,973 Da; and MALDI-TOF showed a Mn=20,972 Da). Hydrazine monohydrate, NH2NH2.H2O, 98% was from Aldrich, FW 50.06, d 1.032.

[0203]H-PEOZ-T-SPA 20K (2.0 gm, 9.1240×10−5 mol, 1 equiv.) was first dissolved in anhydrous dichloromethane (90 mL) and this solution was transferred into an addition funnel. In a 250 mL round bottom flask, hydrazine monohydrate (452 μL, 9.1240×10−3 mol, 100 equiv.) was dissolved in anhydrous dichloromethane (5 mL). Under an argon atmosphere, the H-PEOZ-T-SPA solution in dichloromethane was added to the hydrazine solution drop wise over one hour with rapid stirring. This solution was stirred overnight at ambient temperature and in an argon atmosphere. The white precipitate that was formed in the reaction mixture was filtered out and the filtrate was...

example 2

Conjugation of Heparin by 20 kDa H-PEOZ-Hydrazide Through Carbohydrate Reducing Terminus

[0204]A 50 mg / mL solution of 20 kDa H-PEOZ-Hydrazide (2,222 μL, 5.5556×10−6 mole, 5 equiv.) was added to heparin sodium salt (Grade 1-A, purchased from Sigma-Aldrich, MW 17000-19000 Da, 20 mg, 1.1111×10−6 mole, 1 equiv.) in pH 3.0 acetic acid solution. The solution was allowed to stir at room temperature for 10 minutes. The solution pH was adjusted to 3.0 by adding 0.1 N HCl acid. To this solution, freshly prepared 1 M NaBH3CN in deionized water (111.1 μL, 1.1111×10−4 mole, 100 equiv) was added. The solution was stirred at room temperature for 10 minutes, and then incubated at 37° C. for overnight. The reaction mixture was analyzed by GFC using a BioSEP-SEC-S 4000 column. GFC showed the formation of H-PEOZ-heparin conjugate (data not shown).

example 3

Conjugation of Dynorphin A (1-13) at C-Terminus by 5 kDa M-PEOZ-Hydrazide using EDC as Coupling Agent

[0205]

[0206]Dynorphin A (dynorphin 1-13) (Bachem, 0.55 mg, 2.2133×10−7 mol, 1 equiv.) was dissolved in 250 μL of 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer at pH 3.0. A 109.5 μL aliquot of 50 mg / mL 5 kDa M-PEOZ-Hydrazide 5K (5.5 mg, 1.1066×10−6 mol, 5 equiv) in 50 mM MES buffer at pH 3.0 was prepared and filtered through a 0.2 μm syringe filter. This 5 kDa M-PEOZ-Hydrazide solution was then added to the dynorphin A solution. The solution pH was adjusted to 3.0 by drop by drop addition of a 50 mM HCl acid solution. A solution of N-(3-Dimethylaminopropyl)-N′-ethyl-carbodiimide hydrochloride (EDC, purchased from Fluka) in deionized water (31 μL, 100 mg / mL, 1.6097×10−5 mol, 20 equiv.) was added into the mixture. The solution pH was adjusted to 3.0 by slowly adding 50 mM HCl. The solution was allowed to stir at room temperature for 3 hours. The reaction mixture was analyzed by...

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Abstract

The present disclosure provides POZ derivatives having a range of active functional groups allowing conjugation of POZ derivatives to a variety of target molecules under a wide range of reaction conditions to produce a hydrolytically stable target molecule-POZ conjugate. Furthermore, the present disclosure provides novel methods of synthesis for the disclosed POZ derivatives and hydrolytically stable target molecule-POZ conjugates created using the disclosed terminally activated monofunctional POZ derivatives. In one embodiment, the POZ derivative is a terminally activated monofunctional POZ derivative.

Description

[0001]The present application claims the benefit of U.S. Provisional Application Nos. 61 / 1116,246, filed Nov. 19, 2008 (currently expired), and 61 / 116,252, filed Nov. 19, 2008 (currently expired), and is a continuation of U.S. application Ser. No. 13 / 676,048, filed Nov. 13, 2012 (currently pending). U.S. application Ser. No. 13 / 676,048 is a continuation of U.S. application Ser. No. 13 / 276,910, filed on Oct. 19, 2011 (currently abandoned), which is a continuation of U.S. application Ser. No. 12 / 622,264, filed on Nov. 19, 2009 (currently abandoned) U.S. application Ser. No. 12 / 622,264 is a continuation in part of U.S. application Ser. No. 12 / 529,001, filed on Aug. 27, 2009, now U.S. Pat. No. 7,943,141, issued on May 17, 2011. U.S. Pat. No. 7,943,141 is the National Stage of International Application No. PCT / US2008 / 002626, filed Feb. 28, 2008, (currently expired), which claims the benefit of U.S. Provisional Application No. 60 / 892,212, filed Feb. 28, 2007 (currently expired).FIELD OF T...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K47/48
CPCA61K47/48207A61K31/727A61K31/728A61K38/10A61K38/1719A61K38/1816A61K38/193A61K38/27A61K38/33A61K38/42A61K38/47A61K47/59C12N9/96C12Y302/01017C07K7/08C07K14/47C07K14/505C07K14/535C07K14/62C08G69/48
Inventor HARRIS, J. MILTONBENTLEY, MICHAEL DAVIDYOON, KUNSANGFANG, ZHIHAOVERONESE, FRANCESCO MARIAVIEGAS, TACEYMERO, ANNA
Owner SERINA THERAPEUTICS INC
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