Activated Polyoxazolines and Conjugates and Compositions Comprising the Same

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...

AI Technical Summary

Benefits of technology

[0166]Covalent modification of proteins increases the protein's effective size and reduces renal clearance (Abuchowski et al., 1984; Hershfield, 1987; Meyers et al., 1991). Polymer conjugation also improves in-vivo stability (protection from proteases), improves protein solubility and lowers antigenicity (Katre et al., 1987; Katre, 1990).

[0167]Traditionally these modifications have been made by reacting terminal electrophiles on the polymer with nucleophiles on the protein (non-enzymatically mediated reactions). Conjugation occurs at the α-amino group on the N-terminal amino acid or on the ε-amino groups of lysine within the polypeptide chain. These reactions typically give non-specific attachment of the macromolecule or polymer substrate to the biopharmaceutical and may result in significant loss in bioactivity of the biopharmaceutical. In addition, bonding of the biopharmaceutical to a macromolecule or polymer substrate can be accomplished using enzymatically mediated reactions. The latter approach provides the benefit of increased specificity in the conjugation reactions, which may lead in increased activity in vivo.

[0168]Because of the success of polymer-modified therapeutics and biopharmaceuticals in general, for example EPO, it is of interest to expand the range of polymers suitable for such applications, especially to provide polymers having properties not possessed by polymers of the prior art. The present disclosure provides methodologies of attaching POZ and / or POZ derivatives, to biopharmaceuticals and the resulting POZ-biopharmaceutical conjugates. In one embodiment, the POZ-biopharmaceutical conjugates are hydrolytically stable. One advantage of POZ is that the hydrophilicity of the polymer can be varied by changing the nature of the R7 group, such as, but not limited to, changing an alkyl group present in the R7 position from methyl to ethyl to propyl; these changes can lead to differences in the PK profile and to greater activity as shown by an increase in the area under the activity curve.

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.

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