The following is a discussion of relevant art pertaining to certain prodrug compositions. The discussion is provided only for understanding of the invention that follows. The summary is not an admission that any of the work described below is prior art to the claimed invention.
The fields of medicinal chemistry and biotechnology have yielded a multitude of biologically active compounds having well demonstrated in vitro activity. These compounds, however, need to be effectively delivered to target cells and tissues of interest in order to be useful as research tools or as therapeutic agents in vivo. Many biologically active molecules have in vivo activity profiles that are compromised by virtue of their net negative (anionic) charge, their size, or a combination of both anionic charge and size. Furthermore, the polyanionic backbone of oligonucleotide compounds is recognized by nucleases that rapidly degrade these molecules. Various strategies have been developed in an attempt to overcome these barriers. In the case of oligonucleotide delivery, both viral and non-viral delivery strategies have yielded mixed results, with most having dose limiting toxicity or other safety issues. Such safety issues are representative of the challenge faced in effectively delivering other charged molecules to cells and tissues of interest. These challenges have driven innovation in a divergent approach, that of the prodrug.
A prodrug is a pharmacological substance administered in an inactive or significantly less active form. Once administered, the prodrug is metabolised in vivo into an active metabolite in a process termed bioactivation. The rationale behind the use of a prodrug is generally for absorption, distribution, metabolism, and excretion (ADME) optimization. Anion masking produgs represent a new platform of compounds that provide the advantage of improving ADME properties through a variety of mechanisms. First, the anion masking prodrug neutralizes anionic charge, and therefore overcomes barriers of cellular adsorption and tissue distribution. Second, because certain nucleases rely upon anionic charge to identify their substrates, the anion masking prodrug can circumvent metabolism. Furthermore, the charge masking moiety of the prodrug can also serve as a scaffold for various chemical entities that can confer improved targeting, immunosuppression, or solubility profiles. All of these factors can lead to improved pharmacokinetic and pharmacodynamic properties of a compound or molecule of interest.
Examples of anion masking phosphotriester protecting groups have been disclosed, see for example Lebleu et al., (2000), Russ. J. Bioorg. Chem., 26, 174-182. However, this approach utilizes ultraviolet radiation for deprotection and thus is not amenable to use in vivo. Beaucage et al. (2007), J. Org. Chem., 72, 805-815 describes in vivo bioactivation of certain phosphotriester oligonucleotide prodrugs. However, this approach generates THF as a byproduct upon bioconversion. WO 2010/039543 describes certain anion masking disulfide phosphotriester oligonucleotide prodrugs. Nevertheless, cyclodeesterification of these disulfide prodrugs results in the release of a reactive thiirane species, which can limit the use of such prodrugs due to dose limiting toxicity.
Glutathione (GSH) is a tripeptide that contains an unusual peptide linkage between the amine group of cysteine, which is attached by normal peptide linkage, to a glycine and the carboxyl group of the glutamate side-chain. Glutathione is an important antioxidant, preventing damage to cellular components caused by reactive oxygen species such as free radicals and peroxides. Glutathione thiol groups are reducing agents, existing at a concentration of approximately 5 mM in cells. Glutathione reduces disulfide bonds formed within cytoplasmic proteins to cysteines by serving as an electron donor. In the process, glutathione is converted to its oxidized form glutathione disulfide (GSSG), which is also called L(−)-Glutathione. Once oxidized, glutathione can be reduced back by glutathione reductase, using NADPH as an electron donor. Glutathione is therefore an attractive bioconversion reducing agent for prodrugs.
Certain examples of glutathione activated prodrugs are disclosed in, for example, Gunnarsdottir et al., (2003), Drug Metabolism and Disposition, 32, 321-327 and Tirouvanziam et al., (2006), PNAS, 103, 12, 4628-4633. These glutathione activiated prodrugs, however, do not offer any anionic charge masking capabilities. Accordingly, there exists a need for improved anion masking prodrugs that are amenable to bioconversion in vivo.