FIELD

The present invention relates to the therapeutic use of oligonucleotides and aims at solving the problem of the stability of said oligonucleotides in a composition, in particular in a composition comprising at least one fatty acid and/or at least one emulsifying agent. More specifically, the present invention relates to a sterile composition comprising at least one fatty acid and/or at least one emulsifying agent, a phosphorothioate oligonucleotide, at least one agent containing a thiol group and at least one phosphate compound.

BACKGROUND

Oligonucleotides are commonly used as laboratory tools and increasingly used as therapeutic agents. Oligonucleotides are characterized by their specificity, as they are able to recognize and bind to a specific target, for example through sequence complementarity by virtue of Watson-Crick base pairing. Antisense oligonucleotides, siRNAs and shRNAs, the most common oligonucleotides with therapeutic purposes, are thus able to modulate the expression of a target gene. In particular, antisense oligonucleotides bind to a specific mRNA target and induce its degradation through the recruitment of RNase H, a ubiquitous enzyme that hydrolyzes the RNA strand of RNA/DNA hybrids. Alternatively, some antisense oligonucleotides may act as “steric blockers” as they block the access of cellular machinery to their RNA target. Antisense oligonucleotides are useful in the treatment of many disorders, including cancer, metabolic diseases, inflammatory diseases and angiogenesis related diseases.

Treatments consisting in the administration of an oligonucleotide to a human subject require compositions in which the oligonucleotide is stable. Unmodified oligonucleotides are susceptible to degradation by both intracellular and extracellular nucleases. Chemical modifications of the natural phosphodiester backbone were thus developed, and are currently commonly used to increase the stability of the modified oligonucleotides. In particular, phosphorothioate oligonucleotides are known to be more resistant to degradation by nucleases. Phosphorothioate antisense oligonucleotides are able to activate RNase H activity and thus can induce the degradation of their target mRNA.

Depending on the disease to be treated, different ways of administration and correspondingly different types of composition are used. Phosphorothioate oligonucleotides are very stable in aqueous solutions. However, administration of oligonucleotides has also been envisaged in the form of emulsions, creams or any bi- or multiphasic formulations, in particular for topical administration, in order to ensure a sufficient exposure of the target tissue to the active oligonucleotides. For example, a topical application of an emulsion may be required for the treatment of some diseases of the eyes. Being applied in an emulsion rather than in an aqueous solution may indeed prevent the hydrophilic oligonucleotides from being readily absorbed in the vitreous humor.

However, stability issues arise with phosphorothioate nucleotides in emulsions. Indeed, phosphorothioate oligonucleotides are susceptible to desulfurization through the action of peroxide radicals generated from excipients present in the compositions. WO03005822 presents how the addition of antioxidants which partition into the aqueous phase of a bi- or multiphasic topical formulation prevents desulfurization of phosphorothioate internucleoside linkages.

It should however be noted that the bi- or multiphasic compositions comprising a phosphorothioate oligonucleotide and an antioxidant described in WO03005822 were not submitted to temperatures higher than 40° C. However, some treatments, such as an ophthalmic application, require the administration of a sterile emulsion obtained by autoclaving, i.e., a sterile emulsion obtained by sterilization by saturated steam under pressure (more than 100° C.). Upon autoclaving, the Applicant demonstrated that phosphorothioate oligonucleotides may be subjected to β-elimination following sequential peripheral oxidation, as shown inFIGS. 1 and 2. Without willing to be bound to a theory, the Applicant suggests that exposition of fatty acids and/or emulsifying agents of the emulsion to high temperatures generates highly reactive chemical entities and/or free radicals which could lead to the degradation of the phosphorothioate oligonucleotide.

Thus, there is still a need for a sterile composition comprising at least one fatty acid and/or at least one emulsifying agent wherein the phosphorothioate oligonucleotide is stable.

SUMMARY

The present invention relates to a composition comprising a phosphorothioate oligonucleotide and at least one fatty acid and/or at least one emulsifying agent, wherein said composition is sterile and wherein said composition further comprises at least one phosphate compound and at least one agent comprising a thiol group, preferably said composition is an ophthalmic composition.

In one embodiment, the composition is an emulsion, preferably an oil-in-water emulsion or a water-in-oil-in-water emulsion.

In one embodiment, the agent comprising a thiol group is selected from the group comprising N-acetylcysteine, lipoic acid, DL-cysteine, creatinine, glutathione, 2-mercapto-5-benzimidazole salts, 2-mercaptoethanesulfonic acid salts, Na-edetate, Na-bisulfite and Na-sulfite, preferably the agent comprising a thiol group is lipoic acid, DL-cysteine or N-acetylcysteine.

In one embodiment, the agent comprising a thiol group is selected from the group comprising N-acetylcysteine, lipoic acid, DL-cysteine, creatinine, glutathione, 2-mercapto-5-benzimidazole salts and 2-mercaptoethanesulfonic acid salts, preferably the agent comprising a thiol group is lipoic acid, DL-cysteine or N-acetylcysteine.

In one embodiment, the phosphate compound is selected from the group comprising Na2HPO4, NaH2PO4, Na3PO4, K2HPO4, K3PO4, KH2PO4, (NH4)2HPO4, (NH4)3PO4, Mg(H2PO4)2, Mg3(PO4)2, MgHPO4, MgNH4PO4, Ca(H2PO4)2, CaHPO4, Ca3(PO4)2, Ca8(HPO4)2(PO4)4, H3PO4and phosphate acid/base conjugate pairs selected from the group comprising NaH2PO4/Na2HPO4, KH2PO4/K2HPO4, Na2HPO4/Na3PO4, and K2HPO4/K3PO4.

In one embodiment, the phosphorothioate oligonucleotide is selected from the group comprising antisense oligonucleotides, siRNAs, shRNAs, ribozymes, aptamers, molecular decoys and RNA-DNA hybrid molecules, preferably said phosphorothioate oligonucleotide is an antisense oligonucleotide.

In one embodiment, the phosphorothioate oligonucleotide is an antisense oligonucleotide specific for IRS-1 (insulin receptor substrate-1), preferably the IRS-1 antisense phosphorothioate oligonucleotide comprises a sequence of at least 12 contiguous nucleotides of SEQ ID NO: 1, more preferably the IRS-1 antisense phosphorothioate oligonucleotide is SEQ ID NO: 2 or a function-conservative derivative thereof.

In one embodiment, the function-conservative derivative of SEQ ID NO: 2 comprises from 9 to 50 nucleotides, has at least about 75% identity compared to SEQ ID NO: 2 and conserves the capacity of inhibiting IRS-1 expression as SEQ ID NO: 2, preferably said function-conservative derivative of SEQ ID NO: 2 is selected from SEQ ID NO: 3 to SEQ ID NO: 28.

In one embodiment, the phosphorothioate oligonucleotide is stable for at least 1 day at 25° C., and/or the phosphorothioate oligonucleotide is stable for at least 1 day at 40° C.

The present invention further relates to a pharmaceutical composition comprising the composition of the invention and at least one pharmaceutically acceptable excipient.

The present invention further relates to a medicament comprising the composition of the invention.

The present invention further relates to a method for preventing and/or inhibiting the degradation of a phosphorothioate oligonucleotide in a composition comprising at least one fatty acid and/or at least one emulsifying agent and subjected to autoclaving, wherein said method comprises adding at least one phosphate compound and/or at least one agent comprising a thiol group within the composition.

The present invention further relates to a method for obtaining a sterile composition comprising a phosphorothioate oligonucleotide and at least one fatty acid and/or at least one emulsifying agent, wherein said phosphorothioate oligonucleotide is stable within the sterile composition, and wherein said method comprises adding at least one phosphate compound and/or at least one agent comprising a thiol group within the composition.

In one embodiment, the method comprises the steps of:preparing a bulk emulsion, comprising at least one fatty acid and/or at least one emulsifying agent;sterilizing said bulk emulsion by autoclaving; andadding a phosphorothioate oligonucleotide, at least one phosphate compound and/or at least one agent comprising a thiol group within the sterile bulk emulsion.

In one embodiment, the percentage of the bulk emulsion ranges from about 60% to about 99% in weight to the total weight of the sterile composition and comprises:an oil phase, comprising an oil, an emulsifying agent, a thickening agent, and/or an osmolality modifying agent; andan aqueous phase comprising a viscosity modifying agent, a pH buffering agent, and/or urea; and
the percentage of the phosphorothioate solution ranges from about 1% to about 40% in weight to the total weight of the sterile composition and comprises a phosphorothioate oligonucleotide, at least one phosphate compound and/or at least one agent comprising a thiol group.

The present invention further relates to a composition, pharmaceutical composition or medicament as described hereinabove, wherein the phosphorothioate oligonucleotide is an IRS-1 antisense, for treating an angiogenic disorder.

The present invention further relates to a kit comprising:a bulk emulsion, comprising at least one fatty acid and/or at least one emulsifying agent;a phosphorothioate solution, comprising at least one phosphate compound and/or at least one agent comprising a thiol group; andoptionally, a phosphorothioate oligonucleotide.

