STERILE FORMULATION COMPRISING A STABLE PHOSPHOROTHIOATE OLIGONUCLEOTIDE

Abstract

A composition including a phosphorothioate oligonucleotide and at least one fatty acid and/or at least one emulsifying agent, where the composition is sterile and where the composition includes at least one agent including a thiol group and at least one phosphate compound, and the composition can be an ophthalmic composition. The present invention also relates to a method for obtaining the same and to the therapeutic use thereof.

Claims

1-15 (canceled)

16. 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.

17. The composition according to claim 16, being an ophthalmic composition.

18. The composition according to claim 16, wherein the composition is an emulsion.

19. The composition according to claim 16, being an oil-in-water emulsion or a water-in-oil-in-water emulsion.

20. The composition according to claim 16, wherein 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.

21. The composition according to claim 16, wherein the phosphate compound is selected from the group comprising Na.sub.2HPO.sub.4, NaH.sub.2PO.sub.4, Na.sub.3PO.sub.4, K.sub.2HPO.sub.4, K.sub.3PO.sub.4, KH.sub.2PO.sub.4, (NH.sub.4).sub.2HPO.sub.4, (NH.sub.4).sub.3PO.sub.4, Mg(H.sub.2PO.sub.4).sub.2, Mg.sub.3(PO.sub.4).sub.2, MgHPO.sub.4, MgNH.sub.4PO.sub.4, Ca(H.sub.2PO.sub.4).sub.2, CaHPO.sub.4, Ca.sub.3(PO.sub.4).sub.2, Ca.sub.8(HPO.sub.4).sub.2(PO.sub.4).sub.4, H.sub.3PO.sub.4 and phosphate acid/base conjugate pairs selected from the group comprising NaH.sub.2PO.sub.4/Na.sub.2HPO.sub.4, KH.sub.2PO.sub.4/K.sub.2HPO.sub.4, Na.sub.2HPO.sub.4/Na.sub.3PO.sub.4, and K.sub.2HPO.sub.4/K.sub.3PO.sub.4.

22. The composition according to claim 16, wherein the phosphorothioate oligonucleotide is selected from the group comprising antisense oligonucleotides, siRNAs, shRNAs, ribozymes, aptamers, molecular decoys and RNA-DNA hybrid molecules.

23. The composition according to claim 16, wherein said phosphorothioate oligonucleotide is an antisense oligonucleotide.

24. The composition according to claim 16, wherein the phosphorothioate oligonucleotide is an antisense oligonucleotide specific for IRS-1 (insulin receptor substrate-1).

25. The composition according to claim 24, wherein the IRS-1 antisense phosphorothioate oligonucleotide comprises a sequence of at least 12 contiguous nucleotides of SEQ ID NO: 1.

26. The composition according to claim 24, wherein the IRS-1 antisense phosphorothioate oligonucleotide is SEQ ID NO: 2 or a function-conservative derivative thereof, wherein 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.

27. The composition according to claim 26, wherein said function-conservative derivative of SEQ ID NO: 2 is selected from SEQ ID NO: 3 to SEQ ID NO: 28.

28. The composition according to claim 16, wherein the phosphorothioate oligonucleotide is stable for at least 1 day at 25 C., and/or wherein the phosphorothioate oligonucleotide is stable for at least 1 day at 40 C.

29. The composition according to claim 16, being a pharmaceutical composition and comprising at least one pharmaceutically acceptable excipient.

30. 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.

31. The method according to claim 30, wherein said method is 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.

32. The method according to claim 30, wherein said 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; and adding a phosphorothioate oligonucleotide, at least one phosphate compound and/or at least one agent comprising a thiol group within the sterile bulk emulsion.

33. The method according to claim 31, wherein: 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; and an 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.

34. A method for treating an angiogenic disorder, comprising administering to a subject a composition according to claim 16, wherein the phosphorothioate oligonucleotide is an IRS-1 antisense.

35. 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; and optionally, a phosphorothioate oligonucleotide.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0491] FIG. 1 is a scheme showing the sequential peripheral oxidation of phosphorothioate-derived oligonucleotides.

[0492] FIG. 2 is a scheme showing the sequential peripheral oxidation of phosphorothioate-derived oligonucleotides followed by -elimination reaction.

[0493] FIG. 3 is a set of histograms showing the percentage assay, i. e. , the ratio of purities (intact GS-101 oligonucleotides) versus impurities (degraded GS-101 oligonucleotides), expressed as a percentage. Analysis were carried out on day 0 (T0), after 2 weeks (T2w), after one month (T1m), after 2 months (T2m), after 3 months (T3m) and after 6 months (T6m). FIG. 3A reports the percentage assay results of various formulations of the GS-101 sterile emulsion, incubated at 5 C.; FIG. 3B reports the same results for emulsions incubated at 25 C.; FIG. 3C reports the same results for emulsions incubated at 40 C.

EXAMPLES

[0494] The present invention is further illustrated by the following examples.

Example 1

Accelerated Stability Study of GS-101 in Presence of Phosphate

[0495] The accelerated stability study of GS-101 is reported in this example. A composition containing 0.86 mg GS-101/g emulsion was evaluated in 7 different formulations, comprising phosphate compounds and/or an antioxidant, such as an agent comprising a thiol group. These formulations were kept at different storage conditions, and the stability of GS-101 in the different formulations was evaluated by reversed-phase high-performance liquid chromatography (RP-HPLC).

[0496] Materials and Methods

[0497] Preparation of the Sterile Bulk Emulsion

[0498] 8% w/w* of Miglyol 812, 3.5% w/w* of Gelot 64 and 2% w/w* of cetyl alcohol were mixed in a beaker. The beaker was then placed onto a magnetic stirrer-heater adjusted at 70 C. The resulting oil phase was solubilized and homogenized under continuous stirring (300 rpm) at 70 C. for 10 minutes.

[0499] Parallelly, 0.05% w/w* or 0.1% w/w* of Carbopol 980NF (depending on the formulation as described below in Table 1) were dissolved in 70 mL of ultrapure water in a 100-mL beaker. The mixture was solubilized under magnetic stirring for at least 30 minutes and the pH was adjusted to 7 with 1 N NaOH solution. The resulting aqueous phase was then heated to 70 C.

[0500] * percentages are given in w/w of the total composition.

[0501] The prepared oil phase kept at 70 C. was gradually added to the prepared aqueous phase kept at 70 C., under stirring, at room temperature.

[0502] After cooling down to room temperature, the resulting bulk emulsion was autoclaved for 20 minutes at 121 C. under 1 bar pressure. The sterile bulk emulsions were kept under agitation until their cooling to room temperature.

[0503] Preparation of the GS-101 Sterile Emulsions

[0504] 32 grams of sterile bulk emulsion were supplemented with 8 grams of a GS-101 solution.

[0505] Since the GS-101 solution has to be filtered twice before addition to the sterile bulk emulsion, 10 grams were prepared. These 10 grams can contain one or more of the following substances: [0506] 50 mg (0.1% w/w*) or 125 mg (0.25% w/w*) of N-acetylcysteine (NAC), an agent containing a thiol group (dissolved in 2 M NaOH); [0507] 50 mg (0.1% w/w*) or 125 mg (0.25% w/w*) of sodium metabisulfite, an agent which does not contain a thiol group; [0508] 155 mg of Na.sub.2HPO.sub.4.12H.sub.2O and 43.8 mg of NaH.sub.2PO.sub.4.H.sub.2O (15 mM phosphate final in the total composition); [0509] 43 mg (0.086% w/w*) of GS-101 having the sequence SEQ ID NO: 2 (5-TCTCCGGAGGGCTCGCCATGCTGCT-3).

[0510] * percentages are given in w/w of the total composition.

[0511] The various composition formulations are detailed in Table 1 below.

TABLE-US-00004 TABLE 1 % of 15 mM Aeration % and type Formulation Carbopol phosphate with of antioxidant number 980NF (w/w) buffer? N.sub.2-gas? (w/w) 1 0.05 No No No 2 0.1 Yes No No 3 0.1 Yes Yes No 4 0.1 Yes No 0.1% NAC 5 0.1 Yes No 0.25% NAC 6 0.1 Yes No 0.1% Na- metabisulfite 7 0.1 Yes No 0.25% Na- metabisulfite

[0512] All components of the GS-101 solution were added in the order given above. After the addition of each component, ultrapure water was added until the component was fully dissolved, before proceeding to the addition of the next component. The pH was checked and adjusted to 7.0 using 10% NaOH, if necessary. Finally, ultrapure water was added until a total weight of 10 grams was achieved, and these GS-101 formulations were filtered twice using a sterile Acrodisc 0.2 m filter unit. This filtration step was carried out in a laminar flow cabinet, as the following steps.

[0513] Ultimately, for each of the 7 formulations of Table 1, 32 grams of the sterile bulk emulsion were weighted and 8 grams of the respective sterile GS-101 solutions were added. All mixes were homogenized by shaking vigorously.

[0514] GS-101 formulation #3 was also aerated with Na gas for 90 minutes to get rid of all oxygen that might be present in the sample. In this way, the effect of the presence of oxygen on the stability of the GS-101 (i.e., the amount of air oxidation) could be investigated by making a comparison with GS-101 formulation #2.

[0515] Incubation of the GS-101 Sterile Emulsions

[0516] All GS-101 sterile emulsions were stored overnight at 5 C. The next day, 6 aliquots of each emulsion, of approximately 4 grams each, were placed in climatic rooms for stability testing.

[0517] The GS-101 sterile emulsion formulations were incubated at 25 C./60% RH (relative humidity) or 40 C./75% RH for different time periods. Analysis were carried out as described below on day 0 (T0), 1 week (T1w) and 2 weeks (T2w).

[0518] RP-HPLC

[0519] The percentage assay, i.e., the ratio of purities (intact GS-101 oligonucleotides) versus impurities (degraded GS-101 oligonucleotides), expressed as a percentage, was calculated for each GS-101 sterile emulsions, using chromatographic conditions summarized in Table 2 below.

TABLE-US-00005 TABLE 2 HPLC Waters Acquity H-Class or equivalent system Data- Waters Empower 3 or equivalent acquisition system Column Water Acquity UPLC OST BEH C18 2.1 mm ID 50 mm, 1.7 m particle size Mobile phase A: Water B: 17.2 mM triethylamine, 200 mM hexafluoroisopropanol in water C: MeOH Gradient elution Min 0.0 15.0 15.1 23.0 Elution mode % A 35 30 35 35 % B 50 50 50 50 % C 15 20 15 15 Analytical 15 minutes run time Total run 23 minutes time Flow rate 0.3 mL/minute Injection 20 L (1 g of GS-101) volume Column 50 3 C. temperature Sample 10 5 C. temperature Detection UV detection with analytical (10 mm) flow cell, at = 260 nm Purge solvent Water Needle wash MeOH solvent

[0520] Results

[0521] Addition of Phosphate Buffer Improves the Stability of GS-101

[0522] The RP-HPLC percentage assay results of the 7 different formulations are shown in Table 3 below (in %), at day 0 (T0) at 5 C., and after 1 week (T1w) and 2 weeks (T2w) at 25 C. and 40 C. The evolution of the stability is also reported, as the difference of percentage assay result between T1w and TO (T1w-T0) and between T2w and T0 (T2w-T0). Data have not been standardized and it will appear clearly to the skilled artisan that values remaining within 5% can be considered comparable values, based on measurement and dilution bias.

TABLE-US-00006 TABLE 3 T1 w T2 w T1 w T0 T2 w T0 25 C. 40 C. 25 C. 40 C. 25 C. 40 C. 25 C. 40 C. T0 60% 75% 60% 75% 60% 75% 60% 75% Formulation 5 C. RH RH RH RH RH RH RH RH 1 91.5 82.9 62.6 82.7 49.5 8.6 28.9 8.8 42.0 2 92.4 93.0 82.9 91.0 79.1 +0.6 9.5 1.4 13.3 3 97.8 97.7 87.0 95.2 80.6 0.1 10.8 2.6 17.2 4 98.3 96.9 90.7 97.4 88.5 1.4 7.6 0.9 9.8 5 97.1 96.4 89.7 95.4 89.0 0.7 7.4 1.7 8.1 6 92.9 89.1 70.1 87.3 62.8 3.8 22.8 5.6 30.1 7 99.9 93.0 77.1 90.6 65.8 6.9 22.8 9.3 34.1

[0523] The values for assay of all formulations decrease over time and is more pronounced for the samples kept at 40 C./75% RH.

[0524] However, it is clear that formulation #1, without phosphate buffer, shows a much greater decrease in assay than the other formulations (28.9% after 1 week, 42.0% after 2 weeks). Formulations #6 and #7 were made with a phosphate buffer and metabisulfite as an antioxidant, and show a similar decrease (22.8% after 1 week, 30.1% to 34.1% after 2 weeks), even though formulation #2 and #3 show less of a decrease and don't contain an antioxidant. This means that metabisulfite, which does not comprise a thiol group, is not a suitable antioxidant for this formulation.

[0525] On the other hand, formulations #4 and #5, comprising a phosphate buffer and NAC as an antioxidant, show a decrease of less than 10% at 40 C., after a 2-week incubation.

[0526] The same can be concluded for the samples kept at 25 C., which shows a slightly decreased stability over time, however insignificant (less than 2% over 2 weeks) in formulations #4 and #5.

[0527] Conclusion

[0528] These results show the stabilizing effect of agents comprising a thiol group as antioxidant, compared to agents which do no comprise a thiol group, on the degradation of the phosphorothioate oligonucleotide of the composition.

[0529] The addition of 15 mM phosphate buffer further improves the stability of GS-101 on a short-term.

Example 2

Long-Term Stability Study

[0530] A long-term stability study of GS-101 was initiated and is reported in this example. The emulsion containing 0.86 mg GS-101/g emulsion was evaluated in 5 different formulations, comprising phosphate and/or an agent comprising a thiol group. These formulations were kept at different storage conditions, and the stability of GS-101 in the different formulations was evaluated by RP-HPLC, as described in Example 1.

[0531] Materials and Methods

[0532] Preparation of the GS-101 Sterile Emulsions

[0533] The GS-101 sterile emulsions were prepared similarly to the emulsions of Example 1. These emulsions can contain one or more of the following substances: [0534] 0.1% w/w* or 0.25% w/w* of N-acetylcysteine (NAC); [0535] 0.436% w/w* of Na.sub.2HPO.sub.4.12H.sub.2O and 0.039% w/w* of NaH.sub.2PO.sub.4.H.sub.2O (15 mM phosphate final in the total composition); [0536] 0.086% w/w* of GS-101 having the sequence SEQ ID NO: 2 (5-TCTCCGGAGGGCTCGCCATGCTGCT-3).

[0537] * Percentages are Given in w/w of the Total Composition.

[0538] The various composition formulations are detailed in Table 4 below.

TABLE-US-00007 TABLE 4 % of 15 mM % of Carbopol phosphate antioxidant Formulation number 980NF (w/w) buffer? (w/w) 1 0.05 No No 2 0.1 Yes No 3 0.1 Yes 0.1% NAC 4 0.1 Yes 0.25% NAC 5 (Placebo emulsion GS- 0.1 Yes 0.25% NAC 101-free)

[0539] Incubation of the GS-101 Sterile Emulsions

[0540] The GS-101 sterile emulsion formulations were incubated at 5 C., 25 C./ 60% RH or 40 C./75% RH for different time periods. Analysis were carried out on day 0 (T0), 2 weeks (T2w), one month (T1m), 2 months (T2m), 3 months (T3m) and 6 months (T6m).

[0541] Results

[0542] Addition of phosphate buffer together with an agent comprising a thiol group improves the stability of GS-101

[0543] The RP-HPLC assay results of the 5 different formulations are shown in Table 5 below (in %), and in FIGS. 3A-C.

TABLE-US-00008 TABLE 5 T0 T2 w T1 m T2 m T3 m T6 m 5 C. Formulation 1 95.6 91.6 87.3 82.7 75.6 Formulation 2 95.0 91.0 94.2 87.1 79.6 Formulation 3 93.7 93.5 94.8 93.9 92.1 Formulation 4 94.6 93.8 98.4 95.3 95.0 Formulation 5 0.1 0.0 0.0 0.0 0 25 C./60% Formulation 1 95.6 86.2 74.8 68.1 56.3 35.7 RH Formulation 2 95.0 93.5 86.8 80.7 80.2 72.1 Formulation 3 93.7 95.7 92.0 94.6 90.3 88.0 Formulation 4 94.6 96.6 93.9 94.5 91.5 85.2 Formulation 5 0.1 0.0 0.0 0.0 0.0 0 40 C./75% Formulation 1 95.6 55.9 26.9 5.6 2.2 RH Formulation 2 95.0 85.0 73.9 64.4 62.0 Formulation 3 93.7 92.8 86.1 86.4 77.9 Formulation 4 94.6 95.0 88.9 88.3 81.2 Formulation 5 0.1 0.0 0.0 0.0 0.0

[0544] These results clearly show a major drop of stability of formulation #1 (without phosphate buffer nor NAC), even at 5 C., when compared to the other formulations.

[0545] In comparison, formulation #2, which comprises a phosphate buffer, shows a stabilization of GS-101 and a slowed-down decrease.

[0546] Formulations #3 and #4, which comprise a phosphate buffer and NAC as an antioxidant at different concentrations (0.1 and 0.25% w/w final), show further stabilization of GS-101 when kept at 5 C. or 25 C. even after 6 months of incubation.

[0547] Unlike the assays of Example 1 which showed no difference in the stabilization of GS-101 with the two NAC concentrations at short-term, long-term studies tend to show an increased stability using higher amounts of antioxidant (0.25% w/w final).

[0548] Conclusion

[0549] These results confirm the stabilizing effect of 15 mM phosphate buffer on GS-101 at medium- to long-term.

[0550] Further addition of an agent comprising a thiol group, such as N-acetylcysteine, offers an even greater stability of GS-101, even after several months of incubation at room temperature or 40 C.

[0551] At 4 C., 15 mM phosphate buffer and 0.25% w/w final of N-acetylcysteine fully preserve the integrity of the phosphorothioate oligonucleotide, even after several months.