COMPOSITION FOR ADMINISTERING AND RELEASING OLIGONUCLEOTIDES

Abstract

The present invention relates to a composition comprising an oligonucleotide, at least two copolymers with at least two of the copolymers being Poloxamer 188 and Poloxamer 407, and a medium. The present invention further relates to a method for preparing said composition.

Claims

1. A composition comprising an oligonucleotide, at least two copolymers with at least two of the copolymers being Poloxamer 188 and Poloxamer 407, and a medium.

2. The composition according to claim 1, wherein the oligonucleotide is a native, semi-synthetic, synthetic or modified, single or double stranded nucleic acid molecule of deoxyribonucleotides and/or ribonucleotides and/or modified nucleotides.

3. The composition according to claim 1, wherein the composition comprises two copolymers and said copolymers are Poloxamer 188 and Poloxamer 407.

4. The composition according to claim 3, wherein the ratio of the mass of Poloxamer 188 to the mass of Poloxamer 407 in the composition is from 5:1 to 1:10.

5. The composition according to claim 1, wherein the medium is selected from one or more of the group consisting of water, PBS, and glycine.

6. The composition according to claim 1, wherein a sol-gel transition temperature of the composition is from 25 to 35° C.

7. The composition according to claim 1, wherein the concentration of the oligonucleotide is from 1.0 pM to 10 M.

8. A method of preventing or treating an inflammatory disease or condition of the oral cavity in a mammal, comprising administering to the mammal an amount of the composition of claim 1 to prevent or treat the inflammatory disease or condition of the oral cavity.

9. The method of claim 8, wherein the inflammatory disease or condition of the oral cavity is a periodontal disease or condition.

10. The method of claim 9, wherein the periodontal disease or condition is selected from the group consisting of gingivitis, chronic periodontitis, aggressive periodontitis, periodontitis and peri-implantitis or mukosivitis as a manifestation of systemic disease, necrotizing ulcerative gingivitis, necrotizing ulcerative periodontitis, abscesses of the periodontium, and combined periodontic-endodontic lesions.

11. The method of claim 8, wherein the composition is applied via dermal application.

12. The composition according to claim 1, wherein the composition is a pharmaceutical composition.

13. A method for preparing the composition according to claim 1, comprising the steps of (a) mixing the oligonucleotide, the copolymers, and the medium and (b) stirring the mixture.

14. The method according to claim 13, wherein the temperature of the medium in the step (a) is adjusted to 0° C. to 10° C. before, between, and/or after mixing the medium with the other components of the composition.

15. The method according to claim 13, wherein the step (b) comprises stirring the mixture for 1 min to 96 h at a temperature of from 0° C. to 10° C.

Description

[0038] The figures show:

[0039] FIG. 1: The state of formulation 4 at 18° C. before sol-gel transition.

[0040] FIG. 2: The state of formulation 4 at 37° C. after sol-gel transition.

[0041] FIG. 3: AEX-HPLC traces of the nucleotide sequence according to SEQ ID NO: 1 released from the formulation F4 over time during dialysis.

[0042] FIG. 4: HPLC traces for the unformulated nucleotide sequence according to SEQ ID NO: 1 (FIG. 4).

[0043] FIG. 5: HPLC traces for the nucleotide sequence according to SEQ ID NO: 1 released from a composition according to the present invention (FIG. 5).

[0044] FIG. 6: Uptake and distribution of antisense oligonucleotide of SEQ ID NO: 1 in porcine oral mucosa tissue by the use of the composition according to the present invention. 5-prime radioactively labelled oligonucleotide (α.sup.32P-UTP-labelled phosphorothioate-modified PS—ON) was formulated in the composition according to the present invention and applied to the mucosa for different time points. Cryostat sections of the tissue were performed (left side) and oligonucleotides visualized by contact emulsion autoradiography. Arrangement of porcine oral mucosa (left side) and human mucosa (right side) is similar. Oligonucleotides can be seen as white spots in all layers of the oral epithelium in dark field microscopy (left side).

[0045] FIG. 7: Kinetics of formulated oligonucleotide of SEQ ID NO: 1 delivery in porcine mucosa tissue ex vivo 5-prime radioactively labelled oligonucleotide of SEQ ID NO: 1 (α.sup.32P-UTP-labelled phosphorothioate-modified PS—ON) was formulated in the composition according to the present invention and applied to the mucosa for different time points as indicated. Cryostat sections of the tissue were performed and oligonucleotides visualized by contact emulsion autoradiography (white spots). A homogeneous distribution of the formulated oligonucleotide could be observed after 60 minutes of incubation.

[0046] FIG. 8: Quantification of uptake kinetics of formulated oligonucleotide of SEQ ID NO: 1 in porcine buccal and gingival mucosa tissue. 5-prime radioactively labelled oligonucleotide of SEQ ID NO: 1 (α.sup.32P-UTP-labelled phosphorothioate-modified PS—ON) was formulated in the composition according to the present invention and applied to porcine mucosa tissue ex vivo (buccal: blue line; gingival: red line) for different time points. Samples (at triplicates) were withdrawn at indicated time points and oligonucleotides of SEQ ID NO: 1 were extracted from the tissue and analyzed by PAGE. Quantification was performed using a Phosphorimager (Perkin Elmer®). Quantification revealed half times of drug delivery between approx. 20 minutes (gingival mucosa) and 45 minutes (buccal mucosa). C=full length oligonucleotide not applied to tissue for internal control (size/stability).

[0047] FIG. 9: Stability of the oligonucleotide according to SEQ ID NO: 1 in the absence (left) or presence (right) of human serum in vitro.

[0048] The present invention will be further illustrated in the following examples without being limited thereto.

Materials:

[0049] Poloxamer 188 (CAS No. 9003-11-6) and Poloxamer 407 (CAS No. 9003-11-6) were purchased from Sigma Aldrich. PBS was purchased from Biochrom GmbH. All of these materials had a grade for biology use.

EXAMPLE 1: FORMULATION 0 (F0)

[0050] To 1 mL of water at 4° C. were added 100 mg Poloxamer 188. The mixture was stirred at 4° C. until the polymer was dissolved, then 250 mg Poloxamer 407 were added to the mixture. After 8 h stirring at 4° C. a clear solution was obtained.

EXAMPLE 2: FORMULATION 1 (F1)

[0051] To 7 g of water at 4° C. were added 1 g Poloxamer 188 and 2 g Poloxamer 407. The mixture was stirred for 48 h at 4° C. until the polymers were dissolved and a clear solution was obtained.

EXAMPLE 3: FORMULATION 2 (F2)

[0052] To 7.03 g of 1×PBS at 4° C. were added 1 g Poloxamer 188 and 2 g Poloxamer 407. The mixture was stirred for 48 h at 4° C. until the polymers were dissolved and a clear solution was obtained.

EXAMPLE 4: FORMULATION 3 (F3)

[0053] To 3.50 g of 1×PBS were added 0.30 mg of the nucleotide sequence according to SEQ ID NO: 1 (21 μL; 15 mg/mL). The solution was cooled to 4° C. and then 0.50 g Poloxamer 188 and 1.00 g Poloxamer 407 were added. The mixture was stirred for 48 h at 4° C. until the polymers were dissolved and a clear solution was obtained.

EXAMPLE 5: FORMULATION 4 (F4)

[0054] To 3.29 g of 1×PBS were added 3.15 mg of the nucleotide sequence according to SEQ ID NO: 1 (210 μL; 15 mg/mL). The solution was cooled to 4° C. and then 0.50 g Poloxamer 188 and 1.00 g Poloxamer 407 were added. The mixture was stirred for 48 h at 4° C. until the polymers were dissolved and a clear solution was obtained.

EXAMPLE 6: DETERMINATION OF THE SOL-GEL TEMPERATURES OF FORMULATIONS F1 TO F4

[0055] Sol-gel temperatures were determined for the different formulations. The sol-gel transition point was measured by the magnetic stirrer method. Thereby, the respective formulation is heated to 40° C. in 30 min with constant magnetic stirring (100 rpm) and the temperature at which the stirrer stops rotating is considered as sol-gel transition temperature. The results are summarized in Table 1. The state of formulation 4 before and after sol-gel transition is depicted in FIGS. 1 and 2, respectively.

TABLE-US-00001 TABLE 1 Temperature Temperature Formulation (° C.) (° C.) F1 33.0 33.6 F2 31.0 30.7 F3 30.6 30.0 F4 29.0 29.1

EXAMPLE 7: DETERMINATION OF THE RNA RELEASE BY DIALYSIS FOR THE FORMULATION F4 AND A FORMULATION CONTAINING THE RNA IN PBS

[0056] A vial containing 25 mL of PBS was warmed up to 37° C., and kept at this temperature. A dialysis tube (MWCO 300 kD, Float-A-Lyzer G2, Spectrum Labs) pre-wetted at 37° C., was placed in the PBS vial and 1 mL of formulation F4 (4° C.) was filled in the tube via a syringe. The formulation was dialyzed for 90 min and for each time point 50 μL of the buffer solution was collected. The RNA concentration was determined by AEX HPLC (Dionex Ultimate 3000 UPLC using a DNA Pac PA 200 column) for each sample. The RNA concentration in F4 is 30 μg/mL. The results are summarized in Table 2. AEX-HPLC traces of the nucleotide sequence according to SEQ ID NO: 1 released from the formulation F4 over time are depicted in FIG. 3.

TABLE-US-00002 TABLE 2 Time point Oligonucleotide (min) conc. (μg/mL) % 5 0.15 0.6 10 0.32 1.3 15 0.48 1.9 20 0.63 2.5 30 0.93 3.6 40 1.28 5.0 50 1.65 6.4 60 1.97 7.7 70 2.35 9.2 80 2.68 10.5 90 3.04 11.9 48 h 35.25 100.0

[0057] The same procedure was applied to a solution of the nucleotide sequence according to SEQ ID NO: 1 (1 mg/mL) in PBS as a control. The solution was dialyzed for 240 min at 37° C. and for each time point 50 μL of the buffer solution were collected. The RNA concentration was determined for each sample by UV measurements (Eppendorf BioPhotometer D30). The results are summarized in Table 3.

TABLE-US-00003 TABLE 3 Time point Oligonucleotide (min) conc. (μg/mL) % 5 1.2 3.0 10 1.9 4.7 30 1.6 4.0 60 1.7 4.3 90 2.4 6.0 120 3.1 7.8 180 3.9 9.8 240 5.1 12.8 48 h 27.6 69.0

EXAMPLE 8: COMPARISON OF THE HPLC TRACES OF UNFORMULATED AND FORMULATED NUCLEOTIDE SEQUENCE ACCORDING TO SEQ ID NO: 1

[0058] HPLC traces were recorded for unformulated (i.e. neat) (FIG. 4) and formulated (FIG. 5) nucleotide sequence according to SEQ ID NO: 1. FIGS. 4 and 5 clearly demonstrate that the formulation process does not result in a degradation of the oligonucleotide.

EXAMPLE 9: UPTAKE OF THE NUCLEOTIDE SEQUENCE ACCORDING TO SEQ ID NO: 1 IN HUMAN ORAL MUCOSA TISSUE USING THE COMPOSITION ACCORDING TO THE PRESENT INVENTION

[0059] To investigate uptake and distribution of the nucleotide sequence according to SEQ ID NO: 1 included in the composition according to the present invention in the mucosa, 5-prime radioactively labelled oligonucleotide according to SEQ ID NO: 1 (α.sup.32P-UTP-labelled phosphorothioate-modified PS—ON) was formulated in the composition according to the present invention and applied to porcine mucosa tissue which is very similar to the human gingival ex vivo. After incubation cryostat sections were performed (FIG. 6, left side) and the oligonucleotides visualized by contact emulsion autoradiography. Arrangement of porcine oral mucosa (FIG. 6, left side) and human mucosa (FIG. 6, right side) is similar. Oligonucleotides can be seen as white spots in all layers of the oral epithelium in dark field microscopy (FIG. 6, left side).

[0060] Accumulation of more than 60% of the applied oligonucleotides in the keratinized layer was observed within several minutes (not shown), and the intermediate layer, prickle cell layer and basal cell layer also were reached in a time-dependent manner (FIG. 6). After one to two hours again accumulation of oligonucleotides at the basement membrane occurs indicating an additional protective function of this membrane within the healthy skin (not shown). However, in case of inflammation the basement membrane becomes more flexible and permeable due to the destruction by enzymatic degradation of invasive leukocytes.

EXAMPLE 10: KINETICS OF THE DELIVERY OF THE NUCLEOTIDE SEQUENCE ACCORDING TO SEQ ID NO: 1 IN ORAL MUCOSA TISSUE EX VIVO

[0061] The time course of an uptake of the nucleotide sequence according to SEQ ID NO: 1 included in the composition according to the present invention on oral mucosa tissue (porcine buccal and gingival mucosa tissue) was studied (FIGS. 7 and 8; in FIG. 8: buccal: blue line; gingival: red line). Experiments were performed as described in Example 9 and kinetics were performed by taking samples at indicated time points. Cryostat sections of the tissue were performed and oligonucleotides visualized by contact emulsion autoradiography (FIG. 7, white spots). A homogeneous distribution of the formulated oligonucleotide could be observed after 60 minutes of incubation. Samples (at triplicates) were withdrawn at indicated time points and oligonucleotides were extracted from the tissue and analyzed by PAGE (FIG. 8). Quantification was performed using a Phosphorimager (Perkin Elmer®). The quantification of the uptake kinetics revealed half times of drug delivery between approximately 20 minutes (gingival mucosa) and 45 minutes (buccal mucosa).

EXAMPLE 11: STABILITY OF THE NUCLEOTIDE SEQUENCE ACCORDING TO SEQ ID NO: 1 UNDER EXPERIMENTAL CONDITIONS

[0062] To test stability of the nucleotide sequence according to SEQ ID NO: 1, the oligonucleotide was 5′-radiolabeled and incubated either in the absence or in the presence of human serum (20%) for different time points. Samples were analyzed by PAGE and checked for degradation products. As seen in FIG. 9 no degradation products could be observed in the absence of serum at all, whereas slight degradation was visible in the presence of serum (20%) after 8 to 16 hours (upper arrow: full length oligonucleotide, lower arrow: degradation products).

[0063] The present invention relates to the following nucleotide sequence.

TABLE-US-00004 SEQ ID NO: 1 5′-mA-mU-m(5mC)-mA-G-A-T-G-(5mC)-G-T-G-G-(5mC)- (5mC)-T-mA-mG-mU-mG-3′ [0064] N=2′-deoxy nucleoside; [0065] mN=2′-O-methyl nucleoside; [0066] 5 mC=2′-deoxy-5-methyl-cytidine; [0067] m(5 mC)=2′-O-methyl 5-methyl-cytidine; [0068] all internucleotide phosphates are monophosphorothioate-modified.