Pharmaceutical preparation for increasing stability and bioavailability of Botulinum toxin A and its complex

Abstract

The main aspect of present invention is to provide a pharmaceutical composition to increase the stability of liquid formulation of botulinum toxin and related proteins. The present invention provides a method to stabilize toxin in liquid formulation. Lipid based drug delivery system is known to increase the bioavailability of drugs (Amidon et al., 1995; Jannin et al., 2008). We investigated the stability of BoNT/A toxin and complex. We used two formulations in liquid phase: combination of lipids and liposomes, with two different storage conditions: 4° C. and 25° C. The present invention also provides a method for efficient delivery of botulinum toxin through skin as a topical application.

Claims

1. A pharmaceutical composition comprising proteins with lipids, solution as emulsion or suspension, and/or encapsulated microspheres including liposomes.

2. The pharmaceutical composition as claimed in claim 1, wherein the proteins are botulinum toxin or complex thereof or various associated proteins of botulinum toxin.

3. The pharmaceutical composition as claimed in claim 1, wherein the pharmaceutical composition further comprises proteins of Clostridium botulinum.

4. The pharmaceutical composition as claimed in claim 1, wherein the pharmaceutical composition comprises therapeutic protein including various serotypes of botulinum toxin, vaccines and protein hormones.

5. The pharmaceutical composition as claimed in claim 1, wherein the lipids are charged with one of positive or negative molecules, or the lipids are neutral molecules.

6. The pharmaceutical composition as claimed in claim 1, wherein the lipids are mixture of charged and neutral lipids.

7. The pharmaceutical composition as claimed in claim 1, wherein the lipids are herbal or plant lipids.

8. The pharmaceutical composition as claimed in claim 1 further comprising non-ionic amphiphiles or detergents including one of glutaryl PE, Tween 80, Tween 60, Tween 20, PEGs, Cremophor EL or SPAN 80, or combination thereof to be mixed with the lipids.

9. The pharmaceutical composition as claimed in claim 1, wherein the emulsion is encapsulated microspheres and nanospheres (nanoparticles) containing propylene glycol (0.3-6%), phenoxyethanol (0.1-5%), sodium hyaluronate (0.01-1%), caprylic/capric triglyceride (0.5-10%), hydrogenated castor oil (1-15%) and Span-80 (0.01-6%) with water.

10. The pharmaceutical composition as claimed in claim 1, wherein the lipids are one of microspheres or nanospheres, including nanoparticle solution.

11. The pharmaceutical composition as claimed in claim 1, wherein the lipids are composed of oil solutions including for example, triglyceride, ethyl icosapentate, castor oil, tocopherol nicotinate, teprenone, indomethacin franesil, soy-bean oil, tea oil, sunflower seed oil, vegetable oil, fish oil, sesame oil, soy-bean oil, tea oil, sunflower seed oil, vegetable oil, fish oil, sesame oil, soy-bean oil, tea oil, sunflower seed oil, vegetable oil, fish oil, sesame oil, soy-bean oil, tea oil, sunflower seed oil, vegetable oil, fish oil, sesame oil, Labrafac Lipophile WL 1349 oil, and dronabinol.

12. The pharmaceutical composition as claimed in claim 1, wherein the pharmaceutical composition to be with excipients including oil solution, mixed glycerides, water-soluble co-solvents and surfactants, including Permulen TR1, Permulen TR2, RH-40, Tween-80, Tween-60, etc. and self-made creams.

13. The pharmaceutical composition as claimed in claim 1, wherein the pharmaceutical composition further comprises surface active agents, chelating agents, salicylates, anti-inflammatory agents, antibacterial agents, antifungal agents, antiviral agents or phenothiazines and bioactive peptides including pentapeptide KTTKS, tetrapeptide GQPR, hexapeptide argireline, tripeptide GHK, Snap-8 octapeptide and oligo-peptides.

14. The pharmaceutical composition as claimed in claim 1, wherein the pharmaceutical composition further comprises humectants including propylene glycol or lecithin; and emolliants including zinc oxide or dimethicone.

15. The pharmaceutical composition as claimed in claim 1, wherein the pharmaceutical composition further comprises a stabilizer including human serum albumin or IgG.

16. The pharmaceutical composition as claimed in claim 1, wherein the pharmaceutical composition is a lyophilized powder, lotion, serum or gel.

17. The pharmaceutical composition as claimed in claim 1, wherein the pharmaceutical composition is an encapsulated liposome or emulsified proteins with retinoids, alpha hydroxyl acids, hyaluronic acid and/or sodium salt, resveratrol, stem cells, EGFs (epidermal growth factors), KGFs (keratinocyte growth factors), FGFs (fibroblast growth factors), HGH (human growth hormones), niacinamide, aloe vera, allantoin and therapeutic agents thereof.

18. The pharmaceutical composition as claimed in claim 1, wherein the pharmaceutical composition is stabilized at a pH in between 5.5 and 8.0.

19. A method of treatment administrating effective amount of pharmaceutical composition comprising proteins with lipids, solution as emulsion or suspension, and/or encapsulated microspheres including liposomes.

20. The method claimed in claim 19, wherein the method of administration route includes one of topical, intranasal, injectable and oral, wherein the administration is to treat local and systemic conditions including neuromuscular, gastrointestinal, diabetic, cardiovascular, reproductive issues and skin problems.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0012] Described herein are formulations, pharmaceutical formulations, and methods of preparing and using the stable formulations described herein. These pharmaceutical formulations may be prepared by the processes described herein. In some variations the therapeutic agent is botulinum toxin A and botulinum toxin complex A.

[0013] In some variations the pharmaceutical formulations described herein can be used for the treatment, prevention, inhibition, delaying onset of, or causing regression of one or more neuromuscular diseases and conditions. In some variations the diseases or conditions include neuronal regeneration/sprouting, disease involving muscle movement, various wounds, scars and gastrointestinal symptoms.

[0014] Botulinum neurotoxin is a large protein toxin (approximately 150 kDa) that is able to bind and internalize to motor neurons very specifically. BoNTs are produced by Clostridium botulinum along with several neurotoxins associated polypeptides (NAPs). The toxin with NAPs is termed as complex toxin. Present application provides the method to have a stable liquid formulation. The method used in this application tested three different conditions; a) stability of the formulation in the presence of lipids, b) stability of the formulation in the encapsulated liposome, c) stability of the formulation in emulsion, mixture, or suspension form, and d) stability of the formulation in cellular model (function of the main active therapeutic ingredient). The present invention provides a pharmaceutical formulation of toxin with lipids.

[0015] In the first part of the present application, a lipid solution was made by dissolving a lipid film, made of Dotap: DPCC: Cholesterol (5:5:3), in 10 mM sodium phosphate buffer, pH 7.1. Dissolve the therapeutic proteins, BoNT/a toxin and BoNT/a toxin complex, in the solution of lipids. Activity was performed at different time points after incubating solution at 4° C. and 25° C. Activity of the enzyme was performed against the full-length substrate. Prior to activity reaction, lipid solution, containing enzyme, was incubated with the reaction buffer, 10 mM sodium phosphate (pH 7.4) containing 150 mM NaCl and 1.25 mM DTT (dithiotritol) for 30 mins at 37° C. After incubation proteins were incubated with substrate for 1 hr at 37° C. Reaction was stopped by adding 4×SDS-sample buffer. At 4° C., liquid formulation of both toxin and complex in lipids holds their endopeptidase activity for 8 weeks, as assessed by endopeptidase activity. After 8 weeks, 90% of activity remains for the toxin whereas complex hold 100% of its activity. After 24 weeks, the complex still holds about 57% of its activity, whereas toxin holds 29% of its endopeptidase activity (FIG. 1).

[0016] In the second part, lipid film was resuspended in the buffer, mM sodium phosphate, pH 7.1, containing toxin or complex. Encapsulated liposomes were formed by several freeze-thaw cycle followed by sonication. Unencapsualted proteins were removed from the supernatant using spin columns. The activity of encapsulated proteins were determined as follows. Prior to activity reaction, encapsulated proteins were incubated in the reaction buffer for 30 mins at 37° C. After incubation proteins were incubated with the substrate for 1 hr at 37° C. Reaction was stopped by adding 4×SDS-sample buffer. For botulinum toxin samples, reactions were performed in 10 mM sodium phosphate buffer, pH 7.1, containing 1.25 mM DTT and 0.2% Triton X-100. Whereas for botulinum toxin complex samples, reactions were performed 10 mM sodium phosphate, pH 7.1, containing 150 mM NaCl, 1.25 mM DTT and 0.2% Triton X-100. At 4° C., liquid formulation of both toxin and complex in lipids holds their endopeptidase activity for 8 weeks as assessed by endopeptidase activity (FIG. 2).

[0017] In the third part, proteins were emulsified or suspended with encapsulated microspheres and nanoshperes (nanoparticles) containing propylene glycol (0.3-6%), phenoxyethanol (0.1-5%), sodium hyaluronate (0.01-1%), caprylic/capric triglyceride (0.5-10%), hydrogenated castor oil (1-15%) and span-80 (0.01-6%) in water Protein was emuslsified by rotating the solution at room temperature (25° C.) for 15 min. Activity of emulsified protein was determined as above (FIG. 3).

[0018] In the fourth part, encapsulation or emulsification of protein was performed by using the similar procedure as above. Liposome incubated proteins are dissolved in the serum free media and incubated with M-17 neuroblastoma cells for 48 hrs. After incubation times, cells were detached and lysed using M-per reagent (Thermo Fisher Scientific). SNAP-25 cleavage in M-17 cells wasmonitored using western blot. Anti-SNAP25 monoclonal antibody was used as a primary antibody, and anti-rabbit IgG alkaline phosphatase was used as a secondary antibody for western blot. The blot was developed using BCIP (5-bromo-4-chloro-3-indolylphosphate toluidine; Sigma Aldrich) reagent. The Western blot image showed that both encapsulated toxin and complex had higher activity than the unencapsulated proteins (FIG. 4), indicating better delivery of encapsulated toxin inside the M17 cells. Emulsified formulation is not good for cell morphology that's why cellular assay was not performed in this formulation.

FIGURE DESCRIPTIONS

[0019] FIG. 1: Stability data of Botulinum toxin A and its complex in lipid mixture solution (DOTAP: DPCC: Cholesterol:: 5:5:3). Stability experiments were performed in two conditions; 4° C. and ° C. All the reactions were performed at 37° C. in the 10 mM sodium phosphate buffer, pH 7.4, 150 mM NaCl and 1.25 mM DTT. Prior to reaction, protein solutions were incubated at 37° C. for 30 mins in the reaction buffer.

[0020] FIG. 2: Stability data of liposome encapsulated Botulinum toxin A and its complex. Stability experiments were performed in two conditions; 4° C. and 25° C. All the reactions of liposome encapsulated botulinum toxin A were performed at 37° C. in 10 mM sodium phosphate buffer, pH 7.1, containing 1.25 mM DTT and 0.2% Triton X-100. All the reactions of liposome encapsulated botulinum toxin complex A were performed at 37° C. in 10 mM sodium phosphate buffer, pH 7.1, containing 150 mM NaCl, 1.25 mM DTT and 0.2% Triton X-100. Prior to reaction, protein solutions were incubated at 37° C. for 30 mins in the reaction buffer.

[0021] FIG. 3A and FIG. 3B: Stability data of Botulinum toxin A and its complex in lipid mixture solution in emulsified nanosphere formulations. The stability data of two different nanosphere formulations i.e. HA1 and HA2 are depicted in FIG. 3A and FIG. 3b respectively. Initially, stability experiments were performed in two conditions; 4° C. and 25° C. All the reactions were performed at 37° C. in the 10 mM sodium phosphate buffer, pH 7.4, 150 mM NaCl and 1.25 mM DTT. Prior to reaction, protein solutions were incubated at 37° C. for 30 mins in the reaction buffer. After month 5, samples were divided into two parts and store at −20° C. and −80° C. 13th month stability data was of samples stored at −20° C. and −80° C. A) Weekly stability data of HA1 and HA2. B)4th, 5th and 13th month stability data.

[0022] FIG. 4: Western blot of the cleavage of SNAP-25. Lane 1: Marker, lane 2: control M-17 cells without any treatment, lane 3: encapsulated botulinum toxin complex A treated M-17 cells, lane 4: botulinum toxin complex A (unencapsulated) treated M-17 cells, lane 5: encapsulated botulinum toxin A treated M-17 cells, and lane 6: botulinum toxin (unencapsulated) A treated M-17 cells. Samples for Western blot were prepared after 48 hr of incubation at 37° C. U and C are uncleaved SNAP-25 and cleaved SNAP-27.

REFERENCES

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