Patent Application
20210346292
Botulinum toxins (BoNTs) consist of a primarily two major domains: binding domain (Heavy chain (HC)) and a catalytic domain (Light chain (LC)), linked through a disulfide bond (Montecucco and Schiavo 1995). Upon binding specifically to the presynaptic nerve membrane, BoNT is internalized through endocytosis, and the LC is translocated through a membrane pore formed by the translocation domain (TD) of the HC (Li and Singh 2000). The LC is a zinc-metalloprotease and acts as an endopeptidase with remarkable substrate specificity requiring a substantially long peptide sequence, depending on the serotype (Segelke et al. 2004). This is unique to BoNTs as other microbial metalloproteases can recognize sequences as short as a dipeptide (Segelke et al., 2004; Silvaggi et al. 2007). Serotype A cleaves SNAP-25 (25-kDa synaptosome associated protein) and the light chain of BoNT/A will be examined in this paper (Li and Singh 1999; Kukreja and Singh 2007).
BoNTs are secreted from the Clostridium botulinum bacteria in the form of multimeric complexes, with a set of non-toxic proteins coded for by genes adjacent to the neurotoxin gene (Inoue et al., 1996; Singh et al., 2014). Botulinum complex size ranges from 300 kDa to 900 kDa and exist in three progenitor toxin forms: M (medium), L (large) and LL (extra-large) forms. The M form consists of neurotoxin (150 kDa) and a nontoxic protein component (120 kDa) which is called neurotoxin binding protein (NBP) (Singh et al., 1995) or nontoxic non-hemagglutinin component (NTNH) (East and Collins, 1994) with 12S molecular size (the molecular size of complex forms is expressed as sedimentation equilibrium values). The L form has molecular weight of about 500 kDa and a molecular size of 16S. The LL form is about 900 kDa and 19S.
Currently major BoNT therapeutic products include BoNT/A complex (marketed as Botox® and Dysport®), BoNT/B complex (marketed as Myobloc® and Neurobloc®), and isolated BoNT/A without NAPs (marketed as Xeomin®). Although there is no therapeutic role defined for NAPs, these may play a role in the stability of the BoNT formulation and in diffusion of the injected BoNT for therapeutic purposes (Carli et al., 2009; Shone et al., 2011). In BoNT/A complex preparations adding either sodium chloride (Botox®, Allergan, Inc.) or lactose (Dysport®, Ipsen, Ltd.) protect the steric conformation of BoNT (Panicker and Muthane, 2003). Human serum albumin is also added to prevent loss from surface adsorption. The toxin is then dried either with freezing (Dysport®) or without freezing (Botox®, Allergan, Inc.) (Panicker and Muthane, 2003). These as well as the pure BoNT/A product, Xeomin®, are lyophilized products which are reconstituted with saline solution maintained near physiological pH.
The botulinum toxin type B product (Myobloc®, Neuroblock®) is provided in liquid form at pH 5.6, as opposed to a lyophilized powder that requires reconstitution in saline. It nevertheless is also based on the complex of BoNT/B neurotoxin and NAPs. BoNT/B has shown stability for months when stored appropriately at 2° C. to 8° C., whereas BoNT/A must be stored at −5° C. as a powder and must be used within hours once reconstituted according to the manufacturer's recommendation (Kim et al., 2003).
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.
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.
Another aspect of present invention is the use of lipids.
Another aspect of present invention is the use of lipids in certain ratios.
Another aspect of present invention is the use of different herbal lipids.
Another aspect of the present invention is the lipids are DOTAP (1,2-dioleolyl-3-triethylammonium-propane), DPPC (Dipalmitoylphosphatidylcholine), and cholesterol.
Another aspect of the present invention is the ratio of DOTAP:DPPC:Cholesterol is 5:5:3.
Another aspect of the present invention is the mixing of the lipids with DOTAP/glutaryl PE 99:1 mol/mol) and non-ionic amphiphiles or detergents such as Tween 80 or SPAN-80 in chloroform or chloroform/water mixture.
Another aspect the formation of liposomes.
Another aspect of the present invention is the encapsulation of protein in the liposome.
Another aspect of the present invention is the encapsulated proteins are botulinum toxin A and botulinum toxin complex A.
Another aspect of the present invention is the method of encapsulation of toxin or complex.
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.
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.
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.
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 (
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 (
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 (
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 (
