HK1107670B - Therapeutic composition with a botulinum neurotoxin - Google Patents

Description

Therapeutic compositions comprising botulinum neurotoxin

The present invention relates to pharmaceutical compositions comprising a botulinum neurotoxin from Clostridium botulinum (Clostridium botulinum) which neurotoxin is free of complexing proteins naturally present in the botulinum neurotoxin complex or to pharmaceutical compositions comprising a chemically modified or genetically modified botulinum neurotoxin which modified botulinum toxin is free of complexing proteins naturally forming complexes with botulinum neurotoxin. Furthermore, the pharmaceutical composition of the present invention has good stability and can be advantageously formulated without human serum albumin.

Albumin of human origin has been used in large quantities as carriers and stabilizers for protein active ingredients present in pharmaceutical compositions. Albumin has been shown to stabilize proteinaceous active ingredients in compositions by reducing the adhesion and denaturation of the active ingredient. Furthermore, the albumin is not immunogenic when injected into a human patient.

However, there are significant disadvantages to using albumin in pharmaceutical compositions. Albumin has been thought to contribute to the transmission of certain stable viruses, prions or other infectious or pathogenic compounds, such as Transmissible Spongiform Encephalopathies (TSEs) in humans. As a result, regulatory studies on pharmaceutical compositions comprising human serum albumin are increasing. Similarly, gelatin has been used in some pharmaceutical compositions containing protein active ingredients as an albumin substitute. Gelatin, being a protein of mammalian origin, also poses the same risk of pathogen transmission. Therefore, there is a need for alternatives to protein stabilizers of mammalian origin.

The botulinum toxin complex consists of a mixture of clostridial proteins. They are hemagglutinin of varying molecular weight, non-toxic, non-hemagglutinating proteins (Mr. about 120,000) and neurotoxins (Mr. about 150,000). They form acid-stable complexes which are responsible for oral toxicity in food poisoning. In contrast to pure neurotoxins, the complex resists the aggressive environment in the gastrointestinal tract, making possible the enteral absorption of the neurotoxin, and its passage through the bloodstream and the lymphatic system to the target cells, causing the blocking of neurotransmitter release. This in turn produces muscle paralysis and cessation of various autonomic functions. Poisoned patients die from respiratory muscle failure. The neurotoxin is non-toxic after ingestion as it degrades in the gastrointestinal tract and thus does not undergo intestinal absorption. For parenteral administration, there is no difference in the therapeutic effect of the neurotoxin and the complex, since the complex is broken down into various components in the tissue and only the neurotoxin is taken up by the target cells.

Currently, two products comprising botulinum neurotoxin type a are approved for the treatment of blepharospasm, hemifacial spasm and spastic torticollis: BOTOXAnd DYSPORT. The state of the art is the direct injection of botulinum neurotoxin into dystonic or spastic muscles, releasing the neurotoxin from the complex at physiological pH values, thereby producing the desired pharmacological effect. Clinical trials are currently underway to treat other neurological disorders (e.g., spasticity, migraine, low back pain, cervical spine disorders, salivation). Botulinum toxin type A complex (Mr 900,000) is approved for the treatment of various dystonia. Approved products are also used for cosmetic indications such as hyperhidrosis and significant wrinkles. Other Clostridium botulinum toxin complexes (types B, C1, D, E, F, G) and toxins derived from Clostridium botulinum toxin by chemical modification or genetic manipulation are also suitable for these treatments.

BOTOXAnd DYSPORTAre provided to the clinician in lyophilized form for reconstitution prior to use. Unfortunately, not every patient and indication requires the same dosage. As a result, reconstituted compositions are often frozen or refrigerated for later use. The potency stability of these subsequently reconstituted compositions has been evaluated. BOTOX has been found to be present when reconstituted and stored frozen for 12 hoursAt least 44% of the potency was lost. Furthermore, 70% of the potency was lost when the reconstituted composition was frozen at-70 ℃. Gartlan, m.g., and Hoffman, h.t.crystal prediction of botulin type a (Botox): degradation in location with storage, Otolaryngology-Head and neutral Sun gery 102 (2): 135-140(1992). This instability leads to significant dose variation and product waste. It is therefore an object of the present invention to develop and produce stable liquid and lyophilized formulations of botulinum toxin which have better handling characteristics than existing formulations.

Novel pharmaceutical compositions comprising botulinum neurotoxin (type A, B, C1, D, E, F or G) have been developed which are free of hemagglutinin and other exogenous proteins. This reduces the total protein content of the pharmaceutical composition without reducing the amount of toxins. We found in antigen studies that all types of pure neurotoxins do not induce or at most very little induce the formation of antibodies, in contrast to the type a commercial products and the type B to G complexes. When this newly developed drug (pure A, B, C1, D, E, F or type G neurotoxin) was used therapeutically, there was no treatment failure due to the antibody even after repeated dosing.

However, as noted above in particular, the formulation creates problems. Botulinum toxin is very labile as an active ingredient of a proteinaceous nature. Furthermore, botulinum toxin complexes are highly susceptible to denaturation by surface denaturation, heat and alkaline conditions.

The biological activity of botulinum toxins (which are intracellular peptidases) is dependent, at least in part, on their three-dimensional conformation, due to the enzymes generally contained. Thus, botulinum toxin can be detoxified by heat, various chemicals, surface stretching and surface drying. In addition, it is well known that dilute toxin concentrations used in approved indications result in rapid detoxification of the toxin unless a suitable stabilizing agent is present. Moreover, toxin stability is an important factor for storage. A stabilizer is necessary. To date, stability has been achieved by preparing albumin and gelatin as mammalian derived proteins. As noted above, mammalian derived proteins increase the risk of contamination of the toxin by certain stable viruses, prions or other infectious or pathogenic compounds carried from the donor.

In addition, the conditions of lyophilization, including pH, temperature, dilution, and vacuum pressure cause detoxification of the toxin. To date, mammalian derived proteins such as gelatin and serum albumin have been successfully used to stabilize botulinum toxin and are standard stabilizers.

For example, pharmaceutical compositions BOTOX comprising commercially available botulinum toxin(commercially available from Allergan, Inc., of Irvine, California) consists of purified botulinum toxin type A, albumin and sodium chloride, packaged in sterile, vacuum-dried form. BOTOX per bottleComprising about 100 units (U) of a botulinum toxin complex type A in sterile, vacuum-dried form, 0.5 mg human serum albumin and 0.9 mg sodium chloride, without preservatives.

There have been many attempts in the prior art to stabilize protein compositions. Carpender et al, Interactions of Stabilizing Additives with Proteins DuringFreeze-Thwinging and Freeze-Drying, International Symposium on biological Product Freeze-Drying and Formulation, 24-26October 1990; karger (1992), 225- & 239.

Cellobiose was used as an excipient in combination with albumin and sodium chloride, and toxicity degradation was demonstrated after lyophilization of crystalline botulinum toxin type A with these excipients (10% recovery), compared to toxicity after lyophilization of albumin alone (> 75% to > 90% recovery). Goodnough et al, Stabilization of Botulinum Toxin type A During lysis, App & Envir. Micro.58(10) 3426-.

Furthermore, protein formulations comprising sugars (e.g. glucose or glucose polymers) or carbohydrates are known to be unstable, since proteins and glucose have been shown to interact and undergo Maillard degradation, both due to the reducing nature of glucose and glucose polymers. In contrast, alcohols, such as inositol, mannitol, are non-reducing and have long been used as cryoprotectant excipients to stabilize proteins during lyophilization.

Depending on the instability of botulinum toxin and the risks associated with mammalian derived stabilizers and polysaccharides, appropriate protein stabilizers will be a continuing goal of formulation scientists.

SUMMARY

It is an object of the present invention to provide a non-protein alternative to a mammalian derived protein in a pharmaceutical composition comprising a botulinum toxin or toxin derived from a botulinum toxin by chemical modification or genetic manipulation. When injected into a human patient, the novel formulation must have low, preferably negligible, immunogenicity.

We therefore believe that the specific statements we intend to encompass by our invention may be summarized as follows:

a pharmaceutical composition comprises a botulinum neurotoxin from Clostridium botulinum A, B, C1, D, E, F or Clostridium botulinum G, or a mixture of two or more botulinum neurotoxins, wherein the neurotoxin or mixture of neurotoxins is free of complexing proteins that naturally form complexes with the botulinum neurotoxin, and a non-proteinaceous stabilizer that retains the biological activity of the botulinum neurotoxin in aqueous solution.

A pharmaceutical composition wherein the botulinum neurotoxin has been modified chemically or by genetic manipulation, or a mixture thereof;

such a pharmaceutical composition wherein the non-proteinaceous stabilizing agent is selected from one or more of hyaluronic acid, polyvinylpyrrolidone and polyethylene glycol;

such a pharmaceutical composition comprises a pH buffering agent;

such pharmaceutical compositions wherein the pH buffering agent is sodium acetate;

such pharmaceutical compositions comprise a cryoprotectant;

such pharmaceutical compositions wherein the cryoprotectant is a polyol;

such a pharmaceutical composition, wherein the polyhydric alcohol is selected from one or more of inositol, mannitol, and sorbitol;

such pharmaceutical compositions, are lyophilized;

such a pharmaceutical composition wherein the non-proteinaceous agent is hyaluronic acid and wherein the composition is lyophilized;

a pharmaceutical composition comprising hyaluronic acid and wherein the composition is freeze-dried;

such pharmaceutical compositions are administered to an animal, including a human, in an amount effective to treat the disorder for which botulinum neurotoxin therapy or treatment is indicated;

such pharmaceutical compositions, wherein the disorder for which botulinum neurotoxin therapy or treatment is indicated is selected from the group consisting of cosmetic disorders, blepharospasm, hemifacial spasms, spasmodic torticollis, spasticity, dystonia, migraine, low back pain, cervical spinous process disorders, strabismus, hyperhidrosis, and hypersalivation;

such pharmaceutical compositions, wherein the cosmetic condition is a significant wrinkle;

and a botulinum neurotoxin formulation comprising a botulinum neurotoxin from Clostridium botulinum A, B, C1, D, E, F or Clostridium botulinum G or a mixture of two or more botulinum neurotoxins, wherein the neurotoxin or mixture of neurotoxins is free of complexing proteins which naturally form complexes with the botulinum neurotoxin, and a non-proteinaceous stabilizer which retains the biological activity of the botulinum neurotoxin in aqueous solution;

such a botulinum neurotoxin formulation wherein the botulinum neurotoxin has been modified chemically or by genetic manipulation, or a mixture thereof;

such a botulinum neurotoxin formulation wherein the non-proteinaceous stabilizing agent is selected from one or more of hyaluronic acid, polyvinylpyrrolidone and polyethylene glycol;

such a botulinum neurotoxin formulation wherein the aqueous solution comprises a pH buffer;

such a botulinum neurotoxin formulation wherein the pH buffering agent is sodium acetate;

such botulinum neurotoxin preparations are free of stabilizers of mammalian derived proteinaceous nature;

such a botulinum neurotoxin formulation wherein the formulation is free of stabilizers of a mammalian derived proteinaceous nature, albumin and gelatin;

such a botulinum neurotoxin formulation wherein the non-proteinaceous stabilizer is hyaluronic acid and wherein the composition is freeze-dried;

such a botulinum neurotoxin formulation comprising hyaluronic acid and wherein the composition is freeze dried;

and a method of stabilizing a botulinum neurotoxin from Clostridium botulinum A, B, C1, D, E, F, or Clostridium botulinum G, or a mixture of two or more botulinum neurotoxins, wherein the neurotoxin or mixture of neurotoxins is free of complexing proteins that naturally form complexes with the botulinum neurotoxin, comprising mixing the neurotoxin with a non-proteinaceous stabilizer in an amount effective to retain the biological activity of the neurotoxin in an aqueous solution;

a method wherein the botulinum neurotoxin is modified chemically or by genetic manipulation, or a mixture thereof;

such a method wherein the non-proteinaceous stabilizing agent is selected from one or more of hyaluronic acid, polyvinylpyrrolidone and polyethylene glycol;

such a method, wherein the aqueous solution comprises a pH buffer;

such a method, wherein the pH buffer is sodium acetate;

such a method, wherein the aqueous solution comprises an anti-freeze agent;

such a method, wherein the aqueous solution is freeze-dried;

such a method wherein the non-proteinaceous stabilizing agent is hyaluronic acid and wherein the composition is freeze-dried;

such methods, comprising hyaluronic acid and wherein the composition is freeze-dried;

and a method of treating a botulinum neurotoxin therapy or a condition for which treatment is indicated, comprising the step of administering to a human, or animal in need thereof a composition comprising a botulinum neurotoxin from Clostridium botulinum type A, B, C1, D, E, F, or G, or a mixture of two or more botulinum neurotoxins, wherein the neurotoxin or mixture of neurotoxins is free of complexing proteins that naturally form complexes with the botulinum neurotoxin, and is mixed with a non-proteinaceous stabilizer that retains the biological activity of the neurotoxin in water;

such methods, wherein the botulinum neurotoxin is modified chemically or by genetic manipulation, or mixtures thereof;

such a method wherein the non-proteinaceous stabilizing agent is selected from one or more of hyaluronic acid, polyvinylpyrrolidone and polyethylene glycol;

such a method, wherein the aqueous solution comprises a pH buffer;

such a method, wherein the pH buffer is sodium acetate;

such a process, wherein an aqueous antifreeze agent;

such a process wherein the anti-freeze agent is a polyol;

such a process wherein the polyol is selected from one or more of inositol, mannitol and sorbitol;

such a method wherein the non-proteinaceous stabilizing agent is hyaluronic acid and wherein the composition is lyophilized;

such a method wherein the aqueous solution comprises hyaluronic acid and wherein the composition is freeze-dried;

such a method, wherein the disorder for which botulinum neurotoxin therapy or treatment is indicated is selected from the group consisting of cosmetic disorders, blepharospasm, hemifacial spasm, spasmodic torticollis, spasticity, migraine, low (back) pain, cervical spinous process disorders, strabismus, hyperhidrosis, hypersalivation, and dystonia;

such methods, wherein the cosmetic condition is a significant wrinkle;

and the use of a botulinum neurotoxin formulation for the treatment of a cosmetic condition;

and the use of a botulinum neurotoxin preparation for the preparation of a medicament for the treatment of a condition for which a botulinum neurotoxin therapy is indicated.

Definition of

As used herein, the following words and terms have the following definitions.

A "pharmaceutical composition" is a formulation in which the active ingredient, i.e., botulinum toxin herein, or botulinum toxin free of the hemagglutinin protein of the present invention, or toxin derived from botulinum toxin by chemical modification or genetic manipulation, is stabilized by a substance other than a mammalian-derived protein. The pharmaceutical composition may be suitable for diagnostic or therapeutic administration (i.e., intramuscular or subcutaneous injection) to a human patient. The pharmaceutical composition may be lyophilized or vacuum dried, reconstituted, or in solution. The botulinum toxin active ingredient may be one of botulinum toxin serotypes A, B, C1, D, E, F or G, all of which may be further modified to be free of complexing proteins present in the native neurotoxin or modified by genetic manipulation.

"therapeutic agent" refers to the ability of the agent of the invention to treat/alleviate a condition such as an indication of peripheral muscle hyperactivity (i.e., spasticity).

"Stabilizing", "Stabilizing" or "stabilization" means that the active ingredient, i.e., botulinum toxin or a toxin derived from botulinum toxin by chemical modification or genetic manipulation, present in the reconstituted or aqueous pharmaceutical composition has a toxicity that is about 20% to about 100% higher than the toxicity that the biologically active botulinum toxin would have had prior to incorporation into the pharmaceutical composition.

"cryoprotectant" refers to an excipient that renders the active ingredient, i.e., botulinum toxin or a toxin derived from botulinum toxin by chemical or genetic modification present in a reconstituted or aqueous pharmaceutical composition to have a toxicity that is about 20% to about 100% higher than the toxicity that the biologically active botulinum toxin would have prior to being lyophilized into a pharmaceutical composition.

"pH buffering agent" refers to a chemical substance that is capable of adjusting the pH of a composition, solution, etc. to a certain value or range of pH.

"polyol" refers to an aliphatic or cycloaliphatic carbohydrate that contains more than one hydroxyl functionality and no carbonyl functionality (e.g., of a carbohydrate).

By "free of mammalian-derived protein stabilizers" is meant that the composition or formulation does not contain a detectable amount of stabilizers derived from mammalian proteins.

"chemical modification" refers to methods known in the art that modify any serotype of native botulinum toxin by chemical reaction or the like; it especially refers to a substitution, deletion, insertion, addition or genetic post-translational modification of an amino acid of a botulinum toxin.

"genetic manipulation" refers to methods known in the art for modifying any serotype of native botulinum toxin by modifying the gene encoding the botulinum toxin or a respective nucleic acid, such as DNA or RNA.

Description of the invention

The present invention describes pharmaceutical compositions comprising a stabilized neurotoxin, which compositions can be formulated free of mammal derived proteins or donor pool albumin by incorporating non-proteinaceous stabilizing agents, in particular by incorporating hyaluronic acid or polyvinylpyrrolidone or polyethylene glycol or a mixture of two or more thereof. The present invention relates to the development of a botulinum toxin composition formulated with hyaluronic acid or polyvinylpyrrolidone or polyethylene glycol or a mixture of two or more thereof. Such compositions are safer compositions with significant stability.

Fortunately, the compositions of the present invention are characterized by the absence of formulating botulinum toxin in a stabilizer of a mammalian derived proteinaceous nature or a toxin derived from botulinum toxin by chemical modification or genetic manipulation. It has been determined that compositions of hyaluronic acid or polyvinylpyrrolidone or polyethylene glycol components or mixtures thereof, particularly those incorporating a pH buffer, especially a sodium acetate buffer, and/or a cryoprotectant, can increase the stability and useful shelf life of the pharmaceutical compositions of the present invention.

Furthermore, the pharmaceutical compositions and formulations of the present invention preferably do not contain stabilizers of the nature of mammalian derived proteins and do not contain any stabilizing proteins.

The present invention is not limited to pharmaceutical compositions, but also refers to methods of stabilizing botulinum toxin compositions or toxin compositions derived from botulinum toxins by chemical modification or genetic manipulation. Botulinum toxin or toxins derived from botulinum toxin by chemical modification or genetic manipulation are stabilized by incorporating hyaluronic acid or polyvinylpyrrolidone or polyethylene glycol or mixtures thereof into the composition. In addition, the incorporation of a pH buffer into the pharmaceutical composition enhances stability, thereby stabilizing the pH and facilitating shelf-life of the reconstituted toxin and/or by incorporating a cryoprotectant into the pharmaceutical composition, thereby increasing freeze-drying stability and shelf-life.

Preferably, the nature and amount of the pH buffering agent is such as to stabilize or adjust the pH of the compositions or formulations of the present invention to a range of from about 4 to about 7.5. Suitable pH buffers may be citrate, phosphate and especially acetate buffer systems, especially sodium acetate buffer systems. Surprisingly, it has been found that if an acetate buffer is used in the composition or formulation of the present invention and the composition or formulation is freeze-dried, acetate can be removed from the composition or formulation during the freeze-drying process; the compositions or formulations have an approximately neutral pH (in the range of about 6.5 to about 7.5) after reconstitution or thawing. It is advantageous when administering said composition or formulation to a human or patient in need thereof, especially when injected into a muscle, because the (approximately) neutral botulinum toxin composition or formulation causes less pain than a composition or formulation having an acidic pH, e.g. 4.

A detailed embodiment of the invention may be a pharmaceutical composition suitable for injection into a human patient comprising a botulinum toxin or a toxin derived from a botulinum toxin by chemical modification or genetic manipulation, and hyaluronic acid or polyvinylpyrrolidone or polyethylene glycol, the composition optionally being pH stabilized by a suitable pH buffer, in particular by a sodium acetate buffer, and/or a cryoprotectant polyol.

The pharmaceutical composition is suitable for administration to a human patient to achieve a therapeutic effect, and the neurotoxin can be botulinum toxin serotype A, B, C1, D, E, F, and G, preferably botulinum toxin free of complexing proteins present in the native neurotoxin or a neurotoxin modified by chemical or genetic manipulation. The modified neurotoxin is also free of complexing proteins that naturally form complexes with botulinum neurotoxin.

The neurotoxin derived from a botulinum neurotoxin by chemical modification or genetic manipulation can be located on each portion of the neurotoxin protein, for example on the heavy chain portion and/or the light chain portion of the neurotoxin molecule. One or more modifications may be present. Preferably, the heavy chain of the neurotoxin derived from a botulinum neurotoxin comprises one or more modifications which may reduce or increase the affinity of the neurotoxin for binding to nerve cells compared to the native neurotoxin. The modified neurotoxin may comprise substitutions and/or deletions and/or insertions and/or additions and/or genetic post-translational modifications of amino acids on the heavy chain of at least one neurotoxin, and preferably a neurotoxin.

Whether or not the pharmaceutical composition comprises only hyaluronic acid or polyvinylpyrrolidone or a polyethylene glycol stabilizer in addition to the neurotoxin active ingredient, the composition retains its potency substantially unchanged during 6 months, 1 year, 2 years, 3 years and/or 4 years of storage at a temperature of about +8 ℃ to about-20 ℃. In addition, the composition may have a potency or percent recovery of about 20% to about 100% after reconstitution.

Pharmaceutical compositions within the scope of the present invention may comprise a neurotoxin and hyaluronic acid. Hyaluronic acid stabilizes the neurotoxin. When reconstituted or injected, the pharmaceutical compositions disclosed herein may have a pH of about 4 to 7.5. The hyaluronic acid in the composition of the invention is preferably mixed with the botulinum toxin according to the invention in an amount of 0.1 to 10mg, in particular 1mg hyaluronic acid per ml of 200U/ml botulinum toxin solution. More preferably, the solution further comprises 1-100mM, especially 10mM, sodium acetate buffer.

In another preferred embodiment, the composition may comprise a polyol as an antifreeze agent. Examples of polyols that can be used include, for example, inositol, mannitol, and other non-reducing alcohols.

It is to be understood that the compositions or formulations of the present invention do not contain trehalose or maltotriose or related sugars or polyols which are often used as cryoprotectants.

The polyvinylpyrrolidone in the pharmaceutical composition of the present invention is preferably mixed with the botulinum toxin of the present invention in an amount of 10 to 500mg, especially 100mg polyvinylpyrrolidone per ml of 200U/ml botulinum toxin solution. More preferably, the solution further comprises 1-100mM, especially 10mM, sodium acetate buffer.

The polyethylene glycol in the pharmaceutical composition of the present invention is preferably mixed with the botulinum toxin of the present invention in an amount of 10 to 500mg, especially 100mg polyethylene glycol per ml of 200U/ml botulinum toxin solution. More preferably, the solution further comprises 1-100mM, especially 10mM, sodium acetate buffer.

Thus, the present invention encompasses the formulation of botulinum toxin into a pharmaceutical composition comprising a hyaluronic acid stabilizer or a polyvinylpyrrolidone stabilizer or a polyethylene glycol stabilizer. In addition, the pharmaceutical composition may comprise a sodium acetate buffer system and/or an alcoholic cryoprotectant. The following examples are provided for illustration only and are not intended to be limiting.

The formulations or pharmaceutical compositions of the present invention are useful for treating disorders for which botulinum neurotoxin therapy or treatment is indicated. On the one hand it can be used for the treatment of cosmetic disorders such as wrinkles and pronounced wrinkles. In another aspect it may be used for the treatment of a condition selected from blepharospasm, hemifacial spasm, spasmodic torticollis, spasticity, migraine, low back pain, cervical spinous process disorders, strabismus, hyperhidrosis, hypersalivation and dystonia. Furthermore, the formulation or composition of the invention is also useful for the preparation of a medicament for the treatment of a condition for which botulinum neurotoxin therapy is indicated, preferably selected from the group consisting of cosmetic conditions, blepharospasm, hemifacial spasms, spasmodic torticollis, spasticity, migraine, low back pain, cervical spine disorders, strabismus, hyperhidrosis, hypersalivation and dystonia. In addition, other medical indications which may be treated by the formulations or compositions of the invention are benign angina (benigncraping), essential tremor, mykomia, neurogenic muscular hypertrophy, palatal myoclonus, spinal myoclonus, synkinetic/Vll cranial nerve disorder, Duane's retrobulbar syndrome, nystagmus, therapeutic ptosis for corneal protection, vibratory hallucinations, spasmodic dysphonia, granuloma, pubenophora, posterior glottic stenosis, reba1ancing, stuttering, TEP failure, primary vocal tremor, vocal tic, circumpharyngeal muscle, nocturnal bruxism, masticatory hypertrophy, morbid obesity, achalasia, anal (glottic) fissure, anal spasm, intractable hiccup, severe constipation, anorectal pain, gastroparesis, benign anal disorders, diverticulosis, Oddi's disease, sphincter, perianal sphincter, lacrimation, salivary fistula, salivary gland dribbling, salivary fistula, salivary gland loss, and salivary gland loss Fletch's syndrome, salivation, detritus-spinotor dysnergia, overactive bladder, vaginismus, urinary retention, hyperproliferation, benign hyperplasia, tension headaches, cervicogenic headaches, myofascial pain (cervicogenic pain), eyelid patency, synkinesis secondary to facial paralysis, glottic stuttering, body odor, intrinsic rhinitis.

Examples

The botulinum toxin formulations of the present invention and pharmaceutical compositions thereof and methods of treatment therewith "the subject matter as a whole" have proven to have unique and advantageous properties, as claimed herein, which are not obvious. Botulinum toxin formulations and pharmaceutical compositions thereof have shown the following valuable properties and characteristics in standard, well-established and reliable experimental procedures:

example 1: botulinum toxin preparations

By applying DasGupta&The method based on the Sathyamoorthy procedure yields pure neurotoxin from Clostridium botulinum type A. 2 percent of the total weight in a 201 fermentation tankCulturing Clostridium botulinum type A in a culture medium consisting of peptone, 1% yeast extract, 1% glucose and 0.05% sodium thioglycolate. After 72 hours of growth, the toxin was precipitated by addition of 3N sulfuric acid (final pH 3.5). The precipitated and centrifuged biomass was extracted with 0.2M sodium phosphate buffer at pH 6.0.

After removing nucleic acids by protamine sulfate precipitation, ammonium sulfate was added to precipitate toxins. The precipitate which had been dissolved and dialyzed at pH 6.0 by 50mM sodium phosphate was bound to DEAE-Sephadex at the same pHOn the column and eluted with 150mM NaCl. Followed by QAE-SephadexChromatography on a column which had been equilibrated with 50mM Tris/HCl buffer, pH 7.9. The toxin was eluted via a NaCl gradient. In the last step, the toxin is SP-Sephadex at pH 7.0And (4) performing chromatography. In this case, bound toxin was eluted from the column with a NaCl gradient (0-300 mM). Purification was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE)The toxin of (1), which shows a purity of 95 +/-5%. Biological activity was determined in the mouse LD50 assay: one LD50 unit corresponds to 4.8pg protein.

Example 2: finished pharmaceutical composition comprising hyaluronic acid

The purified neurotoxin of example 1 was used to prepare a solution comprising 200U botulinum toxin preparation and 1mg hyaluronic acid per ml distilled water. The solution was dispensed into small glass vials.

Example 3: finished pharmaceutical composition comprising hyaluronic acid and sodium acetate buffer

The purified neurotoxin of example 1 was used to prepare a solution containing 200U botulinum toxin preparation and 1mg hyaluronic acid per ml distilled water and adjusted to pH 4.5, 5.0 and 5.5 by the addition of 10mM sodium acetate buffer. The solution was dispensed into small glass vials.

Example 4: stability assay for botulinum toxin preparations

The formulation of example 2 was prepared and compared to botulinum toxin formulated in Human Serum Albumin (HSA). After formulation, both preparations had the same activity. At 24 and 48 hours, the stability of the formulation of example 2 was consistent with that of the HAS formulation, with less than 5% of the initial activity present in both samples being lost.

Example 5: stability assay of botulinum toxin preparations at different pH

The formulation of example 3 was prepared and compared to botulinum toxin formulated in Human Serum Albumin (HSA). After formulation, both the as-formulated (instant preparation) and the HAS preparation at each pH point had the same activity. The preparation at pH 4.5 showed about a 50% loss of activity by day 6. The preparations at pH 5.0 and 5.5 lost all activity by day 6.

Example 6: stabilization of botulinum toxin formulations at different pH and lyophilization conditions Qualitative determination

The formulation of example 3 was prepared and compared to botulinum toxin formulated in Human Serum Albumin (HSA). After formulation, the freshly formulated preparation at pH 4.5 had the same activity as the HAS preparation. More importantly, no loss of activity was detected under the lyophilization conditions.

Example 7: finished pharmaceutical composition comprising polyvinylpyrrolidone

The purified neurotoxin of example 1 was used to prepare a solution comprising 200U botulinum toxin preparation and 100mg polyvinylpyrrolidone per ml of distilled water. The solution was dispensed into small glass vials.

Example 8: finished pharmaceutical composition comprising polyvinylpyrrolidone and sodium acetate buffer Article (A)

The purified neurotoxin of example 1 was used to prepare a solution containing 200U botulinum toxin preparation and 100mg polyvinylpyrrolidone per ml of distilled water and adjusted to pH 4.5, 5.0 and 5.5 by the addition of 10mM sodium acetate buffer. The solution was dispensed into small glass vials.

Example 8A: compositions comprising polyvinylpyrrolidone, mannitol and sodium acetate buffer Pharmaceutical composition

The purified neurotoxin of example 1 was used to prepare a solution comprising 200U botulinum toxin preparation per ml of distilled water, 100mg polyvinylpyrrolidone and 20mg mannitol and adjusted to pH 4.5, 5.0 and 5.5 by the addition of 10mM sodium acetate buffer. The solution was dispensed into small glass vials.

Example 8B: comprising polyvinylpyrrolidone, sorbitol and sodium acetate buffer Finished pharmaceutical composition

The purified neurotoxin of example 1 was used to prepare a solution containing 200U botulinum toxin preparation per ml of distilled water, 100mg polyvinylpyrrolidone and 20mg sorbitol, adjusted to pH 4.5, 5.0 and 5.5 by the addition of 10mM sodium acetate buffer. The solution was dispensed into small glass vials.

Example 9: stability assay for botulinum toxin preparations

The formulation of example 7 was prepared and compared to botulinum toxin formulated in Human Serum Albumin (HSA). After formulation, both preparations had the same activity.

Example 10: stability assay of botulinum toxin preparations at different pH

The formulation of example 8 was prepared and compared to botulinum toxin formulated in Human Serum Albumin (HSA). After formulation, the as-formulated and HAS preparations had the same activity at each pH point. The preparations at pH 4.5 and 5.0 and the HAS preparation showed no loss of activity within 24 hours of formulation. The preparation at pH 5.5 lost 20% activity over the HAS preparation and other preparations within 24 hours.

Example 10A: stability assay of botulinum toxin preparations at different pH

The formulation of example 8A was prepared and compared to botulinum toxin formulated in Human Serum Albumin (HSA). After formulation, the as-formulated and HAS preparations had the same activity at each pH point. The preparations at pH 4.5, 5.0 and 5.5 and the HAS preparation showed no loss of activity within 24 hours of formulation.

Example 10B: stability assay of botulinum toxin preparations at different pH

The formulation of example 8B was prepared and compared to botulinum toxin formulated in Human Serum Albumin (HSA). After formulation, the as-formulated and HAS preparations had the same activity at each pH point. The preparations at pH 4.5, 5.0 and 5.5 and the HAS preparation showed no loss of activity within 24 hours of formulation.

Example 11: stabilization of botulinum toxin preparations at various pH and lyophilization conditions Qualitative determination

The formulation of example 8 was prepared and compared to botulinum toxin formulated in Human Serum Albumin (HSA). After formulation, the as-formulated and HAS preparations had the same activity at each pH point. More importantly, all lyophilized formulations detected less than 10% loss of activity up to 6 months.

Example 11A: botulinum toxin preparations at different pH and freeze-drying conditions Stability determination

The formulation of example 8A was prepared and compared to botulinum toxin formulated in Human Serum Albumin (HSA). After formulation, the as-formulated and HAS preparations had the same activity at each pH point. More importantly, all lyophilized formulations detected less than 10% loss of activity up to 6 months.

Example 11B: botulinum toxin preparations at different pH and freeze-drying conditions Stability determination

The formulation of example 8B was prepared and compared to botulinum toxin formulated in Human Serum Albumin (HSA). After formulation, the as-formulated and HAS preparations had the same activity at each pH point. More importantly, all lyophilized formulations detected less than 10% loss of activity up to 6 months.

Example 12: finished pharmaceutical compositions comprising polyethylene glycol

The purified neurotoxin of example 1 was used to prepare a solution comprising 200U botulinum toxin preparation and 100mg polyethylene glycol per ml of distilled water. The solution was dispensed into small glass vials.

Example 12A: pharmaceutical composition comprising polyethylene glycol and mannitol

The purified neurotoxin of example 1 was used to prepare a solution comprising 200U botulinum toxin preparation per ml of distilled water, 100mg polyethylene glycol and 20mg mannitol. The solution was dispensed into small glass vials.

Example 12B: pharmaceutical composition comprising polyethylene glycol and sorbitol

The purified neurotoxin of example 1 was used to prepare a solution comprising 200U botulinum toxin preparation per ml of distilled water, 100mg polyethylene glycol and 20mg sorbitol. The solution was dispensed into small glass vials.

Example 13: stability assay for botulinum toxin formulations

The formulation of example 12 was prepared and compared to botulinum toxin formulated in Human Serum Albumin (HSA). After formulation, both preparations had the same activity.

Example 14: stability assay for botulinum toxin formulations

The formulations of examples 12A and 12B were prepared and compared to botulinum toxin formulated in Human Serum Albumin (HSA). After formulation, both the freshly formulated and the HAS preparations had the same activity. Both the as-formulated preparation as well as the HAS preparation showed less than 20% loss of activity within 24 hours of formulation.

Example 15: application of botulinum toxin pharmaceutical composition

A 50 year old female sought treatment for blepharospasm. Between about 10U and about 20U of the botulinum toxin preparation of example 3 comprising hyaluronic acid is administered to the patient by intramuscular injection. Within 1-7 days, the symptoms of blepharospasm are reduced, and the reduction in symptoms persists for at least about 2 months to about 6 months.

Example 16: application of botulinum toxin pharmaceutical composition

A 50 year old female sought treatment for blepharospasm. Between about 10U and about 20U of the botulinum toxin preparation of example 8 comprising polyvinylpyrrolidone is administered to the patient by intramuscular injection. Within 1-7 days, the symptoms of blepharospasm are reduced, and the reduction in symptoms persists for at least about 2 months to about 6 months.

Example 17: application of botulinum toxin pharmaceutical composition

A 50 year old female sought treatment for blepharospasm. Between about 10U and about 20U of the botulinum toxin preparation of example 12 comprising polyethylene glycol is administered to the patient by intramuscular injection. Within 1-7 days, the symptoms of blepharospasm are reduced, and the reduction in symptoms persists for at least about 2 months to about 6 months.

Example 18: application of botulinum toxin pharmaceutical composition

A 50 year old female sought treatment for blepharospasm. Between about 10U and about 20U of the botulinum toxin preparation of example 12A comprising polyethylene glycol is administered to the patient by intramuscular injection. Within 1-7 days, the symptoms of blepharospasm are reduced, and the reduction in symptoms persists for at least about 2 months to about 6 months.

Example 19: application of botulinum toxin pharmaceutical composition

A 50 year old female sought treatment for blepharospasm. Between about 10U and about 20U of the botulinum toxin preparation of example 12B comprising polyethylene glycol is administered to the patient by intramuscular injection. Within 1-7 days, the symptoms of blepharospasm are reduced, and the reduction in symptoms persists for at least about 2 months to about 6 months.

Claims (36)

1. A pharmaceutical composition comprising a botulinum neurotoxin from clostridium botulinum type a and a non-proteinaceous stabilizer that retains the biological activity of the botulinum neurotoxin in an aqueous solution, wherein the neurotoxin is free of a complexing protein that naturally forms a complex with the botulinum neurotoxin, the stabilizer is hyaluronic acid, and wherein the composition is free of a mammalian-derived proteinaceous stabilizer.

2. The pharmaceutical composition of claim 1, comprising a pH buffering agent.

3. The pharmaceutical composition of claim 2, wherein the pH buffering agent is sodium acetate.

4. The pharmaceutical composition of claim 1, comprising a cryoprotectant.

5. The pharmaceutical composition of claim 4, wherein the cryoprotectant is a polyol.

6. The pharmaceutical composition of claim 5, wherein the polyol is selected from one or more of inositol, mannitol, and sorbitol.

7. The pharmaceutical composition of claim 4 which is lyophilized.

8. The pharmaceutical composition of claim 1, wherein the non-proteinaceous agent is hyaluronic acid and wherein the composition is lyophilized.

9. The pharmaceutical composition of claim 3, which comprises hyaluronic acid and wherein the composition is lyophilized.

10. The pharmaceutical composition of claim 1, administered to an animal, including a human, in an amount effective to treat a condition for which botulinum neurotoxin therapy or treatment is indicated.

11. The pharmaceutical composition of claim 10, wherein the disorder for which botulinum neurotoxin therapy or treatment is indicated is selected from the group consisting of cosmetic disorders, blepharospasm, hemifacial spasms, spasmodic torticollis, spasticity, dystonia, migraine, low back pain, cervical spinous process disorders, strabismus, hyperhidrosis, and hypersalivation.

12. The pharmaceutical composition of claim 11, wherein the cosmetic condition is significant wrinkles.

13. A botulinum neurotoxin formulation comprising a botulinum neurotoxin from clostridium botulinum type a and a non-proteinaceous stabilizer which retains the biological activity of the botulinum neurotoxin in an aqueous solution, wherein the neurotoxin is free of complexing proteins which naturally form complexes with the botulinum neurotoxin, the stabilizer is hyaluronic acid, and wherein the formulation is free of mammalian derived proteinaceous stabilizers.

14. The botulinum neurotoxin formulation of claim 13, wherein the aqueous solution comprises a pH buffer.

15. The botulinum neurotoxin formulation of claim 14, wherein said pH buffering agent is sodium acetate.

16. The botulinum neurotoxin formulation of claim 14, wherein the formulation is free of stabilizers of a mammalian derived proteinaceous nature, albumin and gelatin.

17. The botulinum neurotoxin formulation of claim 13, wherein said non-protein based stabilizer is hyaluronic acid and wherein the composition is freeze dried.

18. The botulinum neurotoxin formulation of claim 15, comprising hyaluronic acid and wherein the composition is freeze dried.

19. A method of stabilizing a botulinum neurotoxin from Clostridium botulinum type A, wherein the neurotoxin is free of complexing proteins which naturally form complexes with the botulinum neurotoxin, comprising mixing the neurotoxin with a biologically active effective amount of a non-proteinaceous stabilizer which retains the neurotoxin in aqueous solution, said stabilizer being hyaluronic acid.

20. The method of claim 19, wherein the aqueous solution comprises a pH buffer.

21. The method of claim 20, wherein the pH buffering agent is sodium acetate.

22. The method of claim 19, wherein the aqueous solution comprises an antifreeze agent.

23. The method of claim 22, wherein the aqueous solution is freeze-dried.

24. The method of claim 19, wherein the non-proteinaceous stabilizing agent is hyaluronic acid and wherein the composition is lyophilized.

25. The method of claim 21, comprising hyaluronic acid and wherein the composition is lyophilized.

26. Use of a botulinum neurotoxin from Clostridium botulinum type A in the manufacture of a medicament for the treatment of a botulinum neurotoxin therapy or a condition adapted for treatment, wherein the neurotoxin is free of complexing proteins which naturally form complexes with the botulinum neurotoxin and is mixed with a non-proteinaceous stabilizer which retains the biological activity of the botulinum neurotoxin in an aqueous solution, the stabilizer being hyaluronic acid, and wherein the medicament is free of a stabilizer of a mammalian derived proteinaceous nature.

27. The use of claim 26, wherein the aqueous solution comprises a pH buffer.

28. The use of claim 27, wherein the pH buffering agent is sodium acetate.

29. The use of claim 26, wherein the aqueous solution comprises a cryoprotectant.

30. The use of claim 29, wherein the antifreeze agent is a polyol.

31. The use of claim 30, wherein the polyol is selected from one or more of inositol, mannitol, and sorbitol.

32. The use of claim 26, wherein the non-proteinaceous stabilizing agent is hyaluronic acid, and wherein the composition is lyophilized.

33. The use of claim 28, wherein the aqueous solution comprises hyaluronic acid and wherein the composition is lyophilized.

34. The use of claim 26, wherein the disorder for which botulinum neurotoxin therapy or treatment is indicated is selected from the group consisting of cosmetic disorders, blepharospasm, hemifacial spasms, spasmodic torticollis, spasticity, migraine, low back pain, spinous process disorders of the cervical spine, strabismus, hyperhidrosis, hypersalivation and dystonia.

35. The use of claim 34, wherein the cosmetic condition is significant wrinkles.

36. Use of a botulinum neurotoxin formulation according to at least one of claims 13 to 18 for the preparation of a medicament for a condition for which a botulinum neurotoxin therapy is indicated.