HK1144691A - Process for providing a temperature - stable muscle relaxant on the basis of the neurotoxic component of botulinum toxin - Google Patents

Description

Method for supplying temperature-stable muscle relaxants based on the neurotoxic component of botulinum toxin

Technical Field

The present invention provides a method for providing a muscle relaxant, wherein the muscle relaxant is a reconstituted solution comprising the neurotoxic component of botulinum toxin free of complexing proteins, which exhibits at least one of the following properties, preferably all of the properties a) to d):

a) stable at storage temperatures above +20 ℃;

b) (ii) stable in the presence of preservatives and/or analgesics;

c) tolerating "freeze and thaw" -cycles;

d) can be stored stably in containers made of different materials.

Background

The botulinum toxin is produced by the bacterium clostridium. There are seven antigenically distinct serotypes of botulinum toxin, namely, botulinum toxin type A, B, C, D, E, F and botulinum toxin type G. Botulinum toxin is typically released from lysed clostridium cultures in the form of a complex, a toxin complex formed by complexing the subunit responsible for the toxicity of botulinum toxin (the so-called "neurotoxic component") with other bacterial proteins. The molecular weight of the complex varies from about 300,000Da to about 900,000 Da. The complex proteins are, for example, various hemagglutinins. The proteins of this toxin complex are not toxic per se, but are believed to provide stability to the neurotoxic component and can lead to oral toxicity in botulinum. Unlike the toxin complex, the neurotoxic component is present in its isolated and purified form, i.e. free of any complex clostridial proteins, which are labile acids and are not resistant to the aggressive environment in the gastrointestinal tract.

The neurotoxic component of the botulinum toxin complex is initially formed as a single chain polypeptide, with a molecular weight of approximately 150kDa in the case of serotype a. In another serotype, the molecular weight of the neurotoxic component was observed to vary between about 145kDa and 170kDa depending on the bacterial origin. In the case of serotype A, for example, enzymatic processing of the polypeptide produces an active polypeptide in the form of a two-chain polypeptide consisting of a heavy chain and a light chain linked by a disulfide bond. In humans, the heavy chain mediates the binding of the endotoxin to the presynaptic cholinergic nerve terminal and translocates the toxin intracellularly. Light chains are thought to have toxic effects, act as zinc-endopeptidase and cleave specific proteins responsible for membrane fusion (SNARE complexes) (see, e.g., Montecoco C1 Shiavo G., Rosetto O: The mechanism of action on cellulose and Botulinum neurotoxins. Arch toxin.1996; 18 (Suppl.): 342. 354)).

By interfering with the intracellular membrane fusion process, botulinum toxin prevents the release of acetylcholine into the synaptic cleft. The overall effect of botulinum toxin at the nerve-muscle junction is to block nerve-muscle conduction and, in effect, denervate the muscle. Botulinum toxin is also active on other peripheral cholinergic synapses, causing a reduction in salivation or sweating.

The term "botulinum toxin" or "botulinum toxins" as used throughout the present invention refers not only to the neurotoxic component free of any other clostridial proteins, but also to the "botulinum toxin complex": the term "botulinum toxin" is used herein when no distinction between toxin complexes and neurotoxic components is necessary or required. The complex usually contains other, so-called "non-toxic proteins", which we will call "complex proteins" or "bacterial proteins".

Despite its toxic effects, botulinum toxin complexes have been used as therapeutic agents for many diseases. Botulinum toxin type A was approved for human use in the United states in 1989 for the treatment of strabismus, blepharospasm and other disorders. Botulinum toxin type A protein complexes are commercially available, for example under the trade name BOTOX (Allergan Inc.) or DYSPORT (Ipsen Ltd.). The compound for therapeutic use may be injected directly into the muscle to be treated. At physiological pH, neurotoxic components are released from the protein complex and produce the desired pharmaceutical effect.

Pharmaceutical compositions comprising the neurotoxic component of botulinum toxin type A are available from Merzpharmaceuticals GmbH, Germany under the trademark "Merzpharmaceuticals GmbHAre commercially available. The preparation of the neurotoxic component of botulinum toxin types A and B is described, for example, in International patent applications WO 00/74703 and WO 2006/133818.

For compositions and pharmaceutical dosages based on botulinum toxin, and pharmaceutical compositions, dosages and frequency of administration based on the neurotoxic component of botulinum toxin, see PCT/EP 2007/005754.

In addition to the above functions, it is speculated that the complex protein also protects the neurotoxic component of the botulinum toxin from harsh environmental conditions, since the neurotoxic component is highly susceptible to degradation and/or inactivation, particularly when subjected to short-term temperature stresses, such as storage and/or transport in warm to hot climates or generally during the summer season, i.e. at temperatures above 20 ℃.

For this reason, in the past, extreme care has generally been taken to prevent the temperature of drugs based on botulinum toxin, and in particular on the neurotoxic component of botulinum toxin, from reaching above +4 ℃, for example close to 20 ℃. In most cases, vials containing solid lyophilizates or reconstituted solutions thereof containing botulinum toxin are stored frozen (lyophilized) on ice or at least in a refrigerator (about +4 ℃) at about-20 ℃. This necessary cooling can result in additional costs in supplying the medicament.

Furthermore, it was believed prior to the present invention that the reconstituted solution comprising the neurotoxic component of botulinum toxin was even more unstable to different storage or transport conditions. It is also believed that freezing and thawing of the reconstituted solution will result in rapid degradation and inactivation of the protein. Thus, physicians recommend reconstituting protein-lyophilisates just prior to administration and/or storing them rigorously at the low temperatures outlined above.

In view of the above, the present inventors have studied the stability of botulinum toxin based muscle relaxants in the form of reconstituted solutions under different environmental conditions:

a) storing at above +20 deg.C;

b) adding a preservative and/or a sedative;

c) freeze and thaw-cycle;

d) storing in containers of different materials.

Surprisingly, it was found that a recombinant solution comprising the neurotoxic component of botulinum toxin free of complexing proteins is much more stable under these conditions than was envisioned in the prior art. The invention described below is based on this finding.

Disclosure of Invention

The present invention provides a method for providing a muscle relaxant, wherein the muscle relaxant is a reconstituted solution comprising the neurotoxic component of botulinum toxin free of complexing proteins, which exhibits at least one of the following properties, more preferably all of the properties a) to d):

a) stable at storage temperatures above +20 ℃;

b) (ii) stable in the presence of preservatives and/or analgesics;

c) tolerating "freeze and thaw" -cycles;

d) can be stored stably in containers made of different materials.

In one embodiment, the present invention provides a muscle relaxant with a supply temperature greater than 30 ℃, wherein the muscle relaxant is a reconstituted solution comprising a botulinum toxin neurotoxic component free of complexing proteins.

In another embodiment, said supply involves storage and/or transport or is a step in a process for preparing said muscle relaxant. In yet another embodiment, the muscle relaxant is stored and/or transported at ambient temperatures above 30 ℃ up to 70 ℃ without any cooling means.

In another embodiment, the muscle relaxant undergoes a "freeze and thaw" -cycle. In yet another embodiment, the "freeze and thaw" -cycles number is from 1 to 20.

In another embodiment, the muscle relaxant remains stable in the presence of a preservative and/or analgesic.

In another embodiment, the reconstituted solution is stored in a container made of plastic, glass or metal or any combination thereof.

In another embodiment, the solution further comprises sucrose or human serum albumin or both.

In another embodiment, the solution further comprises at least one component selected from the group consisting of cryoprotectants other than sucrose and human serum albumin, stabilizers, pH buffers, excipients, and mixtures thereof. In another embodiment, the neurotoxic component is the neurotoxic component of botulinum toxin type A.

Drawings

FIG. 1: storage at +4 ℃ in the Presence of non-preserved saline for reconstitutionAndthe effect of activity. Stored in polyethylene containers.

FIG. 2: storage at +4 ℃ in the Presence of non-preserved saline for reconstitutionAndthe effect of activity. Stored in a polyethylene syringe with a rubber stopper.

FIG. 3: repeated freezing and thawing pair recombinationThe effect of activity.

Detailed Description

The present invention relates to a process for supplying a muscle relaxant at a temperature above +4 ℃, preferably above +6 ℃, more preferably above +20 ℃, wherein the muscle relaxant is a reconstituted solution comprising a botulinum toxin neurotoxic component free of codrugs. In the present invention, the term "supply" includes any supply of the muscle relaxant as defined in the present invention, in particular storage, transport and/or as a step in the preparation of said muscle relaxant. The term "supplying" also includes a step wherein the muscle relaxant undergoes a temperature ramp from a frozen state (e.g. -20 ℃) to +4 ℃ or more, preferably +6 ℃ or more, more preferably +20 ℃ or more.

In the present invention, any form of the neurotoxic component of botulinum toxin can be used, particularly the various serotypes, including serotypes A, B, C, D, E, F and G. In a preferred embodiment of the present invention, the neurotoxic component of the botulinum toxin type B is provided at a temperature greater than 8 ℃. In another embodiment the neurotoxic component of botulinum toxin type B is provided at a temperature greater than 30 ℃. In addition, modifications including mutations, deletions, and the like, as well as the neurotoxic component of a recombinantly produced botulinum toxin, are also within the scope of the invention. For suitable mutations, reference may be made to WO 2006/027207a1 and WO2006/114308a1, and EP 07014785.5, which are incorporated herein by reference in their entirety. Furthermore, in the present invention, mixtures of the various serotypes (either as the neurotoxic component or recombinant form thereof or both forms, e.g., a mixture of botulinum neurotoxins of types A and B) may be employed. However, the invention also relates to chemically modified neurotoxins, such as pegylated, glycosylated, sulfated, phosphated or any other modification, in particular one or more surface or solvent exposed amino acids.

In one embodiment, the chemodenervating agent is a clostridial neurotoxin. In another embodiment, the clostridial neurotoxin is a botulinum toxin. In yet another embodiment, the botulinum toxin is a botulinum toxin of serotypes A, B, C, D, E, F and G that are antigenically distinct. In all references to botulinum toxin types A, B, C, D, E, F and G, known variants of serotypes are also included, such as serotypes A1, A2, A3, B1, B2, B3, C1, C2, C3, D1, D2, D3, E1, E2, E3, F1, F2, F3, or G1, G2, G3. In one embodiment, the botulinum toxin is botulinum toxin type A.

In another embodiment, isoforms, homologs, orthologs, and paralogs of botulinum toxin are also included that exhibit at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% sequence identity. Sequence identity can be calculated by any algorithm suitable for obtaining reliable results, for example, using the FASTA algorithm (W.R.Pearson & D.J.Lipman PNAS (1988) 85: 2444-.

Botulinum toxin, when released from lysed clostridial media, is often accompanied by other bacterial proteins, which together form a toxin complex. In another embodiment, the botulinum toxin does not contain any complexing proteins, e.g., is a botulinum toxin type A pure product. In addition, modifications including mutations, deletions, and the like, as well as the neurotoxic component of a recombinantly produced botulinum toxin, are also within the scope of the invention. For suitable mutations, see WO 2006/027207a1 and WO2006/114308a1, and EP 07014785.5 (patent application filed by Merz on 10/27, 2007), which are incorporated herein in their entirety by reference. Furthermore, in the present invention, mixtures of the various serotypes (either as the neurotoxic component or recombinant form thereof or both forms, e.g., a mixture of botulinum neurotoxins of types A and B) may be employed. However, the invention also relates to chemically modified neurotoxins, such as pegylated, glycosylated, sulfated, phosphated or any other modification, in particular one or more surface or solvent exposed amino acids.

The neurotoxin subunit of the botulinum toxin complexes referred to herein are the "neurotoxic component" or "neurotoxic component free of complexing proteins". The term "neurotoxic component" also includes functional homologs found in other serotypes of clostridium botulinum. In one embodiment of the invention, the neurotoxic component is free of any other clostridium botulinum proteins and, in one embodiment, is free of RNA that may potentially be associated with the neurotoxic component. The neurotoxic component can be a single chain precursor protein of about 150kDa or a proteolytically processed neurotoxic component comprising about 50kDa light chain (L) linked by one or more disulfide bonds_c) And a heavy chain (H) of about 100kDa_c) (see, e.g., Simpson LL, Ann Rev Pharmacol Toxicol.2004; 44: 167-93).

In the present invention, any form of botulinum toxin may be used, particularly the various serotypes, the various complexes of the neurotoxic component of botulinum toxin and its complexed chaperone proteins, and the neurotoxic component of such botulinum toxin. In addition, modifications including mutations, deletions, and the like, as well as the neurotoxic component of a recombinantly produced botulinum toxin, are also within the scope of the invention. For suitable mutations, reference may be made to WO 2006/027207a1, which is incorporated herein in its entirety by reference. Furthermore, in the present invention, mixtures of the various serotypes (either as the neurotoxic component or recombinant form thereof or both forms, e.g., a mixture of botulinum neurotoxins of types A and B) may be employed.

In accordance with the teachings of the present invention, the medicament may be free of proteins found in botulinum toxin complexes other than the neurotoxic component. Precursors of the neurotoxic component may or may not be cleaved, however in one embodiment the precursors are cleaved into the heavy and light chains. As noted elsewhere herein, the polypeptide may be a wild-type sequence or may be modified at one or more residues. Modifications include chemical modifications, for example by glycosylation, acetylation, acylation or amidation, etc., which may be beneficial, for example, for uptake or stability of the polypeptide. However, the polypeptide chain of the neurotoxic component can be selectively modified or otherwise modified by the addition, substitution or deletion of one or more amino acid residues.

The neurotoxic component referred to above in the present invention may be part of a composition or pharmaceutical composition. The pharmaceutical compositions used in the present invention may comprise a botulinum toxin, e.g. with the neurotoxic component as the sole active ingredient or may contain other pharmaceutically active ingredients, e.g. hyaluronic acid and/or polyvinylpyrrolidone and/or polyethylene glycol, which may be stabilized at an optional pH by a suitable pH buffer, especially a sodium acetate buffer, and/or a cryoprotectant polyol.

A "pharmaceutical composition" is a preparation containing or comprising an active ingredient for use as a medicament or diagnostic agent. Such pharmaceutical compositions may be suitable for diagnostic or therapeutic administration (i.e., by intramuscular or subcutaneous injection) to a human patient.

In one embodiment of the invention, the composition may comprise a neurotoxic component and hyaluronic acid or polyvinylpyrrolidone or polyethylene glycol, which may be stabilized at an optional pH by a suitable pH buffer, especially sodium acetate buffer and/or cryoprotectant polyol. Preferably, the composition comprises the neurotoxic component of botulinum toxin type a. The composition is a reconstituted solution of the neurotoxic component of a botulinum toxin. Preferably, the composition further comprises sucrose or human serum albumin or both, more preferably the ratio of human serum albumin to sucrose is about 1: 5. In one embodiment, the composition isMore preferably, the human serum albumin is recombinant human serum albumin. Optionally, the composition is free of mammalian originAnimal proteins, such as human serum albumin. Any of these protocols can provide sufficient neurotoxin stability (nfra) by replacing serum albumin with other non-protein stabilizers.

The composition may include other ingredients, such as pH buffers, excipients, cryoprotectants, preservatives, analgesics, stabilizers, or any combination thereof.

Thus, in a preferred embodiment, the neurotoxic component is formulated with a hyaluronic acid stabilizer or a polyvinylpyrrolidone stabilizer or a polyethylene glycol stabilizer or any combination thereof. In addition, the composition may contain sodium acetate buffer systems or alcohol cryoprotectants or both. In another preferred embodiment, the formulation is albumin free and contains the stabilizing agents hyaluronic acid, polyvinylpyrrolidoneAnd/or hydroxyethyl starch and/or alginate and/or mixtures of two and/or more thereof. In addition to the above stabilizers, the preferred compositions also comprise water and at least one polyol, preferably mannitol or sorbitol or mixtures thereof.

In one embodiment, the mouse LD₅₀The result of test shows that the biological activity of the neurotoxic component is 50 to 250 LD per ng neurotoxic component₅₀Units. In another embodiment, the neurotoxic component has a biological activity of about 150 LD₅₀Units. Where units as described herein refer to units per nanogram. Typically, the pharmaceutical compositions of the present invention comprise a neurotoxic component in an amount of about 6pg to about 30ng by weight. Pharmaceutical compositions comprising a neurotoxic component in isolated form of botulinum toxin type A are commercially available from Merz Pharmaceuticals GmbH, Germany under the trade nameThe preparation of the neurotoxic component of botulinum toxin types A and B is described, for example, in International applications WO 00/74703 and WO 2006/133818.

In one embodiment, the composition includes a neurotoxic component of botulinum toxin type A. The composition is a reconstituted solution of the neurotoxic component of a botulinum toxin. In another embodiment, the composition further comprises sucrose or human serum albumin or both, and in another embodiment, the ratio of human serum albumin to sucrose is 1: 5. In one embodiment, the composition isIn another embodiment, the human serum albumin is recombinant human serum albumin. Optionally, the composition is free of proteins derived from mammals, such as human serum albumin. Any of these protocols can provide sufficient neurotoxin stability (nfra) by replacing serum albumin with other non-protein stabilizers.

For compositions and pharmaceutical dosages based on botulinum toxin, and pharmaceutical compositions, dosages and frequency of administration based on the neurotoxic component of botulinum toxin, see PCT/EP 2007/005754.

The pharmaceutical composition may be lyophilized or vacuum dried, reconstituted, or be the main ingredient in solution. In one embodiment, when reconstituted, the reconstituted solution is prepared by addition of sterile physiological saline (0.9% NaCl).

Such combinations may include other excipients. The term "excipient" refers to substances present in the composition other than the active ingredient. Excipients may be buffers, carriers, antiadherents, binders, disintegrants, fillers, diluents, preservatives, adjuvants, cyclodextrins and/or fillers, for example albumin, gelatin, collagen, sodium chloride. In another embodiment, the excipient may also be an analgesic, a cryoprotectant, and/or a stabilizer.

The term "pH buffer" 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. In one embodiment, the pH may range from 5 to 8, for example from 7 to 8, or from 7.2 to 7.6, or 7.4. The pH ranges given above are only typical examples and the actual values may encompass any interval between the above values. Suitable buffers according to the teachings of the present invention are, for example, sodium phosphate buffer, sodium acetate buffer, TRIS buffer or any buffer suitable for buffering to the above mentioned pH range.

In one embodiment, the composition further comprises 1-100mM sodium acetate buffer, and in another embodiment, 10mM sodium acetate buffer.

The pH ranges given above are only typical examples. Actual values may include any interval between the above values. Suitable buffers according to the teachings of the present invention are, for example, sodium phosphate buffer, sodium acetate buffer, TRIS buffer or any buffer suitable for buffering to the above mentioned pH range.

By "stable", "stabilizing" is meant that the toxicity of the neurotoxic component in the reconstituted solution or aqueous solution of the pharmaceutical composition is greater than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% up to about 100% of the toxicity of the biologically active neurotoxic component prior to addition to the pharmaceutical composition. In one embodiment, the neurotoxic component is referred to as the active ingredient.

Examples of stabilizers are gelatin or albumin, in one embodiment, albumin of human or recombinant origin. Proteins of non-human or non-animal origin are also included. The stabilizer may be modified by chemical means or by genetic recombination. In one embodiment of the present invention, it is envisaged to stabilize proteins during lyophilization with an alcohol, e.g. inositol, mannitol, as a cryoprotective excipient.

In another embodiment of the present invention, the stabilizing agent may be a non-protein stabilizing agent comprising hyaluronic acid or polyvinylpyrrolidone or polyethylene glycol or any combination thereof. In another embodiment, the stabilizer isHydroxyethyl starch and/or alginate. The composition may be stabilized at an optional pH by a suitable pH buffer, especially a sodium acetate buffer or a cryoprotectant or a combination of both. In addition to the above mentioned stabilizers, the composition may comprise water and at least one polyol, such as mannitol or sorbitol or mixtures thereof. It may also include monosaccharides, disaccharides or higher polysaccharides such as glucose, sucrose or fructose. Such compositions are considered to be safer compositions with high stability.

In one embodiment, the hyaluronic acid in the pharmaceutical composition of the invention is used in combination with the neurotoxic component in an amount of 0.1 to 10mg, preferably 1mg, hyaluronic acid per ml in a 200U/ml solution of botulinum toxin.

When polyvinylpyrrolidone is present in the composition of the present invention, it is used in combination with the neurotoxic component in an amount that enables the preparation of a reconstituted solution, for example in an amount of 10 to 500mg, preferably 100mg, of polyvinylpyrrolidone per ml of botulinum toxin solution containing 200U/ml of neurotoxic component. In another embodiment, the reconstitution is performed in up to 8ml of solution. This resulted in a concentration of 12.5mg polyvinylpyrrolidone per ml in a 25U/ml solution of the neurotoxic component. In another embodiment, the resulting solution further contains 1-100mM, preferably 10mM, sodium acetate buffer. This ratio of components applies equally if the concentration of the solution of neurotoxic component is reduced from 25U/ml to lower.

In one embodiment, the polyethylene glycol in the pharmaceutical composition of the present invention is combined with the neurotoxic component in an amount of 10 to 500mg, preferably 100mg, of polyethylene glycol per ml in a 200U/ml solution of botulinum toxin. In another embodiment, the resulting solution further comprises 1-100mM sodium acetate buffer, and in yet another embodiment, 10mM sodium acetate buffer.

In one embodiment, the pharmaceutical composition of the present invention has a potency that remains substantially unchanged for six months, one year, two years, three years, and/or four years at a storage temperature of about +30 ℃ to-20 ℃. In addition, the pharmaceutical composition may have a pharmaceutical effect after reconstitution, or may be restored to 20% to 100%.

"cryoprotectant" refers to an excipient that results in a neurotoxic component of a reconstituted solution or aqueous solution of the composition having a toxicity that is 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% greater than the toxicity of the neurotoxic component of the pharmaceutical composition prior to lyophilization, up to about 100% toxicity.

In another embodiment, the composition may comprise a polyol, for example a polyol as a cryoprotectant. Examples of useful polyols include, for example, inositol, mannitol, and other non-reducing alcohols. Some embodiments of the compositions do not contain protein stabilizers or trehalose, maltose, lactose, sucrose or related sugars that are sometimes used as cryoprotectants.

The terms "preservative" and "preservatives" refer to a substance or class, respectively, that prevents the growth or survival of microorganisms, insects, bacteria, or other contaminating microorganisms in the composition. Preservatives also prevent undesirable chemical changes in the composition. Preservatives which can be used within the scope of this patent are all known to those skilled in the art. Examples of useful preservatives include, inter alia, benzyl alcohol, benzoic acid, benzalkonium chloride, calcium propionate, sodium nitrate, sodium nitrite, sulfites (sulfur dioxide, sodium bisulfite, potassium bisulfite, etc.), disodium ethylenediaminetetraacetic acid (EDTA), formaldehyde, glutaraldehyde, diatomaceous earth, ethanol, methylchloroisothiazolinone, butylhydroxyanisole, and/or butylhydroxytoluene.

The term "analgesic" relates to analgesics which act in a number of ways on the peripheral and central nervous system, including in particular paracetamol (acetaminophen), non-steroidal anti-inflammatory drugs (NSAIDs), such as salicylates, narcotics such as morphine, synthetic drugs with narcotic properties such as tramadol, and other drugs. Also included are any compounds with local analgesic effects, such as lidocaine, benzyl alcohol, benzoic acid and other compounds.

In one embodiment, the analgesic agent is part of a composition, and in another embodiment, the analgesic agent is administered before, during, or after the chemodenervating agent treatment.

The term "freeze-drying" as used herein is used to treat a solution containing the neurotoxic component of a botulinum toxin, wherein the solution is frozen and dried until only the solid components of the composition remain. The lyophilisate obtained in this process is therefore defined as "lyophilisate" in the present application.

In the present application, the term "recombinant" is defined as a method of dissolving the lyophilized composition of the neurotoxic component. This can be done by adding an appropriate amount of sterile water, for example, if all necessary components are already contained in the lyophilizate. Alternatively, if not, sterile saline may be added, for example, alone or, if applicable, including, for example, pH buffers, excipients, cryoprotectants, preservatives, analgesic stabilizers, or any combination thereof. The aforementioned "salt solution" is a salt-solution, more preferably a sodium chloride (NaCl) solution, yet more preferably an isotonic sodium chloride solution (i.e. a sodium chloride concentration of 0.9%). Dissolution in this manner allows the final "reconstituted" solution to be administered to the patient either directly or indirectly, i.e., after dilution, for example. Recombinant neurotoxins in isotonic media are preferred. More preferably in isotonic saline. More preferably, the saline is sterile saline.

The term "freezing and thawing cycle" as used herein refers to the process of freezing and thawing the reconstituted solution. Here, the "freezing" process is defined as the storage of the reconstituted solution at temperatures below 0℃, for example preferably below-20℃ (normal refrigerator temperature), more preferably at or below-80℃ (dry ice temperature). Wherein the "thawing" process is defined as storage at above 0 ℃, preferably above +4 ℃, more preferably above +20 ℃, most preferably above +25 ℃, above +30 ℃ and above +40 ℃ respectively in the temperature range, but not above 50 ℃. Typical and exemplary storage times during and after freezing and thawing can be up to 1 minute, up to 10 minutes, up to 30 minutes, up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to 8 hours, up to 1 day, up to 2 days, up to 3 days, up to 4 days, up to 5 days, up to 6 days, up to 7 days, up to 8 days, up to 9 days, up to 10 days, up to 2 weeks, up to 3 weeks, up to 1 month, up to 2 months, up to 3 months (90 days). The term "freezing and thawing cycle" also includes cycles of cooling and (re) heating that are subsequently performed continuously. The time period mentioned above is only a typical example, and the actual time period may be long or short, including the interval between any of the values mentioned above. A "freeze and thaw cycle" is defined to mean a freezing step and a thawing step performed according to the above conditions. Reference herein to a plurality of "freeze and thaw cycles," i.e., "a plurality of freeze and thaw cycles," means that the reconstituted solution is repeatedly subjected to the "freeze and thaw cycles" at different and the same time intervals and temperatures within the ranges defined above. The above-mentioned repetition is at least 2 times, preferably at least 3 times or at least 4 times, more preferably at least 5 times, at least 6 times, at least 7 times, even more preferably at least 8 times, at least 9 times or at least 10 times, but not more than 20 times.

The term "container" refers to a vessel, such as a vial, syringe, flask, or any other type of container, in which the composition may be stored and/or transported. The container walls are in direct contact with the composition and are constructed of materials suitable for sealing reconstitution solutions, such as various glasses, plastics, metals, ceramics, or any combination thereof, or any material.

As used herein, the term "room temperature" refers to any temperature between +22 ℃ and +25 ℃, preferably any temperature between +20 ℃, +21 ℃, +22 ℃, +23 ℃, +24 ℃ or +25 ℃ and any numerical range thereof.

The term "excipient" in the present application refers to a substance other than the active ingredient present in the composition. The excipient may be a buffer, carrier, antiadherent, binder, disintegrant, filler, diluent, preservative, adjuvant, cyclodextrin and/or filler, such as albumin, gelatin, collagen and/or sodium chloride.

A "cooling device" is defined as any device capable of reducing the temperature of a composition below ambient temperature. Preferably, the "cooling means" is capable of achieving a stable temperature below ambient temperature, typically at or about 6 ℃, in some cases even lower.

Typically, the provision of the muscle relaxant described above comprises storage or transport at elevated temperatures or both, or is a step in the preparation of the muscle relaxant, more preferably a step that is performed after the protein comprising the neurotoxic component of the botulinum toxin has been lyophilized and reconstituted. "high temperature" means +6 ℃ or higher, preferably +20 ℃ or higher, and more preferably +30 ℃ or higher. The term "+ 6 ℃ or higher" means for example +7 ℃, +8 ℃, +9 ℃, +10 ℃, +11 ℃, +12 ℃, +13 ℃, +14 ℃, +15 ℃, +16 ℃, +17 ℃, +18 ℃, +19 ℃ or higher, but not higher than 70 ℃. The term "+ 20 ℃ or higher" means for example +21 ℃, +22 ℃, +23 ℃, +24 ℃, +25 ℃, +26 ℃, +27 ℃, +28 ℃, +29 ℃ or +30 ℃. The term "+ 30 ℃ or higher" means for example +31 ℃, +32 ℃, +33 ℃, +34 ℃, +35 ℃, +36 ℃, +37 ℃, +38 ℃, +39 ℃ or +40 ℃. Preferably the muscle relaxant is not stored above +70 ℃. In some cases, i.e., where the botulinum toxin-based neurotoxic component is stored at temperatures below 0 ℃, the term "high temperature" means a temperature above 0 ℃, preferably above 4 ℃, more preferably above +6 ℃, above +20 ℃, and above +30 ℃ for the above temperature ranges, respectively.

In a preferred embodiment, the muscle relaxant is subjected to a temperature in the range above +6 ℃ up to +40 ℃ for a period of time not exceeding 14 days. As known to those skilled in the art, the time period during which the muscle relaxant is subjected to the respective temperature may be any time interval between a few minutes and 14 days. Generally, the time period should not be less than 10 minutes, taking into account the conditions of the supply of such muscle relaxants, especially at the site of interest for storage and/or transport or both. These short periods are particularly important in certain situations where the muscle relaxant is subjected to direct sunlight after transport in hot climates or after storage, for example at airports or on the street. Thus typical time periods of the invention are up to 10 minutes, up to 30 minutes, up to 1 hour, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to 8 hours, up to 1 day, up to 2 days, up to 3 days, up to 4 days, up to 5 days, up to 6 days, up to 7 days, up to 8 days, up to 9 days, up to 10 days, up to 2 weeks, up to 3 weeks, up to 1 month, up to 2 months, up to 3 months (90 days). Needless to say, the time periods mentioned above are only typical examples, and the actual time period may be longer or shorter and include any interval between the values given above.

For the temperatures to which the muscle relaxant is subjected, a typical lower temperature limit of 20 ℃ or above will occur to those skilled in the art. For the temperatures and temperature ranges specified in the present invention, it will be appreciated by those skilled in the art that the upper temperature limit to which the muscle relaxant/composition is subjected is preferably not higher than 70 ℃. I.e. the temperature to which the muscle relaxant is subjected is preferably in the range of above 20 ℃ to 70 ℃. Thus, in the present invention, the muscle relaxant is subjected to a temperature of 20 ℃ or more, or 25 ℃ or more, or 30 ℃ or more, or 35 ℃ or more, or 40 ℃ or more, or 45 ℃ or more, or 50 ℃ or more, or 55 ℃ or more, or 60 ℃ or more, or 65 ℃ or more, respectively, up to 70 ℃. In addition, any given specific temperature and respective temperature interval between 20 ℃ and above to 70 ℃ may be determined by the environment in which the muscle relaxant is supplied, preferably transported or stored or both, and is within the scope of the present invention.

The following temperatures and time intervals represent preferred embodiments of the present invention. According to a first embodiment, the muscle relaxant is allowed to stand for a period of time but not more than 90 days at a temperature between +30 ℃ and up to +70 ℃, preferably for 10 minutes to 14 days at a temperature between +30 ℃ and up to +70 ℃, more preferably for 10 minutes to 90 days at a temperature between +40 ℃ and up to +60 ℃.

In a preferred embodiment, the temperature is from above 30 ℃ up to 70 ℃, preferably from 40 ℃ to 60 ℃, more preferably from 50 ℃ to 60 ℃ when the time period ranges from 10 minutes to 30 days.

In another preferred embodiment, representing the extreme case, the temperature is in the range of 65 ℃ to 70 ℃ and the muscle relaxant is subjected to said temperature for a period of from 10 minutes to 90 days, preferably from 10 minutes to 3 days.

The experimental results show that the surface contacted by the solution has no influence on the stability of the recombinant neurotoxin of the present invention. Thus, the reconstituted compositions of the invention can be stored in a variety of vessels or containers. The surfaces of these vessels or containers can therefore also be made of various plastics, metals, glass, etc.

In view of this discovery, the present invention is now able to provide the muscle relaxant described above without the use of an artificial cooling device. This finding is particularly important for the transport and/or storage of muscle relaxants. In addition, the present invention is greatly related to an environment of high temperature, possibly accompanied by high humidity.

The invention will now be illustrated in detail by the following non-limiting examples.

Examples

Example is the use of a commercially available productThe method is carried out.Is a lyophilized powder containing botulinum toxin type A (150kDa) as active ingredient. This toxin exists in a gapped (nicked) double-stranded form, i.e., it comprises a heavy chain and a light chain. The toxin was obtained from a Clostridium botulinum culture (ATCC 3205 strain). Has been purified to be free of any complex proteins.Also contains human serum albumin and sucrose.

To evaluateThe biological activity was measured by the mouse diaphragm assay (HDA). In this method, a neuromuscular preparation consisting of the mouse phrenic nerve and the corresponding part of the diaphragm muscle is fixed in a force measuring instrument. The entire phrenic nerve is passed through two electrodes to stimulate the nerve and thus the diaphragm. The composition is immersed in a toxin containing HAD buffer, and the nerve is periodically stimulated with an electrical pulse (frequency 1Hz, stimulation time 0.1ms, stimulation current amplitude 5-50 mA).

The contractile response of the indirectly stimulated muscle is monitored by means of isometric sensors. The signal was amplified and recorded by a personal computer using commercially available software, vitrodatnn 3.4. When measuring the time course of the muscle contraction response, an exponential decrease in the contractile force in the presence of botulinum toxin was observed. This reduction is characterized by a so-called paralysis time. Paralysis time is defined as the time period between the two time points "toxin sample increase" and "half maximal contractile force" and is proportional to the toxin concentration in the organ bath.

The above method is carried out according to the provisions of the European pharmacopoeia for the detection of botulinum toxin activity.

Example 1 "storage at room temperature"

The reconstituted salt solution of (2) was stored in a plastic-syringe at room temperature (+23 ℃) for up to 9 days. The activity of the sample was compared to the activity of the reconstituted control solution just before (no more than 2 hours) the activity assay. No significant decrease in protein activity was detected in the sample and control groups.

EXAMPLE 2 "Plastic Container storage"

The salt solution of recombinant botulinum neurotoxin NT201 was introduced into various plastic containers and applied (see Table 1 below). In addition, recombinant toxins were stored in plastic containers and syringes, respectively, for various periods of up to 14 days before activity was detected. In no case was a significant reduction in protein activity detected.

Table 1:

will contain 100MLD ("intermediate lethal dose" or LD) before use₅₀Unit) the lyophilized powder NT201 was reconstituted in saline solution.

To test the effect of the plastic material on botulinum toxin activity, the recombinant saline solution was introduced into and adapted for use in various syringes using the needles, adapters and stoppers listed above, as well as an organ bath (activity measured by mouse diaphragmatic method in the container) adapted to simulate an intramuscular injection medical procedure using conventional methods.

In addition, recombinant toxins were stored in plastic containers and syringes, respectively, for various periods of up to 14 days before activity was detected.

Experiments were performed in 3 independent series to study the maximum number of different variables and to obtain reliable results. See below for details.

Aliquots of saline were taken according to typical medical practice (series 2 and 3) either directly with a pipette (series 1) or with a 3ml syringe and a 20G needle (BD #300910 and BD # 301300; series 2) or with a 5ml syringe and a 20G needle (B.Braun #4617053V and # 4665503; series 3).

To ensure the accuracy of small sample sizes, saline solution is injected into the centrifuge tube for intermediate storage when the required volume is less than 0.5 ml. In these cases, the pipette is used to transfer the required volume.

Series 1 and 3: 10 vials were carefully inflated with a fine needle and subsequently opened. Saline (1.0ml) was aspirated into the first vial. After the dry pellet (pellet) was fully dissolved by careful mixing, the solution was transferred to a second vial. In this way the liquid is transferred from one vial to the next and up to the last, i.e. 10 th vial. The two toxin solutions were then combined by adding 1.0ml of saline to the first vial and washing the vials one by one according to the previous procedure to collect any residual sample.

These combined solutions (2.0ml) represent approximately 100MLD botulinum neurotoxin cells per 0.2ml of solution. When subsequently treated with syringes, 0.2ml aliquots of the pools were aspirated into microtubes to ensure accurate amounts.

Series 2: exactly 2.5ml of saline was aspirated into a 3ml syringe with a 20G needle (BD #300910 and BD #301300) and injected into five vials containing botulinum toxin at 0.5ml per serving. The vials were carefully mixed until the dry powder mass (pellet) was completely dissolved. The vial was inflated and a fine needle was inserted and the recombinant toxin was drawn into a 10ml syringe with a 21G needle (BD #300912 and BD # 301155). Air was then drawn into the syringe to a total volume of 10ml and the syringe was sealed with a stopper (# 394075). The syringe is then carefully rotated to bring the toxin solution into contact with the entire interior surface of the syringe.

Control sample wells were created as described above except that 0.5ml of saline solution was used instead of 0.2ml each in 5 vials.

The experiments were performed in three units using three different botulinum toxin wells. However, 0.2ml (series 1 and 3) or 0.5ml (series 2) botulinum toxin wells per sample were used relative to 100MLD botulinum toxin in each case. This volume is either filled into microtubes and aspirated into syringes or stored directly in cryovials.

Series 1: in the first experiment, aliquots of botulinum neurotoxin cells were stored in freezer tubes or 1ml syringes with 26G needles (BD #309602, US edition, and B.Braun #4657683) for 7 to 14 days, respectively.

The activity assay of the toxin was started by injecting or pipetting an aliquot of the toxin (0.2ml ═ 100MLD) into a prepared diaphragm organ bath containing 3.6ml of HAD buffer (Eagles balanced salt solution + 0.1% human serum albumin). 0.2ml of fresh saline solution was introduced into the syringe to flush the residual toxin solution and injected into the organ bath. Using a pipette, the microtube was flushed with 0.2ml of fresh saline solution.

Series 2: the second series was performed using several different injection materials from Becton Dickinson.

The control cells were either used immediately or stored in microtubes on ice for up to 1 hour prior to activity determination.

The botulinum toxin wells were stored in closed 10ml syringes at 4 ℃ to 8 ℃ for 1 hour, 3 days and 5 days, respectively. 0.5ml of sample was taken by connecting a 1ml syringe to a 10ml storage syringe using a female/female adaptor (BD #300013 and B.Brown # 5206634). After removal of the sample, the storage syringe is resealed and optionally returned to the refrigerator.

The activity assay of the toxin was started by injecting or pipetting an aliquot of the toxin (0.5ml ═ 100MLD) into a prepared diaphragm organ bath containing 3.5ml of HAD buffer (Eagles balanced salt solution + 0.1% human serum albumin). A30G needle (BD #304000) was used with the 1ml syringe described above. Residual toxin fluid was flushed by introducing 0.5ml of the membrane organ bath solution into a syringe and injecting it back into the organ bath.

Series 3: braun injection material from b.braun was used for the final series.

The control cells were either used immediately or stored in microtubes placed on ice for up to 1 hour prior to activity determination.

An aliquot of the above 0.2ml pool of botulinum toxin was introduced into a 1ml syringe equipped with a 20G needle (B.Braun #9161406V and # 4665503). The syringe then aspirates air to a total volume of 10ml and is sealed with a stopper (b.braun # 4495101). The syringe is then carefully rotated to bring the toxin solution into contact with the entire interior surface of the syringe. Thereafter, the samples were stored at 4 ℃ to 8 ℃ for 1 hour, 3 days and 9 days, respectively. The prepared membrane organ bath containing 3.6ml HAD buffer (Eagles' balanced salt solution + 0.1% human serum albumin) was injected or pipetted with toxin aliquots (0.2ml ═ 100 MLD). A 30G needle (b.braun #4656300) was used with the 1ml syringe described above. Another 0.2ml of fresh saline solution was introduced into the syringe to flush residual toxin solution and injected back into the organ bath.

The results of the activity measurements are listed in the table below.

(¹SD-standard deviation)

The plastic materials used in this study showed no significant effect on the activity of the recombinant neurotoxin preparation.

The paralysis time values of the control samples ranged from 68min to 82min (average: 72.6 min. + -. 5.0min), and the paralysis time values of the plastic-treated samples ranged from 60min to 77min (average: 68.6 min. + -. 4.8 min).

Example 3 "stability in Presence of preservatives"

Will be provided withReconstitution was carried out in sterile saline with or without preservative (benzyl alcohol) and stored for various time periods. No significant decrease in protein activity was detected in the presence of benzyl alcohol.

Each vial of drug product was reconstituted with 1.0ml of saline (with (0.9% v/v) or without preservatives) to a final concentration of 100MLD/ml and stored in polyethylene containers for the appropriate storage time. Samples of all experimental groups were then pooled.

Because of the anesthetic effect of benzyl alcohol on the membrane formulations of the experimental systems, a dialysis step was required to remove the drug prior to bioassay. Thus all experimental samples were dialyzed twice with 250 times excess Earl's buffered saline (EBSS), with or without preservatives, for more than 2 hours each time.

Following the dialysis step, the samples were diluted with EBSS to a final concentration of 25MLD/ml and the residual activity of the samples was determined in a diaphragmatic experiment using a nominal dose of 100MLD per assay.

The results of this example are shown in FIG. 1.

FIG. 1 shows storage of a reconstituted botulinum toxin drug in saline with or without preservatives in polyethylene containers at 4 deg.CThe effect of activity.

FIG. 2 shows the storage of a reconstituted botulinum toxin drug in saline with or without preservatives in a polyethylene syringe with rubber stopper at 4 deg.CThe effect of activity.

FIG. 1 the results show the performance in brine with or without the preservative benzyl alcoholThe recombination had no significant effect on the activity of the botulinum neurotoxin drug.

The drug product remains stable for up to 14 days when reconstituted in saline solution and stored at 4 ℃. The presence or absence of the preservative benzyl alcohol had no effect on stability.

Example 4 "freezing and thawing cycle"

In this example, reconstitution in sterile saline solution without preservativesFreezing and thawing were repeated 5 times. No significant effect of botulinum neurotoxin drugs on paralytic activity was detected.

Sterile saline for injection consists of 0.9% aqueous sodium chloride (w/vol). Using the medicineA sample cell is created.

The total number of the filter screen is 14The vials were divided into two groups of 7, carefully inflated with a needle and subsequently opened. Care was taken to keep the lyophilized product intact at the bottom of the vial.

Pair of 7 bottles with 1.4ml saline solutionThe first vial of the set was reconstituted and the lyophilized product was completely dissolved. The solution was quantitatively transferred to the next vial, where the lyophilizate was completely dissolved again. This procedure was repeated until the group of all 7 vials were dissolved in 1.4ml of sterile saline. Then the two groups of 1.4ml solutions were pooled to obtain 2.8mlAnd (4) a pool.

Will be provided withPool aliquots (0.2ml) were transferred to 12 labeled polyethylene tubes: two controls and 10 freeze-thaw replicate samples numbered 1 to 5 (representing the number of freeze-thaw cycles). Two control tubes were stored at 4 ℃ before diaphragm testing, while the other tube was frozen at-20 ℃ for at least 120 min. Tubes labeled 2-5 were completely thawed at room temperature (about 30min) and then frozen at-20 ℃ for at least 120 min. This procedure is continued until the tube designated 3 is frozen 3 times, the tube designated 4 is frozen 4 times and the tube designated 5 is frozen 5 times.

All samples were thawed at room temperature for 30min and incubated at 37 ℃ for 10min prior to diaphragm testing. They were mixed and centrifuged for 10 seconds. Each sample was transferred to 3.6ml Earl's buffered saline/0.1% HSA using an additional 0.2ml Earl's buffered saline/0.1% HSA. The 4.0ml obtained was subsequently checked in a diaphragm testAnd (4) carrying out measurement.

The results of these examples are shown in FIG. 3.

FIG. 3 results show recombinationRepeated freeze-thawing of (a) has no significant effect on the paralytic activity of the botulinum neurotoxin drug. When the recombination is carried out in a salt solution,stable for at least up to 5 freeze-thaw cycles.

The claims (modification according to treaty clause 19)

1. A method of supplying a muscle relaxant at a temperature above 20 ℃, wherein the muscle relaxant is a reconstituted solution comprising a botulinum toxin neurotoxic component free of complexing proteins.

2. The method of claim 1, wherein the supplying involves storage and/or transport or is a step in a process for preparing the muscle relaxant.

3. The method according to claim 1 or 2, wherein the muscle relaxant is transported or stored or both transported and stored at an ambient temperature of above 20 ℃ up to 70 ℃ without any cooling means.

4. The method of any preceding claim, wherein the muscle relaxant undergoes a "freeze and thaw" -cycle.

5. The method of claim 4, wherein said "freeze and thaw" -cycles are from 1 to 20.

6. The method of any preceding claim, wherein the muscle relaxant remains stable in the presence of a preservative and/or analgesic.

7. The method of any preceding claim, wherein the reconstituted solution is stored in a container made of plastic, glass, or metal, or any combination thereof.

8. The method of any preceding claim, wherein the solution further comprises sucrose or human serum albumin or both.

9. The method according to any preceding claim, wherein the solution further comprises at least one component selected from the group consisting of cryoprotectants other than sucrose and human serum albumin, stabilizers, pH buffers, excipients, and mixtures thereof.

10. The method of any preceding claim, wherein the neurotoxic component is a neurotoxic component of botulinum toxin type a.

Claims (10)

1. A process for supplying a muscle relaxant at a temperature above 6 ℃, preferably above 25 ℃, wherein the muscle relaxant is a reconstituted solution comprising a botulinum toxin neurotoxic component free of complexing proteins.

2. The method of claim 1, wherein the supplying involves storage and/or transport or is a step in a process for preparing the muscle relaxant.

3. A process according to claim 1 or 2, wherein the muscle relaxant is transported or stored or both transported and stored at an ambient temperature above 6 ℃, preferably above 25 ℃ up to 70 ℃, without any cooling means.

4. The method of any preceding claim, wherein the muscle relaxant undergoes a "freeze and thaw" -cycle.

5. The method of claim 4, wherein said "freeze and thaw" -cycles are from 1 to 20.

6. The method of any preceding claim, wherein the muscle relaxant remains stable in the presence of a preservative and/or analgesic.

7. The method of any preceding claim, wherein the reconstituted solution is stored in a container made of plastic, glass, or metal, or any combination thereof.

8. The method of any preceding claim, wherein the solution further comprises sucrose or human serum albumin or both.

9. The method according to any preceding claim, wherein the solution further comprises at least one component selected from the group consisting of cryoprotectants other than sucrose and human serum albumin, stabilizers, pH buffers, excipients, and mixtures thereof.

10. The method of any preceding claim, wherein the neurotoxic component is a neurotoxic component of botulinum toxin type a.