HK1240862A1 - Botulinum toxin prefilled container - Google Patents

Description

Botulinum toxin prefilled container

Technical Field

The present invention relates to prefilled glass containers, such as prefilled glass syringes containing an aqueous botulinum toxin formulation. The aqueous botulinum toxin formulation in the prefilled container is stable over time at low to ambient temperatures. Furthermore, the present invention relates to a kit comprising a botulinum toxin prefilled container, and the use of the botulinum toxin prefilled container in therapeutic and cosmetic applications.

Background

The stability of pharmaceutical products is of paramount importance to ensure safe and effective use over a sufficiently long period of time. Unfortunately, the performance (safety, reliability and efficacy) of most pharmaceutical products deteriorates over time. Causes of drug degradation include chemical degradation (e.g., hydrolysis, oxidation, reduction, and racemization), microbial contamination, and other mechanisms (e.g., precipitation).

Protein active ingredients generally have variable properties and are naturally unstable. This results in a loss of biological activity of the protein-containing pharmaceutical composition during its manufacture, reconstitution and/or storage. These problems observed in proteins can be attributed to chemical instability leading to bond formation or cleavage (e.g., hydrolysis, oxidation, racemization, beta elimination, and disulfide exchange) and/or to physical instability of the secondary or higher structure of the protein without modification by covalent bond cleavage (e.g., denaturation, adsorption to the surface, and non-covalent spontaneous aggregation).

Protein active ingredients are typically formulated as lyophilized (i.e., freeze-dried) products since degradation reactions typically occur most rapidly in aqueous solutions and most slowly in solid dosage forms. However, lyophilized products typically must be reconstituted with a pharmaceutically acceptable liquid (e.g., physiological saline) prior to use. Thus, lyophilized pharmaceutical products are considered less convenient than other dosage forms. Furthermore, the manufacture of lyophilized products is generally more expensive and time consuming. In addition, improper handling during reconstitution can cause inaccurate dosing or sterility problems. All these disadvantages can be overcome by using a pre-filled syringe. Thus, pre-filled syringes have become an increasingly popular delivery device.

However, if proteins are used as active ingredients, the limited stability of the proteins often precludes the formulation scientist from adopting a prefilled syringe format. This applies in particular to very dilute aqueous solutions of botulinum toxin (botulinum neurotoxin, BoNT). Such BoNT solutions are used in the treatment of a variety of debilitating neuromuscular diseases (e.g., cervical dystonia, blepharospasm, spasticity, and hyperhidrosis), as well as in cosmetic medicine (e.g., treatment of facial wrinkles). There are seven homologous serotypes (a-G) of botulinum toxin, produced by different Clostridium species, in particular Clostridium botulinum (c.botulinum), in the form of a complex comprising a neurotoxic polypeptide and other (non-toxic) clostridial proteins, i.e. different hemagglutinins and non-toxic, non-hemagglutinin proteins. The neurotoxic polypeptide has a molecular weight of about 150kDa, is activated by selective proteolytic cleavage, so as to obtain an active double-stranded form comprising a heavy chain (HC; comprising a translocation domain and a receptor binding domain) and a light chain (LC; comprising a catalytic domain), linked by disulfide bonds and by non-covalent interactions.

Botulinum toxin is naturally labile, particularly known to be highly labile at alkaline pH, and heat labile. Furthermore, it is known that there is significant difficulty in diluting the isolated toxin complexes from milligram quantities to much lower toxin concentrations (in the nanogram per milliliter range) for injection solutions, as such large-fold dilution causes rapid loss of specific activity.

Thus, commercial preparations of botulinum toxin are typically vacuum dried or lyophilized materials. Examples thereof include, for example(the Acetonuliumtoxina of Acegagen; Allergan, Inc.) and(Abobotulinumtoxina; Ipsen Ltd.) of Yipusheng, both of which contain botulinum toxin type A. Another example is(botulinum toxin (incobotulinumtoxin; Merz Pharma GmbH, Metz, Inc., Limited pharmaceutical Co., Ltd.)&Kgaa)), which contains pure neurotoxic components of the serotype a type (i.e., neurotoxic polypeptides of molecular weight about 150 kDa), free of any other proteins of the clostridium botulinum (c.botulinum) toxin complex (i.e., different hemagglutinin and non-toxic, non-hemagglutinin proteins).

However, lyophilized toxin products have a number of disadvantages, including the need to reconstitute before use, and its attendant sterility issues. Furthermore, in clinical practice, reconstituted toxin solutions are not usually used in their entirety, as different patients and indications require different dosages. Those reconstituted toxin solutions that are not used can be stored at lower temperatures, but only for shorter periods of time. For example, before use, diluted with normal physiological salineLater, it is recommended to use both within 6 hours and 4 hours, respectively. In a similar manner to that described above,the package insert indicates that after storage for more than 24 hours, the product is reconstitutedThe solution cannot be used for more than 24 hours and should be discarded.

To increase toxin stability, it is common in the art to add a stability protein such as Human Serum Albumin (HSA). Other stabilization strategies include the use of non-protein stabilizers, such as surfactants, polypropylene pyrrolidone (PVP), disaccharides, polyols, and the like. Furthermore, in WO 00/15245, it is disclosed that a high concentration liquid formulation of botulinum toxin type B (about 2500U/mL) is stable for up to 30 months when stored in a glass vial at 5 ℃. However, this long-term stabilization requires buffering the pH of the formulation to an acidic pH between 5 and 6, which causes pain upon injection. Other known methods of increasing toxin stability rely on the addition of various non-protein excipients, but are not suitable or desirable for human use (see, e.g., WO 01/58472, WO 2006/005910, and WO 2007/041664).

Thus, there is currently no injectable botulinum toxin dosage form that is not only stable over a long period of time to provide a sufficiently long shelf life, but is also convenient and easy to use, reduces medication errors, and minimizes the risk of contamination.

Object of the Invention

In view of the above, it is an object of the present invention to provide a pharmaceutical dosage form for administration of botulinum toxin which has a long shelf life and is convenient, safe and simple to use.

Disclosure of Invention

The above object is solved by providing a prefilled container (e.g. a syringe, a bottle, a carpule or an ampoule) of botulinum toxin, said container being characterized in that the liquid botulinum toxin formulation in the container has an excellent long term stability.

In a first aspect, the present invention provides a prefilled glass container (e.g., syringe, bottle, carpule or ampoule) containing an aqueous botulinum toxin formulation, wherein the toxin activity decreases by less than 25%, preferably less than 20%, relative to the initial toxin activity after storage in the prefilled container (e.g., syringe, bottle, carpule or ampoule) via: (a) 12 months at 5 ℃, (b) 12 months at 25 ℃ or (c) 6 months at 30 ℃.

The stability of the aqueous botulinum toxin formulation in the prefilled container (e.g., syringe, bottle, carpule or ampoule) is also excellent with respect to the number of particles visible under a microscope equal to or greater than 10 μm, typically less than 1000/mL after 6 to 24 months (e.g., 6, 9, 12, 15, 18 or 24 months) of storage at 2 ℃ -30 ℃ (e.g., 5 ℃, 25 ℃, or 30 ℃). In addition, the aqueous botulinum toxin formulation also exhibits excellent pH stability, with a pH that does not increase or decrease by more than 10% relative to the initial pH after 6 to 24 months (e.g., 6, 9, 12, 15, 18, or 24 months) storage in a pre-filled container (e.g., a syringe, a bottle, a carpule, or an ampoule) at 2 ℃ -30 ℃ (e.g., 5 ℃, 25 ℃, or 30 ℃).

In another aspect, the present invention provides a kit comprising a prefilled glass container (e.g., a syringe, a vial, a carpule or an ampoule) according to the first aspect of the present invention, and optionally instructions for use of the prefilled glass container.

In another aspect, the present invention provides a prefilled glass container (e.g. syringe, bottle, carpule or ampoule) for use in therapy according to the first aspect of the present invention. For example, the prefilled glass container (e.g., syringe, bottle, carpule or ampoule) may be used to treat a disease or condition caused by, or associated with, hyperactive muscular or exocrine cholinergic innervation of a patient, including, but not limited to, dystonia, spasticity, hyperextensibility, dysfunction, focal spasm, strabismus, tremor, spasm, migraine, sialorrhea and hyperhidrosis.

In another aspect, the present invention relates to the use of a prefilled glass container (e.g. a syringe, bottle, carpule bottle or ampoule) according to the first aspect of the present invention for cosmetic treatment, such as the treatment of skin wrinkles and facial asymmetries, e.g. glabellar lines, crow's feet, upper wrinkles and platysma bands.

In another aspect, the invention provides a method of treating a disease or condition caused by, or associated with, hyperactive cholinergic innervation of muscles or exocrine glands in a patient, the method comprising topically administering an effective amount of botulinum toxin to muscles or exocrine glands of the patient using a prefilled container (e.g., syringe, bottle, carpule or ampoule) according to the first aspect of the invention.

In another aspect, the invention relates to a cosmetic treatment method for the skin, such as the treatment of skin wrinkles and facial asymmetry, comprising topically administering an effective amount of botulinum toxin to a patient by intradermal, subcutaneous or subcutaneous injection with a prefilled container (e.g., syringe, bottle, carpule or ampoule) according to the first aspect of the invention.

Further embodiments of the invention are shown in the appended dependent claims. The invention may be more fully understood by reference to the following detailed description, examples and the accompanying drawings.

Drawings

FIG. 1 shows the stability of liquid botulinum toxin formulations in prefilled syringe configurations A, B, G and H as a function of time at 5 ℃. Structure a: (●), configuration B (■), configuration H: (□), Structure G: (. smallcircle.).

FIG. 2 shows the stability of liquid botulinum toxin formulations in prefilled syringe configurations A, B, G and H as a function of time at 25 ℃. Structure a: (●), configuration B (■), configuration H: (□), Structure G: (. smallcircle.).

FIG. 3 shows the stability of liquid botulinum toxin formulations in prefilled syringe configurations A, B, G and H as a function of time at 30 ℃. Structure a: (●), configuration B (■), configuration H: (□), Structure G: (. smallcircle.).

Detailed Description

The present invention is based on the surprising discovery that liquid botulinum toxin formulations in glass containers (e.g., in the form of syringes, vials, carpule bottles, or ampoules) are stable after prolonged storage at reduced temperatures (e.g., 2-8 ℃) and even at ambient temperatures (e.g., 20-30 ℃, particularly 25 ℃). Thus, the botulinum toxin prefilled containers of the present invention advantageously have a longer shelf life.

In addition, high long-term stability provides resistance to cold chain outages, which may facilitate approval procedures and/or marketization in all climatic regions, including countries with hot climates. Furthermore, the prefilled glass container of the present invention, in particular the syringe form, has several additional advantages compared to other administration forms, such as ease of use, reduced risk of medication errors, high dosage accuracy, low risk of contamination, improved sterility assurance and/or high administration safety.

As used herein, "prefilled container" refers to any device having a partially or fully enclosed space that can be sealed or is hermetically sealed and can be used to contain, store and/or transport a liquid formulation. Within the meaning of the present invention, a "prefilled container" is preferably a closed (or sealed) container made of glass, or made partly or mainly of glass, including for example containers in the form of (i) syringes, (ii) vials, (iii) carpule bottles or (iv) ampoules.

Pre-filled syringes and carpule vials have two openings that are sealed to prevent leakage of the contents (e.g., aqueous formulations). In the case of a pre-filled syringe, the proximal end is sealed by a plunger stopper and the distal end is sealed by a capping device, as described in detail below. In the case of a glass carpule bottle, which is typically a glass column aseptically filled with a pharmaceutical formulation, the proximal end is typically sealed by a rubber stopper. The rubber stopper can be pressed in by the pressure of a plutella syringe puncture, acting as a plunger in the column. The distal end is typically sealed by a puncture membrane. The puncture membrane is pierced for injection.

Within the meaning of the present invention, a "vial" is a vessel, generally tubular or necked, suitable for containing, storing and/or transporting a pharmaceutical formulation. The single opening may be sealed by different vial closure systems. For example, the vials may be closed with screw top caps (screw top vials), cork stoppers, plastic or rubber (lip and roll top vials), and other closure systems such as pull caps or snap caps. In the present invention, "vial" preferably means a glass vessel whose opening is sealed with a vial closure system.

The present invention is described in further detail below. It should be noted that although the terms "prefilled syringe", "prefilled glass syringe", "syringe" or "glass syringe" are used in the embodiments of the present invention, this is not meant to be limited to a specific example of a (glass) syringe as a (glass) container. Indeed, unless otherwise indicated, any reference herein to "prefilled syringe", "prefilled glass syringe", "syringe" or "glass syringe" and the like is to be understood as reference to and disclosure of "container" or "glass container", and also includes or discloses "vial" or "glass vial", "carpule bottle" or "glass carpule bottle", or "ampoule" or "glass ampoule".

In a first aspect, the present invention relates to a prefilled glass syringe containing an aqueous formulation of a botulinum toxin, wherein the toxin activity does not decrease by more than 25% relative to the initial toxin activity upon storage of the prefilled syringe under the following conditions: (a) a standard refrigerator temperature (i.e., 2-8 ℃, such as 5 ℃) for 12 months, (b)25 ℃ for 12 months, or (c)30 ℃ for 6 months. Preferably, toxin activity is reduced by no more than 20% or 15% relative to the initial toxin activity after storage in a pre-filled syringe under the following conditions: (a)2-8 ℃ (e.g., 5 ℃) for 12 months, (b)25 ℃ for 12 months, or (c)30 ℃ for 6 months. More preferably, the toxin activity does not decrease by more than 20% or 15% relative to the initial toxin activity after storage in a pre-filled syringe under the following conditions: (a)2-8 ℃ (e.g., 5 ℃) for 6 months, (b)25 ℃ for 6 months, or (c)30 ℃ for 3 months. Particularly preferably, the toxin activity does not decrease more than 10% relative to the initial toxin activity after storage in a pre-filled syringe under the following conditions: (a)2-8 ℃ (e.g., 5 ℃) for 3 to 6 months, or (b)25 ℃ for 3 to 6 months. It is especially preferred that the toxin activity does not decrease more than 5% relative to the initial toxin activity after storage in a pre-filled syringe under the following conditions: (a)2-8 ℃ (e.g., 5 ℃) for 3 to 6 months, or (b)25 ℃ for 3 to 6 months.

Surprisingly, the aqueous botulinum toxin formulation in the prefilled syringe is also stable for storage periods of up to 24 months or even longer. For example, after storage at 2-8 ℃ (e.g., 5 ℃) or 25 ℃ for up to 24 months (e.g., 15, 18, or 24 months), the toxin activity preferably decreases by no more than 30% or 25%, more preferably no more than 20%, particularly no more than 15%, particularly preferably no more than 10%, most preferably no more than 5% relative to the initial toxin activity.

In particular, the toxin activity preferably decreases by no more than 25%, 20%, 15%, 10% or 5% relative to the initial toxin activity after 24 months of storage of the pre-filled syringe at 2-8 ℃. The toxin activity preferably decreases by no more than 25%, 20%, 15%, 10% or 5% relative to the initial toxin activity after storage of the pre-filled syringe for 18 months at 2-8 ℃. Furthermore, the toxin activity preferably does not decrease by more than 35%, 30%, 25%, 20% or 15% relative to the initial toxin activity after 24 months of storage of the pre-filled syringe at 25 ℃. The toxin activity preferably decreases by no more than 30%, 25%, 20%, 15% or 10% relative to the initial toxin activity after storage of the pre-filled syringe for 18 months at 25 ℃.

In the present invention, the term "toxin activity" is intended to refer to the biological activity of botulinum toxin. "biological activity" may refer to (a) receptor binding, (b) internalization, (c) translocation across the endocytotic membrane into the cytoplasm, and/or (d) endoprotease cleavage of proteins involved in synaptophytic membrane fusion. For example, within the scope of the present invention, any LC (light chain) domain that satisfies the following conditions may be considered "biologically active" or "exhibiting proteolytic activity": exhibit more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and up to 100% of the proteolytic activity of the corresponding wild-type LC domain in the SNAP-25 assay. Furthermore, any HC (heavy chain) domain that satisfies the following conditions is considered "biologically active": is capable of binding to a cellular HC domain receptor, in particular its native HC domain receptor, and of translocating the LC domain to which it is attached.

Biological activity is expressed in Mouse Units (MU). As used herein, 1MU is the amount of neurotoxic component that kills 50% of a given mouse population following intraperitoneal injection, i.e., the intraperitoneal injection of LD into mice, measured according to the method of Schantz and Kauter (Schantz and Kauter, J.Assoc. off. anal. chem.1978,61:96-99(Schantz and Kauter, journal of the American society of analytical chemists, 1978, volume 61, pages 96-99))₅₀. The terms "MU" and "unit" or "U" are used interchangeably herein.

Suitable assays for assessing biological activity include: pearce et al (Toxicol. appl. Pharmacol.128:69-77,1994 (toxicology and applied pharmacology, Vol. 128, pp. 69-77, 1994)) the mouse hemiphrenic partial determination (MHA); according toEt al (Experimental Neurology 147:96-102,1997 (Experimental Neurology 147, Vol. 147, pp. 96-102, 1997)); mouse membrane assay (MDA) according to Dressler et al (Mov. Disord.20:1617-1619,2005 (dyskinesia, Vol. 20, p. 1617-1619, 2005)); a SNAP-25 protease assay (e.g., the "GFP-SNAP 25 fluorescence release assay" described in WO 2006/020748, or the "modified SNAP25 endopeptidase immunoassay" described in Joneset al, 2008, j.immunol.methods 329:92-101(Jones et al, 2008, journal of immunization, v 329, p. 92-101)); electrochemiluminescence (ECL) sandwich ELISA as described in WO 2009/114748; and cell-based assays as described in WO 2009/114748, WO 2004/029576, WO 2013/049508 and in particular WO 2014/207109.

As used herein, the term "initial toxin activity" or "initial potency" generally refers to the activity of botulinum toxin at the beginning of the shelf life, i.e., after production of the final product sterile botulinum toxin prefilled syringe, particularly within one or two days immediately following production or after production. In addition, the term "after storage" as used herein is intended to mean after a particular time of storage. Furthermore, the term "storage period" generally means during the entire storage period.

Furthermore, the aqueous botulinum toxin formulation is highly stable according to the particle count which is only visible under the microscope. Within the meaning of the present invention, "microscopic particles" are generally particles having a diameter of less than 100 μm. In particular, the count (or number) of particles equal to or greater than 10 μm in an aqueous botulinum toxin formulation is typically less than 1000/mL, preferably less than 600/mL, more preferably less than 200/mL during storage at 2-30 deg.C (e.g., 5 deg.C, 25 deg.C, or 30 deg.C) for 6 to 24 months (e.g., 6, 9, 12, 15, 18, or 24 months).

Particle measurements can be made using different methods, such as microfluidic imaging (MFI), Resonance Mass Measurement (RMM), and Nanoparticle Tracking Analysis (NTA). Particle measurements generally follow USP <788 >. In the context of the present invention, preferably microfluidic imaging measurements are used. This measurement method can be performed, for example, using a DPA-5200 particle analyzer system (protein simple, Santa Clara, CA, USA) equipped with a silane-coated high-resolution 100 μm flow cell. Generally, samples are analyzed without dilution.

Alternatively, Resonance Mass Measurements (RMM) may be used to determine particle number using, for example, ARCHIMEDES particle measurement system (ARCHIMEDES Particle Metrology System) equipped with a microsensor (size range 0.3-4 μm), calibrated with 1 μm polystyrene standards (Affinity Biosensors, Santa Barbara, CA, USA). All samples were typically analyzed without dilution. The results can be analyzed using particle lab software (v1.8.570) in bin steps of 10 nm. As an alternative to determining particle count, Nanoparticle Tracking Analysis (NTA) may be performed, for example, using the NanoSight LM20 system (NanoSight LM20 system; NanoSight, Amesbury, UK) in Ammsbury, England. The sample is typically measured without dilution. The movement of particles in the sample can be recorded at ambient temperature in 60 seconds of video and analyzed with appropriate software (e.g., NTA 2.3 software).

Furthermore, the aqueous botulinum toxin formulation exhibits high pH stability, i.e., the pH is substantially stable during storage in the prefilled syringe. Preferably, the pre-filled syringe increases or decreases the pH by no more than 10%, 8% or 6% from the initial pH after storage at 2-30 ℃ (e.g., 5 ℃, 25 ℃ or 30 ℃) for 6 to 24 months (e.g., 6, 9, 12, 15, 18 or 24 months), e.g., 18 months at 25 ℃ or 24 months at 25 ℃. The pH can be measured according to the united states pharmacopeia standardized test method USP <791>, which outlines the pH measurement method for a number of pharmaceutical products. A suitable pH meter, such as a Lab 870pH meter from schottky Instruments, may be used.

As used herein, the term "pre-filled syringe" refers to a syringe that is filled with a pharmaceutical composition (i.e., an aqueous botulinum toxin formulation) prior to distribution to an end user who will administer the drug to a patient. Pre-filled syringes generally comprise: a medicament containing container forming part of the syringe body (i.e. the syringe barrel); a plunger sealing the proximal opening of the syringe and for expelling the medicament; and a sealing means (e.g. an end cap (tip cap) or needle cover) on the outlet end of the syringe (e.g. the syringe tip or the open end of a pre-filled needle (cannula)) which seals the distal outlet opening. As used herein, the term "prefilled glass syringe" refers to a prefilled syringe in which at least the syringe barrel is made of glass.

In the present invention, the prefilled syringe is preferably a straight port (Luer slip) or screw-port (Luer lock) syringe equipped with an end cap (if not prefilled) or a needle cover (if prefilled). Within the meaning of the present invention, a "straight syringe" is a syringe that allows the needle to be pushed to the end of the tip, while a "screw syringe" is a syringe that allows the needle to be rotated into the tip and locked in place. This provides a reliable connection and avoids accidental removal of the needle for injecting the liquid.

The prefilled syringe according to the present invention is generally sterile and therefore ready to use on the fly. Further, the pre-filled syringes described herein are intended for single use and are intended to be disposable. Prior to sterilization, the inside surface of the syringe, and more particularly the glass syringe barrel, is typically coated with a lubricant to facilitate sliding of the plunger stop and expression of the syringe contents. Suitable sterilization methods include, but are not limited to, gamma irradiation, ethylene oxide (ETO) treatment, and moist heat (e.g., autoclaving).

According to the present invention, the aqueous botulinum toxin formulation in the prefilled syringe comprises a concentration of botulinum toxin, for example, from 1U/mL to 3000U/mL, from 10U/mL to 1000U/mL. Preferably, the botulinum toxin is present at a concentration of about 10U/mL to 400U/mL, more preferably about 25U/mL to 200U/mL, most preferably about 40U/mL to 150U/mL (e.g., 50U/mL, 75U/mL, or 100U/mL).

As used herein, the term "botulinum toxin" broadly refers to any form and type of botulinum toxin. Specifically, the botulinum toxin can be selected from botulinum toxin types A, B, C1, D, E, F, G, or mixtures thereof. In the context of the present invention, the botulinum toxin is preferably of the A, B or C1 serogroup, in particular the serogroup A serogroup.

Furthermore, as used herein, the term "botulinum toxin" is intended to include both botulinum toxin complexes ("toxin complexes") and the "neurotoxic component" of botulinum toxin complexes. As used herein, the term "botulinum toxin complex" or "toxin complex" refers to a high molecular weight complex comprising a neurotoxic component of about 150kDa and, in addition, non-toxic proteins of botulinum, including hemagglutinin and non-hemagglutinin proteins. Botulinum toxin type A serotype complexes are commercially available, for example, in(Allergan, Inc.) or(Ipsen, Ltd.) was obtained commercially.

As used herein, the term "neurotoxic component" relates to a neurotoxic polypeptide ("150 KDa" polypeptide) that is free of toxin complexes of any non-toxic protein of interest. Pure neurotoxic component may be, for example, under the trade nameAnd(Merz Pharmaceuticals GmbH) is commercially available. In the present invention, the botulinum toxin is preferably the neurotoxic component of a botulinum toxin complex, for example of the serotype A, B, C1, in particular of the serotype A. In other words, the aqueous botulinum toxin formulation encapsulated in the prefilled glass syringe preferably comprises (only) the neurotoxic component and is free of any other proteins of the botulinum toxin complex.

It is also contemplated that the invention includes isomers, homologs, orthologs, and paralogs that have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and up to 60%, up to 70%, up to 80%, up to 90%, up to 100% sequence identity to a wild-type botulinum toxin, e.g., the neurotoxic component of a botulinum toxin of wild-type botulinum toxin type a or serotype a1 deposited in the GenBank database under accession number AAA 23262. The sequence identity can be calculated by any algorithm suitable for producing reliable results, for example by using the FASTA algorithm (W.R.Pearson & D.J.Lipman PNAS (1988)85: 2444-. Sequence identity can be calculated by comparing two polypeptides or two domains thereof (such as two LC domains or fragments thereof).

Modified and recombinant botulinum toxins are also within the scope of the invention. For suitable mutants, reference may be made to WO 2006/027207, WO 2009/015840, WO 2006/114308, WO 2007/104567, WO 2010/022979, WO 2011/000929 and WO 2013/068476, which are incorporated herein by reference. Furthermore, the present invention also relates to chemically modified botulinum toxins, for example by pegylation, glycosylation, sulfation, phosphorylation or any other modification, in particular to one or more surface or solvent exposed amino acids. Modified, recombinant isomers, homologs, orthologs, paralogs and mutants suitable for use in the present invention are biologically active, i.e., capable of translocating into the nerve and cleaving proteins of the SNARE complex (e.g., VAMP/syntaxin, synaptophysin and SNAP-25), exerting their acetylcholine inhibitory effect, e.g., their muscle paralytic effect.

In the context of the present invention, the aqueous botulinum toxin formulation can include various other pharmaceutically acceptable substances, such as salts (e.g., sodium chloride), stabilizing proteins (e.g., albumin, gelatin), sugars (e.g., glucose, fructose, galactose, trehalose, sucrose, and maltose), carbohydrate polymers (e.g., hyaluronic acid and polyvinylpyrrolidone (PVP)), polyols (e.g., glycerol and sugar alcohols such as mannitol, inositol, lactitol, isomalt, xylitol, erythritol, sorbitol), amino acids, vitamins (e.g., vitamin C), zinc, magnesium, anesthetics (e.g., local anesthetics such as lidocaine), surfactants, tonicity modifiers, and the like. As used herein, the term "pharmaceutically acceptable" refers to a compound or substance that is suitable for contact with the tissues of a mammal (particularly a human).

As used herein, the terms "comprising," including, "and" containing "are intended to include both the open-ended term" comprising "and the closed-ended term" consisting of …. As used herein, the term "made from …" is intended to broadly relate to "made from …," in particular "made primarily from …," and is generally intended to mean "including," "including" (meaning that other substances or materials may be included in some amount). It may also mean "consisting of …".

Preferably, the pH of the aqueous botulinum toxin formulation in the prefilled syringe during storage is between 6.0 and 7.5, between 6.5 and 7.5, between 6.1 and 7.3, between 6.2 and 7.2, between 6.3 and 7.1, and between 6.5 and 7.0. A pH in the range of 6.1 to 7.3 is advantageous because injection of such a neutral or only slightly acidic solution is far less painful than injection of an acidic solution.

As used herein, the term "aqueous formulation" or "aqueous botulinum toxin formulation" is not particularly limited and may refer to aqueous suspensions, aqueous dispersions, aqueous emulsions, and preferably aqueous solutions.

Preferably, the aqueous botulinum toxin formulation is free of buffering materials, such as phosphate buffers, phosphate-citrate buffers, lactate buffers, acetate buffers, and the like. As used herein, the term "buffering agent" means a pharmaceutically acceptable excipient that stabilizes the pH of a pharmaceutical preparation. In addition, the aqueous botulinum toxin formulation may be free of amino acids (e.g., methionine) and/or surfactants (e.g., polysorbates, such as polysorbate 80) and/or animal derived proteins (e.g., Human Serum Albumin (HSA) or Bovine Serum Albumin (BSA)).

Preferred aqueous botulinum toxin formulations for use herein comprise water, a concentration of botulinum toxin (e.g., the neurotoxic component of botulinum toxin, preferably type a) such as from 10 to 150U/mL, a concentration of a salt (e.g., sodium chloride) such as from 0.5% to 1.5% w/v, a concentration of a sugar (e.g., a mono-or disaccharide such as glucose, fructose, galactose, trehalose, sucrose and maltose) such as from 0.1% to 2% w/v, and a concentration of a stabilizing protein (e.g., albumin) such as from 0.01% to 4% w/v, from 0.1% to 3% w/v or from 0.1% to 1% w/v.

Another preferred aqueous botulinum formulation useful herein consists essentially of water, botulinum toxin (e.g., the neurotoxic component of botulinum toxin type a), sodium chloride, sucrose, and albumin (e.g., human serum albumin). The concentration of the components mentioned may be in the following ranges: 10 to 200U/mL or 30 to 125U/mL (botulinum toxin), 0.5% to 1.5% w/v or 0.7% to 1.1% w/v (sodium chloride), 0.1% to 2% w/v or 0.2% to 1% w/v (sucrose), 0.01% to 1% w/v, 0.05% to 0.5% w/v, 01% to 3% w/v or 0.5% to 1.5% w/v (HSA). Yet another preferred botulinum toxin formulation for use herein is, for example, reconstituted with physiological saline (0.9% sodium chloride)A solution comprising 20 to 150U/mL of a neurotoxic component of botulinum toxin type a.

As used herein, the term "consisting essentially of …" is intended to mean that other substances than the indicated substances are contained therein in only trace amounts, such as unavoidable impurities contained in the components used to formulate aqueous botulinum toxin preparations, as well as minor impurities (e.g., very low residual amounts of buffers, chelators, etc.) contained in the isolated botulinum toxin (e.g., the neurotoxic component of botulinum toxin type a) as a result of the purification process.

According to the present invention, the construction of the pre-filled syringe is not particularly limited and typically comprises a fluid receiving cartridge removably capped after filling by a capping device (e.g. by removing or replacing a "cap" (or "end cap") with a needle prior to use, or a sealing means such as a needle cover when using a syringe with a removable needle or a permanent needle) to sealingly close the distal end of the syringe and to seal the fluid receiving cartridge at the proximal end with its plunger or other means in fluid tight engagement with the inner wall of the cartridge. To use a prefilled syringe, the end cap, needle cover or other type of closure device is removed, a needle is optionally attached (if not already present), and the plunger tip or piston is advanced within the syringe barrel to inject the contents of the syringe barrel into the patient.

The prefilled glass syringe according to the present invention preferably comprises:

(a) a syringe barrel made of glass comprising a proximal end and a distal end, and a generally cylindrical wall extending between the proximal end and the distal end and defining a syringe barrel lumen, the syringe barrel having a distally projecting tip with a fluid passageway extending through the distally projecting tip and communicating with the syringe barrel lumen, wherein the generally cylindrical wall has an inner surface, the inner surface optionally coated with a barrier layer,

(b) a capping device having an outlet junction sealingly engaging and closing the distal open outlet end of the syringe, wherein the outlet junction is made of an elastomeric material, optionally having a coating on its surface, and

(c) a plunger rod assembly extending into the proximal end of the syringe barrel and comprising a plunger stopper in sliding fluid-tight engagement between the plunger stopper and the cylindrical wall of the interior chamber of the syringe barrel, wherein the plunger stopper is made of an elastomeric material, and at least a portion of the plunger stopper optionally has a coating thereon that contacts the aqueous botulinum toxin formulation during storage and/or injection.

In general, the primary container closure system (including components such as syringe barrel, sealing device (e.g., end cap or needle cover) and plunger) is likely to interact with the drug formulation in the prefilled syringe. This can result in the release of extractables/leachables from the syringe material that is contacted with the aqueous botulinum toxin formulation. Extractables/leachables may contaminate aqueous botulinum preparations and reduce the stability or activity of botulinum toxin. Thus, the materials of the pre-filled syringe are generally selected to minimize or limit the amount of extractables/leachables.

As used herein, the terms "extractables" and "leachables" refer to chemicals that may be released from the components of a container or prefilled syringe material and/or chemicals that migrate from the syringe material into an aqueous botulinum toxin formulation under typical use or storage conditions. Methods for identifying extractables/leachables are known in the art and are based on suggested industry conventions and International harmonization Conference (ICH) guidelines (see FDA guidelines, Container closure systems for Packaging Human pharmaceuticals and biologicals (Container closure systems for Packaging Human pharmaceuticals and biologicals)), and include, for example, liquid chromatography/mass spectrometry (LC/MS), gas chromatography/mass spectrometry (GC/MS), Inductively Coupled Plasma (ICP), and Infrared (IR).

The inner surface of the glass cylinder is typically coated with a lubricant layer (also referred to herein as the terms "barrier layer" or "barrier coating," and used interchangeably with these terms). The lubricant layer should not only provide a high degree of lubricity, enabling the plunger to slide easily through the sleeve, but also be compatible with the aqueous botulinum toxin formulation and preserve the shelf life of the aqueous botulinum toxin formulation. In the context of the present invention, the lubricant layer may be a silicone-free lubricant layer or a silicone lubricant layer.

Likewise, the inner surface of the glass part of the vial, the inner surface of the glass column of the carpule and the inner surface of the glass ampoule may optionally be coated with a barrier layer, in particular with a silicone-free layer or a silicone layer. Accordingly, all of the descriptions provided below with respect to the silicone-free lubricant layer and the silicone lubricant layer of the glass syringe apply equally to the silicone-free layer and the silicone layer of the glass vial, the glass carpule and the glass ampoule, respectively.

Suitable silicone-free lubricating layers are, for example, fluoropolymer layers (e.g., fluoropolymer (fluorocarbon) layers, such as ethylene-tetrafluoroethylene (ETFE) layers and perfluoropolyether-based (PFPE-based) layers (e.g.,) And silicon oxide based glass PECVD (plasma enhanced chemical vapor deposition) coatings.

The silicone-free lubricant layer can be prepared as known in the art, for example by spraying a glass syringe barrel with perfluoropolyether oil to cause the lubricant to form a thin layer on the interior surface of the syringe, followed by exposing the interior cavity to a downstream inert gas (e.g., argon or helium) plasma. The plasma treatment causes the perfluoropolyether to crosslink, thereby immobilizing the coating and reducing its tendency to migrate out of the target surface, resulting in a reduction of particles that potentially compromise the stability/efficacy of the botulinum toxin drug. An exemplary preparation scheme is described in WO2014/014641a1, the contents of which are incorporated herein by reference. Further, particularly suitable silicone-free barrier coatings useful herein are known in the art asA coating which is a perfluoropolyether coating crosslinked by plasma treatment.

Suitable silicone lubricant layers for use herein may be prepared by a siliconizing process selected from, but not limited to, silicone oil based processes (spray-on siliconizing) or bake-on siliconizing) and vapor deposition processes (e.g., plasma enhanced chemical vapor deposition). Preferably, the silicone lubricant layer is formed by a spray siliconizing method, or more preferably, by a bake siliconizing method.

In the spray siliconizing process, a silicone oil (e.g. DOW with a viscosity of 1000 cSt) is sprayed using e.g. a submerged nozzle (diving nozzle) or a static nozzle (static nozzle)360) Into a syringe (i.e., syringe barrel) to create a thin layer of silicone oil. While silicone oils are excellent lubricants, excessive silicone oils can lead to the formation of unwanted visually and microscopically visible particles of silicone oils. These silicone oil particles can cause adverse interactions with protein drugs, particularly when protein-based drugs are used. For example, microscopically visible silicone oil particles are believed to promote protein aggregation. Thus, the bake siliconization process is particularly preferred for use herein since it produces less silicone oil particles that are visible and visually perceptible under the microscope. This involves using the silicone oil as an emulsion (e.g., DOW)365 siliconized emulsion) and then baked on the glass surface for a specified time at a specified temperature.

The design of the syringe barrel is not particularly limited and typically has a volume adjusted to accommodate the desired fill volume (e.g., 0.5 cm)³、1.0cm³、1.5cm³Or 2.0cm³) Of the inner diameter of (a). Typically, the syringe barrel has graduated markings indicating the volume of fluid in the syringe. Further, the syringe barrel may include a flange-type interface. The design of the flange may for example comply with ISO 11040. The flanged interface may further be compatible with an optionally present handle. Furthermore, in the case of a screw-type syringe, the syringe may be equipped with a screw-type joint of, for example, polycarbonate.

The syringe tip is typically formed integrally with the syringe barrel. The tip is formed with an integral passageway extending axially through the tip and communicating with the chamber for dispensing the contents of the syringe barrel. The tip may have a substantially frustoconical shape that converges from the distal outlet end of the syringe barrel toward the outlet end of the tip. Alternatively, the tip may be characterized as being divergent (i.e., expanding from a smaller diameter to a larger diameter). Furthermore, the tip is typically centrally located with respect to the syringe body (concentric syringe tip), but may also be located at an offset position towards the edge of the body (eccentric syringe tip).

In the meaning of the present invention, "capping device" refers broadly to any means of closing and sealing the open outlet end of a syringe. In the present invention, the term "open outlet end" refers to any distal open end of the syringe that is in fluid communication with the syringe barrel lumen. The capping device typically has a channel with a closed end and an open end sized to receive and effectively seal the open outlet end of the syringe to prevent leakage.

In the case of pre-filled syringes without pre-mounted needles, the capping device is a capping tool commonly referred to as an "end cap". The end cap forms a fluid seal with the tip of the syringe to effectively enclose the syringe barrel and prevent leakage of the contents of the syringe barrel. The end cap is typically removably coupled to the syringe tip or luer collar. The luer collar surrounds the top of the syringe barrel (e.g., the syringe tip). Preferably, the luer collar has internal threads and the end cap has external threads complementary to said luer collar internal threads for coupling the end cap to the syringe barrel. The luer collar is typically a separately molded luer collar that is mounted directly to the top end of the syringe barrel, such as by a snap or interference fit. Prior to use, the tip can be removed and the needle cannula (or needle/needle assembly) can then be securely coupled to the syringe tip.

If the prefilled syringe comprises a removable or non-removable (permanent) cannula (also referred to as a "needle" or "needle assembly") extending from the syringe tip for delivery of an aqueous botulinum toxin formulation from the syringe, the closure device may be referred to as a "needle cover". The needle cover typically has a channel with a closed end and an open end sized to receive and couple with a cannula (needle) mounted on the top end of the syringe. Typically, the (sharp) end of the cannula passes through the closed end of the passage in the needle cover to seal the open end of the cannula.

The cover device (e.g., end cap or needle cover) may be a unitary member and is typically made of a flexible and/or elastic polymeric material (e.g., an elastomer), at least a portion of which contacts and seals the distal opening of the syringe (referred to as the "outlet junction"). Alternatively, the closure device may have an outer cap made of a rigid plastic material coupled to a flexible and/or elastomeric inner cap made of a flexible and resilient polymer material (e.g., an elastomer), wherein at least a portion of the inner cap contacts and seals the distal opening (referred to as the "outlet junction") of the syringe.

In view of the fact that the outlet junction contacts the aqueous botulinum toxin formulation during storage and/or use, the outlet junction is preferably made of a material that has a minimal likelihood of producing unwanted extractables/leachables. To this end, the outlet junction may have a coating thereon to increase compatibility with the aqueous botulinum toxin formulation.

Suitable flexible and/or elastic materials for the means for covering, particularly the outlet junction, include elastomers that do not interfere with the aqueous botulinum toxin formulation and are capable of long term storage. In particular, during extended storage of the aqueous botulinum toxin formulation, the portion of the sealing device that contacts the aqueous botulinum toxin formulation (i.e., the exit junction) should have a low extractable/leachable level. As used herein, the term "elastomer" or "elastomeric material" refers primarily to a crosslinked thermoset rubber polymer that is more deformable than plastic, but is approved for use in medical grade fluids and is not susceptible to leaching or gas migration.

Preferably, the elastomeric material IS selected from isoprene rubber (IS), butadiene rubber (polybutadiene, BR), butyl rubber (copolymer of isobutylene and isoprene, IIR), halogenated butyl rubber (e.g. chlorobutyl rubber (CIIR) and bromobutyl rubber (BIIR)), styrene-butadiene rubber (copolymer of styrene and butadiene, SBR) and the like. Preferably, the elastomeric material is a styrene-butadiene rubber, a butyl rubber, a blend of isoprene rubber and halogenated (e.g. bromo or chloro) butyl rubber, a halogenated butyl rubber, in particular a bromobutyl rubber or a chlorobutyl rubber or a mixture thereof. Inert minerals may also be used to reinforce elastomeric materials. Additionally, the elastomeric material may be cured (e.g., using organic peroxides, phenolic resins, etc.).

Suitable coatings, which may optionally be present on the elastomeric material, are made of materials that do not undesirably interfere with the aqueous botulinum toxin formulation and have low extractables/leachables levels. A preferred example of such a coating is a coating made of a fluoropolymer, i.e. a fluorocarbon coating. Other suitable coatings that may be used herein include, for example, polypropylene, polyethylene,Parylene (e.g., N-type parylene, C-type parylene, and HT-type parylene) and cross-linked silicone (e.g., B2-coating (Daikyo Seiko) or XSi)^TM(BD Co (Becton Dickinson))).

Fluoropolymer coatings include, but are not limited to, fluorinated ethylene-propylene copolymers (e.g., tetrafluoroethylene-hexafluoropropylene copolymer (FEP)), fluorinated ethylene-ethylene copolymers (e.g., ethylene-tetrafluoroethylene copolymer (ETFE), such as) PVA (a copolymer of Tetrafluoroethylene (TFE) and perfluoropropyl vinyl ether (PPVE)), tetrafluoroethylene-perfluoroethylene copolymer, polyvinylidene fluoride (PVDF), polyvinyl fluoride (PVF), Polytetrafluoroethylene (PTFE), and mixtures thereof. Preferably, the coating is made of ETFE, and in particular isAnd (4) coating.

In the case of the carpule bottle of the present invention, the distal end thereof is sealed by a piercing membrane. The puncture membrane may be formed of thin rubber or silicone, thin plastic/polymer, film (such as Mylar), polyolefin (such as polyethylene or polypropylene), metal foil (such as aluminum foil), and the like. The thickness of the film may be between about 0.001mm and 2.0mm, typically between 0.002mm and 0.65 mm. Additionally, the membrane may be made of an elastomeric material and optionally have a coating as described above in connection with the capping device of the prefilled glass syringe.

With respect to the vials of the present invention, the vial closure system (e.g., cap), particularly those portions of the vial closure system that contact or may contact and/or seal the vial (e.g., septum), may be made of an elastomeric material, particularly a thermoplastic elastomeric material, more particularly a styrene block copolymer thermoplastic elastomer, or an elastomeric material as described above in connection with the closure device of the prefilled glass syringe of the present invention. Another suitable material is a silicone material. Furthermore, the material may have an optional coating, in particular a fluoropolymer coating, as defined above with respect to the capping device of the prefilled glass syringe.

In accordance with the present invention, a pre-filled syringe typically includes a plunger rod assembly that extends into the proximal end of the syringe barrel. The plunger rod assembly may include a rod (also referred to as a pushrod) having a tip with a plunger stop (also referred to as a "plunger") in sliding fluid-tight engagement with the cylindrical wall of the syringe barrel lumen. The plunger forms a proximal seal, as well as a dynamic seal that allows for the expression of the liquid botulinum toxin formulation. During storage and/or administration, the plunger stop is contacted with the aqueous botulinum toxin formulation. Thus, the plunger stop should be compatible with the aqueous botulinum toxin formulation and not compromise its long term stability. In particular, the plunger stop should preferably be designed to minimize the amount of extractables/leachables in the event of prolonged storage.

In the present invention, the plunger stop is preferably made of an elastomeric material, optionally having a coating on at least a portion thereof, which contacts the aqueous botulinum toxin formulation during storage and/or use. Suitable plunger stop elastomeric materials for use herein include, but are not limited to, isoprene rubber (IS), butadiene rubber (polybutadiene, BR), butyl rubber (copolymer of isobutylene and isoprene, IIR), halogenated butyl rubber (e.g., chlorobutyl rubber (CIIR) and bromobutyl rubber (BIIR)), styrene-butadiene rubber (copolymer of styrene and butadiene, SBR), and mixtures thereof. Preferably, the plunger stopper material is butyl rubber or halogenated butyl rubber or a mixture thereof, more preferably bromobutyl rubber or chlorobutyl rubber, most preferably butyl rubber. Inert minerals may also be used to reinforce elastomeric materials. Additionally, the elastomeric material may be cured (e.g., using organic peroxides, phenolic resins, etc.).

Preferably, the plunger stop comprises a coating that acts as a barrier film. The coating is typically applied to at least the sealing surface, including the portion of the surface of the plunger stopper that faces the syringe barrel lumen and contacts the aqueous botulinum toxin formulation during storage and/or use. The coating serves to minimize interaction between the plunger and the liquid botulinum toxin formulation and provides good lubricity.

Suitable coatings for plunger stops are generally made of materials that do not undesirably interfere with aqueous botulinum toxin formulations and have low extractables/leachables levels. Such coatings include, but are not limited to, polypropylene, polyethylene, parylene (e.g., N-parylene, C-parylene, and HT-parylene), cross-linked silicone, and preferably fluoropolymer coatings. Examples of suitable crosslinked silicone coatings include B2-coatings (Daikyo Seiko) or XSi^TM(BD Co., Becton Dickinson).

Fluoropolymer coatings include, but are not limited to, fluorinated ethylene-propylene copolymers (e.g., tetrafluoroethylene-hexafluoropropylene copolymer (FEP)), fluorinated ethylene-ethylene copolymers (e.g., ethylene-tetrafluoroethylene copolymer (ETFE), such as) PVA (a copolymer of Tetrafluoroethylene (TFE) and perfluoropropyl vinyl ether (PPVE)), tetrafluoroethylene-perfluoroethylene copolymer, polyvinylidene fluoride (PVDF), polyvinyl fluoride (PVF), Polytetrafluoroethylene (PTFE), and mixtures thereof. Preferably, the coating is made of ETFE, and in particular isAnd (4) coating.

The design of the plunger stop is not particularly limited and may be a nested or pocket stop. Further, the interface with the stem may be threaded to allow installation of the stem after sterilization. Alternatively, the interface with the stem may be designed using a snap-on design. Rods such as plunger stops are typically designed to withstand sterilization but are not otherwise limited in any particular manner. Typically, the stem is made of a plastic material, such as Ethylene Vinyl Acetate (EVA) copolymer or polypropylene.

The rubber stopper of the carpule bottle of the present invention may be made of the same elastomeric material as described above in connection with the plunger stopper of a glass syringe. In addition, the rubber stopper of the carpule bottle may have the same optional coating as defined above for the coating on the plunger stopper. In addition, the coating can be located on at least a portion of the rubber stopper that contacts the aqueous botulinum toxin formulation during storage and/or use.

The pre-filled syringe of the invention complies with the industry standards for extractables, such as defined by the foaming Test, the pH Test, the potassium permanganate reducing substance Test, the UV spectroscopy Test and the evaporative residue Test according to the japanese Pharmacopoeia, No.61, Test Methods for Plastic Containers (2001) (japanese Pharmacopoeia No.61, "Test Methods for Plastic Containers", 2001). In addition, pre-filled syringes and The corresponding components before and after sterilization all meet The standards of The Japanese Pharmacopoeia,14th edition, No.59, Test for Rubber Closure for aqueous infusion of Rubber stoppers ("Japanese Pharmacopoeia" 14th edition, No.59, "aqueous infusion Test of Rubber stoppers").

In another aspect, the invention relates to a kit comprising a prefilled glass container (e.g., a syringe, vial, carpule or ampoule) according to the present invention and optionally instructions for use of the prefilled glass container.

In a further aspect, the invention relates to a prefilled glass syringe according to the invention for use in therapy. In particular, prefilled glass containers (e.g., syringes, vials, carpule bottles, or ampoules) according to the present invention may be used to treat diseases or disorders caused by, or associated with, hyperactive cholinergic innervation of muscles or exocrine glands in a patient.

In the context of the present invention, if the container is not a syringe (e.g., a vial, carpule or ampoule), the contents of these "non-syringe type" containers (i.e., the aqueous botulinum toxin formulation) are typically injected into the desired target site using a suitable injection device (e.g., a syringe) in the same manner as described herein with respect to the prefilled glass syringe. The carpule bottle is inserted into the carpule injection device as known to those skilled in the art. The contents of the vial and ampoule are typically aseptically filled into a syringe and then injected into the target site using a suitable injection device (e.g., syringe) in the same manner as described herein with respect to the prefilled glass syringe.

As used herein, the term "cholinergic innervation" relates to synapses characterized by an abnormally high amount of acetylcholine released into the synaptic cleft. An "abnormally high" relates to an increase of, for example, up to 25%, up to 50% or more relative to a reference activity, which can be obtained, for example, by comparing the release to a release at a synapse of the same type but not in an overactive state, wherein a muscle dystonia can indicate an overactive state. By "up to 25%" is meant, for example, from about 1% to about 25%. Methods for making the desired measurements are known in the art.

In the present invention, diseases or conditions caused by, or associated with, hyperactive cholinergic innervation of muscles include, but are not limited to: dystonia (e.g., blepharospasm, spasmodic torticollis, limb dystonia, and task-specific dystonia (such as writer's cramp)), spasticity (e.g., post-stroke spasticity, spasticity caused by cerebral palsy), hyperextension, movement disorders (e.g., tardive dyskinesia), facial spasticity (e.g., hemifacial spasm), (juvenile) cerebral palsy (e.g., spastic cerebral palsy, dyskinetic cerebral palsy, or ataxia cerebral palsy), strabismus, pain (e.g., neuropathic pain), wound healing, tremor, tics, and migraine.

The prefilled botulinum toxin containers (e.g., syringes, vials, carpule bottles, or ampoules) of the present invention are particularly useful for treating muscular dystonia. Exemplary dystonia include, but are not limited to, dystonia selected from the following types: (1) craniofacial dystonia, including blepharospasm and oromandibular dystonia of the open or closed type, (2) cervical dystonia, including cervical lordosis, retrocervical recline, cervical roll and torticollis, (3) pharyngeal dystonia, (4) laryngeal dystonia, including spastic vocalization, (5) limb dystonia, including arm dystonia such as task-specific dystonia (e.g., writer's cramp), leg dystonia, axial dystonia, segmental dystonia, and (6) other dystonia.

The "hyperactive exocrine glands" to be treated in the context of the present invention is not particularly limited and encompasses any exocrine glands with hyperactive properties. It is therefore envisaged that the invention may be applied to treatments involving any of the glands mentioned in the following documents: sobotta, Johannes, Atlas der Anatomie desmenchen.22. afllage. Sobotta, Johannes, Atlas of human anatomy, 22 nd edition, volume 1 and volume 2, Urban & Fischer press 2005), which is incorporated herein by reference. Preferably, the hyperactive gland is an exocrine gland of the autonomic nervous system. Preferably, the botulinum toxin composition is injected into or near the hyperactive exocrine gland.

In the present invention, the overactive exocrine glands may be selected from sweat, lacrimal, salivary and mucosal glands. Alternatively, the hyperactive glands may also be or may be associated with a disease or condition selected from the following types: fleshing's syndrome, alligator tear syndrome, axillary hyperhidrosis, palmar hyperhidrosis, plantar hyperhidrosis, head and neck hyperhidrosis, body hyperhidrosis, rhinorrhea, or the relative salivation in patients with stroke, parkinson's disease, or amyotrophic lateral sclerosis. In particular, diseases or conditions caused by, or associated with, hyperactive cholinergic innervation may include drooling (salivation ) and excessive sweating (hyperhidrosis).

Administration is not limited to any particular administration regimen, mode, form, dosage and interval. As will be appreciated by those skilled in the art, the amount or dose of botulinum toxin administered will depend on the mode of application, the type of disease, the weight, age, sex and health of the patient and the target tissue selected for injection. Botulinum toxin formulations are typically administered topically, for example, by subcutaneous or intramuscular injection into or near a target tissue (e.g., muscle, skin, exocrine gland).

Furthermore, different muscles, depending on their size, often require different dosages. Suitable doses may range from 10U to 2000U, preferably from 50U to 500U, more preferably from 100U to 350U of botulinum toxin. For the treatment of exocrine glands, the dose is typically in the range of 10U to 500U, preferably 20U to 200U, more preferably 30U to 100U. Such total amounts may be administered on the same day of treatment or on a subsequent day. For example, during a first treatment session, a first portion of the dose may be administered. The remainder of the total dose may be administered during one or more treatment sessions. Further, the frequency of application is not particularly limited, and a suitable administration interval may be 3 months or less (e.g., 4 weeks to 8 weeks) or 3 months or more.

In another aspect, the present invention relates to the use of a prefilled glass container (e.g., syringe, vial, carpule bottle, or ampoule) of the present invention for cosmetic applications, such as for treating facial asymmetry and wrinkles and fine lines of skin (e.g., facial fine lines and facial wrinkles), such as upper wrinkles, platysma, glabellar lines, nasolabial folds, chin folds, marionette lines, buccal commissural lips (buccal comissure), perioral wrinkles, fishtail lines, and mandibular lines. Preferably, a prefilled botulinum toxin container (e.g., a syringe, vial, carpule bottle, or ampoule) of the present invention is used for injection into the glabellar fold, horizontal frontal line, crow's feet, perioral fold, mental fissure, chin and/or platysma.

The amount of botulinum toxin applied in cosmetic applications is typically in the range of 1U to 5U, 5U to 10U, 10U to 20U, or 20U to 50U. Such total amounts may be administered on the same day of treatment or on a subsequent day. For example, during a first treatment session, a first portion of the dose may be administered. The first portion is preferably a sub-optimal portion, i.e., a portion where wrinkles or fine lines of skin are not completely removed. The remainder of the total dose may be administered during one or more treatment sessions.

In another aspect, the invention relates to a method of treating a disease or condition caused by, or associated with, hyperactive cholinergic innervation of muscles or exocrine glands in a patient, the method comprising topically administering an effective amount of botulinum toxin to a muscle or exocrine gland of a patient with a prefilled glass container (e.g., syringe, vial, carpule or ampoule) according to the first aspect of the invention.

As used herein, the term "effective amount" refers to an amount of botulinum toxin sufficient to achieve a beneficial or desired therapeutic, cosmetic, or anesthetic result. In the context of the present invention, the term "topical administration" within the meaning of the present invention preferably refers to subcutaneous or intramuscular injection into or near the target tissue (e.g. muscle, skin, exocrine glands). As used herein, the term "patient" generally relates to a person suffering from a disease or condition caused by, or associated with, overactivity of cholinergic innervation of muscles or exocrine glands of the patient, or to a person in need of cosmetic or anesthetic treatment. As used herein, "patient" is used interchangeably with "subject" or "individual".

Administration is not limited to any particular administration regimen, mode, form, dosage and interval. As used herein, the term "to a muscle or exocrine gland" means that the botulinum toxin can be administered into or near one or more muscles or exocrine glands. Typically, the botulinum toxin is administered by local intramuscular injection. For more details regarding administration (e.g., protocols, modes, forms, dosages, and intervals) and the disease or condition to be treated, the same descriptions as those described above for prefilled glass containers (e.g., prefilled botulinum toxin syringes) for cosmetic and therapeutic applications apply.

In another aspect, the invention relates to a cosmetic treatment method for the skin, such as the treatment of skin wrinkles and facial asymmetry, comprising topically administering to a patient an effective amount of botulinum toxin by intradermal, subdermal, or subcutaneous injection with a prefilled glass syringe according to the first aspect of the invention.

This aspect is on the other hand closely related to the other aspects of the invention described above, and therefore all the explanations, definitions and explanations given above in relation to these other aspects apply equally, unless otherwise stated.

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

Examples

The following examples demonstrate the superior long term stability of aqueous botulinum toxin formulations in different prefilled syringe systems (hereinafter "configurations") according to the present invention.

The results obtained for the different syringe configurations surprisingly show that, contrary to expectations and general belief in the art, aqueous botulinum toxin formulations stored in prefilled syringe systems are stable over long periods of time (e.g., up to 18 months) at standard refrigerator temperatures (2 ℃ to 8 ℃), and remain stable even when stored at elevated temperatures of 25 ℃ for about 9 months. Furthermore, extrapolation of the measured stability data indicates that the prefilled botulinum toxin syringe allows for a shelf life of about 24 months or even longer at 2 ℃ to 8 ℃.

Overall, the results obtained indicate that botulinum toxin can be conveniently used by pre-filled syringes. This is an important contribution to the management of therapeutic and cosmetic indications for a wide variety of botulinum toxin treatments because botulinum toxin prefilled syringes are safer and more convenient for clinicians and patients to use, and provide flexibility and excellent shelf life compared to conventional lyophilized botulinum toxin products.

Materials and methods

The liquid botulinum toxin formulation for the following examples was prepared by dissolving 1.0mg human albumin, 4.7mg sucrose and botulinum toxin type A in 0.9% saline to a concentration of 50U/mL.

The botulinum toxin solution is then loaded into a glass syringe barrel pre-assembled with a screw-type closure comprising a screw-type fitting and an end cap which, when assembled, contacts the opening of the distal syringe tip to seal the syringe barrel. To close the proximal opening, the plunger stopper is inserted into the proximal portion of the syringe barrel. The resulting prefilled syringes are then stored at temperatures of about 5 ℃, 25 ℃ and 30 ℃.

The stability of the botulinum toxin solution was first determined and then the remaining toxin potency, pH and microscopically visible particle levels were measured after one month, three months, six months and nine months of storage.

Potency was determined using the lateralization assay. The assay was performed using mouse neuromuscular preparations maintained in an organ bath containing 4mL of medium. The muscle was connected to a force transducer and electrically stimulated through the phrenic nerve, resulting in an isometric force that remained constant for more than 180 minutes if no toxin was added. After the toxin was introduced into the organ bath, the amplitude of contraction of the neurostimulated muscle gradually decreased. The magnitude of the contraction of the diaphragm is monitored over time. As a readout, the time to half the initial contractile force was determined and referred to as the paralysis time. The duration of paralysis is proportional to the amount of active toxin added to the preparation.

The pH was measured using a pH meter (Lab 870, SCHOTT Instruments ltd (SCHOTT Instruments) according to the united states pharmacopeia standardized test method USP <791>, which outlines the pH measurement method for a large number of pharmaceutical products.

Particle measurements were performed using microfluidic imaging. Microfluidic imaging measurements were performed using a DPA-5200 particle analyzer system (ProteinSimple, Santa Clara, Calif.) equipped with a silane-coated, high-resolution 100 μm flow cell. Samples were analyzed without dilution. The MFI View System Software (MVSS) version 2-R2-6.1.20.1915 was used to perform the measurements, while the MFI View Analysis Suite (MVAS) Software version 1.3.0.1007 was used to analyze the samples.

Four different pre-filled syringe systems (or "syringe configurations") that differ from one another by syringe barrel, end cap and/or plunger stop were examined and summarized in table 1.

TABLE 1 syringe configurations studied A, B, G and H

Gres marine group (Gerresheimer)

BD Co (Becton, Dickinson and Company)

Use after 3 ═365. Polydimethylsiloxane NF emulsion

TELC (tamper resistant screw closure)

5 ═ PRTC (Plastic rigid end cap)

6＝SCF(Sterile、CleaningAnd ready to be filled)

BSCF (sterile, clean and ready-to-fill pouch; use of a pouch (BSCF) stopper)

Results

The results of the stability measurements for configurations A, B, G and H are shown in table 2 below.

TABLE 2 stability in terms of efficacy

Initial absolute toxin activity units ranged from 51U to 56U.

The stability data described above are shown graphically in FIG. 1 (stability at 2 ℃ to 8 ℃), FIG. 2 (stability at 25 ℃) and FIG. 3 (stability at 30 ℃), together with the storage time extrapolated to 24 months. As can be seen from table 2 and fig. 1 to 3, the maximum measured losses of biological activity were only 12%, 20% and 20%, respectively, for temperature conditions of 2 ℃ to 8 ℃ (up to 18 months), 25 ℃ (up to 12 months) and 30 ℃ (up to 6 months). Extrapolation showed less than 5% loss of bioactivity at 2 ℃ to 8 ℃ for all constructs A, B, G and H, and less than 10% loss of bioactivity at 25 ℃ for construct B, after 24 months of storage time.

Furthermore, pH measurements show that the pH value remains very stable over a period of up to 18 months. No trend towards higher or lower values was observed and all measured pH values remained within ± 0.5 of the initial pH (see table 3).

TABLE 3 stability in pH

N.d. ═ undetermined

Furthermore, particle size measurements by microfluidic imaging showed no significant increase in particle count (see table 4).

TABLE 4 stability in terms of particle count visible under microscope

As can be seen from Table 4, the particle count remained well below 1000/mL, in most cases even below 200/mL. Also, particle measurements by means of resonance mass measurement method (using ARCHIMEDES particle measurement system (ARCHIMEDESPARTICLE METHOD SYSTEM); Affinity Biosensors, Santa Barbara, Calif.) and nanoparticle tracking analysis (using NanoSight LM20 system; NanoSight LM20 system; NanoSight, Amesbury, UK) of Santa Barbara, Calif.) showed no relevant particle counts.

Taken together, the above results show that the liquid botulinum toxin formulation in the prefilled syringe is stable over time at temperatures of 2 ℃ to 8 ℃ and even at ambient temperatures (e.g., 25 ℃ to 30 ℃). This finding was unexpected in view of the fact that botulinum toxin is inherently unstable, particularly at low toxin concentrations. In particular, botulinum toxin is known to be highly thermolabile and highly unstable at alkaline pH. Thus, the discovery that botulinum toxin in aqueous solution is highly stable when stored in a pre-filled syringe is highly surprising in view of the labile nature of botulinum toxin.

Thus, the botulinum toxin prefilled syringe according to the present invention provides significant advantages over other means of delivering botulinum toxin, including improved convenience and ease of handling, reduced medication errors, improved dosage accuracy, minimized risk of contamination, improved sterility assurance, and improved safety of administration.

Claims (15)

1. A prefilled glass container comprising an aqueous botulinum toxin formulation, wherein the toxin activity decreases by no more than 25% relative to an initial toxin activity when stored in the prefilled container for 12 months at 5 ℃ or 12 months at 25 ℃ or 6 months at 30 ℃.

2. The prefilled glass container of claim 1, wherein the number of particles that are visible under a microscope equal to or greater than 10 μ ι η is less than 1000/mL during storage at 2 ℃ to 30 ℃ for 6 months to 24 months.

3. The prefilled glass container of claim 1 or 2, wherein the pH increases or decreases by no more than 10% relative to an initial pH after the prefilled container is stored at 5 ℃ or 25 ℃ or 30 ℃ for 6 months to 12 months, or wherein the pH of the aqueous botulinum toxin formulation is maintained within a range of 6.0 to 7.5 during storage, or both.

4. The prefilled glass container of any one of claims 1 to 3, wherein the botulinum toxin is present in the aqueous formulation at a concentration of from 10U/mL to 1000U/mL.

5. The prefilled glass container of any one of claims 1 to 4, wherein the aqueous botulinum toxin formulation in the prefilled container is free of a buffer.

6. The prefilled glass container of any one of claims 1 to 5, wherein the container is (i) a syringe, (ii) a vial, (iii) a carpule bottle, or (iv) an ampoule.

7. The prefilled glass container in the form of a prefilled glass syringe of claim 6, comprising:

(a) a syringe barrel made of glass comprising a proximal end and a distal end, and a generally cylindrical wall extending between the proximal end and the distal end and defining a syringe barrel lumen, the syringe barrel having a distally projecting tip through which a fluid passageway extends and communicates with the syringe barrel lumen, wherein the generally cylindrical wall has an inner surface, the inner surface optionally coated with a barrier layer,

(b) a capping device having an outlet junction sealingly engaging and closing the distally open outlet end of the syringe, wherein the outlet junction is made of an elastomeric material and optionally has a coating on its surface, and

(c) a plunger rod assembly extending into the proximal end of the syringe barrel and comprising a plunger stopper in sliding fluid-tight engagement with the cylindrical wall of the syringe barrel lumen, wherein the plunger stopper is made of an elastomeric material and optionally has a coating on at least a portion of the plunger stopper that contacts the aqueous botulinum toxin formulation during storage and/or injection.

8. The prefilled glass container in the form of a prefilled glass syringe of claim 6 or 7, wherein the elastomeric material of the outlet engagement portion and/or the plunger stopper IS selected from isoprene rubber (IS), Butadiene Rubber (BR), butyl rubber, halogenated butyl rubber, styrene-butadiene rubber and mixtures thereof, or wherein the optional coating on the outlet engagement portion and/or the plunger stopper IS a crosslinked silicone coating or a fluoropolymer coating, or wherein the elastomeric material of the outlet engagement portion and/or the plunger stopper IS selected from isoprene rubber (IS), Butadiene Rubber (BR), butyl rubber, halogenated butyl rubber, styrene-butadiene rubber and mixtures thereof, and the optional coating on the outlet engagement portion and/or the plunger stopper IS a crosslinked silicone coating or a fluorinated silicone coating A fluoropolymer coating.

9. The prefilled glass container in the form of a prefilled glass syringe of any one of claims 6 to 8, wherein the barrier layer of the syringe barrel is a silicone-free layer or a silicone layer.

10. A kit comprising the prefilled glass container of any one of claims 1 to 9, and optionally instructions for use of the prefilled glass container.

11. A prefilled glass container for use in therapy according to any one of claims 1 to 9.

12. The prefilled glass container of any one of claims 1 to 9 for use in treating a disease or condition caused by or associated with hyperactive cholinergic innervation of muscles or exocrine glands in a patient, the disease or condition comprising dystonia, spasticity, hyperextensibility, movement disorders, facial spasm, strabismus, tremor, tics, migraine, sialorrhea and hyperhidrosis.

13. Use of a prefilled glass container according to any one of claims 1 to 9 in a cosmetic application, such as for treating said skin wrinkles and facial asymmetries.

14. A method of treating a disease or condition caused by, or associated with, hyperactive cholinergic innervation of muscles or exocrine glands in a patient, the method comprising locally administering an effective amount of botulinum toxin to a muscle or exocrine gland of the patient by injection using the prefilled glass container according to any one of claims 1 to 9.

15. A cosmetic treatment method for the skin, such as the treatment of skin wrinkles and facial asymmetries, comprising the local administration of an effective amount of botulinum toxin to a patient by intradermal, subdermal or subcutaneous injection using a prefilled glass container according to any one of claims 1 to 9.