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

Proteinaceous active ingredients are often of a labile nature and inherently instable. This leads to loss of biological activity during production, reconstitution and/or storage of protein-containing pharmaceutical compositions. These problems observed with proteins may be due to chemical instability, resulting in bond formation or cleavage (e.g., hydrolysis, oxidation, racemization, β-elimination and disulfide exchange), and/or due to physical instability of the second or higher-order structure of proteins without covalent bond-breaking modification (e.g., denaturation, adsorption to surfaces, and non-covalent self-aggregation).

Since degradation reactions are generally fastest in aqueous solutions and slowest in solid dosage forms, protein active ingredients are often formulated as lyophilized (i.e. freeze-dried) products. However, the lyophilized products have generally to be reconstituted with a pharmaceutically acceptable liquid (e.g., saline) prior to use. Therefore, lyophilized pharmaceutical products are considered less convenient than other dosage forms. Further, lyophilized products are usually more expensive and time-consuming to manufacture. Moreover, mismanagement can occur during the reconstitution process resulting in inaccurate dosing or sterility issues. All these disadvantageous can be overcome by the use of prefilled syringes. Therefore, prefilled syringes have been become increasingly popular as drug delivery devices.

However, if proteins are used as active ingredients, the limited stability of proteins rendered it often impossible for formulation scientists to use a prefilled syringe format. This applies especially to very dilute aqueous solutions of botulinum toxin (botulinum neurotoxin, BoNT). Such BoNT solutions are used in the treatment of a wide range of debilitating neuromuscular diseases (e.g., cervical dystonia, blepharospasm, spasticity, and hyperhidrosis) and in aesthetic medicine (e.g., treatment of facial wrinkles). There are seven homologous serotypes (A-G) of botulinum toxin, which are produced by different Clostridium spp. , in particular C. botulinum, in the form of a complex consisting of a neurotoxic polypeptide and other (non-toxic) clostridial proteins (i.e. different hemagglutinins and a nontoxic, non-hemagglutinating protein). The neurotoxic polypeptide has a molecular weight of about <NUM> kDa and is activated by selective proteolytic cleavage to yield the active two-chain form consisting of a heavy chain (HC; includes the translocation domain and receptor-binding domain) and a light chain (LC; includes the catalytic domain) linked by a disulfide bond and non-covalent interactions.

Botulinum toxins are inherently instable and, in particular, are known to be highly unstable at alkaline pH and heat-labile. Additionally, it is known that dilution of the isolated toxin complex from milligram quantities to the much lower toxin concentrations used in solutions for injection (in the nanograms per milliliter range) presents significant difficulties because of the rapid loss of specific activity upon such great dilution.

Therefore, commercial preparations of botulinum toxin often come as vacuum-dried or lyophilized material. Examples include, for example, Botox® (onabotulinumtoxinA; Allergan, Inc. ) and Dysport® (abobotulinumtoxinA; Ipsen Ltd. ), which both contain the C. botulinum toxin complex of type A. Another example is Xeomin® (incobotulinumtoxin; Merz Pharma GmbH & Co. KGaA), which contains the pure neurotoxic component of serotype A (i.e. the neurotoxic polypeptide of a molecular weight of about <NUM> kDa) and is devoid of any other proteins of the Clostridium botulinum toxin complex (i.e. the different hemagglutinins and the nontoxic, non-hemagglutinating protein).

However, the lyophilized toxin products have a number of drawbacks including the need for reconstitution prior to use and concomitant sterility issues. In addition, the reconstituted toxin solution is often not entirely used in the clinical practice because not every patient and indication requires the same dosage. The unused amount of the reconstituted toxin solution can be stored at lower temperatures, but only for a short period of time. For example, after dilution with normal saline prior to use, Botox® and Dysport® are recommended to be used within <NUM> hours and <NUM> hours, respectively. Likewise, the package leaflet of Xeomin® specifies that after storage for more than <NUM> hours, the reconstituted Xeomin® solution shall no longer be used than for <NUM> hours and is then to be discarded.

To increase toxin stability, stabilizing proteins such as human serum albumin (I) are often added in the art. Other stabilizing strategies involve the use of non-proteinaceous stabilizing agents, for example surfactants, polyvinylpyrrolidone (PVP), disaccharides, polyols and the like. In addition, it was disclosed in <CIT> that a liquid formulation of highly concentrated botulinum toxin type B (about <NUM> U/ml) is stable for up to <NUM> months when stored in glass vials at <NUM>. However, this prolonged stability requires buffering the pH of the formulation down to an acidic pH of between <NUM> and <NUM>, which causes pain upon injection. Other known approaches to increase toxin stability rely on the addition of various non-protein excipients which, however, are unsuitable or undesirable for human use (see, e.g., <CIT>, <CIT>, and <CIT>).

<CIT> discloses improved vitamin supplement compositions formulated for administration to patients, particularly improved vitamin compositions formulated for use in cosmetic or therapeutic applications. The compositions may be used to slow aging process and promote wellness including treating a vitamin deficiency, skin conditions, improving skin appearance, wound healing and scar prevention and hair loss. Botulinum toxin may be present in the composition as an additional bioactive agent. Furthermore, <CIT> discloses a prefilled syringe having a barrel with a lubricated interior portion and a plunger tip that slides along it. The prefilled syringe has a surface, a primer coating or layer of SiOx, SiOxCy or SiNxCy applied to the surface, and a deposit of lubricant applied to the primer coating or layer. The primer coating or layer of SiOx, SiOxCy or SiNxCy is applied by chemical vapor deposition of a polysiloxane or polysilazane precursor in the presence of oxygen. A first deposit of lubricant is applied to the primer coating or layer. The primer coating or layer improves the adhesion or wetting of the lubricant on the surface to be lubricated. The prefilled syringe may contain a fluid that can be a botulinum toxin such as abobotulinumtoxinA.

<CIT> further discloses a botulinum toxin pharmaceutical composition comprising a botulinum toxin and a non-pasteurized recombinant albumin. <CIT> discloses medical components, such as a syringe, tube or medical collection device, having coated surfaces exhibiting low friction and/or low gas/liquid permeability. <CIT> discloses a botulinum toxin pharmaceutical composition free of blood derived albumin, comprising a botulinum toxin, sodium chloride or water and a polysaccharide such as a hydroxyethyl starch and/or an amino acid, the pharmaceutical composition being suitable for therapeutic administration to a human patient. <CIT> discloses a needle injector includes a syringe body along with a needle disposed in fluid communication with said syringe body. A plunger including a plunger head is disposed within said syringe body and a saline solution is disposed in the syringe body. A dried medicament is disposed on an inside surface of the syringe body in a position enabling movement of the plunger head in the syringe body to cause reconstitution of the medicament saline solution and ejection of the reconstituted medicament through the needle.

Thus, there is still no injectable botulinum toxin presentation available which is not only stable over a long period to provide a sufficiently long shelf life, but is also convenient and easy to use, reduces medication errors, and minimizes the risk of contamination.

In view of the above, the objective of the present invention is to provide a medical dosage form for the administration of botulinum toxin, which has a long shelf life and is convenient, safe and easy to use.

The above object is solved by the provision of a botulinum toxin prefilled syringe which is characterized by a superior long-term stability of the liquid botulinum toxin formulation.

In a first aspect, the present invention provides a prefilled glass syringe comprising an aqueous botulinum toxin formulation according to claim <NUM>, which is characterized in that the toxin activity is not reduced by more than <NUM>%, preferably by not more than <NUM>%, relative to the initial toxin activity, upon storage of the prefilled syringe for (a) <NUM> months at <NUM> or (b) <NUM> months at <NUM>.

The stability of the aqueous botulinum toxin formulation in the prefilled syringe in terms of the count of subvisible particles equal to or greater than <NUM> is also excellent and generally below <NUM>/ml during storage for <NUM> to <NUM> months (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM> months) at <NUM>-<NUM> (e.g., at <NUM>, <NUM> or <NUM>). Furthermore, the aqueous botulinum toxin formulation exhibits an excellent pH stability as indicated by a pH value that is generally not increased or decreased by more than <NUM>%, relative to the initial pH value, during storage of the prefilled syringe for <NUM> to <NUM> months (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM> months) at <NUM>-<NUM> (e.g., at <NUM>, <NUM> or <NUM>).

In another aspect, the present invention provides a kit comprising a prefilled glass syringe according to the first aspect of the invention and, optionally, instructions for use of said prefilled glass syringe.

In still another aspect, the present invention relates to the use of the prefilled glass syringe according to the first aspect of the invention for use in non-surgical cosmetic applications, such as for treating wrinkles of the skin and facial asymmetries, e.g. glabellar frown line, crow's feet, upper facial rhytides and platysma bands.

In a still further aspect, the present invention relates to a non-surgical cosmetic method for the treatment of the skin, such as for treating wrinkles of the skin and facial asymmetries, the method comprising locally administering an effective amount of botulinum toxin to a patient by intradermal, subdermal or subcutaneous injection using the prefilled glass syringe according to the first aspect of the present invention.

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

The present invention is based on the surprising finding that a liquid botulinum toxin formulation in a glass syringe is stable after storage for a prolonged period of time at reduced temperature (e.g., <NUM>-<NUM>) and even at ambient temperature (e.g., <NUM>-<NUM>, in particular <NUM>). The botulinum toxin prefilled syringe of the present invention therefore advantageously exhibits an extended shelf life.

Moreover, the high long-term stability provides tolerance against interruptions of the cool chain and may facilitate the approval procedure and/or the commercialization in all climate zones, including countries with hot climate. Furthermore, the prefilled glass syringe presents several additional advantages in comparison to other administration forms, such as easy and convenient use, reduced risk of medication errors, high dosing accuracy, low risk of contamination, improved sterility assurance, and/or high safety in administration.

Prefilled syringes have two openings that are sealed to prevent leakage of the contents (e.g., aqueous formulations). In case of a prefilled syringe, the proximal end is sealed by a plunger stopper and the distal end is sealed by a capping device, as explained in detail herein below.

In a first aspect, the present invention relates to a prefilled glass syringe comprising botulinum toxin in an aqueous formulation, wherein the toxin activity is not reduced by more than <NUM>%, relative to the initial toxin activity, upon storage of the prefilled syringe for (a) <NUM> months at standard refrigerator temperatures (i.e. <NUM>-<NUM>, such as <NUM>), or (b) <NUM> months at <NUM>. Preferably, the toxin activity is not reduced by more than <NUM>% or <NUM>%, relative to the initial toxin activity, upon storage of the prefilled syringe for (a) <NUM> months at <NUM>-<NUM> (e.g., <NUM>), or (b) <NUM> months at <NUM>. More preferably, the toxin activity is not reduced by more than <NUM>% or <NUM>%, relative to the initial toxin activity, upon storage of the prefilled syringe for (a) <NUM> months at <NUM>-<NUM> (e.g., <NUM>), (b) <NUM> months at <NUM>. Particularly preferable, the toxin activity is not reduced by more than <NUM>%, relative to the initial toxin activity, upon storage of the prefilled syringe for (a) <NUM> to <NUM> months at <NUM>-<NUM> (e.g., <NUM>) or (b) <NUM> to <NUM> months at <NUM>. Especially preferable, the toxin activity is not reduced by more than <NUM>%, relative to the initial toxin activity, upon storage of the prefilled syringe for (a) <NUM> to <NUM> months at <NUM>-<NUM> (e.g., <NUM>) or (b) <NUM> to <NUM> months at <NUM>.

Surprisingly, the aqueous botulinum toxin formulation in the prefilled syringe is also stable for even longer storage times of up to <NUM> months or even longer. For example, upon storage for up to <NUM> months (e.g., <NUM>, <NUM> or <NUM> months) at <NUM>-<NUM> (e.g., <NUM>) or <NUM>, the toxin activity is preferably not reduced by more than <NUM>% or <NUM>%, more preferably by no more than <NUM>%, in particular by no more than <NUM>%, particularly preferable by no more than <NUM>%, and most preferable by no more than <NUM>%, relative to the initial toxin activity.

In particular, the toxin activity is preferably not reduced by more than <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>%, relative to the initial toxin activity, upon storage of the prefilled syringe for <NUM> months at <NUM>-<NUM>°. Upon storage of the prefilled syringe at <NUM>-<NUM>° for <NUM> months, the toxin activity is preferably not reduced by more than <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>%, relative to the initial toxin activity. Furthermore, the toxin activity is preferably not reduced by more than <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>%, relative to the initial toxin activity, upon storage of the prefilled syringe for <NUM> months at <NUM>. Upon storage of the prefilled syringe at <NUM> for <NUM> months, the toxin activity is preferably not reduced by more than <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>%, relative to the initial toxin activity.

Within the present invention, the term "toxin activity" is intended to refer to the biological activity of the botulinum toxin. "Biological activity" may refer to (a) receptor binding, (b) internalization, (c) translocation across the endosomal membrane into the cytosol, and/or (d) endoproteolytic cleavage of proteins involved in synaptic vesicle membrane fusion. For example, any LC (light chain) domain, which shows proteolytic activity of more than <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% and up to <NUM>% of the corresponding wild-type LC domain in a SNAP-<NUM> assay may be considered "biological active" or "to exhibit proteolytic activity" within the scope of this invention. Furthermore, any HC (heavy chain) domain that is capable of binding to a cellular HC domain receptor, in particular to its native HC domain receptor, and is capable of translocating an LC domain attached to it, is considered "biologically active".

The biological activity is expressed in Mouse Units (MU). As used herein, <NUM> MU is the amount of neurotoxic component, which kills <NUM>% of a specified mouse population after intraperitoneal injection, i.e. the mouse i. LD<NUM>, as measured in accordance with the method of Schantz and Kauter (<NPL>). The terms "MU" and "Unit" or "U" are used interchangeable herein.

Suitable assays for assessing the biological activity include the mouse hemidiaphragm assay (MHA) described by <NPL>), the hemidiaphragm assay (HDA) according to <NPL>), the mouse diaphragm assay (MDA) according to <NPL>), the SNAP-<NUM> protease assay (e.g., the "GFP-SNAP25 fluorescence release assay" described in <CIT> or the "improved SNAP25 endopeptidase immuno-assay" described in <NPL>), the electrochemiluminescence (ECL) sandwich ELISA described in <CIT>, and cell-based assays as those described in <CIT>, <CIT>, <CIT> and, in particular, <CIT>.

As used herein, the term "initial toxin activity" or "initial potency" generally refers to the activity of the botulinum toxin at the beginning of the storage period, i.e. after manufacture of the final, sterilized botulinum toxin prefilled syringe, in particular directly after manufacture or within one or two days after manufacture. Further, the term "upon storage", as used herein is intended to mean after storage for a certain time period. In addition, the term "during storage" generally means over the course of the entire storage period.

Furthermore, the aqueous botulinum toxin formulation is highly stable in terms of the subvisible particle count. A "subvisible particle" within the meaning of the present invention is typically a particle with a diameter below <NUM>. Specifically, the count (or number) of particles equal to or greater than <NUM> in the aqueous botulinum toxin formulation is typically below <NUM>/ml, preferably below <NUM>/ml and more preferably below <NUM>/ml during storage for <NUM> to <NUM> months (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM> months) at <NUM>-<NUM> (e.g., at <NUM>, <NUM> or <NUM>).

Particle measurements may be conducted by different methods, such as Micro-Flow Imaging (MFI), Resonant Mass Measurement (RMM), and Nanoparticle Tracking Analysis (NTA). The particle measurements usually follow USP <<NUM>>. Within the context of the present invention, the Micro-Flow Imaging measurement is preferably used. This measurement method may, for example, be conducted using a DPA-<NUM> particle analyzer system (ProteinSimple, Santa Clara, CA, USA) equipped with a silane coated high-resolution <NUM> flow cell. Generally, the samples are analyzed undiluted.

Alternatively, Resonant Mass Measurements (RMM) may be employed to determine the number of particles using, for example, the ARCHIMEDES Particle Metrology System (Affinity Biosensors, Santa Barbara, CA, USA) equipped with a microsensor (size range <NUM>-<NUM>) calibrated with <NUM> polystyrene standards. All samples are typically analyzed without dilution. The results may be analyzed using the ParticleLab software (v1. <NUM>) with a size bin step of <NUM>. As another alternative for determining the particle count, Nanoparticle Tracking Analysis (NTA) may be used, for example, using a NanoSight LM20 system (NanoSight, Amesbury, UK). The samples are typically measured undiluted. Movements of the particles in the samples may be recorded as videos for <NUM> seconds at ambient temperature and analyzed using suitable software (e.g., the NTA <NUM> Software).

Moreover, the aqueous botulinum toxin formulation shows high pH stability in that the pH value is essentially stable during storage of the prefilled syringe. Preferably, the pH value is not increased or decreased by more than <NUM>%, <NUM>% or <NUM>%, relative to the initial pH value, upon storage of the prefilled syringe for <NUM> to <NUM> months (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM> months) at <NUM>-<NUM> (e.g., at <NUM>, <NUM> or <NUM>), for example for <NUM> months at <NUM> or for <NUM> months at <NUM>. The pH may be measured in accordance with the US Pharmacopeia standardized test method USP <<NUM>>, which outlines pH measurements for a multitude of pharmaceutical product. Any suitable pH meter may be used, for example the Lab <NUM> pH meter of Schott Instruments.

As used herein, the term "prefilled syringe" refers to a syringe which is filled with a drug composition (i.e. the aqueous botulinum toxin formulation) prior to distribution to the end user who will administer the drug to the patient. A prefilled syringe includes a drug containment container forming part of a syringe body (i.e. a syringe barrel), a plunger to seal the proximal opening of the syringe and for expelling the drug, and a sealing device (e.g., a tip cap or a needle shield) on the outlet end of the syringe (e.g., the open end of the syringe tip or of a pre-mounted needle (cannula)) to seal the distal outlet opening. The term "prefilled glass syringe", as used herein, refers to a prefilled syringe, of which at least the barrel is made of glass.

Within the present invention, the prefilled syringe is preferably a Luer slip or Luer lock syringe equipped with a tip cap (if no needle is pre-mounted) or a needle shield (if the needle is pre-mounted). Within the meaning of the present invention, a "luer slip syringe" is a syringe that allows a needle to be pushed on to the end of the tip, whereas a "Luer-Lock syringe" is a syringe that allows a needle to be twisted onto the tip and then locked in place. This provides a secure connection and prevents accidental removal of the needles of the injection of fluids.

The prefilled syringe according to the present invention is generally sterilized and, thus, ready-to-use. Further, the prefilled syringe described herein is usually intended for single use and intended to be disposable. Prior to sterilization, the syringe, more specifically the inner surface of the glass syringe barrel, is typically coated with a lubricant to ease gliding of the plunger stopper and extruding of the syringe content. Suitable methods for sterilization include, but are not limited to, gamma radiation, ethylene oxide (ETO) treatment and moist heat (e.g., autoclaving).

In accordance with the present invention, the aqueous botulinum toxin formulation in the prefilled syringe contains the botulinum toxin at a concentration of, for example, <NUM> U/ml to <NUM> U/ml, <NUM> U/ml to <NUM> U/ml. Preferably, the botulinum toxin is present at a concentration of about <NUM> U/ml to <NUM> U/ml, more preferably about <NUM> U/ml to <NUM> U/ml, and most preferably about <NUM> U/ml to <NUM> U/ml (e.g., <NUM> U/ml, <NUM> U/ml or <NUM> U/ml).

The term "botulinum toxin", as used herein, refers to botulinum toxin of serotype A.

Furthermore, the term "botulinum toxin", as used herein, is intended to include both the botulinum toxin complex (the "toxin complex") and the "neurotoxic component" of a botulinum toxin complex. As used herein, the term "botulinum toxin complex" or "toxin complex" refers to a high molecular weight complex comprising the neurotoxic component of approximately <NUM> kDa and, in addition, non-toxic proteins of Clostridium botulinum, including hemagglutinin and non-hemagglutinin proteins. The botulinum toxin serotype A complex is commercially available, for example, as Botox® (Allergan, Inc. ) or as Dysport® (Ipsen, Ltd.

The term "neurotoxic component", as used herein, relates to the neurotoxic polypeptide of the toxin complex (the "<NUM> kDa" polypeptide) without any associated non-toxic proteins. The pure neurotoxic component is, for example, commercially available under the trade names Xeomin® and Bocouture® (Merz Pharmaceuticals GmbH). Within the present invention, the botulinum toxin is preferably the neurotoxic component of a botulinum toxin complex of serotype A. In other words, the aqueous botulinum toxin formulation contained in the prefilled glass syringe preferably contains (only) said neurotoxic component and is devoid of any other protein of the Clostridium botulinum toxin complex.

It is also contemplated that the present invention encompasses isoforms, homologs, orthologs and paralogs of botulinum toxin that show at least <NUM>% sequence identity to wild-type botulinum toxin, e.g. wild-type botulinum toxin A or the neurotoxic component of botulinum toxin of serotype A1 deposited with the GenBank database under the accession number AAA23262. The sequence identity can be calculated by any algorithm suitable to yield reliable results, for example by using the FASTA algorithm (<NPL>). Sequence identity may be calculated by comparing two polypeptides or two domains such as two LC domains or fragments thereof.

Modified and recombinant botulinum toxins are also within the scope of the present invention. With respect to suitable mutants, reference is made to <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT> and <CIT>. Furthermore, the present invention also refers to botulinum toxins, which are chemically modified, e.g. by pegylation, glycosylation, sulfatation, phosphorylation or any other modification, in particular of one or more surface or solvent exposed amino acid(s). The modified, recombinant, isoforms, homologs, orthologs, paralogs and mutants suitable for use in the present invention are biologically active, i.e. able to translocate into neurons and cleave proteins of the SNARE complex (e.g., VAMP/syntaxin, synaptobrevin, and SNAP-<NUM>) to exert its acetylcholine inhibitory effects, e.g., its muscle paralyzing effects.

Within the context of the present invention, the aqueous botulinum toxin formulation comprises water, botulinum toxin of serotype A, sodium chloride, sucrose, and human serum albumin. It may comprise various other pharmaceutically acceptable substances, for example, sugars (e.g., glucose, fructose, galactose, trehalose, and maltose), carbohydrate polymers (e.g., hyaluronic acid and polyvinylpyrrolidone (PVP)), polyols (e.g. glycerol and sugar alcohols like mannitol, inositol, lactitol, isomalt, xylitol, erythritol, sorbitol), amino acids, vitamins (e.g. vitamin C), zinc, magnesium, anesthetic agents (e.g., local anesthetic agents like lidocaine), surfactants, tonicity modifiers, and the like. The term "pharmaceutically acceptable", as used herein, refers to those compounds or substances which are suitable for contact with the tissues of mammals, especially humans.

The term "comprise", as used herein, is intended to encompass both the open-ended term "include" and the closed term "consist (of)". The term "made of", as used herein, is intended to broadly relate to "produced of/from", in particular mainly produced from, and generally means "comprising" (indicating that other substances or materials may be included in some amounts). It may also mean "consisting of".

The pH of the aqueous botulinum toxin formulation in the prefilled syringe is between <NUM> to <NUM>. The pH may also be from <NUM> to <NUM>, from <NUM> to <NUM>, and from <NUM> to <NUM> during storage. A pH within the range of <NUM> to <NUM> is advantageous in that injections of such neutral or only slightly acidic solutions are much less painful upon injection than acidic solutions.

The term "aqueous formulation" or "aqueous botulinum toxin formulation", as used herein, is not particularly limited and is preferably an aqueous solution.

Preferably, the aqueous botulinum toxin formulation does not contain a buffer substance like a phosphate buffer, a phosphate-citrate buffer, a lactate buffer, an acetate buffer and the like. The term "buffer" as used herein denotes a pharmaceutically acceptable excipient, which stabilizes the pH of a pharmaceutical preparation. Furthermore, the aqueous botulinum toxin formulation may be free of amino acids (e.g., methionine) and/or surfactants (e.g., polysorbates such as polysorbate <NUM>).

A preferred aqueous botulinum formulation for use herein essentially consists of water, botulinum toxin (e.g. the neurotoxic component of botulinum toxin type A), sodium chloride, sucrose, and albumin (e.g., human serum albumin; HSA). The concentration of the mentioned components may be in the following ranges: <NUM> to <NUM> U/ml or <NUM> to <NUM> U/ml (botulinum toxin), <NUM>% to <NUM>% w/v or <NUM>% to <NUM>% w/v (sodium chloride), <NUM>% to <NUM>% w/v or <NUM>% to <NUM>% w/v (sucrose), <NUM>% to <NUM>% w/v, <NUM>% to <NUM>% w/v, <NUM>% to <NUM>% w/v or <NUM>% to <NUM>% w/v (HSA). A further preferred botulinum toxin formulation for use herein is a Xeomin® solution, e.g., reconstituted with physiological saline (<NUM>% sodium chloride), including <NUM> to <NUM> U/ml of the neurotoxic component of botulinum toxin type A.

The term "essentially consists of", as used herein is intended to mean that substances other than those indicated are only contained in trace amounts, e.g. unavoidable impurities contained in the components used for formulating the aqueous botulinum toxin formulation, and low amounts of impurities included in the isolated botulinum toxin (e.g., the neurotoxic component of botulinum toxin type A) as a result of the purification procedure (e.g., very low residual amounts of buffers, chelating agents and the like).

In accordance with the present invention, the configuration of the prefilled syringe is not particularly limited and commonly comprises a fluid-receiving barrel that, after filling, is removably capped by a capping device to sealingly close the distal end of the syringe (e.g., by a "cap" (or "tip cap") that is removed and replaced by a needle prior to use, or a sealing means like a needle shield in case of a syringe with a removable or permanent needle), and is closed at the proximal end by its plunger or any other means that is in fluid-tight engagement with the inner wall of the barrel. To use the prefilled syringe, the tip cap, needle shield or other type of capping device are removed, optionally a needle is attached (if not already present), and the plunger tip or piston is advanced in the barrel to inject the contents of the barrel into a patient.

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

Generally, primary container closure systems, including components such as syringe barrels, sealing devices (e.g., tip caps or needle shields) and plungers, have the potential to interact with the drug formulation in the prefilled syringe. This may lead to the release of extractables/leachables from the syringe materials that contact the aqueous botulinum toxin formulation. The extractables/leachables have the potential to contaminate the aqueous botulinum toxin formulation and to impair the stability or activity of the botulinum toxin. Therefore, the materials of the prefilled syringe are generally selected to minimize or limit the amount of extractables and leachables.

As used herein, the terms "extractable(s)" and "leachable(s)" refer to chemical species that can be released from a container or component of material of the prefilled syringe and/or has migrated from syringe materials into the aqueous botulinum toxin formulation under normal conditions of use or storage. Methods for identification of extractables/leachables are known in the art and based on recommended industry practices and International Conference for Harmonisation (ICH) guidelines (see FDA guidance, Container Closure Systems for Packaging Human Drugs and Biologics), and include, e.g., Liquid Chromatography/Mass Spectrophotometry (LC/MS), Gas Chromatography Spectroscopy/Mass Spectrophotometry (GC/MS), Inductively Coupled Plasma (ICP) and Infrared (IR).

The inside surface of the glass barrel is coated with a lubricant layer (herein also referred to, and interchangeably used with, the term "barrier layer" or "barrier coating"). The lubricant layer should not only provide high lubricity, enabling the plunger to easily glide through the barrel, but also be compatible with the aqueous botulinum toxin formulation and protect its shelf life. Within the context of the present invention, the lubricant layer is a silicone lubricant layer.

A suitable silicone lubricant layer for use herein may be prepared by a siliconization method selected from, but not limited to, silicone oil-based methods (e.g., spray-on siliconization or baked-on siliconization) and vapor deposition methods (e.g., plasma enhanced chemical vapor deposition (PECVD)). Preferably, the silicone lubricant layer is formed by spray-on siliconization or, more preferably, by baked-on siliconization.

In the spray-on siliconization method, a silicone oil (e.g. DOW CORNING® <NUM> with a viscosity of <NUM> cSt) is sprayed into the syringe (i.e. the barrel) using, e.g., a diving or static nozzle to produce a thin silicone oil layer. While silicone oil is an excellent lubricant, excess silicone oil can lead to the formation of unwanted visual and subvisual silicone oil particles. With protein-based drugs, in particular, these silicone oil particles may lead to undesirable interactions with protein drugs. For example, subvisual silicone oil particles are thought to promote protein aggregation. Therefore, since it results in fewer sub-visual and visual silicone oil particles, the baked-on siliconization processes is particularly preferred for use herein. It involves the application of silicone oil as an emulsion (e.g., DOW CORNING® <NUM> siliconization emulsion), which is then baked on the glass surface at a specific temperature and for a specific time.

The design of the syringe barrel is not particularly limited and typically has an inside diameter adjusted to accommodate the desired fill volume of, e.g., <NUM><NUM>, <NUM><NUM>, <NUM><NUM> or <NUM><NUM>. Usually, the syringe barrel has graduated marks indicating the volume of fluid in the syringe. In addition, the syringe barrel may include a flange-style interface. The design of the flange may, for example, be compatible with ISO11040. The flange-style interface may further be compatible with an optionally present handle. Furthermore, in case of a Luer-Lock syringe, the syringe may be equipped with a Luer-Lock adaptor of, e.g., polycarbonate.

The syringe tip is usually integrally formed with the syringe barrel. The tip is formed with an integral passage extending axially through the tip and being in communication 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 towards the tip's outlet end. Alternatively, the tip may be characterized as divergent (i.e., expanding from a smaller diameter to a larger one). Furthermore, the tip is usually located centrally in relation to the body of the syringe (concentric syringe tip) but may also be located offset towards the edge of the body (eccentric syringe tip).

The "capping device" within the meaning of the present invention broadly refers to any means for closing and sealing the open outlet end of a syringe. Within the present invention, the term "open outlet end" refers to any distal open end of a syringe that is in fluid communication with the barrel lumen. The capping device generally has a channel with a closed end and an open end having a dimension for receiving and efficiently sealing the open outlet end of the syringe to prevent leakage.

In case of prefilled syringes without pre-mounted needles, the capping device is a capping means commonly known as "tip cap". The tip cap forms a fluid-tight seal with the tip of the syringe to efficiently close the syringe barrel and to prevent leakage of the contents of the syringe barrel. The tip cap is usually removable coupled to the syringe tip or a luer collar. The luer collar surrounds the top of the syringe barrel (e.g., syringe tip). Preferably, the luer collar has internal threads and the tip cap has external threads complementing said internal threads of said luer collar for coupling the tip cap to the syringe barrel. The luer collar is typically a separately molded luer collar that is mounted directly to the tip of the syringe barrel, e.g., by a snap or interference fit. Prior to use, the tip can be removed, and a needle cannula (or needle/needle assembly) can then be securely coupled to the syringe tip.

If the prefilled syringe includes a removable or non-removable (permanent) cannula (also referred to as "needle" or "needle assembly") extending from the syringe tip for delivering the aqueous botulinum toxin formulation from said syringe, the capping device may be referred to as "needle shield". Said needle shield generally has a channel with a closed end and an open end having a dimension for receiving and coupling with the cannula (needle) mounted on the tip of the syringe. Typically, the (sharpened) end of the cannula penetrates the closed end of the channel in the needle shield to seal the open end of the cannula.

The capping device (e.g., tip cap or needle shield) may be a unitary member and is usually made from a flexible and/or resilient 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 engaging portion"). Alternatively, the capping device may have an outer cap made of a rigid plastic material that is coupled to a flexible and/or resilient inner cap made of a flexible and resilient polymeric material (e.g., an elastomer), wherein at least a portion of the inner cap contacts and seals the distal opening of the syringe (referred to as the "outlet engaging portion").

In view of the fact that the outlet engaging portion contacts the aqueous botulinum toxin formulation during storage and/or use, it is preferably made of a material having a minimized potential for unwanted extractables/leachables. To this end, the outlet engaging portion may have a coating thereon to increase compatibility with the aqueous botulinum toxin formulation.

Suitable flexible and/or resilient materials of the capping device, in particular of the outlet engaging portion, include elastomers that do not interfere with the aqueous botulinum toxin formulation and enable long-term storage. In particular, the part of the sealing device that contacts the aqueous botulinum toxin formulation, i.e. the outlet engaging portion, should exhibit low extractable/leachable levels during prolonged storage of the aqueous botulinum toxin formulation. As used herein, the term "elastomeric" or "elastomeric material" refers primarily to crosslinked thermosetting rubbery polymers that are more easily deformable than plastics but that are approved for use with pharmaceutical grade fluids and are not readily susceptible to leaching or gas migration.

The elastomeric material is selected from styrene-butadiene rubber, a blend of isoprene rubber and a halogenated (e.g. bromo or chloro) butyl rubber, and a halogenated butyl rubber, particularly a bromo butyl rubber or a chloro butyl rubber. The elastomeric material may also be reinforced with an inert mineral. Further, it may be cured (e.g., with organic peroxide, phenolic resins, etc.).

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

The 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 FluroTec®), PVA (a copolymer of tetrafluoroethylene (TFE) and perfluoropropylvinylether (PPVE)), tetrafluoroethylene-perfluoroethylene copolymers, polyvinylidene fluoride (PVDF), polyvinyl fluoride (PVF), polytetrafluoroethylene (PTFE), and mixtures thereof. Preferably, the coating is made of ETFE and, particularly, is a FluroTec® coating.

In accordance with the present invention, the prefilled syringe commonly includes a plunger rod assembly, which extends into the proximal end of the syringe barrel. The plunger rod assembly may include a rod (also known as pushrod) with a plunger stopper at its tip (also known as "plunger") in sliding fluid-tight engagement with the cylindrical wall of the barrel lumen. The plunger forms the proximal seal and the dynamic seal that allows for extrusion of the liquid botulinum toxin formulation. The plunger stopper contacts the aqueous botulinum toxin formulation during storage and/or administration. Therefore, the plunger stopper should be compatible with the aqueous botulinum toxin formulation and not impair its long-term stability. In particular, the plunger stopper should preferably be designed to minimize the amount of extractables/leachables upon long-time storage.

Within the present invention, the plunger stopper is preferably made of an elastomeric material, which has a coating on at least a portion of the plunger stopper that contacts the aqueous botulinum toxin formulation during storage and/or use. Suitable plunger stopper elastomeric materials for use herein are selected from halogenated butyl rubber (e.g., chloro butyl rubber, CIIR; and bromo butyl rubber, BIIR), styrene-butadiene rubber (copolymer of styrene and butadiene, SBR), and a mixture of isoprene rubber and a halogenated butyl rubber. Preferably, the plunger stopper material is a halogenated butyl rubber, more preferably a bromo butyl rubber or a chloro butyl rubber, and most preferably a butyl rubber. The elastomeric material may also be reinforced with an inert mineral. Further, it may be cured (e.g., with organic peroxide, phenolic resins, etc.).

The plunger stopper comprises a coating acting as a barrier film. The coating is usually applied to at least the seal surfaces, including the surface portion of the plunger stopper facing the barrel lumen and contacting the aqueous botulinum toxin formulation during storage and/or use. The coating serves the purpose of minimizing interaction between the plunger and the liquid botulinum toxin formulation and to provide good lubricity.

Suitable coatings of the plunger stopper are generally made of a material that does not undesirably interfere with the aqueous botulinum toxin formulation and exhibits low levels of extractables/leachables. Within the present invention the coating on at least a portion of the plunger stopper that contacts the aqueous botulinum toxin formulation during storage and/or injection is a fluoropolymer coating.

The design of the plunger stopper is not particularly limited and may be a nested or bagged stopper. Further, the interface to the rod may be threaded to allow installation of the rod after sterilization. Alternatively, the interface to the rod may be designed with a snap-on design. The rod, like the plunger stopper, is generally designed to withstand sterilization but is not otherwise limited in any particular way. Typically, the rod is made of a plastic material such as an ethylene vinyl acetate (EVA) copolymer or a polypropylene.

The prefilled syringe of the present invention meets the industry standard with regard to extractables, such as defined by the foaming test, pH test, potassium permanganate-reducing substances test, UV spectrum test and residue on evaporation test according to The Japanese Pharmacopoeia, No. <NUM>, Test Methods for Plastic Containers (<NUM>). Furthermore, the prefilled syringe and the respective component before and after sterilization satisfy the standards of The Japanese Pharmacopoeia, 14thEdition, No. <NUM>, Test for Rubber Closure for Aqueous Infusions.

In another aspect, the present invention relates to a kit comprising a prefilled glass syringe according to the present invention and, optionally, instructions for use of said prefilled glass syringe.

In still another aspect, the present invention relates to the use of the prefilled glass syringe of the present invention for cosmetic applications, such as for treating facial asymmetries and wrinkles and lines of the skin (e.g., facial lines and facial wrinkles), such as upper facial rhytides, platysma bands, glabellar frown lines, nasolabial folds, chin folds, marionette lines, buccal commissures, perioral wrinkles, crow's feet, and jawlines. Preferably, the prefilled botulinum toxin container (e.g., a syringe, vial, carpule or ampoule) of the present invention is used for injection into glabellar frown lines, horizontal forehead lines, crow's feet, perioral folds, mental ceases, popply chin, and/or platysmal bands.

The amounts of botulinum toxin administered for cosmetic application are usually in the range of <NUM> to <NUM> U, <NUM> to <NUM> U, <NUM> to <NUM> U or <NUM> to <NUM> U. Such total amounts may be administered on the same day or on a subsequent day of treatment. For example, during a first treatment session a first fraction of the dose may be administered. This first fraction is preferably a suboptimal fraction, i.e. a fraction, which does not remove the wrinkles or skin lines completely. During one or more treatment sessions, the remaining fraction of the total dose may be administered.

In a still further aspect, the present invention relates to a method for the cosmetic treatment of the skin, such as for treating wrinkles of the skin and facial asymmetries, the method comprising locally administering an effective amount of botulinum toxin to a patient by intradermal, subdermal or subcutaneous injection using the prefilled glass syringe according to the first aspect of the present invention.

This still further aspect is closely related to other aspects of the present invention described above and, thus, all comments, definitions and explanations given above in relation to these other aspects equally apply, unless otherwise stated.

The present invention will now be further illustrated by the following, nonlimiting examples.

The following examples demonstrate the superior long-term stability of an aqueous botulinum toxin formulation in different prefilled syringe systems (in the following referred to as "configurations") according to the present invention.

The results obtained for the different syringe configurations surprisingly show that, contrary to expectation and common belief in the art, an aqueous botulinum toxin formulation stored in a prefilled syringe system is stable for a prolonged time period (e.g., up to <NUM> months) at standard refrigerator temperature (<NUM>-<NUM>) and is even stable when stored for about <NUM> months at an elevated temperature of <NUM>. Furthermore, extrapolation of the measured stability data indicates that the prefilled botulinum toxin syringe allows the provision of a shelf life at <NUM>-<NUM> of about <NUM> months and even longer.

Overall, the results obtained show that botulinum toxin can be conveniently used via prefilled syringes. This is an important contribution to the management of a wide variety of botulinum toxin-treated therapeutic and cosmetic indications since botulinum toxin prefilled syringes are safer and more convenient to use for clinicians and patients compared to conventional lyophilized botulinum toxin products and offer flexibility and excellent shelf life.

The liquid botulinum toxin formulation used in the following examples was prepared by dissolving <NUM> human albumin, <NUM> sucrose and incobotulinumtoxinA in <NUM>% saline to a concentration of <NUM> U/ml.

The botulinum toxin solution was then filled into a syringe glass barrel preassembled with a Luer-Lock-type closure comprising a Luer-Lock adaptor and a tip cap which, when fitted, contacts the opening of the distal syringe tip in order to seal the syringe barrel. A plunger stopper was inserted into the proximal end portion of the barrel in order to close the proximal opening. The resulting prefilled syringe was then stored at a temperature of about <NUM>, <NUM>, and <NUM>.

The stability of the botulinum toxin solution was determined initially and after a storage time of one month, three months, six months and nine months by measuring the remaining toxin potency, the pH value, and the subvisible particles level.

The potency was determined using a hemidiaphragm assay. The assay is conducted using a murine nerve muscle preparation which is maintained in an organ bath containing <NUM> of medium. The muscle is attached to a force transducer and electrically stimulated via the phrenic nerve resulting in an isometric contraction force which remains constant for more than <NUM> if no toxin is added. Upon introduction of toxin to the organ bath, the contraction amplitude of the nerve-stimulated muscle gradually declines. The contraction amplitude of the diaphragm is monitored over time. As a read-out, the time at which half the initial contraction force is reached is determined and referred to as paralysis time. The paralysis time is proportional to the amount of active toxin added to the preparation.

The pH measurements were performed in accordance with the US Pharmacopeia standardized test method USP <<NUM>>, which outlines pH measurements for a multitude of pharmaceutical product, using a pH meter (Lab <NUM>, Schott Instruments).

Particle measurements were conducted using Micro-Flow Imaging. The Micro-Flow Imaging measurements were conducted using a DPA-<NUM> particle analyzer system (ProteinSimple, Santa Clara, CA, USA) equipped with a silane coated high-resolution <NUM> flow cell. The samples were analyzed undiluted. MFI View System Software (MVSS) version <NUM>-R2-<NUM>. <NUM> was used to perform the measurements, and MFI View Analysis Suite (MVAS) software version <NUM>. <NUM> was used to analyze the samples.

Four different prefilled syringe systems (or "syringe configurations"), which differ from each other by the syringe barrel, tip cap and/or plunger stopper, were examined and are summarized in Table <NUM>.

The results of the stability measurements for configurations A, B, G, and H are shown in Table <NUM> below.

The above stability data are, together with an extrapolation to a storage time of <NUM> months, graphically shown in <FIG> (stability at <NUM>-<NUM>), <FIG> (stability at <NUM>), and <FIG> (stability at <NUM>). As can be seen from Table <NUM> and <FIG>, the maximum measured loss of biological activity is only <NUM>%, <NUM>%, and <NUM>% for the temperature conditions <NUM>-<NUM> (up to <NUM> months), <NUM> (up to <NUM> months), and <NUM> (up to <NUM> months), respectively. Extrapolations indicate that the loss in biological activity after a storage time of <NUM> months is less than <NUM>% at <NUM>-<NUM> for all configurations A, B, G, and H, and less than <NUM>% at <NUM> for configuration B.

Furthermore, the pH measurements revealed that the pH remained exceptionally stable over a period of up to <NUM> months. No trend towards higher or lower values was observed and all measured pH values remained within ±<NUM> of the initial pH (see Table <NUM>).

Moreover, the particle size measurements by Micro-Flow Imaging showed no significant increase in the particle count (see Table <NUM>).

As can be seen from Table <NUM>, the particle counts stay well below <NUM>/ml and in most cases even below <NUM>/ml. Likewise, particle measurements by means of the resonant mass measurement method (using the ARCHIMEDES particle methodology system; Affinity Biosensors, Santa Barbara, CA, USA) and nanoparticle tracking analysis (using a NanoSight LM20 system; NanoSight, Amesbury, UK) revealed no relevant particle counts.

In conclusion, the results presented above show that liquid botulinum toxin formulations in prefilled syringes are stable for a prolonged period at temperatures of <NUM>-<NUM> and even at ambient temperatures (e.g., <NUM> to <NUM>). In view of the fact that botulinum toxins are inherently instable, in particular at low toxin concentrations, this finding was unexpected. In particular, botulinum toxins are known to be highly heat-labile and highly unstable at alkaline pH. Therefore, given the labile nature of botulinum toxins, the finding that botulinum toxin in aqueous solution is highly stable, when stored in prefilled syringes, was highly surprising.

Claim 1:
A prefilled glass syringe comprising an aqueous botulinum toxin formulation, the glass syringe comprising:
(a) a syringe barrel made of glass including a proximal end and a distal end, and a generally cylindrical wall extending therebetween and defining a barrel lumen, the syringe barrel having a distally projecting tip with a fluid passage extending therethrough and communicating with the barrel lumen, wherein the generally cylindrical wall has an interior surface coated with a barrier layer,
(b) a capping device having an outlet engaging portion sealingly engaging and closing the distal open outlet end of the syringe,
(c) a plunger rod assembly which extends into the proximal end of the syringe barrel and includes a plunger stopper in sliding fluid-tight engagement with the cylindrical wall of the barrel lumen, wherein the plunger stopper has a coating on at least a portion of the plunger stopper that contacts the aqueous botulinum toxin formulation during storage and/or injection,
characterized in that
the barrier layer is a silicone layer,
the outlet engaging portion is made of an elastomeric material selected from halogenated butyl rubber, styrene-butadiene rubber, and a mixture of isoprene rubber and halogenated butyl rubber,
the plunger stopper is made of an elastomeric material selected from halogenated butyl rubber, styrene-butadiene rubber, and a mixture of isoprene rubber and halogenated butyl rubber,
the coating on at least a portion of the plunger stopper is a fluoropolymer coating,
the aqueous botulinum toxin formulation comprises water, botulinum toxin of serotype A, sodium chloride, sucrose, and human serum albumin,
the aqueous botulinum toxin formulation has a pH value of between <NUM> and <NUM> during storage, and
the activity of the botulinum toxin in the aqueous botulinum toxin formulation is not reduced by more than <NUM>%, relative to the initial toxin activity, after storage of the prefilled syringe for <NUM> months at <NUM> or <NUM> months at <NUM>.