KR102404217B1 - Botulinum toxin prefilled syringe formulation - Google Patents

Abstract

Provided is a botulinum toxin prefilled syringe formulation that is easy to control and is stable in dispensing rate.

Description

Botulinum toxin prefilled syringe formulation with easy and stable discharge rate control

The present invention relates to a botulinum toxin prefilled syringe formulation that is easy to control and is stable in dispensing rate.

Botulinum toxin (BoNT) is one of the most potent toxins known and works by blocking the release of acetylcholine from peripheral cholinergic neurons. Botulinum toxin has been used to reduce overactive muscles and exocrine glands by administering very small doses to the subject. For example, botulinum toxin is used for cosmetic purposes as well as for the treatment of neuromuscular diseases and hyperactive exocrine diseases.

Botulinum toxin is inherently labile, particularly at alkaline pH, and is known to be highly labile and heat-labile. A medical dosage form that overcomes these shortcomings is the prefilled syringe, which has become increasingly popular as a drug delivery device in recent years. However, when a protein is used as an active ingredient, the protein's limited stability has made it a particularly difficult task for formulation scientists to use the prefilled syringe method. In particular, this applies to very dilute aqueous botulinum toxin solutions.

To increase the stability of pharmaceutical botulinum toxin compositions, stabilizing proteins such as human serum albumin (HSA) are often added. It is also known to add non-proteinaceous stabilizers, such as surfactants, polyvinylpyrrolidone (PVP), disaccharides, polyols and the like.

However, despite the above background art, there is still a need for a toxin prefilled syringe formulation with increased storage stability and easy control of the ejection rate.

One aspect is to provide a botulinum toxin prefilled syringe formulation that is easy and stable to control the syringe ejection rate.

One aspect provides a botulinum toxin prefilled syringe formulation comprising a syringe comprising a syringe barrel, a plunger rod and a plunger stopper, and a botulinum toxin liquid formulation filled within the syringe.

In the formulation, the amount of increase in the force acting on the end of the plunger rod may decrease as the rate at which the botulinum toxin liquid formulation is discharged increases.

As used herein, the term “prefilled syringe” means that it can be sealed or has a sealed, partially or completely enclosed space that can be used to receive, store, and/or transport a liquid formulation. indicates a syringe. Said syringe refers to a closed or sealed container made of plastic, such as an organic polymer, or partly or predominantly made of plastic.

The prefilled syringe has two openings that are sealed to prevent leakage of the contents. The prefilled syringe has a proximal end sealed by a plunger stopper and a distal end sealed by a capping device.

The barrel may include a proximal end and a distal end, and a generally cylindrical wall extending therebetween and defining a barrel lumen. The barrel may have a distally projecting tip extending therethrough and having a fluid passageway in communication with the barrel lumen. The proximal and distal ends each have a proximal end developing outlet and a distal end open outlet. The tip may have the same material as the rest of the barrel.

The barrel may be, after filling, by a capping device for sealingly closing the distal end of the syringe (eg, a “tip cap” that is removed prior to use and replaced with a needle, or prefilled with a removable or permanent needle). Removably capped (by a sealing means, such as a needle shield in the case of a closed syringe), and closed at the proximal end by a plunger or by any other means fluid-tightly coupled to the inner wall of the barrel to contain the fluid. To use a prefilled syringe, remove the tip cap, needle shield or other type of capping device, optionally attach a needle (if not already), and advance the plunger tip or piston within the barrel to That is, an aqueous botulinum toxin formulation) is injected into the patient.

The inner surface of the barrel may or may not be coated. The inner surface of the bagel may be coated, for example, with a barrier layer for lubrication purposes (hereinafter also referred to as "lubricant layer"). The lubricant layer should not only provide a high lubricity to allow the plunger to easily glide through the barrel, but should also be compatible with the aqueous botulinum toxin formulation and ensure its shelf life. The lubricant layer may be a silicone-free lubricant layer or a silicone lubricant layer. The inner surface of the wall may be coated with silicone. That is, the barrier layer may be a silicon layer.

The silicone coating may be prepared by silicone oil-based methods (eg, spray-on siliconization or baked-on siliconization) and vapor deposition methods (eg, plasma enhanced chemical vapor deposition (PECVD)). It can be prepared by a siliconization method selected from. The silicone coating may be formed by, for example, spray-on siliconized, or baked-on siliconized coating. The silicone coating may be a polydimethylsiloxane coating.

In the spray-on siliconeization method, silicone oil (eg, DOW CORNING® 360 having a viscosity of 1000 cSt) is injected with a syringe (i.e., using, for example, a diving or static nozzle) barrel) to create a thin layer of silicone oil. The baked-on siliconization process can include applying silicone oil to the barrel as an emulsion (eg, Dow Corning® 365 Siliconeized Emulsion), followed by baking on the plastic surface at a specific temperature for a specific time. The baked-on siliconization process can result in fewer invisible silicone oil particles and fewer visible silicone oil particles compared to the spray-on siliconization process.

The material of the syringe barrel may include a plastic material. The material may be glass, COC, COP, or a mixture thereof. The COP may be coated with silicone on its surface. COC is produced by polymerization of ethane with a cyclic monomer such as norbornene, while COP is produced by hydrogenation after ring-opening metathesis of a cyclic monomer. COC, COC and COP/COC materials exhibit a number of desirable characteristics including high transparency, low density, excellent moisture barrier ability, and resistance to aqueous and polar organic media. Specific examples include Topas _® COC and DaikyoCrystal Zenith _® .

The tip of the barrel may be integrally formed with the barrel. For example, the tip may be molded integrally with the barrel. The barrel may include an integrally formed luer lock tip or a luer slip tip. The tip may include an integral passageway extending axially through the tip and communicating with the chamber for dispensing the contents of the barrel. The tip may have a substantially frustoconical shape converging from the distal exit end of the barrel toward the exit end of the tip.

The barrel may have an inner diameter adapted to accommodate a fill volume of, for example, 0.5 ml, 1.0 ml, 1.5 ml, or 2.0 ml. The barrel may have a graduated mark indicating the volume of fluid in the syringe. Also, the barrel may include a flange-style interface. The design of the flange may be, for example, compatible with ISO11040. The flange-style interface may optionally be compatible with an existing handle.

The ratio of the length to the inner diameter of the syringe barrel may be 10 to 22, 12 to 20, or 14 to 16.

The syringe barrel has an inner diameter of 3 to 7 mm, 3.5 to 6.5 mm, 4 to 6 mm, or 4.5 to 5.5 mm, and a length of 30 to 130 mm, 50 to 110 mm, 60 to 100 mm, or 70 to 90 mm. can be The syringe may have an inner diameter of 3.5 to 6.5 mm and a length of 60 to 100 mm. In addition, the syringe may have an inner diameter of 3 to 7 mm of the barrel, and a length of 30 to 130 mm. In addition, the syringe may have an inner diameter of 4 to 6 mm and a length of 50 to 110 mm. In addition, the syringe may have an inner diameter of 4.5 to 5.5 mm and a length of 70 to 90 mm.

The plunger rod and the plunger stopper may be combined to form a plunger rod and a plunger stopper assembly. The assembly may extend into a proximal end of the barrel. The assembly may include a rod having a plunger stopper at its tip in sliding fluid tight engagement with the cylindrical wall of the barrel lumen. The assembly forms a proximal seal and a dynamic seal that allows extrusion of the botulinum toxin liquid formulation. The stopper is in contact with the botulinum toxin liquid formulation during storage and/or administration.

The stopper may be made of an elastomeric material. The stopper may optionally have a coating on at least a portion thereof that comes into contact with the botulinum toxin liquid formulation during storage and/or injection.

The elastomer may include isoprene rubber (IS), butadiene rubber (polybutadiene, BR), butyl rubber (copolymer of isobutylene and isoprene, IIR), halogenated butyl rubber (eg, chlorobutyl rubber, CIIR; and bromobutyl rubber, BIIR), styrene-butadiene rubber (copolymer of styrene and butadiene, SBR), or mixtures thereof. In one embodiment, the stopper material may be butyl rubber, halogenated butyl rubber, or a mixture thereof. In one embodiment, the stopper material may be bromobutyl rubber or chlorobutyl rubber. The elastomer may also be reinforced with inert minerals. ㄸ In addition, the elastic polymer may be cured by, for example, organic peroxide, phenol resin, or the like.

The stopper may be coated or uncoated. The coating is typically applied to at least the seal surface, including the surface portion of the plunger stopper that faces the barrel lumen and is in contact with the botulinum toxin formulation during storage and/or use. The coating may be one that provides good lubricity while minimizing interaction between the plunger stopper and the liquid botulinum toxin formulation.

A suitable coating of the plunger stopper may generally be one made of a material that does not undesirably interfere with the botulinum toxin formulation and exhibits low levels of extractables/leachables. The coating may be one comprising polypropylene, polyethylene, parylene (eg, Parylene N, Parylene C, and Parylene HT), crosslinked silicone, a fluoropolymer, or mixtures thereof. Examples of crosslinked silicone coatings include B2-coating (Daikyo Seiko) or XSi ^™ (Becton Dickinson).

The fluoropolymer coating may be a fluorinated ethylene-propylene copolymer (eg, tetrafluoroethylene-hexafluoropropylene copolymer (FEP)), a fluorinated ethylene-ethylene copolymer (eg, ethylene tetrafluoro Roethylene copolymer (ETFE), such as Flurotech ^® ), PVA (copolymer of tetrafluoroethylene (TFE) and perfluoropropylvinyl ether (PPVE)), tetrafluoroethylene-perfluoroethylene copolymer, polyvinylidene fluoride (PVDF), polyvinyl fluoride (PVF), polytetrafluoroethylene (PTFE) (Teflon), or mixtures thereof. For example, the coating is made of ETFE, in particular a PLUROTEC® coating. In one embodiment, the plunger stopper may be uncoated.

The design of the plunger stopper is not particularly limited and may be a nested or bagged stopper. In addition, 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 in a snap-on design. Rods, such as plunger stoppers, are generally designed to withstand sterilization, but are not otherwise limited in any particular way. The rod may be made of a plastic material such as ethylene vinyl acetate (EVA) copolymer or polypropylene.

The prefilled syringe may include a “capping device”. The capping device broadly refers to any means for closing and sealing the distal open outlet end of a syringe. As used herein, the term “open outlet end” or “distal open outlet end” refers to any distal open end of a syringe in fluid communication with the barrel lumen. The capping device generally has a channel having a closed end and an open end dimensioned to receive and effectively seal the open outlet end of the syringe to prevent leakage.

In the case of prefilled syringes without a preloaded needle, the capping device is a capping means commonly known as a “tip cap”. The tip cap forms a fluid tight seal with the tip of the syringe, effectively closing the syringe barrel and preventing leakage of the syringe barrel's contents. The tip cap is typically removably coupled to a syringe tip or luer collar. A luer collar surrounds the top of the syringe barrel (eg, syringe tip). The luer collar may have an internal thread and the tip cap may have an external thread complementary to the internal thread of the luer collar for coupling the tip cap to the syringe barrel. For the prefilled syringes of the present invention, the luer collar is generally formed integrally with the syringe barrel. Prior to use, the tip may be removed and then the needle cannula (needle assembly) may be securely coupled to the syringe tip.

A prefilled syringe may be a removable or non-removable (i.e. permanent) cannula or needle cannula (also referred to as a “needle” or “needle assembly”) extending from the syringe tip to deliver the aqueous botulinum toxin formulation from the syringe. referred to), the capping device may be referred to as a “needle shield”. The needle shield generally has a channel having a closed end and an open end, dimensioned for receiving and coupling a cannula (needle) mounted on the tip of a syringe. Typically, the (sharp) end of the cannula passes through the closed end of the channel in the needle shield to seal the open end of the cannula.

The capping device (eg, tip cap or needle shield) may be a single member and may typically be made of a flexible and resilient polymeric material (eg, elastomeric), or coupled to a flexible and resilient inner cap. It may have an outer cap made of a rigid plastic material, or a material comprising or made of, for example, an elastomer, at least a portion of which contacts and seals the distal opening of the syringe. Generally, at least the outlet engagement portion that contacts the distal tip opening to form a fluid tight seal is made from a flexible and/or resilient material (eg, an elastomeric material), wherein the engagement portion is botulinum toxin during storage and/or use. toxin formulation. The outlet engagement portion may be made of a material that minimizes the likelihood of unwanted extractables/leachables. To further reduce the amount of extractables and/or leachables and increase compatibility with the botulinum toxin formulation, the outlet binding moiety may have a coating thereon.

Suitable flexible and/or resilient materials of the capping device, in particular of the outlet engaging portion, include elastomers that allow long-term storage without interfering with the aqueous botulinum toxin formulation. In particular, the portion of the sealing device that comes into contact with, or is configured to contact, the aqueous botulinum toxin formulation (ie the outlet engagement portion) should exhibit low extractable/leachable levels during long-term storage of the aqueous botulinum toxin formulation. As used herein, the term “elastomeric” or “elastomeric material” refers to a cross-linked material that is more readily deformable than plastic but approved for use with pharmaceutical grade fluids and that does not readily leach or outgas. It refers mainly to thermosetting rubbery polymers.

The elastomeric material may include isoprene rubber (IS), butadiene rubber (polybutadiene, BR), butyl rubber (copolymer of isobutylene and isoprene; IIR), halogenated butyl rubber (eg, chlorobutyl rubber, CIIR; and bromobutyl rubber: BIIR), styrene-butadiene rubber (copolymer of styrene and butadiene, SBR), and mixtures thereof. Preferably, the elastomeric material is butyl rubber or halogenated butyl rubber, in particular bromo butyl rubber or chloro butyl rubber, or mixtures thereof. The elastomeric material may also be reinforced with inert minerals. In addition, the elastomeric material can be cured (eg, with organic peroxides, phenolic resins, etc.).

A suitable coating, which may optionally be present on the outlet binding portion made, for example, from the elastomeric material mentioned above, generally does not undesirably interfere with the aqueous botulinum toxin formulation and has low levels of extractables/leachables. It can be made of a material representing Coatings for use in the present invention include polypropylene, polyethylene, parylene (eg, Parylene N, Parylene C and Parylene HT), crosslinked silicone, and preferably, fluoropolymer coatings. but is not limited thereto. Examples of suitable crosslinked silicone coatings include B2-coating (Daikyo Seiko) or XSi™ (Becton Dickinson).

Fluoropolymer coatings include a fluorinated ethylene-propylene copolymer (eg, tetrafluoroethylene-hexafluoropropylene copolymer (FEP)), a fluorinated ethylene-ethylene copolymer (eg, ethylene tetra Fluoroethylene copolymers (ETFE), such as Flurotech®), PVA (copolymer of tetrafluoroethylene (TFE) and perfluoropropylvinyl ether (PPVE)), tetrafluoroethylene-perfluoro ethylene copolymers, polyvinylidene fluoride (PVDF), polyvinyl fluoride (PVF), polytetrafluoroethylene (PTFE), and mixtures thereof. Preferably, the coating is made of ETFE, in particular a PLUROTEC® coating.

The botulinum toxin liquid formulation may be an aqueous formulation. The corrective formulations may include aqueous suspensions, aqueous dispersions, aqueous emulsions, or aqueous solutions.

The botulinum toxin liquid formulation has a concentration of, for example, about 1 to about 3,000 U/mL, about 10 to about 1,000 U/mL, about 10 to about 400 U/mL, about 10 to about 200 U/mL , about 10 to about 100 U/mL, about 10 to about 70 U/mL, about 20 to about 60 U/mL, or about 40 U/mL.

The dose of the botulinum toxin liquid formulation may be 10 to 80, 10 to 50, 20 to 80, 20 to 60, 20 to 50, 30 to 80, 40 to 80, 20 to 40, or 25 to 35 units/syringe. .

As used herein, the term “botulinum toxin” broadly refers to any form or type of botulinum toxin. More specifically, the botulinum toxin may be selected from botulinum toxin types A, B, C1, C2, D, E, F, G, or mixtures thereof. For example, the botulinum toxin may be of serotype A, B or C1.

Also, the term “botulinum toxin” is intended to include both the botulinum toxin complex (“toxin complex”) and the “neurotoxic component” of the botulinum toxin (complex). As used herein, the term "botulinum toxin complex" refers to a high molecular weight complex comprising a neurotoxic component of approximately 150 kDa and a non-toxic protein of Clostridium botulinum, also comprising hemagglutinin and non-hemagglutinin proteins. do. Botulinum toxin serotype A complex is commercially available, for example, as Medytoxin (Medytox).

As used herein, the term "neurotoxic component" relates to a neurotoxic polypeptide of the toxin complex ("150 kDa" polypeptide; usually its two-chain form), free of any associated non-toxic proteins. The pure neurotoxic component may be, for example, Coretox Injection (Medytox). For example, the term “botulinum toxin” refers to the neurotoxic component of a botulinum toxin complex of a given serotype (eg serotype A, B or Cl, in particular serotype A). In other words, the botulinum toxin formulation contained in the prefilled syringe may contain only the neurotoxic component and free of any other protein of the Clostridium botulinum toxin complex.

The botulinum toxin is a wild-type botulinum toxin, for example, at least 50%, 60%, relative to the amino acid sequence of the neurotoxic component of wild-type botulinum toxin A or serotype A1 botulinum toxin deposited in the GenBank database under accession number AAA23262 Activation of a botulinum toxin exhibiting a sequence identity of greater than, greater than 70%, greater than 80%, greater than 90%, and less than 60%, less than 70%, less than 80%, less than 90%, or less than 99% (i.e., Biological activity) is also contemplated to encompass isoforms, homologs, orthologs, paralogs, and fragments.

Sequence identity can be calculated by any algorithm suitable for producing reliable results, for example using the FASTA algorithm (WR Pearson & DJ Lipman, PNAS 85:2444-2448, 1988). Sequence identity can be calculated by comparing two domains, such as two polypeptides, or two LC domains or fragments thereof.

The botulinum toxin includes modified and recombinant botulinum toxin.

The botulinum toxin liquid formulation may contain other pharmaceutically acceptable substances such as salts (eg sodium chloride), stabilizing proteins (eg albumin, gelatin), sugars (eg glucose, fructose, galactose, trehalose, sucrose and maltose), carbohydrate polymers (e.g. hyaluronic acid and polyvinylpyrrolidone (PVP)), polyols (e.g. mannitol, inositol, lactitol, isomalt, xylitol, eryth glycerol and sugar alcohols such as ritol and sorbitol), amino acids, vitamins (eg vitamin C), zinc, magnesium, anesthetics (eg, local anesthetics such as lidocaine), surfactants, tonicity modifiers, etc. may include The term “pharmaceutically acceptable,” as used herein, refers to such compounds or substances suitable for contact with tissue of a mammal, particularly a human.

The botulinum toxin liquid formulation may not contain animal ingredients. The botulinum toxin liquid formulation may not contain albumin. For example, the botulinum toxin liquid formulation may include botulinum toxin, an amino acid, a surfactant, and a tonicity adjusting agent in water. The liquid formulation of botulinum toxin contains, for example, 10 to 70 U/ml of botulinum toxin, 0.05 to 0.7 g/L amino acid, and 0.10 to 0.5 g/L of surfactant and 0.10 to 1.0 g/L of NaCl in water. may include. The botulinum toxin may be a botulinum toxin type A, B, C1, C2, D, E, F, G, or a mixture thereof. The amino acids are glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, methionine, asparagine, glutamine, lysine, arginine, histidine, aspartic acid, glutamic acid, pharmaceutically thereof acceptable salts, or mixtures thereof. The amino acid may be, for example, glycine, proline, phenylalanine, tyrosine, tryptophan, lysine, arginine, a pharmaceutically acceptable salt thereof, or a mixture thereof. The surfactant may be a nonionic surfactant. The nonionic surfactant may be a polysorbate, a poloxamer, or a mixture thereof. The polysorbate may be polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, or a mixture thereof. The NaCl may be added at a physiologically acceptable concentration, for example, 0.9 g/L.

The botulinum toxin liquid formulation may further contain a buffer to maintain the pH at 5.5 to 7.5. The buffer may be citrate, histidine, HEPES, arginine, acetic acid, phosphoric acid, salts thereof, or a mixture thereof.

The pH of the botulinum toxin preparation may be maintained in the range of 5.5 to 7.5, 6.0 to 7.5, 6.5 to 7.5, 6.1 to 7.3, or 6.2 to 7.2 during storage.

The botulinum toxin liquid formulation exhibits an LD ₅₀ titer recovery rate of 75 to 125%, 75 to 120%, or 80 to 125% compared to the initial value when stored for 2, 4, or 6 months at 25°C, an accelerated test condition. it could be

The liquid formulation of botulinum toxin exhibits a protease activity recovery rate of 75 to 125%, 75 to 120%, or 80 to 125% compared to the initial value when stored for 2, 4, or 6 months at 25°C, an accelerated test condition. can

The botulinum toxin liquid formulation may have a pH change within ± 1.5, 1.0, or 0.5 compared to the initial value when stored for 2, 4, or 6 months at 25° C., an accelerated test condition.

According to the botulinum toxin prefilled syringe formulation according to an aspect, the LD ₅₀ titer, the botulinum toxin endopeptidase titer, and the pH are stably stored at 25° C., an accelerated test condition, for 2, 4, or 6 months. can be maintained

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1 : Botulinum toxin Stability of prefilled liquid syringe formulations: COC or syringes with barrels made of COP material

1. Preparation of Botulinum Toxin Prefilled Liquid Formulations

In this example, a liquid botulinum toxin formulation was filled in a syringe for use in a highly viscous drug (HVD) (hereinafter also referred to as 'HVD syringe'), and stability according to the storage period was measured. The HVD syringe had an inner diameter of 5.00 mm, an outer diameter of 9.40 mm, a barrel length of 80.0 mm, and a volume to be filled with the liquid formulation of 0.8 mL. For reference, as an example of a conventionally known syringe, SCHOTT's TopPac ^TM syringe has an inner diameter of 6.50 mm, an outer diameter of 9.40 mm, a barrel length of 64.5 mm, and a volume filled with a liquid formulation of 1 mL. That is, the HVD syringe has a smaller inner diameter and a longer length compared to the TopPac ^TM syringe. The TopPac ^™ syringe includes a barrel, plunger, plunger rod and tip cap, the barrel is made of COC material and has a Luer Lock. The inner surface of the barrel is crosslinked by curing after siliconization with a reactive silicone mixture. The tip cap is made of rubber (Datwyler). HVD syringe contains COC material made of TOPAS 6015 material and has improved heat resistance.

In addition, in the HVD syringe, stability according to the material of the barrel, the material of the plunger stopper and the material used for its coating, and the composition of the aqueous liquid botulinum toxin formulation filled in the barrel were measured. wherein the syringe includes a plastic syringe barrel, a capping device, and a plunger rod assembly. The barrel includes a proximal end and a distal end, and a generally cylindrical wall extending therebetween and defining a barrel lumen. The barrel has a distally projecting tip extending therethrough and having a fluid passageway in communication with the barrel lumen, the generally cylindrical wall having an interior surface optionally coated with a barrier layer. In this embodiment the inner surface of the wall is coated with silicone, ie the barrier layer is a silicone layer. In addition, in this embodiment, the barrel has a COC or COP material.

The capping device has an outlet engagement portion sealingly engaging and closing the open outlet of the distal end of the barrel. The capping device for the distal end of the barrel consists of a cap and a rubber tip cap, the cap being made of polycarbonate material, and the rubber tip cap being made of the same material as the plunger. Here, the rubber tip cap corresponds to the outlet coupling portion. The outlet engaging portion is made of an elastomeric material having an optional coating on its surface. The outlet coupling portion and the capping device are coupled in a luer-lock configuration.

The plunger rod assembly extends into the proximal end of the barrel and includes a plunger stopper in sliding fluid tight engagement with the cylindrical wall of the barrel lumen, the stopper being made of an elastomeric material, and wherein the stopper is made of an elastomeric material, wherein the stopper is during storage and/or injection. optionally having a coating on at least a portion of said stopper in contact with the aqueous liquid botulinum toxin formulation. In this embodiment, the material of the stopper is bromobutyl rubber (BIIR) or chlorobutyl rubber (CIIR), which are optionally coated with Teflon.

Table 1 shows a botulinum toxin prefilled syringe formulation prefilled with a liquid botulinum toxin formulation in water used in this example.

Formulation name A B C D E F G H barrel material COC COC COC COC COC COC COP COP plunger material BIIR BIIR BIIR BIIR CIIR CIIR CIIR CIIR plunger coating doesn't exist doesn't exist teflon teflon doesn't exist doesn't exist teflon teflon L-Met (g/l) 0.1 0.2 0.1 0.2 0.1 0.2 0.1 0.2 T20 (g/l) 0.3 0.15 0.30 0.15 0.3 0.15 0.3 0.15 NaCl (g/l) 9 9 9 9 9 9 9 9 Toxin concentration (U/ml) 40 40 40 40 40 40 40 40 Water fill volume (ml) 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8

In Table 1, COC represents a cyclic olefin copolymer, and COP represents a cyclic olefin polymer. BIIR and CIIR stand for bromo and chloro butyl rubber (IIR), respectively. The plunger material indicates the material of the rubber part. Here, butyl rubber (IRR) is a copolymer of isobutylene and isoprene. Teflon coating refers to coating of BIIR or CIIR surfaces with FluroTec ^R film. L-Met, T20, Nacl, and toxin represent the amounts of L-methionine, polysorbate 20, NaCl, and botulinum toxin type A components dissolved in water, that is, water for injection. The formulation engages the plunger rod assembly into the lumen of the barrel such that the stopper of the plunger seals the liquid formulation while in contact with the liquid formulation. An outlet engaging portion of the distal end of the barrel also sealingly engaged the capping device to close the open outlet. The syringe and the capping device are integrally formed, and the capping device is removed immediately before use. At this time, the liquid comes into contact with the rubber tip cap inside the capping device.

That is, the prefilled syringe botulinum liquid formulation is in a state in which the syringe is coupled to the cappip device without the injection needle being coupled thereto.

As a result, the material of the syringe barrel is selected from COC and COP, and the stopper of the plunger is 2 concentrations of methionine and polysorbate 20 in 4 syringes selected from bromobutyl rubber and chlorobutyl rubber and its Teflon coating. A total of eight prefilled syringe botulinum liquid formulations were prepared differently.

2. Long-term stability testing of prefilled syringe formulations

(1) potency test

The prepared eight formulations, ie, formulations A to H, were stored at 4° C. in a storage room for stability samples, and the half lethal dose (LD ₅₀ ) of mice at 0, 3, 6, 9, 12, 18, and 24 months was measured.

After serial dilution of each of the liquid samples of the eight formulations, they were administered intraperitoneally to 4-week-old female ICR mice having a body weight of 17 internal 22 g. The lethality was observed for each diluted sample for 3 days after administration, and the half lethal dose was calculated using the statistical program PROBIT. The half lethal dose at month 0 was set as 100%, and the tendency to change in the half lethal dose obtained at each time point was expressed as a percentage. Table 2 is a table showing the half lethal dose according to the storage period of formulations A, B, C, D, E, F, G, and H.

LD ₅₀ (recovery lethal value relative to initial value, %) designation Time tested (months) 0 3 6 9 12 18 24 A 100 99 94 89 83 77 75 B 100 96 90 90 92 89 85 C 100 96 75 95 86 76 73 D 100 102 75 84 86 77 77 E 100 100 73 91 97 79 77 F 100 96 76 80 80 78 78 G 100 97 99 - - - - H 100 79 92 - - - -

As shown in Table 2, the recovered lethal dose values of Formulations A to F at 24 months were measured to be 73% or more, but the recovered lethal dose values of Formulations G and H were not measured from 9 months. This indicates that botulinum toxin is stable for 24 months if the barrel is made of COC material and silicone coated, regardless of whether the plunger stopper material is BIIR or CIIR, or whether the plunger stopper surface is Teflon coated. On the other hand, when the barrel material is COP, the recovery titer was not measured from 9 months in this syringe.

(2) botulinum toxin endopeptidase titer analysis

The eight pre-filled syringe formulations A to H were stored in a stability chamber to measure the activity of BoNTA/LC at month 0, 3, 6, 9, 12, 18, and 24 months.

100 ul of a SNAP25 (self-made) fragment consisting of 70 amino acid sequences was put into each well of a 96-well plate, and incubated at 2 to 8° C. for 16 hours. The SNAP25 fragment contains 10 or more contiguous amino acid sequences including cleavage sites at amino acids 197 and 198.

BONTA samples, which are standard samples from which a quantitative curve can be obtained, are prepared using BoNTA standards (Innotox, Medytox), and in HEPES solution (Sigma) so that the result values of formulations A to H can be included in the range of the quantitative curve. Test samples were prepared by diluting BONTA. Each of the standard samples and test samples A to H was put into a well containing SNAP25 and reacted at room temperature for 1 hour. Next, an anti-SNAP25 polyclonal antibody (manufactured in-house) was added to each well and reacted at room temperature for 1 hour. After the HRP-conjugated secondary antibody was reacted at room temperature for 1 hour, a coloring reagent 3,3',5,5'-tetramethylbenzidine (3,3',5,5'-Tetramethylbenzidine: TMB) was added and incubated. It gave a color reaction.

The absorbance was measured at 450 nm in a plate reader (Microplate Reader, TECAN) to quantify the degree of color development according to the activity of BoNT/LC, and a quantitative curve was obtained using the absorbance value of a standard sample. By substituting the absorbance values of samples A to H into the quantitative curve, the BoNT/LC activity of each sample was calculated. The activity of BoNT/LC at month 0 was set to 100%, and the tendency to change the activity of BoNT/LC obtained at each time point was expressed as a percentage. Table 3 is a table showing the endopeptidase activity of BoNTA contained in Formulations A to H according to the storage period.

Protease activity (Recovered activity relative to initial value, %) designation Tested period (months) 0 3 6 9 12 18 24 A 100 106 109 103 121 123 141 B 100 102 105 103 119 115 130 C 100 107 111 101 127 107 124 D 100 100 104 94 121 101 118 E 100 99 107 92 119 99 111 F 100 108 104 96 130 111 130 G 100 107 112 - - - - H 100 100 109 - - - -

As shown in Table 3, the recovery titers of Formulations A to F at 24 months were maintained at 92 to 141% compared to the initial stage, but the recovery titers of Formulations G and H were not measured from 9 months.

(3) pH stability

Eight prefilled syringes At month 0, 3, 6, 9, 12, 18, and 24 months of Formulations A to H, each sample was placed in a 15 mL disposable tube at a volume of 3 mL or more and measured with a pH meter (METTLER TOLEDO). The pH of each sample was measured. Table 4 is a table showing the pH change of formulations A to H according to the storage period.

pH designation Tested period (months) 0 3 6 9 12 18 24 A 6.7 6.9 7.0 7.3 7.2 7.3 7.4 B 6.8 7.1 7.1 7.3 7.3 7.3 7.3 C 6.0 6.5 6.6 6.6 6.5 6.5 6.8 D 6.1 6.5 6.5 6.5 6.5 6.6 6.8 E 6.3 6.6 6.5 6.6 6.5 6.7 6.8 F 6.2 6.6 6.5 6.6 6.7 6.7 6.6 G 5.9 5.7 5.5 - - - - H 5.9 5.6 5.3 - - - -

As shown in Table 4, at 24 months, the pH of Formulations A to G was maintained between 6.0 and 7.4, while Formulations G and H were maintained at pH 5.3 to 5.9.

Example 2 : Botulinum toxin Stability of prefilled liquid syringe formulations: syringes with barrels made of glass or COP material

1. Preparation of Botulinum Toxin Prefilled Liquid Formulations

Botulinum toxin prefilled liquid formulations in Table 5 below were prepared. At this time, the syringe is an HVD syringe listed in Table 1, with an inner diameter of 5.00 mm, an outer diameter of 9.40 mm, a barrel length of 80.0 mm, and a volume of 0.8 mL filled with a liquid formulation.

Formulation name I J K L M N barrel material glass glass COP COP COP COP plunger stopper material BIIR BIIR BIIR BIIR i-coating i-coating L-Met (g/l) 0.05 0.1 0.05 0.1 0.05 0.1 T20 (g/l) 0.3 0.3 0.3 0.3 0.3 0.3 NaCl (g/l) 9 9 9 9 9 9 Toxin concentration (U/ml) 40 40 40 40 40 40 Water fill volume (ml/syringe) 0.8 0.8 0.8 0.8 0.8 0.8

In Table 5, the barrels of formulations I and J are products of POONGLIM Pharmatech Inc. (Cat. No., Art.69, Korea), and the barrels of formulations K and L are SiO2 Medical Products Inc. (Cat. No., 850009-100-04, USA), and the barrels of formulations M and N are products of TERUMO (catalog number, PJ-B1L9BFTF1, Japan). The botulinum toxin is BTX1301 (API batch number) from Medytox, BoNTA. In Table 5, COC represents a cyclic olefin copolymer, and COP represents a cyclic olefin polymer. BIIR and CIIR stand for bromo and chloro butyl rubber (IIR), respectively. Here, butyl rubber (IRR) is a copolymer of isobutylene and isoprene. L-Met, T20, Nacl, and toxin represent the amounts of L-methionine, polysorbate 20, NaCl, and botulinum toxin type A components dissolved in water. The liquid containing these components had a pH of 6.0 to 7.0.

The formulation engages the plunger rod assembly into the lumen of the barrel such that the stopper of the plunger seals the liquid formulation while in contact with the liquid formulation. An outlet engaging portion of the distal end of the barrel also sealingly engaged the capping device to close the open outlet. That is, the prefilled syringe botulinum liquid formulation is in a state in which the syringe is coupled to the cappip device without the injection needle being coupled thereto.

As a result, the material of the syringe barrel is selected from glass and COP, and the plunger stopper is 3 types of syringes selected from bromobutyl rubber and i-coating. prefilled syringes of botulinum liquid formulations I to N were prepared.

2. Stability Test of Prefilled Syringe Formulations I to N

(1) potency test

The prepared six formulations, that is, I to N formulation samples were stored at 25° C. in a thermo-hygrostat (Binder) for experiments under accelerated test conditions. For long-term storage testing, the samples were stored at 4°C in a stability specimen storage room. The half lethal dose (LD ₅₀ ) of mice at 0, 2, 3, 4, and 6 months after storage was measured.

After serial dilution of liquid samples of the six formulations, they were administered intraperitoneally to 4-week-old female ICR mice having a body weight of 17 internal 22 g. The lethality was observed for each diluted sample for 3 days after administration, and the half lethal dose was calculated using the statistical program PROBIT. The half lethal dose at month 0 was set as 100%, and the tendency to change in the half lethal dose obtained at each time point was expressed as a percentage. Table 6 is a table showing the half lethal dose according to the storage period of formulations I, J, K, L, M, and N.

LD ₅₀ (recovery lethal value relative to initial value, %) designation Time tested (months) 0 2 3 4 6 I Acceleration 100 97 - 87 89 long time 100 - 93 - 94 J Acceleration 100 98 - 86 89 long time 100 - 89 - 94 K Acceleration 100 100 - 90 97 long time 100 - 92 - 97 L Acceleration 100 105 - 97 102 long time 100 - 100 - 108 M Acceleration 100 100 - 92 91 long time 100 - 96 - 102 N Acceleration 100 93 - 85 86 long time 100 - 90 - 93
As shown in Table 6, at 6 months, the six prefilled syringe formulations I to N maintained their titers in the range of 85 to 125% relative to their initial titers.

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(2) botulinum toxin endopeptidase titer analysis

The six prefilled syringe formulations I to N were stored in a stability chamber to measure the activity of BoNTA/LC at 0, 2, 3, 4, and 6 months time points.

100 ul of a SNAP25 (self-made) fragment consisting of 70 amino acid sequences was put into each well of a 96-well plate, and incubated at 2 to 8° C. for 16 hours. The SNAP25 fragment contains 10 or more contiguous amino acid sequences including cleavage sites at amino acids 197 and 198.

BONTA samples, which are standard samples from which a quantitative curve can be obtained, are prepared using BoNTA standards (Innotox, Medytox), and in HEPES solution (Sigma) so that the result values of formulations A to H can be included in the range of the quantitative curve. Test samples were prepared by diluting BONTA. Each of the standard samples and test samples A to H was put into a well containing SNAP25 and reacted at room temperature for 1 hour. Next, an anti-SNAP25 polyclonal antibody (manufactured in-house) was added to each well and reacted at room temperature for 1 hour. After the HRP-conjugated secondary antibody was reacted at room temperature for 1 hour, a coloring reagent 3,3',5,5'-tetramethylbenzidine (3,3',5,5'-Tetramethylbenzidine: TMB) was added and incubated. It gave a color reaction.

The absorbance was measured at 450 nm in a plate reader (Microplate Reader, TECAN) to quantify the degree of color development according to the activity of BoNT/LC, and a quantitative curve was obtained using the absorbance value of a standard sample. By substituting the absorbance values of samples I to N into the quantitative curve, the BoNT/LC activity of each sample was calculated. The activity of BoNT/LC at month 0 was set to 100%, and the tendency to change the activity of BoNT/LC obtained at each time point was expressed as a percentage. Table 7 is a table showing the endopeptidase activity of BoNTA contained in Formulations I to N according to the storage period.

Protease activity (Recovered activity relative to initial value, %) designation Tested period (months) 0 2 3 4 6 I Acceleration 100 81 - 54 53 long time 100 - 93 - 80 J Acceleration 100 68 - 67 65 long time 100 - 89 - 83 K Acceleration 100 76 - 63 70 long time 100 - 92 - 83 L Acceleration 100 78 - 82 78 long time 100 - 100 - 90 M Acceleration 100 82 - 79 81 long time 100 - 96 - 96 N Acceleration 100 86 - 83 84 long time 100 - 90 - 94

As shown in Table 7, the recovery titers of Formulations I to N at 6 months were maintained at 68 to 100% compared to the initial level.

(3) pH stability

Six pre-filled syringes At certain time points of formulations I to N, each sample was placed in a 15 mL disposable tube so as to be 3 mL or more, and the pH of each sample was measured with a pH meter (Mettler Toledo). Table 8 is a table showing the pH change of Formulations I to N according to the storage period. The time points are 0, 2, 4, and 6 months for accelerated condition experiments, and 0, 3, and 6 months for long-term preservation experiments.

pH designation Tested period (months) 0 2 3 4 6 I Acceleration 6.5 7.8 7.3 7.9 long time 6.5 7.6 7.7 J Acceleration 6.7 7.8 7.4 7.8 long time 6.7 7.6 7.8 K Acceleration 6.6 7.6 7.2 7.6 long time 6.6 7.4 7.5 L Acceleration 6.5 7.5 7.1 7.4 long time 6.5 7.2 7.4 M Acceleration 6.2 7.4 6.7 7.3 long time 6.2 7.2 7.3 N Acceleration 5.9 7.3 6.7 7.1 long time 5.9 7.1 7.1

As shown in Table 8, the pHs of Formulations I to N were maintained at 5.9 to 7.9 by 6 months and maintained at ±1.0 at the initial pH.

Example 3: Botulinum toxin Stability of Prefilled Liquid Syringe Formulations: Effect of Albumin

1. Preparation of Botulinum Toxin Prefilled Liquid Formulations

As shown in Table 9 below, formulation O containing human serum albumin but not containing L-Met and polysorbate 20 and formulation P containing L-Met and polysorbate 20 instead of containing human serum albumin were prepared. prepared. In Table 9, the solvent is water, and Toxin is a BoNTA type product manufactured by Medytox.

The O, and P formulations were filled into the HVD syringe. Here, the HVD syringe is a syringe B of Table 1, the barrel is made of COC material, and the plunger material is made of siliconized BIIR.

designation NaCl L-Met Tween20 HSA Toxin 0.9% 0.02% 0.015% 0.02% (30 Unit) O + - - + + P + + + - +

2. potency test : LD ₅₀ (accelerated test)

Two prepared, i.e., O and P formulation samples were prefilled in each syringe with syringe formulations stored in a stability chamber, and half lethal dose (LD50) of mice at 0, 2, 3, 7, 14, 28, and 56 time points. was measured.

Samples 1 and 2 were sequentially diluted with water, and then administered intraperitoneally to 4-week-old female ICR mice weighing 17 to 22 g. The lethality was observed for each diluted sample for 3 days after administration, and the half lethal dose was calculated using the statistical program PROBIT. The half lethal dose at month 0 was set to 100%, and the tendency to change the half lethal dose obtained at each time point was expressed as a percentage. Shelf life was calculated using the Arrhenius equation (https://met.uk.com/medical-device-packaging-testing/4a-medical-accelerated-ageing) showing the relationship between the reaction rate constant and temperature. Calculated.

Table 10 is a table showing the storage stability of the albumin-containing formulation O and the albumin-free formulation P as LD ₅₀ values.

Stability test period (days) 0 2 5 7 14 28 56 LD50 (%) STD 97 97 103 101 103 105 96 O 92 68 50 48 25 N/A N/A P 109 98 91 94 86 86 52 normalization O 100 74 55 53 28 N/A N/A P 100 90 84 87 79 79 48 Validity (days) 0 30 60 90 170 340 680

In Table 10, STD is Medytoxin strain (Medytoxin) as a standard sample, normalization represents the relative titer when the data value is 100 at day 0, and N/A is the test result LD50 in the previous cycle. Since the value was so low as 20%, it was meaningless to proceed with the test.

The three prepared formulations, namely, standard sample (STD), O, and P formulations, lost their recovery potency on the 28th day of the test for the albumin formulation O under accelerated conditions of 56 days, that is, about 2 months, but the non-albumin formulation P at 28 days. A recovery titer of about 80% at , and about 50% at day 56 was maintained. For the accelerated condition, samples were stored at 40° C., and samples were recovered at 0, 2, 5, 7, 14, 28, and 56 days.

3. Injection force test

Albumin Formulation O and albumin-free Formulation P described in Table 9 were respectively filled into 1 ml of the SHOTT TopPac syringe and HVD syringe described in Section 1 of Example 1, i.e., the B syringe of Table 1, for a total of 4 prefilled syringes. Botulinum liquid formulations Q, R, S, and T, i.e., four formulations were prepared.

For the above four formulations, the plunger injection force according to the plunger movement speed of the syringe was measured using a MultiTest 2.5 (Mecmesin, UK) tensile compression device. The injection force is an average injection force.

Specifically, first, a 30 G needle was coupled to a syringe containing each of the four formulations. Next, the syringe was fixed to the jig of the tensile compression device and the plunger rod was adjusted to be centered on the load cell of the device. The jig is the syringe holding part of the device.

After setting the measuring distance in consideration of the length of each syringe, the speed value was input in the program built into the device, and the device was operated by pressing the start button to perform the test. The measurement distance is about 40 mm high when filling 1 mL because the HVD syringe has a small inner diameter and a long length, and the TopPac syringe is filled to a height of about 30 mm when 1 mL is filled, so it is 5 mm for safety when measuring injection force. The measurement distance was set to press the plunger from 0 to 35 mm for the HVD syringe and 0 to 25 mm for the TopPac syringe. After the measurement was completed, the sample was removed from the jig, and the measurement was repeated 3 times per sample to obtain the injection force result. Table 11 is a table showing the injection force according to the plunger moving speed.

designation Q R S T formulation O O P P syringe type TopPac 1ml HVD 1ml TopPac 1ml HVD 1ml
Average plunger injection force (N)
10 mm/min 1.45 1.68 1.13 1.35 50 mm/min 2.72 2.72 2.52 2.53 100 mm/min 4.24 3.79 3.08 3.30 200 mm/min 6.25 4.72 4.84 4.39

As shown in Table 11, formulations R and T with HVD syringe had the same or higher injection force compared to formulations Q and S with TopPac syringe at low plunger movement rates of 10 and 50 mm/min, but 100 and At a high plunger movement speed of 200 mm/min, the injection force was lower compared to formulations Q and S with TopPac syringes. This indicates that formulations R and T containing the HVD syringe had higher plunger compression force at low plunger travel speeds and lower plunger compression force at high plunger travel speeds, compared to formulations Q and S, including TopPac syringes. Thus, HVD syringes Formulations containing

Claims (19)

a syringe comprising a syringe barrel, a plunger rod and a plunger stopper, and
It contains a liquid formulation of botulinum toxin filled in the syringe,
The botulinum toxin liquid formulation is a botulinum toxin prefilled syringe formulation that does not contain albumin,
The material of the syringe barrel is a cyclic olefin copolymer (cyclic olefin copolymer, COC),
The botulinum toxin liquid formulation comprises botulinum toxin, an amino acid, a surfactant and an isotonic agent, wherein the amino acid is methionine, and the surfactant is polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, Or a formulation that is a poloxamer. a syringe comprising a syringe barrel, a plunger rod and a plunger stopper, and
It contains a liquid formulation of botulinum toxin filled in the syringe,
The botulinum toxin liquid formulation is a botulinum toxin prefilled syringe formulation that does not contain animal ingredients,
The material of the syringe barrel is a cyclic olefin copolymer (cyclic olefin copolymer, COC),
The botulinum toxin liquid formulation comprises botulinum toxin, an amino acid, a surfactant and an isotonic agent, wherein the amino acid is methionine, and the surfactant is polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, Or a formulation that is a poloxamer. delete delete delete The formulation of claim 1 or 2, wherein the botulinum toxin liquid formulation further contains a buffer to maintain the pH between 5.5 and 7.5. The formulation of claim 6, wherein the buffer is citrate, histidine, HEPES, arginine, acetic acid, phosphoric acid, a salt thereof, or a mixture thereof. The formulation of claim 1, wherein the dose of the botulinum toxin liquid formulation is 10 to 50 units/syringe. delete delete delete The formulation of claim 1 or 2, wherein the material of the plunger stopper is isoprene rubber (IS), butadiene rubber (BR), butyl rubber, halogenated butyl rubber, styrene-butadiene rubber, or a mixture thereof. The formulation of claim 1 or 2, wherein the plunger stopper is uncoated. The botulinum toxin prefilled syringe formulation according to claim 1 or 2, wherein the amount of increase in the force acting on the end of the plunger rod decreases as the rate at which the botulinum toxin liquid formulation is discharged is increased. The dosage form according to claim 1 or 2, wherein the ratio of the length to the inner diameter of the syringe barrel is from 10 to 22. The formulation of claim 1 or 2, wherein the syringe barrel has an inner diameter of 3.5 to 6.5 mm and a length of 60 to 100 mm. The formulation of claim 1 or 2, wherein the botulinum toxin liquid formulation exhibits an LD ₅₀ titer recovery rate of 80 to 125% compared to the initial value when stored for 2, 4, or 6 months at 25°C, an accelerated test condition. The formulation of claim 1 or 2, wherein the botulinum toxin liquid formulation exhibits a protease activity recovery rate of 80 to 125% compared to the initial value when stored for 2, 4, or 6 months at 25°C, an accelerated test condition. The formulation of claim 1 or 2, wherein the botulinum toxin liquid formulation has a pH change within ±1.0 compared to the initial value when stored for 2, 4, or 6 months at 25°C, an accelerated test condition.