Albumin-free botulinum toxin formulations.
Abstract
The present invention relates to a method for the stabilization of a botulinum toxin formulation comprising a botulinum toxin, a non-reducing disaccharide or a non-reducing trisaccharide, a non-ionic surfactant, and a physiologically compatible buffer, the method comprises: combining botulinum toxin, non-reducing disaccharide or non-reducing trisaccharide, non-ionic surfactant, and physiologically compatible buffer thereby forming a liquid formation of botulinum toxin; wherein the concentration of the non-reducing disaccharide or the non-reducing trisaccharide is in the range of 10% to 40% (w / v); and where the concentration of the nonionic surfactant is in the range of 0.005% to 0.5%) w / v).

Term
3.8 yearsleft in the term
Expires 25 June 2030.
- Priority
- Filed
- Granted
- Today
- Expires
40 claims: 1 independent, 39 dependent
- 1REIVINDICACIONES 1. Un método para la estabilización de una formulación de toxina botulínica que comprende una toxina botulínica, un disacárido no reductor o un trisacárido no reductor, un tensoactivo no iónico, y un amortiguador fisiológicamente compatible, el método comprende:combinar la toxina botulínica, el disacárido no reductor o el trisacárido no reductor, el tensioactivo no iónico, y el amortiguador fisiológicamente compatible formando con ello una formulación líquida de toxina botulínica;en donde la concentración del disacárido no reductor o el trisacárido no reductor está en el intervalo de 10% a 40% (p/v);y en donde la concentración del tensioactivo no iónico está en el intervalo de 0.005% a 0.5% (p/v).
- 2El método de acuerdo con la reivindicación 1, en donde el pH de la composición líquida está en el intervalo de pH 4.5 a 6.5.
- 3El método de acuerdo con la reivindicación 1, en donde la cantidad del disacárido no reductor o el trisacárido no reductor y la cantidad del tensioactivo no iónico proporcionan una composición sólida no cristalina, amorfa al secar la composición líquida en una manera que excluya la formación de cristales.
- 4El método de acuerdo con la reivindicación 3, que comprende además la etapa de secado de la composición líquida para producir una composición sólida.
- 5El método de acuerdo con la reivindicación 4, en donde la etapa de secado comprende I¡ofiIización o secado al vacío de la composición líquida para producir la composición sólida.
- 6El método de acuerdo con la reivindicación 5, en donde la etapa de secado comprende liofilización para producir la composición sólida.
- 7El método de acuerdo con la reivindicación 1, en donde la etapa de combinación se efectúa sin agregar excipientes proteicos derivados de animales.
- 8El método de acuerdo con la reivindicación 7, en donde la etapa de combinación se efectúa sin agregar excipientes proteicos derivados de animales que comprende albúmina.
- 9El método de acuerdo con la reivindicación 1, en donde la etapa de combinación comprende además una estructura cargada positivamente seleccionada de (a) un péptido cargado positivamente con una secuencia de aminoácidos seleccionada de RKKRRQRRR-G(K)i 5 -G-RKKRRQRRR (SEQ ID NO:1), RGRDDRRQRRR-G-(K)i 5 -GRGRDDRRQRRR (SEQ ID NO: 2), o YGRKKRRQRRR-G-(K)i 5 -GYGRKKRRQRRR (SEQ ID NO: 3);o (b) un polipéptido cargado positivamente o un polímero distinto a peptidilo que tiene unido covalentemente a ello al menos un grupo de eficiencia cargado positivamente que tiene una secuencia de aminoácidos seleccionada de -(gly)ni-(arg)n2 (SEQ ID NO: 4), en donde el subíndice n1 es un número entero de 0 a 20 y el subíndice n2 es independientemente un número entero impar desde 5 a 25;(gly) p -RGRDDRRQRRR-(gly) q (SEQ ID NO 5);(gly) p -YGRKKRRQRRR-(gly) q (SEQ ID NO 6);(gly) p RKKRRQRRR-(gly) q , (SEQ ID NO 7);en donde los subíndices p y q son cada uno independientemente un número entero de 0 a 20.
- 10El método de acuerdo con la reivindicación 9, en donde la estructura cargada positivamente es un polipéptido cargado positivamente el cual es polilisina.
- 11El método de acuerdo con la reivindicación 9, en donde la estructura cargada positivamente es un polímero distinto a peptidilo cargado positivamente el cual es una polialquilenoimina.
- 12El método de acuerdo con la reivindicación 10, en donde el disacárido o trisacárido no reductor usado para preparar la composición se selecciona del grupo que consiste de dihidrato de trehalosa, trehalosa anhidra, sacarosa, rafinosa y combinaciones de los mismos.
- 13El método de acuerdo con la reivindicación 10, en donde el disacárido no reductor usado para preparar la composición se selecciona de sacarosa, dihidrato de trehalosa o trehalosa anhidra.
- 14El método de acuerdo con la reivindicación 13, en donde el tensioactivo no iónico usado para preparar la composición se selecciona del grupo que consiste de polisorbatos, ésteres de sorbitan, óxido de octilfenol etileno (TRITON™ X-100), etoxilato de nonilfenol (NP-40), poloxámeros y combinaciones de los mismos.
- 15El método de acuerdo con la reivindicación 14, en donde el tensioactivo no iónico usado para preparar la composición se selecciona del grupo que consiste de polisorbato 20, polisorbato 40, polisorbato 60, polisorbato 80, monolaurato de sorbitán (SPAN® 20), monoestearato de sorbitán (SPAN® 60), triestearato de sorbitán (SPAN® 65), y monooleato de sorbitán (SPAN® 80).
- 16El método de acuerdo con la reivindicación 15, en donde el amortiguador fisiológicamente compatible se selecciona del grupo que consiste de ácido cítrico, ácido acético, ácido succínico, ácido tartárico, ácido maleico, histidina, citrato/acetato, citrato/histidina, citrato/tartrato, maleato/histidina, succinato/histidina, o sales de los mismos, y amortiguador de fosfato.
- 17El método de acuerdo con la reivindicación 16, en donde la estructura cargada positivamente es el péptido con una secuencia de aminoácidos seleccionada de RKKRRQRRR-G-(K)is-G-RKKRRQRRR (SEQ ID NO:1), RGRDDRRQRRR-G-(K)i 5 -G-RGRDDRRQRRR (SEQ ID NO: 2), o YGRKKRRQRRR-G-(K)i 5 -G-YGRKKRRQRRR (SEQ ID NO: 3).
- 18El método de acuerdo con la reivindicación 16, en donde la estructura cargada positivamente es polilisina que tiene unida a ella al menos un grupo de eficiencia cargado positivamente seleccionado de secuencia de aminoácidos (gly) p -RKKRRQRRR-(gly) q (SEQ ID NO:5);(gly)p-RGRDDRRQRRR-(gly) q (SEQ ID NO: 6);o (gly) P YGRKKRRQRRR-(gly) q (SEQ ID NO: 7);en donde los subíndices p y q son cada uno independientemente un número entero de 0 a 20.
- 19El método de acuerdo con la reivindicación 9, en donde el subíndice n1 es un número entero de 2 a 5;y el subíndice n2 es independientemente un número entero impar desde 7 a 13.
- 20El método de acuerdo con la reivindicación 18, en donde los subíndices p y q son cada uno independientemente un número entero de 2 a 5.
- 21El método de acuerdo con la reivindicación 17, en donde la toxina botulínica en la composición es toxina botulínica del serotipo A.
- 22El método de acuerdo con la reivindicación 21, en donde la toxina botulínica en la composición es una toxina botulínica de 150 kD.
- 23El método de acuerdo con la reivindicación 21, que comprende además agregar a la composición un agente gelificante, un agente modificador de la viscosidad, o una combinación de los mismos.
- 24El método de acuerdo con la reivindicación 23, en donde el agente gelificante es un agente gelificante de base celulosa y/o el agente modificador de la viscosidad se selecciona de uno o más de polietilenglicol, un poloxámero, un ácido poliacrílico, una poliamida, o una goma vegetal.
- 25El método de acuerdo con la reivindicación 24, en donde el agente modificador de la viscosidad es un poloxámero.
- 26El método de acuerdo con la reivindicación 25, en donde el agente modificador de la viscosidad es un poloxámero seleccionado de poloxámero 181, poloxámero 188, o poloxámero 407.
- 27El método de acuerdo con la reivindicación 22, en donde el disacárido no reductor o el trisacárido no reductor está presente en la composición en una cantidad de 10% a 25% (p/v).
- 28El método de acuerdo con la reivindicación 27, en donde el disacárido no reductor o el trisacárido no reductor está presente en la composición en una cantidad de 15% a 20% (p/v).
- 29El método de acuerdo con la reivindicación 27, en donde el tensoactivo no iónico está presente en la composición en una cantidad de 0.01% a 0.2% (p/v).
- 30El método de acuerdo con la reivindicación 29, en donde el tensioactivo no iónico está presente en la composición en una cantidad de 0.02% a 0.1% (p/v).
- 31El método de acuerdo con la reivindicación 30, en donde el tensioactivo no iónico está presente en la composición en una cantidad de 0.05% a 0.08%.
- 32El método de acuerdo con la reivindicación 29, en donde la toxina botulínica está presente en la composición en una cantidad en el intervalo desde 400 U a 3,000 U.
- 33El método de acuerdo con la reivindicación 29, en donde la toxina botulínica está presente en la composición en una cantidad cosméticamente o terapéuticamente efectiva en el intervalo desde 1,000 U a 50,000 U adecuada para administración tópica.
- 34El método de acuerdo con la reivindicación 30, en donde el péptido cargado positivamente tiene la secuencia de aminoácidos RKKRRQRRR-G-(K) 15 -G-RKKRRQRRR (SEQ ID NO:1).
- 35El método de acuerdo con la reivindicación 30, en donde el péptido cargado positivamente tiene la secuencia de aminoácidos RGRDDRRQRRR-G-(K)i5-G-RGRDDRRQRRR (SEQ ID NO:2).
- 36El método de acuerdo con la reivindicación 30, en donde 5 el péptido cargado positivamente tiene la secuencia de aminoácidos YGRKKRRQRRR-G-(K) 15 -G-YGRKKRRQRRR (SEQ ID NO:3).
- 37El método de acuerdo con la reivindicación 5, en donde la composición sólida es un polvo.
- 38El método de acuerdo con la reivindicación 5, en donde 10 la composición sólida es una torta liofilizada.
- 39El método de acuerdo con la reivindicación 13, en donde el disacárido no reductor es sacarosa.
- 40El método de acuerdo con la reivindicación 13, en donde el disacárido no reductor es trehalosa.
Independent claims40
155 paragraphs in 2 sections, as filed
BOTULIN FREE TOBIN FORMULATIONS
Cross Reference to Related Application
This application claims the priority for US Provisional Patent Application No. 61 / 220,433, filed on June 25, 2009, the content of which is incorporated by reference in its entirety.
Field of the Invention
This invention relates to pharmaceutical formulations containing botulinum toxin. Particularly, this invention relates to botulinum toxin formulations that are stabilized by non-protein excipients. Background of the Invention
Botulinum toxins (also known as botulinum toxin or botulinum neurotoxins) are neurotoxins produced by Clostridium botulinum bacteria. Botulinum toxins cause muscle paralysis preventing synaptic transmission or release of acetylcholine through the neuromuscular joint. The action of botulinum toxins essentially blocks the signals that would normally cause muscle spasms or contractions, resulting in paralysis.
There are eight naturally occurring botulinum toxins serologically related, seven of which are known to cause paralysis (ie, botulinum neurotoxin serotypes A, B, C, D, E, F and G). Each of these serotypes is distinguished by neutralization with type-specific antibodies. However, the molecular weight of the botulinum toxin 5 protein molecule is approximately 150 kD for the seven of these active botulinum toxins. As released by the Clostridium botulinum bacteria, the botulinum toxin is present in a complex comprising the 150 kD botulinum toxin protein molecule along with proteins not associated with the toxin. The total size of the complex may vary. For example, the botulinum toxin type A complex can be produced by Clostridium botulinum bacteria as complexes of 900 kD, 500 kD and 300 kD. Botulinum toxin type B and C complexes are produced only as 700 kD or 500 kD complexes. The 15 botulinum toxin type D complexes are produced as 300 kD and 500 kD complexes. Botulinum toxin complexes type E and F are produced only as 300 kD complexes. The complexes are believed to contain the hemagglutinin protein not related to the toxin and the protein other than the hemagglutinin 2ü not related to the toxin and non-toxic. It is believed that these two proteins not related to the toxin (which together with the botulinum toxin molecule comprise the relevant neurotoxin complex) provide stability against denaturation to the botulinum toxin molecule and protection against digestive acids when ingested the toxin
Although botulinum toxin is the most deadly natural toxin known to man, it has found extensive use as a therapeutic and cosmetic agent. For example, in 1986, the feasibility of using botulinum toxin type A for the treatment of wrinkles associated with movement in the eyebrow area, was first demonstrated by Schantz and Scott, in Lewis GE (<sup>Ed</sup>) B_ipmedical Aspects of Botulinum. NY: Academic Press, 143-150 (1981). The use of botulinum toxin type A for the treatment of wrinkles was published in 1992 (Schantz and Scott, in Lewis GE (Ed) Biomedical Aspects of Botulinum, NY: Academic Press, 143-150 (1981)), and in 1994 Other wrinkles associated with movement in the face were treated with botulinum toxin type A (Scott, Ophthalmol, 87: 1044-1049 (1980)). Demand for cosmetic treatments with botulinum toxin has grown steadily over the years, with current annual sales of botulinum toxin in the United States that exceeded $ 1 billion per year.
A challenging aspect of commercially made botulinum toxin formulations is the stabilization of botulinum toxin. Like many proteins, botulinum toxin can be degraded or denatured by environmental factors, such as heat, alkaline conditions, mechanical shear forces, or contact with surfaces or reactive substances. In addition, the difficulty of stabilizing botulinum toxin in commercial formulations is exacerbated by the extreme toxicity of the toxin, which allows only minute amounts of the toxin to be used for therapeutic purposes. If the botulinum toxin formulation is not stabilized correctly, tiny amounts of botulinum toxin may experience unwanted reactions and / or adhere to the internal surfaces of their storage containers, thus leading to the unacceptable loss of botulinum toxin or activity.
Commercial botulinum toxin formulations are often distributed as freeze-dried powder (i.e., freeze-dried) or vacuum-dried, to prevent degradation and make botulinum toxin formulation easier to handle and less expensive to transport. Before use, botulinum toxin powder formulations are reconstituted with a liquid carrier, such as water or a saline solution. For example, a commercially available botulinum toxin formulation is sold under the trademark BOTOX® (Allergan, Inc., Irvine, Calif.). The BOTOX® formulation is distributed as a vacuum-dried powder stored in individual bottles containing approximately 100 units (U) of type A botulinum toxin complex Clostridium botulinum, 0.5 milligrams of human serum albumin and 0.9 milligrams of sodium chloride It has been reported that the commercial botulinum toxin formulation should be stored at a temperature of -10 ° C or less to maintain the designated potency for a shelf life of one year.
In commercial botulinum toxin formulations, human serum albumin is frequently added as a bulk carrier and stabilizer. Generally, albumin can stabilize a therapeutic protein (eg, botulinum toxin) by one or more of the following mechanisms: (1) reducing the adherence of the therapeutic protein to the internal surfaces of storage or distribution containers, which include objects glass, storage bottles, and syringes used to inject the pharmaceutical composition; and (2) reducing the denaturation of the therapeutic protein, especially after reconstitution to prepare a solution of the therapeutic protein. Human serum albumin has the added advantage of being minimally immunogenic, which decreases the likelihood of a human patient developing antibodies against the botulinum toxin formulation.
Although human serum albumin has been adopted as a stabilizer in commercial botulinum toxin formulations, there are still significant problems associated with this method. A serious problem is that albumin is derived from blood and, therefore, is susceptible to containing the pathogens or infectious agents transported in the blood. For example, human serum albumin may contain the human immunodeficiency virus (HIV). Albumin may also contain prions, which are the infectious protein agents that are responsible for causing a neurodegenerative disorder known as Creutzfeldt-Jakob disease. Prions 5 cause the incorrect folding of proteins in the brain, resulting in dementia, memory loss, speech impairment, loss of motor coordination, and death, often within the period of months after the initial onset of symptoms. .
io Attempts to replace human serum albumin with non-protein stabilizers have generally faced difficulties. A non-protein polymer may be reactive towards botulinum toxin or it may contain reactive impurities that degrade and / or denature botulinum toxin. For example, some studies have used a poloxamer as a non-protein stabilizing compound for botulinum toxin. However, these studies report that reconstituted botulinum toxin formulations stabilized with poloxamer demonstrate low activity of botulinum toxin, which suggests that the poloxamer excipient cannot properly stabilize botulinum toxin and / or induced undesired degradation reactions that occur .
Therefore, it would be highly desirable to have a botulinum toxin formulation be stabilized, but without a protein excipient, especially without any animal protein based excipient. In addition, it would be highly desirable to have a non-protein stabilizing excipient that by itself does not react with botulinum toxin.
Brief Description of the Invention
In one aspect, this invention relates to botulinum toxin formulations that are stabilized by non-protein excipients. Particularly, in preferred embodiments, this invention relates to botulinum toxin formulations that are stabilized without albumin or other excipients derived from animal proteins.
An aspect of the invention is to provide a liquid composition comprising a botulinum toxin, a non-reducing disaccharide or a non-reducing trisaccharide, a non-ionic surfactant, and a compatible physiological buffer capable of maintaining the pH between 4.5. and 6.5. The concentration of the non-reducing sugar in the liquid composition is in the range of 10% to 40% (w / v) and the concentration of the non-ionic surfactant is in the range of 0.005% to 0.5% (w / v).
Another aspect of the invention is to provide a powder composition by drying the liquid composition described above.
This invention also provides a method for stabilizing a botulinum toxin formulation. The method comprises combining a botulinum toxin, a non-reducing disaccharide or a non-reducing trisaccharide, a non-ionic surfactant, and physiologically compatible buffer components capable of maintaining the pH between 4.5 and 6.5 to form a liquid composition. The concentration of the non-reducing sugar in the liquid composition is in the range of 10% to 40% (w / v) and the concentration of the non-ionic surfactant is in the range of 0.005% to 0.5% (w / v). Optionally, the method further comprises drying the liquid composition to produce a stabilized powder composition.
Another aspect of the invention is to provide a liquid composition comprising a botulinum toxin, a non-reducing disaccharide or a non-reducing trisaccharide, a non-ionic surfactant, a thickening agent and a compatible physiological buffer. In these embodiments, the concentration of the non-reducing disaccharide compositions is in the range of 0.50 to 3.0% (w / v), the concentration of the thickening agent is in the range of 1.5% to 7.5%, the concentration of the non-ionic surfactant is in the range of 0.005% to 0.5% (w / v); and the pH of the composition is in the range of 4.5 to 6.5. Also, the amount of non-reducing disaccharide or non-reducing trisaccharide relative to the amount of thickening agent is selected such that the thickening agent does not crystallize when the liquid composition dries. The invention also expressly contemplates powder formulations prepared by drying such liquid compositions.
The invention also provides a method for stabilizing a botulinum toxin formulation comprising combining a botulinum toxin, a non-reducing disaccharide or a non-reducing trisaccharide, a non-ionic tective agent, a thickening agent and a compatible physiological buffer to form a liquid composition The concentration of the non-reducing disaccharide compositions is in the range of 0.50 to 3.0% (w / v), the concentration of the thickening agent is in the range of 1.5% to 7.5%, the concentration of the non-ionic surfactant is in the range from 0.005% to 0.5% (w / v); and the pH of the composition ίο is in the range of 4.5 to 6.5. Optionally, the method further comprises drying the liquid composition to produce a stabilized powder composition. Also, the amount of non-reducing disaccharide or non-reducing trisaccharide relative to the amount of thickening agent is selected such that <sup>15</sup> The thickening agent does not crystallize when the liquid composition dries.
This invention also provides a liquid composition comprising a botulinum toxin, a non-reducing sugar, a non-ionic surfactant, a compatible physiological buffer, a positively charged peptide and an optional thickening agent. The positively charged peptide has an amino acid sequence selected from the group consisting of RKKRRQRRR-G- (K)<sub>15</sub>-GRKKRRQRRR, RGRDDRRQRRR-G- (K)<sub>15</sub>-G-RGRDDRRQRRR, and YGRKKRRQRRR-G- (K)<sub>15</sub>-G-YGRKKRRQRRR. The pH of the liquid composition is in the range of 4.5 to 6.5. The invention also expressly contemplates powder formulations prepared by drying such liquid compositions.
Still another aspect of the invention is to provide a method for stabilizing a botulinum toxin formulation comprising combining a botulinum toxin, a non-reducing sugar, a non-ionic surfactant, a compatible physiological buffer, a positively charged peptide and an optional thickening agent. The positively charged peptide has an amino acid sequence selected from the group consisting of RKKRRQRRR-G- (K)<sub>15</sub>-G-RKKRRQRRR, RGRDDRRQRRR-G- (K)<sub>15</sub>G-RGRDDRRQRRR, and YG R KKR RQ R RR-G- (K)<sub>15</sub>-GYGRKKRRQRRR. The pH of the liquid composition is in the range of 4.5 to 6.5. Optionally, the method further comprises drying the liquid composition to produce a stabilized powder composition.
Brief Description of the Drawings
Figure 1: Activity recovered from botulinum toxin according to time after storage of a lyophilized botulinum toxin at 40 ° C.
Figure 2: Activity recovered from botulinum toxin according to time after storage of a lyophilized botulinum toxin at different temperatures.
Figure 3: Activity recovered from botulinum toxin according to the time after storage of a lyophilized botulinum toxin at different temperatures.
II
Detailed description of the invention
This invention relates to botulinum toxin formulations that are stabilized without the addition of protein excipients derived (ie, purified) from animal sources, such as albumin. As such, the botulinum toxin formulations of the invention do not suffer the potential problems associated with pathogens or other types of infectious agents transported in the blood. This invention also provides methods for preparing botulinum toxin formulations or without the addition of animal derived protein excipients. In preferred embodiments, the formulations contain absolutely no protein excipient. However, in certain embodiments, protein excipients not derived from animal sources (eg, recombinant albumin or recombinant gelatin) may be present in the formulations of the invention.
As used herein with respect to the botulinum toxin formulations of the invention, the term "stabilizes" and variations thereof (eg, "stabilization," "stabilize," stabilized, etc.) refer to retention. of the biological activity of botulinum toxin in the formulation for a specified period as measured by an LD analysis<sub>50</sub> of 20 mice (see LB Pearce, RE Borodic, Tox. Appl. Pharmacol, v128, p69, 1994 e
ICC VAM / NIC EATM / ECVAM, Scientific Workshop on Altérnate
Methods to Refine, Reduce and Replace the Mouse LD50 Assay for Botulinum Toxin Testing, November 14, 2006). In the preferred embodiments, there is a 100% retention of the biological activity of the botulinum toxin during the specified period. However, in other modalities, there is at least a retention of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the biological activity of the botulinum toxin during the specified period. In certain modalities, the duration of the specified period can be chosen to be consistent with the duration of the manufacturing processes of the botulinum toxin product. For example, the duration of the specified period can be chosen to be sufficient to stabilize the botulinum toxin while undergoing one or more phases of treatment. In other embodiments, the specified time will be in the order of weeks, months or even years, such as the case when non-toxin formulation components are selected to allow long-term stabilization of the botulinum toxin formulation for the storage. In certain embodiments, the specified time 20 is at least two weeks, at least one month, at least three months, at least six months, at least nine months, at least one year, at least 18 months or at least two years. In other embodiments, the specified time is coincident with the time for the botulinum toxin to retain a desired amount of bioactivity using the formulations of the invention, the time of which includes, for example, the duration of the activities indicated above. The components used to stabilize the formulation can be selected to allow stabilization at low temperatures (for example, -5 to 10 C) or at room temperature, as described herein.
In some embodiments, the botulinum toxin formulations of the invention are provided in solid form. By way of example only, the formulations can be lyophilized or dried under vacuum to produce a solid formulation. When a botulinum toxin formulation of the invention is provided in solid form, and the specified period during which the stability of the botulinum toxin formulation is measured is one week or less, the activity observed during reconstitution is the activity observed. during initial recovery. In certain preferred embodiments of the invention, there is at least a 70%, 80%, or 90% retention of activity during the initial recovery. In other modalities, there is at least a retention of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the activity during the initial recovery. In a particularly preferred embodiment, the activity during the initial recovery is greater than 99% and may even be 100%.
In other embodiments, the specified time during which stability is measured is greater than 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months at the given storage temperature . When a botulinum toxin formulation of the invention is provided in solid form, and the specified period during which the stability of the botulinum toxin formulation is measured is twelve months or more, the activity observed during reconstitution is the activity. observed during long-term storage. In certain preferred embodiments, there is at least 70%, 80%, or 90% retention of botulinum toxin activity during long-term storage. In certain modalities there is a retention of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the activity of botulinum toxin during long-term storage . In certain modalities, there is at least 90% retention of botulinum toxin activity after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months of storage at 25 ° C; at least 91% retention of botulinum toxin activity after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months of storage at 25 ° C ; at least 92% retention of botulinum toxin activity after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months of storage at 25 ° C ; at least 93% retention of botulinum toxin activity after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months of storage at 25 ° C ; at least 94% retention of botulinum toxin activity after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, or 24 months storage at 25 ° C; at least 95% retention of botulinum toxin activity after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months of storage at 25 ° C ; at least 96% retention of botulinum toxin activity after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months of storage at 25 ° C ; at least 97% retention of botulinum toxin activity after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months of storage at 25 ° C ; at least 98% retention of botulinum toxin activity after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months of storage at 25 ° C ; or at least 99% retention of botulinum toxin activity after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months of storage at 25 ° C:
In some embodiments of the invention, botulinum toxin formulations are stored in liquid form, rather than in solid form. In certain preferred embodiments, the liquid formulations of the botulinum toxin according to the invention retain at least 60%, 70%, 80%, or 90% of the activity of the botulinum toxin for up to 8 hours at room temperature. In a particularly preferred embodiment, the liquid botulinum toxin formulations according to the invention retain 100% of the activity of the botulinum toxin for up to 8 hours at room temperature.
The excipients in the botulinum toxin formulations of the invention advantageously reduce or eliminate the mechanisms by which the botulinum toxin is lost during manufacture and storage. Without wishing to be bound by theory, it is believed that the non-protein excipients of the invention reduce or eliminate unwanted adhesion of botulinum toxin to the containers used for the manufacture, storage, or supply of the formulation. Non-limiting examples of such containers include those made of glass, polystyrene, polypropylene, and other polymers. Furthermore, and again without wishing to be bound by theory, it is believed that the non-protein excipients of the invention reduce or prevent unwanted reactions between botulinum toxin and the objects or substances found by botulinum toxin during manufacture, storage, or supply. In preferred embodiments, non-protein excipients are inert to the other components of the formulation.
In some embodiments, botulinum toxin formulations are prepared in a powdered form for ease of handling, transport, or storage. The powder form can be prepared by any method known in the art. Non-limiting examples of such methods include freeze drying, vacuum drying, centrifugation and spray drying, where lyophilization and vacuum drying are particularly preferred.
The term "botulinum toxin as used herein means that it refers to any of the known types of botulinum toxin, produced by bacteria or by recombinant techniques, as well as any type that can be discovered later that includes the variants or proteins of Fusion designed. As mentioned above, currently, seven different botulinum neurotoxins have been immunologically characterized, that is, botulinum neurotoxin serotypes A, B, C, D, E, F and G, which are distinguished by neutralization with antibodies specific for the kind. The 10 botulinum toxin serotypes are available from Sigma-Aldrich and Metabiologics, Inc. (Madison, Wis.), As well as from other sources.
The different botulinum toxin serotypes vary in the animal species they affect and in the severity and duration of the paralysis they evoke.
The botulinum toxin used in the compositions of this invention may alternatively be a derivative of botulinum toxin, that is, a compound that has the activity of botulinum toxin but contains one or more chemical or functional alterations anywhere or in any related chain. with natural or recombinant native botulinum toxins. For example, the botulinum toxin can be a modified neurotoxin (for example, a neurotoxin that has at least one of its amino acids suppressed, modified or substituted, with respect to a natural neurotoxin, or recombinantly produced or a derivative or fragment of the same). For example, botulinum toxin can be one that has been modified in a way that, for example, improves its properties or decreases undesirable side effects, but still retains the desired activity of botulinum toxin. Botulinum toxin can be any of the botulinum toxin complexes produced by bacteria, as described above. Alternatively, the botulinum toxin can be a toxin prepared using recombinant or synthetic chemical techniques (for example, a recombinant peptide, a fusion protein, or a hybrid neurotoxin, as prepared from the subunits or domains of different toxin serotypes botulinum (see, for example, US Patent No. 6,444,209)). The botulinum toxin can also be a portion of the whole molecule that has been shown to possess the activity of the necessary botulinum toxin, and in that case it can be used by itself or as part of a combination or a conjugated molecule, for example a fusion protein . In addition, botulinum toxin may be in the form of a precursor to botulinum toxin, which may itself be non-toxic, for example, a non-toxic zinc protease that becomes toxic during proteolytic division.
The term "botulinum toxin complex" or "toxin complex" as used herein refers to the approximately 150 kD botulinum toxin protein molecule (which belongs to any botulinum toxin serotype AG), together with endogenous proteins different toxin associates (i.e. hemagglutinin protein and non-hemagglutinin protein unrelated to the toxin produced by Clostridium botulinum bacteria). Note, however, that botulinum toxin complex 5 does not need to be derived from Clostridium botulinum bacteria as a unit toxin complex. For example, botulinum toxin or modified botulinum toxin can first be prepared recombinantly and then subsequently combined with proteins not related to the toxin. Recombinant botulinum toxin can also be purchased (for example, from List Biological Laboratories, Campbell, CA) and then combined with non-toxin-related proteins.
This invention also contemplates "reduced botulinum toxin complexes", wherein botulinum toxin complexes have reduced amounts of non-toxin-related protein compared to naturally found amounts in botulinum toxin complexes produced by Clostridium bacteria. botulinum In one embodiment, the reduced botulinum toxin complexes are prepared using any conventional protein separation method to extract a fraction of the hemagglutinin protein or non-hemagglutinin protein unrelated to the toxin of the botulinum toxin complexes derived from the bacteria. Clostridium botulinum. For example, reduced botulinum toxin complexes can be produced by dissociating botulinum toxin complexes through exposure to red blood cells at a pH of 7.3 (for example, see EP 1514556 A1, incorporated herein by reference) . 5 HPLC, dialysis, columns, centrifugation, and other methods can be used to extract proteins from proteins. Alternatively, when reduced botulinum toxin complexes must be produced by combining synthetically produced botulinum toxin with non-toxin-related proteins, simply less hemagglutinin protein or non-toxin-related hemagglutinin protein can be added to the mixture. of which would be present for natural botulinum toxin complexes. Any of the non-toxin-related proteins (for example, hemagglutinin protein or non-toxin-related hemagglutinin protein or both) in the reduced botulinum toxin complexes according to the invention can be independently reduced to any amount. In certain exemplary embodiments, one or more non-toxin-related proteins are reduced to at least about 0.5%, 1%, 3%, 5%, 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, or 90% compared to the amounts normally found in botulinum toxin complexes. In one embodiment, substantially all non-toxin-related protein (for example,> 95% of the hemagglutinin protein or non-toxin-related hemagglutinin protein) that would normally be found in botulinum toxin complexes derived from bacteria Clostridium botulinum, is removed from the botulinum toxin complex. In another embodiment, only the pure botulinum toxin molecule is used, without any hemagglutinin protein or protein other than hemagglutinin unrelated to the toxin. In addition, although the amount of endogenous proteins not related to the toxin can be reduced to the same amount in some cases, this invention also contemplates reducing each of the endogenous proteins not related to the toxin to different amounts, as well as reducing them by at least one of the endogenous proteins not related to the toxin, but not of the others.
This invention also contemplates the general use of combinations and mixtures of botulinum toxins, although due to their different nature and properties, mixtures of botulinum toxin serotypes are not generally administered at this time in the medical or cosmetic care industry.
Botulinum toxin formulations of the invention comprise a non-ionic surfactant. Generally, this invention contemplates the use of any non-ionic surfactant that has the ability to stabilize botulinum toxin and that is convenient for pharmaceutical use. In certain embodiments, the nonionic surfactant is a polysorbate, whose non-limiting examples include polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80. In other embodiments, the nonionic surfactant is a sorbitan ester, whose non-limiting examples include Span 20, Span 60, Span 65, and Span 80. The invention also contemplates the use of poloxamers, whose non-limiting examples include poloxamer 181, poloxamer 188, and poloxamer 407. The invention also contemplates using Triton X-100 or NP-40 as non-ionic surfactants. Furthermore, the invention contemplates the modalities in which combinations of different non-ionic surfactants are used in conjunction. In certain preferred embodiments, the non-ionic surfactant is selected from the group consisting of polysorbates, poloxamers, and sorbitan, wherein polysorbates and sorbitan are particularly preferred. In preferred embodiments, the concentration of the nonionic surfactant is in the range of 0.005% to 0.5%, or in the range of 0.01% to 0.2%, or in the range of 0.02% to 0.1% or in the range of 0.05 to 0.08% This invention also contemplates formulations where the concentration of the nonionic surfactant is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12% , 0.13%, 0.14%, or 0.15%.
The botulinum toxin formulations of the invention comprise a non-reducing sugar. In preferred embodiments, non-reducing sugar has a glass transition temperature of above 55 ° C, 57 ° C or 60 ° C. Without wishing to be bound by theory, it is believed that such glass transition temperatures are high enough to suppress undesirable molecular movements that cause botulinum toxin to become denatured. In certain particularly preferred embodiments, non-reducing sugar is a disaccharide, whose non-limiting examples include trehalose and sucrose. In other embodiments, the non-reducing sugar is a trisaccharide, whose non-limiting example is raffinose. Generally, the concentration of non-reducing sugar in the botulinum toxin formulations of the invention is in the range of 10% to 40% (w / v), preferably 10% to 25% (w / v), more preferably 15% to 20% (p / v). In some preferred embodiments, the concentration of non-reducing sugar is 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% (w / v). When the non-reducing sugar is trehalose, the hydrated form of trehalose (ie trehalose dihydrate) is preferably used to prepare the formulation, although the use of the anhydrous form of trehalose is also contemplated.
In addition, one aspect of this invention is the recognition that the choice of non-reducing sugar can be used to adapt the stability of the botulinum toxin formulation according to the temperature. For example, when the botulinum toxin formulation is subjected to conditions without refrigeration, it is advantageous to use trehalose as the non-reducing sugar, because trehalose confers stability to botulinum toxin at room temperatures. Thus, the trehalose-containing formulations of the invention can be advantageously processed at room temperatures during various manufacturing processes without appreciable loss of botulinum toxin activity. In situations where the botulinum toxin formulation will be refrigerated (for example, during long-term storage, for months or even years), sucrose can be chosen as the non-reducing sugar. Mixtures of sugars are also contemplated by the invention. For example, in certain embodiments, sucrose and trehalose are added to the botulinum toxin formulation. The specific ratio of the components of the sugar mixture will depend on the effect that will be achieved and can be determined by routine experimentation. Also within the scope of this invention is the use of different non-reducing sugars, such as disaccharides, alone or in combination during different stages of purification, manufacturing, or storage. Thus, for example, trehalose can be used during the initial process and / or purification at higher temperatures (for example, room temperature), followed by the removal and replacement of trehalose with sucrose. The removal and substitution of a non-reducing sugar, such as a disaccharide, by another can be achieved by, for example, dialysis, chromatography, or other methods known in the art.
Another aspect of the invention is the recognition that the non-reducing sugar (or sugars) specified herein may, for certain embodiments, act as the primary thickening agent for solid botulinum toxin formulations. In particular, it has been discovered that non-reducing sugars, when added in the amounts specified herein, can form a stable and mechanically robust cake during lyophilization or vacuum drying of the botulinum toxin formulation. Without wishing to be bound by theory, it is believed that non-reducing sugars in such formulations form an amorphous solid in which the hydroxyl groups in the non-reducing sugar randomly orient the non-reducing sugar to maximize hydrogen bonding, which stabilizes the Botulinum toxin and imparts mechanical strength to solid cake. In some preferred embodiments, the non-reducing sugars described herein are used as the sole thickening agent for botulinum toxin formulations. In other preferred embodiments, a non-reducing sugar acts as the primary thickening agent, but small amounts of secondary thickening agents are added to the botulinum toxin formulation. The secondary thickening agents are not particularly limited, and can be any thickening agent that does not have an unacceptably adverse effect on the stability of the botulinum toxin formulation. When using secondary thickening agents that have a tendency to crystallize, it is preferable to add them in sufficiently low concentrations, so that they cannot crystallize. Without wishing to be limited by theory, it is believed that the crystallization of such secondary thickening agents would have an adverse effect on the stability of the solid cake obtained after lyophilization or vacuum drying.
A further aspect of the invention is the unexpected result that non-reducing sugars, at the concentrations described herein, can be used not only to stabilize lyophilized botulinum toxin formulations, but also botulinum toxin formulations in the phase of solution.
In preferred embodiments, the botulinum toxin formulation comprises a buffer. Generally, any compatible physiological buffer capable of maintaining the pH in the range of 4.5 to 6.5, preferably in the range of 5 to 6, and more preferably about 5.5, is suitable for the botulinum toxin formulations of the invention. Non-limiting examples of such buffers include those involving the salts of citric acid, acetic acid, succinic acid, tartaric acid, maleic acid, and histidine. Non-limiting examples of convenient buffer concentrations include buffer concentrations in the range of 0.400% to 0.600%; 0.450% to 0.575%, or 0.500% to 0.565%. The invention also contemplates botulinum toxin formulations comprising a mixture of buffer salts, the non-limiting examples of which include citrate / acetate, citrate / histidine, citrate / tartrate, maleate / histidine, or succinate / histidine. In certain preferred embodiments, the buffer is phosphate buffer.
In certain embodiments, botulinum toxin formulations comprise a thickening agent (in addition to non-reducing sugars) that make it easier to manipulate lyophilized forms of botulinum toxin formulations. In certain preferred embodiments, the thickening agents crystallize under the conditions of Iofi Iiza ci or n and do not mix well with the other excipients when they are in the solid state. However, in other preferred embodiments, the thickening agent remains amorphous under lyophilization conditions, even if it is known that they have a tendency to crystallize during freezing (eg, mannitol or glycine). As will be understood by one skilled in the art, if a thickening agent crystallizes or remains amorphous during lyophilization is controlled by several factors, including the type of thickening agent, amount of thickening agent relative to the other components of the formulation, and the speed at which the formulation is lyophilized. In certain preferred embodiments, the amount of non-reducing sugar relative to the thickening agent is adjusted to suppress crystallization of the thickening agent. When this is the case, the ratio of the non-reducing sugar to the thickening agent, on a weight percentage basis, may be greater than 0.33 to 1; 0.5 to 1, 0.75 to 1; 1: 1; 2: 1, 3: 1, or 4: 1. Non-limiting examples of the thickening agents contemplated by the invention include sorbitol, mannitol, glycine, arginine, and histidine. In some embodiments, the concentration of the thickening agent may be in the range of 1% to 10%, 2% to 6%, 3% 5 to 5% or 4% to 4.5% (w / v). In certain preferred embodiments, when a thickening agent is used, the concentration of the non-reducing sugar can be reduced from the range of 10% to 40% (w / v) to a range of 0.5% to 3.0% (w / v). In addition, in certain preferred embodiments, the ratio of the non-reducing sugar to the thickening agent is in the range of 0.07 to 2.0, preferably in the range of 0.4 to 0.6. Thus, by way of example only, the formulation may comprise mannitol as a thickening agent and trehalose dihydrate as non-reducing sugar, wherein mannitol is present in a concentration range of 1.5% to 7.5% (w / v ) and trehalose dihydrate is present in a concentration range of 0.5% to 3.0%
<td></td><td>(p / v). Preferably, the</td><td>thickener</td><td>do not</td><td>it's chloride</td><td>from</td>
<td></td><td>sodium.</td><td></td><td></td><td></td><td></td>
<td></td><td>The formulations of</td><td>Botulinum toxin</td><td>from</td><td>the invention</td><td>I know</td>
<td> 20</td><td>can administer via</td><td>any route</td><td>from</td><td colspan="2">administration</td>
<td></td><td>conventional. In some</td><td>modalities of</td><td>the</td><td>invention,</td><td>the</td>
Botulinum toxin formulations are administered to the subject by intramuscular or subcutaneous injection. In other embodiments, botulinum toxin formulations are administered topically. Regardless of whether the botulinum toxin formulations are administered topically or by injection, the formulations may include a positively charged carrier that has positively charged efficiency groups, as described herein, to facilitate the penetration of the botulinum toxin into The applications of interest. It should be noted, however, that the positively charged carrier is not required for stabilization and that the invention contemplates the modalities in which the formulations do not comprise such a positively charged carrier. The botulinum toxin formulations of the invention can also be administered via a drug delivery device, the non-limiting example of which is a dermal patch.
When botulinum toxin formulations should be administered topically, it is advantageous to include in the 15 formulations a positively charged carrier with positively charged branching groups, as described herein, to promote transdermal penetration of botulinum toxin (see also US Patent Applications Nos. 09 / 910,432; 1 1 / 073,307; 1 1 / 072,026; and 10 / 793,138, which are incorporated by reference in their entirety). Without the use of the positively charged carrier or some other means to improve transdermal transport, the transdermal flow of botulinum toxin applied topically would be expected to be very low. It should be noted that the invention also contemplates the use of other methods to improve transdermal transport, in addition to the use of a positively charged carrier as described herein, with the botulinum toxin formulations of the invention. Non-limiting examples of such methods include transdermal delivery of botulinum toxin formulations using liposomes, iontophoresis, micelles, and the like.
In addition, when botulinum toxin formulations must be administered topically, it is often advantageous to mix them with gelling agents and / or viscosity modifying agents to increase the viscosity of the formulation, to make the use of botulinum toxin easier and more accurate. . In addition, if a positively charged carrier is used, these agents help prevent the botulinum toxin / carrier formulation from drying out, which tends to cause a decrease in the activity of the botulinum toxin. Particularly preferred agents are those that are not charged and do not interfere with the activity of the botulinum toxin or efficacy of the toxin-carrier complexes when combined in the skin. The gelling agents may be certain cellulose-based gelling agents, such as, for example, hydroxypropyl cellulose (HPC). In some embodiments, the botulinum / carrier toxin complex is formulated in a composition that has 2-4% HPC. Alternatively, the viscosity of a solution containing a botulinum toxin / carrier complex can be altered by adding polyethylene glycol (PEG). In other embodiments, the botulinum toxin / carrier solution is combined with viscous agents mixed previously, such as Cetaphil® moisturizer. In other embodiments, the viscosity modifying agent is a poloxamer, whose non-limiting examples include poloxamer 188 and poloxamer 407. The viscosity modifying agent may also be a polyacrylic acid, polyamide, or a vegetable gum (e.g. gum guar). In addition, botulinum toxin formulations as described herein may be mixed with the excipients to form emulsions (including, microemulsions), suspensions, creams, lotions, gels, powders, or other common solid or liquid compositions used for use. in the skin and other tissues where the compositions can be used. Such compositions may also contain other ingredients commonly used in such products, such as antimicrobials, moisturizers and moisturizing agents, penetrating agents, preservatives, emulsifiers, natural or synthetic oils, solvents, surfactants, detergents, emollients, antioxidants, fragrances, fillers, thickeners, waxes, odor absorbers, dyes, coloring agents, powders, and optionally anesthetics, anti-itching additives, botanical extracts, conditioning agents, dimming or lighting agents, diamantine, humectants, mica, minerals, polyphenols, silicones or derivatives thereof, sun blockers, vitamins, and phytomedicines.
This invention also provides the kits for preparing and / or administering the botulinum toxin formulations of the invention. In some embodiments, the kit comprises botulinum toxin, as well as such additional excipients that are necessary to produce a stabilized formulation according to the invention. The kit may also comprise a pre-mix that can in turn be used to produce such a formulation. In other embodiments, the kit comprises a botulinum toxin formulation according to the invention that has been lyophilized and a device for delivering the formulation, the non-limiting example of which is a syringe.
The invention provides formulations that provide a "therapeutic amount of botulinum toxin to the subject during treatment." As used herein, the term "therapeutic amount" refers to an amount of botulinum toxin that is sufficient to produce the desired effect (eg, muscle relaxation, wrinkle treatment, pain treatment, or reduction of the activity of an active gland 20, such as a sweat gland). The "therapeutic amount" of botulinum toxin is implicitly understood as a safe amount that does not cause unwanted paralysis or other unwanted or harmful side effects. The specific amount of botulinum toxin that is administered will depend on several factors, including the route of administration, site of administration, indication to be treated, and serotype (or serotypes) of the botulinum toxin in the specific formulation. For example, when the botulinum toxin type A serotype is selected, a therapeutic amount may be in the range of 10 U to 150 U or 1,000 U to 2,500 L). In other preferred embodiments, the therapeutic amount ranges from 400 U to 800 U and 1,000 U to 50,000 U, preferably from 2,000-35,000 U, more preferably from 3,000 U-30,000 U, and most preferably from 4,000 U to 25,000 U. In certain embodiments, the therapeutic amount ranges from 4,000 U to 8,000 U, 9,000 U to 19,000 U, or 20,000 U to 40,000 U.
The compositions of the invention are applied to cause a desired effect, which may be a cosmetic or therapeutic effect. The desired effects include the relaxation of certain muscles with the aim of, for example, reducing the appearance of fine lines and / or wrinkles, especially on the face, or adjusting the facial appearance in other ways such as widening the eyes, lifting the corners of the mouth, or smoothing the lines that distribute the upper lip, or general relief of muscle tension. The last effect, general relief of muscle tension, can be performed on the face or elsewhere. The compositions of the invention may contain an appropriate effective amount of botulinum toxin for use as a single dose treatment, or they may be concentrated, for dilution at the site of administration or for use in multiple applications. The stabilized botulinum toxin complexes or reduced stabilized botulinum toxin complexes can be administered to a subject to treat conditions such as undesirable facial muscle spasm or other spasms, hyperhidrosis, acne, or muscle conditions elsewhere in the body where it is You want pain relief or muscle spasms. Botulinum toxin is administered by supply to the muscles or other structures associated with the skin. Administration can be done, for example, to the face, legs, shoulders, back (including, lower back), armpit, palms, feet, neck, groin, back of hands or feet, elbows, forearms, knees, thighs, Buttocks, torso, pelvis, or any other part of the body where botulinum toxin administration is desired.
In certain preferred embodiments, the botulinum toxin formulations of the invention comprise a positively charged carrier with positively charged efficiency groups. When this is the case, the positively charged carrier is present in an amount sufficient to facilitate the penetration of botulinum toxin into the tissues of interest. As used herein, the term "positively charged" means that the carrier has a positive charge under at least some conditions of the solution phase, more preferably under at least some physiologically compatible conditions. More specifically, "positively charged" means that the group in question contains the functionalities that are charged under all pH conditions, for example, a quaternary amine, or contains a functionality that can acquire the positive charge under certain conditions of the phase of solution, such as pH changes in the case of primary amines. More preferably, "positively charged" as used herein refers to those groups that have the behavior of associating with anions under physiologically compatible conditions. Polymers with a plurality of positively charged portions need not be homopolymers, as will be apparent to the expert. Other examples of positively charged portions are well known in the prior art and can be easily used, as will be apparent to those skilled in the art.
Generally, the positively charged carrier comprises the positively charged structure, which is commonly a chain of atoms, or with the groups in the chain that carry a positive charge at the physiological pH, or with the groups that carry a positive charge attached to the side chains that extend from the structure. Preferably, the positively charged structure itself will not have a defined enzymatic or therapeutic biological activity. The linear structure is a hydrocarbon structure that, in some modalities, is interrupted by the heteroatoms selected from nitrogen, oxygen, sulfur, silicon and phosphorus. Most structure chain atoms are usually carbon. In addition, the structure will be a polymer of repeated units (for example, amino acids, poly (ethyleneoxy), poly (propylenamine), polyalkyleneimine, and the like) but often it can be a heteropolymer. In a group of modalities, the positively charged structure is a polypropylene amine in which a number of amine nitrogen atoms are present as the ammonium (tetra-substituted) groups that carry a positive charge. In another modality, the structure
<td colspan="5">positively charged is a polymer other than peptidyl, which</td>
<td>It can be a</td><td>hetero-</td><td>u homopolymer</td><td>such as</td><td>a</td>
<td>polyalkyleneimine,</td><td>by</td><td colspan="2">example, a polyethyleneimine</td><td> 0</td>
<td>polypropyleneimine,</td><td>what</td><td>It has a weight</td><td>molecular</td><td>from</td>
<td>approximately</td><td> 100</td><td>to approximately</td><td> 2,500,000</td><td>D,</td>
preferably from about 250 to about 1,800,000 D, and more preferably from about 1000 to about 1,400,000 D. In another group of embodiments, the structure has a plurality of side chain portions that include positively charged groups (eg, ammonium groups, pyridinium groups, phosphonium groups, sulfonium groups, guanidinium groups, or amidinium groups). The side chain portions in this group of modalities can be placed in the separations along the structure that are consistent with the separations or variables. In addition, the length of the side chains may be similar or different. For example, in a group of modalities, the side chains may be linear or branched hydrocarbon chains having one to twenty carbon atoms and ending at the distal end (away from the structure) in one of the positively charged positively charged groups. . In all aspects of the present invention, the association between the carrier and the botulinum toxin is by non-covalent interaction, whose non-limiting examples include ionic interactions, hydrogen bonding, van der Waals forces, or combinations thereof.
In a group of modalities, the positively charged structure is a polypeptide having multiple positively charged side chain groups (eg, lysine, arginine, ornithine, homoarginine, and the like). Preferably, the polypeptide has a molecular weight of from about 100 to about 1,500,000 D, preferably from about 250 to about 1,200,000 D, more preferably from about 1000 to about 1,000,000 D. One skilled in the art will appreciate that when amino acids are used in this portion of the invention, the side chains may have the form D or L (R or S configuration) at the center of the junction. In certain preferred embodiments, the polypeptide has a molecular weight of about 500 to about 5000 D, preferably about 1000 to about 4000 D, more preferably about 2000 to about 3000 D. In other embodiments, the polypeptide has a molecular weight of at least about 10,000.
In another embodiment, the structure portion is a polylysine and the efficiency groups, as discussed herein, bind to the polylysine. The polylysine can have a molecular weight of about 100 to about 1,500,000 D, preferably about 250 to about 1,200,000 D, and more preferably about 1000 to about 3000 D. In one embodiment, the positively charged carrier with the positively charged efficacy groups is a peptide with the amino acid sequence RKKRRQRRR-G (K)<sub>15</sub>-G-RKKRRQRRR, RGRDDRRQRRR-G- (K)<sub>15</sub>-GRGRDDRRQRRR, or YGRKKRRQRRR-G- (K)<sub>15</sub>-G-YGRKKRRQRRR. It can also be any of the commercially available polylysines (Sigma Chemical Company, St. Louis, Mo., USA) such as, for example, polylysine having a MW of> 70,000 D, polylysine having a MW of 70,000 to 150,000 D, polylysine that has a MW of 150,000 to 300,000 D and polylysine that has a MW of> 300,000 D. The selection of an appropriate polylysine will depend on the remaining components of the composition and will be sufficient to provide a total net positive charge to the composition and provide a length that is preferably one to four times the combined length of the negatively charged components.
Alternatively, the structure may be an analogue of a polypeptide such as a peptoid. See, for example, Kessler, Angew, Chem. Int. Ed. Enql. 32: 543 (1993); Zuckermann et al., Chemtracts - Macromol. Chem. 4:80 (1992); Y
Simon and collaborators, Proc. Nat'l Acad. Sci. USA 89: 9367 (1992)). Briefly, a peptoid is a polyglycine in which the side chain binds to the nitrogen atoms of structure 5 instead of the alpha carbon atoms. In accordance with the foregoing, a portion of the side chains will commonly terminate in a positively charged group to provide the positively charged structure component.
The synthesis of peptoids is described in, for example, ίο US Patent No. 5,877,278, which is incorporated herein by reference in its entirety. As the term used herein, positively charged structures that have a peptoid structure construction are considered
<td></td><td>"No peptides" because they are not composed</td><td>from</td><td>the amino acids</td>
<td> 15</td><td>that have natural side chains</td><td>in</td><td>the location of</td>
<td></td><td>carbon a.</td><td></td><td></td>
<td></td><td>A variety of other structures</td><td>I know</td><td>can use</td>
using, for example, steric or electronic mimics of the polypeptides wherein the peptide amide bonds are replaced by substituents such as ester bonds, thioamides (--CSNH-), reverse thioamide (--NHCS--) , aminomethylene (--NHCH<sub>2</sub>-) or inverse methyleneamine groups (-CH<sub>2</sub>NH--), keto-methylene groups (--COC H<sub>2</sub>-), phosphinate (--PO<sub>2</sub>RC H<sub>2</sub>-), phosphonamidate and phosphonamidate ester (--PO<sub>2</sub>RNH--), reverse peptide (-NHCO--), trans-alkene (--CR = CH--), fluoroalkene (-40 ur., '
CF = CH-), dimethylene (--CH<sub>2</sub>CH<sub>2</sub>-), thioether (-CH<sub>2</sub>S-), hydroxyethylene (-CH (OH) CH<sub>2</sub>-), methylene oxy (-CH<sub>2</sub>O-), tetrazole (CN<sub>4</sub>), sulfonamido (--SO<sub>2</sub>NH--), Methylene Sulfonamido (-CHRSO<sub>2</sub>NH--), reverse sulfonamide (--NHSO<sub>2</sub>-), and structures with the malonate and / or gem-diamino-alkyl subunits, for example, as reviewed by Fletcher et al. ((1998) Chem. Rev. 98: 763) and detailed by the references cited in the same. Many of the above substitutions give rise to approximately isospheric polymer structures related to the formed structures of the a-amino acids.
In each of the structures provided above, the side chain groups can be added to carry a positively charged group. For example, sulfamide-linked structures (--SO<sub>2</sub>NH-- and --NHSO<sub>2</sub>-) may have side chain groups attached to the nitrogen atoms. Similarly, the hydroxyethylene bond (-CH (OH) CH<sub>2</sub>-) can carry a side chain group attached to the hydroxy substituent. One skilled in the art can easily adapt the other link chemistries to provide positively charged side chain groups using standard synthetic methods.
In some embodiments of the invention, the positively charged carrier comprises the positively charged efficacy groups. Non-limiting examples of efficacy groups include - (gly)<sub>n</sub>i- (arg)<sub>n2</sub>, wherein the subscript n1 is an integer from 0 to 20, preferably from 0 to 8, even preferably from 2 to 5, and the subscript n2 is independently an odd integer from about 5 to about 25, preferably from about 7 to about 17, and more preferably about 7 to about 13, HIV-TAT or fragments thereof, or Antennapedia PTD or a fragment thereof. Preferably the side chain or branching groups have the general formula • (gly) ni- (arg)<sub>n2</sub> as described above. Other preferred embodiments are those in which the branching or efficacy groups are the HIV-TAT fragments having the (gly)<sub>p</sub>-RGRDDRRQRRR- (gly)<sub>that</sub>, (gly)<sub>p</sub>-YGRKKRRQRRR- (gly)<sub>that</sub>, or (gly) p-RKKRRQRRR- (gly)<sub>that</sub>, where the subscripts pyq each independently are an integer from 0 to 20 and the fragment binds to the carrier molecule via terminal C or terminal N of the fragment. The lateral branching groups may have the form D or L (R or S configuration) at the center of junction. Preferred fragments of HIV-TAT are those in which the subscripts p and q each are independently integers from 0 to 8, preferably from 2 to 5. Other preferred embodiments are those in which the branching groups are the Antennapedia PTD groups or fragments thereof that retain the activity of the group. They are known in the art, for example, from Consolé et al., J. Biol. Chem. 278: 35109 (2003). Preferably, the positively charged carrier includes the positively charged side chain branching groups in an amount of at least about 0.05%, as the percentage by weight of the carrier, preferably from about 0.05 to about 45% by weight, and more preferably from about 0.1 to about 30% by weight. For positively charged branching groups that have the formula - (gly) m- (arg)<sub>n2</sub>, the most preferred amount is about 0.1 to about 25%.
The following examples are given to provide non-limiting illustrations of various embodiments of the invention. As one skilled in the art will recognize, modifications can be made without departing from the spirit and scope of the invention. Examples
Example 1: Preparation of a botulinum toxin formulation
An exemplary botulinum toxin formulation of the invention was prepared by combining the appropriate amounts of trehalose dihydrate, polysorbate 20, histidine, and histidine HCI to produce a 2x formulation stock solution containing 35% trehalose dihydrate, 0.05% polysorbate 20, and 1,126% histidine at a pH of 5.5. The solution was cooled to 4 ° C. The botulinum toxin API was granulated by centrifugation of a mother suspension of ammonium sulfate. The toxin granule was dissolved in 0.56% histidine buffer at pH 5.5 with 0.05% polysorbate 20. This solution was further diluted with histidine / polysorbate 20 solution to give a 1074 pg / ml stock toxin stock solution. solution.
A stock solution of the carrier peptide RKKRRQRRR-G- (K)<sub>15</sub>-GRKKRRQRRR was prepared by dissolving enough peptide in water to produce a solution of 6.0 mg / ml peptide. The 5 toxin stock solution, the peptide stock solution, 0.56% histidine buffer, and the 2x formulation stock solution were combined to produce a bulk drug formulation product consisting of 18% trehalose dihydrate, 0.025 % polysorbate 20, 0.56% histidine buffer, 55 ng / ml of toxin and 150 pg / ml of carrier ίο peptide.
Example 2: Lyophilization of a botulinum toxin formulation
This example provides a lyophilization process for a botulinum toxin formulation according to the invention. 200 pl of aliquots of the bulk drug product of formulation described in Example 1 were transferred to each of the fifty-five 2 ml glass bottles. Gray butyl rubber lyophilization caps were freely placed on the glass bottles. The jars were placed in the lyophilizer and the lyophilization started.
The lyophilization process comprised three main stages: (1) 20 a freezing stage; (2) a primary drying stage; and (3) a secondary stage of drying. Each of these three main stages contained one or more individual process stages, which were performed at temperatures and pressures as indicated below. The primary drying time required to complete the primary drying varied by 25 depending on the number of bottles and filling volume in the bottles.
Freezing
<td>Stage</td><td>Speed / Permanence</td><td>° C</td><td>Minutes</td>
<td> 1</td><td>H</td><td> 5</td><td> 50</td>
<td> 2</td><td>R</td><td> -45</td><td> 60</td>
<td> 3</td><td>H</td><td> -45</td><td> 120</td>
Freeze, condense and evacuate
<td></td><td>Freezing temperature:</td><td> -40 1</td><td>° C, shelf or average product</td>
<td> 10</td><td>Additional time of</td><td> 0</td><td>minutes</td>
<td></td><td>freezing:</td><td></td><td></td>
<td></td><td>Start Permission of</td><td> -45</td><td>° C average</td>
<td></td><td>empty:</td><td></td><td>condensed</td>
<td></td><td>Start Permission of</td><td> 200</td><td>mTorr</td>
<td> 15</td><td>hot:</td><td></td><td></td>
Primary drying
MTorr minutes
150 150
2520 150
990
275
240
300
50
150
150
150
<td>Stage</td><td>Speed / Permanence</td><td>° C</td><td>Minutes</td><td>mTorr</td>
<td> 7</td><td>R</td><td> 25</td><td> 1 80</td><td> 150</td>
<td> 8</td><td>H</td><td> 25</td><td> 120</td><td> 150</td>
Secondary drying
Stage
<td> 1</td><td>H</td><td> 27</td><td> 160</td><td> 100</td>
Example 3: Stability of the formulation of botulinum toxin with trehalose dihydrate
Figures 1 and 2 show the results of stability studies in which the botulinum toxin formulation bottles prepared at two different concentrations of trehalose dihydrate toxin (11 ng / bottle and 1.1 ng / bottle) were prepared as described. previously, they stored at 4 ° C, 25 ° C and 40 ° C and tested at the indicated time points to determine the biological activity of botulinum toxin for a duration of up to 18 months. Activity was measured using LD analysis.<sub>50</sub> in mice and the activities were reported in Figures 1 and 2 as equivalent units of botulinum toxin per bottle. At the beginning of the experiment (t = 0), the activity observed was 1802 units of botulinum toxin per bottle for formulation in units of Figure 1 and 192 units of Botulinum toxin per bottle for formulation in Figure 2. The observed variability of botulinum toxin activity in Figures 1 and 2 is believed to result from the interference inherent in the data obtained using LD analysis.<sub>50</sub> in mice and the small sample number (n = 1 bottle / temperature-time point). Despite this variability, the data indicate that there was no tendency for the loss of activity recovered during this eighteen month study, even at a storage temperature of 40 ° C. These data provide an example of the stabilization of an exemplary botulinum toxin formulation of the invention.
io Example 4: Stability of botulinum toxin formulation with sucrose
Figure 3 shows the results of stability studies in which the botulinum toxin formulation bottles with sucrose at 11 ng / bottle were prepared using the protocol described above for formulations containing trehalose, stored at 4 ° C and 25 ° C and tested at the indicated time points to determine the biological activity of botulinum toxin for a duration of up to 18 months. Activity was measured using LD analysis.<sub>50</sub> in mice 20 as described above and the activities are reported in Figure 3 as equivalent units of botulinum toxin per bottle. At the beginning of the experiment (t = 0), the activity observed was 2299 units of botulinum toxin per bottle. The observed variability of botulinum toxin activity in Figure 3 is believed to be the result of the interference inherent in the data obtained using the LD50 analysis in mice and small number of samples (n = 1 bottle / temperature-time point) . Despite this variability, the data indicate that there was no tendency for the loss of activity recovered during this eighteen month study, even at a storage temperature of 25 ° C. These data provide an example of the stabilization of an exemplary botulinum toxin formulation of the invention.
Example 5: Lyophilization of a botulinum toxin formulation with thickening agents
In this example, the botulinum toxin formulation was prepared by combining appropriate amounts of trehalose dihydrate, mannitol, polysorbate 20, histidine, and histidine HCI to produce a formulation stock solution containing 3% trehalose dihydrate, 7.5% mannitol , 0.05% polysorbate 20, and 0.563% histidine at a pH of 5.5. The solution was cooled to 4 ° C. The API botulinum toxin was granulated by centrifugation of a stock suspension of ammonium sulfate. The toxin granule was dissolved in the stock formulation solution. The concentration of this solution was 117 pg / ml. This solution was further diluted with the same solution to a final concentration of toxin stock solution of 11.7 pg / ml. A sufficient amount of a carrier peptide that had the amino acid sequence RKKRRQRRR-G- (K)<sub>15</sub>-G-RKKRRQRRR was dissolved in water to prepare 20 mg / ml of carrier peptide stock solution. These solutions were combined to create a bulk drug product formulation with a final composition of 3% trehalose dihydrate, 7.5% mannitol, 0.05% polysorbate 20, and 0.563% histidine at pH 5.5, 75 pg / ml of carrier peptide, and 5.5 ng / ml of toxin.
200 pl of aliquots of the bulk drug formulation product were added to each of the 192 glass bottles of 2 ml. The gray butyl rubber caps were freely placed on top of the jars. The jars were then placed in the lyophilizer and lyophilization was started.
As in the previous example, the lyophilization process comprised three main stages: (1) a freezing stage; (2) a primary drying stage; and (3) a secondary drying stage. Each of these three main stages contained one or more individual process stages, which were performed at temperatures and pressures as indicated below:
Freezing
<td>Stage</td><td>Speed / Permanence</td><td>° C</td><td>Minutes</td>
<td> 1</td><td>H</td><td> 5</td><td> 30</td>
<td> 2</td><td>R</td><td> -45</td><td> 50</td>
<td> 3</td><td>H</td><td> -45</td><td> 60</td>
<td> 4</td><td>R</td><td> -20</td><td> 25</td>
<td> 5</td><td>H</td><td> -20</td><td> 240</td>
<td>Stage</td><td>Speed / Permanence</td><td>° c</td><td>Minutes</td>
<td> 6</td><td>R</td><td> -45</td><td> 25</td>
<td> 7</td><td>H</td><td> -45</td><td> 60</td>
Freeze, condense and evacuate
<td>Freezing temperature:</td><td> -45</td><td>° C, shelf or average product</td>
<td>Additional freezing time:</td><td> 1</td><td>minutes</td>
<td>Start Permission of empty:</td><td> -40</td><td>° C condensed average</td>
<td>Start Permission of hot:</td><td> 300</td><td>mTorr</td>
Primary drying
<td>Stage</td><td>Speed / Permanence</td><td>° C</td><td>Minutes</td><td>mTorr</td>
<td> 1</td><td>H</td><td> -45</td><td> 30</td><td> 1 50</td>
<td> 2</td><td>R</td><td> -28</td><td> 80</td><td> 150</td>
<td> 3</td><td>H</td><td> -28</td><td> 900</td><td> 1 50</td>
<td> 4</td><td>R</td><td> -15</td><td> 20</td><td> 1 50</td>
<td> 5</td><td>H</td><td> -15</td><td> 180</td><td> 150</td>
<td> 6</td><td>R</td><td> -5</td><td> 20</td><td> 150</td>
<td> 7</td><td>H</td><td> -5</td><td> 1 80</td><td> 1 50</td>
<td> 8</td><td>R</td><td> 27</td><td> 100</td><td> 1 50</td>
Secondary drying
Contents2
7 sheets
Sheet 1 Sheet 2 Sheet 3 Sheet 4 Sheet 5 Sheet 6 Sheet 7
41 members in 13 offices
Priority claims7
| Document | Office | Kind | Date |
|---|---|---|---|
| 22043309 | United States of America | P | |
| 22043309 | United States of America | P | |
| 61220433 | United States of America | – | |
| 2010040104 | United States of America | W | |
| 2010040104 | United States of America | W | |
| US20090220433P | – | – | – |
| WO2010US40104 | – | – | – |
Members41
| Document | Office | Kind | |
|---|---|---|---|
| CA2766521A1 | Canada | A1 | |
| WO2010151840A2 | World Intellectual Property Organization (WIPO) | A2 | |
| US2010330123A1 | United States of America | A1 | |
| AU2010265888A1 | Australia | A1 | |
| SG177357A1 | Singapore | A1 | |
| IL217156A0 | Israel | A0 | |
| IL217156D0 | Israel | D0 | |
| EP2445521A2 | European Patent Office (EPO) | A2 | |
| US2012107361A1 | United States of America | A1 | |
| MX2012000174A | Mexico | A | |
| CO6491076A2 | Colombia | A2 | |
| KR20120102569A | Republic of Korea | A | |
| JP2012531442A | Japan | A | |
| CN102869373A | China | A | |
| WO2010151840A3 | World Intellectual Property Organization (WIPO) | A3 | |
| EP2445521A4 | European Patent Office (EPO) | A4 | |
| US9340587B2 | United States of America | B2 | |
| US2016256532A1 | United States of America | A1 | |
| BRPI1015938A2 | Brazil | A2 | |
| JP6043627B2 | Japan | B2 | |
| MX344583B | Mexico | B | |
| CN102869373B | China | B | |
| KR101804279B1 | Republic of Korea | B1 | |
| KR20170133530A | Republic of Korea | A | |
| US10111939B2 | United States of America | B2 | |
| KR101975051B1 | Republic of Korea | B1 | |
| KR20190047736A | Republic of Korea | A | |
| MX366344BThis record | Mexico | B | |
| MX2019008126A | Mexico | A | |
| US2019290740A1 | United States of America | A1 | |
| IL268980A | Israel | A | |
| CA2766521C | Canada | C | |
| KR102192618B1 | Republic of Korea | B1 | |
| KR20200141546A | Republic of Korea | A | |
| US2021330766A1 | United States of America | A1 | |
| KR102328155B1 | Republic of Korea | B1 | |
| KR20210141783A | Republic of Korea | A | |
| US11351232B2 | United States of America | B2 | |
| IL268980B | Israel | B | |
| US11911449B2 | United States of America | B2 | |
| US2024382571A1 | United States of America | A1 |
Numbers
- Publication
- 366344
- Publication, DOCDB
- 366344
- Publication, EPODOC
- MX366344
- Application
- 20160002034
- Application, DOCDB
- 2016002034
- Application, EPODOC
- MX20160002034
Titles2
- Spanish
- FORMULACIONES DE TOXINA BOTULINICA LIBRES DE ALBUMINA.
- English
- BOTULINIC TOXIN FORMULATIONS FREE OF ALBUMIN.
Classification
- CPC, 33
- A61K39/08
- A61K38/4893
- A61K8/4973
- A61K8/4993
- A61K8/602
- A61K8/64
- A61Q19/008
- A61Q19/08
- C07K14/33
- C12Y304/24069
- A61P17/00
- A61P17/10
- A61P21/00
- A61P21/02
- A61P29/00
- A61K8/60
- A61K9/14
- A61K9/19
- A61K8/90
- A61K47/26
- A61K8/4946
- A61K8/022
- A61K8/99
- A61K9/0019
- A61K9/08
- A61K31/7016
- A61K31/702
- Y02A50/30
- A61K8/66
- A61K8/84
- A61K47/22
- A61K47/34
- A61K47/42
- IPC, 5
- A61K39 08
- A61K8 99
- A61P21 02
- A61P29 00
- A61Q19 08