JP2011157331A - Botulinus toxin formulation capable of high-dose administration - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a botulinus toxin formulation capable of extremely safer high-dose administration than existing botulinus toxin A-type formulation. <P>SOLUTION: This invention relates to a botulinus toxin type-C<SB>1</SB>, D, E or F formulation extremely wider in therapeutic range than existing botulinus toxin type-A formulation and capable of safely administrating much more effective amount. For example, botulinus toxin type-C<SB>1</SB>can be safely administered to a total of about 1,925 U/kg body weight, and the effect is equivalent to about 250 U/kg body weight in terms of botulinus toxin type-A formulation. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

The present invention relates to a C ₁ , D, E or F type botulinum toxin preparation which has a much wider therapeutic range than existing type A botulinum toxin preparations and can safely administer a larger effective dose.

Clostridium botulinum , an anaerobic gram-positive bacterium, produces botulinum toxin, a potent polypeptide neurotoxin that causes a neuroparalytic disorder called botulism in humans and animals. So far, the botulinum toxin is serologically distinct 7 serotypes are classified into _{(A, B, C 1,} D, E, F and G), A botulinum toxin type Among them, the humanity It is the most lethal natural biological substance known. One unit (U) of botulinum toxin is defined as the LD ₅₀ when injected intraperitoneally into mice. The active center protein of botulinum toxin is a neurotoxin component (NTX) with a molecular weight of about 150 kDa in all seven known botulinum toxin serotypes. Interestingly, these botulinum toxins are released by Clostridium bacteria as a complex containing a 150 kDa botulinum toxin protein molecule with associated non-toxin proteins. For example, a botulinum toxin type A complex can be produced by Clostridium bacteria in the form of 900 kDa (LL toxin), 500 kDa (L toxin) and 300 kDa (M toxin). B, C ₁ and Botulinum toxin type D is produced as M toxins and L toxins. E and F botulinum toxins are produced only as M toxins. G-type botulinum toxin is produced only as L toxin. These complexes (ie, LL, L, or M toxins) are thought to include non-toxin hemagglutinin protein (HA protein) and non-toxin and non-hemagglutinin protein (NTNH protein). These two non-toxin proteins (which together with the botulinum toxin molecule make up the associated neurotoxin complex) provide the botulinum toxin molecule with stability against degeneration and from gastric acid when the toxin is ingested. It is thought to act to provide protection.

All botulinum toxin serotypes appear to inhibit the release of the neurotransmitter acetylcholine at the neuromuscular junction, but such inhibition acts on various neurosecretory proteins and / or makes these proteins different sites It is done by cutting at. For example, botulinum toxin type A and botulinum toxin type E both cleave a 25 kDa synaptosome-associated protein (SNAP-25), but these toxins target different amino acid sequences within the protein. Botulinum toxins of type B, D, F and G act on vesicle-associated proteins (VAMP, also called synaptobrevin), which are cleaved at different sites depending on their serotype. Finally, C ₁ botulinum toxin type to cleave both syntaxin and SNAP-25 has been demonstrated. These differences in mechanism of action are thought to affect the relative potency and / or duration of action of various botulinum toxin serotypes.

Botulinum toxin type A can be obtained by cultivating Clostridium botulinum in a culture vessel according to known procedures and then collecting and purifying the fermentation mixture. All serotypes of the botulinum toxin component (NTX) are initially synthesized as inactive single chain proteins, but become active when cleaved or nicked by proteases. Bacterial strains that produce type A and G botulinum toxin serotypes have endogenous proteases, so that type A and type G serotypes can be recovered from bacterial cultures primarily in their active form. In contrast, C ₁ type, since D and E botulinum toxin serotypes are synthesized by protein nondegradable strain, when recovered from culture are therefore typically unactivated. Since B and F serotypes are produced by both proteolytic and non-proteolytic strains, they can be recovered in either active or inactive form.

  Furthermore, in all serotypes of botulinum toxin complexes, NTX and non-toxin proteins dissociate under alkaline conditions (pH 7.2 and above), so this property is used to isolate only 150 kDa NTX. can do. This is known to have an effect of suppressing immunogenicity as a botulinum toxin preparation.

  Botulinum toxin is a toxin that blocks whole-body neurotransmission in botulism and causes human death, but conversely, its activity is actively used to cause abnormal myotonic hypertrophy, such as dystonia Or it is used as a therapeutic agent which relieves local muscle tone by administering directly into the muscle of a spastic patient (nonpatent literature 1). Among all serotypes of botulinum toxins, botulinum toxin type A is known to have the highest specific activity, and therefore botulinum toxin type A is most widely used as a therapeutic agent. Botulinum toxin type A (BOTOX®: Allergan Inc.) is approved by the US Food and Drug Administration (FDA) for the treatment of eyelid cramps, strabismus and unilateral facial cramps, cervical dystonia, and wrinkles between the eyebrows. ing. While botulinum type A products have been confirmed to be effective and safe in various neurological diseases, the treatment of diseases such as spasticity after stroke or cerebral palsy, where a relatively high dose of botulinum toxin is required, Some side effects similar to botulism such as dyspnea, general weakness, dysphagia, and dysphonia have been reported and are problematic (Non-patent Document 2). Although the cause of such side effects has not been clarified, it is considered that the administered toxin diffuses to the non-administration site via blood, lymph or other routes.

  Botulinum toxin type B (MYOBLOC®: Solstice Neurosciences Inc.) has also been approved by the FDA for the treatment of cervical dystonia. It has been reported that botulinum toxin type B has lower efficacy per unit and a lower safety margin than botulinum toxin type A (Non-patent Document 3). However, it has been reported that when the same effective amount of type A and type B botulinum toxin preparations are administered to humans, the time course of each effect is similar. Furthermore, botulinum toxin type B can exert its therapeutic effect sufficiently for patients who have neutralizing antibodies against botulinum toxin type A and cannot expect therapeutic effects for various diseases with botulinum toxin type A. It has characteristics.

  Although there is literature on the use of botulinum toxin for many neurological diseases, such as limb spasticity or spasticity treatment in children and adults, safety, efficacy and dosage have not yet been established. According to the FDA, pediatric patients diagnosed with botulism following local injections of BOTOX and MYOBLOC were reviewed for post-marketing cases obtained from the Adverse Event Reporting System (AERS) database and medical literature, resulting in pediatric botulism Cases have occurred in patients younger than 16 years, and reported symptoms ranged from dysphagia requiring light bowel supplementation to respiratory failure requiring artificial respiration. In these cases, the most commonly reported use of botulinum toxin was treatment of limb muscle spasticity associated with cerebral palsy. The range of doses used was 6.25-32 U / kg for BOTOX and 388-625 U / kg for MYOBLOC. Similarly, in adult botulism cases, symptoms such as difficulty in lifting the head, dysphagia, and drooping eyelids have been reported. There have also been reports of systemic effects that occurred at sites remote from the injection site, including lower extremity weakness and numbness. Some serious adult cases were hospitalized, but no cases required intubation or artificial respiration, and no deaths were reported. The range of doses used was 100-700 U for BOTOX and 10,000-20,000 U for MYOBLOC. In addition, 400 U is recommended as the upper limit of the total dose administered to adults with BOTOX (Non-patent Document 4).

  Against this background, highly purified A-type botulinum toxin preparations derived from infant botulism-causing bacteria have been developed as botulinum toxin preparations that are less diffusible than currently marketed therapeutic A-type botulinum preparations (patents) Reference 1).

On the other hand, therapeutic C ₁ , D, E and F type botulinum toxin preparations are not commercially available like type A and B type botulinum toxin preparations, but some characteristics have been revealed.

For C type ₁ botulinum toxins, relatively low doses (eg, 15 U) of C type ₁ botulinum toxin are as effective as 1 type of botulinum toxin and are also significant for spreading to nearby muscles It has been reported that there is no difference (Non-Patent Documents 5, 6, and 7).

Botulinum toxin type F, as well as C ₁ botulinum toxin type, relatively potency per titer 1U of botulinum toxin type F low dose (e.g., 15 U) is comparable to botulinum toxin type A, to the neighboring muscle It has been reported that there is no significant difference in the diffusion of. However F-type toxin is known to be short duration of the effect than A and type C ₁ toxin.

  For botulinum toxins D and E, little is known about efficacy or safety.

  As described above, existing A or B type botulinum toxin preparations have a high risk of systemic side effects, and diseases that cannot be safely treated beyond the recommended upper limit dose, such as limb trunk spasticity and dystonia Until now, there has been no idea of using botulinum toxins of serotypes other than types A and B in order to safely carry out treatment for these diseases.

WO 2008/050866 A1

Arimitsu H., et al., Infect. Immun., 71 (3): 1599-1603,2003 Cote T.R., et al., J Am Acad Dermatol., 53 (3): 407-415, 2005 Aoki K.R., Toxicon., 40: 923-927,2002 Brin MF, Muscle Nerve., 6: 208-220, 1997 Morbiato L., et al., Eur J Neurosci., 25: 2697-2704, 2007 Eleopra R., et al., Movement Disorders. 19 (8): S53-S59, 2004 Eleopra R., et al., Neurotoxicity Research. 9 (2,3): 127-131, 2006

  Botulinum toxin is known as a drug that exerts therapeutic effects on various neurological diseases. However, administration of existing botulinum toxin type A or B causes side effects that are thought to affect remote muscles other than the administered muscle. There have been reports of deaths associated with dysphagia, pneumonia, severe weakness, and respiratory problems. From these side effect reports, existing botulinum toxin preparations have an upper limit of administration, which is sufficient for symptoms that require administration of high doses of botulinum toxin exceeding the upper limit of administration, such as spasticity of limb trunk and dystonia. There is a problem that the therapeutic effect cannot be expected. Therefore, there is a social need for a botulinum toxin preparation that is highly safe and capable of exerting a sufficient therapeutic effect even in treatments that require relatively high doses of botulinum toxin, such as spasticity of the extremities and dystonia. It has been. The present invention provides a botulinum toxin preparation that can be safely administered at a higher dose than existing botulinum preparations.

In view of the situation as described above, the present inventors focused on serotype botulinum toxins (C ₁ , D, E or F) that have not been used in existing botulinum toxin preparations, and these serotype botulinum toxins However, the present inventors have found that the therapeutic range is much wider than existing type A botulinum toxin preparations, and that a larger effective dose can be safely administered, and the present invention has been completed.

That is, the present invention includes the following inventions (1) to (10).
(1) A botulinum toxin selected from C ₁ , D, E or F type botulinum toxin, characterized in that a larger effective amount can be safely administered compared to a botulinum toxin type A active ingredient As a pharmaceutical composition.
(2) The pharmaceutical composition according to (1) above, wherein an effective amount of botulinum toxin exceeding the upper limit of administration of the botulinum type A preparation can be administered.
(3) The pharmaceutical composition according to (1) or (2) above, wherein the botulinum toxin is a neurotoxin having a molecular weight of about 150 KDa from which non-toxic components have been removed.
(4) The pharmaceutical composition according to any one of (1) to (3) above, which contains albumin, amino acid, sugar or surfactant as a stabilizer.
(5) the pharmaceutical composition containing the type C ₁ botulinum toxin pharmaceutical composition according to any of safely administrable above to 1,925 U per body weight 1 kg (1) (4).
(6) The pharmaceutical composition according to any one of (1) to (4) above, wherein the pharmaceutical composition containing the botulinum toxin type D can be safely administered up to 182,165 U per kg body weight.
(7) The pharmaceutical composition according to any one of (1) to (4) above, wherein the pharmaceutical composition containing the E-type botulinum toxin can be safely administered up to 250 U / kg body weight.
(8) The pharmaceutical composition according to any one of the above (1) to (4), wherein the pharmaceutical composition containing the F-type botulinum toxin can be safely administered up to 18,860 U / kg body weight.
(9) The pharmaceutical composition according to any one of (1) to (8) above, strabismus, blepharospasm, unilateral facial convulsions, spastic torticollis, paralysis after stroke, cerebral palsy, spastic dysphonia, spasticity, piece Headaches such as headaches, chronic pain, low back pain, pelvic pain, postherpetic neuralgia, neuropathic pain, stiff shoulders, muscle relaxation failure at the onset of Parkinson's disease and multiple sclerosis, fascial pain syndrome, masticatory muscle spasm, Chronic anal fissure, detrusor / sphincter dysfunction, overactive bladder, prostate disorder, hyperhidrosis, bruxism, facial myokemia, tics, local dystonia, chalazion, stye, carpal tunnel syndrome, hypertrophic lip deformity, Hallux valgus, hallux valgus, thumb anomaly, hard thigh, idiopathic clubfoot, pepsin ulcer, gastroesophageal reflux disease, esophageal achalasia, gastric paralysis, vaginal spasticity, arthropathy, epicondylitis, rotation Muscle cap disease, fasciopathy, tennis elbow, thorax Syndrome, acne, schizophrenia due to neurotransmitter overactivity, Alzheimer's disease, mania, depression, cardiovascular disease, acromegaly, giantism, Cushing's disease, hypersexuality, hyperthyroidism, gonadotropin related diseases, Suppression of ovulation, suppression of sperm production, Hashimoto's disease, thyroiditis, hypothyroidism and hypercalcemia, hypocalcemia, penile erectile dysfunction, hypermucus secretion, hypersalivation, bone tumor, diabetes, pancreatic disorder, tinnitus Use for cochlear nerve abnormalities, Meniere's disease, cancer, hair growth, alopecia areata, autonomic dysfunction, pressure ulcers, sputum, weight loss, uterine disorders, levator ani syndrome, or diabetic neuropathy.
(10) A strabismus, blepharospasm, unilateral facial convulsions, spastic torticollis, paralysis after stroke, cerebral palsy, comprising the pharmaceutical composition according to any one of (1) to (8) above, Spastic vocal dysfunction, spasticity, migraine and other headaches, chronic pain, low back pain, pelvic pain, postherpetic neuralgia, neuropathic pain, stiff shoulders, muscle relaxation deficiency at the onset of Parkinson's disease and multiple sclerosis, fascia Pain syndrome, masticatory muscle spasm, chronic anal fissure, detrusor / sphincter dysfunction, overactive bladder, prostate disorder, hyperhidrosis, bruxism, facial myokiemia, tics, local dystonia, chalazion, stye, carpal tunnel syndrome, blood Increased lip deformity, hallux valgus, hallux valgus, thumb anomaly, hard hallux, sudden clubfoot, pepsin ulcer, gastroesophageal reflux disease, esophageal achalasia, gastric failure, vaginal spasticity, joint , Epicondylitis, rotator cuff Fasciopathy, tennis elbow, thoracic outlet syndrome, acne, schizophrenia due to excessive neurotransmitter activity, Alzheimer's disease, mania, depression, cardiovascular disease, acromegaly, giantism, Cushing's disease, hypersexuality, Hyperthyroidism, gonadotropin-related disease, ovulation suppression, sperm production suppression, Hashimoto's disease, thyroiditis, hypothyroidism and hypercalcemia, hypocalcemia, penile priapism, hypermucus secretion, hypersalivation, Treatment of bone tumors, diabetes, pancreatic disorders, tinnitus, cochlear nerve abnormalities, Meniere's disease, cancer, hair growth, alopecia areata, autonomic neuropathy, pressure ulcers, epilepsy, weight loss, uterine disorders, levator ani syndrome, or diabetic neuropathy Agent.

The C ₁ , D, E or F botulinum toxin preparation of the present invention is significantly less reactive at the site remote from the administered site than the existing type A or B botulinum toxin preparation used as a botulinum toxin preparation It is a formulation. Therefore, it is particularly useful as a drug for the safe and sufficient treatment of diseases that require administration of high doses of botulinum toxin exceeding the upper limit of administration of existing botulinum toxin preparations, such as spasticity of the extremities and dystonia. .

The figure which shows the CMAP amplitude value in the administration side muscle of A1 type LL toxin. The figure which shows the CMAP amplitude value in the administration side muscle of A2 type NTX. The figure which shows the CMAP amplitude value in the administration side muscle of B type NTX. It shows the CMAP amplitude value in administration side muscle type C ₁ NTX. The figure which shows the CMAP amplitude value in the administration side muscle of D type NTX. The figure which shows the CMAP amplitude value in the administration side muscle of E type NTX. The figure which shows the CMAP amplitude value in the administration side muscle of F type NTX. The figure which shows the CMAP amplitude value in the non-administration side muscle of A1 type LL toxin. The figure which shows the CMAP amplitude value in the non-administration side muscle of A2 type NTX. The figure which shows the CMAP amplitude value in the non-administration side muscle of B type NTX. It shows the CMAP amplitude value in non-administration side muscle type C ₁ NTX. The figure which shows the CMAP amplitude value in the non-administration side muscle of D type NTX. The figure which shows the CMAP amplitude value in the non-administration side muscle of E type NTX. The figure which shows the CMAP amplitude value in the non-administration side muscle of F type NTX.

Various aspects of the invention are described in detail below.
The C ₁ , D, E or F type botulinum toxins of the present invention are Sakaguchi et al. (Biochemical aspects of botulism: Purification and oral toxicities of Clostridium botulinum progenitor toxins., 21-34, Lewis GE., 1981, Academic Press, New York) It can be purified by the method.

The therapeutic agent of the present invention is preferably a pharmaceutical composition comprising a botulinum toxin type C ₁ , D, E or F and a botulinum toxin stabilizing substance.

  The botulinum toxin stabilizing substance is not particularly limited as long as it can stabilize the botulinum neurotoxin under the conditions in which the above composition is stored and does not impair the toxin activity of the botulinum toxin. For example, examples of botulinum toxin stabilizing substances include human serum albumin, amino acids, sugars, surfactants, or alternatives thereof.

A preferred pharmaceutical composition in the present invention can be produced by a step of mixing C ₁ , D, E or F type botulinum toxin with albumin.

C ₁ , D, E or F-type botulinum toxin can be purified by appropriately combining ion exchange chromatography, gel filtration, hydrophobic chromatography and the like. Specifically, the culture solution of Clostridium botulinum is sterilized by filtration, and the resulting M toxin is concentrated by a method such as UF membrane. Subsequently, the M toxin can be separated into a neurotoxin and a nontoxic protein by adjusting the pH to 7.2 or higher. Thereafter, for example, crude purification is performed by cation exchange chromatography, and fractions having toxin activity are collected and further purified by gel filtration. Toxin activity is measured by, for example, the intraperitoneal injection method of mice (method of determining the toxin activity from LD ₅₀ by intraperitoneal injection), and the mouse LD ₅₀ is defined as 1 U.

The steps after purification of the toxin are not particularly limited. For example, the C ₁ , D, E or F type botulinum toxin and the botulinum toxin stabilizing substance are dissolved in a solvent, subjected to sterile filtration, and filled into ampoules, vials, etc. A composition can be produced. Alternatively, the botulinum toxin can be dissolved in a liquid containing a botulinum toxin stabilizing substance in advance, and then aseptically filtered to fill an ampoule or the like. As the solvent, distilled water for injection, physiological saline, 0.01 M to 0.1 M phosphate buffer, and the like can be used, and ethanol, glycerin, and the like can be mixed as necessary.

Furthermore, the C ₁ , D, E or F type botulinum toxin and the botulinum toxin stabilizing substance are dissolved in a solvent, aseptically filtered, filled into a vial or the like, and lyophilized to produce the composition of the invention. Alternatively, the botulinum toxin and the botulinum toxin stabilizing substance can be mixed and then aseptically filled into a vial or the like to produce the pharmaceutical composition of the present invention.

Specifically, purified C ₁ , D, E or F type botulinum toxin is converted into a botulinum toxin stabilizing substance, preferably human serum albumin, more preferably human serum albumin for therapeutic use that ensures safety in humans. In addition, the final concentration may be 0.1 to 5 mg / mL, preferably 0.5 to 2 mg / mL, followed by refrigerated storage, frozen storage, or lyophilization.

  If necessary, the therapeutic agent of the present invention can be further mixed with additives such as saccharides such as mannitol, glucose, sucrose, fructose, and lactose. The pH of the pharmaceutical composition according to the present invention in a dissolved state is usually 3 to 8, preferably 4 to 7, and more preferably 5 to 7.

In the therapeutic agent of the present invention, the C ₁ , D, E or F type botulinum toxin may be contained in an amount effective for the purpose of use of the present invention. When a botulinum toxin stabilizing substance is included, the botulinum toxin stabilizing substance only needs to be included in an amount sufficient to stabilize the botulinum neurotoxin.

  The therapeutic agent of the present invention is less reactive at the non-administration site compared to existing type A or type B botulinum toxin preparations, and the recommended upper limit of administration of existing botulinum toxin preparations (for example, 400 U for adults) It is particularly useful as a drug for safely and sufficiently treating diseases that require administration of high doses of botulinum toxin exceeding 4 tons, such as spasticity of limb trunk and dystonia.

  The therapeutic agent of the present invention is administered in a therapeutically effective amount. When administered to humans, the dosage form is preferably local administration, more preferably intramuscular injection. In addition, the administration timing and dose thereof are not particularly limited, and vary depending on the degree of symptoms. The dosage varies depending on the degree of symptoms, age, sex, body weight, administration site and form.

For example, in the case of C ₁ botulinum toxin type formulations, for an adult weighing 60 kg, in one treatment, the total 0.77～115,500 U, preferably at 7.7～115,500 U, more preferably a 3,100～115,500 U, More preferably, 4,600-115,500 U is injected intramuscularly, more preferably 7,700-115,500 U.
In the case of a botulinum toxin type D preparation, a total of 290 to 10,929,900 U, preferably 2,900 to 10,929,900 U, more preferably 120,000 to 10,929,900 U, more preferably, in one treatment for an adult weighing 60 kg 1,800,000 to 10,929,900 U, more preferably 2,900,000 to 10,929,900 U is injected intramuscularly.
In the case of a botulinum toxin type E preparation, a total of 1.2 to 15,000 U, preferably 12 to 15,000 U, more preferably 4,900 to 15,000 U, more preferably, in one treatment for an adult weighing 60 kg 7,300-15,000 U is more preferably injected intramuscularly, 12,000-15,000 U.
In the case of a botulinum toxin type F preparation, for a 60 kg adult, a total of 6.7 to 1,131,600 U, preferably 67 to 1,131,600 U, more preferably 27,000 to 1,131,600 U, more preferably, in one treatment 40,000 to 1,131,600 U, more preferably 67,000 to 1,131,600 U is injected intramuscularly.

  After the injection, treatment is performed while confirming that there are no serious side effects, no significant reduction in local tension outside the site to be treated, and that the function of the muscle to be treated has been improved.

The present invention relates to C ₁ , D, E or F type botulinum toxins for diseases requiring administration of high doses of botulinum toxin exceeding the upper limit of administration of existing type A botulinum toxin preparations such as limb trunk spasticity and Provided is a therapeutic method characterized by being used as a therapeutic agent for dystonia or the like.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

Example 1: Purification of each serotype botulinum toxin
BOTOX (registered trademark: Allergan) was used as the A1 type LL toxin. Botulinum A2, B, C ₁ , D, E and F type botulinum toxin neurotoxins (NTX) are described in Sakaguchi et al. (Biochemical aspects of botulism: Purification and oral toxicities of Clostridium botulinum progenitor toxins., 21-34, Lewis GE., 1981, Academic Press, New York). Chiba-H strain, Okra strain, CB-19 strain, 1873 strain, 35396 strain and Langeland strain were used as A2, B, C ₁ , D, E and F type botulinum toxin production strains, respectively. A PYG medium containing 2% peptone, 0.5% yeast extract, 0.5% glucose and 0.025% sodium thioglycolate was used as the medium for A2, B, E and F botulinum. The medium C ₁ and D type Clostridium botulinum, to 0.8% glucose, 0.5% starch, 1.0% yeast extract, to produce a basal medium containing 1.0% ammonium sulfate and 0.1% cysteine hydrochloride, basal medium 100 mL, A medium containing 6 g of cooked meat and 0.5% calcium carbonate was used. In each case, after static culture, the complex toxin was purified by acid precipitation, protamine treatment, ion exchange chromatography and gel filtration. Furthermore, each type of M toxin (about 500 kDa) was adsorbed on a DEAE Sepharose column equilibrated with 10 mM phosphate buffer (pH 7.5) and then eluted with a 0 to 0.3 M NaCl concentration gradient in the same buffer. The product was separated into a neurotoxin component (NTX) having a molecular weight of about 150 kDa and a nontoxic protein (NTNH). The obtained NTX was stored at -70 ° C until use.

<< Example 2: Potency test of each serotype toxin >>
LD ₅₀ was measured by the intraperitoneal administration method of mice. The LD ₅₀ test was performed with 7 doses, interval between doses x 1.25, each 20 doses, the cumulative mortality was calculated from life and death 96 hours after administration, and LD ₅₀ was calculated by the probit method. As a mouse, a female ICR / CD-1 mouse (4 weeks old, about 20 g, Nippon Charles River) was used. The mouse breeding room was light and dark controlled, and was kept in an environment where food and water could be ingested at will.

Example 3 Evaluation of efficacy and safety of botulinum toxins A to F by rat test
Each toxin was diluted with saline containing 0.5 w / v% human serum albumin, A1 and A2 type is 0.1~300 U / mL, B type 100-100000 U / mL, C ₁ type is 1 to 1 10,000 U / mL, D type 300 to 100,000 U / mL, E type 1 to 3000 U / mL and F type 10 to 100,000 U / mL, respectively. Toxins were administered intramuscularly by 0.1 mL into the gastrocnemius muscle of the rat left hind limb (n = 5 for each group). CMAP measurement was performed as follows using the Nicolet Viking Quest (Nikolay). The rats used were female S / D rats (8 weeks old, approximately 200 g, Nippon Charles River). Rats were kept in an environment where light and dark were controlled, and food and water could be voluntarily consumed. Rats were anesthetized by intraperitoneally administering about 40 mg / kg of pentobarbital sodium (Somnopentyl, Kyoritsu Pharmaceutical). After confirming the disappearance of the eyelid reflex, the measurement was performed with the rat in the prone position. The electrodes were placed on the root of the spinal cord, the recording electrode (+) on the gastrocnemius muscle of the hind limb, the recording electrode (−) on the gastrocnemius tendon of the hind limb, and the ground electrode on the ridge. Electrical stimulation was performed at 25 mA and 0.2 msec, and CMAP amplitude was measured twice on both the administration side and non-administration side, and the average was used for the data. CMAP measurement was performed before administration and 1, 2, 4, 7 and 14 days after administration, and changes over time were examined.

In order to compare the efficacy of each type of NTX, regression analysis was performed with the CMAP amplitude value at the time of administration side CMAP amplitude value after toxin administration being minimized and the amount of toxin activity on the horizontal axis, and the administration side before administration The amount of toxin activity that reduces the CMAP amplitude value by 50% was defined as the 50% effective amount (ED ₅₀ ). Similarly, the non-administration side CMAP amplitude value at the time when the non-administration side CMAP amplitude value is minimized is also analyzed on the non-administration side CMAP amplitude value on the vertical axis and the toxin activity level on the horizontal axis. The toxin activity amount that reduces the urine concentration by 20% was defined as a 20% toxic dose (TD ₂₀ ), and this TD ₂₀ was used as a safety indicator. Furthermore, the value obtained by dividing TD ₂₀ by ED ₅₀ for each toxin was defined as the “treatment area” of each toxin. This therapeutic window is a suitable index for evaluating how many times the effective dose can be safely administered for each toxin.

The time course of the CMAP amplitude value of the left and right gastrocnemius of each toxin is shown in FIGS. Table 1 shows the ED ₅₀ and TD ₂₀ (U / head) and treatment area of each toxin obtained.

From the above results, the following became clear.
The therapeutic range of botulinum toxin type ₁ was found to be about 40 times wider than that of type A botulinum toxin and about 103 times wider than that of type B botulinum toxin. That, C ₁ botulinum toxin type about 40 times greater than botulinum toxin type A, about 103 times greater than botulinum toxin type B has been shown to be administered safely many effective amount. If the sum is a TD ₂₀ of the study extrapolated upper limit of the total dose in humans, C ₁ botulinum toxin type weighing approximately 0.2 kg per 385 U (1,925 U per 1 kg), in a human weighing 60 kg 115,500 U can be administered. From the ED ₅₀ results, 115,500 U of type ₁ botulinum toxin is considered to be a dose equivalent to 14,972 U of botulinum toxin type A.

The therapeutic range of botulinum toxin type D was about 10 times wider than that of type A botulinum toxin and about 25 times wider than that of type B botulinum toxin. In other words, it was shown that botulinum toxin type D can be safely administered in an effective amount that is about 10 times that of type A botulinum toxin and about 25 times that of type B botulinum toxin. If we extrapolate the TD ₂₀ of this study as the upper limit of the total dose in humans, botulinum toxin type D is 36,433 U (182,165 U per kg) and a total of 10,929,900 U in humans weighing 60 kg. Can be administered. From the ED ₅₀ results, botulinum toxin type D 10,929,900 U is considered to be a dose equivalent to 3,714 U of type A botulinum toxin.

It was revealed that the therapeutic range of botulinum toxin type E was about 3 times wider than that of type A botulinum toxin and about 8 times wider than that of type B botulinum toxin. In other words, it was shown that E-type botulinum toxin can be safely administered in an effective amount that is about 3 times that of botulinum toxin A and about 8 times that of botulinum toxin B. If we extrapolate the TD ₂₀ of this study as the upper limit of the total dose in humans, botulinum toxin E is 50 U / 250 kg (250 U / kg) and 15,000 U / 60 kg. Can be administered. From the ED ₅₀ results, 15,000 U of botulinum toxin type E is considered to be a dose equivalent to 1,235 U of botulinum toxin type A.

The therapeutic range for botulinum toxin type F was found to be approximately 45 times wider than that of type A botulinum toxin and about 115 times wider than that of type B botulinum toxin. In other words, it was shown that botulinum toxin type F can be safely administered in an effective amount approximately 45 times that of botulinum toxin type A and approximately 115 times that of botulinum toxin type B. If the TD ₂₀ of this study is extrapolated as the upper limit of the total dose in humans, botulinum toxin type F is 3,772 U (18,860 U per kg) and a total of 1,131,600 U in a human weighing 60 kg. Can be administered. From the ED ₅₀ results, botulinum toxin type F 1,131,600 U is considered to be a dose equivalent to 16,962 U of type A botulinum toxin.

Considering the duration of the treatment zone and effect, believed to type C ₁ is suitable for most large doses, followed by F-type, E, is considered suitable in order type D.

Claims (10)

Contains as an active ingredient a botulinum toxin selected from C ₁ , D, E or F botulinum toxin, characterized in that it can be safely administered in a larger effective amount compared to botulinum toxin type A Pharmaceutical composition. The pharmaceutical composition according to claim 1, wherein an effective amount of the botulinum toxin exceeding the upper limit of administration of the type A botulinum preparation can be administered. The pharmaceutical composition according to claim 1 or 2, wherein the botulinum toxin is a neurotoxin having a molecular weight of about 150 KDa from which non-toxic components have been removed. The pharmaceutical composition according to any one of claims 1 to 3, comprising albumin, amino acid, sugar or surfactant as a stabilizer. Pharmaceutical compositions containing the type C ₁ botulinum toxin pharmaceutical composition according to any one of 1 kg body weight per 1,925 U to the safely administrable claims 1-4. The pharmaceutical composition according to any one of claims 1 to 4, wherein the pharmaceutical composition containing the botulinum toxin type D can be safely administered up to 182,165 U / kg body weight. The pharmaceutical composition according to any one of claims 1 to 4, wherein the pharmaceutical composition containing botulinum toxin type E can be safely administered up to 250 U per kg body weight. The pharmaceutical composition according to any one of claims 1 to 4, wherein the pharmaceutical composition containing the botulinum toxin type F can be safely administered up to 18,860 U / kg body weight. 9. A pharmaceutical composition according to any one of claims 1 to 8, headache such as strabismus, blepharospasm, unilateral facial convulsions, spastic torticollis, paralysis after stroke, cerebral palsy, spastic vocal disturbance, spasticity, migraine, chronic Pain, low back pain, pelvic pain, postherpetic neuralgia, neuropathic pain, stiff shoulders, muscle insufficiency at the onset of Parkinson's disease or multiple sclerosis, fascial pain syndrome, masticatory muscle spasm, chronic anal fissure, detrusor / Sphincter dysfunction, overactive bladder, prostate disorder, hyperhidrosis, bruxism, facial myokemia, tics, local dystonia, chalazion, stye, carpal tunnel syndrome, hypertrophic lip deformity, hallux valgus, hallux valgus , Abnormal toe, hard thigh, idiopathic clubfoot, pepsin ulcer, gastroesophageal reflux disease, esophageal achalasia, gastric paresis, vaginal spasm, arthropathy, epicondylitis, rotator cuff, fascia , Tennis elbow, thorax exit syndrome, Schizophrenia due to excessive activity of neurotransmitters, Alzheimer's disease, mania, depression, cardiovascular disease, acromegaly, giantism, Cushing's disease, hypersexuality, hyperthyroidism, gonadotropin-related disease, ovulation suppression , Sperm production suppression, Hashimoto's disease, thyroiditis, hypothyroidism and hypercalcemia, hypocalcemia, penile priapism, hypersecretion of mucus, hypersalivation, bone tumor, diabetes, pancreatic disorder, tinnitus, cochlea Use for neurological abnormalities, Meniere's disease, cancer, hair growth, alopecia areata, autonomic neuropathy, pressure ulcers, epilepsy, weight loss, uterine disorders, levator ani syndrome, or diabetic neuropathy. A strabismus, blepharospasm, hemifacial spasm, spastic neck, paralysis after stroke, cerebral palsy, spastic dysphonia, spasticity, comprising the pharmaceutical composition according to any one of claims 1 to 8. Migraine and other headaches, chronic pain, low back pain, pelvic pain, postherpetic neuralgia, neuropathic pain, stiff shoulders, muscle relaxation disorder that occurs at the onset of Parkinson's disease and multiple sclerosis, fascial pain syndrome, masticatory muscle spasm , Chronic anal fissure, detrusor / sphincter dysfunction, overactive bladder, prostate disorder, hyperhidrosis, bruxism, facial myokia, tics, local dystonia, chalazion, stye, carpal tunnel syndrome, blood volume increased lip deformity , Hallux valgus, hallux valgus, thumb anomaly, hard thigh, idiopathic clubfoot, pepsin ulcer, gastroesophageal reflux disease, esophageal achalasia, gastric paralysis, vaginal spasticity, arthropathy, epicondylitis, Rotator cuff, fasciopathy, teni Elbow, thoracic outlet syndrome, acne, schizophrenia due to neurotransmitter overactivity, Alzheimer's disease, mania, depression, cardiovascular disease, acromegaly, giantosis, Cushing's disease, hyperthyroidism, hyperthyroidism, Gonadotropin related diseases, ovulation suppression, sperm production suppression, Hashimoto's disease, thyroiditis, hypothyroidism and hypercalcemia, hypocalcemia, persistent penile erectile dysfunction, hypermucus secretion, hypersalivation, bone tumor, diabetes, Therapeutic agent for pancreatic disorder, tinnitus, cochlear nerve abnormality, Meniere's disease, cancer, hair growth, alopecia areata, autonomic disorder, pressure ulcer, epilepsy, weight loss, uterine disorder, levator ani syndrome, or diabetic neuropathy.

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