+

WO1996039166A1 - Analogues de toxine de botulinium et compositions pharmaceutiques de toxine de botulinium - Google Patents

Analogues de toxine de botulinium et compositions pharmaceutiques de toxine de botulinium Download PDF

Info

Publication number
WO1996039166A1
WO1996039166A1 PCT/US1996/000075 US9600075W WO9639166A1 WO 1996039166 A1 WO1996039166 A1 WO 1996039166A1 US 9600075 W US9600075 W US 9600075W WO 9639166 A1 WO9639166 A1 WO 9639166A1
Authority
WO
WIPO (PCT)
Prior art keywords
botulinum toxin
toxin
botulinum
neurotoxin
analog
Prior art date
Application number
PCT/US1996/000075
Other languages
English (en)
Inventor
Eric A. Johnson
Michael C. Goodnough
Original Assignee
Wisconsin Alumni Research Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wisconsin Alumni Research Foundation filed Critical Wisconsin Alumni Research Foundation
Priority to AU47460/96A priority Critical patent/AU4746096A/en
Publication of WO1996039166A1 publication Critical patent/WO1996039166A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to novel analogs of the botulinum toxin and pharmaceutical compositions containing botulinum toxin.
  • botulism The most serious form of bacterial food poisoning is botulism which is caused by toxins produced by Clo ⁇ tridi um botulinum.
  • the toxins are usually preformed by the causative organism in foods and subsequently absorbed through the intestinal tract and transported via the circulatory system to motor nerve synapses where their action blocks normal neural transmissions.
  • Seven known serotypes of botulinum toxins (type A to G) with similar toxic activity but which differ antigenically have been isolated and characterized. Serotype A toxin is the predominant cause of botulism in the United States while type B toxin is most prevalent in Europe.
  • a single batch of crystalline type A botulinum toxin complex was prepared in 1979 by E. J. Schantz of the Food Research Institute/Department of Food Microbiology and Toxicology at the University of Wisconsin-Madison, U.S.A. which has been used medically to treat hyperactive muscle disorders such as strabismus, blepharospasm, spasmodic torticollis, and many other diseases (Jankovic and Hallet "Therapy with Botulinum Toxin” (1994)). Treatment involves injection of nanogram quantities of the toxin complex directly into the neuromuscular tissue of hyperactive muscles.
  • the botulinum neurotoxin in the toxin complex inhibits the release of acetylcholine across the synaptic junction causing a decrease in the activity of the injected muscles.
  • Type A neurotoxin produced by C. botulinum is present as part of a toxin complex of at least seven different noncovalently bound proteins . Both the toxin complex and the purified neurotoxin can be used in medicine and both are referred to as "botulinum toxin" herein.
  • High quality type A toxin complex has a specific toxicity of 3 x 10 7 mouse intraperitoneal 50% lethal doses (LD 50 ) per mg.
  • the purified type A neurotoxin that is the neurotoxin that has been chromatographically separated from the other proteins of the toxin complex, has a specific toxicity of 9 X 10 7 to 1 x 10 8 LD 50 per mg. In the medical field, a unit (U) is considered to be 1 mouse LD 50 .
  • Toxin titers are determined in female, white mice, 18-22g in weight according to the method of Schantz and Kautter as described in Association of Official and Analytical Chemistry, vol. 61, p. 96, (1978). Evidence is accumulating that different types of botulinum toxin may bind to different acceptors than type A and may have differences in their mode of action and clinical applications .
  • a major drawback to the use of pharmaceutical compositions containing either botulinum toxin complex or the pure neurotoxin in treatment of hyperactive muscle disorders is that the duration of action in neuromuscular tissue of botulinum toxin in the available products currently is relatively short (i.e., a few days to several months, depending on the indication being treated) and thus frequent injections are required by the patients .
  • botulinum toxin containing compositions which have longer half-lives in. vivo in neuromuscular tissue than known botulinum toxin containing compositions. This would enable patients to be injected less often and would reduce the incidence of antibodies since immunologic tolerance to botulinum toxin is correlated with cumulative doses .
  • compositions containing botulinum toxin which have longer half-lives than known compositions.
  • compositions containing novel analogs of natural botulinum neurotoxins that have more resistant amino acids in degradable sites outside of the active toxin sites have longer half-lives in neuromuscular tissue than the corresponding natural botulinum toxins.
  • compositions containing a botulinum toxin and a Ca ++ chelating agent prevents the degradation of the neurotoxin in neuromuscular tissue resulting in longer half-lives .
  • the use of chelating agents or the preventing of the uptake of Ca++ in neuromuscular tissue will prolong the half-life of toxin activity.
  • botulinum toxin preparations with high specific toxicities e.g. a properly prepared complex of about 30U/mg; or pure neurotoxins of about 90-100U/mg
  • the use of the botulinum toxin preparations with high specific toxicities is preferred because it reduces the amount of toxin required to obtain the necessary number of active U per vial as mandated by the United States Food and Drug Administration. This also reduces the amount of inactive toxin (toxoid) in each vial and thereby lessens the possibility of antibody formation after injection of the preparation into patients.
  • the preferred pharmaceutical compositions of the present invention have the following composition:
  • Serum albumin 0.5-1 mg. per vial. It may also be desired to include 1-5 mg of a chelating agent for Ca ++ (e.g. 1-5 mg of EDTA and EGTA) and trehalose for shelf stability.
  • a chelating agent for Ca ++ e.g. 1-5 mg of EDTA and EGTA
  • trehalose for shelf stability.
  • the Hall A strain of type A C. botulinum (deposited with the ATCC) is used to produce type A botulinum toxin. This strain is routinely used for production of type A botulinum toxin because of high toxin titers and the rapid onset of cell lysis (usually within 48 h).
  • the terms "Type A toxin” and “Type A neurotoxin” as used herein are intended to cover the toxin and neurotoxin of all strains because they exhibit only minor variations in sequence.
  • TPM toxin production medium
  • NZ amine or TT Sheffield Laboratories, Norwich, NY U.S.A.
  • yeast extract Difco
  • dextrose pH 7.37.4, for 5-7 days at 37oC.
  • Type A crystalline complex is purified by a modification of the method of Duff et al, J. Bacteriol, 13_, pp. 42-47 (1957).
  • type A toxin is then purified according to the method described in the
  • toxin complex is recovered from DEAE-Sephadex A50 (Sigma Chemical Co., St. Louis, MO U.S.A.), pH 5.5, column and is precipitated by addition of 39 g of solid ammonium sulfate/lOOml .
  • the precipitated toxin complex is collected by centrifugation, dialyzed against 25 mM sodium phosphate, pH 7.9, and applied to a DEAE-Sephadex A50 column equilibrated with the same buffer.
  • Toxin is separated from the non-toxic proteins of the complex and eluted from the column with a linear 0-0.5M sodium chloride gradient.
  • Partially purified neurotoxin is recovered from the DEAE-Sephadex A50 column at pH 7.9 and dialyzed against 25 mM sodium phosphate, pH 7.0.
  • the dialyzed toxin is applied to SP-Sephadex C50 (Sigma Chemical Co.) in 25 mM sodium phosphate, pH 7.0. Contaminating material does not bind to the column under these conditions.
  • the neurotoxin is eluted with a linear 0-0.25 M sodium chloride gradient.
  • the neurotoxin can be further purified by metal affinity chromato- graphy, gel filtration, high pressure liquid chromatography or other methods of protein chromatograph .
  • the Ca ++ chelating agents preferred for use in the compositions of the present invention are EDTA and EGTA.
  • the amount of chelating agent to be included is about 1 mg to about 5 mg for each 100U of the botulinum neurotoxin that is present either alone or calculated to be present in the botulinum toxin complex.
  • trehalose allows for the recovery of active type A neurotoxin following lyophilization and storage at ambient temperatures (37°C) which are in excess of those required for storage of the commercially available type A toxin complex (-10°C).
  • the amount of trehalose that should be present is the amount that is necessary to stabilize the composition so that it can be stored at ambient temperatures.
  • the composition will contain about 10 mg of trehalose per vial of (50 to 100 mg/ml prelyophilized solution) .
  • compositions of the preferred compositions will also contain an effective amount of a stabilizing protein, such as human serum albumin.
  • a stabilizing protein such as human serum albumin.
  • the serum albumin will be present in about 0.5 mg per vial (1/10 mg/ml of prelyophilized solution).
  • other known stabilizing proteins including bovine serum albumin, can be used in the compositions of the present invention.
  • the preferred analogs of the botulinum neurotoxin of the present invention are those which possess the beneficial medicinal activity of the natural botulinum neurotoxin serotype and which differ from the natural botulinum neurotoxin only in that they have been genetically modified to resist proteolytic degradation in neuromuscular tissue (e.g. a threonine residue at the 540 amino acid position in place of the tryosine residue of the natural product).
  • the genetically modified product is more resistant to proteolytic breakdown.
  • Other residues in sensitive sites can also be modified by analogous site specific mutagenesis .
  • botulinum neurotoxin which differ from the natural neurotoxin in that they contain modification of susceptible amino acid pairs (e.g. tyrosine, arginine, tryptophan pairings) in the sites subject to degradation in neuromuscular tissue, are more resistant to proteolytic degradation.
  • susceptible amino acids e.g. tyrosine, arginine, tryptophan pairings
  • the susceptible amino acids are replaced with residues that resist proteolytic degradation (e.g. tyrosine is replaced by a threonine).
  • modified toxins possess improved half-lives in neuromuscular tissue as compared to natural botulinum toxin.
  • the method for preparing the proteolytic degradation resistant botulinum neurotoxin analogs basically comprises replacing an amino acid residue in a pairing at a site(s) subject to degradation outside the active sites in the natural neurotoxin with another amino acid residue which is more resistant to acid and/or proteolytic attack or degradation.
  • the method of the present invention may be practiced by using cloning, site-specific mutagenesis and/or cassette mutagenesis to modify the gene encoding the natural or wild type neurotoxin and by subsequent introduction of the modified gene into a microorganism that overproduces the modified neurotoxin.
  • the analogs of the neurotoxin are prepared by the use of site-directed mutagenesis.
  • An especially preferred method comprises preparing a DNA transfer vector containing a DNA sequence which encodes for the neurotoxin but which has an amino acid residue which is more resistant to degradation in a site which is subject to degradation in vivo in the natural neurotoxin; transforming the DNA transfer vector into a microorganism, such as .E. coli; and then culturing the microorganism under conditions suitable for the expression of the botulinum neurotoxin analog.
  • Type A botulinum neurotoxin that can be made in the light chain (the catalytic region of botulinum neurotoxin) are the following: (a) An analog having a threonine residue in place of the tyrosine residue at site 540.
  • compositions of the present invention are preferably supplied as lyophilized products.
  • a solution containing the active crystalline toxin, neurotoxin or genetically modified toxin or analog, a protective protein, such as serum albumin and the water for injection (USP) is placed in glass vials, Teflon lined screw cap closures are fastened loosely, and the solutions quickly frozen in liquid nitrogen.
  • the formulations may include a Ca ++ chelating agent and a carbohydrate, such a trehalose for shelf stability.
  • the frozen solutions in the vials are placed into a lyophilization vessel which is then immersed in liquid nitrogen. The vessel is then connected to a freeze-drier. When the pressure drops below ca.
  • the lyophilized preparations were usually reconstituted by the addition of 1.0 ml of distilled water or 0.85% saline for injection.
  • compositions also differ from the commercially available products in that they do not contain sodium chloride which has been found to reduce the recovery of active toxin following lyophilization.
  • the primary advantage of preferred compositions of the present invention is their longer half-lives in the presence of tissue proteolytic enzymes .
  • Example 1 The invention is further illustrated by the following examples .
  • Example 1 The invention is further illustrated by the following examples .
  • Example 1
  • a suitable plasmid is digested with restriction of enzymes, such as EcoRi and BamHI .
  • a DNA fragment, containing the entire type A neurotoxin gene and its promoter region is isolated and subcloned into the EcoRi/BamHI sites of the plasmid.
  • the oligonucleotide primers obtained from Genetic Designs, Inc., (Houston, TX U.S.A.), are designed to be complementary to the single-strand template DNA and to contain appropriate mismatches. Synthesis of genes for the analogs and selection are performed using a kit of Oligonucleotide-directed Mutagenesis System, version 2 (Amersham) . Nucleotide sequences of the mutant genes obtained can be confirmed by the dideoxy sequencing method.
  • the enzyme (EcoRI/BamHI ) fragments, containing the mutant genes are subcloned from each recombinant into the EcoRI and BamHI sites of vectors, and introduced into a suitable E . coli strain, such as HB101 (ATCC 33694).
  • the E_. coli cells, carrying appropriate recombinant plasmids that specify the mutant analogs are cultivated overnight in LB media, containing 100 ⁇ q/ml A picillin, at 37°C with vigorous shaking. Cells are harvested by centrifugation at 5,000 x g, washed and suspended in 50 mM MPOS buffer (pH 7.0) containing 10 mM MgSO and 1 mM CoCl 2 . For preparation of cell extracts, cells are broken by two passages through a French pressure cell at 18,000 p.s.i., and the debris removed by centrifugation at 12,000 x g for 20 in. The cell extracts are stirred at 85°C for 20 min.
  • Example 2 The light chain of botulinal toxin (the chain that causes poisoning) was purified and incubated with brain extract of rabbits. Using conventional techniques it was shown that the toxin was cleaved by proteases present in the nerve extract. The cleavage was found to be dependent on calcium. Using a purified Ca ++ -dependent protease it was shown that the light chain was cleaved at only one site (540) yielding two fragments of about the same size. The cleavage was Ca ++ dependent and it was found that it could be prevented by using chelators that bind CA, including EGTA and EDTA.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Cette invention concerne de nouveaux analogues de toxine de botulinium qui contiennent des résidus d'acides aminés plus résistant à la dégradation dans les tissus neuromusculaires que les résidus d'acides aminés contenus dans les toxines naturelles correspondantes. Cette invention concerne également des compositions pharmaceutiques à base de toxine de botulinium, qui contiennent des agents chélateurs pour ions métalliques, et qui ont une demi-vie plus longue dans les tissus neuromusculaires que celle des préparations courantes.
PCT/US1996/000075 1995-06-06 1996-01-11 Analogues de toxine de botulinium et compositions pharmaceutiques de toxine de botulinium WO1996039166A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU47460/96A AU4746096A (en) 1995-06-06 1996-01-11 Analogs of botulinum toxin and pharmaceutical compositions o f botulinum toxin

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US46938795A 1995-06-06 1995-06-06
US08/469,387 1995-06-06

Publications (1)

Publication Number Publication Date
WO1996039166A1 true WO1996039166A1 (fr) 1996-12-12

Family

ID=23863594

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/000075 WO1996039166A1 (fr) 1995-06-06 1996-01-11 Analogues de toxine de botulinium et compositions pharmaceutiques de toxine de botulinium

Country Status (2)

Country Link
AU (1) AU4746096A (fr)
WO (1) WO1996039166A1 (fr)

Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002008268A3 (fr) * 2000-07-21 2003-02-20 Allergan Inc Motif a base de leucine et neurotoxines clostridiales
WO2002040506A3 (fr) * 2000-11-17 2003-03-27 Allergan Inc Neurotoxines clostridiales modifiees a persistance biologique alteree
WO2006116302A2 (fr) * 2005-04-22 2006-11-02 Botulinum Toxin Research Associates, Inc. Preparations de toxines botulinum de haute puissance
US7491403B2 (en) 2002-12-20 2009-02-17 Botulinum Toxin Research Associates Pharmaceutical botulinum toxin compositions
US7491799B2 (en) 2000-07-21 2009-02-17 Allergan, Inc. Modified botulinum neurotoxins
US7691983B2 (en) 2000-07-21 2010-04-06 Allergan, Inc. Chimera botulinum toxin type E
US7888469B2 (en) 2000-11-17 2011-02-15 Allergan, Inc. Post-translation modification and clostridial neurotoxins
EP2445521A4 (fr) * 2009-06-25 2015-08-26 Revance Therapeutics Inc Formulations à base de toxine botulique sans albumine
WO2015132004A1 (fr) 2014-03-05 2015-09-11 Merz Pharma Gmbh & Co. Kgaa Neurotoxines clostridiales de recombinaison d'un nouveau type présentant une durée d'effet accrue
WO2016198163A1 (fr) 2015-06-11 2016-12-15 Merz Pharma Gmbh & Co. Kgaa Nouvelles neurotoxines clostridiales recombinées présentant une durée d'effet accrue
WO2017125487A1 (fr) 2016-01-20 2017-07-27 Merz Pharma Gmbh & Co. Kgaa Nouvelles neurotoxines clostridiales recombinantes à durée d'effet augmentée
JP2018502580A (ja) * 2015-01-09 2018-02-01 イプセン・バイオイノベーション・リミテッドＩｐｓｅｎ Ｂｉｏｉｎｎｏｖａｔｉｏｎ Ｌｉｍｉｔｅｄ 陽イオン性神経毒
US9901627B2 (en) 2014-07-18 2018-02-27 Revance Therapeutics, Inc. Topical ocular preparation of botulinum toxin for use in ocular surface disease
US9950042B2 (en) 2008-12-04 2018-04-24 Revance Therapeutics, Inc. Extended length botulinum toxin formulation for human or mammalian use
WO2018233813A1 (fr) 2017-06-20 2018-12-27 Merz Pharma Gmbh & Co. Kgaa Nouvelles toxines botuliniques recombinantes présentant une durée d'effet accrue
WO2019101308A1 (fr) 2017-11-22 2019-05-31 Merz Pharma Gmbh & Co. Kgaa Nouvelles toxines botuliniques recombinées ayant une durée d'effet accrue
JP2019206582A (ja) * 2019-08-09 2019-12-05 イプセン バイオイノベーション リミテッド 陽イオン性神経毒
US10857215B2 (en) 2012-04-12 2020-12-08 Revance Therapeutics, Inc. Use of botulinum toxin for the treatment of cerebrovascular disease, renovascular and retinovascular circulatory beds
US11155802B2 (en) 2017-07-06 2021-10-26 Merz Pharma Gmbh & Co. Kgaa Recombinant botulinum neurotoxins with increased duration of effect
US11484580B2 (en) 2014-07-18 2022-11-01 Revance Therapeutics, Inc. Topical ocular preparation of botulinum toxin for use in ocular surface disease
WO2023156385A1 (fr) * 2022-02-15 2023-08-24 Merz Pharma Gmbh & Co. Kgaa Formulation de toxine botulique liquide et son utilisation
TWI838804B (zh) * 2021-11-11 2024-04-11 南韓商伊妮寶有限公司 肉毒桿菌毒素乾餅的製備方法
US11969461B2 (en) 2016-03-02 2024-04-30 Merz Pharma Gmbh & Co. Kgaa Composition comprising botulinum toxin
US12144847B2 (en) 2016-09-13 2024-11-19 Allergan, Inc. Non-protein clostridial toxin compositions

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994000481A1 (fr) * 1992-06-23 1994-01-06 Associated Synapse Biologics Composition pharmaceutique contenant un complexe de botulinum b
EP0593176A2 (fr) * 1992-09-28 1994-04-20 Wisconsin Alumni Research Foundation Compositions pharmaceutiques contenant la toxine de botulinum et procédé de préparation
WO1995005842A1 (fr) * 1993-08-26 1995-03-02 The Johns Hopkins University Procede et dispositif pour traiter les troubles musculaires gastro-intestinaux et autres dysfonctionnements des muscles lisses

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994000481A1 (fr) * 1992-06-23 1994-01-06 Associated Synapse Biologics Composition pharmaceutique contenant un complexe de botulinum b
EP0593176A2 (fr) * 1992-09-28 1994-04-20 Wisconsin Alumni Research Foundation Compositions pharmaceutiques contenant la toxine de botulinum et procédé de préparation
WO1995005842A1 (fr) * 1993-08-26 1995-03-02 The Johns Hopkins University Procede et dispositif pour traiter les troubles musculaires gastro-intestinaux et autres dysfonctionnements des muscles lisses

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GOODNOUGH, M.C. AND E.A. JOHNSON: "Stabilization of Clostridium-Botulinum Neurotoxin during Lyophilization", 205TH ACS (AMERICAN CHEMICAL SOCIETY) NATIONAL MEETING, DENVER, COLORADO, USA, MARCH 28-APRIL 2, 1993. ABSTR. PAP. CHEM. SOC. 205 (1-2). 1993, ABSTR. NO. 149, XP002002967 *
SCHANTZ, E.J. AND E.A. JOHNSON, JANKOVIC, J. AND M. HALLETT (ED.). NEUROLOGICAL DISEASE AND THERAPY, VOL. 25. THERAPY WITH BOTULIMUN TOXIN. XXVI+608P. MARCEL DEKKER, INC.: NEW YORK, NEW YORK, USA; BASEL, SWITZERLAND, 1994, pages 41 - 49, XP002002969 *
SCHANTZ, E.J. AND E.A. JOHNSON, MICROBIOLOGICAL REVIEWS, vol. 56, 1992, pages 80 - 99, XP002002968 *

Cited By (51)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8206723B2 (en) 2000-07-21 2012-06-26 Allergan, Inc. Leucine-based motif and clostridial neurotoxins
US7705124B2 (en) 2000-07-21 2010-04-27 Allergan, Inc. Leucine-based motif and clostridial neurotoxins
WO2002008268A3 (fr) * 2000-07-21 2003-02-20 Allergan Inc Motif a base de leucine et neurotoxines clostridiales
US8017741B2 (en) 2000-07-21 2011-09-13 Ester Fernandez-Salas Chimera botulinum toxin type E
AU2001280703B2 (en) * 2000-07-21 2006-08-31 Allergan, Inc. Leucine-based motif and clostridial neurotoxins
US8008465B2 (en) 2000-07-21 2011-08-30 Allergan, Inc. Nucleic acids encoding chimera botulinum toxin type E
US7723480B2 (en) 2000-07-21 2010-05-25 Allergan, Inc. Leucine-based motif and clostridial neurotoxins
EP1849801A1 (fr) * 2000-07-21 2007-10-31 Allergan Sales, Inc. Motif à base de tyrosine et neurotoxines clostridiales
US7393925B2 (en) 2000-07-21 2008-07-01 Allergan, Inc. Leucine-based motif and Clostridial neurotoxins
CN100457777C (zh) * 2000-07-21 2009-02-04 阿勒根公司 基于亮氨酸的基序和梭菌神经毒素
US7705125B2 (en) 2000-07-21 2010-04-27 Allergan, Inc. Leucine-based motif and Clostridial neurotoxins
US7491799B2 (en) 2000-07-21 2009-02-17 Allergan, Inc. Modified botulinum neurotoxins
US7534863B2 (en) * 2000-07-21 2009-05-19 Allergan, Inc. Leucine-based motifs and enhanced biological persistence of clostridial neurotoxins
US7671177B2 (en) 2000-07-21 2010-03-02 Allergan, Inc. Leucine-based motif and clostridial neurotoxins
EP2174949A3 (fr) * 2000-07-21 2014-02-19 Allergan Sales, Inc. Motif à base de Léucine et neurotoxines clostridiales
US7691983B2 (en) 2000-07-21 2010-04-06 Allergan, Inc. Chimera botulinum toxin type E
US7691974B2 (en) 2000-07-21 2010-04-06 Allergan, Inc. Leucine-based motif and clostridial neurotoxins
AU2002219850B2 (en) * 2000-11-17 2006-08-10 Allergan, Inc. Modified clostridial neurotoxins with altered biological persistence
US8119767B2 (en) 2000-11-17 2012-02-21 Allergan, Inc. Post-translational modifications and clostridial neurotoxins
WO2002040506A3 (fr) * 2000-11-17 2003-03-27 Allergan Inc Neurotoxines clostridiales modifiees a persistance biologique alteree
US7888469B2 (en) 2000-11-17 2011-02-15 Allergan, Inc. Post-translation modification and clostridial neurotoxins
US7893202B1 (en) 2000-11-17 2011-02-22 Allergan, Inc. Post-translational modifications and Clostridial neurotoxins
JP2004536778A (ja) * 2000-11-17 2004-12-09 アラーガン、インコーポレイテッド 変化した生物学的持続性を有する改変クロストリジウム神経毒
US7691394B2 (en) 2002-05-28 2010-04-06 Botulinum Toxin Research Associates, Inc. High-potency botulinum toxin formulations
US7491403B2 (en) 2002-12-20 2009-02-17 Botulinum Toxin Research Associates Pharmaceutical botulinum toxin compositions
WO2006116302A2 (fr) * 2005-04-22 2006-11-02 Botulinum Toxin Research Associates, Inc. Preparations de toxines botulinum de haute puissance
WO2006116302A3 (fr) * 2005-04-22 2007-05-24 Botulinum Toxin Res Ass Inc Preparations de toxines botulinum de haute puissance
US9950042B2 (en) 2008-12-04 2018-04-24 Revance Therapeutics, Inc. Extended length botulinum toxin formulation for human or mammalian use
EP2445521A4 (fr) * 2009-06-25 2015-08-26 Revance Therapeutics Inc Formulations à base de toxine botulique sans albumine
US10857215B2 (en) 2012-04-12 2020-12-08 Revance Therapeutics, Inc. Use of botulinum toxin for the treatment of cerebrovascular disease, renovascular and retinovascular circulatory beds
WO2015132004A1 (fr) 2014-03-05 2015-09-11 Merz Pharma Gmbh & Co. Kgaa Neurotoxines clostridiales de recombinaison d'un nouveau type présentant une durée d'effet accrue
US11484580B2 (en) 2014-07-18 2022-11-01 Revance Therapeutics, Inc. Topical ocular preparation of botulinum toxin for use in ocular surface disease
US9901627B2 (en) 2014-07-18 2018-02-27 Revance Therapeutics, Inc. Topical ocular preparation of botulinum toxin for use in ocular surface disease
JP2018502580A (ja) * 2015-01-09 2018-02-01 イプセン・バイオイノベーション・リミテッドＩｐｓｅｎ Ｂｉｏｉｎｎｏｖａｔｉｏｎ Ｌｉｍｉｔｅｄ 陽イオン性神経毒
US12037369B2 (en) 2015-01-09 2024-07-16 Ipsen Bioinnovation Limited Cationic neurotoxins
US10603353B2 (en) 2015-06-11 2020-03-31 Merz Pharma Gmbh & Co. Kgaa Recombinant clostridial neurotoxins with increased duration of effect
WO2016198163A1 (fr) 2015-06-11 2016-12-15 Merz Pharma Gmbh & Co. Kgaa Nouvelles neurotoxines clostridiales recombinées présentant une durée d'effet accrue
US11357821B2 (en) 2015-06-11 2022-06-14 Merz Pharma Gmbh & Co. Kgaa Recombinant clostridial neurotoxins with increased duration of effect
WO2017125487A1 (fr) 2016-01-20 2017-07-27 Merz Pharma Gmbh & Co. Kgaa Nouvelles neurotoxines clostridiales recombinantes à durée d'effet augmentée
US11078472B2 (en) 2016-01-20 2021-08-03 Merz Pharma Gmbh & Co., Kgaa Recombinant clostridial neurotoxins with increased duration of effect
US11969461B2 (en) 2016-03-02 2024-04-30 Merz Pharma Gmbh & Co. Kgaa Composition comprising botulinum toxin
US12171816B2 (en) 2016-09-13 2024-12-24 Allergan, Inc. Non-protein Clostridial toxin compositions
US12144847B2 (en) 2016-09-13 2024-11-19 Allergan, Inc. Non-protein clostridial toxin compositions
US11952601B2 (en) 2017-06-20 2024-04-09 Merz Pharma Gmbh & Co. Kgaa Recombinant botulinum toxin with increased duration of effect
WO2018233813A1 (fr) 2017-06-20 2018-12-27 Merz Pharma Gmbh & Co. Kgaa Nouvelles toxines botuliniques recombinantes présentant une durée d'effet accrue
US11155802B2 (en) 2017-07-06 2021-10-26 Merz Pharma Gmbh & Co. Kgaa Recombinant botulinum neurotoxins with increased duration of effect
WO2019101308A1 (fr) 2017-11-22 2019-05-31 Merz Pharma Gmbh & Co. Kgaa Nouvelles toxines botuliniques recombinées ayant une durée d'effet accrue
JP2019206582A (ja) * 2019-08-09 2019-12-05 イプセン バイオイノベーション リミテッド 陽イオン性神経毒
JP2021104047A (ja) * 2019-08-09 2021-07-26 イプセン バイオイノベーション リミテッド 陽イオン性神経毒
TWI838804B (zh) * 2021-11-11 2024-04-11 南韓商伊妮寶有限公司 肉毒桿菌毒素乾餅的製備方法
WO2023156385A1 (fr) * 2022-02-15 2023-08-24 Merz Pharma Gmbh & Co. Kgaa Formulation de toxine botulique liquide et son utilisation

Also Published As

Publication number Publication date
AU4746096A (en) 1996-12-24

Similar Documents

Publication Publication Date Title
WO1996039166A1 (fr) Analogues de toxine de botulinium et compositions pharmaceutiques de toxine de botulinium
US10883096B2 (en) Carrier for targeting nerve cells
JP7354197B2 (ja) クロストリジウム・ヒストリチクム（ｃｌｏｓｔｒｉｄｉｕｍ ｈｉｓｔｏｌｙｔｉｃｕｍ）酵素およびその使用のための方法
JP5833039B2 (ja) 神経細胞に化合物を導入するために用いられる輸送タンパク質
US7981432B2 (en) Proteins within the type E botulinum neurotoxin complex
KR20080106451A (ko) 페그화된 돌연변이 클로스트리디움 보툴리눔 독소
Lee et al. Involvement of NH2-terminal pro-sequence in the production of active aqualysin I (a thermophilic serine protease) in Escherichia coli
Cortina et al. Role of tryptophan 26 in the NAD glycohydrolase reaction of the S-1 subunit of pertussis toxin
US7008783B1 (en) Gene encoding chondroitinase ABC and uses therefor
EP4461307A1 (fr) Composition de protéine de toxine botulique
JPH10215885A (ja) 新規ｌｅｐ
CN100580082C (zh) 一种重组葡激酶冻干制剂、制备方法和应用
JP3518868B2 (ja) バシラスリケニフォーミス菌株から由来したアミノペプチダーゼ及び天然蛋白質の製造方法
Hernández et al. Streptokinase: about of a thrombolytic patented in Cuba
US20240141308A1 (en) Organophosphorus nerve agent hydrolyzing enzymes
WO1998001754A1 (fr) Nouvelles proteines dans le complexe neurotoxine botulique de type e
Lew Botulinum toxin type B (Myobloc™, NeuroBloc™): a new choice in cervical dystonia
JPH07135971A (ja) リパーゼを活性化する新規タンパク質及びその製造法並びにリパーゼの製造法

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IS JP KE KG KP KR KZ LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG UZ VN AZ BY KZ RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载