# US 10,369,235 B2 · Non-diffusive botulinum toxin causing local muscle paralysis, and purification method thereof

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,369,235 B2
- Document ID: US10369235B2
- Status: Granted
- Kind: B2
- Application number: 15/422,129
- Filed: 2017-02-01
- Published: 2017-05-25
- Granted: 2019-08-06
- Earliest claimed: 2010-05-31
- Assignees: Medexgen Co., Ltd.
- Applicants: Medexgen Incorporated
- Inventors: Yong Hoon Chung; Hyun Sub Lee
- Web page: https://patentgrep.com/patent/US10369235B2

## CPC

- A61K 49/0004: {Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions}
- A61K 38/4893: {Botulinum neurotoxin (3.4.24.69)}
- A61K 49/00: Preparations for testing in vivo
- A61P 17/00: Drugs for dermatological disorders
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- A61P 23/02: Local anaesthetics
- C12N 9/52: derived from bacteria {or Archaea}
- C12Y 304/24069: Bontoxilysin (3.4.24.69), i.e. botulinum neurotoxin
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- G01N 30/96: using ion-exchange (G01N30/02, G01N30/90 take precedence)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present invention relates to a method for purifying a non-spreading botulinum toxin that causes local muscle paralysis and a non-spreading botulinum toxin obtained thereby. The method comprises the steps of: subjecting a purified botulinum toxin type A product to ion-exchange chromatography using a controlled pH of buffer, concentration of sodium chloride (NaCl), thereby separating the botulinum toxin type A product into subfractions; and collecting a subfraction having an A260/A280 value in a specific range from the separated subfractions.

## Claims

### Claim 1

A method for determining a non-spreading botulinum toxin, comprising:
injecting the non-spreading botulinum toxin into the either left or right hind limb calf muscle of mouse (4-6 wk old, weighing 18-22 g) in an amount equivalent to 1.5-3 times the LD50 of the toxin in the volume ranges of 20-50 μL; and
assessing paralysis conditions of the hind limb muscles and respiratory muscles of the injected mouse and a survival rate of the injected mouse to determine a non-spreading botulinum toxin.

### Claim 2 (depends on 1)

The method of claim 1, wherein the non-spreading botulinum toxin shows a survival rate at least 80 % at 96 hours after injection of 2 U toxin preparation in the volume 50 μL either left or right hind limb calf muscle of the mouse.

### Claim 3 (depends on 2)

The method of claim 2, wherein the paralysis conditions comprises:
limb appearance and walking of normal mice;
mice walk with dragging, but toes are not put together;
mice walk with dragging, toes are put together;
mice walk with dragging, toes are put together, foot joints are bent inward; and
mice walk with dragging, toes are put together, foot joints are bent inward, feet come into contact with leg muscles, and legs are lame.