Pharm/Biotech
Resources

Outsourcing Guide

Cont. Education

Software/Reports

Training Courses

Web Seminars

Jobs

Buyer's Guide

Home Page

Pharm Patents /
Licensing

Pharm News

Federal Register

Pharm Stocks

FDA Links

FDA Warning Letters

FDA Doc/cGMP

Pharm/Biotech Events

Consultants

Advertiser Info

Newsletter Subscription

Web Links

Suggestions

Site Map
 

 

 

 

Title:  Medicament for treatment of Duchenne muscular dystrophy

United States Patent:  6,653,467

Issued:  November 25, 2003

Inventors:  Matsuo; Masafumi (3-31, Kitaochiai 5-chome, Suma-ku, Kobe-shi, Hyogo 654-0151, JP); Takeshima; Yasuhiro (Kobe, JP)

Assignee:  JCR Pharmaceutical Co., Ltd. (Hyogo, JP); Matsuo; Masafumi (Hyogo, JP)

Appl. No.:  667327

Filed:  September 22, 2000

Abstract

Antisense oligonucleotides comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:1 or NO:2 are disclosed. The antisense oligonucleotides are used for treatment of specific types of Duchenne muscular dystrophy which is attributed to a change in number of the nucleotides composing one or more exons adjacent to exon 43 or 53, respectively, in human dystrophin mRNA, wherein the change is due to deletion of one or more nucleotides from the normal nucleotide sequence for the exons, wherein the net of the change in number of the nucleotides is expressed as a reduction of (3xN+1) nucleotides, wherein N is zero or a natural number.

SUMMARY OF THE INVENTION

Upon the above background, the present inventors successfully identified new SES's within exons 43 and 53, respectively, of dystrophin gene, and thereupon created new means for treatment of Duchenne muscular dystrophy.

Thus the present invention provides an oligonucleotide selected from the group consisting of an RNA having the nucleotide sequence set forth under SEQ ID NO:1 or NO:2 in the Sequence Listing and a DNA having a nucleotide sequence complementary to a complementary sequence to said nucleotide sequence set forth under SEQ ID NO:1 or NO:2 in the Sequence Listing. Of these oligonucleotides, the RNAs correspond to the SES (set forth under SEQ ID NO:1 in the Sequence Listing) within exon 43 and the SES (set forth under SEQ ID NO:2 in the Sequence Listing) within exon 53, respectively, of human dystrophin pre-mRNA. These RNAs and DNAs are used as templates for production of antisense nucleotides as therapeutic agents for Duchenne muscular dystrophy discussed below.

The present invention further provides antisense oligonucleotides comprising nucleotide sequences complementary to the nucleotide sequences set forth under SEQ ID NO:1 or 2 in the Sequence Listing.

The antisense oligonucleotides, when administered, can induce skipping of exon 43 or 53 in the splicing process of human dystrophin mRNA as they are complementary to the SES (set forth in the form of an equivalent DNA under SEQ ID NO:1) within exon 43 or the SES (set forth in the form of an equivalent DNA under SEQ ID NO:2) within exon 53, respectively, of human dystrophin mRNA. Therefore, these antisense oligonucleotides can be used as therapeutic agents against particular types of Duchenne muscular dystrophy based on correcting the shift of reading frame.

The above antisense oligonucleotides may be DNAs having the nucleotide sequences set forth under SEQ ID NO:3 and NO:4, respectively, in the Sequence Listing and may also be phosphorothioate DNAs having the same nucleotide sequences as them. The sequences are complementary to sequences which include the SES and adjacent nucleotide sequences at both ends thereof within exon 43 and 53, respectively. A DNA comprising one of these sequences can therefore more strongly hybridize with the SES within exon 43 or 53, respectively, and block their functions.

The present invention further provides use of the antisense oligonucleotide having a nucleotide sequence complementary to the nucleotide sequence set forth under SEQ ID NO:1 in the Sequence Listing for the manufacture of a medicament for treatment of Duchenne muscular dystrophy which is attributed to a change in number of the nucleotides composing one or more exons adjacent to exon 43 in human dystrophin mRNA, wherein said change is due to deletion of one or more nucleotides from the normal nucleotide sequence for said exons, wherein the net of said change in number of the nucleotides is expressed as a reduction of (3xN+1) nucleotides, wherein N is zero or a natural number. The antisense oligonucleotide may be a DNA selected from the group consisting of the DNA having the nucleotide sequence set forth under SEQ ID NO:3 in the Sequence Listing and a phosphorothioate DNA having the same nucleotide sequence as the nucleotide sequence set forth under SEQ ID NO:3.

The present invention further provides a medicament for treatment of Duchenne muscular dystrophy which is attributed to a change in number of the nucleotides composing one or more exons adjacent to exon 43 in human dystrophin mRNA, wherein said change is due to deletion of one or more nucleotides from the normal nucleotide sequence for said exons, wherein the net of said change in number of the nucleotides is expressed as a reduction of (3xN+1) nucleotides, wherein N is zero or a natural number, said medicament comprises one of the above antisense oligonucleotides against the SES within exon 43, in a pharmaceutically acceptable injectable medium.

The present invention further provides use of the antisense oligonucleotide having a nucleotide sequence complementary to the nucleotide sequence set forth under SEQ ID NO:2 in the Sequence Listing for the manufacture of a medicament for treatment of Duchenne muscular dystrophy which is attributed to a change in number of the nucleotides composing one or more exons adjacent to exon 53 in human dystrophin mRNA, wherein said change is due to deletion of one or more nucleotides from the normal nucleotide sequence for said exons, wherein the net of said change in number of the nucleotides is expressed as a reduction of (3xN+1) nucleotides, wherein N is zero or a natural number. The antisense oligonucleotide may be a DNA selected from the group consisting of the DNA having the nucleotide sequence set forth under SEQ ID NO:4 in the Sequence Listing and a phosphorothioate DNA having the same nucleotide sequence as the nucleotide sequence set forth under SEQ ID NO:4.

The present invention further provides a medicament for treatment of Duchenne muscular dystrophy which is attributed to a change in number of the nucleotides composing one or more exons adjacent to exon 53 in human dystrophin mRNA, wherein said change is due to deletion of one or more nucleotides from the normal nucleotide sequence for said exons, wherein the net of said change in number of the nucleotides is expressed as a reduction of (3xN+1) nucleotides, wherein N is zero or a natural number, said medicament comprises one of the above antisense oligonucleotides against the SES within exon 53, in a pharmaceutically acceptable injectable medium.

Furthermore, the present invention provides a method of treatment of a human patient of Duchenne muscular dystrophy comprising administering to said patient an therapeutically effective amount of an antisense oligonucleotide comprising a nucleotide sequence complementary to the nucleotide sequence set forth under SEQ ID NO:1 or NO:2 in the Sequence Listing, wherein said Duchenne muscular dystrophy is attributed to a change in number of the nucleotides composing one or more exons adjacent to exon 43 or 53, respectively, in human dystrophin mRNA, wherein said change is due to deletion of one or more nucleotides from the normal nucleotide sequence for said exons, wherein the net of said change in number of the nucleotides is expressed as a reduction of (3xN+1) nucleotides, wherein N is zero or a natural number. The antisense oligonucleotide comprising a nucleotide sequence complementary to the nucleotide sequence set forth under SEQ ID NO:1 may be selected from the group consisting of a DNA having the nucleotide sequence set forth under SEQ ID NO:3 in the Sequence Listing and a phosphorothioate DNA having the same nucleotide sequence as the nucleotide sequence set forth under SEQ ID NO:3, and the antisense oligonucleotide comprising a nucleotide sequence complementary to the nucleotide sequence set forth under SEQ ID NO:2 may be selected from the group consisting of a DNA having the nucleotide sequence set forth under SEQ ID NO:4 in the Sequence Listing and a phosphorothioate DNA having the same nucleotide sequence as the nucleotide sequence set forth under SEQ ID NO:4.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, "oligonucleotide" includes not only oligoDNA and oligoRNA but also a phosphorothioate analogue such as phosphorothioate oligoDNA. Phosphorothioate DNAs are nucleotides in which an oxygen atom in the phosphate group is replaced with a sulfur atom. They are nucleotide analogues more resistant to various nucleotide decomposing enzymes and therefore widely used in the field of genetic engineering, e.g., for site specific substitution in genes. The method of their production, their properties and their various application are well known to those skilled in the art. Phosphorothioate DNAs, which form base pairs as natural DNAs do, are employed in the present invention with particular advantage as they are more resistant to various decomposing enzymes. "Phosphorothioate analogue" herein is of a structure in which one or more phosphorodiester groups between the nucleotides in a DNA chain are replaced with phosphorothioate groups.

The medicament of the present invention preferably contains 0.05-5 .mu.mol/ml of one of the antisense oligonucleotides, 0.02-10 w/v % of at least one carbohydrate or polyalcohol, and 0.01-0.4 w/v % of at least one pharmaceutically acceptable surfactant. A more preferred concentration range for the antisense oligonucleotide is 0.1-1 .mu.mol/ml.

For the above carbohydrate, monosaccharide and/or disaccharide are preferred. Examples of the carbohydrates and polyalcohols include glucose, galactose, mannose, lactose, maltose, mannitol, and sorbitol. They may be employed alone or in combination.

Examples of preferred surfactants include polyoxyethylene sorbitan mono- to tri-ester, alkyl phenyl polyoxyethylene, sodium taurocholate, sodium cholate, and polyalcohol esters. A particularly preferred one of them is polyoxyethylene sorbitan mono- to tri-ester, and particularly preferred esters are oleate, laurate, stearate, and palmitate. They may be employed alone or in combination.

The medicament of the present invention preferably further contains 0.03-0.09 M of at least one pharmaceutically acceptable neutral salt, for example, sodium chloride, potassium chloride, and/or calcium chloride.

The medicament of the present invention preferably may further contain 0.002-0.05 M of a pharmaceutically acceptable buffering agent. Examples of preferable buffering agents include sodium citrate, sodium glycinate, sodium phosphate, and tris(hydroxymethyl)aminomethane. The buffering agents may be employed alone or in combination.

The above medicaments may be supplied in liquid forms. Considering, however, for cases in which they have to be stored for certain periods of time, it generally is preferred that they are provided in a lyophilized form of composition, in order to stabilize the antisense oligonucleotide and thereby preventing reduction of its therapeutic effect. Prior to use, such a composition is reconstituted, i.e., turned back into a liquid form to be injected, using a solvent (e.g., injectable distilled water). Therefore the medicaments of the present invention include those provided in lyophilized form which are intended to be reconstituted prior to use with a solvent in order to make the concentrations of their ingredients fall within predetermined ranges. For greater stability of such lyophilized compositions, albumin or amino acids such as glycine may be added.

Claim 1 of 2 Claims

What is claimed is:

1. An oligonucleotide selected from:

an RNA consisting of the nucleotide sequence set forth as SEQ ID NO: 1, or

a DNA consisting of the nucleotide sequence set forth as SEQ ID NO: 1 modified such that each uracil nucleotide of SEQ ID NO: 1 is replaced with thymine.




____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]