Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Liposomes
Reexamination Certificate
2001-08-13
2004-08-24
Nguyen, Dave T. (Department: 1635)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Liposomes
C536S022100, C536S023100, C536S026740, C536S026800, C536S027300, C514S04400A
Reexamination Certificate
active
06780429
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a chain-shortened polynucleotide particularly useful as a medicine, and a method for preparing the same. More specifically, the present invention relates to a synthetic chain-shortened polynucleotide or salts thereof, wherein the proportion of a 2′-5′ phosphodiester bond is up to 3% based on the whole phosphodiester bonds, i.e., the rate of phosphate groups rearranged from 3′ position to 2′ position based on the whole phosphate groups of phosphodiester bonds (the phosphate rearrangement rate) is up to 3%, and a method for preparing the same.
TECHNICAL BACKGROUND
Polynucleotides typified by polyinosinic—polycytidylic acid, i.e., poly(I)·poly(C), are well-known compounds in the art, and the potentiality as a medicine for treating hepatitis or cancer have been investigated in view of their interferon inducing action, immune activating action, and the like.
The pharmacological action of these polynucleotides has high correlation with the chain length, and longer the chain length, stronger the interferon inducing action and the like. On the other hand, longer the chain length, stronger the toxicity manifested.
Recently, there has been an approach for reducing toxicity with maintaining useful pharmacological action of a polynucleotide, by a method wherein a synthetic polynucleotide having a relatively shorter chain prepared by the hydrolysis of a polynucleotide is enclosed in a carrier such as cationic liposome effective for introducing a medicament into a cell (e.g., PCT W099/20283, PCT WO99/4853 1).
It is known that when a polynucleotide is hydrolyzed to shorten the chain length as described above, some phosphate groups cause intramolecular rearrangement from 3′ position to 2′ position through a mechanism called pseudo rotation simultaneously with the chain-shortening (see, e.g., “Protein Nucleic acid Enzyme”, Vol. 40, No. 10, pp. 1323 to 1332 (1995)). As a result, a portion of 3′-5′ phosphodiester bonds in the chain-shortened polynucleotide molecule are replaced by 2′-5′ phosphodiester bonds. It has never been known whether or not such a phosphate rearrangement phenomenon would affect the pharmacological action.
DESCRIPTION OF INVENTION
An object of the present invention is to provide, firstly, a chain-shortened polynucleotide or salt thereof and a double stranded chain-shortened polynucleotide or salt thereof, which are safe and effective as a medicine.
The present inventors have been intensively studied and found that the problems described above can be solved by a chain-shortened polynucleotide which contains 2′-5′ phosphodiester bond produced mainly in a chain-shortening reaction only at a particular proportion or less, or a salt thereof, and accomplished the present invention.
An aspect of the present invention is a chain-shortened polynucleotide containing a 2′-5′ phosphodiester bond at a proportion of 3% or less, preferably 2% or less, based on the whole phosphodiester bonds, or salt thereof.
The present invention includes also as an embodiment a double stranded chain-shortened polynucleotide or salt thereof, which is formed from two chain-shortened polynucleotides or salts thereof capable of forming a double strand, which is inclusive in the above-described chain-shortened polynucleotides containing a 2′-5′ phosphodiester bond at a proportion of 3% or less, preferably 2% or less, based on the whole phosphodiester bonds. Further, the present invention also includes a composition comprising a complex formed from a carrier effective for introducing a medicament into a cell and the above-described chain-shortened polynucleotide or salt thereof wherein the proportion of a 2′-5′ phosphodiester bond is 3% or less based on the whole phosphodiester bonds, or the double stranded chain-shortened polynucleotide or salt thereof formed from two chain-shortened polynucleotides or salts thereof capable of forming a double strand as an essential ingredient.
The polynucleotide used in the present invention is a compound comprising at least about 20 nucleotides, which is formed by polymerizing linearly through a phosphodiester bond and includes synthetic and natural compounds. Specific examples include polyinosinic acid [namely, poly(I)] or an analogue thereof, polycytidylic acid [namely, poly(C)] or an analogue thereof, polyadenylic acid [namely, poly(A)] or an analogue thereof, and polyuridylic acid [namely, poly(U)] or an analogue thereof.
The polyinosinic acid analogue is a homopolymer in which all or a part of inosinic acid is chemically modified or a copolymer of inosinic acid with other nucleotide, for example, poly(7-deazainosinic acid) and poly(2′-azidoinosinic acid). The polycytidylic acid analogue is a homopolymer in which all or a part of cytidylic acid is chemically modified or a copolymer of cytidylic acid with other nucleotide, for example, poly(5-bromocytidylic acid), poly(2-thiocytidylic acid), poly(cytidine-5′-thiophosphoric acid), poly(cytidylic acid, uridylic acid), poly(cytidylic acid, 4-thiouridylic acid) and poly(1-vinylcytidylic acid). The polyadenylic acid analogue and polyuridylic acid analogue are defined likewise. Among them, polyinosinic acid and polycytidylic acid are suitable in the present invention.
The average chain length of the chain-shortened polynucleotide of the present invention is suitably from 0.1 k bases to 1 k base. The term “base” means the number of base and “1 k base” indicates a base number of 1000, and hereinafter, “base(s)” is abbreviated to “b”. The mean chain length is preferably from 200 b to 800 b, and more preferably from 300 b to 600 b. The average chain length can be determined easily, for example, by gel permeation chromatography (hereinafter, referred to as “GPC”) as described hereinafter in Experiment 5.
In the chain-shortened polynucleotide of the present invention, the phosphate rearrangement rate is 3% or less, preferably 2% or less or between 0.1% and 2%, and more preferably 1% or less or between 0.1% and 1%.
The rearrangement of phosphate group from 3′ position to 2′ position in polynucleotide can be confirmed easily, for example, by a method as described in Experiment 6. Namely, a polynucleotide is degraded with nuclease P
1
, which specifically hydrolyzes a 3′-5′ phosphodiester bond to such levels as a nucleoside, nucleotide and oligonucleotide, and then treated with an alkaline phosphatase, which specifically hydrolyzes a terminal phosphate group, to convert the whole nucleotides into nucleosides. On the other hand, an oligonucleotide having a 2′-5′ phosphodiester bond, which is not hydrolyzed by the nuclease P
1
is not degraded till nucleoside even by the treatment with an alkaline phosphatase because an intramolecular 2′-5′ phosphodiester bond is not hydrolyzed. The phosphate rearrangement rate can be calculated by determining nucleosides and oligonucleotides (most of them are dimers) by liquid chromatography, or the like.
Examples of a pair of chain-shortened polynucleotides capable of forming a double strand in connection with the present invention include polyinosinic acid and polycytidylic acid, polyadenylic acid and polyuridylic acid, polyinosinic acid analogue and polycytidylic acid, polyinosinic acid and polycytidylic acid analogue, polyinosinic acid analogue and polycytidylic acid analogue, polyadenylic acid analogue and polyuridylic acid, polyadenylic acid and polyuridylic acid analogue, and polyadenylic acid analogue and polyuridylic acid analogue. Therefore, examples of the double stranded chain-shortened polynucleotide formed between two chain-shortened polynucleotides capable of forming a double strand include polyinosinic-polycytidylic acid, polyadenylic-polyuridylic acid, polyinosinic analogue-polycytidylic acid, polyinosinic-polycytidylic acid analogue, polyinosinic analogue-polycytidylic acid analogue, polyadenylic analogue-polyuridylic acid, polyaden
Ishiyama Kouichi
Matsuyama Shinji
Ohgi Tadaaki
Seki Junzo
Angell J. Eric
Greenberg & Traurig, LLP
Nguyen Dave T.
Nippon Shinyaku Co. Ltd.
Rzucidlo Eugene C.
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