JPS59112999A - Preparation of poly(i)-poly(c)-poly-l-lysine complex - Google Patents

Abstract

PURPOSE:To obtain easily the titled complex, resistant to ribonucleases, and useful as a remedy for viral infectious diseases, etc., by adding a specific amount of an aqueous solution of poly-L-lysine to an aqueous solution of poly(I)-poly(C) (polyinosinic acid-polycytidylic acid), adjusting the pH, and solubilizing insoluble substances in the mixture solution. CONSTITUTION:(B) An aqueous solution of poly-L-lysine is added to (A) an aqueous solution of poly(I)-poly(C) (polyinosinic acid-polycytidylic acid) to give 1: 0.35 or above weight ratio between the components (A) and (B), and both components are mixed. The pH of the resultant mixture solution is then adjusted to >=8 with an aqueous solution of sodium dihydrogenphosphate, and the mixture solution is stirred to solubilize insoluble substances in the mixture solution. Thus, a transparent poly(I)-poly(C)-poly-L-lysine complex is obtained. The resultant complex is preserved as an aqueous solution or a hypotonic solution and preferably diluted with a physiologically saline solution or isotonic vehicle for use.

Description

Detailed Description of the Invention The present invention provides poly(I)-poly(C)-polyL-
The present invention relates to a novel method for preparing a lysine complex (hereinafter abbreviated as [poly (ICL) J).

Synthetic double-stranded RNA poly(I) and poly(C) (
Polyinosonic acid (polycytidylic acid) is effective as an interferon inducer in rodents and induces various RNA or DNA in these animals.
It is useful as a medicine for the prevention and treatment of viral infections (proc, HallAcad, 3ci, U
, S, A Vol. 58, pp. 1004-1010 (19
67)). However, poly(I) and poly(C
) shows very weak interferon-inducing ability in primates including humans because it is inactivated by RNAse in the animal body (JI).
nfDIS Volume 132, Page 434, (1975)
).

In order to improve the activity of poly(I)-poly(C) against primates, attempts have been made to prepare ribonuclease-resistant complexes, and poly(I)-poly(C) is one of the most deviant complexes. A king complex (BOIJ (ICLC)) of poly(C), poly-L-lysine, and carboxymethylcellulose (CMC) is known (Japanese Patent Laid-Open Nos. 112882-1982 and 86484-1984).
(see publication). However, the absorption and metabolism of CMC in the body have not yet been elucidated in detail, which remains a problem in developing the complex described above as a medicine.

On the other hand, in order to increase the poly(1)/poly(C) glue bonuclease resistance, poly(I)/poly(
Complexes of C) and polycations such as poly-L-lysine may be prepared, and CMC is used to make these complexes into more stable aqueous solutions. Bo IJ (IC
L) is a stable aqueous solution without forming a precipitate in an extremely dilute solution state (JMed, f3io1 No. 15)
Vol., pp. 256-267 (1966)), C1.
Inf, DiS Volume 132, Nos. 484-439
page (i975)). Transparent practical density IJ (ICL)
Methods for obtaining solutions include poly(■), poly(C),
Adjust the aqueous solutions of poly-L-lysine to pH 7 and 8, respectively, and add the poly(■) solution to the Bo'J(C) solution for 5 to 1
After stirring and mixing for 0 minutes, the poly-L-lysine solution was added dropwise with stirring, and left to stand for about 24 hours with gentle stirring to obtain an almost transparent solution.Additionally, 5M sodium chloride was added to this to a final concentration of 0.15M. In addition, it has been reported that the mixture is left to stir at room temperature for 1 to 2 days (proc, 3o
cExp, f3ioIMed Volume 169, No. 183
~188 pages (1982)). However, this method has many inconveniences in terms of preparation, such as the resulting solution is not a completely transparent liquid and requires a long period of stirring for its preparation.

Another way to obtain a poly(ICL) solution is to mix poly(I) and poly(C) of a particular molecular size into a volume of IJ-
A method is known in which the compound is mixed with 1,-lysine in an isotropic vehicle (JP-A-56-58621). However, the mixing ratio of poly-L-lysine to poly(I) and poly(C) is as small as 0.3;
It has the disadvantage that sufficient bonuclease resistance cannot be obtained. There were also problems in preparation, such as the need for 72 hours of stirring for solubilization and the fact that a small amount of insoluble solid remained.

The present invention aims to solve the conventional technical problems and to develop a method for obtaining a poly(ICL) solution in a short time that has a practical concentration as a pharmaceutical, is clear, and has high ribonuclease resistance. It is complete. That is,
In the present invention, when preparing a poly(I, CL) solution, an aqueous solution of poly(1) and poly(C) is added with poly(L-
After adding and mixing an aqueous solution of lysine so that the weight ratio of poly(I)/poly(C):boly-L-lysine is 1:0.85 or more, I) adjust to H8 or more and stir. ,
Solubilize insoluble matter in the mixture to create a transparent IJ (ICL)
This method is characterized by obtaining a solution.

The poly('ICL) solution prepared according to the present invention has a poly(I)/poly(C) concentration of 0.5 to 2 my/
ml, the weight ratio of poly(I)/poly(C):boly-L-lysine is 1:0.85 or more, preferably 1:03
Produced to have a color of ~0.55. If the weight ratio of poly-L-lysine to poly(I)/poly(C) is less than 0.3, sufficient bonuclease resistance of the poly(ICL) product cannot be obtained, and if it exceeds 0.55, The method of the invention also makes it extremely difficult to prepare clear solutions. In addition, it is poly (I) ・Pomare.

Therefore, unless water content is corrected, the optimum value and limit value of the mixing weight ratio may vary due to these differences in water content. Strictly speaking, the above values are values that are used as a guideline when handling products that are currently on the market.

The poly(ICL) solution is prepared with BoIJ(I) Bo! of known concentration and appropriate molecular weight. It is prepared by mixing an aqueous solution of J(C) with an aqueous solution of Bo1)-L-lysine of known concentration and appropriate molecular weight.

Poly(I) and poly(C) used in the method of the present invention
Solution of poly (I), poly CO,) a degree, poly
The molecular sizes of (I) and poly(C), and the mixing ratio of poly(I) and poly(C) are determined according to the desired poly(ICL).
arbitrarily selected according to the standards of The poly(I)/poly(C) concentration of the poly(■)/poly(C) solution is usually about 0.5 to 4 m9/ltl. In addition, poly (■) and poly (
The molecular size of C) is in the range of about 4 to 125, and the mixing ratio is poly(I):poly(C) in molar ratio.
=a~1:1 can be applied.

IJ-L-IJ resin is usually used as a suitable acid addition salt such as hydrobromide or hydrochloride. Polly L-
The molecular weight of lysine is 8. It is selected from OOO to about 70,000 Daltons. The concentration of the poly-L-lysine aqueous solution is usually 0.25 to 2 m9/yptl.

There is no need to particularly adjust I)H in these aqueous solutions.

When unadjusted, the pH is usually 55-60.

There are no particular restrictions on the method of combining the poly(I)/poly(C) aqueous solution and the poly-KL-IJ resin solution. For example, a poly-L-lysine aqueous solution is added dropwise to a poly(I)/poly(C) aqueous solution without stirring,
Just mix.

After the rainwater solution is mixed uniformly, an alkaline aqueous solution such as sodium hydroxide or potassium hydroxide or an alkaline hypotonic buffer (such as a phosphate buffer) is added to this mixture to adjust the pH to 8 or higher, preferably. pH8-1
0, more preferably pH 8 to 9. After pH adjustment, at room temperature or low temperature conditions (4-8°C), it is usually 20°C.
Completely transparent polyester can be obtained by stirring and mixing for 1 minute to 1 hour.
(I CL ) solution can be obtained. The I)H conditions and the stirring time after pH adjustment vary depending on the molecular weight of the poly(I)IJ resin and the weight ratio to poly(I)/(C). The higher the adjusted pH value, the shorter the time required for solubilization.

In this way, a clear poly(ICL) solution can be obtained and can be stably stored in an aqueous or hypotonic solution. When used as a medicine, it is diluted with physiological saline or an isotropic vehicle (e.g., phosphate buffered saline (PBS), etc.) and used for injection.Also, the pH is adjusted to around neutrality. If necessary, the pH may be adjusted using a weak acid (for example, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen citrate, etc.).

Next, the structure and effects of the present invention will be explained in more detail with reference to experimental examples and examples.

Experimental Example (Influence of the amount of poly L-lysine in poly(ICL)) Poly(1)/pol) (C) of 50 mFI was dissolved in 25 ml of purified distilled water. 10 to 27.5 m
9 poly-L-lysine (molecular weight, 26,000>
C) Aqueous solution (2mf/ml) Gradually added to 25 ml with stirring. After adding the entire amount, the pH was adjusted to 8.5 with a trace amount of IN sodium hydroxide, and the mixture was stirred for 1 hour.

Furthermore, 0.1 M sodium dihydrogen phosphate was added,
+1) H was set to 7.8.

The properties of each IJ (ICL) solution obtained are shown in Table 1. The physicochemical properties of each shoe were measured as follows.

■ Turbidity measurement volume) (I CL ) Measure the turbidity of the solution at a wavelength of 650 nm.
It was measured as the absorbance at . That is, an undiluted poly(ICL) solution was placed in a cell with an optical path length of 1α, and the absorbance at 55 Q nm was measured using a Hitachi spectrophotometer model 101.

■ Measurement of thermal transition midpoint (Tnl) Poly(ICL) solution (lay/ml) was mixed with 0.08 M sodium chloride in 1/150 M potassium phosphate laminate buffer (pH 7.0) at 80 pQ/rxl. dilute,
The sample was placed in a quartz cell with an optical path length of 1 scratch, and the temperature was increased at a rate of 1°C per 2 minutes using a Tm measuring device, and the absorbance at OD 248 was measured to determine the Tm.

■ Measurement of ribonuclease (RNase) resistance Poly(ICL) solution (1 mti/rat) 0.6 gt
lc ria.

Add 19.8 ml of acid-buffered saline (PBS) and add pancreatic nuclease (/my/shoulder 1) 0
．． After adding 1yIl and reacting at 37°C for 1 hour,
Measure the absorbance of the reaction solution at OD248, and calculate the OD after the reaction.
The increase rate of 248 □□□ was calculated to indicate RNase resistance.

Table 1 As is clear from Table 1, poly(ICL) in poly(ICL)
) Sufficient RNase resistance can be obtained when the weight ratio of poly-L-lysine to poly(C) exceeds 0.3.

Example hol) (I) Bo! 200 mg of J (C) was completely dissolved in 100 mt of purified distilled water at room temperature, and sterilized and filtered using a membrane filter. Separately molecular weight 2
6000 Polly L-Rigi 7100179 100ml
It was dissolved in purified distilled water and filtered for sterilization. Poly (1)
・, jf IJ (C) solution (2Q/1ttl) 1
00 mlcpoU-L-IJ lysine solution (1mg/
s/) was gradually added under aseptic stirring. When the entire amount was added, the mixture became slightly cloudy;
A trace amount of insoluble matter was produced, but when a trace amount of IN sodium hydroxide was added to adjust the pH to 8.5 and stirring was continued for 30 minutes, the insoluble matter was dissolved and a clear solution was obtained. Furthermore, 0.1M sodium dihydrogen phosphate was added to this to adjust the pH to 7.6. The obtained solution contains poly(I)/poly(
C) 1■/shoulder t, poly L-lysine (15Q/ml
A poly(ICL) solution containing

The physical properties of Kono solution are OD 650 ” O, OO2, T
m = 87.2°C, RNaSeNaSe resistance increased. Note that these measurements were performed by the method shown in the experimental example.

In addition, the ultraviolet absorption curve of this specimen is shown in Figure 1, and the absorption curve in distilled water and the absorption curve in PBS are almost the same. It showed almost the same pattern as poly(C). In other words, this specimen contains poly (I) and poly
Unlike (C), it is suggested that a double chain is formed regardless of the presence or absence of salts in the solvent.

[Brief explanation of drawings]

FIG. 1 is an ultraviolet absorption curve of a BoIJ(I)/poly(C)-poly-luridine complex preparation obtained in an example of the present invention. Figure 2 shows the control Bo IJ (I) and Bo IJ (
It is an ultraviolet absorption curve of C). In both FIGS. 1 and 2, the absorption curve in phosphate buffered saline is shown by a solid line, and the absorption curve in distilled water is shown by a broken line. Patent applicant (677) Yamasa Soy Sauce Co., Ltd. 1iI1 Figure Z

Claims (1)

[Claims] 1) When preparing a poly(I)/poly(C)-polyL-lysine complex, an aqueous solution of poly-L-1) lysine is added to an aqueous solution of poly(I)/poly(C). Poly(I)
・(C): Weight ratio of borly L-lysine is 〇, 3
After adding and mixing to a pH of 5 or higher, the pH is adjusted to 8 or higher and stirred to solubilize insoluble matter in the mixture to obtain a transparent poly(1)-poly(C)-poly-luridine solution. Poly (I) and poly (C) characterized by
- Method for preparing poly-L-lysine complex. 2) The poly(I)-poly(C)-poly-luridine solution is stored as an aqueous or hypotonic solution and diluted with saline or an isotropic usable vehicle at the time of use. The method described in section.