JPH0296533A - Absorption preventing agent for polypeptides - Google Patents

Abstract

PURPOSE:To obtain a safe absorption-preventing agent containing glycyrrhitins as active ingredients and capable of efficiently preventing absorption to equipment, vessel, etc., for polypeptides. CONSTITUTION:The aimed absorption-preventing agent containing glycyrrhitinic acid or salt thereof and glycyrrhetinic acid which is agricon thereof and beta- aminophosphorus based triterpenoid or salt thereof as active ingredients. Blend of the above-mentioned compounds are preferably carried out at an amount of >=0.01W/V%, especially 0.1-2W/V% based on water solution or aqueous suspension containing polypeptidescontaining substance and the above-mentioned compounds are blended in producing, storing or using preparation for parenteral use or reagent for measuring immunity. When physiologically active peptides are administered as a medicine, the dose can be accurately maintained and the effectiveness and safety is ensured and accurate determination is made possible in the case of measurement of immunity.

Description

DETAILED DESCRIPTION OF THE INVENTION "Field of Industrial Application" The present invention relates to an anti-adsorption agent for preventing adsorption of polypeptides to instruments, containers, etc.

``Prior art'' Physiologically active polypeptides such as calcitonin, barathyroid hormone (PTH), and insulin have physiological activities including hormonal activity, and when they are administered as pharmaceuticals, their effectiveness and safety are important. To ensure sex, it is necessary to follow the dosage more strictly than with other drugs. It has been known that when physiologically active polypeptides are stored in an aqueous solution, they are adsorbed to glass or plastic instruments, containers, etc. It has been reported that this phenomenon is more likely to occur when the concentration of
1979)). Since these physiologically active polypeptides usually exhibit activity in extremely small amounts, the concentration of the administered solution is extremely low, and as described above, it can be said that they are in a state where adsorption is likely to occur. Furthermore, the loss rate due to adsorption becomes a very serious problem since the initial amount of the compound is very small, and is an important issue to be solved in order to ensure the effectiveness of the drug.

Various substances are added for the purpose of preventing adsorption of bioactive polypeptides, and substances added for the purpose of preventing adsorption (hereinafter referred to as adsorption inhibitors)
Conventionally, methylcellulose, POE-hydrogenated castor oil and its derivatives (e.g. HCO-60), lecithin, hydroxypropylcellulose, ethylene oxide propylene oxide copolymer, POE-sorbitan alkyl ester I (e.g. POE-sorbitan monooleate, TWEEN80, POE-sorbitan monolaurate), methylcyclodextrin, sorbitan fatty acid ester, POE-octylphenyl ether, POE-lauryl ether, bacitracin, polyethylene glycol 6.000, benzalkonium chloride, gelatin, BSA, H3A (human serum albumin), urea, casein, and other surfactants and polymeric substances are known.

Furthermore, similar to bioactive polypeptides, various polypeptides are used as reagents in immunoassay reactions, but these polypeptides also have a tendency to adsorb to instruments, containers, etc. in immunoassay systems. This is one of the causes of inaccurate quantification and limits on measured concentrations.

"Problems to be Solved by the Invention" Parenterally administered preparations such as injections must be safe and stable, and immunoassays also require accuracy. However, it could not be said that these conditions were necessarily satisfied in the preparations. For example, HCO-60, TWEEN80 for bioactive polypeptides
It is widely known that the addition of benzalkonium chloride, etc., tends to cause side effects such as a drop in blood pressure and hemolysis due to the release of histamine. In addition, many polymeric substances that have anti-adsorption effects are not approved as pharmaceuticals.
Human serum albumin and the like are difficult to use due to problems with AIDS and hepatitis, and there is a need for the development of more useful anti-adsorption agents.

"Means for Solving the Problems" As a result of research into the adsorption prevention effect of various substances in order to solve the above-mentioned problems, the present inventors found that glycyrrhizins can achieve the intended purpose. The present invention has now been completed.

That is, the present invention relates to an adsorption inhibitor containing glycyrrhizin as an active ingredient for preventing adsorption of polypeptides.

The polypeptides in the present invention have a molecular weight of 10
00-30000, more preferably molecular weight 1000
-10,000 or derivatives thereof. Preferably, the polypeptides are physiologically active polypeptides that require prevention of adsorption when administered as pharmaceuticals, such as calcitonin, ACTH (adrenocorticotropic hormone), and PT.
H (barathyroid hormone), insulin, secretin, oxytocin, growth hormone, angiotensin, pradekinin, β-endorphin, substance P, glucagon, dynorphin, pap pressin, somatostatin, gastrin, thyroid hormone (TS)
H), prolactin, luteinizing hormone-releasing hormone (
LH-RH), enkephalin, neurotensin, atrial natriuretic peptide (ANP), interferon, interleukin, G-C3F, glutathione peroxidase, superoxide diismutase (S
OD) etc. and one or two types while retaining their activities.
Examples include analogs (mutins) in which more than one amino acid is substituted, and derivatives and salts thereof. Furthermore, other polypeptides that are the object of the present invention include peptide antigens used in immunoassays, such as various known peptide hormones or fragments thereof that are to be measured in immunoassays, or similar A body is exemplified.

Among physiologically active polypeptides, calcitonin is a general term for bebutide hormones having a serum calcium-lowering effect, and natural calcitonin or its analogues are known as those having extremely important physiological activities. Natural calcitonin includes eel calcitonin, salmon calcitonin, pig calcitonin, human calcitonin, chicken calcitonin, and the like, and functions as a calcium-regulating hormone secreted from endocrine cells. Also, as an analogue, elcatonin ((A
su'・7] eel calcitonin), [Asu'・7]
Known examples include human calcitonin.

In addition, PTH (barathyroid hormone) is a peptide 1 hormone that has the effect of increasing serum calcium.
It has a sequence of 4 amino acids, and natural PTH or its analogues are known. Examples of these include human parathyroid hormone (1-84>(human pa
rathyroid hormon; h-PTH) (
Biochemfstry 17.5723 (19
78)), h-PTH(1-34)(Hoppe 5
eyler's Z, Physiol, Chem
, 355.415 (1974)), h-PTH (
1-34) NHt (Japanese Unexamined Patent Publication No. 58-96052)
(N 1 e””) h-PTH (1-34)
, [Nl e"8. Tyr") h-PTH
(1-34) (Unexamined Japanese Patent Publication No. 113753/1983)
, (Nle”1ll)h-PTH(1-34)NHt
(Japanese Unexamined Patent Application Publication No. 6124598), (Nlea,
lll, Ty, 34)h-PTH(1-34)NHz
(Unexamined Japanese Patent Publication No. 60-34996), Rat-PT
H(1-84) (J, Biol, Chem,,
259 (5).

3320 (1984), rat-PTH (1-3
4) (Endocrinol,, 117 (
3).

1230 (1985)), bovine-PTH ('l-84
) (Am, J. Med,, 50. 639
(1971)), bovine-PTH(1-34), bovine-PTH(1-34) NHz, etc. (Pthobiol
.

gy annual, 11. 53 (19
81)) etc.

The glycyrrhizins used in the present invention include glycyrrhizic acid or its salts, which are known to be extracted from the roots of licorice or its coexisting plants, and glycyrrhetinic acid, which is an aglycone thereof and a β-amyrin triterpenoid compound, or its salts. salts, as well as analogs of these and m8
means the body or its salts. Furthermore, the salt is preferably a non-toxic salt, such as potassium salt, sodium salt, ammonium salt, calcium salt, etc. When the cation is monovalent, mono-salt, di-salt, and tri-salt are exemplified. . Furthermore, when using glycyrrhizins, one or more types selected from the group consisting of these substances may be used.

In the present invention, the device for adsorbing polypeptides, container walls, etc. are container walls made of glass or plastic, such as various glasses, polypropylene, polyethylene, polystyrene acrylonitrile, polystyrene, polycarbonate, polymethylpentyl, etc. Examples include those made of minute porous materials. These instruments and containers include ampoules, syringes, infusion bottles, infusion tubes, reaction containers for immunoassays, etc., and are used, for example, during the production, storage, and use of parenteral preparations and immunoassay reagents. It is used for.

The amount of glycyrrhizin necessary to prevent adsorption can vary depending on the type of polypeptide, the contact area of the container wall, and its material, so it is desirable to determine the amount appropriately, but it does not necessarily have to be determined depending on each case. 0.01 (W/V) for aqueous solutions or suspensions containing polypeptides for boat fishing.
)% or more, and an upper limit of about 5% is sufficient. Preferably, it may be used at a concentration of about 0.1% to about 2%, which exhibits a remarkable adsorption prevention effect.

When using the anti-adsorption agent of the present invention, polypeptides and glycyrrhizin do not necessarily need to coexist in the same composition from the beginning, but rather a composition in which they are combined for the purpose of mixing at the time of use. It's okay.

Further, these compositions may be in a solution state or a dry solid state, except when the polypeptide alone is in a solution state. That is, several types of forms shown below can be mentioned.

(2) A composition in which polypeptides and glycyrrhizin are blended so that they coexist directly in the same aqueous solution or suspension.

(2) A composition in which polypeptides and glycyrrhizin are blended so that they coexist directly in the same dry solid, or a dry composition for dissolution before use.

(2) A composition in which polypeptides and glycyrrhizin are provided as separate dry solids and combined in such a way that they are mixed at the time of use.

(2) A composition in which polypeptides are provided as a dry solid, glycyrrhizin is provided as an aqueous solution or suspension, and the two are mixed together at the time of use.

Furthermore, freeze-drying is advantageously used to obtain the above composition in a dry solid state. It goes without saying that when using a composition in a dry solid state, it may be dissolved in distilled water for injection, physiological saline, reaction solution for immunoassay, etc. before use.

In the form described above, the adsorption inhibitor of the present invention further includes suitable stabilizers, surfactants, solubilizing agents, buffers,
Isotonicity agents, bulking agents, etc. may be appropriately selected and used in combination.

Preferable preparations of bioactive polypeptides for the present invention include injections, drip preparations, nasal preparations, eye drops, external preparations, etc. It can be administered by injection, externally in liquid, jelly, or powder form, or nasally.

Next, the present invention will be described in more detail below with reference to Examples, but the present invention is not limited thereto.

Examples 1 to 3 Adsorption inhibitor; Glycyrrhizic acid (manufactured by Maruzen Kasei Co., Ltd.), monoammonium glycyrrhizinate (manufactured by Maruzen Pharmaceutical Co., Ltd.), and dipotassium glycyrrhizinate (manufactured by Tokiwa Phytochemical Research Institute) were dissolved in physiological saline solution, and NaOH or The pH was adjusted to about 6 with HCl, and the concentrations were adjusted to 2% (W/v), 0.1%, and 0.01%, respectively, and used as adsorption prevention samples. Separately, a physiological saline solution was prepared as a control sample.

Test method; Salmon calcitonin (manufactured by Sigma) was dissolved in physiological saline to prepare a solution with a concentration of 200 μg/ml, and 10 μl of this solution was added to 1 ml of each of the above adsorption prevention samples.
Adjust the pH to 5.5 with aOH or HCi and add the following
The drug solutions of Examples 1 to 3 were prepared as shown in the table.

Each drug solution was injected into a polypropylene test tube and left to stand for 30 minutes, and then the residual rate of salmon calcitonin was determined by high performance liquid chromatography or a biological activity measurement test.

The conditions for high performance liquid chromatography are YMCP
acked AM-3020DS, wavelength 220nm
, the moving layer was a mixture of CHffCN-0, 1
ient method was used.

Results: As is clear from the results shown in Table 1, when glycyrrhizin was added as an adsorption inhibitor, the adsorption of salmon calcitonin to the container wall was prevented.

Table 1 Adsorption prevention effect Example 4 h-PTH (1-34) (manufactured by Toyo Jozo Co., Ltd.) 1 mg 1 mannit 0.6 g and monoammonium glycyrrhizinate 0.2 g were dissolved in distilled water (or sterilized water). , total amount 2
00 ml. The mixture was aseptically dispensed into 1 ml ampoules and sealed by melting to obtain an injection.

Example 5 Insulin (manufactured by Sigma) 4000 units, dipotassium glycyrrhizinate 0.2 g, phenol 0.2 g, Na
(1! 1.6 g was dissolved in distilled water (or sterilized water), adjusted to pH 3.0 with hydrochloric acid, and the total amount was sealed at 200 m/m. Aseptically dispensed into 1 m/ampules, melted and sealed, and injected. obtained the drug.

Example 6 10,000 units of ACTH (manufactured by Funakoshi), monoammonium glycyrrhizinate 1, Qg, NaC14, Og were dissolved in distilled water (or sterilized water) to make a total amount of 500 mβ. The mixture was aseptically dispensed into 1 mj2 ampoules and sealed by melting to obtain an injection.

Example 7 0.4 mg of eel calcitonin (manufactured by Sigma), 0.1 g of glycyrrhizic acid, and 10.9 g of NaC were dissolved in distilled water (or sterilized water) and adjusted to pH 5.5 with NaOH to make the total volume 100 ml. The solution was aseptically dispensed into ampoules at 1 mA each and melted and sealed to obtain an injection.

Example 8 0.4 mg of elcatonin (manufactured by Toyo Jozo Co., Ltd.), dipotassium glycyrrhizinate O, Ig, and Na (1.09 g) were dissolved in distilled water (or sterilized water) and the total amount was made into LOOml.Aseptically, 1 mA each was added into ampoules. The mixture was dispensed and melted and sealed to obtain an injection.

Example 9 0.4 mg of secretin (manufactured by Funakoshi), 0.2 g of dipotassium glycyrrhizinate, 8 g of 10° NaC, and distilled water (
or sterile water) to make the total volume 100ml. Aseptically 1m7! The mixture was dispensed into ampoules and sealed by melting to obtain an injection.

Example 10 Oxytocin (manufactured by Wako Pure Chemical Industries, Ltd.) 0.8 mg, monoammonium glycyrrhizinate 0.5 g, Na (1!0.8 g
was dissolved in distilled water (or sterilized water) to make the total amount 100 mA. The solution was aseptically dispensed into ampoules at 1 mA each and melted and sealed to obtain an injection.

Example 11 0.1 mg of human growth hormone (manufactured by Funakoshi), dipotassium glycyrrhizinate Ig, and Na (1.07 g) were dissolved in distilled water (or sterilized water) to make a total volume of 100 mA. The mixture was dispensed and melted and sealed to obtain an injection.

Example 12 0.2 mg of angiotensin (manufactured by Wako Pure Chemical Industries, Ltd.), 0.2 g of dipotassium glycyrrhizinate, 9 g of NaCno were dissolved in distilled water (or sterilized water) and the total amount was 100 mjl! Closed. The mixture was aseptically dispensed into ampoules in 1 ml portions and sealed by melting to obtain an injection.

Example 13 0.4 mg of bradykinin (manufactured by Wako Pure Chemical Industries, Ltd.), 0.1 g of glycyrrhizic acid, and 10.9 g of NaC were dissolved in distilled water (or sterilized water), the pH was adjusted to 5.5 with NaOH, and the total amount was dissolved. It was set to 100mA. The solution was aseptically dispensed into ampoules at 1 mA each and melted and sealed to obtain an injection.

Example 14 β-endorphin (manufactured by Sigma) O, 1 mg, monoammonium glycyrrhizinate 0.5 g, NaC 10.9
Dissolve g in distilled water (or sterilized water) and make the total amount 100mf
And so. The solution was aseptically dispensed into ampoules at 1 mA each and melted and sealed to obtain an injection.

Example 15 0.4 mg of glucagon (manufactured by Funakoshi), 0.05 g of monoammonium glycyrrhizinate, and 10.9 g of NaC were dissolved in distilled water (or sterilized water), and the total amount was 1 OOm/bin. The mixture was aseptically dispensed into ampoules in 1 ml portions and sealed by melting to obtain an injection.

Example 16 Dynorphin (manufactured by Funakoshi) 0, 2 mg
, monoammonium glycyrrhizinate 0.1g, Nac
Dissolve 9g of fo in distilled water (or sterilized water) and reduce the total amount to 1
It was set to 00mA. The mixture was aseptically dispensed into ampoules in 1 ml portions and sealed by melting to obtain an injection.

Example 1 Finterferon (Type T, manufactured by Sigma) 10,000 units, dipotassium glycyrrhizinate 0.05 g, NaC10
, 9g in distilled water (or sterilized water) and make the total amount 100g.
mj! And so. The solution was aseptically dispensed into ampoules at 1 mA each and melted and sealed to obtain an injection.

"Effects of the Invention" According to the present invention, by adding glycyrrhizin, adsorption of physiologically active polypeptides such as cacitonin etc. to instruments, containers, etc. can be efficiently prevented, and pharmaceutical When administering these as
Its efficacy and safety are ensured without making the dosage inaccurate. Furthermore, if the present invention is used during production or storage,
There is a great economic advantage in preventing these expensive bioactive polypeptides from being lost due to adsorption. Furthermore, in immunoassays, polypeptides involved in the measurement system are prevented from adsorbing to the immunoreaction container, allowing accurate immunoassays to be performed.

Patent applicant: Toyo Jozo Co., Ltd.

Claims (4)

[Claims] (1) An adsorption inhibitor containing glycyrrhizin as an active ingredient for preventing adsorption of polypeptides. (2) Polypeptides have a molecular weight of 1000 to 3000
The adsorption inhibitor according to claim 1, which is a peptide or a derivative thereof falling within the range of 0. (3) The adsorption inhibitor according to claim 1, wherein the glycyrrhizin is one or more selected from the group of glycyrrhizinic acid, glycyrrhetinic acid, derivatives thereof, and non-toxic salts thereof. (4) The amount of glycyrrhizin compounded is 0.01% with respect to the aqueous solution or aqueous suspension containing the polypeptides.
4. The anti-adsorption agent according to claim 3, wherein the amount is 5%.