JP2646048B2 - Lysozyme-containing pharmaceutical composition - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a pharmaceutical composition containing lysozyme or a salt thereof.

[0002]

2. Description of the Related Art Lysozyme is a protein having a molecular weight of about 14,000 and is found in all living organisms. Lysozyme or a salt thereof has pharmacological actions such as anti-inflammatory, hemostatic, anti-viral, anti-conjunctivitis, and anti-ocular inflammation, and is useful as a pharmaceutical. It is also used in cosmetics.

[0003] Egg white is usually used as a raw material for lysozyme.

[0004] In order to produce egg white lysozyme, typically, egg white is uniformly dispersed in water at around neutrality, and then contacted with a weakly acidic cation exchange resin to adsorb lysozyme to the resin. The lysozyme is eluted with an appropriate salt solution, and salting out is performed to extract lysozyme as crystals.

[0005] Literature relating to a method for producing or purifying egg white lysozyme is disclosed in, for example, JP-A-55-71490.
JP, JP-A-58-201986, JP-A-58-201986
JP-A-209977, JP-A-58-209981, and JP-A-58-212784.

[0006]

However, since lysozyme is a protein extracted from egg white and a heterogeneous protein for humans, when lysozyme is used as a drug such as eye drops, it must be a purified product. In some cases, lysozyme commercially available under the trade name may cause allergies.

[0007] The present inventors have studied whether the allergy caused by lysozyme is actually caused by lysozyme itself or by impurities contained in lysozyme. Considering that it may depend on the molecular weight,
He concluded that if a protein of a higher molecular weight than 4,000 lysozyme was contaminated, the protein could seem like an allergy caused by lysozyme itself.

[0008] Then, when lysozyme was obtained by various methods and analyzed, lysozyme commercially available as a purified product was found to have avidin (molecular weight of about 64000) detected in an electrophoretogram and avidin to be analyzed by electrophoresis. While avidin degradation products generated by the degradation in the process are also detected, some highly purified lysozyme high-purity products have substantially no avidin or avidin degradation products detected in the electropherogram. I found something.

[0009] The present inventors have found that allergies when this avidin was administered lysozyme - hypothesized that or not is one of the causes of, as a result of intense study to confirm it, to the following The invention described has been reached.

[0010]

That is, the present invention relates to a pharmaceutical composition containing lysozyme or a salt thereof,
As the lysozyme or a salt thereof, a highly purified avidin-free lysozyme or a salt thereof which does not substantially detect avidin and avidin degradation products in an electrophoretogram is used.

Hereinafter, the present invention will be described in detail.

Although lysozyme or a salt thereof can be produced from various raw materials, it is advantageous to produce it from egg white.

The lysozyme salts include, in addition to the hydrochloride,
Various inorganic or organic acid salts such as carbonate, phosphate, hexametaphosphate, sulfate, glutamate, glycerophosphate, and gluconate are exemplified. Especially the hydrochloride,
That is, lysozyme chloride is important.

As described in the section of the prior art, the method for producing lysozyme is to uniformly disperse egg white in water near neutrality and then contact it with a weakly acidic cation exchange resin to convert lysozyme to the resin. A method is employed in which lysozyme is adsorbed, the adsorbed lysozyme is eluted with an appropriate salt solution, and salting out is performed to extract lysozyme as crystals.

The advanced purification of lysozyme, for example,
(A) a method of removing impurities with a saline solution or water many times during a washing operation after adsorbing lysozyme to the resin by contacting the resin with the weakly acidic cation exchange resin; When eluting the lysozyme adsorbed on the surface with a salt solution or a buffer solution, the pH, concentration, stirring conditions, etc. are devised, and (c) an operation of further contacting the aqueous solution containing the eluted lysozyme with a weakly acidic cation exchange resin. (D) a method in which pH, concentration, stirring conditions, cooling conditions, etc. are devised when salting out by adding seed crystals after elution, (e) a method in which the salting-out operation is repeated a plurality of times, (f) lysozyme Dissolving the crystals in water, adding pulp, cotton, and other adsorbents while controlling the pH, and adsorbing impurities to this adsorbent, etc. Ri is achieved.

In any case, the lysozyme or a salt thereof used in the present invention is required to be highly purified to substantially no avidin. However,
As long as the gist of the present invention is not impaired, the present invention is not excluded from the present invention even if traces of avidin are contained, and `` contains substantially no avidin '' means that the presence or absence of avidin is easily confirmed. In the electrophoretogram of the electrophoresis method, which is one of the possible measurement methods, it is meant that almost no peak or shoulder indicating the presence of avidin and avidin degradation product is observed.

FIG. 1 shows the highly purified lysozyme used in the present invention.
(100 μg) is an electrophoretogram showing only a main peak based on lysozyme (1), and no shoulder or peak based on avidin or avidin degradation product.

FIG. 2 is an electrophoretogram of commercially available purified lysozyme (100 μg), in which a shoulder based on avidin (2) is recognized at the rising part of the main peak based on lysozyme (1).
Further, on the left side, a peak based on the avidin decomposition product (3) is also observed.

FIG. 3 shows the commercially available purified lysozyme (100 μm).
g) to avidin (2μg) is an electrophoretogram,
A peak based on avidin (2) is observed at the rising portion of the main peak based on lysozyme (1), and a peak based on avidin degradation product (3) is observed on the left side thereof.

FIG. 4 shows the commercially available purified lysozyme (100 μm).
g) plus avidin (4 μg) is an electropherogram of
A peak based on avidin (2) is observed at the rising portion of the main peak based on lysozyme (1), and a peak based on avidin degradation product (3) is observed on the left side thereof. The peak based on avidin (2) is higher than in FIG.

From the comparison between FIG. 2 and FIGS. 3 and 4, it can be confirmed that the shoulders and peaks in FIG. 2 are avidin and avidin degradation products, respectively.

The lysozyme-containing pharmaceutical composition obtained by using highly purified lysozyme or a salt thereof substantially free of avidin thus obtained hardly causes allergic symptoms, which will be described in detail in the examples below. Will be described.

The composition of the present invention is used as a medicament in the form of eye drops, injections, nasal drops, ear drops, suppositories, tablets, capsules, granules, powders, liquid preparations, creams and the like. It has pharmacological effects such as anti-inflammatory, anti-conjunctivitis, hemostasis, anti-virus. In particular, eye drops are important.

These pharmaceuticals can be produced using any conventional method for producing pharmaceuticals for convenience. Of course, as long as it does not contradict the purpose of the present invention, an active ingredient other than lysozyme is added, or an appropriate additive such as an excipient, a bulking agent, a binder, a wetting agent, a disintegrant, a lubricant, a preservative, A stabilizer, an emulsifier, a suspending agent, a fragrance, a dye, and the like may be allowed to coexist.

[0025]

Highly purified lysozyme or a salt thereof substantially containing no avidin hardly produces antibodies due to low antigenicity, and therefore has little possibility of causing allergic symptoms.

That is, an animal is sensitized with highly purified lysozyme substantially free of avidin, commercially available purified lysozyme in which avidin is detected, and avidin, respectively.
When the antibody titer is measured, the results are obtained such that the antibody titer of the avidin group is the highest, the commercially available purified lysozyme has the highest antibody titer, and the highly purified lysozyme has the lowest antibody titer.

[0027] As described above, commercially available purified lysozyme containing avidin has higher antibody production performance than highly purified lysozyme containing substantially no avidin, and furthermore, avidin alone shows high antibody production performance. ,
It can be seen that avidin is greatly involved in antibody production.

Next, these antibody-bearing animals were instilled,
Observation of the induced allergic symptoms revealed that animals sensitized with highly purified lysozyme substantially free of avidin were significantly weaker in allergic symptoms than animals sensitized with commercially available purified lysozyme containing avidin. It was considered to have low originality.

However, this result is considered to be due to the difference in the antibody titer between the two groups, which resulted in a difference in the induced allergic symptoms. Therefore, when the animals sensitized with avidin itself, which was considered the antigenic causative substance, were instilled and allergic symptoms were observed, in the group in which avidin was instilled, when the above highly purified lysozyme, commercially available purified lysozyme was instilled Severely stronger symptoms appeared, and the commercially available purified lysozyme instillation was weaker, while the highly purified lysozyme instillation showed only a weaker reaction.

This result indicates not only the possibility that the difference in symptoms between the highly purified lysozyme sensitized group described above and the commercially available purified lysozyme sensitized group is due to a difference in antibody titer, but also causes symptoms in animals having antibodies. This indicates a difference in the ability to induce, that is, a different inducibility. It is shown that the use of highly purified lysozyme reduces antigenicity and inducibility.

Thus, it has been revealed that the expression of allergic symptoms can be reduced by highly purifying lysozyme until avidin is substantially not detected, thereby producing a clinically safer lysozyme-containing drug. .

[0032]

The present invention will be further described with reference to the following examples.

<Experimental materials and methods> Production of highly purified lysozyme 3 g of commercially available lysozyme obtained from egg white was dissolved in water to make 1 liter, 30 g of sodium chloride was dissolved in it, and then dissolved in water to make 1 liter. 1N sodium hydroxide was added to adjust the pH to 9.5, and a 5% -concentration isoelectric crystal lysozyme suspension prepared in advance (5% saline)
0.4 g was added, and the mixture was kept at 20 ° C. for 4 hours, and then the crystallization temperature was lowered to 2 ° C., and kept at this temperature for another 4 hours. And filtered under conditions of a gauge pressure of 1.5 kg / cm ² . The operation up to filtration was performed under stirring conditions using an agitator.

The operation of dissolving the crystal lysozyme thus obtained in water and then recrystallizing it was further performed twice to obtain the desired highly purified lysozyme.

Next, the aqueous solution of the highly purified lysozyme was adjusted to an acidic pH of 3 to 3.5 with hydrochloric acid, and salt was added for salting out to obtain highly purified lysozyme chloride. Hereinafter, the term “highly purified lysozyme” refers to this highly purified lysozyme chloride.

In order to sensitize the test substance animals and to observe the occurrence of allergic reactions,
As the test substance, the above-mentioned highly purified lysozyme, commercially available purified lysozyme, and avidin (manufactured by Sigma) were used.

FIG. 1 is an electrophoretic diagram of highly purified lysozyme, and FIG. 2 is an electrophoretic diagram of commercially available purified lysozyme. The highly purified lysozyme has no shoulder at the position of avidin found in commercially available purified lysozyme, indicating that avidin is not substantially contaminated.

FIGS. 3 and 4 show that commercially available purified lysozyme (100 μg) was mixed with avidin (2 μg, 4 μg) at various concentrations.
FIG. 2 is an electrophoretogram of a sample to which avidin (μg) was added, and it can be seen that the shoulder seen in FIG. 2 is avidin.

Sensitization method Weight: 580 g to 70 purchased from Shizuoka Experimental Animal Cooperative
23 g of a male Hartley guinea pig of 0 g were grouped with 5 highly purified lysozyme groups, 5 group of commercially available lysozyme groups,
And 13 groups of avidin.

Highly purified lysozyme and commercially available purified lysozyme were each dissolved in physiological saline to give a 5% solution. Avidin was dissolved in physiological saline so as to be a 0.5% solution.

These solutions were mixed with Freund complete adjuvant (manufactured by Sigma) at a ratio of 1: 1 to prepare emulsions.

In each group, the first injection of 0.8 ml of each of the emulsions into the guinea pig's limb footpad was 0.8 ml per animal. Two weeks later, the same emulsion was prepared again, and 0.2 ml of the emulsion was injected into four portions of the back skin at a total of 0.8 ml per animal.

[0043] off the claws of the hind leg of one week after the guinea pigs of the antibody titer measured second time sensitization, blood was each 1ml collected in the separator Rapid tube mini S (manufactured by Sekisui Medical Co., Ltd.). The collected blood was centrifuged at 3000 rpm for 10 minutes, and the obtained serum was subjected to a PCA reaction for 4 hours using guinea pigs (15) prepared separately.
The antibody titer was measured.

That is, in each group, 0.1 ml of the diluted antiserum was injected into the back skin of the guinea pig, which had been shaved the day before, and after 4 hours, a solution containing 0.5% of antigen and 0.5% of Evans blue (physiological saline) 1 ml) was injected from the dorsal vein of the hind limb, and after 30 minutes, the major axis and minor axis of the pigment leakage spot on the skin were measured. The case where the average value of the major axis and the minor axis was 5 mm or more was defined as positive, and the maximum dilution that showed a positive reaction was defined as the antibody titer of the antiserum.

Induction method The day after the antibody titer was measured, the antibody was induced by eye drops. Eight eyes of sensitized guinea pigs of five highly purified lysozyme groups were instilled with 0.5% highly purified lysozyme dissolved in physiological saline, and two eyes were instilled with physiological saline.

Regarding the commercially available purified lysozyme group, 0.5% dissolved in physiological saline was added to 8 eyes of 5 sensitized guinea pigs.
Commercially purified lysozyme was instilled, and two eyes were instilled with physiological saline.

In the avidin group, 0.5% highly purified lysozyme was added to 8 eyes of 13 sensitized guinea pigs and 0.5 to 8 eyes.
% Commercial purified lysozyme was dissolved in saline in 8 eyes
0.5% avidin was instilled in two eyes with physiological saline.

[0048] Each of the animals was started with instillation 1, 2,
Observations were made after 4, 7, 24, 48 and 72 hours.
Observations were made on corneal opacity, conjunctival congestion, conjunctival edema and secretions according to the following scoring criteria.

The criteria for scoring the degree of eye disorder are as follows. The total score is a perfect score of 15. In the case of a symptom that falls in the middle of each score, an intermediate score was given as 0.5 or 1.5.

Corneal opacity 0 ... transparent 1 ... opacity enough to clearly see the details of the iris 2 ... opacity obscuring the details of the iris 3 ... opacity and opacity where the iris is not visible 4 ... complete opacity, bleeding and abscess May accompany

Conjunctival redness 0: no hyperemia 1: blood vessels around the cornea slightly dilated 2 ... dilation of blood vessels becomes noticeable 3 ... reddish viman in general 4 ... marked reddish overall

Conjunctival edema 0 ... no swelling 1 ... slight edema tendency 2 ... obvious swelling 3 ... begins to cover the cornea due to edema 4 ... covers a significant part of the cornea due to edema

Secretion 0: No secretion 1: Some amount different from normal 2: Wet eyelid and hair in contact with eyelid 3: Wet eyelid hair and considerable area around eye

<Experimental Results> Table 1 shows the antibody titers of each sensitization group.

[0055]

The highly purified lysozyme group has a width of × 100 to 800 and has an average of × 320, and the commercially available purified lysozyme group has × 80.
In the range of 0 to 1600, the average is × 1440, and the avidin group is ×
The average was × 5420 in the range of 3200 to 6400.
As described above, the antibody titer of the highly purified lysozyme group was determined by the t test (St test).
As a result, the antibody titer of avidin was significantly higher than that of the other two groups.

After the measurement of the antibody titer, a solution of highly purified lysozyme, a commercially available purified lysozyme, and avidin was instilled 8 times at an interval of 1 hour at a time of 5 hours to observe the occurrence of local allergic reaction by eye drops. And the results are shown in Table 2. Table 3 shows the results of the analysis of variance by the cumulative method.

Table 2 (Total scores of eye symptoms of various sensitized guinea pigs by various origin eyes) Observation time Sensitized group Induced animal No. 1 hr 2 hr 4 hr 7 hr 24 hr 48 hr 72 hr 1R 0 0 0 1 0 0 0 High High 1L 0 0 0 1 0 0 0 Refine 2R 0 0 0 0 0 0 0 Zo Zo 2L 0 0 0 0 0 0 0 Chi 3R 0 0 0 0 0 0 0 | | 3L 0 0 0 1 0 0 0 Mum 4R 0 0 0 0 0 0 0 Group 5R 0 0 0 1 0 0 0 Eye Average 0 0 0 0.5 0 0 0 City 6R 0 0 0 2 0 0 0 Resale Resale 6L 0 0 0 2 0 0 0 Zo Zo ZR 7R 1 1 1 1 0 0 0 H 7L 1 1 2 2 0 0 0 | | 8R 0 1 1 2 0 0 0 Mum 8L 0 0 1 2 0 0 0 9R 0 0 0 1 0 0 0 Eye 10R 0 0 0 0 0 0 0 Average 0.25 0.38 0.63 1.50 0 0 0 High 11R 1 1 1 0 0 0 0 Refine 12R 1 1 2 2 0 0 0 Zo 13R 0 0 0 0 0 0 0 H 14R 0 0 0 0.5 0 0 0 | 15R 0 0 0 0 0 0 0 16R 0 0 0 0 0 0 0 Point 17R 0 0 0 0 0 0 0 Eye 18R 0 0 0 0 0 0 0 Average 0.25 0.25 0.38 0.31 0 0 0 City 11L 1 2 2 2 1 1 0 Bill sales 12L 1 2 2 1 1 0 0 Zo 13L 0 1 1 0 0 0 0 Chi 14L 0 0 1 1 0 0 0 19R 0 0 0 0 0 0 0 time 20R 0 0 0 1 0.5 0 0 point 21R 0 0 0 1 0 0 0 eye 22R 0 0 2 2 1 1 0 average 0.25 0.63 1.0 1.0 0.44 0.25 0 15L 0 0 0 0 0 0 group 16L 0 1 2 2 2 2 2 Bi 17L 1 0 0 0 0 0 0 Di 18L 0 1 2 2 1 1 0 In 19L 0 0 0 1 0 0 0 Point 20L 0 0 1 3 3 2 0 Eye 21L 0 0 0 2 0 0 0 23L 0 1 1 1 1 1 0 Average 0.12 0.38 0.75 1.38 0.88 0.75 0.25 Note1. In the animal No., R is the right eye and L is the left eye. Note2. The right eye or the left eye (two in each sensitization group) of the animal No. not shown in the table was instilled with physiological saline. All of these did not show any symptoms.

[0059]Table 3 (Results of significance test between each group (Analysis of variance by cumulative method)) Commercially purified lyso avidin sensitization Avidin sensitization Avidin sensitization Team sensitization Commercially purified lyso Highly purified lysochi Commercially purified lyso avidin ophthalmic solution Team instillation team instillation team instillation Highly purified lysozyme Sensitization p <0.002 n.s. p <0.001 p <0.001 Highly purified lysozymeEye drops Commercially purified lysozyme sensitization n.s.n.s.n.s.Mu eye drops Avidin sensitization Highly purified lysozyme p <0.04 p <0.04Eye drops Avidin sensitization Commercially purified lysozyme n.s.Mu eye drops (N.s. has no significant difference)

In the group sensitized with highly purified lysozyme and induced by instillation with highly purified lysozyme, no symptoms were observed until 4 hours after the start of instillation, but blood vessels around the cornea were observed in 4 out of 8 eyes 7 hours later. Mild hyperemia of the bulbar conjunctiva was seen, which was slightly dilated. However, they recovered normally after 24 hours.

In the group sensitized with commercially available purified lysozyme and induced by instillation with commercially available purified lysozyme, mild congestion of bulbar conjunctiva was observed in 2 out of 8 eyes 1 hour after the start of instillation, and 4 hours later, Seven hours later, 2 eyes had mild hyperemia of bulbar conjunctiva and 5 eyes had marked dilation of blood vessels. However, after 24 hours everything had recovered.

Next, the animals sensitized with avidin were instilled with the respective solutions. In highly purified lysozyme instillation, mild congestion of bulbar conjunctiva began to appear in 2 eyes 1 hour after the start of instillation.
The eyes became severe with the instillation, but after 24 hours no symptoms were observed.

In the case of commercially available purified lysozyme instillation, mild conjunctival hyperemia of the bulbar conjunctiva began to appear in 2 eyes 1 hour after the start of instillation, and the symptoms worsened with the instillation. Symptoms were observed in 5 eyes. Seven hours later, the number of cases further increased, and symptoms were observed in six eyes. 4 of them
The eyes remained symptomatic after 24 hours, and were still observed in two eyes after 48 hours, but in both cases the symptoms resolved after 72 hours.

With avidin instillation, only one eye showed conjunctival hyperemia 1 hour after instillation, but increased with instillation, and after 7 hours, there were 6 eyes including 4 hyperemic eyes.
Their hyperemia was still visible in 4 eyes after 48 hours and did not recover even after 72 hours. In addition, conjunctival edema of luminance was observed in one eye after 7 hours and in one eye after 24 hours, and remained until 48 and 72 hours, respectively.

The allergic symptoms observed this time were hyperemia and edema of the bulbar conjunctiva, and no corneal opacity or secretion was observed. In addition, no symptom due to instillation of physiological saline was observed in any of the sensitized groups.

<Example of prescription>

[0067]

Formulation Example 3 (suppository) Highly purified lysozyme 50 mg cacao butter 1.5 g (these are made into one suppository by a conventional method)

[0069]

The composition of the present invention has low antigenicity despite the use of lysozyme, and therefore has almost no risk of causing allergic symptoms.

Therefore, the composition of the present invention is useful as a lysozyme-containing pharmaceutical composition.

[Brief description of the drawings]

FIG. 1 is an electrophoretogram of highly purified lysozyme used in the present invention.

FIG. 2 is an electrophoretogram of a commercially available purified lysozyme.

FIG. 3 is an electrophoretogram of a commercially available purified lysozyme obtained by adding avidin.

FIG. 4 is an electrophoretogram of the commercially available purified lysozyme with added avidin.

[Explanation of symbols]

 (1) lysozyme, (2) avidin, (3) avidin degradation product

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Kazuya Yoshida 4-26-5 Ikenoba, Taito-ku, Tokyo (72) Inventor Mineo Hasegawa 61-870, Uchikoshi-cho, Hachioji-shi, Tokyo

Claims (2)

(57) [Claims] 1. A pharmaceutical composition containing lysozyme or a salt thereof, wherein the lysozyme or a salt thereof is avidin-free highly purified lysozyme or a salt thereof which does not substantially detect avidin and avidin degradation products in an electrophoretogram. A lysozyme-containing pharmaceutical composition, characterized in that: 2. The pharmaceutical composition according to claim 1, which is an eye drop.