KR0152082B1 - A composition comprising somatotropin - Google Patents

Abstract

The present invention is to provide a somatotropin composition, characterized in that the somatotropin having in vivo activity evenly dispersed in the choline derivatives in combination with one or two or more tocopherol acetate or tocopherol and derivatives thereof. According to the present invention, not only can maximize the sustained effect of somatotropin in vivo, but also has no side effects in vivo and can exert a synergistic effect with somatotropin having in vivo activity and moreover, be applied to the composition. The advantage is that the somatotropin with biological activity exhibits an excellent sustaining effect equivalent to or greater than that of the metal or transition metal without binding.

Description

Somatotropin composition.

1 is a graph showing the weight gain effect when administering the bovine somatotropin composition according to the present invention,

2 is a graph showing the weight gain effect when administering the pig somatotropin composition according to the present invention,

3 is a graph showing the weight gain effect when administering the pig somatotropin composition according to the invention,

4 is a graph showing the weight gain effect when administering the bovine somatotropin composition according to the present invention,

5 is a graph showing the effect of weight gain upon administration of the bovine somatotropin composition according to the present invention,

Figure 6 is a graph showing the weight gain effect when administering the bovine somatotropin composition and pig somatotropin composition according to the present invention,

7 is a graph showing the effect on the acid flow rate when administering the somatotropin composition according to the present invention,

8 is a graph showing changes in the concentration of somatotropin in plasma when the somatotropin composition according to the present invention is administered.

The present invention relates to a somatotropin composition, and more particularly to the continuous release of such somatotropin when parenteral administration of somatotropin, generally known to have a short in vivo half life, to humans and animals. It relates to a composition.

Since most of somatotropin with in vivo activity has a short half-life in vivo, in order to exhibit sufficient biological effects and maintain the effect for a desired period of time, an overdose or frequency of administration should be increased. Relatively high doses can be caused to the subject.

Therefore, when the somatotropin is administered in vivo, it is necessary to maintain the effect for a long time in one administration. Therefore, according to such a requirement, a composition is developed to continuously release a substance having in vivo activity. In order to minimize the damage of the subject to be minimized while minimizing the amount of labor and labor associated with frequent administration has been conducted in various ways.

As an example, Korean Patent Publication No. 89-2631 discloses a thickening vehicle formed by adding an excipient and a moisturizer using vegetable oils or mineral oils, especially peanut oil and sesame oil, and then combining transition metal complexes, especially zinc. The growth hormone was mixed with the thickening vehicle to extend the sustained release of the active ingredient. However, the composition according to this method has a lasting effect of only about two weeks, growth hormone cannot be used as it is, and must be used in combination with a metal or transition metal through a complicated process.

For example, in order to form a complex of growth hormone and zinc ion, zinc chloride is added to growth hormone under limited conditions to form a suspension, and a series of exaggerations are repeated several times to prevent aggregation of the precipitate with sterile deionized water and then centrifugation. It must be lyophilized, which is very demanding and time consuming.

In addition, Korean Patent Publication No. 90-6886 describes a method of implanting a body coating in the form of a barrier coating that partially covers the formulation to suppress the release of somatotropin, in which case The sustaining effect may be somewhat prolonged, but this method requires the use of surgical methods or special instruments when administering to animals, and the foreign body feels larger than the liquid phase.

In European Patent No. 193,917, animal growth hormones were mixed with carbohydrate polymers, such as dextran, textine, starch, glycogen, cellulose, and chitosan, which were administered to cows and sheep. As short as 7 days and dextran and carbohydrate polymers may act as a fragrance source in some cases and thus exhibit a susceptible reaction, which is not preferred as a pharmaceutical composition.

European Patent No. 314,421 also describes a composition using oil as a main ingredient, an adjuvant and a carbohydrate polymer, but has essentially the same problem as similar to European Patent No. 193,917.

In addition, Korean Patent Application Publication No. 87-1825 also attached zinc to growth hormone and prepared an oil vehicle using peanut oil, and then the oil vehicle 8 mg and the growth hormone 40 mg were administered to pigs, but about 9 days. Only a short lasting effect was seen.

As described above, when somatotropin having biological activity using the Confucius composition is administered in vivo, the initial release is excessively excessive or the loss in vivo is high. There are problems such as side effects and somatotropin to be administered in advance to be combined with a metal or transition metal in advance.

On the other hand, the present inventors have disclosed a composition in the form of a mixture of animal growth hormone by adding a retardation aid to copherol acetate in Korean Patent Application No. 90-1689.

However, although the composition is surprisingly improved compared to other compositions in terms of sustainability and stability to the living body, the remaining ingredients are increased even after the viscosity increases or the active ingredient is exhausted in vivo. There was a problem that can remain in vivo for a long time.

Accordingly, the present inventors have intensively studied a composition which is most suitable for administering somatotropin having a biological activity in vivo and having excellent sustaining effect, so that the somatotropin having a biological activity is evenly dispersed in the choline derivative. When combined with tocopherol acetate or tocopherol and its derivatives, it is possible to prevent anomalous increase in viscosity, optimize initial release and maximize the sustained effect during in vivo administration, and to be somatoto with in vivo activity without any side effects in vivo. Synergistic effect with pins and moreover, it is found to be more stable because it exhibits a superior sustained effect equivalent to or greater than that of the metal or transition metal to somatotropin having biological activity to be applied to the composition. The present invention has been completed.

That is, an object of the present invention is to provide a very stable somatotropin composition while parenterally administering somatotropin having a bioactivity while continuously releasing the active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention is to provide a somatotropin composition, characterized in that the somatotropin having in vivo activity evenly dispersed in the choline derivatives in combination with one or two or more tocopherol acetate or tocopherol and derivatives thereof. will be.

Such a present invention will be described in more detail as follows.

The present invention is a mixture of choline derivatives and somatotropin having an in vivo activity to pass through a microfluidizer and to manipulate to obtain excellent properties during lyophilization and then tocopherol acetate or tocopherol and derivatives thereof It is a technique that allows somatotropin having in vivo activity to be continuously released in vivo when the composition thus obtained is administered parenterally.

At this time, somatotropin having an in vivo activity that can be used in the present invention is bovine somatotropin (BST: Bvine Somatotropine), porcine somatotropin (PST: Porcine Somatotropine) growth hormone of various animals, As human growth hormones and other hormones, both can be preferably used for somatotropin having a relatively short half-life in vivo.

In the present invention, the form of somatotropin is sufficient even in chemically unbound form, so that it may be used to lower the solubility of somatotropin in a complex ion metal or other water-soluble fluid in the field of the present invention. Somatotropin administered by dispersing with choline derivatives according to the present invention even if not chemically combined with

However, depending on the purpose of use, the combined form of somatotropin may be used. Somatotropin used in the present invention is a growth hormone, in particular, bovine somatotropin and pig somatotropin, such growth hormone can be obtained by extraction and concentration from the pituitary gland of various animals, prepared by recombinant DNA method Growth hormone is also suitable.

The content of somatotropin having in vivo activity in the composition according to the present invention is in a fairly wide range, the lower limit of which is the minimum requirement of somatotropin required in vivo, and the upper limit of the somatotropin such as tocopherol acetate in the composition. Any amount that can contain virtually all of the totrophin is acceptable but there is no need to use excess somatotropin unless there is a clear purpose.

On the other hand, tocopherol acetate or tocopherol and its derivatives used in the composition according to the present invention has pharmacological activity in itself, so in rats, mice, guinea pigs, pigs or poultry, the normal production process, lamb, calf or Dogs prevent muscle development abnormalities, and chicks can prevent encephalomalacia, muscle activity irregularities, muscle spasms, ataxia and tonic spasms.

And in mink, pigs, or cats, it helps to prevent steatitis.

In the composition of the present invention, choline derivatives are used in addition to tocopherol acetates in order to further delay the release of somatotropin. Such choline derivatives are well known, and one or two or more of them may be used in combination.

Particularly preferred choline derivatives in the present invention are phosphatidylcholine, lysophospholipid, plasmaogen, spinomyelin and the like.

The composition according to the present invention generally comprises 90 to 98% by weight, preferably 93 to 98% by weight of tocopherol acetate, when the combined weight of choline derivative and tocopherol acetate is 100, and the rest of the It is good to include.

In the case of the conventionally known composition, the lasting effect was not sufficient by itself, so that the desired purpose could not be achieved unless the solubility of somatotropin in the body was lowered. In addition to the addition of a metal, but also had to be administered in excess of somatotropin to compensate for the loss of excessive initial release, the composition according to the present invention, unlike the prior art in addition to the advantage of excellent release sustainability in vivo activity The use of somatotropin with chemically unbound state, rather than in the form of metal lead or complex compounds, has a surprisingly long lasting effect.

In addition, since choline derivatives are dispersed in somatotropin and mixed and dispersed in tocopherol acetate to prepare a composition showing excellent sustaining effect, the preparation process is very simple, the initial release is gentle, and the bioavailability of somatotropin is also increased. Since it is much improved compared to the prior art, it is possible to achieve a desired effect even if a small amount is given. Moreover, the metals or transition metals used to bind somatotropin in the prior art may cause side effects in the living body, but in the present invention, there is no fear of such side effects, but tocopherol acetate acts as a useful pharmacological agent in the living body. It also acts to prevent or mitigate biorejection of foreign substances.

Hereinafter, the present invention will be described in more detail based on the following examples.

Example 1

In a 500 ml beaker, 300 ml of bovine somatotropin solution (Lucky's recombinant bovine somatotropin) and 3 g of L-α-phosphatidylcholine (L-α-lecithin) extracted from soybeans of type IV-S After homogeneously mixing in a homomixer for 10 minutes, it was put into a microfluidizer maintained at 4 ° C and operated for 5 minutes.

The solution was collected, put into a lyophilization bottle, and rapidly frozen at −70 ° C. using dry ice and acetone, followed by freeze drying for at least 12 hours.

Lyophilized bovine somatotropin was put in a bowl and ground evenly. Tocopherol acetate was added to bovine somatotropin mixed with L-α-phosphatidylcholine and dispersed in a homomixer for 5 minutes.

When the bovine somatotropin composition thus obtained was administered, the effect of weight gain was tested as follows.

Female SD rats of about 80 to 100g were used for the experiment. Two weeks after surgery, the pituitary gland was removed using the Parapharyngeal method, which is a method of the pituitary gland removal (Hypophysectomy). The rats without weight change were selected by measuring their weight at the same time every day for 1 week, and these were used for the experiment.

Three rats with no change in body weight were administered with the bovine somatotropin composition prepared above in an amount of 0.1 ml per horse subcutaneously. The body weight was measured at the same time every day, compared with the weight of 3 days before giving, and the gain rate is shown in Table 1 and FIG.

On the other hand, tocopherol acetate was administered to three rats having no change in body weight as a control group.

Example 2

A bovine somatotropin mixed with L-α-phosphatidylcholine, which was prepared in the same manner as in Example 1 but did not pass through the microfluidizer, was experimented using three pituitary gland rats prepared in the same manner as in Example 1. The results are shown in Table 1 and FIG.

Example 3

200 ml of porcine somatotropin solution and 1.58 g of L-α-phosphatidylcholine were added and prepared in the same manner as in Example 1. Porcine somatotropin mixed with L-α-phosphatidylcholine was added to 1 ml of tocopherol acetate. The experiment was carried out in the same manner as in Example 1 and the results are shown in Table 2 and FIG.

Example 4

Example 1 was added in the same manner as in Example 3 was mixed with L-α-phosphatidylcholine pork somatotropin and 0.5 ml of tocopherol acetate, 0.5 ml of sesame oil and the results were tested in the same manner as in Example 1. And in Figure 2.

Example 5

Same as Example 4, using the same amount of peanut oil instead of sesame oil and was carried out in the same manner as in Example 1 and the results are shown in Table 2 and FIG.

Example 6

33 mg of L-α-phosphatidylcholine was added to 1 ml of tocopherol acetate and dispersed with a homomixer. Then, lyophilized pork somatotropin was added and experimented in the same manner as in Example 1. The results are shown in Tables 3 and 3 below. It is shown in the figure.

Example 7

As in Example 3, the amount of tocopherol acetate was adjusted to 2 ml, and the same concentration of pig somatotropin was injected into the pituitary gland rat, and the experiment was conducted in the same manner as in Example 1. The results are shown in Table 3 and FIG. Indicated.

Example 8

As in Example 3, using the same amount of peanut oil instead of tocopherol acetate in the same manner as in Example 1 and the results are shown in Table 3 and FIG.

Example 9

400 ml of bovine somatotropin solution and 3.102 g of L-α-phosphatidylcholine were added and prepared in the same manner as in Example 1, and bovine somatotropin mixed with L-α-phosphatidyl was added to 1 ml of tocopherol acetate. Experiments were carried out in the same manner as in Example 1, and the results are shown in Table 4 and FIG.

Example 10

The experiment was performed in the same manner as in Example 1 using bovine somatotropin mixed with L-α-phosphatidylcholine and shown in Example 4, and the results are shown in Table 4 and FIG.

Example 11

Lyophilized bovine somatotropin, which was not mixed with L-α-phosphatidylcholine, was mixed with 1 ml of tocopherol acetate in the same manner as in Example 1, and the results are shown in Table 4 and FIG.

Example 12

Add 1 ml of tocopherol acetate and 20 mg of aluminum monostearate, heat it to 150 ° C, heat it for 5 minutes, cool it sufficiently at room temperature, and add lyophilized bovine somatotropin unmixed with L-α-phosphatidylcholine. Experiments were carried out in the same manner as in Example 1, and the results are shown in Table 4 and FIG.

Example 13

As in Example 12, bovine somatotropin was added to bovine somatotropin and zinc-bonded bovine somatotropin was tested in the same manner as in Example 1, and the results are shown in Tables 4 and 4 below.

Example 14

20 mg of polyethylene glycol-75 lanolin (PEG-75 Lanolin or Solan E) was dispersed in 1 ml of tocopherol acetate with a homomixer, and zinc-typed bovine somatotropin was added and tested in the same manner as in Example 1. Are shown in Table 5 and FIG.

Example 15

As in Example 14, using Aracel 165 (glyceryl monostearate and PEG-100 stearate combined) instead of polyethylene glycol-75 lanolin was tested in the same manner as in Example 1 and the results are shown in Table 5 And in FIG.

Example 16

As in Example 12, using the spray dried bovine somatotropin instead of the lyophilized bovine somatotropin in the same manner as in Example 1 and the results are shown in Table 5 and FIG.

Example 17

As in Example 12, the aluminum monostearate was reduced to 10 mg, and instead of 10 mg of cholesterol was tested in the same manner as in Example 1, and the results are shown in Tables 5 and 5 below.

Example 18

As in Example 12, lyophilized bovine somatotropin and zinc-coupled bovine somatotropin were added and tested in the same manner as in Example 1. The results are shown in Table 6 and FIG.

Example 19

As in Example 12, instead of bovine somatotropin, zinc-coupled pig somatotropin was used, and the experiment was performed in the same manner as in Example 1. The results are shown in Table 6 and FIG.

[Experiment I]

Experiments were performed with three Holstein cows in lactating steam. 7.5 ml of the well-formed somatotropin composition mixed with L-α-phosphatidylcholine prepared in the same manner as in Example 1 was administered, and three cows were injected with the same amount of tocopherol acetate as a control. Six dairy cows were measured daily for two weeks prior to injection and used as an indicator for comparison with after injection.

The filled composition was injected subcutaneously over the scapula of the cow, the daily milk flow was measured after the injection, and the daily milk flow was summarized in the following Table 7 and the graph of the increase in milk flow rate over time is shown in FIG. The unit is expressed in kg / day.

Then, bovine blood was collected from the vein of the cow before and after the injection on the 3rd, 7th, 14th, 21st and 28th days, and the serum bovine somatotropin concentration was measured by radioimmunoassay. 8 is shown in FIG. 8 and the graph of the time-dependent trend for Table 8 is shown in FIG.

Experiment II

Fifteen castrated pigs weighing about 60 kg were used in the experiment. Five of the pigs were given pig somatotropin mixed with L-α-phosphatidylcholine three times every two weeks in an amount of 1.2 ml per horse (administration group 1), and the other five pigs were L-α- Porcine somatotropin mixed with phosphatidylcholine was given 1.8 ml per horse twice every three weeks (administration group 2), and the other five pigs were used as a control without administering anything.

The body weight of the pigs was measured before administration, and measured once per week at the same time after administration, along with feed efficiency and back fat thickness.

The weight of the pigs is expressed as the average daily weight gain (ADG, unit: kg / day), and the feed efficiency (FE = feed intake / weight, lower FE is better feed efficiency) and back fat thickness. It is shown in Table 9 below.

Experiment III

Pig somatotropin composition prepared as in Example 19 was injected once every three weeks in the amount of 0.9ml per 5 pigs (administration group 1), 3 weeks in the amount of 1.8ml per horse to the other 5 pigs Injected once (administration group 2), another 5 pigs were injected daily at an amount of 6 ml per animal (daily administration group), and the other 5 pigs were used as controls without administering anything. Body weight was measured every two weeks, and feed efficiency and back fat thickness were also measured.

The weight of pigs is expressed as the average daily weight gain (ADG, unit: kg / day), and the feed efficiency (FE = feed intake / weight, lower FE is better feed efficiency) and back fat thickness. It is shown in Table 9 below.

Claims (7)

A somatotropin composition, characterized in that the somatotropin having in vivo activity is evenly dispersed in choline derivatives and combined with one or two or more tocopherol acetates or tocopherols and derivatives thereof. The somatotropin composition of claim 1, wherein the somatotropin is an animal growth hormone or a human growth hormone. The somatotropin composition according to claim 2, wherein the animal growth hormone is bovine somatotropin or porcine somatotropin. The somatotropin composition according to claim 1, wherein the somatotropin is a complexed metal attached thereto or not attached thereto. According to claim 1, when the sum of the remaining components excluding somatotropin is 100% by weight of the choline derivative 2 to 7% by weight, tocopherol acetate or tocopherol acetate and derivatives containing 93 to 98% by weight Somatotropin composition characterized in that. The somatotropin composition according to claim 1, wherein the choline derivative is phosphatidylcholine, lysophospholipid, plasmagen or spinomyelin. The somatotropin composition according to claim 6, wherein the phosphatidylcholine is extracted from soybean and bovine liver, bovine heart, egg yolk and the like.