KR100236468B1 - Feed additive type gastrointestinal delivery system of recombinant proteins for fish growth stimulation - Google Patents

Abstract

The present invention relates to a feed-added oral administration composition for promoting growth of fish containing a recombinant animal growth hormone as a main ingredient, comprising a animal growth hormone protein, a mucoadhesive agent, and a non-reducing sugar excipient prepared by genetic recombination technology. The composition of the present invention can be usefully used in the fish farming industry because it has an excellent effect of promoting fish growth, significantly improves the stability and absorbency of the drug during storage, and is easy to administer.

Description

Oral administration composition for promoting fish growth using recombinant protein

The present invention relates to an oral administration composition for promoting fish growth comprising a recombinant growth hormone protein, and more particularly, animal growth hormone and mucoadhesive agent having an excellent effect on promoting growth of aquaculture fish, produced by genetic recombination technology. And a non-reducing sugar excipient, the present invention relates to a feed-added oral administration composition for promoting fish growth, which improves the stability of the drug and its absorption in the body.

In conventional protein hormone formulations, glucose, sucrose, mannose, and the like are mainly used as dissolution aids or excipients (see Korean Patent Application No. 95-48340 of the applicant). However, when glucose, mannose, and the like are added to the protein preparation as reducing sugars, their carbonyl groups (RCH = O) react with free amino groups (eg, R'NH ₃ groups of lysine) on protein molecules. This results in a so-called Maillard reaction, which forms a Schiff base (RCH = NR '), resulting in browning of the protein (Musetta A, Hanson and S. Kathryn, Edmond Rouan, Introduction to formulation). of protein pharmaceuticals in Pharmaceutical Biotechnology, Vol. 3: Stability of protein pharmaceuticals part B (Tim J. Ahern and Mark C. Manning), Plenum press, New York: London, 201-233 (1992). This phenomenon leads to denaturation and degradation of the composition during long and short term storage of the protein formulation, thereby lowering the efficacy, and therefore, the sustained and improved stability of the composition is a challenge.

Another important requirement to be considered in addition to stability in the preparation of the composition is its absorption in the body. Typically, binders or mucoadhesive agents are used to enhance intestinal absorption of gastrointestinal degradable drugs such as proteins. For example, the Korean Patent Application No. 95-48340 of the present applicant uses a binder such as sodium polyacrylate. Once the composition is administered via a particular route, its intestinal absorption efficiency will depend on the concentration of the binder surrounding the protein.

Kazuhiro Morimoto et al tested the rectal administration of insulin in rats, using a polyacrylate water-soluble gel to promote intestinal absorption of the drug, and the optimal polyacrylate water-soluble gel to absorb the drug in the intestine of rats. The concentration was reported to be 0.1% (Kazuhiro Morimoto et al., Pharmaceutical studies of polyacrylic acid aqueous gel bases: Absorption of insulin from polyacrylic acid aqueous gel bases following rectal administration in alloxan diabetics rats and rabbits, J. Pharm.Dyn. , 3, 24-32 (1980)). In this case, since the concentration of the gel containing the protein is put into the rectum, the concentration of the gel in the formulation can be generally maintained as it is, but since the formulation is administered through the anus, there are many difficulties in daily use in actual target animals. Therefore, the ease and efficiency of administration should be considered together with the absorption in the body.

Accordingly, the present inventors have continued to prepare a composition for promoting growth of aquaculture fish, which is excellent in stability and absorption in the body and is easy to administer, and as a result, mannitol instead of the conventional reducing sugar as an excipient in the recombinant animal growth hormone Long- and short-term storage of the composition by using non-reducing sugars such as sorbitol, sucrose, trehalose, etc., and using 1-10% of sodium polyacrylate as a mucoadhesive agent administered to maximize intestinal absorption. Denatured and degraded protein is significantly reduced, it was found that a feed-added oral dosage form excellent in body absorption can be obtained to complete the present invention.

SUMMARY OF THE INVENTION An object of the present invention is to provide an oral administration composition for promoting fish growth, which has an excellent effect of promoting fish growth and has improved stability and absorption in the body.

1A and 1B are graphs showing changes in body length and weight when the composition of the present invention containing 1% sodium polyacrylate as a mucoadhesive agent is administered to a flounder,

2A and 2B are graphs showing changes in body length and weight when the composition of the present invention containing 4% sodium polyacrylate as a mucoadhesive agent is administered to a flounder,

3A and 3B are graphs showing changes in body weight and body weight when the composition of the present invention containing 10% sodium polyacrylate as a mucoadhesive agent is administered to a flounder,

Figures 4a, 4b and 4c is a graph showing the long-term storage stability at 4, 25 and 40 ℃ of the composition according to the present invention,

Figures 5a, 5b and 5c is a graph showing the change in concentrations of bovine growth hormone and IGF-I in the blood after administration of the composition of the present invention containing bovine growth hormone in a low and high doses of the flounder.

In order to achieve the above object, the present invention provides a feed additive oral dosage composition for promoting fish growth comprising a recombinant animal growth hormone, mucoadhesive agent and non-reducing sugar excipient.

Hereinafter, the present invention will be described in detail.

The components constituting the composition of the present invention improve the protein absorption efficiency in the intestinal mucosa along with the role of the binding agent to the feed when the protein, which is found to have an excellent effect on promoting fish growth, when administered in a feed additive form Mucoadhesive agents and dissolution aids or excipients.

In the composition of the present invention, the protein component is a mass production of growth hormones of chickens or mammals or growth hormones of cultured fish itself, which are already known to be effective in promoting growth of cultured fish (Jacqueline A. Gill, et al., Recombinant chicken and bovine growth hormone accelerate growth in aquacultured juvenile pacific salmon Oncorhynchus kisutch, BIO / TECHNOLOGY, 3, 643-646 (1985); Shunsuke Moriyama & Hiroshi Kawauchi, Growth stimulation of juvenile salmonids by immersion in recombinant salmon growth hormone, Nippon Suisan Gakkaishi, 56 (1), 31-34 (1990)). The route by which they are administered and absorbed into the bloodstream has been partially identified (JHWM Rombout, et al., Uptake and transport of intact macromolecule in the intestinal epithelium of carp (Cyprinus carpio) and the possible immunological implications, Cell Tissue Res., 239). , 519-530 (1985).

According to the applicant's Korean Patent Application No. 95-49165, the growth hormone protein as described above may promote the growth of cultured fish, and considering the economics, the same effect may be obtained even if it is less purified. Thus, the protein components included in the composition of the present invention are protein hormones in high purity or low purity form, which are mass produced by genetic recombination techniques depending on the purpose of administration. Specific examples of recombinant growth hormone proteins usable in the present invention include bovine growth hormone, pig growth hormone, chicken growth hormone, flounder growth hormone, eel growth hormone, salmon growth hormone, and the like. The content of these recombinant proteins in the composition is preferably 5 to 50% by weight.

Another component used in the composition of the present invention is a mucoadhesive agent that promotes intestinal absorption upon administration to fish as well as a binder that assists in adhering the composition to the feed when added to the feed. Rate is preferred. Polyacrylate is one of the high molecular weight materials which is charged and gelled by moisture (Lee Seung-jin, Development of Drug Delivery System and Pharmaceutical Additives, 87-95, Pharmaceutical Additives and Korean Pharmaceutical Association). They surround the protein components and release the drug that is enclosed in close contact with the intestinal mucosa, thus increasing absorption and reducing digestive enzyme activity. In addition, the first pass effect can be avoided by the gastrointestinal tract, there is an advantage that the change in conditions is small and easy to remove.

The concentration of sodium polyacrylate in the feed composition according to the present invention is optimal for the absorption of protein in the intestinal tract, and the ease and efficiency of the method of administration of the feed addition composition should also be taken into consideration. Therefore, the content of sodium polyacrylate in the composition of the present invention has a growth promoting effect, and can be easily used as a general feed additive composition regardless of the type of fish, the weight, the form and administration of various feeds, and the feeding rate. As determined through many experiments, the range of 1 to 10% of the composition is preferred.

When the content of the sodium polyacrylate is less than 1%, it may not be effective as a binder and a mucoadhesive agent during drug administration, and when it exceeds 10%, it is very difficult to administer because of the high viscosity of the sodium polyacrylate.

As the excipient which is another component included in the composition of the present invention, non-reducing sugars such as mannitol, sorbitol, sucrose, trehalose and the like are used. Excipients increase the volume of the drug when the feed is added so that the drug can be evenly distributed throughout the feed. In the non-reducing sugar used in the present invention, the reducing sugar, which has been conventionally used as an excipient, reacts with an amino group in the protein to form a Schiff base, resulting in browning, whereas the denatured or deamidation reaction of the protein in the frozen state during lyophilization of the protein solution. In addition to acting as a cryoprotectant that prevents the degradation process, it also plays a role in protecting the protein from denaturation and degradation during long and short term storage of the protein dried body and its composition after lyophilization.

The content of the non-reducing sugar excipient in the composition of the present invention is preferably at least two times the content of the protein component.

The feed-added oral administration composition for promoting fish growth of the present invention as described above can be applied to all cultured fish, including halibut, trout, carp, salmon, jopibolic (Urug), eel, growing and ornamental fish. The composition of the present invention can be used in addition to the feed in an absolute amount of 100 mg per kg of fish to be administered.

Hereinafter, the present invention will be described in more detail based on the following examples. However, these Examples are only for illustrating the present invention, the present invention is not limited to these Examples.

In the following Examples, the recombinant growth promoting protein is Korean Patent Registration Nos. 53945, 86911, 77698 and Patent Application Nos. 95-44346, 95-41475, No. Note that it is one of the proteins prepared using the method described in 96-4156 et al. In addition, the component ratio of sodium polyacrylate in the method of manufacturing the composition described in the following examples can be used as a universal feed additive composition regardless of the type of feed, administration method, feed rate, etc. while maintaining the effects of growth promoting proteins Note that it was determined through a lot of experimentation.

Example 1 Preparation and Confirmation of Composition Containing 1% Sodium Polyacrylate (Genetic Recombination Growth Promoting Protein: Sodium Polyacrylate: D-Mannitol = 10: 1: 89)

After purification, 10 g of the lyophilized transgenic bovine growth hormone was milled in a ball mill (ball mill, C-BMM, Cheil Science Industry Co., Ltd.) for 1 hour, followed by 1 g of sodium polyacrylate and D-mannitol ( 89 g of D-Mannitol) was added thereto and further milled for 2 hours to prepare 100 g of a fine powder composition.

The composition was administered to flounder reaching an average body weight of approximately 5.5 g at the start of administration. That is, 150 mg of the composition per 1 kg of body weight to be administered is dissolved in 2 ml of water, added to 8 g of feed to be fed once a day, left for 10 minutes to be absorbed well, and the remaining feed is added and dispersed to drop into grains. Was administered. The frequency of administration was administered once a week for 4 weeks, and the weight and body length were measured for one more week after the end of the administration to investigate the effect of promoting the growth of the composition (Sebosan: Yeonhwa-ri, Sanyang-eup, Tongyeong, Gyeongnam).

1A and 1B are graphs of the body weight and body weight during the breeding period of the composition-administered group and no-administration control group of the present invention containing 1% sodium polyacrylate as a mucoadhesive agent.

The growth-promoting effect was calculated as follows: The total weight gain of the (AB) / B × 100) administration group and the control group by comparing the average weight and body length (A) at the end of the test and the average weight and body length (B) at the beginning of the test. Was calculated (represented as C and D, respectively), and the growth promoting effect (E) of the composition was calculated by comparing the total growth rate of the administration group with the control group (CD) / D × 100), and the results are shown in the following table. 1 is shown.

This Example 1 is a test conducted in a real-world fish farm with good breeding conditions and good feed quality and breeding techniques. As can be seen from the results of Table 1, the weight of the fry increased as compared to the non-administered control group. It increased to 92% for 5 weeks including the administration period. In the case of the body length is also increased to 69%, when the breeding conditions are good at the fry period, the growth promoting effect of the present composition can be confirmed that more than 1.5 times more than the non-administered group.

Example 2 Preparation and Confirmation of Composition Containing 4% Sodium Polyacrylate (Gene Recombinant Growth Promoting Protein: Sodium Polyacrylate: D-Mannitol = 15: 4: 81)

After purification, 15 g of the lyophilized recombinant bovine growth hormone (G-BMM) was milled for 1 hour in a beads mill (C-BMM), followed by 4 g of sodium polyacrylate and 81 g of D-Mannitol. Further milling for 2 hours to prepare 100 g of the fine powder composition.

The composition was administered to broiler flounder with an average body weight of approximately 135 g at the start of administration. 100 mg of the composition per kg of the body to be administered was sprayed on the feed to be fed once a day, mixed well, and then sprayed with a little water with a spray and mixed again to allow the drug to slightly bind to the surface of the feed, and then administered to the flounder. The number of administrations was administered once a week for four weeks, and the body and body length were measured for one more week after the end of the administration to investigate the growth promoting effect of the composition according to the method described in Example 1 (own tank experiment). The results are shown in Table 1.

Figures 2a and 2b is a graph of the body weight and body weight during the breeding period of the administration of the composition of the present invention containing 4% sodium polyacrylate as a mucoadhesive to the flounder and the composition administration group and no administration control group.

Example 2 is to test the effect of the present composition when raised in a harsh environment. In particular, in this test, the flounder at the stage of growing fish was gradually adapted to the water temperature of 16 ° C. from 2 weeks before the start of the test in order to investigate the growth rate and the administration effect of the flounder in the tank maintained at low temperature (16 ° C.). In spite of the slow growth rate during the life cycle of the flounder, the total increase in body weight increased by 24% and 18% in body length compared to the 5 week-free control group, including the administration period.

Example 3 Preparation and Confirmation of Composition Containing 10% Sodium Polyacrylate (Gene Recombination Growth Promoting Protein: Sodium Polyacrylate: D-Mannitol = 10: 10: 80)

After purification, 10 g of the lyophilized recombinant bovine growth hormone (G-BMM) was milled for 1 hour in a ball mill (C-BMM), followed by 10 g of sodium polyacrylate and 80 g of D-Mannitol. Further milling for 2 hours to prepare 100 g of the fine powder composition.

The composition was administered to flounder with an average body weight of approximately 6 g at the start of administration. 150 mg of the composition per kilogram of body weight to be administered was sprayed on the feed to be fed once a day and mixed well, followed by spraying only a little water with a spray and mixing again to allow the drug to slightly bind to the surface of the feed, and then to the flounder. The frequency of administration was administered once a week for 4 weeks, and body weight and body length were measured over 3 weeks after the end of the administration to investigate the growth promoting effect of the composition according to the method described in Example 1 (own tank experiment). The results are shown in Table 1.

3A and 3B are graphs of the body length and body weight of the composition of the present invention comprising 10% sodium polyacrylate as a mucoadhesive agent and on the flounder during the breeding period of the composition-administered group and the non-administered control group.

In Example 3, although the growth rate during the life cycle of the flounder is very fast, the water temperature is changed, the quality of the feed is not good, and the effect of the present composition on the fry in an environment of low breeding technology is confirmed. The 8-week growth rate, including the duration of the administration, was increased by 38% and 16%, respectively. After 4 weeks, the growth rate of the administration group increased more than the no-administration group. Can be.

Sodium Polyacrylate Content Start date (B) End date (A) Total growth rate (A-B) / B × 100 dose group: C control group: D Growth Promotion Effect (E) (C-D) / D × 100 (%) One% Control weight 5.6 14.5 158.9 Length 8.1 11.8 45.6 Administration group weight 5.3 21.5 305.7 92 Length 7.9 14.0 77.2 69 4% Control weight 134.5 181.5 34.9 Length 23.6 25.9 9.8 Administration group weight 130.8 187.4 43.2 24 Length 23.4 26.1 11.6 18 10% Control weight 6.1 44.1 628.2 Length 8.9 16.8 87.5 Administration group weight 5.9 56.9 866.2 38 Length 8.9 17.9 101.2 16

Example 4 Confirmation of Stability of Composition

In order to confirm the stability of the composition of the present invention, each of the compositions prepared in Examples 1 to 3 (FA002, 003, 004) is stored for 6 months at various temperatures of 4, 25 and 40 ℃ bovine growth hormone Degradation or denaturation resulted in a drop in purity.

Figures 4a, 4b and 4c is a graph showing the purity of bovine growth hormone over time periods of long-term storage at 4, 25 and 40 ℃ of the composition according to the present invention. As can be seen from the above, all of the compositions of the present invention was found to be stable for at least 6 months at a storage temperature of 4 ℃, it was confirmed that both of the accelerated experiment results at 25 ℃ and 40 ℃ to maintain stability.

Example 5 Confirmation of Growth Promoting Effect by Delivery of Recombinant Protein in Blood after Administration of Composition

In order to confirm that the compositions of the present invention are delivered to the blood in the body from the digestive tract of the fish after administration to show the growth promoting effect of the fish, the composition prepared in Examples 1 to 3 orally administered to the flounder at low and high doses of blood We examined the concentration of bovine growth hormone (BST) in the blood and IGF-I. At this time, low dose means 20mg of hormone per 1kg body weight (BST20), high dose means 60mg of hormone amount per 1kg body weight (BST20).

Figures 5a, 5b and 5c is a graph measuring the concentrations of calcination hormone and IGF-I in the blood after the administration of the composition of the present invention containing bovine growth hormone to the flounder at low and high doses, respectively. As can be seen, the serum concentrations of bovine growth hormone reached peak levels in 9 and 12 hours in the low-dose (BST20) and high-dose (BST60) -administered groups, respectively, and the concentration of IGF-I was increased at 24 and 21 hours, respectively. The peak was reached, with a delay of about 9 to 15 hours. In addition, bovine growth hormone absorbed into the blood was found to recover to almost normal levels within a short time after reaching the blood peak (see FIG. 5C).

As described above, the fish growth promoting oral composition of the present invention comprising a recombinant animal growth hormone protein having a fish growth promoting effect, sodium polyacrylate as a mucoadhesive agent and a non-reducing sugar excipient has excellent growth promoting effects. While the stability of the drug during the storage period and the absorption in the body is significantly improved and easy to administer it can be usefully used in the fish farming industry.

Claims (7)

A feed additive oral administration composition for promoting fish growth, comprising a recombinant animal growth hormone, a mucoadhesive agent, and a non-reducing sugar excipient. The composition of claim 1, wherein the recombinant growth hormone protein is bovine growth hormone, pig growth hormone, chicken growth hormone, flounder growth hormone, eel growth hormone or salmon growth hormone. The composition of claim 2, wherein said recombinant growth hormone protein content is 5 to 10% of the composition. The composition of claim 1 wherein said mucoadhesive agent is sodium polyacrylate. 5. A composition according to claim 4, wherein the content of sodium polyacrylate is 1 to 10% of the composition. The composition of claim 1 wherein the non-reducing sugar excipient is mannitol, sorbitol, sucrose or trehalose. 7. The composition of claim 6, wherein the content of the excipient is at least twice the content of the recombinant protein in the composition.

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