CZ255293A3 - Somatotropic agent with controlled release, and process for preparing thereof same - Google Patents

Description

The invention relates to a controlled-release somatotropic composition having a long in vivo half-life and which has little side effects on animals. The present invention also relates to a process for preparing the composition.

BACKGROUND OF THE INVENTION

Somatotropic agents, event. the most biologically active formulations have a relatively short in vivo half-life. In order to achieve sufficiently high biological effects and to maintain the prolonged biological activity of somatotropin, the composition has been administered in excess and / or very often, which may, however, cause serious difficulties to the animals receiving the composition. Therefore, benign somatotropin administration (in vivo administration of somatotropin) requires a benign pattern of prolongation of its effect; To meet these requirements, various methods have been developed for the preparation of such a somatotropic composition having prolonged release properties while at the same time being able to administer lower doses and lower dosing frequencies.

For example, in U.S. Pat. No. 4,985,440 discloses a composition comprising a transition metal complex, in particular a zinc-somatotropin complex in an oil-suspended vehicle made from a vegetable or mineral oil mixed with an ointment base and an adjuvant. This process is complicated and requires a somatotropin metal complex or a transition metal somatotropin complex. For example, a zinc-somatotropin complex is prepared by adding zinc chloride and a somatotropin solution under strictly defined conditions, and the excess zinc chloride is then eluted from the slurry containing the desired complex in several steps by centrifugation. Then the final solution is lyophilized, whereby the whole process becomes very time consuming and time consuming.

European Patent Publication no. 216,485 discloses a composition comprising a zinc-somatotropin complex dispersed in an oil, such as a peanut oil. Typically, 8 mg of the composition and 40 mg of somatotropin are administered to pigs, with a prolonged release effect of only 9 days. In addition, the process is similarly complicated as in the case of the aforementioned US Patent 4,985,404.

U.S. Pat. No. 4,863,736 provides a method for making implantable pills comprising somatotropin fibers with partial baffles of protective polymers such as shellac, beeswax and cellulose. These pills release somatotropin in vivo. Similarly, European Patent no. No. 374,120 provides a method of manufacturing an implantable somatotropin-containing composition in an osmotic device. European Patent Publication no. 246,540 discloses a method of synthesizing implantable compositions comprising a polypeptide and a fatty acid.

U.S. Pat. No. 4,786,501 illustrates a method of making an implantable pill formulation, the method comprising the steps of mixing somatotropin, sucrose and ethylcellulose, followed by coating with porous and non-porous polyethylene. U.S. Pat. No. 1,761,203 discloses a method for making an implantable formulation, comprising mixing somatotropin and water-insoluble polymers, adding a water-insoluble solvent, and drying. Although these implantable formulations have a lasting effect, they require surgery or a special apparatus for administration, thus requiring relatively complicated and unconventional routes of administration.

On the other hand, European Patent Publication No. 193,917 a composition comprising water-dispersible and water-soluble carbohydrate, carbohydrate polymers and a biologically active growth hormone macromolecule. European Patent Publication no. 314,421 shows a composition comprising an oil-dispersed polypeptide with a carbohydrate polymer such as dextran as a carrier material. European Patent Publication no. No. 211,691 discloses a composition comprising a polypeptide dispersed in a plurality of waxes as carrier materials, but has similar defects to said composition in European Patent Publication No. WO 97/18511. 193,917. With these compositions, animals often respond to a carbohydrate polymer, such as dextran, as an antigen, and responsive, making it impossible to sustain the use of the compositions.

Korean Patent Laid-open 91-15302 discloses a composition comprising animal somatotropin dispersed in tocopherol acetate with sustained release adjuvants. Because of its high viscosity, this composition is difficult to inject and there is a tendency to remain in administration and application in the tissue, which does not favor the tissue.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method for preparing a somatotropic composition having a long in vivo half-life, with little side effects on the animals to which the composition is administered.

According to a further aspect of the present invention there is provided a composition which is a somatotropic composition prepared according to the method of the present invention.

In accordance with the present invention, there is provided a process for the preparation of a somatotropic composition whose in vivo release is time-controlled, the process comprising the following steps:

dissolving lecithin and somatotropin in an aqueous solution to form a liposomal somatotropic solution, lyophilizing or lyophilizing said liposomal somatotropic solution under conditions customary for lyophilization; and suspending said lyophilized liposomal somatotropin in the tocopherol derivative to form a novel composition of the invention.

Description of the picture

This invention can be fully understood with reference to the accompanying drawings, in which:

Figures 1 and 2 show the effect of the composition of the invention on milk production when applied to cows giving milk.

Figure 3 shows the effect of serum concentration of somatotropin on the growth of pigs when the novel formulation is applied.

Figure 4 shows the effect of serum urea nitrogen concentration upon application of the composition of the invention on pig growth.

Figure 5 depicts the serum concentration of insulin-like growth factor I when administered to pigs as a result of the effect of pig growth.

According to the invention, the somatotropin solution is mixed with lecithin in a homogenizer and the resulting solution is processed in a microfluidizer to form a homogeneous mixture. This mixture is then lyophilized according to conventional methods and suspended in the tocopherol derivative to give the composition of the invention. Many types of somatropin can be used in the present invention as long as they have the desired biological activity. Such types include, for example: bovine somatotropin, porcine somatotropin, sheep somatotropin and the like. Although the use of somatotropin metal or transition metal complexes or modified somatotropin with low hydrogenation is not required according to the present invention, these types of somatotropin may also be used. The field of useful types of somatotropin includes those produced using recombinant DNA technology from host cells such as E. coli and yeast as well as natural somatotropin extracted from the pituitary gland of such animals as bovine and porcine cattle and sheep.

The tocopherol derivatives that may be used in the present invention include, for example: dl-oC-tocopherol acetate, dl-oC-tocopherol acetate, dl-oC-tocopherol succinate, dl-eC-tocopherol phosphate, 1 - << - tocopherol erol, 1- t -tocopherol acetate, 1-α-tocopherol succinate, 1-α-tocopherol phosphate, and α-2-tocopherol derivatives; with C-tocopherol acetate being preferred. The tocopherol derivatives support the normal reproductive mechanism of animals such as rats, mice, guinea pigs, pigs and poultry, helping to prevent defects in muscle development in animals; and also play an important role as carriers in the time-controlled release of somatotropin in vivo. In addition, tocopherol derivatives tend to prevent irregular muscle movements or muscle stiffness in chickens, as well as steatosis in mink, pig or cat. The amount of tocopherol derivatives used in the composition ranges from 85 to 99% by weight, preferably from 90 to 99% by weight based on the total weight of the composition.

The biologically active somatotropin used in the present invention is mixed with lecithin in a ratio ranging from 7: 3 to 8: 2 by weight. If the somatotropin content is less than the lower limit (ie 70% by weight), its biological activity is difficult to manifest. If the somatotropin content exceeds the upper limit (i.e. 80% by weight), it is difficult to form a liposomal somatotropic composition.

The lecithin present in the composition of the invention controls the time of somatotropin release in the animal body. The lecithin suitable for use in the composition of the invention may be, for example: phosphatidylcholine, lysophospholipid, plasmalogen and sfin gomyelin.

In addition, a suitable vehicle and / or adjuvant, i.e. ointment bases and excipients, may be added to the composition of the invention according to various types of application.

As already discussed, known formulations need to be somatotropin complexed with a metal or with a transition metal, which complicates methods such as dialysis, since its in vivo solubility must be lower in order to obtain the desired effect of release time control . Also, somatotropin must be applied in excess to achieve the initiating rupture effect.

In contrast, the formulation prepared according to the invention comprises a lyophilized liposomal somatotropin suspended in a tocopherol vehicle, releases biologically active somatotropin with controlled release time when administered to animals without the need for complex formation, i.e. the formulation and method of administration is very simple and economical. Very importantly, the formulation of the invention exhibits an excellent release profile along with low initiation release or somatotropin rupture in vivo.

While the metal or transition metal used in the somatotropin-metal complex formulation according to methods known in the art exhibits undesirable side effects, the composition of the invention exhibits only minor side effects. In addition, the tocopherol derivatives used herein have certain transfer pharmacological effects in vivo and ameliorate or prevent negative body responses and reactions when using the composition.

The following examples are intended to illustrate the invention in further detail without limiting it in any way, both in terms of content and scope.

Example 1

300 ml bovine somatotropin solution (30.03 mg / ml bovine somatotropin solution produced by recombinant DNA technology from Lucky Ltd.) is mixed with 3 g of L-α-phosphatidylcholine IV-S (L-β-lecithin) extracted from soybean in homogenizer for 20 minutes. The solution was treated in a microfluidizer for 5 minutes at 4 ° C, then poured into a flask, cooled rapidly to -70 ° C, dried in an ice-acetone bath and then lyophilized. 133.3 mg of the lyophilized powder containing L-cis-phosphatidylcholine and bovine somatotropin are mixed homogeneously with 1 ml of oC-tocopherol acetate in a homogenizer for 10 minutes.

The following test was performed on ten Holstein cows in the middle lactation period. The formulation is injected at a rate of 5 ml per head six times every two weeks. Five cows were used for testing and five cows were in the comparator group that did not receive the injection. The formulation was applied subcutaneously in the suprascapular region. Milk production was measured daily after injection as shown in Table 1 and Figure 1.

Table 1

Effect of the composition of the invention on daily milk production (kg)

Control group Group with injections Before injections 21.50 23.91 1st injection 21.66 27.75 2. injection 21.84 28.18 3. injection 21.51 28.10 4. injection 20.76 27.73 5. injection 19.59 28.06 6. injection 18.51 27.17 2 weeks after the 6th injection 17.94 23.45

Example 2

The formulations were prepared in the same manner as in Example 2 except that 333.35 mg of lyophilized bovine somatotropin and 37.02 mg of lecithin were mixed with 1 ml of tok-tocopherol acetate.

testing is performed on twelve Holstein cows at their mean lactation time. The formulation is injected at a rate of 2 ml per head every two weeks six times. Six cows were in the comparator group and six cows were in the test group. The test was performed in the same manner as in Example 1 and the result is shown in Table 2 and Figure 2.

Table 2

Effect of the composition of the invention on daily milk production (kg)

j Control group Injection group i Before injections 26.14 25.50 1. injection 22.6 23.8 2. injection 23.8 25.6 J 3. injection 22.3 24.8; 4. injection 22.0 24.5 5. injection 20.7 23.0 6. injection 21.0 23.2 2 weeks after 6. injection 18.9 19.3

Example 3

Pig somatotropin formulations were prepared in the same manner as in Example 1. The test was performed on fourteen pigs, each weighing 60 kg. The formulation was injected at 1.5 ml per head twice every three weeks, to seven pigs. The other seven pigs were in the comparator group that did not receive the injections. Feed consumption was measured three weeks after injection. Ofeed efficiency (FE) was calculated. Table 3 shows the average daily gain (ADG, kg / day) and feeding efficiency (FE = feed intake / weight gain).

Table 3

Daily increment and feeding efficiency

Control group Group with injections After the first ADG 0.94 0.97 three weeks FE 3.55 3.33 After the second ADG 0.81 0.96 three weeks FE 4.23 3.31

Example 4

Pig somatotropin formulations were prepared in the same manner as in Example 1. The test was performed on six pigs, each weighing 60 kg. The formulation is injected at 1.5 ml per head to three pigs. The other three pigs are in the control group without injections. They were measured: serum concentration of porcine somatotropin (PST), blood urea nitrogen (BUN) and insulin-like growth factor I (IGF-I concentration). The results are shown in Table 4 and in Figures 4 and 5.

Table 4

Serum concentration of PST, BUN and insulin-like growth factor I

day PST serum (ng / ml) IGF-I (ng / ml) BUN (mg / dl) PST serum (ng / ml) IGF-I (ng / ml) BUN (mg / dl) 0 1.43 343 14.5 1.44 441 15.0 1 1.52 306 15.7 325 891 4,5 3 1.15 308 17.4 46.9 1135 8.0 6 1.42 356 15.0 8.30 1018 6.0 10 1.44 351 19.8 3.10 692 16.5 14 1.46 354 19.7 1,02 506 19.0 21 1.80 348 23.3 1.10 438 17.7

As can be seen from the above, the formulation prepared according to the present invention exhibits a long half-life in vivo when administered to animals, showing very little side effects and is suitable for injection as its low viscosity facilitates its use. Therefore, the composition can be applied without surgery or a special apparatus. It is furthermore very advantageous that the process for preparing the composition is very simple and economical.

The present invention discloses the above-mentioned specific embodiment, however, various modifications and variations are possible in the application of the present invention without limiting the scope thereof to the extent that the invention is set forth below. claims.

Industrial applicability

The composition of the invention is prepared according to the process of the invention and can be used in the field of pharmaceutical industry in the industry.

Claims (10)

1. A process for the preparation of a controlled-release somatotropic composition comprising the steps of: homogenously mixing the somatotropin with lecithin to form a mixture; dissolving said mixture in an aqueous solution to form a liposomal somatotropic solution; lyophilizing said liposomal somatotropic solution under the conditions customary to lyophilize; and suspending said lyophilized liposome somatotropin in the tocopherol derivative to form said controlled release composition. The method of claim 1, wherein said somatotropin is selected from the group consisting of bovine somatotropin, porcine somatotropin and sheep somatotropin. The method of claim 1, wherein said somatotropin is prepared by recombinant DNA technology. The method of claim 1, wherein said tocopherol derivative is selected from the group consisting of: dl-α-tocopherol, dl-α-tocopherol acetate, dl-α-tocopherol succinate, dl-α-tocopherol phosphate 1-alpha-tocopherol, 1-alpha-tocopherol acetate, 1-alpha-tocopherol succinate, l - <? C-tocopherol phosphate, E * 2 ^_ tocopherolic derivatives. The method of claim 1, wherein said tocopherol derivative is dl-α-tocopherol acetate. The method of claim 1, wherein the lecithin is selected from the group consisting of: phosphatidyl choline, lysophospholipid, plasmogen, and sphingomyelin. The method of claim 1, wherein the somatotropin is mixed with lecithin in a ratio ranging from 7: 3 to 8: 2 (by weight). The process according to claim 1, wherein the amount of lyophilized liposomal somatotropin is in the range of 1 to 15% by weight, and the tocopherol derivative is in the range of 85 to 99% by weight, based on the total weight of the composition. A controlled-release somatotropic composition, characterized in that it is prepared according to the method of claim 1. The composition of claim 9, further comprising an ointment base and / or adjuvant.