JPS61251627A - Water-dispersable and water-soluble polymer composition for non-oral administration - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to biologically active compositions comprising aqueous solutions of biologically active macromolecular compounds and carbohydrates.

It has been discovered that the compositions of the present invention provide sustained release of growth hormone when administered parenterally as solutions, dispersions and pastes.

The present invention provides water-soluble or water-dispersible carbohydrate polyhydric acid.
- a mixture of polymers of 1 or carbohydrates, and a biologically active macromolecular compound, and water or a buffer solution;
The present invention relates to a composition for parenteral administration comprising a pharmaceutically or pharmacologically acceptable solvent. The present invention, which includes a method of administering a molecular compound and maintaining its blood, also encompasses a method of increasing milk production in a dairy cow, which comprises parenterally administering to the cow a composition of the invention. do.

Biologically active macromolecular compounds of the invention include, for example, growth hormone, somatomedin, growth factors, and other biologically active fragments. These macromolecular compounds include growth hormones such as bovine growth hormone, ovine growth hormone, equine growth hormone, porcine growth hormone and human growth hormone. However, other biologically active macromolecular compounds, such as insulin,
may also be used within the scope of the invention.

Carbohydrates, such as dextrans and starches, have been considered to date to be inert with respect to their ability to adsorb high molecular weight substances, such as proteins.

As an example of this, dextran is a covalently cross-linked sphere [Sephadex, =7y-Macia (
pharmacia) AB]. These spheres are used for protein purification and chemical characterization when very high requirements are required for low non-specific adsorption of proteins to matrices.

However, as described in this invention, when certain carbohydrates are in solution, strong and previously undescribed interactions between carbohydrates and proteins are obtained. These interactions are very strong and require high concentrations of strong dissociative substances to disrupt interactions between carbohydrates and proteins.

Preferred polymers for use in the present invention are polymers of carbohydrates, such as dextones, dextrins, alginates, starches and fractionated starches (fr.
actionated 5 tarches), glycogen, pullulan, agarose,
Cellulose, chitosan, carrageenan, and synthetic biopolymers, as well as gums, such as xanthan gum,
Includes guar gum, locust bean gum, gum arabic, and gum karaya, their derivatives and mixtures thereof. These carbohydrate polymers, many of which are classified as polysaccharides or oligosaccharides or their derivatives, have the desirable property of being inert to biological systems; they are well characterized and nontoxic. and because of their water-soluble and water-dispersible properties, they can be easily administered in the form of aqueous compositions. The term water-soluble for these polymers is meant to encompass colloidal solutions and dispersions.

Suitable solvents for use in the compositions of the invention include diphosphate buffered saline (PBS);
This physiological saline is NaH2P adjusted to pH 7.1.
Oa (0,025 mol), Na2 HPO4 (0,
0.025 mol), and NaCl (0.15 mol); carbonate-buffered saline (CBS), which was adjusted to pH 9.4 and containing N &2 CO3 (0.025 mol);
% Le), NaHcO* (0,025 mol), and NaCl (0,15 mol); and physiological saline; alone or in other pharmaceutically acceptable solvents. combination with a pharmaceutically or pharmacologically acceptable solvent.

Pharmaceutically or pharmacologically acceptable solvents frequently used in biological preparations for parenteral administration include various liquid alcohols, glycols, esters,
and amides.

As such, these solvents have utility in the compositions of the present invention.

The present invention encompasses a method of preparing such a composition, which method includes a hydrophobic or hydrophilic compound that can interfere with hydrophobic or hydrophilic interactions in the complex. It involves the formation of complexes of proteins and carbohydrates in aqueous solution, along with other substances.

Furthermore, the formation of the conjugate can be carried out by methods already well known in the literature describing the groovedization of carbohydrates with various hydrophobic or charged moieties. Examples of hydrophobic derivatives are cholesterol or Cibachrome blue.
(blue), or charged groups, such as sulfonate or amino groups.

Additionally, stabilizers and preservatives 1 such as acids.

Ester a, organic ammonium halides, sulfides, alcohols, amines, anilides or organic mercury compounds are added to the compositions of the invention in concentrations of up to 0.2% on a 1 weight to volume basis to stabilize them. Preferred stabilizers for the compositions of the present invention that can improve properties include: dehyde acetate and their salts, the sodium salt being most preferred; salicylanilide; sorbic acid and its salts. , potassium salts are most preferred;
Sodium nitride and sodium nitrate.

By using detergents that disrupt hydrophobic interactions, we now show that it is this type of interaction that predominates for certain carbohydrates. The complex is
According to the invention, although having a size of less than 50 nanometers, the complex binding protein nevertheless
SA determination cannot be detected with the help of antibodies.

The inability of polyclonal antibodies to bind to proteins means that proteins are masked by carbohydrates (h
idcien).

Surprisingly, despite the aforementioned concealment, the protein still retains its biological activity, as the complex dissociates continuously and uniformly over 10 days after injecting it into animals. This has now been discovered. In-vivo dissociation is caused by a detergent system within the two bodies.
sy s t em), i.e. with the aid of systems that influence hydrophobic or hydrophilic interactions in the complex. Alternatively, when preparing the complex, such substances can be added to facilitate dissociation of the complex.

A preferred composition of the invention is 0.25/l1 on a weight basis.
O-100, 0/1.0, is composed of a polymer of soluble carbohydrates paired with a biologically active polymeric compound. The polymer to solvent ratio of soluble carbohydrate will, of course, depend on the solubility of the carbohydrate and the solvent used: 1.
These compositions having a ratio of polymer to biologically active macromolecular compound of soluble carbohydrates of from 0/1.0 to 100/1 can be prepared as an aqueous paste or solution in water or buffered saline. can be administered. Higher ratios of water-soluble polymers can of course also be used. The amount of water or buffered saline solution will vary depending on the carbohydrate polymer and its solubility properties, as well as the pH of the buffered saline sample. 0.
The aqueous composition of the present invention having a water to carbohydrate polymer ratio of 83/1 to 4.0/l and administered in the pH range of approximately pH 3.0 to pH 1 O. It has been found to be effective for administering and maintaining biologically active macromolecular compounds.

Particularly preferred compositions of the invention include bovine growth hormone;
and water, an aqueous buffer solution, or a saline solution;
Dextrin; Dextran; Glucose, galactose, mannose, glucuronic acid and fucose [Biopolymer PS-
87, v Bar Ken Brothers Company (Lever
Brothers Company)], U.S. Patent No. 4
, 357,423; contains a mixture of xanthan gum and locust bean gum.

The invention is further illustrated by the following examples.

Low viscosity corn dextrin (18 g) with acidic properties as a 25% dispersion with a viscosity of 50 centipoise at 25°C.

and bovine growth hormone (0,18 g) t, 15 ml carbonate buffer solution, pH 9,4 (Na2 C03,0
,025% Jlz; NaC0,,0,025 mol; and 0.15 mol) until a homogeneous paste is obtained.

Divide 9 milk-producing cows into 3 groups of 3 cows each. All cows received the same proportions of corn silage, alfalfa hay throughout the trial.

and 25) feed dairy cow concentrates adequate to produce Cg ~ 30 kg 7 days of milk. Cows are untreated for one week and daily milk production levels and bovine growth hormone blood levels are obtained for each group of three animals. The experimental treatments described in Table ■ below were performed at weeks 2, 43, and 4, and blood levels of bovine growth hormone were then established daily at 2 hours, 4 hours, and 6 hours after injection. determined by a radioimmunoassay procedure.

Treatment A, Control - 3 animals B, bGH in dextrin, 175 mg bGH1
times/week - 3 heads C, @ bGH in buffered saline, 25 mg bGH
7th - 3 heads.

All injections are administered subcutaneously.

The results of these experiments, summarized in Table H below, demonstrate the effectiveness of the compositions of the present invention in providing sustained release of bovine growth hormone compared to other compositions of the present invention. gives comparable results.

Table m below summarizes the milk production of these animals and demonstrates the effectiveness of the compositions of the invention for increasing milk production in animals treated with these compositions.

Table 1 Average (1) bovine growth hormone blood level in ng/ml
(nanograms/m1) B (0th, C) A 7.14th day after treatment (1 dish daily -
-(vs. 1Q-1 肚と一一一 shottu 12 hours 2.9
7.1 23.04 hours 3.3 9
．． 9 9.96 hours 2.8 21.6
5.71 days 3.2 65.4 4.42
Days 2.9 3B, 4 4.53 days 2.
8 16.3 4.64 days 2.5 1
6.5 4.65 days 3.2 26.1
7.66 days 2.7 12.9 3.17
Days 4.5 17.6 5.314 days 3
．． 9 22.9 8.718 days 8.1
31.6 10.7 (1) Average of 3 animals rounded to nearest 0.1.

V
ZeC~ Fruit” 11] Using the same woody procedure as in Example 1,
The compositions listed in Table 1 below are administered to two groups of sheep (3 sheep/group). Daily blood samples are taken for 5 days and assayed for blood levels of bGH as described above. The results of these experiments, summarized in Table V below, demonstrate that the compositions of the present invention are effective in maintaining high levels of growth hormone in the blood over extended periods of time.

Table 1 Administered composition Drum wheel 1 p DanbGH 30mg
30mg Dextrin 3g 1.5g Carboxymethyl None 0.163g Cellulose Carbonated Buffered 5ml 5ml Physiological Saline Friend-X Average (1) blood level of bovine growth hormone in sheep;
n g/m 1 1 MD E 0 hours 4.1 2゜6 2 hours 35.0 19.0 4 hours 39.0 23.0 6 hours 71.0 23.0 1 day 161.5 149.0 2 Day 17.7 22.2 3rd 12.0 13.8 4th, 51st, 9th 9th, 85th 11th
3.1 31.4 (1) Average of 3 animals.

Utilizing essentially the same procedure as in Example 2,
The compositions listed in Table 1 below are administered to two groups of sheep (3 sheep/group). Daily blood samples are taken for 5 days and then periodically and assayed for blood levels of bGH as described above. The results of these experiments, summarized in Table ■ below, demonstrate that the compositions of the present invention are effective in maintaining high levels of growth hormone in the blood over extended periods of time.

friend! Administered composition! uU7 ヱ 9bGH5B
mg 63mg Heteropolysaccharide 100mg 100mg (Biopolymer PS-87 Lever Brothers Company) Water 5ml
- Carbonated Buffered Saline (CBS) 70% Sorby - Thor/CB5 1! (Continued) Administered Composition 1: 1 bGH 23.8 mg Heteropolysaccharide 100 mg (Biopolymer PS-87 Lever Brothers 6 Company) Water - Carbonated Buffered Saline (CBS) 70% Sorbi 5 g Thor/CBS ■ Average (1) bovine growth hormone blood level in sheep,
ng/m1 ALJEJF G G1 day
2.4 l. 3 4.52 days 152.4 10
3.4 140.93 days 38.2 32.9
53.85 days 20.0 23.1 36.76
Sun 18.1 17.3 30.77 Sun 17
．． 1 14.7 25.18th 10.8 1
2.3 28.49 days 1g, 3 22.9
29.610 days 97.2 18.8 26.0
13 14.7 16.2 11.915
9.8 11.6 9.617 7.
7 11.0 8.320 9.1
9.2 5.8 (1) Average of 3 animals.

Utilizing the same woody procedure as in Example 2,
The compositions listed in Table 1 below are administered to two groups of sheep (3/group). Daily blood samples are taken for 5 days and then periodically assayed for blood levels of bGH as described above. The results of these experiments, summarized in Table ■ below, demonstrate that the compositions of the present invention are effective in maintaining high levels of growth hormone in the blood over extended periods of time.

■ Administered composition UL! ! 1 1 1bGH64,
4mg 35mg Guar gum 250mg -Carrageenan 125mg Saturated boric acid solution
5ml pH 9,4- water
5 ml ■ Average (1) bovine growth hormone blood level in sheep,
ng/ml Tadasoko I J 0 days 4.4 4.5 1 day 5.9 75.0 2 days 6.4 32.2 3 days 6.1 19.7 4 days 4.9 14.9 5 days 6.8 8.5 6th 15.4 11.1 8th 46.6 8.6 10th 58.3 16.7 13th 28.7 12.7 15th 38.4 - 17th 23.7 20 .0 (1) Average of 3 animals.

Utilizing the same woody procedure as in Example 2,
The compositions listed in Table X below are administered to two groups of sheep (3/group). Daily blood samples are taken for 5 days and assayed for blood levels of bGH as described above. The results of these experiments, summarized in Table XI below, demonstrate that the compositions of the present invention are effective in maintaining high levels of growth hormone in the blood over extended periods of time.

Administered composition composition 3
Nb5TH51.8mg 51.8mg 51°xantanga J, 150mg
150mg 15 carob gum
lQOmg 100mg 10 water
5g weight None
70% Sorbitol/Water None
5g of polyethylene glycol
None None 5gCB5/70% Sorbitol None None (2
) bGH dispersed/dissolved in water, N OP dispersed in polyethylene glycol 8mg 51.11mg 23.8m5r
64.4mg0mg 150ng 125m
g NoneOHg loomg None
250mg and 2350m5r None 5 ml None None None 2350mg None
2) Add 5g of rubber and aqueous solution to the suspension.

Table "Average (1) Bovine Growth of L Sheep n g/m Composition Day K
L Mo 2.4
4.1 1.21 148.5 160.
7 14.002 60.6 45.5 5
．． 33 49.6 29.8 5.44
35.6 28.0 5.85 29.2
23.4 7,86 24.8 20.
2 10,88 17.2 14.8 15
．． 810 8.7 9,9 12.513
7.7 6.0 6.715
9.1 5.2 8.017 11.8
4.4 2.9 Hormone Blood Level N OF 1.9 2.9 2.7 108.7 107.9 125.1 25.8 3.9.3 44.5 18.0 24.2 19.2 14.1 26.8 15.5 11.8 18.7 28.0 8.5 19.1 40.9 9.5 19.2 48.9 5.5 14.5 47.9 3.9 7. 5 34.2 4.4 7.3 31.8 4.7 6.2 51.6 111 Divide milk-producing cows into groups of 4 or 5.

Throughout the study, all cows were fed the same proportions of corn silage, alfalfa hay, and 25 kg to 3
Feed dairy cows a daily concentrate feed suitable to produce 0 kg/day of milk. Cows are untreated for two weeks and daily milk production levels are obtained for each group. The experimental treatments described in Table X■ below are carried out for three weeks and milk production data are recorded daily for groups of treated and untreated (control) animals.

LX” Actions taken 1
EbGH 350mg 350+ Xanthan gum -
− Locust bean gum −
-Heteropolysaccharide 500mg
400: (Biopolybou PS-87 Lever Brothers Company) Water
25g to 20g 170% sorbitol/
Physiological saline -- Polyethylene glycol/Physiological saline -- Once A Total ng 350mg 350mg 350m
g-300mg 300mg -200mg 200mg ng 200mg 400mg -This lOg--
The results of these experiments, summarized in Table X■ below, summarize the milk production of these animals and show that it is possible to increase milk production over an extended period of time in the treated animals. The compositions of the invention have proven effective.

(3) 2 (3) 30
0.75-0.49 5 1 10.52 7.35 6 2 12.10 8.84 13 3 10.60 8.05 11 4 9.74 13.02 12 5 10.81 13.10 9 6 3 .47 4.48 6 7 5.16 3.18 3 8 1.24 1.17 4 9 0.03 0.55-1 10 -3.26 -2.02 -2ti
1.51 -0.56 -012 -0.80 -3
．． 44 -313 -1.12 -2.45 -51
4 -1.27 0.22-5 (3) Average of 4 animals.

(4) Average of 5 animals.

Daily average percentage increase, 30 1.85 1.41. 97 8.95 9.14. 14 10.40 11.22 . 96 7.79 14.85. 29 5.1511.49. 79 1.80 3.79. 92 6.26 1.27. 46 5.29 6.99. 50 8.37. 9.49. 57 7.37 9.78. 14 5.78 6.11. 54 6.25 2,44. 89 3.35 3.50. 42 5.17-0.32. 17 4.08-1.04 1.2g of Dextran PZ/9 [Repp
e) Dissolve Vaxjo in 1 ml of water by heating. Add 100 g of bovine growth hormone 7 (150 mg/ml) to the solution at room temperature. After careful mixing, bovine growth hormone is quantified with the aid of the ELISA method.

The results show that only 5% of the added hormone was detectable in the detergent Triton.
When adding X100 in increments, the more detergent added, the more hormone could be detected. At a detergent concentration of 4% all of the added hormones could be detected. This indicates the formation of a strong complex between protein and carbohydrate. The detergent Triton x-too is used in biochemical studies when one requires the dissociation of strong hydrophobic interactions.

Cleaning agents that do not have this ability [e.g.
en) 80] is added.

No corresponding dissociation of the complex is seen.

When the mixture prepared by the procedure of Example 8 was kept in physiological buffer (PBS) for 5 days without the addition of any detergent, only 1% of the hormone was detected in the fifth dead weight. I can do it. Now, if a 4% Triton solution is added, as in Example 5, 1% of the added hormone
00% of the strains.

When the mixture of Example 8 was held for 4 days with various successively increasing concentrations of Triton, 0.0
It is shown that at a concentration of 5%, approximately 5% of the hormone is dissociated and detected by ELISA assay. 0
．． At a concentration of 0.08%, approximately 15% of the hormone is released after 5 days, at a concentration of 0.25%, approximately 20% of the hormone is dissociated, and at a concentration of 1%, approximately 30% of the hormone is complexed. Dissociated from.

Real m yu Dextran T500 (Pharmacia AB.

A dissociation similar to that described in Example 9 is observed when L. rapsara) is used as carbohydrate at a concentration of 0.35 g/ml.

Shiganbanyu J Radioactively labeled hormones are incorporated as described in Examples 8 or 11 and the radioactivity determined after filtration through a 50 nanometer filter shows that 50% of the radioactivity passes through the filter. This means that even though half of the hormone passes through, only 10% of this fraction is EL.
This shows that 90% of the hormone in this fraction is hidden and can be detected by the ISA method.
and means that it cannot be reached by antibodies in ELISA. As these results show, hormones are
Since it is not possible to obtain dissociation between the protein and the antibody,
Masked or hidden from detection.

Text fl Hypoxic rats that do not produce growth hormone
x rat) with the mixture according to Example 8 in a single injection, continuous growth over a period of 7 days is observed. The injection amount of hormone in this case is 2 mg.

When injected with the same amount without complexing with carbohydrates, rats show rapid growth during the first 24 hours, but then lose weight.

Claims (1)

Claims: 1. A biologically active composition comprising an aqueous solution of a complex between a protein and a carbohydrate. 2. A biologically active slow-release composition characterized by a mixture of a water-soluble or water-dispersible carbohydrate polymer or mixture of carbohydrate polymers and a biologically active macromolecular compound. 3. The composition according to claim 2, wherein the biologically active polymer compound is present as a complex. 4. The composition according to claim 2, wherein the mixture is present as a composite. 5. The composition according to claim 4, further comprising a hydrophobic or hydrophilic substance capable of interfering with hydrophobic or hydrophilic interactions in the complex. 6. The macromolecular compound is a growth hormone, somatomedin, growth factor or other biological compound in water, an aqueous buffer solution, physiological saline or a pharmaceutically or pharmacologically acceptable solvent or a mixture thereof. 3. A composition according to claim 2, which is a therapeutically active fragment. 7. The composition according to claim 4, 5 or 6, wherein the carbohydrate is starch or dextran. 8. The carbohydrate polymer is dextran, dextrin, alginate, starch, fractionated starch, glycogen, pullulan, agarose. , cellulose, chitosan, carrageenan, synthetic biological polymers, or the gums of xanthan gum, guar gum, carob gum, gum arabic, gum tragacanth or gum karaya, derivatives or mixtures thereof: and the growth hormone is bovine growth hormone, human growth hormone, 7. The composition of claim 6 which is ovine growth hormone, equine growth hormone or porcine growth hormone. 9. The carbohydrate polymer is dextrin; the growth hormone is bovine growth hormone; the solvent is water or an aqueous buffer solution; the weight ratio of dextrin to bovine growth hormone is 0.25/1.0-100; 9. The composition of claim 8, wherein the ratio of water or aqueous buffer solution to carbohydrate polymer is in the range 0.83/1 to 40/1. 10. The carbohydrate polymer is a heteropolysaccharide of glucose, galactose, mannose, glucuronic acid and fucose; the growth hormone is bovine growth hormone; the weight ratio of heteropolysaccharide to bovine growth hormone is 0.2.
ranges from 5/1.0 to 100/1, and the ratio of water or aqueous buffer solution to carbohydrate polymer is 0.83/1.
9. The composition of claim 8, wherein the ratio is 40/1. 11. The mixture of carbohydrate polymers is a mixture of xanthan gum and carob gum; the growth hormone is bovine growth hormone; the weight ratio of heteropolysaccharide to bovine growth hormone is 0.25/1.0 to 100/1; 9. The composition of claim 8, wherein the ratio of water or aqueous buffer solution to carbohydrate polymer is from 0.83/1 to 40/1. 12. A water-soluble or water-dispersible carbohydrate polymer or mixture of carbohydrate polymers; growth hormone, somatomedin, in an amount effective to maintain high blood levels;
biologically active macromolecular compounds of growth factors or other biologically active fragments; and water, aqueous buffer solutions, saline or pharmaceutically or pharmacologically acceptable solvents or their a composition comprising a mixture of biologically active macromolecular compounds of growth hormone, somatomedin, growth factors or other biologically active fragments in animals, characterized in that the composition consisting of a mixture is administered parenterally to animals; Methods of administering and maintaining blood levels. 13. The method of claim 12, wherein said polymer is a carbohydrate polymer or a mixture of carbohydrate polymers and said biologically active component is growth hormone. 14. The carbohydrate polymer may be dextran, dextrin, alginate, starch, fractionated starch, glycogen, chitosan, carrageenan, synthetic biopolymer substances, or the gums of xanthan gum, guar gum, locust bean gum, gum arabic, gum tragacanth or gum karaya. 14. The method according to claim 13, which is a derivative or a mixture of. 15. The carbohydrate polymer is dextrin;
15. The method of claim 14, wherein the growth hormone is bovine growth hormone and the solvent is water or an aqueous buffer solution. 16. The method of claim 14, wherein the carbohydrate polymer is a heteropolysaccharide of glucose, galactose, mannose, glucuronic acid and fucose, and the growth hormone is bovine growth hormone. 17. Claim 1, wherein said mixture of carbohydrate polymers is a mixture of xanthan gum and locust bean gum, and said growth hormone is bovine growth hormone.
The method described in Section 4. 18. A water-soluble or water-dispersible carbohydrate polymer or mixture of carbohydrate polymers; an effective amount of growth hormone, somatomedin, growth factor or other biologically active fragment to maintain increased weight gain. a biologically active macromolecular compound; and water, an aqueous buffer solution, physiological saline or a pharmaceutically or pharmacologically acceptable solvent or a mixture thereof, administered parenterally to an animal. A method of increasing weight gain in an animal, the method comprising administering. 19. Producing milk in dairy cows by parenterally administering a composition of a carbohydrate polymer or a mixture of carbohydrate polymers and an amount of bovine growth hormone effective to increase milk production. How to increase production. 20. The carbohydrate polymer is dextran, dextrin, alginate, starch, fractionated starch, glycogen, pullulan, agarose, cellulose, chitosan, carrageenan, synthetic biopolymer substances, or xanthan gum, guar gum, carob gum, gum arabic, gum tragacanth. or Karaya gum, derivatives or mixtures thereof. 21. The method of claim 20, wherein the carbohydrate polymer is dextrin. 22. The method of claim 20, wherein the carbohydrate polymer is a heteropolysaccharide of glucose, galactose, mannose, glucuronic acid and fucose. 23. The method of claim 20, wherein said mixture of carbohydrate polymers is a mixture of xanthan gum and locust bean gum. 24. Complexing the protein to the carbohydrate in an aqueous solution, optionally containing hydrophobic and/or hydrophilic substances capable of interfering with hydrophobic or hydrophilic interactions in the complex. A method of preparing a slow-release biologically active composition.