JPH0597701A - Kidney growth promoter - Google Patents

Abstract

PURPOSE:To provide a biological kidney growth promoter capable of activating the kidney reduced in nephrocyter or the function and expecting nephrocyte increase and the improvement of kidney function improvement with excellent safety. CONSTITUTION:The objective kidney growth promoter is derived from a natural material, containing, as the active component, a hybrid containing luteinzing hormone (LH).subunit, preferably a gonadotropic hormone hybrid containing LH alpha- or beta-subunit, showing the above-mentioned pharmacological effects and has excellent safety. In addition, e.g. chorionic gonodotropin(CG) or follicle- stimulating hormone(FSH), especially(CG) can be used as the above-mentioned gonadotropic hormone. LH, CG, FSH, ETC., as the raw materials are obtained from natural materials by extraction method or an artificial production method and the subunits separated therefrom by known method can be used. Further, the above-mentioned hybrid can be prepared therefrom by suitably using a known combination method.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a renal growth promoter derived from a biological substance. More specifically, the present invention is
The present invention relates to a renal growth-promoting agent containing a hybrid containing a luteinizing hormone subunit as an active ingredient.

In the present invention, among hybrids of luteinizing hormone and other hormones having different activities, a hybrid containing luteinizing hormone alpha-subunit or beta subunit has renal growth promoting activity. It is confirmed that administration of the renal growth-promoting agent of the present invention activates a kidney with reduced renal cells or a kidney with reduced renal function, and can be expected to increase renal cells or enhance renal function. , There are great things that contribute to the pharmaceutical world.

[0003]

2. Description of the Related Art The inventors of the present invention thought that there may be some factor that controls renal growth in the pituitary gland because the decrease in kidney weight is remarkable in the mouse from which the pituitary gland was previously removed. As a result of diligent research for a renal growth factor existing in the pituitary gland, a hormone-like component that promotes renal growth was separated from the pituitary gland and named as RGF (or lenotropin). (Japanese Clinical Volume 44, No. 1 (January, 1986), Supplement, pages 84 to 88)

Further, it has been clarified that the RGF obtained here is a mixture of luteinizing hormone isoforms.

The present inventors conducted further research to obtain RGF as a more purified substance, and separated and purified the fraction obtained as a luteinizing hormone / isoform mixture in advance by the difference in isoelectric point. However, surprisingly, they have found that the active ingredient is an aggregate of isoforms isolated in a single band. (Japanese Patent Laid-Open No. 63-26453
0)

Conventionally, luteinizing hormone (hereinafter abbreviated as LH)
And chorionic gonadotropin (hereinafter abbreviated as CG), and
Follicle stimulating hormone (abbreviated as FSH below) is a dimer in which an alpha (hereinafter abbreviated as α) subunit and a beta (hereinafter abbreviated as β) subunit are associated with each other as a molecule. However, due to the so-called micro-heterogeneity due to differences in the length of the amino acid chains that make up the molecule in each of the α subunit and β subunit, and the difference in the constituent sugars of the linked sugar chain, LH, CG It is known that FSH and FSH are aggregates of molecular species having different fine structures.

In the present invention, among LH, CG and FSH, LH, which is fractionated by the difference in their respective microstructures,
CG and FSH molecular species or an assembly of molecular species is called an isoform.

Luteinizing hormone is composed of two subunits, α and β subunits, and the α subunit contains 2 subunits.
It is known that it is a glycoprotein hormone in which one sugar chain is bound to the β subunit. Similarly, chorionic gonadotropin (CG) and follicle stimulating hormone (FSH) are known as similar glycoprotein hormones formed by associating two subunit proteins of α and β. These are substances that are considered to be a family of gonadotropins from their biological activity.

It is generally known that these hormones lose their biological activity when they are separated into α and β subunits alone, and that their activity is restored upon reassociation.

Biological activity of luteinizing hormone (LH activity)
Is not revived in the hybrid that re-associates LHα with CGβ and FSHβ, but LHβ with CGα and FSH
It is known that a hybrid with α will recover. For example, Thomas et al. Used pig and sheep-derived LH and human CG (hereinafter abbreviated as hCG) to prepare hybrids combining the α subunit and β subunit, and these also retain LH activity. Have reported that. (Endocrinology 109, 6, 1
933-1942, 1981).

However, they have not reported the presence of renal growth promoting activity in such hybrids.

From the results of primary structural analysis of these hormones, the amino acid sequence of the α subunit of LH was
As it turned out that it was almost the same as the α subunit of CG and FSH and that it differed in the β subunit, LH
It is presumed that the β subunit of LH is directly involved in the activity.

Hitherto, it has not been known at all that a hybrid containing LH has a renal growth promoting action.

The present inventors have also confirmed that CG and FSH have no renal growth promoting action.

[0015]

SUMMARY OF THE INVENTION An object of the present invention is to develop a novel renal growth promoter of natural origin, which is excellent in safety, that is, a bio-based renal growth promoter.

[0016]

The present invention has been made in order to achieve the above-mentioned object, but even if a hybrid containing LH is known, its action, especially the renal growth promoting action, is completely known. In the hybrid of these glycoprotein hormones, LH of the hybrid level of these glycoprotein hormones was dared to challenge the technical level of the art that it does not exist, and the knowledge that other gonadotropins such as CG and FSH also have no renal growth promoting effect. As a result of various studies focusing on those bound to contain α subunit or β subunit, it was found that, unexpectedly, the hybrid could express a renal growth promoting action, and based on this new finding, As a result, the present invention has been completed.

That is, the present invention relates to a renal growth-promoting agent whose important point is to use a hybrid body containing LH subunit as an active ingredient.

In the present invention, a hybrid is prepared by associating with LH subunit, and as a partner for association, hormones, lymphokines and various other physiologically active substances, and proteins are appropriately used. Examples of hormones include gonadotropins such as follicle stimulating hormone (FSH) and chorionic gonadotropin (CG), and thyroid stimulating hormone (hereinafter abbreviated as TSH).

As described above, according to the present invention, it becomes possible to produce a hybrid with a hormone or a physiologically active substance having another activity, so that an unnecessary activity is eliminated or a plurality of activities are expressed. And the possibility of further enhancing the activity.

In the present invention, the raw materials LH, CG,
To obtain FSH and the like, an extraction method from a naturally occurring raw material or an artificial production method can be used. As the extraction method, a hormone extract obtained from urine, blood or organs of mammals such as mouse, sheep and pig is used. As an artificial production method, mammalian LH, CG or F
A cell into which a gene designed to produce a SH dimer or a subunit monomer of each hormone is introduced is cultured and extracted from the culture system. Alternatively, it can be applied to a product obtained from a synthetic chemical reaction system.

For example, in the case of LH, when obtained from an organ, an aqueous ammonium sulfate solution is added to the pituitary gland to homogenize it, centrifuge it, purify the supernatant, freeze-dry and dissolve the freeze-dried product. Then, SP Sephadex chromatography, Sephadex G100 chromatography, CM cellulose chromatography,
LH crude product is obtained by repeating purification treatments such as conA chromatography and Sephadex G100 chromatography.

Using the already known extraction method, C
Extracts such as G and FSH can be obtained.

Further, if necessary, the obtained crude LH-purified product is subjected to a conventional method such as a treatment for separation / purification according to the difference in isoelectric point to obtain a further purified substance having a renal growth promoting activity. It can be obtained as several single bands.

The LH crudely purified product or purified product thus obtained can be separated into its constituent α and β subunits and used for the preparation of a hybrid. Similarly, CG, FSH and the like can be separated into subunits by a known method and used.

In order to prepare a hybrid with LH by associating these with each other, a known association method such as incubating both in a buffer in the presence of sodium azide is appropriately used, and the hybrid of each hormone subunit is prepared. Can be adjusted.

As a method for directly producing a hybrid artificially, cells into which a gene designed to produce a hybrid of LH, CG and FSH is introduced are cultured and extracted by the culture system.

Next, examples of the present invention will be described.

[0028]

Example 1

[0029]

[(1) Preparation of sheep luteinizing hormone]

Sheep Pituitary Powder (Waiki NZ
Refrigerating LTD) 150 g to 0.
The suspension was suspended in 1.5 l of a 15 M ammonium sulfate aqueous solution, 10 mM of phenylmethylsulfonyl fluoride was added, and then homogenized for 2 minutes with a Waring blender while cooling. After adjusting the pH to 4.0, the mixture was allowed to stand for a while under cooling, then centrifuged to collect the supernatant, and further to 0.
After adjusting the pH to 3.0 with a 5 M aqueous solution of metaphosphoric acid, centrifugation was performed and the supernatant was collected. After the pH of the supernatant was adjusted to 6.5 to 7.0, ammonium sulfate was added until it became 0.5 saturated, and the solution was sufficiently left to stand under cooling again. After centrifugation, the precipitate was collected and suspended in 50 to 100 ml of a 0.2 M dipotassium phosphate solution. After transferring to a dialysis tube and performing sufficient dialysis under cooling, the content liquid was taken out, the pH was adjusted again to 8.5 with 1N sodium hydroxide, and freeze-dried.

7 g of the obtained lyophilized powder was added to 0.01M
After dissolving with a disodium phosphate solution, SP-Sephadex (Pharmaci) equilibrated with the same buffer solution was used.
a) It was applied to a column and 0.1 M disodium phosphate elution fractions were collected. The relevant fraction was freeze-dried.

This lyophilized powder was dissolved in a small amount of 0.05 M ammonium hydrogen carbonate solution, and then Sephadex G-100 (Pharmaci) using the same buffer solution was used.
a) Fractionation by molecular sieve was performed using a column. After collecting about 40,000 fractions, freeze-drying was performed.

The dry powder was dissolved in a small amount of 10 mM Tris-hydrochloric acid (pH 7.5) and 0.3 M sodium chloride solution, and then Concanavalin using the same buffer solution was used.
Fractionation was carried out using an A-Sepharose (Pharmacia) column, and fractions eluted with 0.2 M methyl mannoside were collected. The relevant fraction was sufficiently dialyzed under cooling and then lyophilized to obtain 17 mg of LH crude purified product.

Then, the crude product was subjected to fractional purification by isoelectric point. LKB column for isoelectric focusing (LKB
Producter AB, Bromma, Swe
den) (440 ml volume) was used. Ampholine pH 3.5 ~ 1 as carrier ampholite
0 (LKB), Ampholine pH 9-11 (L
KB) and Ampholine pH 7-9 (LKB) were used. Create a sorbitol density gradient of 5-50%,
5 mg of the crude product was added to 2 ml of dense solution.
After it was dissolved in on, it was overlaid on the central part of the column. A 1M sodium hydroxide solution and a 0.01M acetic acid solution were used as electrode solutions. Energization was performed at 4 ° C. and 600 V to 1400 V for 24 hours. After the completion of energization, a 5 ml fraction was collected using a fraction collector for fractionation. Immediately p of each fraction
H (4 ° C.) was measured. (COM made by Denki Kagaku Co., Ltd.
-11, electrode type CE 105C, standard buffer solution manufactured by Wako Pure Chemical Industries, Ltd.). At the same time, the protein in each fraction was quantified. Each of the respective fractions was collected and pI> 9.8.
5, pI 9.85 to 9.60, pI 9.60 to 9.1
0, pI 9.10 to 8.60, pI 8.60 to 7.3
0, pI <7.30.

Each fraction was sufficiently dialyzed under cooling to remove Ampholine and sorbitol, and then freeze-dried to give a powder.

The present inventors have already disclosed in Japanese Patent Laid-Open No. 63-2645.
As described in No. 30, it was confirmed whether any of these fractions and the crude LH purified product had a renal growth promoting action.

[0037]

[(2) Purification of human chorionic gonadotropin] Purification of human chorionic gonadotropin (hCG) was performed by Bell e
t al, Endocrinology 84, 29
8, (1969).

Commercially available crude hCG (Organon, O
4 g of ss, Netherlands) was dissolved in 1 liter of 0.05 M ammonium acetate buffer (pH 5.0), and Sephadex C-50 equilibrated with the same buffer.
After being adsorbed on 0.1 M, it was eluted with 0.1 M ammonium acetate buffer (pH 5.0) and 0.5 M sodium chloride. Fractions forming the main peak were collected, dialyzed and then freeze-dried. The obtained powder (500 mg) was dissolved in 0.05 M ammonium hydrogen carbonate solution (50 ml), and the solution was passed through a Bio-Gel P-150 column equilibrated with the solution,
The main peak fractions were collected, dialyzed and lyophilized. next,
400 mg of the obtained powder was dissolved in 75 ml of 0.02 M ammonium hydrogen carbonate solution, and the pH was adjusted to 8.5 with aqueous ammonia. DEAE-Se equilibrated with the same solution
It was applied to a Phasex A-50 column and eluted with an ammonium hydrogen carbonate solution (pH 8.5) having a concentration gradient of 0.02 M to 0.4 M. Fractions forming the main peak were collected, dialyzed, and lyophilized to obtain purified hCG powder.

[0039]

[(3) Separation into α and β subunits] LH (crude product or LH isoform) was separated and fractionated into both α and β subunits by high performance liquid chromatography.

Reversed phase column (Baker Bond Wi)
de pore butyle C4: Baker Re
search Products), 0.1%
In the presence of TFA, water-organic solvent (acetonitrile: isopropanol, 3: 7) linear gradient system (organic solvent 10% to 60%) was used for separation. The α subunit was eluted relatively early and the β subunit was eluted later. Then both fractions were dried under reduced pressure to give a powder.

Purified hCG was also subjected to the same operation to separate and fractionate both α and β subunits.

[0042]

[(4) Preparation of hybrid] The subunits obtained from the LH crude purified product and the subunits obtained from the purified hCG were mixed in equal amounts, and a small amount of 0.5 M acetate buffer (pH 6.5), In a 0.01% NaN ₃ aqueous solution,
Incubation at 37 ° C. for 48 hours allowed re-association. Subsequently, in order to remove subunit monomers which are not associated with each other as much as possible, the product was purified by high performance liquid chromatography using a TSK gel G3000SW column (Tosoh Corp.), dialyzed and freeze-dried.

[0043]

[Measurement of Renal Growth Promoting Activity] It was determined which of the above hybrids had renal growth promoting activity.

The effect of promoting renal growth was determined by promoting the ability of renal cells to synthesize DNA.

Male CD rats (Charles River Japan Co., Ltd.) born on the same day with an average body weight of 100 g were subjected to hypophysectomy and castration, and were bred for 9 days. After confirming the rats for which surgery was properly performed, 5 including the control group
Each experiment section was established by grouping one or more rats.

Hybrids, 85 μg each for 15
0 μl (50 mM boric acid, buffer pH 7.5, 0.1%
It was dissolved in bovine serum (argomin) and subcutaneously injected into rats.
After 8 hours, the animals were decapitated, blood was removed, the left and right kidneys were immediately removed, the capsules were peeled off, and corticomedu was used.
Two slices each were made along the lally axis. 2 slices of 2 ml Krebs-Ring
Incubation was carried out at 37 ° C. for 2 hours in er bicarborate buffer (pH 7.5).
The buffer solution was previously aerated with 95% oxygen and 5% carbon dioxide gas mixture, and 1,2-methyl 3H-thymidine (New England Nuclear Corp.) was used.
s. ) Was added at 2 μCi / ml. After completion of the incubation, the slice was rinsed with a 4 mM thymidine solution and then stored at -20 ° C.

To the stored slices, 4 ml of distilled water was added, and homogenized using an Ultra Disperser (Yamato Scientific Co., Ltd.), and then Flick A and Munr.
o HN (1962, Biochim. Biophy.
s. DNA was extracted according to the method of Acta 55, 571), and Wannemacher JRWandB was used.
anks JWL. Wunner WH (1965, A
DNA is hydrolyzed according to the method of Nal Biochem 11: 320, and Burton K (195
6, Biochem. J. 62: 315) in accordance with the method of calf thymus DNA (type I, Sigma Chemi).
cal Co. ) Was used as a standard DNA, and DNA quantification was performed. At the same time, the radioactivity in 0.5 ml of the extracted DNA was measured by a conventional method.

The DNA synthesizing ability of renal DNA was calculated by radioactivity / DNA amount, and was calculated as a percentage of the experimental group with the control group as 100%.

As a result, LHα-hCGβ 127%,
An increase of 131% was observed for hCGα-LHβ, and hCGα-LHβ
-HCGβ and each subunit alone had no ability to promote DNA synthesis (95 to 103%).

The renal growth-promoting agent of the present invention comprises a hybrid body containing an LH subunit as an active ingredient, and if necessary, various conventional reagents are used to prepare various dosage forms according to a conventional method, and the preparation is orally or non-orally administered. Orally administer. Since the hybrid according to the present invention is of natural origin, it is extremely safe and may be administered according to the usual dosage of hormones.

[0051]

EFFECTS OF THE INVENTION The administration of the renal growth-promoting agent of the present invention can be expected to activate kidneys with reduced renal cells or kidneys with reduced renal function, and to increase renal cells or renal function.

Claims (3)

[Claims] 1. A renal growth-promoting agent comprising a hybrid body containing a luteinizing hormone subunit as an active ingredient. 2. A renal growth promoting agent comprising a luteinizing hormone / alpha subunit or a gonadotropin hybrid containing a beta subunit as an active ingredient. 3. The renal growth promoting agent according to claim 2, wherein the gonadotropin is chorionic gonadotropin.