WO1994012203A1

WO1994012203A1 - DRUGS CONTAINING reg PROTEIN AS ACTIVE INGREDIENT

Info

Publication number: WO1994012203A1
Application number: PCT/JP1993/001746
Authority: WO
Inventors: Hiroshi Okamoto
Original assignee: Shionogi Seiyaku Kabushiki Kaisha
Priority date: 1992-12-01
Filing date: 1993-12-01
Publication date: 1994-06-09
Also published as: JPH06219963A; JP3570557B2

Abstract

A cell-proliferating agent and a human sugar level depressant both containing a human or rat reg protein as the active ingredient. Because these drugs enable regeneration of insulin-producing pancreatic β cells, it is possible to resort to the causal therapy of regeneration and activation of the pancreatic β cells of a patient with diabetes himself instead of resorting to the conventional symptomatic therapy of insulin administration. In addition, because the reg protein can proliferate Langerhans' islands, the drugs are useful as a human sugar level depressant.

Description

TECHNICAL FIELD The present invention relates to a drug containing reg protein K as an active ingredient, and more particularly, to a sugar level lowering agent in body fluid and an agent for cells. Background art

The reg protein is a polypeptide encoded by the regII gene. The regit gene is found as a gene that is specifically expressed in rat regenerative Langerhans β cells, and the human gene corresponding to the regit gene of this rat The regit gene has already been sculpted, and the production of reg proteins by the gene recombination method using yeast has been reported (JP 132388, JP 1-137994, JP 2-207007). )

However, no clear biological activity of this reg protein has been reported yet, and its use has not been established.

As mentioned above, the clear physiological activity of reg protein has not been reported yet, but the expression of reg gene in various groups other than the 8th occupation has been reported. There is a need for clear elucidation of biological activity and its application to medicine.

Related to the invention

The present inventors have attempted to elucidate the clear physiological activity of the reg protein. First, when reg protein: B was administered to a 90X knee resection rat, significant blood and urinary glucose levels were observed compared to the 90¾ 脬 resection rat without reg protein. I got a little. In addition, Based on this, we anticipated that reg protein would be involved in cell culture of Langerhans islets, and cultivated Langerhans islets at the invite mouth in the presence of reg proteins. Compared with cells cultured in the absence of protein K, significant increase in cell proliferation was observed. From such a fact, the inventors have completed the present invention.

The present invention relates to a saccharide in a body comprising, as an active ingredient, a polypeptide having any amino acid sequence selected from the following amino acid sequence groups (1) to (1): Value depressant:

(1) Sequence No. 1 in the sequence listing, amino acid E from 21st Gin to 1S6th Asn listed in S3:

(2) Amino acid sequence from Gin at position 23 to Asn at position 166 described in column number 1 in the column list;

(3) Amino acid sequence from lie at position 34 to Asn at position 166 listed in S column in £ column number 1 in the sequence listing;

(4) Amino acid sequence from G1n at position 20 to Ala at position 165 listed in column number 2 of column E:

(5) Amino acid sequence from the 22nd Gin to the U5th Ala described in column No. 2 of the sequence listing: and

(6) Amino acid sequence from lie at position 33 to Ala at position 165 described in SEQ ID NO: 2 in Sequence Listing,

I will provide a.

The present invention further provides, as an active ingredient, a polypeptide having any one of the amino acid sequences selected from the following amino acid sequence E in (1) to (5). Cell proliferating agent: (1) Amino acid sequence from the 21st Gin to the 166th Asn, which is not shown in £ column number 1 of the £ column list:

(2) Described in column E of column No. 1 in column © © Amino acid sequence from Gin at position 23 to Asn at position 166;

(3) Amino acid sequence from lie at position 34 to Asn at position 166 listed in SEQ ID NO: 1 in column E;

(4) Amino acid sequence from Gin at position 20 to Ala at position 165 listed in SEQ ID NO: 2 in the sequence listing;

(5) Amino K column from the 22nd Gin to the 155th A listed in SEQ ID NO: 2 in the column list; and

(6) Amino acid sequence from 1 le at the 33rd position to Ala at the 165th position shown in SEQ ID NO: 2 in column E,

I will provide a.

“Reg protein” means a polypeptide having any amino acid sequence selected from the above amino acid sequence group 0

Among the above amino acid sequences, (1) to (3) are the amino acid sequences of the human reg protein. It is known that human reg protein undergoes various degradations in a living body, and there are those having the above three types of amino acid sequences. However, there may be those having an amino acid sequence other than these due to degradation in vivo, and these are also included in the reg protein of the present invention.

Of the above amino acid sequences, (to (6) are amino acid sequences of rat reg proteins. These amino acid sequences correspond to human reg proteins (1) to (3). Corresponding ones are also included in the present invention. In addition, there may be other proteins having an amino acid K sequence due to degradation in the living body. Reg protein.

In a preferred embodiment of the present invention, the amino acid sequences of (2) and (5) are used.

In the present specification, “body fluid” means all body fluids in a living body such as blood, urine, and aqueous humor, and blood or urine is a typical example.

Drawing ffl nasty light

FIG. 1 shows the count of thymidine incorporation into Langerhans islet cell DNA at various reg protein concentrations. Marauders are mean values (η- I, I is standard error mean. *, ** and *** are p <, respectively, compared to values at reg protein concentration 0 / g / nl. 0.05, p-0.01 and p-0.001 represent statistically significant differences.

FIG. 2 shows the growth rate of Langerhans islet cells at various reg protein B concentrations based on the growth rate at a reg protein concentration of 0 ;: g / ml.讕 represents the average value, and I represents the difference average.

The human reg protein used in the present invention can be obtained by an extraction method, a synthesis method, or a gene recombination method. For example, see Japanese Patent Application Laid-Open No. 1-137334. It can be produced by the gene recombination method described above. Rat reg proteins can be easily manufactured by those skilled in the art by replacing the human regit gene with the rat regit gene according to this method. The sugar level-lowering agent containing the reg protein of the present invention as an active ingredient is preferably administered as an injection. For injections, reg protein is dissolved or dissolved in distilled water, physiological saline, aqueous acetic acid, vegetable oil, glycols, etc., and if necessary, stabilizers and preservatives are used. It is prepared by adding a tonicity agent, a soothing agent, a buffer and the like. Or freeze-dried by adding stabilizers, preservatives, excipients, etc.

DD

Then, at the time of use, it may be dissolved or distilled in distilled water, physiological saline, an acid aqueous solution, brobilling alcohol, or the like. It may be administered parenterally, preferably subcutaneously, intramuscularly or intravenously.

The glucose ffi-lowering effect of the reg protein of the present invention can be measured, for example, by administering a glucose-lowering agent of the present invention to a 90% excised rat and measuring blood glucose or urine level. Can be evaluated more. As shown in Example 1 below, administration of the sugar level lowering agent of the present invention containing a reg protein as an active ingredient significantly reduces urinary glucose and blood glucose utilization.

Since the blood concentration of reg protein K in healthy adults is reported to be about 100 ng / nl, the concentration of the reg protein in the present invention is 100 ng / ml or more. The dose may be maintained to maintain the above value, and for adults, 0.1 mg to lg, preferably lng to 500 mg, of reg protein may be administered per day.

The cell culture agent containing reg protein S of the present invention as an active ingredient can be used both in vitro and in vivo. When using for in vitro cell culture, add reg protein to the growth medium of cells containing fetal serum, glucose, etc. Reg protein is used by dissolving or suspending it in distilled water, physiological saline, acetic acid aqueous solution, propylene glycol or the like. When used in vivo, it is preferably used in the same manner as the above sugar level lowering agent.

增 ¾Evaluation of the effect is, for example, Francis. P. J..Southage,

J 丄 and Bone, AJ. Diabetologia. 35: 238-242 (1992) by determining the reproductive activity using Langerhans islands isolated as described in S. Can be

As shown in Example 2 below, cells cultured in a medium to which reg protein »was added had significantly higher crucible activity than those cultured in a medium without the addition.

The cell confluent of the present invention is preferably used at a concentration of 0.1 to 20 g / ral medium, and particularly preferably at a concentration of 0.3 to 10 tf g / inl medium.

The drug provided by the present invention, which contains rat or human reg protein as an active ingredient, is involved in cell growth and enables regeneration of insulin-producing 緙 9 cells. I do. This will enable a diabetic patient's own 厣) regenerative activation of 3 cells to be replaced by a regenerative therapy, instead of the conventional symptomatic treatment of insulin administration.

Furthermore, by administering a drug containing rat or human reg protein K as an active ingredient of the present invention, the sugar level in a body fluid can be reduced.

(Example)

The present invention will be described in more detail with reference to the following examples. It does not limit the invention.

[Illinois jl] Measurement of decrease in sugar level in body fluid

A. Preparation of a sugar level lowering agent containing rat reg protein as an active ingredient Rat reg protein was prepared by wrapping a human regit gene in accordance with the method described in JP-A-1-137994. The expression was replaced with the reg gene, and the product was isolated and purified. The rats _re g protein has a § Mi Roh acid E string at 22 th Gin or et al. 165 th Ala between the圮載in SEQ ID NO: 2. The purified rat reg protein was dissolved in 50 mM citric acid to a concentration of 0.91 ng / nil to prepare a preparation for administration.

B.90X 脖 Create resection rat

The 90X long excision rat was prepared by the method described in JP-A-1-132388. That is, the Wistar male rat was excised from 90X in the profession according to the method of Foglia, VG (Rev. Soc. Argent. Biol .. 20: 21-37 (1944)).

Administration of sugar lowering drugs

On pig ablation rats each, 1 B 1 times, over K formulations 0. 1ml (r _eg protein and to / zg), was administered intraperitoneally to 20 days continuous gun after 90X脬切removal. As a control group, 0.1 ml of 50ιηΜΒί acid was similarly administered to the 脬 resection rat. The rat's nursing conditions were as follows for both the administration group and the control group.

Temperature: 26 ± 2 · (:, humidity: 55 ± 5¾, water: tap water, feed: Chi catcher Lumpur's Li bar CRF1 - 1 ₀

D. Measurement of blood glucose and urine glucose

90Χ The above formulation or 50 mM L-acid was continuously applied for 20 consecutive days after resection. On day 21, the body weight, urinary glucose, blood glucose, and urine volume were measured. Urine and blood glucose levels were measured by the glucose- and oxidase method. Urine volume was measured by placing 30¾ 驿 resected rats individually in metabolic cages. The results are shown in Table 1.

(Table 1 reitt group / rat / B "0B, Ip)

Deadline l »

Weight weight turn blood glucose urine output

(c) (I) (l B) (■ l / dl) (ll)

red 31 &) 10 4.020 1ZS to

rei2 11 "s 7. US 101 110

ruS 25t 308 0.48S 114 10

2S0 290 0.110 "30

reiS 215 3 «0 0 .:" 11Z 10

regt 2SL S10 s.uo is SO Average 280.S ± 20.S .2 ± ie 2.7ί ± 2.J7 102.1 ± 21.3

(NS) (P <0.01) (P <0. OS)

WXS group

At resection

Weight weight me place

(c) (I) (ai / dl) (ii)

eontl S10 310 T.I0 12S 180 cont2 Z3S 340 22.71 3 SO eontS 300 310 7. "US 95 con" 290 S40 7.80 12i 120 eontS 245 szs IB.SO 212 110 eon "2SS death death death death average Z7S ± J1.1 S2S ± 1S 12.ί5 ± 7 189.1 ± 101.1 As is clear from Table 1, at day 21 after resection of 90X 咩, there was no significant difference in body weight in the reg-administered group compared to the control group! g was not observed, and significant ecchi levels were observed in urine glucose (high 0.01) and blood glucose (high 0.05).

[Example 2] Determination of proliferation activity using rat isolated Langerhans islet primary culture system

Α. Rat reg protein, which is a cell proliferating agent containing rat reg protein as an active ingredient, can be prepared by rat-regulating a rat according to the method described in JP-A-1-1379S4. It was expressed by substituting the reg gene, and isolated and purified. This rat reg protein has an amino acid sequence from the 22nd Gin to the 165th Ala described in Seq. ID No. 2 in the sequence listing. The purified rat reg protein K was dissolved at 0.91 ng / ml in 50 raM sodium hydroxide to obtain a cell crucible.

B. Isolation and culture of Langerhans islets

Langerhans β was isolated from the Wistar male rat (body weight 250-350 g) and incubated overnight in RPMI1640 medium containing 10% FCS and 11.ΙΒΜ glucose. Cultured. After culturing, the cells are subjected to a microscopic uptake under ffl microfluidics to remove exocrine cells and Langerhans S, which have a poor state of swelling. The cells were further cultured in RPM # 640 medium containing the yeast.

Cultivation in medium supplemented with cell culture

A culture medium in which various concentrations of the cell preparation prepared as described in Section A above were added to RPM11640 medium containing 2% FSC and 2.7 nM glucose, and no reg protein was used as a control. The supplemented medium was adapted and converted. Was used as a test medium.

In each well of a 24-well culture dish, 50 Langerhans S in the medium cultured as in the above KB section were divided.

Immediately, the medium was replaced with a test medium (In 1 / ゥ: 1), and the cells were cultured for 4B hours.

D. Addition of tritium and thymidine

The test medium was replaced with a new test medium, tritium 'thymidine was added (10 Ci / ral), and the cells were further cultured for 24 hours.

E. Recovery and crushing of Langerhans S

The Langerhans islets were collected, washed with Hanks' solution, and then sonicated in 350 ^ 1 ΙΟηΜ tris-acid buffer (pH 8.0) containing 5m EDTA. This was stored frozen until quantification.

F. Quantification of incorporation of tritium and thymidine into DNA

Trichloric acid is added to the sonicated product obtained as described in section E above, and precipitated, and the amount of tritium incorporated in the insoluble fraction is removed. It was measured by the filter method. Figures 1 and 2 show the results.

As is evident from Figs. 1 and 2, the group cultivated in the culture solution containing the cell crucible had a higher amount of thymidine incorporation and a higher cell growth activity than the control group. high. Furthermore, it can be seen that the cell proliferation activity depends on the concentration of the reg protein: S used. (Sequence listing)

E column number: 1

Array length: 1 66

Sequence type: amino acid

Topology: Straight «

Column Type: Peptide

Array

Met Ala Gin Thr Ser Ser Tyr Phe Met Leu I le Ser Cys Leu Met Phe

1 5 10 15

Leu Ser Gin Ser Gin Gly Gin Glu Ala Gin Thr Glu Leu Pro Gin Ala

20 25 30

Arg lie Ser Cys Pro Glu Gly Thr. Asn Ala Tyr Arg Ser Tyr Cys Tyr

35 40 45

Tyr Phe Asn Glu Asp Arg Glu Thr Trp Yal Asp Ala Asp Leu Tyr Cys

50 5S 60

Gin Asn Met Asn Ser Gly Asn Leu Val Ser Yal Leu Thr Gin Ala Glu 65 70 75 80

Gly Ala Phe Yal Ala Ser Le lie Lys Glu Ser Gly Thr Asp Asp Phe

85 90 95

Asn Yal Trp I le Gly Leu His Asp Pro Lys Lys Asn Arg Arg Trp His

100 105 110

Trp Ser Ser Gly Ser Leu Val Ser Tyr lys Ser Trp Gly lie Gly Ala

115 120 125

Pro Ser Ser Val Asn Pro Gly Tyr Cys Val Ser Leu Thr Ser Ser Thr

130 135 140

Gly Phe Gin Lys Trp Lys Asp Val Pro Cys Glu Asp Lys Phe Ser Phe 145 150 155 160

Yal Cys Lys Phe Lys Asn

165 ■ Ζ1 ·

S9I

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091 SSI OST SH

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S6 06 S8

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09 SL 01 S9

^ io nio BIV «10 naq IBA Jes Λ naq j / χ jas usv 13W usv

09 SS 09

uio s Q arid nsi dsy BIV nig BJV d "" S SIH dsv nig ^ ayi aqj

S 0, 9 £

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OE SZ OZ

3JV «IV -las oid nai dsy nio" ID «IV niO" 19 ^ IO "10 J8S 0 J3S

SI 0Ϊ S T nai IBA nais 3 JQS na na j sqj j 丄 s i usy 8J Jiu ¾aw

M "S

: S »ofii3 m: one a # 4 Ϊ: ^ ο½3

S9Τ: $ ^ C9½3

wio / e6 «irxDJ marauder ^{/ w 0Λ}

Claims

gff request IS

1. In a bodily fluid, a polypeptide having any amino acid sequence selected from the amino acid group E in the following (ι) to (6) is used as an active ingredient. Sugar level depressants:

c (1) The 21st Gin to the 166th G

Amino acid sequence up to Asn:

(2) Amino acid sequence from 23rd Gin to 1S6th Asn listed in column No. 1 of the sequence listing;

(3) Amino acid sequence from Ue at position 34 to 10 Asn at position 166 listed in SEQ ID NO: 1 in column E;

U) Amino acid sequence from Gin at position 20 to Ala at position 165 described in column number 2 of the sequence listing;

(5) In column S No. 2 of array ら, from Gin at position 22 of SH 戧 to position 1S5

Amino acid sequence up to A1a; and

15 (6) Amino acid sequence from the 33rd e to the 165th Ala listed in column No. 2 of column E.

2. The sugar level lowering agent according to claim 1, wherein the body fluid is blood or urine.

3. The amino acid sequence according to claim 1, wherein the amino acid sequence is an amino acid E sequence from the 23rd 20th Gin to the 166th Asn described in SEQ ID NO: 1 in the column list. Agent.

4. The amino acid sequence according to claim 1, wherein the amino acid sequence is an amino acid E sequence from Gin at the 22nd position to Ala at the 165th position listed in column No. 2 of the column list. Depressant.

5. A cell culture agent comprising, as an active ingredient, a polypeptide having any one of the amino acid sequences selected from the following amino acid sequence groups (1) to (6):

(1) Amino acid sequence from the 21st Gin to the 166th _c Asn in SS in SEQ ID NO: 1 in the column list;

Five

(2) Amino acid sequence from 23rd Gin to 6th Asn listed in K in SEQ ID NO: 1 in the sequence listing;

(3) Amino acid sequence from [le] at position 34 to Asn at position 166 described in SEQ ID NO: 1 in the sequence listing;

10 (4) Amino acid sequence from Gin at position 20 to Ala at position 165 described in SEQ ID NO: 2 in Sequence Listing;

(5) Amino acid sequence from Gin at position 22 to Ala at position 165 listed in SEQ ID NO: 2 in the column list: and

) From column 33 lie to column 165 in column no.

Amino acid sequences up to 15 Ala.

6. The pre-β cell is a Langerhans islet.

7. The amino acid sequence is an amino acid sequence from the 23rd Gin to the 166th Asn described in SEQ ID NO: 1 in the sequence listing.

Item 20. 5 Cell culture agent.

8. The cell according to claim 5, wherein the amino acid sequence is an amino acid E column from Z2nd Gin to 165th Ala described in E column number 2 in the sequence listing. Culture agent.