JPS6041616A - Remedy for hepatic disease - Google Patents

Abstract

PURPOSE:The titled remedy comprising a substance obtained by removing a complement fixation site from a monoclonal antibody to be fixed selectively specifically to a hepatic glomerular basement membrane and/or endothelial cell membrane of hepatic glomerulus to give a site, linking an inhibitor for blood platelet aggregation, etc. to the site as a main component. CONSTITUTION:A complement fixation site is removed from a monoclonal antiboty to be fixed selectively specifically to a hepatic glomerular basement membrane and/or endotherlial cell membrane of hepatic glomerulus to give a site [Fa,b, F(ab')2, or one obtained by decomposing finely it, still having an antigen fixation site)], an inhibitor for blood platelet aggregation an anticoaglulant, or a thrombolytic drug is fixed to the site to give a substance, and the substance is used as a main component to give a remedy for hepatic diseases. It solves or all eviates side effects of a drug such as an anticoagulant, etc., and medicinal action of the drug is more effectively extracted. It will not act on other normal regions, and a dose is small.

Description

[Detailed description of the invention] This invention relates to therapeutic agents for renal diseases.

It is believed that immune mechanisms are involved in the onset of many cases of kidney disease. The main cases are cases in which autoantibodies against kidney tissue are produced in the living body and cause lesions when they combine with kidney tissue, and antigens unrelated to kidney tissue, such as foreign antigens such as bacteria and viruses. Antibodies are produced that combine with antigenic components in the circulating blood to form antigen-antibody complexes (ICs).
This is a case in which the glomerulonephritis is deposited in the kidneys while circulating and causes lesions, and it is thought that most cases of glomerulonephritis belong to this case.

In any of these cases, when an antigen-antibody reaction occurs, complement is activated and various undesirable phenomena occur. For example, in glomerulonephritis, immune complexes deposited on the renal glomerular basement membrane activate complement and activate the coagulation system.
This is said to cause local circulation disorders. That is, it is thought that activation of complement causes platelet adhesion and, subsequently, thrombus formation, resulting in decreased renal function and proteinuria.

As described above, in many cases, immune mechanisms are involved in the onset of kidney diseases and the progression of lesions, and various investigations have been conducted from an immunological standpoint regarding their treatment. Typical examples include immunosuppressants that suppress the immune reaction itself by suppressing the production of antibodies, and anticoagulants that reduce the temperature of tissue damage that occurs as a result of antigen-antibody reactions.

These immunosuppressants, anticoagulants, etc. each have excellent effects on renal diseases, but they also have drawbacks. In other words, since immunosuppressants act suppressively not only on antibody-producing cells but also on cells throughout the body, they act as a defense for the body by suppressing normal immune reactions. , there is a risk of side effects such as increased susceptibility to Bakuto syndrome and hemorrhoids. Also, anticoagulants, etc., act not only on the affected area but also on the whole body, and if used in large quantities, they may cause bleeding as a side effect. It includes problems such as

The present invention was developed with this background in mind, and its purpose is to effectively exert its medicinal effects by acting intensively only on diseased areas. The purpose is to provide treatment for renal diseases.For this purpose, the 7t3 therapeutic drug according to the present invention, which has been added to : Base IF (
membrane) 2 and/or selective IF for renal glomerular endothelial cell membranes,
From the monoclonal antibody that matches 〒27 to 〒27, supplementary Itoyoshiai f1 ``7.
The gist of this is to have a platelet Mε Hayabusa inhibitor, a 11i'C aggregation agent, or a thrombolytic agent combined with an A. In addition to eliminating the side effects of anticoagulants and other drugs and reducing FV, their pharmacological effects were brought to the fore.

As mentioned above, such anticoagulants, aggregation inhibitors, thrombolytic agents, etc. are originally intended to act only on the disease r*shc, and are intended to act on other diseases. i Rina r'X
B (For <L, it is preferable to have the effect of one of these drugs.This willpower) If the drug is administered to L, then the accumulation of The drug concentrates in the disease r'BI IJ'5 and exerts its pharmacological action there. It is expected that the effects of 6111 can be greatly reduced or even completely eliminated.

Therefore, the present inventor focused on the fact that autoantibodies produced in vivo in autoimmune diseases selectively and specifically bind to body components, and developed anti-glomerular basement membrane membranes. The idea was to utilize the property of antibodies that appear in nephritis patients who develop autoantibodies to specifically bind to the renal glomerular basement membrane as an antigen. Then, antibodies of the same type as this antibody are separately made from K14, and they are subjected to predetermined processing, and
By combining this with a platelet aggregation inhibitor, an anticoagulant, and a thrombolytic agent, they completed a potential treatment for renal diseases.

In the present invention, the monoclonal antibody is specifically sensitized with human myeloma cells and 8173 bulbs of nephritis patients in which anti-thread 3'Je body basement membrane autoantibodies appear, or intraglomerular internal and external III follicles. human IFzlit
8173 cells were cultured in a predetermined incubator using a cell fusion cell, and from among them, one cell n', "f It can be obtained by selecting clones that produce antibodies of the same type as the antibody.However, since these monoclonal antibodies contain a portion that has complement fixing properties as they are,
Administering this to patients may cause an adverse immune response.

Therefore, in the present invention, Fab is obtained by removing the complement-fixing portion from such a monoclonal antibody.

1'(a+]')2. Fab', or they are further degraded leaving the antigen-binding site intact and combined with ff4i1, which has traditionally been used as a treatment for renal diseases.
By combining a platelet aggregation inhibitor, an anti-l''X15 solid agent, and a thrombolytic agent, a therapeutic agent was created.

As a means of removing the complement-binding portion from a monoclonal antibody, it is preferable to use a protease. In other words, the protein component 111T1 element is used to digest the Fr portion of the protein (j1 double bonding property). At this time, monovalent Fab is obtained by using papain, and divalent F(ab') is obtained by using pepsin. 2 are obtained respectively, and this F(a
b') 2 culm 1 nojizu λshiba 11 songs IF a b'
is obtained. In addition to these proteases, trypsin and plasmin'-:γ can be used.

However, the method for preparing monoclonal antibodies described above, Fa
The preparation method of b, etc. is just one specific example, and other methods are also possible, and these preparation methods are not required in the present invention, and those obtained by genetic engineering techniques etc. However, it is possible to apply the present invention.

Next, Fab, F(ab')2. As means for binding these platelet aggregation inhibitors and the like to Fab' etc., periodic acid oxidation method, carbodiimide method, glutaraldehyde method, diazo method, cyanogen bromide method and other methods can be used. Here, the periodic acid oxidation method refers to the above-mentioned drug having a diol, which is oxidized with periodic acid to convert the diol into dialdehyde, which is then combined with Fab etc. to a pH of 9 to 9.
．． 5 to convert dialdehyde groups and -NH2 of Fab etc.
This is to condense the groups. Since this periodic acid oxidation method, the above-mentioned carbodiimide method, and glutaraldehyde method are also known methods, detailed explanations thereof will be omitted here.

In this way, the platelet aggregation inhibitor, anticoagulant,
When a thrombolytic agent is administered to a patient with renal disease, these Fabs, etc., along with a platelet aggregation inhibitor, selectively and specifically bind to the patient's renal glomerular basement membrane or renal glomerular endothelial cells. do. In other words, these drugs conventionally used for kidney diseases bind to diseased areas through Fab and other media, and exert their unique pharmacological effects in the diseased areas. In other words, when only an anticoagulant or the like is administered to a patient, only a portion of the drug acts on the diseased area, whereas in the case of the drug according to the present invention, the administered drug only acts on the diseased area. All of these drugs are concentrated in the diseased and dense areas and used for recovery, so even if the dose is small, it will have a significant effect on alleviating the disorder or recovering it. Moreover, these drugs according to the present invention do not act on other normal areas and require a small dosage, so they do not cause harmful side effects such as bleeding.

In addition, when administering to a patient a combination of the above-mentioned Fab or the like with a platelet aggregation inhibitor, an anticoagulant, or a thrombolytic agent, it is possible to administer these alone or in combination of two or more.

Next, in order to clarify the excellent effects of the present invention, below are examples of long-term use of the drug according to the present invention on patients.
A detailed explanation will be given along with an example of the preparation of the co-agent.

(A) Preparation example of drug (a) Preparation example of monoclonal antibody homologous to anti-glomerular basement membrane antibody or anti-glomerular endothelial cell membrane antibody Non-secreting human myeloma cells (8-azaguanine ( az
Aguanine) resistant strain human eloma cells and nephritis in which anti-glomerular basement membrane autoantibodies appear, B lymphocytes of U are mixed to produce Porkroid mitogeny (Pok
After stimulation with eweed tnitogen), these were combined with cell rrlA in polyethylene glycol to produce hybridomas.

These hybridoma cells were poured into tissue i' culture plates and cultured in I (A'I' medium).
Since the cell group consists of a heterogeneous (heterogeneous) cell group, a cell group that produces the desired anti-glomerular basement membrane antibody must be selected from among these. Therefore, EIA (enZy+nQimmuno as
The supernatant from the above culture was dispensed into a plate (say), and which colonies produced the desired antibody was analyzed by ELISA using an anti-immunoglobulin antibody conjugated with peroxidase (Peroxidase). . Anti-immunoglobulin antibodies bind to the target antigen-antibody complex when it exists, so if the enzyme activity of the enzyme bound to this is examined after treatment with the anti-immunoglobulin antibody, it is possible to produce the target antibody. This will tell you which colony the hyperdoma belongs to.

If a colony that is determined to be positive by supernatant analysis or a colony that produces the desired antibody is found, it is dispensed onto another tissue culture plate and cultured. If you perform selective culture by repeating the analysis and dispensing of the specific antibody in the supernatant,
Finally, monoclonal 111TI cells which produce antibodies of the same type as the target anti-renal glomerular group 11 membrane autoantibodies are obtained.

On the other hand, regarding monoclonal antibodies homologous to anti-renal glomerular endothelial cell membrane autoantibodies, human F' I! 1 cell is sensitized with the glomerular endothelial cell membrane in a test tube, a hybridoma is created using the T lymphocytes and human myeloma cells, and the hybridoma is obtained by subjecting it to the same treatment as above. I can do it.

(o) Fab, F(ab')2. Preparation of Fab' etc. (
i) Preparation of (Fab')2 1150 weight of pepsin was added to the monoclonal antibody obtained in (a), and p II 4. Q~5.0.37
After digestion of the Fc portion of the antibody by treatment for 8 to 18 hours at
O-100) (Product name Near 1 Aden, Pharmacia fliu) t JTBz -c? The mixture was separated by filtration and F(ab')2 was a)).

(ii) Preparation of F a b'<I)-cThe obtained F(al)')2 was treated with 0.01M 2-mercabutoecnol p)13. After reduction at O, the free radical S I-I group was alkylated with monoyo-1'acetamide to obtain Fab'.

(1st item) Fab toning ν The monochrome reel antibody prepared in 11) in (a) above,
1/100 weight of papain, synutein 0.01 M,
El) TA, 0.002M Na-phosphorus lπ buffer (0,1Δ(, soluble at pI(7,0)) and 37°
The Fc portion of the antibody was digested by treatment with C for 16 hours, (1)
The Fab portion was isolated in the same manner as described above.

(c) Platelet antiagulant 1i for the above F a l), etc.
(1 Simultaneous 11, Example of combination of anticoagulant and thrombolytic agent Periodate oxidation method (References - Wcaklcy, F-,
+3゜ctal, Bio also ecbnol, Bioeng
, Vol. 15, 1189j7 (1973)), heparin (anticoagulant), etc.
Combined. That is, this is oxidized with periodic acid to form sia pdehyde, which is then converted into the F obtained in (b) above.
ab, F(ab')2. pu9~ with Fab' etc.
9.5 to react with the aldehyde group and -NH of Fab etc.
The two groups were condensed. The Schiff salt formed at this time was ternary added to stabilize the bond.

Warfarin (anti-HL)
solid agent), urokinase (
thrombolytic agents), prostaglandins (pros tag
land 1n) E, No. 12 + ■2 + D2 and their derivatives (e.g. ticlopidine (ticlopidine)
idine), ifenprozi/l/ (1fcnp
rodil), dilazeg (dilazcp), trimethacin (tri+ncLazinc), bencyclolene (bcncylane), aspirin (asp
irin), flurbiprofen (flurbip
rofen), tofupiji)v(t, rapiciil
), pyridimole) -)v (pyri former nol), dipyridamole/L/(dipyridamole), gliclazide, etc.) (hereafter "-"
Platelet aggregation inhibitors, anticoagulants, and thrombolytic agents such as 'Im aggregation inhibitors) are added to the above-mentioned periodate oxidation method, carbodiimide method (References - Carraway, KL, Ct.
al: Met, hods Enzymol, No. 25
Vol., p. 616 (1972)), glugulaldehyde method (Reference - Avrameas 8; Immunocl.
+emistry, Vol. 6, p. 43 (1969)),
Diazo method (References - Landst, eincr, T(,
: The sp θcifici also ofscr
olo decal reactions, Ncw Yo
rk: Dorer.

1962.330PP), cyanogen bromide method (Literature-Axe
n, R, et, al, : Nature 214.
I 802 (1967)) et al.

In addition, those which do not have a carboxyl group but have a hydroxyl group can form a carboxyl group by reacting this with conolic acid anhydride, and those which have a carbonyl group A carboxyl group can be formed by converting this into (0-p-boxymethyl)oxime from (0-carboxymethyl).These can be bonded to Fab etc. using the carbodiimide method.

(B) Administration to patients, treatment example The drug obtained in (A) was administered to patients with renal disease. The specific contents are as follows.

(Part 1) Patient: Ni, N. , female, 38 years old, 48 kg Symptoms: Proteinuria and edema appeared 3 months ago, and renal function rapidly declined.

Drug dosage: 500 mg of the above-mentioned Ji'ab obtained from a monoclonal antibody of the same type as the anti-glomerular basement membrane antibody conjugated to urokinase was administered twice.

Result: (Part 2), Φ: Y, K. , male, 58 years old, 68 kg Symptoms:
The patient had rapidly progressive nephritis, and one month ago, he started exhibiting proteinuria, high blood pressure, and decreased renal function.

Drug dosage: 500 mg of the above-mentioned F a 13 obtained from a monoclonal antibody of the same type as the anti-glomerular basement membrane antibody coupled with hel<phosphorus was administered twice.

(Part 3) Patient: A, G. , female, 25 years old, 55 kg Symptoms: Rapidly progressive nephritis, presenting with proteinuria, decreased renal function, and hematuria for 2 months.

Drug dosage: 500 mg of warfarin conjugated to the above F(ab')2 obtained from the same monoclonal antibody as the anti-glomerular endothelial cell membrane antibody.

Two doses were administered.

Results: (Part 4) Patient: H. M, Male, 34 years old, 48 kg Symptoms = 1
Several months ago, the patient presented with proteinuria, hematuria, and decreased renal function, and was diagnosed with chronic nephritis.

Drug dosage: 500 mg of the Fab obtained from the same monoclonal antibody as the anti-glomerular basement membrane antibody bound to prostaglandin E1 was administered twice.

Results: (Part 5) Patients:, S, T. , female, 28 years old, 54 kg Symptoms: 2
The patient had been exhibiting proteinuria and decreased renal function for several years, and was diagnosed with chronic nephritis.

Drug dosage: F a b' obtained from a monoclonal antibody homologous to the renal glomerular basement membrane antibody was conjugated with dipyridamole, and 500 mg of it was administered twice.

Results: The results shown below indicate that the auxiliary agent according to the present invention did not have any excellent effects on renal diseases.

Applicant: Media Co., Ltd. Toshimitsu Niwa

Claims (2)

[Claims] (1) Monoclonal antibodies that selectively and specifically bind to renal glomerular basement membrane and/or renal glomerular endothelial cell membrane;
1. A therapeutic agent for renal diseases characterized by having as a main component a platelet aggregation inhibitor, an anticoagulant, or a thrombolytic agent bound to a portion other than the complement binding site. (2) The portion excluding the complement binding site is Fab. F(ab')2. Fab' or those broken down into smaller pieces that still bind to antigen! 911. The therapeutic agent according to claim 1, which has position 911.