Process for treatment of nephritis with imidazoquinazolines

An agent for treatment and prevention of nephritis comprising at least one imidazoquinazoline compound represented by formula ##STR1## wherein R.sub.1 represents a dialkylamino group having 1 to 6 carbon atoms in each alkyl moiety, or an unsubstituted or substituted cyclic amino group; and R.sub.2 represents a hydrogen atom, a halogen atom, an alkyl group having 1 to 6 carbon atoms, or an alkoxy group having 1 to 6 carbon atoms, or a salt thereof as an active ingredient.

FIELD OF THE INVENTION 
This invention relates to an agent for treatment and prevention of 
nephritis. More specifically, this invention relates to an agent for 
treatment and prevention of nephritis comprising at least one 
imidazoquinazoline compound or a salt thereof. 
BACKGROUND OF THE INVENTION 
Imidazoquinazoline compounds are known to have an inhibitory activity on 
blood platelet agglutination and an inhibitory activity on metastasis of 
cancers as described in U.S. Pat. Nos. 4,596,806 and 4,610,987, and 
Japanese Patent Application (OPI) Nos. 4186/85 and 152416/85 (the term 
"OPI" as used herein refers to a "published unexamined Japanese patent 
application"). 
Clin. Exp. Immunol., Vol. 61, 388-396 (1985) describes a relation between 
blood platelets and nephritis of acute serum sickness and protection of 
nephritis by dipyridamole 
[2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido[5,4,-d]pyrimidine, Merk 
Index 10th Ed.] as a coronary vasodilator of FUT-175 [2-(6-amino)naphthyl 
4-guanidinobenzoate dihydrochloride] as a Clr and Cl esterase inhibitor, 
but does not refer to effectiveness of imidazoquinazolines according to 
this invention as an agent for treatment and prevention of nephritis. 
SUMMARY OF THE INVENTION 
This invention relates to an agent for treatment and prevention of 
nephritis comprising at least one imidazoquinazoline compound represented 
by formula (I) hereinafter described or a salt thereof as an active 
ingredient. 
Formula (I) is represented by 
##STR2## 
wherein R.sub.1 represents a dialkylamino group having 1 to 6 carbon atoms 
in each alkyl moiety, or an unsubstituted or substituted cyclic amino 
group; and R.sub.2 represents a hydrogen atom, a halogen atom, an alkyl 
group having 1 to 6 carbon atoms, or an alkoxy group having 1 to 6 carbon 
atoms. 
As a result of extensive investigations on pharmacological activities of 
the compounds represented by formula (I), it has now been confirmed that 
they exhibit an activity to decrease protein in proteinuria accompanying 
nephritis. 
DETAILED DESCRIPTION OF THE INVENTION 
The term "cyclic amino group" as used herein for the compounds of formula 
(I) means a 1-pyrrolidinyl group, a 1-piperidinyl group, a 1-piperazinyl 
group or a 4-morpholinyl group, and examples of substituents on the cyclic 
amino group include 1 or 2 alkyl groups having 1 to 6 carbon atoms. 
The compounds represented by formula (I) and the pharmaceutically 
acceptable salts thereof used in the present invention are disclosed in 
U.S. Pat. Nos. 4,596,806 and 4,610,987, and the most preferred compound 
for use in the present invention is a 7-piperidino compound and a 
pharmaceutically acceptable salt thereof as disclosed in U.S. Pat. No. 
4,596,806. 
The present invention will be described below in detail particularly with 
reference to 7-piperidino-1,2,3,5-tetrahydroimidazo[2,1-b]quinazolin-2-one 
dihydrochloride monohydrate (hereinafter referred to as "DN-9693") 
disclosed in U.S. Pat. No. 4,596,806 as a typical example of the 
imidazoquinazoline compounds of formula (I), but it is to be noted that 
other compounds of formula (I) above are also effective as well. 
I. Effect on GVHR-Induced Nephritis in Mouse 
When an animal of a first filial generation is implanted with lymphatic 
cells of its parent's line, a graft-versus-host reaction (hereinafter 
abbreviated as GVHR) takes place to thereby produce an auto-antibody and 
induce chronic immune-complex (IC) type nephritis. 
In this experimentally induced nephritis model, DN-9693 did not influence 
the production of an auto-antibody but exhibited an effect to decrease 
protein in urine. This fact suggests that DN-9693 possesses no immune 
inhibitory activity but anti-phlogistically acts on the inflammatory 
process to inhibit auto-immune IC type glomerulo-nephritis. 
DN-9693 was noted to produce a urinary protein decreasing effect at a low 
dose level of 1 mg/kg of body weight in the experimental nephritis model 
and is considered highly promising as a treating agent for nephritis in 
which an immune-complex takes part. 
II. Therapeutic Effect on Lupus Nephritis in Mouse 
DN-9693 was administered to NZB/WF.sub.1 mice that spontaneously present 
symptoms resembling general lupus erythematosus in human at a time when 
the mice showed a high degree of proteinuria. Measurements of protein in 
urine with time after the administration revealed a reduction of protein 
in the urine. This result suggests that DN-9693 is also effective on lupus 
nephritis of spontaneous auto-immune patients. 
The present invention will now be illustrated by way of experiment 
examples. In these examples, the drug of interest was dissolved in a 0.5 
wt% aqueous solution of carboxymethyl cellulose and administered to test 
animals in an amount of 0.1 ml per 10 g of the body weight.

EXPERIMENT EXAMPLE 1 
Into BDF.sub.1 mice (female; 7-week-old; Nippon Charles River) were 
intravenously implanted twice spleen cells (6.times.10.sup.7 cells) and 
thymus cells (3.times.10.sup.7 cells) taken from DBA/2 mice (female; 
7-week-old; Nippon Charles River) of their parent's line on the 0 day and 
the 5th day to thereby induce GVHR. DN-9693 was orally given at a dose 
level of 1 mg/kg in a single dose per day for consecutive 44 days from the 
6th day to the 49th day. After 2 weeks from the first implantation of 
cells, the protein in urine and the anti-erythrocyte antibody titre were 
determined at one week intervals. The protein in urine and the antibody 
titre were determined by the modified one step Exton method and the direct 
Coombs' test method, respectively, in accordance with the following 
procedures: 
Modified One Step Exton Method 
1. Test urine and an albumin standard preparation were placed in a 
microplate in 10 .mu.l portions. 
2. To each hole was added 200 .mu.l of a reagent prepared by dissolving 15 
g of sulfosalicylic acid, 20 g of anhydrous sodium sulfate, 5 mg of 
Bromophenol Blue, and 0.1 g of carboxymethyl cellulose in one liter of 
water. After stirring to mix, the system was allowed to stand at room 
temperature. 
3. Ten minutes later, the absorbance at 690 nm was measured using the 
system comprising the reagent alone as a blank. 
4. The protein in urine was calculated from a calibration curve. 
Direct Coombs' Test Method 
1. Blood samples (50 .mu.l) were obtained from the orbital veniplex of the 
experimental animals (mice) using a heparinized hematocrit tube. 
2. The erythrocytes were washed three times by centrifugation (2,500 r.p.m. 
for 6 minutes) with a phosphoric acid-buffered physiological saline 
containing 0.3 wt% bovine serum albumin (0.3% BSA-PBS). 
3. The erythrocytes were suspended in 0.3% BSA-PBS to prepare a 1 wt% mouse 
erythrocyte suspension. 
4. The erythrocyte suspension (50 .mu.l) was added to 50 .mu.l of rabbit 
anti-mouse Ig (MBL) in a two-fold serial dilution placed in a microplate. 
After shaking, the system was allowed to stand at 37.degree. C. for 1 hour 
and then at room temperature for 2 hours. The state of agglutination was 
observed. 
5. The maximum dilution of the rabbit anti-mouse Ig that gave a positive 
agglutination was expressed as 10.times.2.sup.n, and the value n was taken 
as an antibody titre. 
The results of these tests are shown in Tables 1 and 2. As is shown in 
Table 1, DN-9693 significantly inhibits an increase of urinary protein at 
a dose of 1 mg/kg. From Table 2 showing changes of the auto-antibody 
production, it can be seen that DN-9693 does not decrease the Coombs 
antibody titre, indicating no influences upon production of an 
auto-antibody. DN-9693 was thus proved to have a potential activity to 
lower protein in urine. 
TABLE 1 
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Influence on GVHR-Induced Proteinuria in Mouse 
Number 
of Urinary Protein Score (Mean .+-. S.E.)* 
Group Animal 4th Week** 
5th Week 
6th Week 
7th Week 
______________________________________ 
Control 
8 0.2 .+-. 0.1 
0.8 .+-. 0.4 
1.8 .+-. 0.7 
2.3 .+-. 0.6 
DN-9693 
7 0.3 .+-. 0.1 
0.1 .+-. 0.1 
0.4 .+-. 0.4 
0.6 .+-. 
0.4*** 
______________________________________ 
Note: 
*0: Urinary protein concentration = not more than 1 mg/ml 
0.5: Urinary protein concentration = from 1 to 3 mg/ml 
1: Urinary protein concentration = from 3 to 10 mg/ml 
2: Urinary protein concentration = from 10 to 30 mg/ml, or not less than 
10 mg/ml and ascitic positive 
3: Urinary protein concentration = not less than 30 mg/ml, or from 10 to 
30 mg/ml and ascitic positive 
4: Urinary protein concentration = not less than 30 mg/ml and ascitic 
positive 
**Reckoned from the first implantation of cells. 
***Significantly different from the control (P &lt; 0.05) 
TABLE 2 
__________________________________________________________________________ 
Influence on Auto-Antibody 
Production in Mouse Induced by GVHR 
Number 
of Anti-Erythrocyte Antibody Titre (Mean .+-. S.E.)* 
Group 
Animal 
3rd Week** 
4th Week 
5th Week 
6th Week 
7th Week 
__________________________________________________________________________ 
Control 
8 6.3 .+-. 1.0 
5.4 .+-. 0.6 
4.9 .+-. 1.3 
2.9 .+-. 1.5 
1.9 .+-. 1.2 
DN-9693 
7 6.6 .+-. 0.6 
5.6 .+-. 0.2 
3.6 .+-. 1.1 
3.9 .+-. 1.1 
3.1 .+-. 1.0 
__________________________________________________________________________ 
Note: 
*n: 10 .times. 2.sup.n, n .gtoreq. 1 
**Reckoned from the first implantation of cells. 
EXPERIMENT EXAMPLE 2 
Ten animals clearly showing proteinuria were chosen from 32-week-old 
NZB/WF.sub.1 mice (female, Jackson) and divided into two groups (5 animals 
per group) taking the protein level in urine as a guide. 
DN-9693 was continuously administered orally one or two times per day at a 
dose of 1 mg/kg, and influences on changes of urinary protein level were 
examined. The results obtained are shown in Table 3. 
In comparing the changes in protein level between the control group and the 
DN-9693 group taking the level before the administration (32-week-old) as 
a standard, it can be seen that the increase of urinary protein level is 
inhibited by the administration of DN-9693. Thus, the activity to lower 
protein in urine was recognized also in spontaneously occurring lupus 
nephritis. 
TABLE 3 
______________________________________ 
Influences on Urinary Protein in NZB/WF.sub.1 Mouse 
Number Urinary Protein Index* (Mean .+-. S.E.) 
of 0 Week 
Group Animal (32)** 1 Week (33) 
2 Weeks (34) 
______________________________________ 
Control 
5 1.0 (5)*** 
7.0 .+-. 5.9 (5) 
4.8 .+-. 2.9 (4) 
DN-9693 
5 1.1 (5) 1.2 .+-. 0.2 (5) 
1.6 .+-. 0.6 (5) 
______________________________________ 
Note: 
*Protein level after administration/protein level before administration 
(32week-old) 
**Period (week) after the commencement of administration. Values in the 
parentheses are week ages of test animals. 
***Values in the parentheses are numbers of survivors. 
As is proved by the foregoing experimental results, the imidazoquinazoline 
compounds of the present invention are effective on various nephritides 
involving an increase of protein in urine, such as glomerulonephritis, 
nephrosis, lupus nephritis, and the like. 
The agent for treatment and prevention of nephritis according to the 
present invention is effective through oral administration. It is 
considered possible to formulate the agent of the present invention into 
injectable solutions. 
The recommended oral dose of the compound of the present invention is from 
5 to 50 mg per day for adult human in 1 to 3 divided doses. As the 
compounds of the invention are rapidly adsorbed when orally administered, 
and the changes in blood level are close to those when administered 
intravenously, dose levels similar to those of oral administration are 
considered applicable to injectable solutions. The LD.sub.50 of DN-9693 in 
mice (male, i.v.) was 314.5 mg/kg. 
The compounds of the present invention can be formulated into various 
dosage forms, such as tablets, capsules, powders, liquid preparations, 
injectable solutions, and the like in a known manner, together with 
carriers and/or excipients conventionally used in pharmaceutical 
preparations. A formulation example is described below for illustrative 
purposes only. 
FORMULATION EXAMPLE 1 
______________________________________ 
DN-9693 30 mg 
Lactose 626 mg 
Corn starch 300 mg 
Hydroxypropyl cellulose 
40 mg 
Magnesium stearate 4 mg 
Total: 1,000 mg 
______________________________________ 
The above components were mixed, granulated, and compressed into tablets 
each weighing 100 mg. 
While the invention has been described in detail and with reference to 
specific embodiments thereof, it will be apparent to one skilled in the 
art that various changes and modifications can be made therein without 
departing from the spirit and scope thereof.