GB2171012A

GB2171012A - Nephritis treating agent

Info

Publication number: GB2171012A
Application number: GB08601014A
Authority: GB
Inventors: Yoshiyuki Ohsugi; Toshiaki Nakano; Koko Katagiri
Original assignee: Chugai Pharmaceutical Co Ltd
Current assignee: Chugai Pharmaceutical Co Ltd
Priority date: 1985-01-22
Filing date: 1986-01-16
Publication date: 1986-08-20
Also published as: FR2580928B1; IT1191988B; GB2171012B; FR2580928A1; IT8667046A0; JPS61267515A; GB8601014D0; DE3601780A1

Abstract

The agent contains N-2'-carboxyl-3',6'-dimethylphenyl-4-chloroanthranilic acid or a salt thereof as active ingredient.

Description

SPECIFICATION Nephritis treating agent The present invention relates to a nephritis treating agent containing N-2'-carboxyl-3',6-dimethylphenyl-4-chloroanthranilic acid or a salt thereof as the active ingredient.

It is well known that N-2'-carboxyl-3',6'-dimethylphenyl-4-chloroanthranilic acid is useful as a medicine and has the capability of lowering the liver triglyceride level (U.S. Patent 4,092,426) and activity against peptic ulcer (U.S. Patent 4,447,453).

There are two types of nephritis: anti-glomerular basement membrane nephritis and immune complex glomerulonephritis. In either type of the disease, the glomerular basement membrane becomes highly permeable to cause extensive loss of plasma proteins, and among the complications of the disease are the excretion of a large amount of protein-uria, hypoproteinemia, protein edema and hyperlipemia. The expectant treatment is traditionally applied to patients with nephritis and it involves the use of steroidal antiinflammatory agents, non-steroidal antiinflammatory agents, immunosuppressants and platelet aggregation inhibitors.

The aforementioned agents currently administered to patients with nephritis are solely employed in the regimen of expectant treatment and many of them cause serious side-effects. It has therefore been strongly desired to develop a pharmaceutical agent that will effectively work against the specific cause of nephritis and which has less side-effects.

The present inventors administered N-2'-carboxyl-3',6'-dimethylphenyl-4-chloroanthranilic acid to rats in which nephrotoxic serum (NTS) nephritis had been induced, and found that this compound had the capabilities of inhibiting the excretion of urinary proteins. The inventors also found that this compound increased the renal blood flow, and had low toxicity. The present invention has been accomplished on the basis of these findings.

The compound used as the active ingredient of the nephritis treating agent of the present invention may be prepared by the Ullmann reaction from 2,4-dichlorobenzoic acid and 3,6dimethylanthranilic acid. In preparing dosage forms, this compound may optionally be converted to an alkali metal salt such as a sodium or potassium salt.

The aforementioned compound is effective when administered to humans for the purpose of treating disorders of kidneys such as rapidly progressive glomerulonephritis, chronic glomerulonephritis, IgA nephropathy and nephrotic syndrome.

Figure 1 shows the cl se-dependent ability of N-2'-carboxyl-3'-6'-dimethylphenyl-4-chloroanthranilic acid to inhibit the appearance of proteinuria 4 days after NTS injection; Figure 2 shows the dose-dependent ability of the compound to lower the total amount of protein excreted into urine 4 days after NTS injection; Figure 3 shows the time-dependent change in the protein level in urine for the period of 2-4 days after NTS injection, with the dose of the compound being taken as a parameter; Figure 4 shows the time-dependent change in the total amount of protein excreted into urine for the period of 2-4 days after NTS injection, with the dose of the compound being taken as a parameter; Figure 5 shows the dose-dependent ability of the compound to inhibit increases in the level of blood cholesterol 4 days after NTS injection;; Figure 6 shows the dose-dependent ability of the compound to inhibit decreases in the level of blood albumin; and Figure 7 shows the dose-dependent ability of the compound to increase the renal blood flow.

This compound may be formulated in such dosage forms as tablet, film coated tablet, capsule, powder, granule, suspension and injection after being mixed with pharmaceutically acceptable adjuvants and carriers by routine procedures. Carriers preferred for use in the preparation of tablets, film coated tablets, capsules, powders, granules and suspensions include lactose, starch, dextrin, sucrose, crystalline cellulose, kaolin, calcium carbonate, talc, magnesium stearate, aliphatic acid esters of sucrose, glycerin, hydroxypropylmethyl cellulose, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose, and hydroxypropyl cellulose. Injections are preferably prepared by dissolving the compound in distilled water or solutions of salts such as sodium chloride and potassium chloride.

The compound may be administered in an amount sufficient to cause enhancement and normalization of renal functions. For oral administration to humans, the daily dose will range from 5 to 100 mg, preferably 10-30 mg; for intravenous injection, the daily dose will be in the range of 1-20 mg, preferably 2-6 mg.

The N-2'-carboxyl-3',6'-dimethylphenyl-4-chloroanthranilic acid or salts thereof used in the present invention have the ability to inhibit urinary protein excretion and other activities. The following experiments and example are provided for further illustrating the present invention but are by no means intended as limiting.

Experiment 1 To the tail veins of SD male rats (5-week old, five animals per group), 1 ml of NTS (rabbit antiserum against the homogenate of the renal cortex of a rat) was injected, so as to induce nephrotoxic serum nephritis. Starting one day before the injection of NTS, N-2'-carboxyl-3',6'dimethylphenyl-4-chloroanthranilic acid was administered orally in selected doses until the third day of the NTS injection on a one-dose-per-day basis. Only water was administered to the control group. The animals were put in individual cages, and samples of 24-hour urine were taken at 2 and 4 days of the NTS injection.The measurement of urinary protein in these samples was made by the following procedures: the sample was centrifuged (3,000 rpmX 10 min); the supernatant was diluted with distilled water; 4 ml of 3% sulfosalicylic acid was added to 1 ml of the dilution; and using a bovine serum albumin standard solution, the protein level was determined in terms of the turbidity of the mixture.

At 4 days of the NTS injection, the rats were anesthetized with ether and samples of the whole blood were taken from the inferior artery. The levels of cholesterol and albumin in blood were measured with UNIKIT(fi)-Cholesterol-E and UNIKIT()-Albumin-S (both available from Chugai Seiyaku Kabushiki Kaisha), respectively. The dosages of N-2'-carboxyl-3',6'-dimethylphenyl-4-chloroanthranitic acid were 10 mg/kg, 50 mg/kg and 100 mg/kg. The results are shown in Figs. 1 to 6. The three actions of the compound, i.e., lowering the excretion of urinary protein, lowering the level of cholesterol in blood and increasing the level of albumin in blood, were dependent on the dosage and increased in a progressive manner from 10 mg/kg through 50 mg/kg to 100 mg/kg.

Experiment 2 SD male rats (7-week old, 5 animals per group) were starved for 19 hours and PSP (phenolsulfonphthalein) was injected intravenously in an amount of 4.8 mg. Fifteen minutes later, the animals were anesthetized with ether and blood samples were taken from the inferior artery for quantitative determination of the PSP level in each sample. One hour before the PSP injection, a predetermined amount of the test compound was administered orally. The percent acceleration of PSP excretion for each of the dosages used was calculated by the following formula. The results are shown in Table 1. Only water was administered to the control group.

A-B Acceleration of PSP excretion= x 100 (%) A where A: the concentration of PSP in blood samples from the control group; and B: the concentration of PSP in blood samples from the treated group.

Table 1

Doses Blood PSP level Acceleration of (mg/kg) (Ug/ml) PSP excretion (%) Control 50.4 + 2.6 0 12.5 42.6 + 1.3* 15.5* 25 32.1 + 3.3** 36.3** 50 25.5 + 1.5** 49.4** 100 21.0 + 0.7** 58.3** (*P < 0.05, **P < 0.01) Experiment 3 The test compound of the present invention was administered orally in an amount of 100 mg/kg as in Experiment 2, and the concentration of PSP in blood as a function of the lapse of time after the administration was examined. The percent acceleration of PSP excretion was calculated as in Experiment 2 and the results are shown in Table 2. Only water was added to the control group.

Table 2

Time Blood PSP level Acceleration of (hr) (g/ml) PSP excretion (8) Control 43.8 i 0.72 0 0.5 21.2 + 1.2** 51.6** 1 21.8 + 1.9** 50.2** 2 19.8 + 2.5** 54.8** 4 19.8 i 1.9** 54.8** (**P < 0.01) Experiment 4 To SD male rats (7-week old, 5 animals per group), 100 mg/kg of BEA (bromoethylamine hydrobromide) was injected intraperitoneally to cause renal insufficiency in the animals.Three days after the injection, the test compound of the present invention were administered orally in selected doses. One hour after the administration, PSP was injected intravenously at a dose of 4.8 mg per rat. Fifteen minutes, 30 minutes or 60 minutes after the intravenous injection, the rats were anesthetized with ether and blood samples were taken from the inferior artery and the PSP levels were quantitatively determined as in Experiment 2. The percent acceleration of PSP excretion for each of the doses used was calculated by the following formula, and the results are shown in Table 3: A-B Acceleration of PSP excretion = X 100 (%) A where A: the concentration of PSP in blood samples from the control group; and B: the concentration of PSP in blood samples from the treated group.

Table 3

15 minutes 30 minutes 60 minutes Treatment Blood Acceleration Blood Acceleration Blood Acceleration PSP of PSP PSP of PSP PSP of PSP level excretion level excretion level excretion ( g/ml) (%) ( g/ml) (%) ( g/ml) (%) No treatment 42.7 # 1.6 - 20.4 # 1.3 - 6.9 # 0.5 With BEA only 54.2 # 2.8 0 34.6 # 2.6 0 19.2 # 2.1 0 (control) With BEA + test compound 30.0 # 3.7* 44.6* 18.2 # 3.1* 47.4* 9.4 # 2.8* 51.0* (50 mg/kg) With BEA + test compound 27.2 # 2.4* 49.8* 15.0 # 1.9* 56.6* 7.0 # 1.1* 63.5* (100 mg/kg) (*P < 0.05) Experiment 5 As in Experiment 4, renal insufficiency was caused in SD male rats (7-week old, 5 animals per group) except that kanamycin was used as an inducer instead of EBA. A PSP excretion test was conducted 15 minutes after the intravenous injection of PSP. The results are shown in Table 4.

Table 4

Blood Blood PSP level Acceleration of Treatment (g/ml) PSP excretion (%) No treatment 59.7 i 3.8 With kanamycin 71.2 + 3.6 0 only (control) With kanamycin + test compound 28.4 + 1.3** 60.0** (50 mg/kg) (**P < 0.05) Experiment 6 Pentobarbital-anesthetized Wister-lmamichi male rats (15-week old, 7 animals per group) were excised at the left renal artery, which was connected to a sensor through the test compound of the present invention was injected intravenously at doses of 10 mg/kg and 30 mg/kg.The renal blood flow was measured with a pulse Doppler hemodromometer. The percent increase in renal blood flow was calculated for each rat and the results are shown in Fig. 7.

Experiment 7 Acute toxicity: A potassium salt of the test compound of the present invention administered orally to SD male rats (5-week old, 5 animals per group) had LD50 values ranging from 1,600-4,096 mg/kg.

Experiment 8 The test compound of the present invention, both in its free and sodium salt forms, was administered orally to Sprague-Dawley male rats (5-week old, 5 animals per group) for 14 consecutive days on a one-dose-per-day basis. The results are shown in Table 5.

Table 5

Test compound Dose (mg/kg) Fatality 220 0/5 Free form 440 0/5 880 0/5 250 0/5 Sodium salt 500 0/5 1000 0/5 Advantages As will be seen from the above data, the compound of the present invention has low toxicity and displays the following actions in rats in which nephrotoxic serum nephritis was induced: lowering the excretion of urinary protein, inhibiting increases in the level of cholesterol in blood, inhibiting decreases in the level of albumin in blood, accelerating the excretion of PSP, and increasing the renal blood flow. Because of these advantages it has, the compound of the present invention has potential utility as a nephritis treating agent.

Example (preparations) a) Tablet Ten grams of disodium N-2'-carboxyl-3',6'-dimethylphenyl-4-chlornanthrnnilate that had been passed through a 50-mesh screen was intimately mixed with 50 g of lactose, 30 g of crystalline cellulose, 9 g of corn starch and 1 g of magnesium stearate. The mix was fed into a tableting machine to make tablets with a diameter of 7 mm, each weighing 100 mg.

b) Film coated tablet The tablets prepared in a) were-pan-coated with a 10% aqueous solution containing hydroxypropylmethyl cellulose, talc and glycerin at a ratio of 1:1:0.2.

c) Capsule Fifty grams of N-2'-carboxyl-3',6'-dimethylphenyl-4-chloroanthranilic acid was intimately mixed with 148 g of lactose and 2 g of magnesium stearate. The mix was encapsulated to make capsules each containing 200 mg of the mix.

d) Powder To 40 g of ground N-2'-carboxyl-3',6'-dimethylphenyl-4-chloroanthranilic acid, 600 g of lactose and 360 g of corn starch were added, and the mix was fed into a fluidized bed granulator, where a powder was prepared under water spraying.

e) Granule To 10 g of N-2'-carboxyl-3',6'-dimethylphenyl-4-chloroanthranilic acid that had been passed through a 32-mesh screen, 450 g of lactose and 40 g of calcium carboxylmethyl cellulose were added, and an intimate mixture was prepared using 100 g of a 10% aqueous solution of hydroxypropyl cellulose. The mix was fed into an extruder type granulator. The resulting granules were dried and passed through a 14-mesh screen.

f) Suspension Sucrose (200 g) was dissolved in water to make a total of 400 ml. To the solution, 10 g of crystalline cellulose and 0.75 g of sodium carboxymethyl cellulose were added, and a uniform suspension was prepared. Five grams of N-2'-carboxyl-3',6'-dimethylphenyl-4-chloroanthranilic acid were ground in a ball mill together with 0.5 g of an aliphatic acid ester of sucrose and 20 ml of water. The ground mixture was combined with the previously prepared suspension to make a total of 500 ml. The resulting mixture was agitated until a uniform suspension was obtained.

g) Injection A purified powder (10 g) of disodium N-2'-carboxyl-3',6'-dimethylphenyl-4-chlornanthrnnilate and sodium chloride (18 g) were dissolved in injectionable distilled water to make a total of 2,000 ml. The solution was filtered through a membrane filter (0.22 ,am) and charged into colorless ampules each having a capacity of 2 ml. The ampules were fused and sterilized by heating at 100"C for 30 minutes to prepare injections.

Reference Example (synthesis) A three-necked round bottomed flask (300 ml) was charged with 150 ml of isoamyl alcohol, 8.72 g (0.0457 mole) of 2,4-dichlorobenzoic acid, 7.56 g (0.0458 mole) of 3,6-dimethylanthranilic acid, 15 g of anhydrous potassium carbonate, 200 mg of a copper powder and a trace amount of iodide, and the stirred mixture was heated under reflux for 5 hours. The reaction mixture was cooled to room temperature, poured into 500 ml of water and subjected to filtration. The mother liquor was concentrated under vacuum to remove isoamyl alcohol and the residue was rendered acidic with 6 N HCI. The resulting crystal was dissolved in tetra-hydrofuran (THF) and the solution was treated with activated carbon. By concentrating the solution under vacuum, THF was removed and the resulting crystal was suspended in methanol. The suspension was heated under reflux for 3-4 hours and cooled to room temperature. The resulting crystal was recovered by filtration; it was identified as N-2'-carboxyl-3',6'-dimethylphenyl-4chloroanthranilic acid having a melting point of 283"C (with decomposition). Yield: 7 g. Elemental analysis for the molecular formula: C16Hl4CLNO4 C H N Cl Calculated (%): 60.10 4.41 4.38 11.09 Found (%): 59.88 4.42 4.21 10.93

Claims

1. A nephritis treating agent containing N-2'-carboxyl-3',6'-dimethylphenyl-4-chloroanthranilic acid or a salt thereof as the active ingredient.

2. A nephritis treating agent according to Claim 1 wherein the nephritis is rapidly progressive glomerulonephritis.

3. A nephritis treating agent according to Claim 1 wherein the nephritis is chronic glomerulonephritis.

4. A nephritis treating agent according to Claim 1 wherein the nephritis is IgA nephropathy.

5. A nephritis treating agent according to Claim 1 wherein the nephritis is nephrotic syndrome.

6. For use in treating nephrites, N-2'-carboxyl-3',6'-dimethylphenyl-4-chloroanthranilic acid or salt thereof.