Antiprotozoal composition containing a 2-nitroimidazole derivative and method thereof

Disclosed herein is an antiprotozoal composition containing a 2-nitroimidazole derivative as its active component. This drug is especially useful against Trypanosoma cruzi. Process for the preparation is also disclosed.

BACKGROUND OF THE INVENTION 
i) Field of the Invention 
This invention relates to an antiprotozoal composition which is especially 
effective against Trypanosomatidae and a process for preparing the same. 
ii) Background Art 
In developing countries especially in South America, high death rate due to 
diseases caused by protozoa is a big issue. Not only the native people, 
foreign people sent by other countries to help develop the country are 
also exposed to danger of getting suffered from such diseases. 
Among the diseases caused by protozoa, those caused by Trypanosoma cruzi 
are fatal because currently no effective antiprotozoals are known. 
Especially in the case of acute infections, it is not rare that the 
patients die from myocardosis. Meanwhile, tens of millions of people 
suffer from chronic infectious diseases caused by Trypanosoma cruzi. 
Prevention and treatment of these diseases, therefore, has been given top 
priority by the WHO. 
It is sbelieved that the development of an effective antiprotozoal, 
especially against Trypanosoma cruzi is significant for the mankind 
welfare. 
SUMMARY OF THE INVENTION 
A primary object of the invention is to provide an antiprotozoal 
composition which contains as its active component a 2-nitroimidazole 
derivative of the following formula (I): 
##STR1## 
wherein R is --CH.sub.2 OCH.sub.2 CH.dbd.CHCH.sub.2 OH, --CH.sub.2 
OCH(CH.sub.2 OH).sub.2 or 
##STR2## 
Another object of the invention is to provide an antiprotozoal composition 
which contains as its active component a 2-nitroimidazole derivative of 
formula (I): 
##STR3## 
wherein R is --CH.sub.2 OCH.sub.2 CH.dbd.CHCH.sub.2 OH, --CH.sub.2 
OCH(CH.sub.2 OH).sub.2 or 
##STR4## 
and an anionic polymer. 
A further object of the invention is to provide a process for preparing 
such compositions. 
The above and other objects, features and advantages of the present 
invention will become apparent from the following description. 
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS 
The 2-nitroimidazole derivative (I), which is an active component of the 
antiprotozoal of this invention, is a known compound, and is prepared, for 
example, by the reaction steps: 
##STR5## 
wherein X is a halogen atom, R' is --CH.sub.2 OCH.sub.2 CH.dbd.CHCH.sub.2 
OAc--CH.sub.2 OCH(CH.sub.2 OAc).sub.2 or 
##STR6## 
In other words, reaction between 2-nitroimidazole (II) and a halide (III) 
produces a compound (IV), and deacetylation of the compound (IV) produces 
the 2-nitroimidazole derivative (I). 
The reaction between 2-nitroimidazole (II) and a halide (III) is preferably 
carried out in the presence of a base such as triethylamine and in a 
solvent such as dimethylformamide. The deacetylation of compound (IV) is 
preferably carried out in a mixture solvent of acetonitrile-water in the 
presence of a base such as triethylamine, pyridine or the like. After 
completion of the reaction, purification of compound (I) is carried out by 
condensing the reaction mixture, separating through means such as column 
chromatography, and then condensing again for recrystalization. 
The thus obtained 2-nitroimidazole derivatives (I) have excellent 
anti-protozoan actions as will hereafter be illustrated in Examples, and 
are very safe, too. Accordingly, they are useful as a drug against 
protozoa, namely as an antiprotozoal. 
Dosage of the antiprotozoal containing 2-nitroimidazole derivative (I) 
according to this invention differs depending on the patient's age, sex, 
weight, administration route, body conditions and the illness. In the case 
of oral administration, 30 mg-1500 mg/day is suggested and in the case of 
non-oral administration, 10-500 mg/day is suggested, both for an adult. 
Further, the anti-protozoan activity of compound (I) is remarkably enhanced 
when it is used in combination with an anionic polymer. Especially, it is 
noted that such combination completely clears up the protozoa from the 
body of the infected patient, which enables to prevent the symptoms from 
becoming chronic. 
The usable anionic polymers are any of natural origin or synthesized ones. 
Exemplary natural anionic polymers include mucopolysaccharides such as 
hyaluronic acid, chondroitin sulfuric acid and their salts; hemicelluloses 
such as alginic acid, carboxymethylcellulose, hydroxymethylcellulose, 
hydroxyethylcellulose, hydroxypropylcellulose, 
hydroxypropylethylcellulose, methylcellulose, carboxymethyl amylose and 
their salts. Exemplary synthetic anionic polymers include polymers 
obtained from the polymerization of the following monomers: 
(a) Unsaturated carbonic acid monomers such as acrylic acid, methacrylic 
acid, maleic acid, etc. 
(b) Unsaturated sulfonic acid monomers such as styrene sulfonic acid, etc. 
(c) Unsaturated phosphoric acid monomers such as vinyl sulfonic acid, acid 
phosphoxyethyl methacrylate, etc. 
Polymers obtained form the copolymerization of the above monomers and other 
polymerizable monomers may also be used. An example of such copolymer is 
an acrylic acid-diisobutylvinyl ether copolymer. 
The above-mentioned anionic polymers are preferably water-soluble. The 
proportion of the anionic polymers to compound (I) ranges form 1/1000 to 
1/1 on the weight basis. 
The antiprotozoal composition of this invention can take various forms. By 
conventional processes, it is formed into tablets, granule, powder, 
capsules, suspensions, injections, suppositories, and the like. 
In order to make a solid preparation, compound (I) is first blended with 
pharmaceutically acceptable carriers or additives such as an excipient, 
and if necessary, with a binder, disintegrator, lubricant, coloring agent, 
taste and smell modifiers, filler, coating material or the like and then 
formed into tablets, granule, powder, capsules and suppositories by known 
methods. Here, the excipient includes saccharides, starch, inorganic 
excipient and plant powders; the binder includes starch paste solution, 
gum arabic, gelatin, polyvinyl pyrrolidone and polyvinyl alcohol; the 
disintegrator includes starch, agar, gelatin powder, calcium carbonate and 
sodium hydrogencarbonate; the lubricant includes magnesium stearate, talc, 
hydrogenated vegetable oils and silicone oils; and the coating material 
includes sugars, glues (gelatin, glycerol, sorbitol) and film coating 
materials. 
To prepare an injection drug, compound (I) is dissolved, dispersed or 
emulsified in an aqueous medium such as distilled water in advance, or is 
made into a powder, which is dissolved in an aqueous medium upon use. As 
for the injection route, intravenous, intra-arterial, intra-portal, 
intraperitoneal, intramuscular and subcutaneous routes are mentioned.

EXAMPLES 
This invention will now be explained by way of Examples, which however 
should not be construed as limiting the invention thereto. 
EXAMPLE 1 
Anti-protozoan action 1 (in vitro) 
A204 cells (derived from a rhabdomyoma), which had been infected in advance 
with Trypanosoma cruzi and maintained by subculture at 37.degree. C., were 
cultured in a 24-well plate for tissue culture. The protozoa in the number 
of about 10.sup.5 were confirmed to be present. After the cells were 
treated with 50 .mu.mol, 100 .mu.mol and 500 .mu.mol of compounds 1) to 
3), they were cultured at 37.degree. C. for 24 hours. Life and death of 
the protozoa was observed under microscope and scored. Criteria for the 
scoring and the results are given below. The medium used was Dulbecco 
Modified Eagle Medium manufactured by Dainihon-Seiyaku K. K. added with 
10% bovine serum. Control was free of any of compounds 1 to 3). 
Compound 1): Compound (I), wherein 
R is --CH.sub.2 OCH.sub.2 CH.dbd.CHCH.sub.2 OH 
Compound 2): Compound (I), wherein 
R is --CH.sub.2 OCH(CH.sub.2 OH).sub.2 
Compound 3): Compound (I), wherein 
##STR7## 
TABLE 1 
______________________________________ 
Results 
Concentration 
Compounds 50 .mu.mol 100 .mu.mol 
500 .mu.mol 
______________________________________ 
Compound 1) .+-. - - 
Compound 2) ++ ++ - 
Compound 3) ++ ++ + 
______________________________________ 
Criteria for Scoring 
+++ : same number of protozoa alive as control 
++ : slightly less number alive than control 
+ : less number alive than control 
.+-. : only a few alive 
- : all dead 
The above results show that compounds 1) to 3) have an excellent 
anti-protozoan action. 
EXAMPLE 2 
Anti-protozoan action 2 (in vitro) 
Trypanosoma cruzi was cultured at 27.degree. C. in a GIT medium (serum-free 
medium) manufactured by Nippon Seiyaku K. K. which was modified to contain 
12.4 mol of hemine. 50 .mu.mol, 100 .mu.mol, 250 .mu.mol and 500 .mu.mol 
of each of compounds 1) to 3) were added thereto. 120 hours later, life or 
death of the protozoa was observed under microscope and scored according 
to the criteria in Example 1. The results are shown in Table 2. Control 
was free of any of compounds 1) to 3). 
TABLE 2 
______________________________________ 
Concentration 
Compounds 50 .mu.mol 
100 .mu.mol 
250 .mu.mol 
500 .mu.mol 
______________________________________ 
Compound 1) 
++ ++ + - 
Compound 2) 
+++ +++ +++ +++ 
Compound 3) 
+++ +++ +++ + 
______________________________________ 
EXAMPLE 3 
Anti-protozoan action (in vivo) 
Trypanosoma cruzi (Tulahuen strain) in the number of 400,000 was 
intraperitoneally administered to a group of 4 week old male ICR mice. At 
each of fifth, sixth, seventh, eighth and ninth day of the administration, 
200 mg/kg of compound 1) was given to the mice. Control group was given 
saline solution instead of compound 1). The anti-protozoan effect of 
compound 1) was determined based on the days of survival of the mice. 
As a result, 2 mice out of 3 mice of control survived 14 days and the other 
15 days, while all of 3 mice administered with compound 1) survived more 
than 50 days. Compound 1) was thus proved to have an excellent in vivo 
anti-protozoan action. 
EXAMPLE 4 
Anti-protozoan action (in vivo) 
Trypanosoma cruzi (Tulahuen strain) in the number of 200,000 was 
intraperitoneally administered to a group of 5 week old male C3H/Hecrj 
mice. At each of fourth, fifth, sixth, seventh and eighth day of the 
administration, 200 mg/kg of compound 1) was given to the mice. Control 
group was given saline solution instead of compound 1). The anti-protozoan 
effect of compound 1) was determined based on the days of survival of the 
mice. 
As a result, 2 mice of the control group survived 12 days while 4 mice 
administered with compound 1) survived more than 20 days. Compound 1) was 
thus proved to have excellent anti-protozoan action. 
EXAMPLE 5 
Five groups (Groups 1 to 5) of four week old ICR male mice, each group 
consisting of 3 mice, were provided. Group 2 and Group 5 mice were given 
4.4 mg of acrylic acid-diisobutylvinyl ether copolymer (monomer ratio=1:1, 
average molecular weight=1.29.times.10.sup.5) as dissolved in a 0.2 ml 
saline. On the second day, all animals were intraperitoneally given 
Trypanosoma cruzi (Tulahuen Strain) in the number of 40,000 for each. On 
the fifth, sixth, seventh, eighth and ninth day of the administration of 
the Tripanosoma, Group 1 and Group 2 mice were given saline, and Group 3 
and Group 5 mice were given compound 1) (200 mg/kg) as dissolved in 
saline. Group 4 mice were intraperitoneally given 4.4 mg of acrylic 
acid-diisobutyl vinyl ether copolymer (monomer ratio=1:1, average 
molecular weight=1.29.times.10.sup.5) and compound 1) (200 mg/kg) on the 
fifth day of the administration of the Trypanosoma, and on the sixth, 
seventh, eighth and ninth day of the administration, compound 1) (200 
mg/kg) was intraperitoneally administered. Observation was continued until 
60 th day of administration of Trypanosoma. The mice which were alive on 
the 60th day were killed and dissected to check the presence or absence of 
the protozoa in blood, the heart and in the liver under microscope. The 
results are shown in Table 4. 
TABLE 4 
______________________________________ 
Presence of Protozoa 
Number of (60th day) 
Mice Group No. 
Dead Samples Blood Heart Liver 
______________________________________ 
1. 1 on the 13th day 
+ + + 
of Trypanosoma 
Administration 
1 on the 14th day 
of Trypanosoma 
Administration 
2. 1 on the 17th day 
+ + + 
of Trypanosoma 
Administration 
1 on the 18th day 
of Trypanosoma 
Administration 
3. 0 + + + 
4. 0 .+-.* .+-.* 
.+-.* 
5. 0 -- -- -- 
______________________________________ 
*.+-. means that although protozoa were observed, the number of the same 
was very small. 
The data in Table 4 shows that there was no death in the groups (Group Nos. 
3, 4 and 5) which were given compound 1), which indicates effective 
anti-Trypanosoma action of compound 1). Further almost no protozoa was 
observed in the body of the animals of the groups (Group Nos. 4 and 5) 
which were given compound 1) and an anionic copolymer. 
EXAMPLE 6 
Acute toxicity 
ICR mice were intraperitoneally administered with compound 1) for the acute 
toxicity test. Observation was continued for 14 days after the 
administration. The results are shown in Table 3. 
TABLE 3 
______________________________________ 
Compounds LD.sub.50 (mg/kg) 
______________________________________ 
Compound 1) 830 
Compound 2) 4,300 
Compound 3) &gt;5,000 
______________________________________ 
The antiprotozoal composition according to this invention has an excellent 
anti-protozoan action, and is very useful for the prevention or treatment 
of acute and chronic infectious diseases caused by various protozoa, 
especially Trypanosoma cruzi.