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Effect of purine derivatives on Trypanosoma cruzi in vitro
Effect
of Purine Mitsuru
Division
Derivatives on Trypanosoma in Vitro1e2
Nakamuras
of Pharmacology University
and
Magdelena
cruzi
B. James
and Experimental Therapeutics, School of Medicine, of California, San Francisco, California
(Submitted
for publication
14 July
1951)
The study of chemotherapeutic agents against Trypanosoma cruzi, the etiological agent of Chagas’ disease, has been rather limited because of the ineffectiveness of drugs teAed against this trypanosome. Infection with T. cruzi is very resistant to drugs efficacious in treating other trypanosomiases, and at present no satisfact,ory chemotherapy is available in Chagas’ disease (Kofoid et al., 1937; von Brand et al., 1946). Kofoid et al. (1937) tested some arsenicals against T. cruzi in tissue culture and found arsenious trit,hiosalicylic acid to be quite toxic for the trypanosomes, but stated that, previous experiments had proved that this drug is also toxic t#o tissue in vitro and highly toxic to mammals. McIvor et al. (1942) studied the chemot’herapeutic effectiveness of certain sulfonamides, organic antimony, arsenic and bismuth compounds against experimental T. crz~z’ infections in mice. Tolerated doses of three agents prolonged the life of the animals, but in all mice Leishman-Donovan bodies remained in the heart muscle. Mazza et al. treated human cases of Chagas’ disease with 7602 (AC) Bayer, an aminoquinoline of the “surphen” group which is trypanocidal, and also with 9736 (As) Bayer. They found that dialilmalonildiamid-2-methyl, 4, G-diaminoquinoline caused the trypanosomes to disappear from the circulation (Mazza, Basso, and Basso, 1941; Mazza, Freire, and Salica, 1942; Mazza, Basso, and Basso, 1942; Mazza, Basso, and Basso, 1945). Talice and LopezFernandez treated Chagas’ disease with p-arsenophenylbutryic acid (1945). Goble (1949) studied S-aminoquinolines against T. cruzi in1 With the assistance of Miss D. Swanman and Mrs. B. Mythen. 2 Supported in part by the Division of Research Grants and Fellowships, National Institutes of Health, United States Public Health Service, Bethesda, Md., and the Abbott Laboratories, North Chicago, Illinois. 3 Present address: Dept. of Biological Sciences, New England College of Pharmacy, Boston, Mass. 19
20
MITSURU NAKAMURA AND MAGDBLENA B. JAMES
fections in mice and found 8-(isopropylaminoamylamino)-6-methoxyquinoline (pentaquine) highly trypanocidal. Pizzi (1951) found that primaquine showed suppressive action in the early stages of T. cruxi infection in mice. Tyrocidine, in cultures, had growth-suppressing effect in concentrations of l/10,000 (Amrein, 1951). Recently Goodwin, et al. (1950) have reported some success against mouse infections with methyl phenanthridinium derivatives. It was shown that 2-amino-9-p-carbethoxyaminophenyl-lO-methylphenanthridium bromide in a concentration of l/150,000 killed 90% of the trypanosomes in vitro in 24 hours (Lock, 1950). Purine and pyrimidine derivatives have been studied by Hitchings and his coworkers (Hitchings, et al., 1945; Hitchings et al., 1948; Hitchings et aE., 1950; Elion and Hitchings, 1950; Elion, et al., 1951) against Lactobacillus casei. They believe that t,hese derivatives act primarily as antimetabolites and give several interesting theories as to their mode of action. Kidder and his group (Kidder and Dewey, 1949; Kidder et al., 1949; Kidder, et al., 1950) have reported that substituted purines inhibited the growth of Tetrahymena geleii. The effect of such compounds against Endamoeba histolytica has also been studied in vitro (Brackett and Bliznick, 1947). Thompson et al. (1951) have reported on the activity of 5-(2,4-dichlorophenoxy)-4-hydroxy-2-mercaptopyrimidine against vaccinia virus. This presentation is a report of some observed effects of purine derivatives on T. cruzi in vitro. The purine derivatives used in this study were generously supplied by Dr. George H. Hitchings, Wellcome Research Laboratories, Tuckahoe, New York. MATERIALS
AND METHODS
The culture of Trypunosoma cruzi (Culbertson strain) was obtained from Dr. C. W. Rees, National Institutes of Health, Bethesda, Md. a. Maintenance of Stock Culture. The medium consisted of a blood-agar base with the following ingredients: Bacto agar (Difco) 2.5 g, Trypticase (BBL) 2.0 g, NaCl 0.5 g, and water 85.0 ml. The ingredients were dissolved in water and adjusted to a pH of 7.0 to 7.2 with 0.1 N NaOH. This was autoclaved at 121°C at 15 pounds for 15 to 20 minutes and before the medium solidified, 10.0 ml of sterile defibrinated rabbit’s blood was added. After thoroughly mixing the medium 25.0 ml each was dispensed into 250 ml Erlenmeyer flasks. The medium was allowed to cool and harden. The overlay consisted of Trypticase (BBL) 2.0 g, dextrose 1.0 and NaCl 1.6 g dissolved in 200.0 ml of water and adjusted to pH 7.0 to 7.2 with 0.1 N NaOH. This was autoclaved and 50 ml was overlayed on each blood-agar base (von Brand, et al., 1946; Phillips and Rees, 1950). The flasks were inoculated with 4.0 ml of a 14-day-old culture of !f. cruzi with an approximate count, as de-
PURINE
DERIV.%TIVES
termined by the hemocytometer, were incubated at 25°C.
AND
TRYPANOSOMA CRUZI
of 25,000,OOO organisms per milliliter.
21
The flasks
b. Technic Used, for Studies of Oxygen Consumption. The effect of the various agents was studied on the oxygen uptake of T. cruzi. The trypanosomes were harvested after 14 days incubation which had an approximate count of 25,000,OOO per ml. The trypanosomes were centrifuged and washed with Krebs-Ringer phosphate buffer solution twice and resuspended in this buffer so that the count approximated 250,000,OOOper ml. (Chen and Geiling, 1945; von Brand et al., 1946; Marshall, 1948, Umbreit et al., 1949). Into each 10.0 ml. Warburg flask was placed 1.0 ml of this t,rypanosome suspension, 0.1 ml of a 2% glucose solution, and aliquots of the drug solution in order to obtain the desired final concentrations. The oxygen uptake was measured for a period of 2 hours. The gas phase was air and the equilibration period was 10 minutes. All tests were made at 37.2”C. Readings were taken at 15 minut.e intervals and t,he manometers were shaken at t.he rate of 100 shakes per minut,e. At the end of the two-hour run, a small sample was removed from each flask and examined microscopically for motility to determine the activity of the trypanosomes. c. Technic for the Study of Druys under Aerobic and Anaerobic Conditions. The effect of substituted purines on T. cruzi cultures under aerobic conditions was studied in the diphasic blood agar medium. The agents to be tested were dissolved in suitable solvent,s and sterilized by Seit.z filtration. The drugs were added aseptically to the medium so as to achieve the desired final concentration, and then 1.0 ml of a 14.day old culture, containing approximately 25,000,OOO trypanosomes, was inoculated into the medium having an overlay of 10.0 ml. These flasks were incubated at room temperature and at O-hour, 1, 2, 3, 4, 7, 10, and 14 days, 0.25 ml of the culture was removed and counted in the hemocytometer. The concent,ration of drugs used was in a range t,hat was found to reduce markedly the oxygen uptake of the trypanosomes when studied manometrically. For studying the effect of these agents on the trypanosomes under anaerobic conditions 10.0 ml of the trypanosomes (with a count of approximately 25,000,000/ml) growing in the diphasic blood agar for 14 days were placed in a series of tubes. A range of concentrat,ion of each agent was studied by adding the drugs to the tubes to make up t.he desired final concentration and the tubes were sealed with 1.25 cm of solid petrolatum. The tubes were incubated at 25°C for a period of 5 days and then examined microscopically. That concentration at. which no trypanosomes were detected was recorded as t,rypanocidal. d. Purine Derivatives Studied. 2,6-Diamino purine hydrate (DP); 2,6-diamino7-methyl purine (DMP); 2,6-diamino-S-methyl purine sulfate (DMPS); 2,6-diamino-8-methylmercapto purine (DMMP) ; 2,6-diamino-8-mercaptopurine (DM) ; 2,6-diamino-S-o-chlorophenyl purine basic hydrochloride (DCPo); 2,6-diamino-8m-chlorophenyl purine basic hydrochloride (DCPm); 2,6-diamino-S-p-chlorophenyl purine hydrochloride (DCPp) ; 2,6-diamino-8-m-nitrophenyl purine hydrochloride hydrate (DNPH) ; and 2-thio-4-oxy-5-o-p-dichlorophenoxy pyrimidine (TODP). I~~IJLT~
;1s noted in Table I, oxygen consumption was markedly influenced by all of the derivatives studied. Some of the compounds appeared more
Effect of Various
TABLE I Derivatives on Oxygen
of T. cruzi
Inhibition of oxygen uptake after 120 minutes exposure. %
Concentration
Compound
Uptake
Motility”
-
l/75,000 1/5o,ooo 1/3o,ooo
1/‘3,f300
16 47 87 97
+++ ++ -
DMP
1/2o,ooo 1/1o,oca 1/5,ooo l/l,~
6 57 71 96
+++ + -
DMPB
l/~,fJoo l/l,~ l/500
14 38 71
+++ -I-+ -
DMMP
1/25,ow
l/2,500 1/1,m l/400
0 20 75 95
+++ +++ -
DM
1/10,000 1/1,~ l/750 l/380
0 46 56 72
+++ + -
DCPO
1/25,ooa 1/1o,ooo f/5,000 l/l ,ooo
13 37 66 96
+++ +++ + -
DCPm
1/50,000 1/=,ooO l/~,ooo l/7,500
36 ;i 96
++ + -
DCPp
l/25,000 1/15,ow 1/1o,ooo l/l,ooO
10 32 63 97
+++ -t-f -
DNPH
l/25,000 1/5,6fJJ 1/1,~ 1/5oCJ
2 41 71 9!
+++ + -
TODP
1/25,ooO l/7,600 l/4,800 1/3,@JJ
3 45 77 97
+++ + -
0
0
+++
DP”
Controls o - - no motility; controls. * For key to full
* name
- ~lusgish
motility;
of derfwtive.
++
P slight
BBB bfaterlale
22
and
motility; Method
+++ ssotion.
= highly
motile.
aimilm
to
PURINE
DERIVATIVES
AND
TRYPANOSOMA CRVZI
23
active than others in that a greater inhibition was produced with less drug. Drugs DP and DCPm appeared to be trypanocidal and caused approximately 8701,inhibition (DP) at a concentration of l/30,000 and TABLE -
Effect
II
of Purine Derivatives on the Growth of T. cruei under Aerobic Conditions Hemocytometer
Compound
Trypanocidal concentrations
DP DMP DMPS DMMP DM DCPo DCPm DCPp DNPH TODP Control gl Control #2 __-. Trypanocidal
l/30,000 1/10,000 l/l,CJfJo l/750 l/750 l/4,000 l/15,000 117,500 l/l,oc@ l/5,000 0 0
1 day
335 270 255 300 370 260 250 295 255 315 290 250
305 275 330 250 300 220 195 220 360 315 440 340
2
days
250 240 240 215 250 160 185 110 265 270 500 800
4
X 101
days
1 days
165 180 155
45 105 110 50 155 25 35 5 55 160 2200 1950
110
210 95 105 50 175 205 1500 1350
14 days
4 35 10 10 65 10 10 1 5 100 29ou 2650
TABLE III Concentrations of Purine Derivatives in Petrolatum Sealed Cultures of T. cruzi Compound
Concentration
DP DMP DMPS DMMP DM DCPo DCPm DCPp DNPH TODP
l/10,000 to l/20,000 l/10,000 l/l,ooo l/750 l/750 l/5,000 l/20,000 l/5,000 to l/10,000 l/l,ooO l/5,000
about 78y0 inhibition motility
O-hour
count/ml
(DCPm) at a concentration of l/20,000. The
of the organisms
corresponded
with
the inhibition
of respira-
tion; the trypanosomes became non-motile when from 70 to 75% inhibition of oxygen uptake occurred. Concentrations which showed slight inhibition had actively motile organisms.
24
MITSURU
NAKAMURA
AND MAQDALENA
B. JAMES
Table II records the effect of purine derivatives on aerobic cultures of T. cruzi. The population steadily decreasedwhen trypanocidal levels of
drugs were added, while in control cultures organisms increased until the peak was reached in 14 days. Trypanosomes remaining in the control flasks after the 14 day period were transferred to fresh media not containing drugs and organisms grew rapidly and reached a peak count in 14 days. Organisms exposed to the derivatives of purine did not multiply and at the end of 14 days only a few viable trypanosomes were seen. The tyrpanocidal concentrations determined under anaerobic culture conditions may be seenin Table III. The concentrations listed are those which produced a total kill of all trypanosomes, i.e. at these concentrations no motile trypanosomes were observed microscopically. Upon subculture to fresh media, without drugs added, no viability was observed. Control (untreated) cultures were maintained during the five day period of the drug test at room temperature. DISCUSSION
Although further work with the derivatives of purine is necessary, our experiments have indicated that some possesstrypanocidal properties in vitro. The ‘tidal concentration ranged from I/750 to l/30,000 depending upon the agent studied. Brackett (1947) found pyrimidine derivatives to be amebastatic against Endamoeba histolytica in concentrations of l/1,000 to l/100,000. The compounds varied a great deal in their activity and one contributing factor seemsto be the point of substitution and whether halogens are incorporated into the molecule. In our study the compounds containing chlorine appeared more active than the others tested except for the 2,G-diamino purine. Hitchings et al. (1945, 1948, 1950) and Elion (1951) studied derivatives of 2,6diamino purine against La&bacillus casei and found the inhibitory concentrations to range from l/10,000 to l/500,000. They postulated that these compounds acted as antimetabolites since the inhibition was reversible. Kidder et al. (1949 and 1950) also believed that purine and pyrimidine derivatives acted as anti-metabolites against Tetrahymena geleii. Our experiments showed that these derivatives inhibited the trypanosomes both in vitro (over 5-14 days) and in the manometric oxygen uptake studies (over 2 hours). Further study of the purine and pyrimidine derivatives against Trypanosoma cruzi seems desirable. SUMMARY
One pyrimidine and nine purine derivatives were studied in vitro against T. cruzi. Concentrations of l/750 to l/30,000 inhibited the try-
PURINE
DERIVATIVES
AND
2X YPANOSOMA
CR WI
25
panosomesstudied manometrically and also in cultures both aerobically and anaerobically. 2,6-diamino purine hydrate appeared to be the most active of the agents studied. kJKNOWLEDGEMENT
Acknowledgement is made to Professor Hamilton H. Anderson, Dean of the Medical Faculty, American University of Beirut, Beirut, Lebanon (1950-1951) for his unending encouragement and advice. REFERENCES AMREIN,
Y. A. 1951. Effects of tyrocidine
on Trypanosoma cruzi cultures. Proc.
Sot. Exp. Biol. Med. 76, 744-746. BRACKETT, S., AND BLIZNICK, A. 1947. The rate of multiplication
of Endamoeba histolytica and its relation to in vitro drug t,esting and possibly to nutritional studies. J. Parasitol. 33, 154-166. VON BRAND, T., JOHNSON, E. M., AND REES, C. W. 1946. Observations on the respiration of Trypanosoma cruzi in culture. J. Gen. Physiol. 30, 163-175. CHEN, G., AND GEILING, E. M. I<. 1945. The determination of antitrypanosome effect of antimonials in vitro. J. Infect. Diseases 7’7, 139-143. ELION, G. B., AND HITCHINGS, G. II. 1950. Antagonists of nucleic acid derivatives. IV. Reversal studies with 2-aminopurine and 2,6-diaminopurine. J. Biol. Chem. 187, 511-522. ELION, G. B., HITCHINGS, G. H., AND VAXDERWERFF, H. 1951. Antagonists of nucleic acid derivatives. VI. Purines. J. Biol. Chem. 192, 505-518. GOBLE, F. C. 1949. Chemotherapeutic activity of certain S-aminoquinolines, particularly pentaquine, in experimental Chagas’ disease. J. Parasitol. 36, 375-378. GOODWIN, L. G., Goss, M. D., LOCK, J. A., AND WALLS, L. P. 1950. The chemocruzi. therapeutic action of phenanthridine compounds. II. Trypanosoma Brit.
J. Pharmacol.
Chemotherapy
6, 277-286.
G. H., FALCO, E. A., AND SHERWOOD, hi. 8. 1945. The effects of pyrimidines on the growth of Lactobacillus casei. Science 102,251-252. HITCHINGS, G. H., ELION, G. B., VANDERWERFF, H., AND FALCO, E. A. 1948. Pyrimidine derivatives as antagonists of pteroylglutamic acid. J. Biol. Chem. 174, 765-766. HITCHINGS, G. I-I., ELION, G. B., FALCO, E:. A., RUSSELL, P. B., SHERWOOD, M. B., AND VANDERWERFF, H. 1950. Antagonists of nucleic acid derivatives. I. The Lactobacillus casei model. J. Biol. Chem. 183, 1-9. KIDDER, G. W., AND DEWEY, V. C. 1949. The biological activity of substituted purines. J. Biol. Chem. 179, 181-187. KIDDER, G. W., DEWEY, V. C., AND PARKS, R. E. 1949. Purine metabolism in Tetrahymena and its relation to malignant cells in mice. Science 109, 511-514. KIDDER, G. W., DEWEY, V. C., PARKS, R. E., AXD HEINRICH, RI. R. 1950. Further studies on the purine ant1 pyrimidine met~abolism of ‘I’etrahymena. PFOC. HITCHINGS,
Natl.
Acad. Sci. 36, 431-439.
C. A., MCNEIL, E., AND WOOD, P. D. 1937. Effects of arsenicals on Z’rypanosoma cruzi in tissue culture. J. Ph,armacol. Exptl. Therap. 69, 424-428.
KOFOID,
26
MITSURU
NAKAMURA
AND
MAGDALENA
B.
JAMES
LOCK, J. A. 1950. The chemotherapeutic action of phenanthridine compounds. IV. Activity in vitro. Brit. J. Pharmacol. Chemotherapy 6, 398408. MCIVOR, B., ANDERSON, H. H., LUDUENA, F. P., AND LEAKE, C. D. 1942. Chemotherapeutic effectiveness of certain sulfonamide, organic antimony, arsenic and bismuth compounds in experimental Chagas’ disease. Federation Proc. 1,160. MARSHALL, P. B. 1948. The glucose metabolism of Trypanosoma evansi and the action of trypanocides. Brit. J. Pharmacol. Chemotherapy 8, 8-14. MAZZA, S., BASSO, G., AND BASSO, R. 1941. Comprobacion por biopsia de la naturaleea chagasica de la esquizotripanide eritematosa polimorfa. Publ. Mis. Est. Patol. Region. Argentina 66, 3-28. (Biol. Abstracts 1942, 16, 433). MAZZA, S., BASSO, G., AND BASSO, R. 1942. Ensayos terapeuticos de1 product0 9736 (As) Bayer y de su action comparada con el7602 (AC) Bayer, en la Enfermedad de Chagas. Publ. Mis. Esl. Patol. Region. Argentina 61, 3-76. (Biol. Abstracts 1943,17, 169). MAZZA, S., BASSO, G., AND BASSO, R. 1945. Investigations on Chagas’ disease. Publ. Mis. Est. Patol. Region. Argentina 70, 1-81. (Biol. Abstracts 1946, 20, 1756). MAZZA, S., FREIRE, R. S., AND SALICA, P. N. 1942. Investigaciones sobre Enfermedad de Chagas: Formas meningoencefalicas. Publ. Mis. Est. Patol. Region. Argentina 80, 3-35. (Biol. Abstracts 1942, 16, 2358). PHILLIPS, B. P., AND REES, C. W. 1950. The growth of Endamoeba histolyticu with live and heat-treated Trypanosoma cruzi. Am. J. Trop. Med. 30, 18&191. PIZZI, T. 1951. Effect of primaquine on experimental Trypanosoma cruzi infection. Proc. Sot. Exptl.
Biol.
Med. 78, 643-644.
TALICE, R. V., AND LOPEZ-FERNANDEZ,J. 1945. Primeros ensayos de tratamiento de las formas agudas de enfermedad de Chagas con un nuevo arsenical. Arch. Uruguayos Med., Cir. y Espec. 27, 3346. (Biol. Abstracts 1946, 20, 1249). THOMPSON, R. L., MINTON, JR., FALCO, E. A., AND HITCHINOS, G. H. 1951. Federalion Proc. 10, in press. UYBREIT, W. W., BURRIS, R. H., AND STAUFFER,J. F. 1949. Manometric Techniques and Tissue Metabolism. Burgess Publishing Co., Minneapolis.
of Purine Mitsuru
Division
Derivatives on Trypanosoma in Vitro1e2
Nakamuras
of Pharmacology University
and
Magdelena
cruzi
B. James
and Experimental Therapeutics, School of Medicine, of California, San Francisco, California
(Submitted
for publication
14 July
1951)
The study of chemotherapeutic agents against Trypanosoma cruzi, the etiological agent of Chagas’ disease, has been rather limited because of the ineffectiveness of drugs teAed against this trypanosome. Infection with T. cruzi is very resistant to drugs efficacious in treating other trypanosomiases, and at present no satisfact,ory chemotherapy is available in Chagas’ disease (Kofoid et al., 1937; von Brand et al., 1946). Kofoid et al. (1937) tested some arsenicals against T. cruzi in tissue culture and found arsenious trit,hiosalicylic acid to be quite toxic for the trypanosomes, but stated that, previous experiments had proved that this drug is also toxic t#o tissue in vitro and highly toxic to mammals. McIvor et al. (1942) studied the chemot’herapeutic effectiveness of certain sulfonamides, organic antimony, arsenic and bismuth compounds against experimental T. crz~z’ infections in mice. Tolerated doses of three agents prolonged the life of the animals, but in all mice Leishman-Donovan bodies remained in the heart muscle. Mazza et al. treated human cases of Chagas’ disease with 7602 (AC) Bayer, an aminoquinoline of the “surphen” group which is trypanocidal, and also with 9736 (As) Bayer. They found that dialilmalonildiamid-2-methyl, 4, G-diaminoquinoline caused the trypanosomes to disappear from the circulation (Mazza, Basso, and Basso, 1941; Mazza, Freire, and Salica, 1942; Mazza, Basso, and Basso, 1942; Mazza, Basso, and Basso, 1945). Talice and LopezFernandez treated Chagas’ disease with p-arsenophenylbutryic acid (1945). Goble (1949) studied S-aminoquinolines against T. cruzi in1 With the assistance of Miss D. Swanman and Mrs. B. Mythen. 2 Supported in part by the Division of Research Grants and Fellowships, National Institutes of Health, United States Public Health Service, Bethesda, Md., and the Abbott Laboratories, North Chicago, Illinois. 3 Present address: Dept. of Biological Sciences, New England College of Pharmacy, Boston, Mass. 19
20
MITSURU NAKAMURA AND MAGDBLENA B. JAMES
fections in mice and found 8-(isopropylaminoamylamino)-6-methoxyquinoline (pentaquine) highly trypanocidal. Pizzi (1951) found that primaquine showed suppressive action in the early stages of T. cruxi infection in mice. Tyrocidine, in cultures, had growth-suppressing effect in concentrations of l/10,000 (Amrein, 1951). Recently Goodwin, et al. (1950) have reported some success against mouse infections with methyl phenanthridinium derivatives. It was shown that 2-amino-9-p-carbethoxyaminophenyl-lO-methylphenanthridium bromide in a concentration of l/150,000 killed 90% of the trypanosomes in vitro in 24 hours (Lock, 1950). Purine and pyrimidine derivatives have been studied by Hitchings and his coworkers (Hitchings, et al., 1945; Hitchings et al., 1948; Hitchings et aE., 1950; Elion and Hitchings, 1950; Elion, et al., 1951) against Lactobacillus casei. They believe that t,hese derivatives act primarily as antimetabolites and give several interesting theories as to their mode of action. Kidder and his group (Kidder and Dewey, 1949; Kidder et al., 1949; Kidder, et al., 1950) have reported that substituted purines inhibited the growth of Tetrahymena geleii. The effect of such compounds against Endamoeba histolytica has also been studied in vitro (Brackett and Bliznick, 1947). Thompson et al. (1951) have reported on the activity of 5-(2,4-dichlorophenoxy)-4-hydroxy-2-mercaptopyrimidine against vaccinia virus. This presentation is a report of some observed effects of purine derivatives on T. cruzi in vitro. The purine derivatives used in this study were generously supplied by Dr. George H. Hitchings, Wellcome Research Laboratories, Tuckahoe, New York. MATERIALS
AND METHODS
The culture of Trypunosoma cruzi (Culbertson strain) was obtained from Dr. C. W. Rees, National Institutes of Health, Bethesda, Md. a. Maintenance of Stock Culture. The medium consisted of a blood-agar base with the following ingredients: Bacto agar (Difco) 2.5 g, Trypticase (BBL) 2.0 g, NaCl 0.5 g, and water 85.0 ml. The ingredients were dissolved in water and adjusted to a pH of 7.0 to 7.2 with 0.1 N NaOH. This was autoclaved at 121°C at 15 pounds for 15 to 20 minutes and before the medium solidified, 10.0 ml of sterile defibrinated rabbit’s blood was added. After thoroughly mixing the medium 25.0 ml each was dispensed into 250 ml Erlenmeyer flasks. The medium was allowed to cool and harden. The overlay consisted of Trypticase (BBL) 2.0 g, dextrose 1.0 and NaCl 1.6 g dissolved in 200.0 ml of water and adjusted to pH 7.0 to 7.2 with 0.1 N NaOH. This was autoclaved and 50 ml was overlayed on each blood-agar base (von Brand, et al., 1946; Phillips and Rees, 1950). The flasks were inoculated with 4.0 ml of a 14-day-old culture of !f. cruzi with an approximate count, as de-
PURINE
DERIV.%TIVES
termined by the hemocytometer, were incubated at 25°C.
AND
TRYPANOSOMA CRUZI
of 25,000,OOO organisms per milliliter.
21
The flasks
b. Technic Used, for Studies of Oxygen Consumption. The effect of the various agents was studied on the oxygen uptake of T. cruzi. The trypanosomes were harvested after 14 days incubation which had an approximate count of 25,000,OOO per ml. The trypanosomes were centrifuged and washed with Krebs-Ringer phosphate buffer solution twice and resuspended in this buffer so that the count approximated 250,000,OOOper ml. (Chen and Geiling, 1945; von Brand et al., 1946; Marshall, 1948, Umbreit et al., 1949). Into each 10.0 ml. Warburg flask was placed 1.0 ml of this t,rypanosome suspension, 0.1 ml of a 2% glucose solution, and aliquots of the drug solution in order to obtain the desired final concentrations. The oxygen uptake was measured for a period of 2 hours. The gas phase was air and the equilibration period was 10 minutes. All tests were made at 37.2”C. Readings were taken at 15 minut.e intervals and t,he manometers were shaken at t.he rate of 100 shakes per minut,e. At the end of the two-hour run, a small sample was removed from each flask and examined microscopically for motility to determine the activity of the trypanosomes. c. Technic for the Study of Druys under Aerobic and Anaerobic Conditions. The effect of substituted purines on T. cruzi cultures under aerobic conditions was studied in the diphasic blood agar medium. The agents to be tested were dissolved in suitable solvent,s and sterilized by Seit.z filtration. The drugs were added aseptically to the medium so as to achieve the desired final concentration, and then 1.0 ml of a 14.day old culture, containing approximately 25,000,OOO trypanosomes, was inoculated into the medium having an overlay of 10.0 ml. These flasks were incubated at room temperature and at O-hour, 1, 2, 3, 4, 7, 10, and 14 days, 0.25 ml of the culture was removed and counted in the hemocytometer. The concent,ration of drugs used was in a range t,hat was found to reduce markedly the oxygen uptake of the trypanosomes when studied manometrically. For studying the effect of these agents on the trypanosomes under anaerobic conditions 10.0 ml of the trypanosomes (with a count of approximately 25,000,000/ml) growing in the diphasic blood agar for 14 days were placed in a series of tubes. A range of concentrat,ion of each agent was studied by adding the drugs to the tubes to make up t.he desired final concentration and the tubes were sealed with 1.25 cm of solid petrolatum. The tubes were incubated at 25°C for a period of 5 days and then examined microscopically. That concentration at. which no trypanosomes were detected was recorded as t,rypanocidal. d. Purine Derivatives Studied. 2,6-Diamino purine hydrate (DP); 2,6-diamino7-methyl purine (DMP); 2,6-diamino-S-methyl purine sulfate (DMPS); 2,6-diamino-8-methylmercapto purine (DMMP) ; 2,6-diamino-8-mercaptopurine (DM) ; 2,6-diamino-S-o-chlorophenyl purine basic hydrochloride (DCPo); 2,6-diamino-8m-chlorophenyl purine basic hydrochloride (DCPm); 2,6-diamino-S-p-chlorophenyl purine hydrochloride (DCPp) ; 2,6-diamino-8-m-nitrophenyl purine hydrochloride hydrate (DNPH) ; and 2-thio-4-oxy-5-o-p-dichlorophenoxy pyrimidine (TODP). I~~IJLT~
;1s noted in Table I, oxygen consumption was markedly influenced by all of the derivatives studied. Some of the compounds appeared more
Effect of Various
TABLE I Derivatives on Oxygen
of T. cruzi
Inhibition of oxygen uptake after 120 minutes exposure. %
Concentration
Compound
Uptake
Motility”
-
l/75,000 1/5o,ooo 1/3o,ooo
1/‘3,f300
16 47 87 97
+++ ++ -
DMP
1/2o,ooo 1/1o,oca 1/5,ooo l/l,~
6 57 71 96
+++ + -
DMPB
l/~,fJoo l/l,~ l/500
14 38 71
+++ -I-+ -
DMMP
1/25,ow
l/2,500 1/1,m l/400
0 20 75 95
+++ +++ -
DM
1/10,000 1/1,~ l/750 l/380
0 46 56 72
+++ + -
DCPO
1/25,ooa 1/1o,ooo f/5,000 l/l ,ooo
13 37 66 96
+++ +++ + -
DCPm
1/50,000 1/=,ooO l/~,ooo l/7,500
36 ;i 96
++ + -
DCPp
l/25,000 1/15,ow 1/1o,ooo l/l,ooO
10 32 63 97
+++ -t-f -
DNPH
l/25,000 1/5,6fJJ 1/1,~ 1/5oCJ
2 41 71 9!
+++ + -
TODP
1/25,ooO l/7,600 l/4,800 1/3,@JJ
3 45 77 97
+++ + -
0
0
+++
DP”
Controls o - - no motility; controls. * For key to full
* name
- ~lusgish
motility;
of derfwtive.
++
P slight
BBB bfaterlale
22
and
motility; Method
+++ ssotion.
= highly
motile.
aimilm
to
PURINE
DERIVATIVES
AND
TRYPANOSOMA CRVZI
23
active than others in that a greater inhibition was produced with less drug. Drugs DP and DCPm appeared to be trypanocidal and caused approximately 8701,inhibition (DP) at a concentration of l/30,000 and TABLE -
Effect
II
of Purine Derivatives on the Growth of T. cruei under Aerobic Conditions Hemocytometer
Compound
Trypanocidal concentrations
DP DMP DMPS DMMP DM DCPo DCPm DCPp DNPH TODP Control gl Control #2 __-. Trypanocidal
l/30,000 1/10,000 l/l,CJfJo l/750 l/750 l/4,000 l/15,000 117,500 l/l,oc@ l/5,000 0 0
1 day
335 270 255 300 370 260 250 295 255 315 290 250
305 275 330 250 300 220 195 220 360 315 440 340
2
days
250 240 240 215 250 160 185 110 265 270 500 800
4
X 101
days
1 days
165 180 155
45 105 110 50 155 25 35 5 55 160 2200 1950
110
210 95 105 50 175 205 1500 1350
14 days
4 35 10 10 65 10 10 1 5 100 29ou 2650
TABLE III Concentrations of Purine Derivatives in Petrolatum Sealed Cultures of T. cruzi Compound
Concentration
DP DMP DMPS DMMP DM DCPo DCPm DCPp DNPH TODP
l/10,000 to l/20,000 l/10,000 l/l,ooo l/750 l/750 l/5,000 l/20,000 l/5,000 to l/10,000 l/l,ooO l/5,000
about 78y0 inhibition motility
O-hour
count/ml
(DCPm) at a concentration of l/20,000. The
of the organisms
corresponded
with
the inhibition
of respira-
tion; the trypanosomes became non-motile when from 70 to 75% inhibition of oxygen uptake occurred. Concentrations which showed slight inhibition had actively motile organisms.
24
MITSURU
NAKAMURA
AND MAQDALENA
B. JAMES
Table II records the effect of purine derivatives on aerobic cultures of T. cruzi. The population steadily decreasedwhen trypanocidal levels of
drugs were added, while in control cultures organisms increased until the peak was reached in 14 days. Trypanosomes remaining in the control flasks after the 14 day period were transferred to fresh media not containing drugs and organisms grew rapidly and reached a peak count in 14 days. Organisms exposed to the derivatives of purine did not multiply and at the end of 14 days only a few viable trypanosomes were seen. The tyrpanocidal concentrations determined under anaerobic culture conditions may be seenin Table III. The concentrations listed are those which produced a total kill of all trypanosomes, i.e. at these concentrations no motile trypanosomes were observed microscopically. Upon subculture to fresh media, without drugs added, no viability was observed. Control (untreated) cultures were maintained during the five day period of the drug test at room temperature. DISCUSSION
Although further work with the derivatives of purine is necessary, our experiments have indicated that some possesstrypanocidal properties in vitro. The ‘tidal concentration ranged from I/750 to l/30,000 depending upon the agent studied. Brackett (1947) found pyrimidine derivatives to be amebastatic against Endamoeba histolytica in concentrations of l/1,000 to l/100,000. The compounds varied a great deal in their activity and one contributing factor seemsto be the point of substitution and whether halogens are incorporated into the molecule. In our study the compounds containing chlorine appeared more active than the others tested except for the 2,G-diamino purine. Hitchings et al. (1945, 1948, 1950) and Elion (1951) studied derivatives of 2,6diamino purine against La&bacillus casei and found the inhibitory concentrations to range from l/10,000 to l/500,000. They postulated that these compounds acted as antimetabolites since the inhibition was reversible. Kidder et al. (1949 and 1950) also believed that purine and pyrimidine derivatives acted as anti-metabolites against Tetrahymena geleii. Our experiments showed that these derivatives inhibited the trypanosomes both in vitro (over 5-14 days) and in the manometric oxygen uptake studies (over 2 hours). Further study of the purine and pyrimidine derivatives against Trypanosoma cruzi seems desirable. SUMMARY
One pyrimidine and nine purine derivatives were studied in vitro against T. cruzi. Concentrations of l/750 to l/30,000 inhibited the try-
PURINE
DERIVATIVES
AND
2X YPANOSOMA
CR WI
25
panosomesstudied manometrically and also in cultures both aerobically and anaerobically. 2,6-diamino purine hydrate appeared to be the most active of the agents studied. kJKNOWLEDGEMENT
Acknowledgement is made to Professor Hamilton H. Anderson, Dean of the Medical Faculty, American University of Beirut, Beirut, Lebanon (1950-1951) for his unending encouragement and advice. REFERENCES AMREIN,
Y. A. 1951. Effects of tyrocidine
on Trypanosoma cruzi cultures. Proc.
Sot. Exp. Biol. Med. 76, 744-746. BRACKETT, S., AND BLIZNICK, A. 1947. The rate of multiplication
of Endamoeba histolytica and its relation to in vitro drug t,esting and possibly to nutritional studies. J. Parasitol. 33, 154-166. VON BRAND, T., JOHNSON, E. M., AND REES, C. W. 1946. Observations on the respiration of Trypanosoma cruzi in culture. J. Gen. Physiol. 30, 163-175. CHEN, G., AND GEILING, E. M. I<. 1945. The determination of antitrypanosome effect of antimonials in vitro. J. Infect. Diseases 7’7, 139-143. ELION, G. B., AND HITCHINGS, G. II. 1950. Antagonists of nucleic acid derivatives. IV. Reversal studies with 2-aminopurine and 2,6-diaminopurine. J. Biol. Chem. 187, 511-522. ELION, G. B., HITCHINGS, G. H., AND VAXDERWERFF, H. 1951. Antagonists of nucleic acid derivatives. VI. Purines. J. Biol. Chem. 192, 505-518. GOBLE, F. C. 1949. Chemotherapeutic activity of certain S-aminoquinolines, particularly pentaquine, in experimental Chagas’ disease. J. Parasitol. 36, 375-378. GOODWIN, L. G., Goss, M. D., LOCK, J. A., AND WALLS, L. P. 1950. The chemocruzi. therapeutic action of phenanthridine compounds. II. Trypanosoma Brit.
J. Pharmacol.
Chemotherapy
6, 277-286.
G. H., FALCO, E. A., AND SHERWOOD, hi. 8. 1945. The effects of pyrimidines on the growth of Lactobacillus casei. Science 102,251-252. HITCHINGS, G. H., ELION, G. B., VANDERWERFF, H., AND FALCO, E. A. 1948. Pyrimidine derivatives as antagonists of pteroylglutamic acid. J. Biol. Chem. 174, 765-766. HITCHINGS, G. I-I., ELION, G. B., FALCO, E:. A., RUSSELL, P. B., SHERWOOD, M. B., AND VANDERWERFF, H. 1950. Antagonists of nucleic acid derivatives. I. The Lactobacillus casei model. J. Biol. Chem. 183, 1-9. KIDDER, G. W., AND DEWEY, V. C. 1949. The biological activity of substituted purines. J. Biol. Chem. 179, 181-187. KIDDER, G. W., DEWEY, V. C., AND PARKS, R. E. 1949. Purine metabolism in Tetrahymena and its relation to malignant cells in mice. Science 109, 511-514. KIDDER, G. W., DEWEY, V. C., PARKS, R. E., AXD HEINRICH, RI. R. 1950. Further studies on the purine ant1 pyrimidine met~abolism of ‘I’etrahymena. PFOC. HITCHINGS,
Natl.
Acad. Sci. 36, 431-439.
C. A., MCNEIL, E., AND WOOD, P. D. 1937. Effects of arsenicals on Z’rypanosoma cruzi in tissue culture. J. Ph,armacol. Exptl. Therap. 69, 424-428.
KOFOID,
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MITSURU
NAKAMURA
AND
MAGDALENA
B.
JAMES
LOCK, J. A. 1950. The chemotherapeutic action of phenanthridine compounds. IV. Activity in vitro. Brit. J. Pharmacol. Chemotherapy 6, 398408. MCIVOR, B., ANDERSON, H. H., LUDUENA, F. P., AND LEAKE, C. D. 1942. Chemotherapeutic effectiveness of certain sulfonamide, organic antimony, arsenic and bismuth compounds in experimental Chagas’ disease. Federation Proc. 1,160. MARSHALL, P. B. 1948. The glucose metabolism of Trypanosoma evansi and the action of trypanocides. Brit. J. Pharmacol. Chemotherapy 8, 8-14. MAZZA, S., BASSO, G., AND BASSO, R. 1941. Comprobacion por biopsia de la naturaleea chagasica de la esquizotripanide eritematosa polimorfa. Publ. Mis. Est. Patol. Region. Argentina 66, 3-28. (Biol. Abstracts 1942, 16, 433). MAZZA, S., BASSO, G., AND BASSO, R. 1942. Ensayos terapeuticos de1 product0 9736 (As) Bayer y de su action comparada con el7602 (AC) Bayer, en la Enfermedad de Chagas. Publ. Mis. Esl. Patol. Region. Argentina 61, 3-76. (Biol. Abstracts 1943,17, 169). MAZZA, S., BASSO, G., AND BASSO, R. 1945. Investigations on Chagas’ disease. Publ. Mis. Est. Patol. Region. Argentina 70, 1-81. (Biol. Abstracts 1946, 20, 1756). MAZZA, S., FREIRE, R. S., AND SALICA, P. N. 1942. Investigaciones sobre Enfermedad de Chagas: Formas meningoencefalicas. Publ. Mis. Est. Patol. Region. Argentina 80, 3-35. (Biol. Abstracts 1942, 16, 2358). PHILLIPS, B. P., AND REES, C. W. 1950. The growth of Endamoeba histolyticu with live and heat-treated Trypanosoma cruzi. Am. J. Trop. Med. 30, 18&191. PIZZI, T. 1951. Effect of primaquine on experimental Trypanosoma cruzi infection. Proc. Sot. Exptl.
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Med. 78, 643-644.
TALICE, R. V., AND LOPEZ-FERNANDEZ,J. 1945. Primeros ensayos de tratamiento de las formas agudas de enfermedad de Chagas con un nuevo arsenical. Arch. Uruguayos Med., Cir. y Espec. 27, 3346. (Biol. Abstracts 1946, 20, 1249). THOMPSON, R. L., MINTON, JR., FALCO, E. A., AND HITCHINOS, G. H. 1951. Federalion Proc. 10, in press. UYBREIT, W. W., BURRIS, R. H., AND STAUFFER,J. F. 1949. Manometric Techniques and Tissue Metabolism. Burgess Publishing Co., Minneapolis.