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Physiological disposition of pyrimethamine-H3
EXPERIMENTAL
PARASITOLOGY 11, 152-155
Physiological
Disposition
Leo E. Gaudette,l Laboratory
of Parasite
(1961)
Edward Chemother;apy, Diseases, NIH,
(Submitted
of Pyrimethamine-H”
D. Titus,
and G. Robert
N#ational Institute of Allergy Bethesda, Maryland
for publication,
21 February
Coatney and Infectious
1961)
The physiological dispcsition of tritiated pyrimethamine and some pyrimethamine salts have been investigated in laboratory animals. After oral administration to rabbits, mice and a monkey, a major portion of the drug was not absorbed and 50 to 80% was excreted unchanged in the feces. In the rat, maximal absorption was achieved only when the antimalarial was present in the intestine as the free base.
Schmidt, Hughes and Schmidt (1953) have suggested that the variability in response of different individuals to pyrimethamine may be due to variations in the physiological disposition of the drug. They concluded that absorption of pyrimethamine from the gastro-intestinal tract is essentially complete. Variations in toxicity arise from individual and species differences. The conclusion that pyrimethamine absorption is complete was based on the finding, in rhesus monkeys, that urinary elimination was the same after oral or intramuscular administration of the drug. Since only 20% of the administered drug could be recovered from the urine in either case, and since the fate of the remaining 805% was not determined, the complete absorption of pyrimethamine does not appear to be an established fact. The present paper presents the results of a study on the physiological disposition of pyrimethamine in the rabbit after oral and intravenous administration, and in the mouse and monkey after oral administration. The paper also describes experiments on absorption of pyrimethamine and its salts from the intestine of the rat. MATERIALS
AND
through the kindness of Dr. George Hitchings of Burroughs-Wellcome Laboratory. Tritiated pyrimethamine was prepared by the New England Nuclear Corporation according to the Wilzbach process with an exposure time of 2 weeks, at a pressure of 0.39 atmosphere of gas. After a 24-hour exposure to initial exchange conditions, pyrimethamine was obtained with a specific activity of 2.63 mc/mg. Methods Purification of pyrimethamine-H3. Pyrimethamine-H” was initially exposed to 200 ml of hot, dilute hydrochloric acid for 24 hours, and precipitated with ammonium hydroxide. The recovered precipitate was further purified by selective dissolution and precipitation according to the method of Hitchings (personal communication). This material was dissolved in 100 ml of warm water with the addition of glacial acetic acid. After standing at room temperature for 24 hours, the solution was made alkaline with 10N sodium hydroxide and allowed to stand another 24 hours. The precipitate was recovered by filtration and dissolved in 400 ml of 50% ethanol. The preparation was filtered, concentrated to a small volume in vacua and chilled. The recovered precipitate was dried in a vacuum desiccator. A recovery of 80% was obtained providing a compound with a specific activity of 2.2 mc/mg. This material was diluted with authentic pyrimethamine and gave one spot by paper chromatography using Whatman No. 1 in the solvent systems butanol-acetic acid-water (4 : 1: 5 ) and butanol-2 55% NHBwater (30:1:20).
METHODS
Materials Pyrimethamine (2,4-d i am i n o-S-p-chlorophenyl-6-ethylpyrimidine) w a s ob t a i n ed 1 Present address: Laboratory of Biochemical Pharmacology, Joseph E. Seagram & Sons, Inc., Cranbury, New Jersey. 152
PHYSIOLOGICAL
DISPOSITION
Analytical method. Aqueous solutions of pyrimethamine were extracted into benzene from alkaline pH. An aliquot of the benzene was reacted with 1 ml of 0.0.5$ bromthymol blue, the dye was returned to O.lN NaOH and its absorption determined in a Bausch & Lomb calorimeter at 610 mp. Optical densities were proportional to concentrations. An optical density of 0.276 was obtained when 20 yg of pyrimethamine was carried through the method. Intestinal perfusion method. The recirculation technique of Schanker et al. (1958) was employed for intestinal perfusion studies in the rat. Male, Sprague-Dawley rats, weighing about 150 g, were anesthetized with pentobarbital. Cannulae were inserted either at the ileocecal and duodenal junctions for intestinal absorption studies, or esophageal and ileocecal cannulae were employed in those studies when perfusion of the stomach and the small intestine was desired. The drugs were dissolved in Krebs-Ringer bicarbonate buffer pH 7.2 and perfused with a Bowman infusion pump at a rate of 0.5 ml per minute for 3 hours. The amount of drug absorbed was determined by measuring the drug concentration in the perfusate before and at the end of its perfusion, by the bromthymol blue method. RESULTS
Physiological Disposition of PyrimethamineII3 ajter Oral and Intravenous Administration Pyrimethamine was poorly absorbed following oral administration. A comparative study was made of its physiological disposition in two rabbits after oral and intravenous administration. Young, adult, male albino rabbits were given 650 pg of pyrimethamine in 4 ml of SOY, ethanol. At the end of 24 hours following oral administration of the drug, 65.4% was found in the feces and 18.5% was found in the blood, while none was found in the feces and 73.17~ was found in the blood following intravenous administration (Table I). The fact that pyrimethamine did not appear in the feces after intravenous administration is convincing evidence that its occurrence in the feces following oral administration was due to failure of absorption.
153
OF PYRIMETHAMINE-H3
Disposition
of
TABLE I Labeled Pyrimethumine and IV Administration
Rabbit
Sample Urine Feces Blood Totals
l-1.5 Oral
Total counts (corr.) wm 1.8 x 1.7 x 4.8 X 2.36 x
lo” 106 lo” 106
kg
Rabbit
after
Oral
Z-l.3 I.V.
kg
Per cent
Total counts (corr.)
Per cent
6.9 65.4 18.5 90.8
wm 5.4 x 10s 0 1.9 x 106 2.44 X 106
20.8 0 73.1 93.9
Rabbits were given orally or intravenously 6.50 pg of pyrimetbamine-H3 equivalent to 2.6 X 106 cpm. Samples of feces, urine and blood were taken at the end of 24 hours, acidified to pH 1, centrifuged and planchets made of the clear supernatant liquids.
The radioactive substances in the blood, urine and feces of each rabbit were isolated at the end of 24 hours and the concentrated extracts subjected to paper chromatography on Whatman paper No. 1 in a butanol-ammonia:water (30: 1: 20) system. For extraction biological materials were initially adjusted to pH 1 with 6N HCl and extracted into 5 volumes of washed chloroform by mechanical shaking for one hour. The phases were separated by centrifugation, the chloroform recovered and concentrated in vacua. The aqueous phase was adjusted to pH 10, and the extraction repeated. The concentrated chloroform extracts were chromatographed for 16 hours, the paper dried and scanned for radioactive peaks. The radioactive substances in the blood and feces gave only one compound which was identical with pyrimethamine in characteristics of alkaline extraction and behavior on paper chromatography (Rf 0.95). The alkaline extract of urine contained some unchanged pyrimethamine and an additional peak with an R, of 0.81. Also, at least one acidic metabolite of pyrimethamine was present in urine with an Rf of 0.90. In studies with mice, rabbits, and a monkey, fecal excretion of drug after oral administration was determined for the first 24 hours. The amount of drug excreted in the feces by individual animals of each species
154
GAUDETTE,
TITUS,
varied from 50 to 80%. Four rabbits, which received 500 ug of pyrimethamine, excreted 52 to 78% of the drug in the feces. The fecal excretion in six mice, which received 100 yg of the tritiated drug, ranged from 41 to 57% of the administered dose. A 14-kg rhesus monkey which was given 75 mg of the tritiated pyrimethamine, excreted 51% of the dose in the feces. Absorption of Pyrimethamine and Pyrimethamine Salts from the Small Intestine and Stomach of the Rat The rate of absorption of various salt derivatives of pyrimethamine by the rat’s small intestine and stomach in situ, was investigated. Pyrimethamine base and 14 salts of pyrimethamine were studied by this method. When the base was perfused through the small intestine, 70% was absorbed. When various salts of pyrimethamine were perfused through the small intestine 10 to 21% absorption was observed. Among the salts showing relatively good absorption were pyrimethamine succinate ( 2 1% ) , pyrimethamine malate (2O%), and pyrimethamine glutamate (19%). Among the salts showing relatively poor absorption were pyrimethamine lactate ( 10% ), pyrimethamine sulfamate ( 10% ) , pyrimethamine hydrochloride (10%)) pyrimethamine hydroiodide ( 10% ) , pyrimethamine hydrobromide ( 11$X) , pyrimethamine salicylate ( 11% ), pyrimethamine sulfate ( 12% ) , pyrimethamine tartrate ( 12% ) , and pyrimethamine oxalate ( 13% ) . Pyrimethamine acetate was intermediate with 15% absorption. All the compounds were perfused at an initial concentration of 20 pg per ml. When the solutions were passed through the stomach and small intestine, absorption of the free base was reduced to 10% but there was no change in the extent of absorption of the various salts, pyrimethamine succinate (19oJo), pyrimethamine malate (18%), pyrimethamine glutamate (22 % ) , pyrimethamine lactate ( 10% ) , pyrimethamine sulfamate (10% ), pyrimethamine hydrochloride (10% ), pyrimethamine hydroiodide (lo%), pyrimethamine hydrobromide ( 10% ) , pyrimethamine salicylate ( 13% ), pyrimethamine sulfate (12%), pyrimethamine tartrate ( 10% ) , and pyrimethamine oxalate ( 117. ) .
AND
COATNEY
DISCUSSION
The results in experimental animals indicate that a major portion of orally administered pyrimethamine was not absorbed but excreted unchanged in the feces. When radiolabeled pyrimethamine was administered orally to individuals of various species, a large part of the radioactivity was found in fecal material. Since no radioactivity occurred in the feces after intravenous administration, it is concluded that its occurrence in the feces after oral administration was due to failure of absorption. Isolation of the radioactive substances in the blood and feces by extraction into chloroform and paper chromatography gave only one compound which was identical with pyrimethamine. The results found in blood support the conclusion of Smith and Ihrig (1957) that antimalarial activity can probably be attributed to unchanged pyrimethamine. The urine contained at least two metabolites, as well as unchanged pyrimethamine. The results of perfusion studies in the rat indicate that maximal absorption is achieved only if pyrimethamine reaches the intestine as the free base. In those instances where salts of the drug were perfused through the intestine or the free base was perfused through the stomach before reaching the intestine, minimal absorption occurred. In contrast to the widely quoted conclusion of Schmidt et al. (1953) that pyrimethamine is slowly but completely absorbed by the rhesus monkey which was based on inference rather than direct observation, the present animal studies indicate only a partial absorption of the drug except when the free base is introduced into the small intestine without traversing the stomach. These variations of absorption in experimental animals suggest a possible explanation for the variable effectiveness and toxicity of pyrimethamine observed in different individuals. It would seem most likely that a group of individuals receiving the same dose of pyrimethamine would include some individuals who would absorb considerably more, and others who would absorb considerably less than the average for the group. Those absorbing relatively little would exhibit a failure of treatment and those absorbing rela-
PHYSIOLOGICAL
DISPOSITION
tively more would experience the toxic effects of the drug. Coated tablets of pyrimethamine would possibly permit disposition of the drug in the small intestine as the free base, and permit maximal absorption of the drug. Such a program, currently under investigation, requires re-evaluation of pyrimethamine toxicity, taking into consideration the increased plasma levels of the compound. Finally, it is suggested that when pyrimethamine (=I Daraprim) is used for the treatment of malaria, alkalosis be considered as a potential cause of drug toxicity.
OF
155
PYRIMETHAMINE-H”
REFERENCES
L. H., HVG~ES, H. B., AND SCHMIDT, I. G. 1953. The pharmacological prcperties of 2,4-diamino-5-p-chlorophenyl-6-ethylpyrimidine (Daraprim). Journal of Phamacology G Experimental Therapeutics 107, 92-130.
SCHMIDT,
&HANKER, L. S., Tocco, HOGBEN, C. A. M.
D. J., BRODIE,
B. B., AND
1958. Absorption of drugs from the rat small intestine. Journal of Pharmacology G Experimental Therapeutics 123, 81-88.
C. C., AND IHRIG, J, 1957. The pharmacological basis for the prolonged antimalarial activity of pyrimethamine. Awzericen Journal of Tropical Medicine G Hygiene 6, 50.57.
SMITH,
PARASITOLOGY 11, 152-155
Physiological
Disposition
Leo E. Gaudette,l Laboratory
of Parasite
(1961)
Edward Chemother;apy, Diseases, NIH,
(Submitted
of Pyrimethamine-H”
D. Titus,
and G. Robert
N#ational Institute of Allergy Bethesda, Maryland
for publication,
21 February
Coatney and Infectious
1961)
The physiological dispcsition of tritiated pyrimethamine and some pyrimethamine salts have been investigated in laboratory animals. After oral administration to rabbits, mice and a monkey, a major portion of the drug was not absorbed and 50 to 80% was excreted unchanged in the feces. In the rat, maximal absorption was achieved only when the antimalarial was present in the intestine as the free base.
Schmidt, Hughes and Schmidt (1953) have suggested that the variability in response of different individuals to pyrimethamine may be due to variations in the physiological disposition of the drug. They concluded that absorption of pyrimethamine from the gastro-intestinal tract is essentially complete. Variations in toxicity arise from individual and species differences. The conclusion that pyrimethamine absorption is complete was based on the finding, in rhesus monkeys, that urinary elimination was the same after oral or intramuscular administration of the drug. Since only 20% of the administered drug could be recovered from the urine in either case, and since the fate of the remaining 805% was not determined, the complete absorption of pyrimethamine does not appear to be an established fact. The present paper presents the results of a study on the physiological disposition of pyrimethamine in the rabbit after oral and intravenous administration, and in the mouse and monkey after oral administration. The paper also describes experiments on absorption of pyrimethamine and its salts from the intestine of the rat. MATERIALS
AND
through the kindness of Dr. George Hitchings of Burroughs-Wellcome Laboratory. Tritiated pyrimethamine was prepared by the New England Nuclear Corporation according to the Wilzbach process with an exposure time of 2 weeks, at a pressure of 0.39 atmosphere of gas. After a 24-hour exposure to initial exchange conditions, pyrimethamine was obtained with a specific activity of 2.63 mc/mg. Methods Purification of pyrimethamine-H3. Pyrimethamine-H” was initially exposed to 200 ml of hot, dilute hydrochloric acid for 24 hours, and precipitated with ammonium hydroxide. The recovered precipitate was further purified by selective dissolution and precipitation according to the method of Hitchings (personal communication). This material was dissolved in 100 ml of warm water with the addition of glacial acetic acid. After standing at room temperature for 24 hours, the solution was made alkaline with 10N sodium hydroxide and allowed to stand another 24 hours. The precipitate was recovered by filtration and dissolved in 400 ml of 50% ethanol. The preparation was filtered, concentrated to a small volume in vacua and chilled. The recovered precipitate was dried in a vacuum desiccator. A recovery of 80% was obtained providing a compound with a specific activity of 2.2 mc/mg. This material was diluted with authentic pyrimethamine and gave one spot by paper chromatography using Whatman No. 1 in the solvent systems butanol-acetic acid-water (4 : 1: 5 ) and butanol-2 55% NHBwater (30:1:20).
METHODS
Materials Pyrimethamine (2,4-d i am i n o-S-p-chlorophenyl-6-ethylpyrimidine) w a s ob t a i n ed 1 Present address: Laboratory of Biochemical Pharmacology, Joseph E. Seagram & Sons, Inc., Cranbury, New Jersey. 152
PHYSIOLOGICAL
DISPOSITION
Analytical method. Aqueous solutions of pyrimethamine were extracted into benzene from alkaline pH. An aliquot of the benzene was reacted with 1 ml of 0.0.5$ bromthymol blue, the dye was returned to O.lN NaOH and its absorption determined in a Bausch & Lomb calorimeter at 610 mp. Optical densities were proportional to concentrations. An optical density of 0.276 was obtained when 20 yg of pyrimethamine was carried through the method. Intestinal perfusion method. The recirculation technique of Schanker et al. (1958) was employed for intestinal perfusion studies in the rat. Male, Sprague-Dawley rats, weighing about 150 g, were anesthetized with pentobarbital. Cannulae were inserted either at the ileocecal and duodenal junctions for intestinal absorption studies, or esophageal and ileocecal cannulae were employed in those studies when perfusion of the stomach and the small intestine was desired. The drugs were dissolved in Krebs-Ringer bicarbonate buffer pH 7.2 and perfused with a Bowman infusion pump at a rate of 0.5 ml per minute for 3 hours. The amount of drug absorbed was determined by measuring the drug concentration in the perfusate before and at the end of its perfusion, by the bromthymol blue method. RESULTS
Physiological Disposition of PyrimethamineII3 ajter Oral and Intravenous Administration Pyrimethamine was poorly absorbed following oral administration. A comparative study was made of its physiological disposition in two rabbits after oral and intravenous administration. Young, adult, male albino rabbits were given 650 pg of pyrimethamine in 4 ml of SOY, ethanol. At the end of 24 hours following oral administration of the drug, 65.4% was found in the feces and 18.5% was found in the blood, while none was found in the feces and 73.17~ was found in the blood following intravenous administration (Table I). The fact that pyrimethamine did not appear in the feces after intravenous administration is convincing evidence that its occurrence in the feces following oral administration was due to failure of absorption.
153
OF PYRIMETHAMINE-H3
Disposition
of
TABLE I Labeled Pyrimethumine and IV Administration
Rabbit
Sample Urine Feces Blood Totals
l-1.5 Oral
Total counts (corr.) wm 1.8 x 1.7 x 4.8 X 2.36 x
lo” 106 lo” 106
kg
Rabbit
after
Oral
Z-l.3 I.V.
kg
Per cent
Total counts (corr.)
Per cent
6.9 65.4 18.5 90.8
wm 5.4 x 10s 0 1.9 x 106 2.44 X 106
20.8 0 73.1 93.9
Rabbits were given orally or intravenously 6.50 pg of pyrimetbamine-H3 equivalent to 2.6 X 106 cpm. Samples of feces, urine and blood were taken at the end of 24 hours, acidified to pH 1, centrifuged and planchets made of the clear supernatant liquids.
The radioactive substances in the blood, urine and feces of each rabbit were isolated at the end of 24 hours and the concentrated extracts subjected to paper chromatography on Whatman paper No. 1 in a butanol-ammonia:water (30: 1: 20) system. For extraction biological materials were initially adjusted to pH 1 with 6N HCl and extracted into 5 volumes of washed chloroform by mechanical shaking for one hour. The phases were separated by centrifugation, the chloroform recovered and concentrated in vacua. The aqueous phase was adjusted to pH 10, and the extraction repeated. The concentrated chloroform extracts were chromatographed for 16 hours, the paper dried and scanned for radioactive peaks. The radioactive substances in the blood and feces gave only one compound which was identical with pyrimethamine in characteristics of alkaline extraction and behavior on paper chromatography (Rf 0.95). The alkaline extract of urine contained some unchanged pyrimethamine and an additional peak with an R, of 0.81. Also, at least one acidic metabolite of pyrimethamine was present in urine with an Rf of 0.90. In studies with mice, rabbits, and a monkey, fecal excretion of drug after oral administration was determined for the first 24 hours. The amount of drug excreted in the feces by individual animals of each species
154
GAUDETTE,
TITUS,
varied from 50 to 80%. Four rabbits, which received 500 ug of pyrimethamine, excreted 52 to 78% of the drug in the feces. The fecal excretion in six mice, which received 100 yg of the tritiated drug, ranged from 41 to 57% of the administered dose. A 14-kg rhesus monkey which was given 75 mg of the tritiated pyrimethamine, excreted 51% of the dose in the feces. Absorption of Pyrimethamine and Pyrimethamine Salts from the Small Intestine and Stomach of the Rat The rate of absorption of various salt derivatives of pyrimethamine by the rat’s small intestine and stomach in situ, was investigated. Pyrimethamine base and 14 salts of pyrimethamine were studied by this method. When the base was perfused through the small intestine, 70% was absorbed. When various salts of pyrimethamine were perfused through the small intestine 10 to 21% absorption was observed. Among the salts showing relatively good absorption were pyrimethamine succinate ( 2 1% ) , pyrimethamine malate (2O%), and pyrimethamine glutamate (19%). Among the salts showing relatively poor absorption were pyrimethamine lactate ( 10% ), pyrimethamine sulfamate ( 10% ) , pyrimethamine hydrochloride (10%)) pyrimethamine hydroiodide ( 10% ) , pyrimethamine hydrobromide ( 11$X) , pyrimethamine salicylate ( 11% ), pyrimethamine sulfate ( 12% ) , pyrimethamine tartrate ( 12% ) , and pyrimethamine oxalate ( 13% ) . Pyrimethamine acetate was intermediate with 15% absorption. All the compounds were perfused at an initial concentration of 20 pg per ml. When the solutions were passed through the stomach and small intestine, absorption of the free base was reduced to 10% but there was no change in the extent of absorption of the various salts, pyrimethamine succinate (19oJo), pyrimethamine malate (18%), pyrimethamine glutamate (22 % ) , pyrimethamine lactate ( 10% ) , pyrimethamine sulfamate (10% ), pyrimethamine hydrochloride (10% ), pyrimethamine hydroiodide (lo%), pyrimethamine hydrobromide ( 10% ) , pyrimethamine salicylate ( 13% ), pyrimethamine sulfate (12%), pyrimethamine tartrate ( 10% ) , and pyrimethamine oxalate ( 117. ) .
AND
COATNEY
DISCUSSION
The results in experimental animals indicate that a major portion of orally administered pyrimethamine was not absorbed but excreted unchanged in the feces. When radiolabeled pyrimethamine was administered orally to individuals of various species, a large part of the radioactivity was found in fecal material. Since no radioactivity occurred in the feces after intravenous administration, it is concluded that its occurrence in the feces after oral administration was due to failure of absorption. Isolation of the radioactive substances in the blood and feces by extraction into chloroform and paper chromatography gave only one compound which was identical with pyrimethamine. The results found in blood support the conclusion of Smith and Ihrig (1957) that antimalarial activity can probably be attributed to unchanged pyrimethamine. The urine contained at least two metabolites, as well as unchanged pyrimethamine. The results of perfusion studies in the rat indicate that maximal absorption is achieved only if pyrimethamine reaches the intestine as the free base. In those instances where salts of the drug were perfused through the intestine or the free base was perfused through the stomach before reaching the intestine, minimal absorption occurred. In contrast to the widely quoted conclusion of Schmidt et al. (1953) that pyrimethamine is slowly but completely absorbed by the rhesus monkey which was based on inference rather than direct observation, the present animal studies indicate only a partial absorption of the drug except when the free base is introduced into the small intestine without traversing the stomach. These variations of absorption in experimental animals suggest a possible explanation for the variable effectiveness and toxicity of pyrimethamine observed in different individuals. It would seem most likely that a group of individuals receiving the same dose of pyrimethamine would include some individuals who would absorb considerably more, and others who would absorb considerably less than the average for the group. Those absorbing relatively little would exhibit a failure of treatment and those absorbing rela-
PHYSIOLOGICAL
DISPOSITION
tively more would experience the toxic effects of the drug. Coated tablets of pyrimethamine would possibly permit disposition of the drug in the small intestine as the free base, and permit maximal absorption of the drug. Such a program, currently under investigation, requires re-evaluation of pyrimethamine toxicity, taking into consideration the increased plasma levels of the compound. Finally, it is suggested that when pyrimethamine (=I Daraprim) is used for the treatment of malaria, alkalosis be considered as a potential cause of drug toxicity.
OF
155
PYRIMETHAMINE-H”
REFERENCES
L. H., HVG~ES, H. B., AND SCHMIDT, I. G. 1953. The pharmacological prcperties of 2,4-diamino-5-p-chlorophenyl-6-ethylpyrimidine (Daraprim). Journal of Phamacology G Experimental Therapeutics 107, 92-130.
SCHMIDT,
&HANKER, L. S., Tocco, HOGBEN, C. A. M.
D. J., BRODIE,
B. B., AND
1958. Absorption of drugs from the rat small intestine. Journal of Pharmacology G Experimental Therapeutics 123, 81-88.
C. C., AND IHRIG, J, 1957. The pharmacological basis for the prolonged antimalarial activity of pyrimethamine. Awzericen Journal of Tropical Medicine G Hygiene 6, 50.57.
SMITH,