Aria pharmacol. et toxicol. 1979, 45, 265-271.
From the Departments of Paediatrics, Experimental Research, and Clinical Pharmacology, University of Lund, Malmo General Hospital, Malmo Sweden
In Vivo Absorption of Phenytoin from Rat Small Intestine and its Inhibition by Phlorizin BY
6. Johansson, T. Lindberg and A. Melander (Received January 5, 1979; Accepted May 9, 1979)
Absrrarr: In vivo absorption of phenytoin from the small intestine was studied by an in vivo closed segment technique. Phenytoin in concentrations of 1000, 2000, and 4000 pmol/l was administered in dissolved form. Polythylene glycol 4000 was used as a non-absorbable marker. The concentrations of phenytoin in the intestinal lumen. in the mucosa, and in cardiac blood were measured both by spectrophotometry and by gas chromatography. Phenytoin was absorbed very rapidly, and the proportion absorbed increased with increasing dose. Thus, during the first 10 min. about 85 per cent of the largest dose but only 25 per cent of the smallest dose had been absorbed. The phenytoin concentration in mucosa and serum increased in an analogous way; maximum values were observed within the first ten minutes. The concentrations in mucosa and serum were dose dependent during the first ten minutes. 0.01 mmol/l and 1 mmol/l phlorizin significantly reduced the transfer of phenytoin (4000 and 2000 pmol/l) from the gut lumen to the mucosa. No inhibition was observed when the initial phenytoin dose was 1000 pmol/l. The results suggest that an active transport mechanism, sensitive t o phlorizin, is involved in the intestinal absorption of phenytoin in the rat. Key-words: Phenytoin - intestinal absorption - phlorizin - rat.
Uniform dosage of phenytoin yields large interindividual variations in blood concentrations and in effects (Booker 1978). There is evidence that interindividual differences in metabolism and elimination of the drug are major contributors to these phenomena (Booker 1978; Lund et d.1974; Glazko 1975). However, variations in absorption of the drug from the gut might also be important. I t has been shown that from some preparations the absorption of phenytoin is variable, incomplete, and prolonged (Meinardi ef a/. 1975). We recently found that food can accelerate phenytoin absorption (Melander at a/. 1979). This aroused interest in the early phase of phenytoin absorption and the influence of dietary components. The present report describes the early in vivo absorption of phenytoin at various concentrations
in the rat small intestine and the influence of phlorizin, an inhibitor of active transport of glucose, there on. Our study includes measurements of the drug concentrations in intestinal content, mucosa, and in blood.
Materials and Methods Male rats (Sprague-Dawley strain), 200-250 g, raised i n the laboratory, were used. Before the experiment, the rats fasted for about 16 hours in metabolic cages with free access t o water. Phenytoin (free acid) was purchased from AB Leo, Helsingborg, Sweden, polyethylene glycol 4000 (PEG) and D-glucose from AB KEBO, Stockholm, Sweden, and phlorizin from Fluka AG, Buchs. Switzerland. Experimental design. Under ether anaesthesia, the abdo-
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men was incised and a proximal jejunal segment (about 12 cm in length, 5 cm from the duodenal-jejunal flexure) was isolated. Polyethylene tubings were inserted and secured by ligatures at both ends. During the entire procedure, particular care was taken not to interferewith blood flow. The intestinal loop was washed with isotonic saline and emptied by air from a syringe. It was then filled with 2 ml of a solution (37") of phenytoin at known concentration, containing 2.5 mg PEG/ml in NaOH 0.05 mol/l, final pH 10. Phlorizin was added to this solution to give final concentrations of 0.01 mmol/l and I mmol/l. The experiment was ended by excision of the segment. The content was immediately transferred to a test-tube and stored at -20" until the assays were made. PEG was used as a non-absorbable marker. This made it possible to correct for the net flux of water across the mucosa during the experiment. The difference between initial and calculated final phenytoin concentration indicated the amount of phenytoin absorbed. The mucosa was dried with soft paper, scrapedoffwith a glass slide, and weighed. After mechanical homogenization for 5 min. (Colworth Stomacher 80)and extraction in 6 ml redistilled water, the phenytoin content was estimated in 1 ml aliquots and expressed in relation to mucosal wet weight. The remaining part of the intestinal wall was treated in the same way. The homogenates were stored at -20" until analysis. Histological examination of the mucosa from sections of intestine showed normal morphology. Blood was obtained by cardiac puncture immediately after the intestinal loop was removed. The serum concentrations of phenytoin were determined in 0.5 ml samples, stored at -20". Measurements ofphenytoin. Quantitative assays of phenytoin were performed by spectrophotometry according t o Dill er al. (1956).Standard curves were made for the range 0 - 200 pmol/l. At higher concentrations, the analyses were made after dilution with NaOH 0.05 mol/l. Tissue homogenates had a slight non-specific optical density (at 550 nm). Therefore, special standard curves were obtained by the use of standard solutions prepared from mucosal homogenates from control rats with known amounts of phenytoin added. The adequacy of the results obtained with the spectrophotometric method was checked by subsequent gas chromatographic analyses (Berlin et a/. 1972) of randomly chosen samples from intestinal content, mucosa, and serum. The correlation coefficient for intestinal content and serum was found to be 0.89 (n=92), and for mucosa 0.94 (n=79). Measurements of polyethylene g(ycal.1000 (PEG). Quantitative assays of PEG were performed by the turbidimetric method according to Boulter & McMichael(l970). As the standard curve proved to be dependent on the concentration of arabic gum, the latter was excluded; the readings were instead made after exactly 1 hr in 20". The slope of the standard curve was dependent on the concentration of phenytoin; therefore, standard curves were made for different ranges of phenytoin.
MINUTES
Fig. 1. Absorption of various doses of phenytoin from isolated in vivo segment of rat jejunum. Absorption expressed as cumulated percentage of initial dose disappeared from gut lumen. Mean values f S . E . M . indicated. Number of rats per group given within bars. I 1 4000 pmol/l,l 12000 pmol/l, 1000 pmol/l
Phlorizin did not interfere with the determinations of phenytoin and of PEG. Sfatisfirs.The significance of differences was calculated by Student t-tests.
Results Fig. 1, 2 and 3 show the absorption of phenytoin studied as its disappearance from the gut lumen, its uptake in intestinal mucosa, and its concentration in serum, respectively. Phenytoin was added at concentrations of 1000, 2000, and 4000 pmol/l equalling about 2.5, 5 , and 10 mg/kg body weight. Fig. I , which expresses the absorption as cumulated percentage of dose disappearing from the luminal content, shows that there was an initial phase of rapid absorption: of the 4000 pmol/l dose, not less than 29 per cent had disappeared within 1 min., 31 per cent within 3 min., 73 per cent within 5 min., and 87 per cent within 10 min. The absorption was rapid also at the other two doses, but the proportions absorbed were smaller. The corresponding figures at 3 and 10 min. for the 2000 prnol/l dose were 20 per cent (P
From the Departments of Paediatrics, Experimental Research, and Clinical Pharmacology, University of Lund, Malmo General Hospital, Malmo Sweden
In Vivo Absorption of Phenytoin from Rat Small Intestine and its Inhibition by Phlorizin BY
6. Johansson, T. Lindberg and A. Melander (Received January 5, 1979; Accepted May 9, 1979)
Absrrarr: In vivo absorption of phenytoin from the small intestine was studied by an in vivo closed segment technique. Phenytoin in concentrations of 1000, 2000, and 4000 pmol/l was administered in dissolved form. Polythylene glycol 4000 was used as a non-absorbable marker. The concentrations of phenytoin in the intestinal lumen. in the mucosa, and in cardiac blood were measured both by spectrophotometry and by gas chromatography. Phenytoin was absorbed very rapidly, and the proportion absorbed increased with increasing dose. Thus, during the first 10 min. about 85 per cent of the largest dose but only 25 per cent of the smallest dose had been absorbed. The phenytoin concentration in mucosa and serum increased in an analogous way; maximum values were observed within the first ten minutes. The concentrations in mucosa and serum were dose dependent during the first ten minutes. 0.01 mmol/l and 1 mmol/l phlorizin significantly reduced the transfer of phenytoin (4000 and 2000 pmol/l) from the gut lumen to the mucosa. No inhibition was observed when the initial phenytoin dose was 1000 pmol/l. The results suggest that an active transport mechanism, sensitive t o phlorizin, is involved in the intestinal absorption of phenytoin in the rat. Key-words: Phenytoin - intestinal absorption - phlorizin - rat.
Uniform dosage of phenytoin yields large interindividual variations in blood concentrations and in effects (Booker 1978). There is evidence that interindividual differences in metabolism and elimination of the drug are major contributors to these phenomena (Booker 1978; Lund et d.1974; Glazko 1975). However, variations in absorption of the drug from the gut might also be important. I t has been shown that from some preparations the absorption of phenytoin is variable, incomplete, and prolonged (Meinardi ef a/. 1975). We recently found that food can accelerate phenytoin absorption (Melander at a/. 1979). This aroused interest in the early phase of phenytoin absorption and the influence of dietary components. The present report describes the early in vivo absorption of phenytoin at various concentrations
in the rat small intestine and the influence of phlorizin, an inhibitor of active transport of glucose, there on. Our study includes measurements of the drug concentrations in intestinal content, mucosa, and in blood.
Materials and Methods Male rats (Sprague-Dawley strain), 200-250 g, raised i n the laboratory, were used. Before the experiment, the rats fasted for about 16 hours in metabolic cages with free access t o water. Phenytoin (free acid) was purchased from AB Leo, Helsingborg, Sweden, polyethylene glycol 4000 (PEG) and D-glucose from AB KEBO, Stockholm, Sweden, and phlorizin from Fluka AG, Buchs. Switzerland. Experimental design. Under ether anaesthesia, the abdo-
266
0 . JOHANSSON ET AL.
men was incised and a proximal jejunal segment (about 12 cm in length, 5 cm from the duodenal-jejunal flexure) was isolated. Polyethylene tubings were inserted and secured by ligatures at both ends. During the entire procedure, particular care was taken not to interferewith blood flow. The intestinal loop was washed with isotonic saline and emptied by air from a syringe. It was then filled with 2 ml of a solution (37") of phenytoin at known concentration, containing 2.5 mg PEG/ml in NaOH 0.05 mol/l, final pH 10. Phlorizin was added to this solution to give final concentrations of 0.01 mmol/l and I mmol/l. The experiment was ended by excision of the segment. The content was immediately transferred to a test-tube and stored at -20" until the assays were made. PEG was used as a non-absorbable marker. This made it possible to correct for the net flux of water across the mucosa during the experiment. The difference between initial and calculated final phenytoin concentration indicated the amount of phenytoin absorbed. The mucosa was dried with soft paper, scrapedoffwith a glass slide, and weighed. After mechanical homogenization for 5 min. (Colworth Stomacher 80)and extraction in 6 ml redistilled water, the phenytoin content was estimated in 1 ml aliquots and expressed in relation to mucosal wet weight. The remaining part of the intestinal wall was treated in the same way. The homogenates were stored at -20" until analysis. Histological examination of the mucosa from sections of intestine showed normal morphology. Blood was obtained by cardiac puncture immediately after the intestinal loop was removed. The serum concentrations of phenytoin were determined in 0.5 ml samples, stored at -20". Measurements ofphenytoin. Quantitative assays of phenytoin were performed by spectrophotometry according t o Dill er al. (1956).Standard curves were made for the range 0 - 200 pmol/l. At higher concentrations, the analyses were made after dilution with NaOH 0.05 mol/l. Tissue homogenates had a slight non-specific optical density (at 550 nm). Therefore, special standard curves were obtained by the use of standard solutions prepared from mucosal homogenates from control rats with known amounts of phenytoin added. The adequacy of the results obtained with the spectrophotometric method was checked by subsequent gas chromatographic analyses (Berlin et a/. 1972) of randomly chosen samples from intestinal content, mucosa, and serum. The correlation coefficient for intestinal content and serum was found to be 0.89 (n=92), and for mucosa 0.94 (n=79). Measurements of polyethylene g(ycal.1000 (PEG). Quantitative assays of PEG were performed by the turbidimetric method according to Boulter & McMichael(l970). As the standard curve proved to be dependent on the concentration of arabic gum, the latter was excluded; the readings were instead made after exactly 1 hr in 20". The slope of the standard curve was dependent on the concentration of phenytoin; therefore, standard curves were made for different ranges of phenytoin.
MINUTES
Fig. 1. Absorption of various doses of phenytoin from isolated in vivo segment of rat jejunum. Absorption expressed as cumulated percentage of initial dose disappeared from gut lumen. Mean values f S . E . M . indicated. Number of rats per group given within bars. I 1 4000 pmol/l,l 12000 pmol/l, 1000 pmol/l
Phlorizin did not interfere with the determinations of phenytoin and of PEG. Sfatisfirs.The significance of differences was calculated by Student t-tests.
Results Fig. 1, 2 and 3 show the absorption of phenytoin studied as its disappearance from the gut lumen, its uptake in intestinal mucosa, and its concentration in serum, respectively. Phenytoin was added at concentrations of 1000, 2000, and 4000 pmol/l equalling about 2.5, 5 , and 10 mg/kg body weight. Fig. I , which expresses the absorption as cumulated percentage of dose disappearing from the luminal content, shows that there was an initial phase of rapid absorption: of the 4000 pmol/l dose, not less than 29 per cent had disappeared within 1 min., 31 per cent within 3 min., 73 per cent within 5 min., and 87 per cent within 10 min. The absorption was rapid also at the other two doses, but the proportions absorbed were smaller. The corresponding figures at 3 and 10 min. for the 2000 prnol/l dose were 20 per cent (P
