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Characterization of mitomycin C-induced gastrointestinal damage
roxlcoLoa
AND
APPLIED
PHARMACOLOGY
Characterization
97,415-423
(1989)
of Mitomycin C-Induced
Gastrointestinal
Damage
I. In Situ Recirculation Experiment MASAYUKI
MIZUNO, MITSURU
SHIGEKI KAWABATA, TAKESHI HAMAURA, HASHIDA, AND HITOSHI SEZAKI’
Department Vj’Basic Pharmaceutics. Faculty ofPharmuceutical Sciences, Kyoto University. kjnto 606, Japun
Received February 5. 1%‘; accepted October 16. 1988 Characterization of Mitomycin C-Induced Gastrointestinal Damage. I. In Situ Recirculation Experiment. MIZUNO, M., KAWABATA, S., HAMAURA, T.. HASHIDA. M.. AND SEZAKI, H. ( 1989). Tosicol. Appl. Pharmucol. 97, 4 15-423. The effects of mitomycin C (MMC), an antitumor agent, on the intestinal absorption of various drugs in rats were investigated. Based on microscopic observations, preadministration of a single intravenous dose of MMC (3 mg/kg) caused serious degeneration of epithelial cells. villous atrophy, and mitotic arrest in crypts at 48 hr after pretreatment. At this time point, absorption of sulfanilamide, salicylic acid, cephalexin, and L-tryptophan was shown to be significantly decreased by means of an in situ recirculation technique. The histological changes and the decrease in absorption of sulfanilamide. a model for passively absorbed drugs, were shown to depend on the timing of MMC pretreatment. Maximal effects were observed 48 hr after dosing. The MMC-induced reduction in the absorption of drugs was not a result of differences between treated and control animals with respect to pH of the drug solution, binding of drugs with intraluminal macromolecules, or intestinal mucosal blood flow. The absorption of sulfanilamide from the small intestine in the in situ system correlated well with the wet weight of the small intestine regardless of pretreatment dose or route. This suggests that the change in absorptive surface area of the intestinal mucosa may play a major role in the MMC-induced decrease in absorption capacity ofthe intestine. IB 1989 Academic Press. Inc
In the treatment of neoplastic diseases, the use of antitumor drugs is often interrupted due to indiscriminate action on both tumor and normal cells (Lazo and Hacker, 1985). The gastrointestinal tract is often a target of chemotherapeutic agents because it is a rapidly proliferating tissue. Severe gastrointestinal effects such as malabsorption, anorexia, atrophy of the tracts, and diarrhea are often limiting factors in therapy with these agents. Mitomycin C (MMC) is widely used in cancer chemotherapy, but its utility is limited by the side effects of severe bone marrow suppression and gastrointestinal damage (Lown,
1979). In several mammalian species high doses or prolonged continuous MMC treatment have caused injury to the small intestines, characterized by villous atrophy and glandular distortion (Philips et al., 1960). It is not known if these morphological alterations in the gastrointestinal tract following MMC administration are associated with alterations in absorptive function. A number of studies have shown that changes in the absorption of nutrients and drugs following treatment with antitumor drugs are explained in terms of a decrease in the active transport capacity (Robinson ef al., 1966; Jolly and Fletcher, 1977; Cape1 et al., 1979; Chen, 1982), diminished water flux
’ To whom correspondence should be addressed. 415
004 1-008X/89 $3.00 Copyright Ci 1989 hy Academic Press, Inc. All rights of reproduction in any form reserved.
416
MIZUNO
through the intestinal membrane (Venho, 1976) loss of mucosal surface area (Komuro et al., 1974: Senius et al.. 198 l), and impaired mucosal integrity (Siber et al., 1980; Cobden et al., 198 1). However, little is known about the mechanisms of disordered absorption. Therefore, the present investigation was aimed at the elucidation of disorders in intestinal absorptive functions induced by MMC pretreatment. Several factors that could influence the drug absorption process, such as intraluminal interactions, mucosal permeability, and circulatory changes, were investigated. An in situ preparation was used to explore the possible mechanisms in reduced absorptive capacity. METHODS Materials. MMC and cephalexin were kindly supplied by Kyowa Hakko Kogyo Company, Tokyo, Japan, and Takeda Chemical Industries, Osaka, Japan, respectively. Fluorescein isothiocyanate-conjugated dextran (FITCD. average molecular weight 65.600) and bovine serum albumin were purchased from Sigma Chemical Company, St. Louis, Missouri. 6-Carboxyfluorescein (h-CF) was obtained from Eastman Kodak Company, Rochester. New York. All other reagents used in these experiments were of reagent grade and were used without further purification. ,4nimal.s. Age (6-7 weeks old)- and weight ( 180-220 g)-matched groups of male Wistar rats (Shiruoka Agricultural Cooperative Association for Laboratory Animals, Shizuoka. Japan) were used. Animals had free access to water and food for at least 3 days before the experiment, and were maintained on 12-hr light-dark cycles. After this period. rats were weighed and given a bolus intravenous injection of MMC (in physiological saline) into the left femoral vein under light ether anesthesia. Unless otherwise stated, the MMC dose was 3 mg/kg. In some experiments. the animals were submitted to an overnight fast to remove gastric contents. They were then lightly anesthetized with ether and administered 10 mg/ kg MMC by gastric intubation [dissolved in isotonic bicarbonate buffer. pH 8.3, to protect MMC against gastric acidity(Wakaki el al., 1958)]. Control rats received comparable volumes of physiological saline. All injections were given between 1200 and 1300 hr. Both groups were freely provided with food and water after MMC pretreatment. Preparation of‘dn~g solution. The isotonic phosphate buffer solution (pH 6.5) was prepared from 0.123 M Na2HP0, and 0.163 M NaHLPOJ. Drugs were dissolved
ET
AL.
in this buffer solution at the stated concentrations for the absorption experiments. Absorption esprriments. The procedure for the in situ absorption study on rat small intestine was the same as that described by Kakemi et ul. (I 967). Briefly, each animal was anesthetized with sodium pentobarbital(20 mg/ kg, ip) and the small intestine was cannulated for in situ recirculation. The entire length of the small intestine. from the pylorus to the ileocecal junction. was used for the absorption studies. The bile duct was ligated in all experiments. Rectal temperature was maintained at 37°C. Forty milliliters ofdrug solution (pH 6.5) was recirculated in the small intestine at the rate of 5 ml/min. At the end of the recirculation period, the perfusate was recovered and the intestine was washed with phosphate buffer, pH 6.5. The absorbed amount of drug was calculated from the difference in the amount of drug in the initial and final solutions. Afeusuremmt yfintestinul mucosal hloodflo~~. Intestinal mucosal blood flow was measured by the hydrogen gas clearance method (PHG 201. Unique Medical Co.. Ltd.. Tokyo, Japan) (Yamamoto et ~1.. 1984b) in rats under anesthesia with sodium pentobarbital(20 mg/kg. ip). The theoretical considerations were based largely on Kety’s approach to blood-tissue exchange of inert gases (Kety and Schmidt. 1945). Intestinal blood flow is expressed as milliliters per minute per 100 g tissue weight. of’ protein
DctcrFniFlUtiWI
and
mucin
in thr
intertinu/
The operation and recirculation techniques were the same as those used in the absorption experiments. After recirculation of pH 6.5 buffer solution for I hr in control and MMC-treated rats, the perfusate was collected as completely as possible. After centrifugation ofthe perfusate at 3000 rpm for IO min. the supernatant was assayed for protein and mucin. Protein was determined according to the method of Lowry el al. ( 195 I ) using bovine serum albumin as standard. Mucin was determined spectrophotometrically at 620 nm by the anthrone method (Roe, 1955) using D-ghCOSe as standard. /ztminul
I?indiqy thrperlksatr.
.rolution.
of drug.\
with
mucromokrdar
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irl
Drugs tested for binding were salicylic acid. sulfanilamide. and cephalexin. To estimate the binding ofdrugs with macromolecular substances in intestinal luminal solution in vitro. two methods were adopted (Yamamoto (I/ ul.. 1984a). In the equilibrium dialysis method. each drug was dissolved in pH 6.5 buffer solution at a concentration of I mM. Visking cellulose tubing containing the intestinal perfusate was immersed in a drug solution and equilibrated for 72 hr at 4°C. After equilibration, the concentration of drugs in the external medium was determined as described below. In the ultrafiltration method, ultrafiltration was performed by using the “Micropartition System” (MPS-I. Amicon. Lexington. MA). This filtration system is purported to efficiently retain macromolecular substances with molecular weights greater than 10,000. The filtrate obtained was assayed to determine the free drug concentration.
MITOMYCIN
C EFFECT
ON
Histological ohwrvatiorz. Specimens were fixed in 10% (v/v) formaldehyde, sectioned, and stained with hematoxylin and eosin. Histological examination was performed by light microscopy. .Itzaiytical methods. Salicylic acid was extracted into chloroform after adding HCI to the test samples. It was then extracted with 0.1 N NaOH, and determined spectrophotometrically at 295 nm. Sulfanilamide was diazotized and coupled with 2-diethylaminoethyl-I-naphthylamine. The colored derivative was extracted into isoamyl alcohol by the addition of NaCI. The complex present in the organic phase was determined spectrophotometrically at 555 nm. D- and L-Tryptophan were determined colorimetritally (600 nm) according to the method of Spies and Chambers (1948). A reversed-phase high-pressure liquid chromatography (HPLC) method (Kimura et al., 1983) was used to determine cephalexin. FITC-D and 6-CF were determined spectrofluorimetrically. Excitation and emission wavelengths were 490 and 520 nm, respectively, for both drugs. Statistical ona~~.~ls. Results were analyzed statistically with Student’s t test, Differences with p < 0.05 were considered significant,
INTESTINAL
ABSORPTION
417
mg/kg), absorption of both 6-CF, which is poorly absorbed, and the high-molecular weight compound FITC-D, was not altered. Absorption of sulfanilamide, salicylic acid, and cephalexin, and of the actively transported L-tryptophan and its optical isomer Dtryptophan significantly decreased irrespective of the degree of ionic dissociation at pH 6.5. In the case of L-tryptophan, the ratio of percentage absorbed in the treated to that in the control animals decreased as the initial concentration of L-tryptophan in the perfusate rose: 1 mM, 0.783 + 0.067; 5 mM, 0.662 -t 0.053; 50 mM, 0.622 + 0.095. To ascertain that the observed changes were derived from pretreatment with MMC, the dose and time dependency of the decrease in drug absorption was characterized using sulfanilamide as a model for passiveIy absorbed drugs (Tables 2 and 3). Table 2 shows that the applied doses (1.2, 2.4, and 3.0 mg/ kg) of MMC caused significant decreases in sulfanilamide absorption. However. within RESULTS the dose ranges examined, no correlation was found between the magnitude of the decrease The general responses of animals to the in sulfanilamide absorption and the dose of MMC treatment were compared with those MMC. In Table 3, the effect of MMC treatreported by Philips et al. (1960). MMCment times on sulfanilamide absorption is treated rats decreased their food intake and presented. At 48 hr after MMC pretreatment. body weight within 48 hr of drug administrathe maximal decrease in sulfanilamide abtion, as previously reported (Mizuno et al., sorption was observed (p < 0.05). At 72 hr the 1986a; Mizuno et ul., 1986b). Light microsabsorption had already returned to the concopy of intestinal tissue taken from MMCtrol level. This time course of the change in treated rats (at 48 hr) showed serious degener- sulfanilamide absorption paralleled the patation of the epithelial cells, villous atrophy, tern of morphological changes in the intestiand mitotic arrest in the crypts, which were nal mucosa. coupled with some disorders in submucosal Table 4 summarizes the effect of MMC tissue (Fig. 1B). At 120 hr the villous epithepretreatment on the various physiological lium started to recover (Fig. 1C) and the gen- and experimental parameters. Wet weights of eral architecture and height of the villi whole small intestines were significantly reseemed to be restored to those of normal tis- duced in the MMC-treated rats (to about 65% of the control). The mean pH of the luminal sue (Fig. IA). The intestinal absorption of drugs possess- solution, measured 1 hr after recirculation of ing various physicochemical properties was the buffer solution (pH 6.5), was virtually measured in control and MMC-treated rats identical in both groups. The lengths of the by means of the in situ recirculation tech- intestines used for perfusion were not signifinique. The results are summarized in Table cantly different among the experimental 1. Forty-eight hours after MMC treatment (3 groups. Intestinal blood flow was significantly
418
MIZUNO
ET AL.
FIG.1, Intestinal muc :osal tissue (X 100) 48 hr after the intravenous administration of physiological saline (A) or a single dose of 3 mg/kg mitomycin C (B) and 120 hr after 3 mg/kg mitomycin C (C). Each section was stained with hematc xylin-eosin.
MITOMYCIN
C EFFECT ON INTESTINAL
419
ABSORPTION
TABLE I EFFECT OF MITOMYCIN
C ON INTESTINAL
Concentrationh (mW
Time’ (min)
Control
I 1 1 1 5 50 1 10 1 0.1 mg/ml
60 60 60 30 30 30 60 60 60 60
33.0 5 0.4 (5)’ 47.9 t 2.2 (6) 67.6 2 2.5 (5) 63.6 k 2.8 (4) 49.9 + 2.7 (5) ll.9+-0.7(6) 62.8 ri- 3.6 (6) 20.5 I 1.8 (7) 1.7 + 0.4 (6) I.1 +-0.4(6)
ABSORPTION
OF DRUGS”
Percentage of drug absorbed d DrUgS Cephalexin Sulfanilamide Salicylic acid L-Tryptophan D-Tryptophan 6-CF FITC-D
MMC-treated 22.3 f I .7 (6) 36.6 i 2.5 (6) 60.8 f I.1 (7) 49.4 f 5.1 (4) 33.0 f 2.7 (5) 7.3? 1.1 (6) 49.3 -+ 2.8 (6) 13.6 f 2.0 (5) 0.9 f 0.3 (6) 1.7 f 0.6 (8)
P
-co.01 -co.02 <0.02 <0.05
’ Mitomycin C (3 mg/kg) or physiological saline was given intravenously 48 hr before the absorption experiments. Intestinal absorption of each drug was determined by means of in situ recirculation technique. ’ Initial concentration ofdrugs in the perfusate. ’ Intestinal absorption of each drug was determined in 30 or 60 min. d Values are Xr& SE. ’ Number of animals per group in parentheses. ’ Not significantly different from control.
reduced 48 hr after dosing with MMC; mean values at this time were about 85% those from the control rats. The amount of intraluminal protein released during recirculation of the pH 6.5 buffer solution increased nearly twofold in MMC-treated rats. Intraluminal mu-
DOSAGE EFFECTOF INTRAVENOUS MITOMYCIN CON INTESTINAL ABSORPTION OF SULFANILAMIDE IN RATS”
Control I.2 2.4 3.0
Percentage of drug absorbed’ 47.9 39.5 36.1 36.6
?I 2.2c(6)d ?I 2.2 (5) i 2.7 (4) i 2.5 (6)
TABLE 3 TIME COURSE OF CHANGE IN INTESTINAL ABSORPTION OF SULFANILAMIDE AFTER ADMINISTRATION OF MITOMYCIN C”
TABLE 2
Dose of MMC (w/kg)
cus release, measured by employing the sugar content originating from mucin as an index, significantly increased in MMC-treated rats. Binding of salicylic acid, sulfanilamide and
P <0.05
0 Absorption of sulfanilamide was determined using the in situ recirculation technique at 48 hr after iv injection of 1.2,2.4. or 3 mg/kg mitomycin C. h Intestinal absorption of sulfanilamide is expressed as the percentage of drug absorbed within 60 min. ’ Values are .U+ SE. d Numbers ofanimals per group in parentheses.
Time after MMC (hr) Control 24 48 72 96
Percentage of drug absorbed b 47.9 49.3 39.5 41.8 46.4
+ f f f f
2.2’16)” 2.3 (4) 2.2 (5) 2.8 (4) 1.5 (5)
P NS’ to.05 NS NS
a Absorption of sulfanilamide was determined using the in situ recirculation technique at 24, 48, 72, and 96 hr after iv administration of mitomycin C ( 1.2 mg/kg). ’ Intestinal absorption of sulfanilamide is expressed as percentage of drug absorbed within 60 min. ’ Values are .Ir? SE. d Numbers of animals per group in parentheses. ‘Not significantly different from control.
420
MIZUNO
ET
TABLE
AL.
4
Control Intestinal wet weight(g) Intestinal length (cm) pH of the perfusate’ Intestinal blood flow (ml/mitt/100 g tissue) Mucus and protein content (fig/ml) Mucus Protein
MMC-treated
5.75 i- O.l4h(43)’ 101.6 * 3.0 (7) 6.4 + 0.02 (9) 102+7.7
P
3.76&0.18(37) 97.9 -t- 2.5 (4) 6.4 f 0.02 (6)
(II)
171 k 13.5(10)
<0.05
of perfusate 20.2 2 1.99 168.9 f 14.8
(8) (5)
31.7*4.48(j) 314.2 ir 47.9 (6)
r? These values were measured 48 hr after intravenous administration ’ Values are I+ SE. ’ Numbers ofanimals per group in parentheses. ‘Not significantly different from control. ” pH values were measured at the end ofthe absorption experiment:
cephalexin with macromolecular substances in the perfusate was unaffected by MMC treatment (Table 5). The mean value of the binding of these drugs was less than 6% in all cases. To correlate the reduction in absorptive surface area of the intestine with the decrease TABLE
of MMC
initial
(3 mg/kg)
pH was 6.5.
in drug absorption, the percentage absorption of sulfanilamide was plotted against intestinal wet weight. Figure 2 shows that intestinal absorption of sulfanilamide decreased with a reduction in intestinal wet weight (correlation coefficient 0.8846, p < 0.05) regardless of the route of exposure to MMC. 5
BINDINGOFDRUGSWITH MACROMOLECULARSUBSTANCESIN-WEPERFUSATEOEXAINED FROMCONTROLANDMITOMYCINC-TREATEDRATS' Percentage Drug Salicylic
acid
Sulfanilamide Cephalexin
Treatment
LJltrafiltration’
Control MMC’ Control MMC Control MMC
0.50 i 0.25 3.27 f 0.34 4.12?6.37(4) 5.77 * 4.37 6.67 t I .45 0.23 + 0.23
of bound
drug (%)” Equilibrium
(4)” (4)” (4) (4) (4)’
dialysis”
1.46 zlz 0.37 (5) 2.17-+0.55(5) 1.21 -+0.31(5) 1.16-tO.45(5) 0.54 -t 0.17 (5) 3.12 & 0.77 (5)X
’ Binding of drugs with macromolecular substances in intestinal solution was estimated ilz pi/n, by two methods. Drugs tested for binding were salicylic acid, sulfanilamide. and cephalexin. * Values are .iY? SE. ’ Ultrafiltration method: Ultrahltration was performed by using the “Micropartition System” (MPS- I. Amicon). d Equilibrium dialysis method: Visking cellulose tubing containing the intestinal perfusate was immersed in a drug solution and equilibrated for 73 hr at 4°C. ’ Numbers of experiments per group in parentheses. ’ MMC (3 mg/kg) was injected intravenously 48 hr before the experiments, x Significantly different from control (p < 0.02).
MITOMYCIN
C EFFECT ON INTESTINAL
ABSORPTION
421
and are absorbed via different transport systems, significantly decreased 48 hr after MMC pretreatment, although absorption of poorly absorbable (Mizuno et al., 1985) 6CF and high-molecular-weight FITC-D remained unchanged. Siber et al. (1980) reported that the gastrointestinal absorption in vivo of f’4C]polyvinylpyrrolidone (average molecular weight 11,000) and poorly absorbable tobramycin increased in patients receiving 5-fluorouracil therapy. Considering the postulation by Hughes ( 197 1) that fever and I infection during cancer therapy might be 6.0 L.0 5.0 3.0 caused by viable bacteria or their products Intestinal wet weight (g) leaking across damaged bowel epithelium, FIG. 2. Relationship between intestinal wet weight and the barrier permeability of intestinal epitheintestinal absorption of sulfanilamide under several exlium to macromolecules should be carefully perimental conditions. The percentages of sulfanilamide characterized. absorbed in 60 min are plotted against the wet weight of each intestine. (a) Control; (b) animals 48 hr after iv Kakemi el al. ( 1970) reported that the abadministration of I .2 mg/kg MMC: (c) 48 hr. iv. 2.4 mg/ sorption rate of L-tryptophan determined by kg MMC: (d) 48 hr. iv, 3 mg/kg MMC; (e) 48 hr. po, the in situ perfusion method could be de10 mg/kg MMC. Vertical and horizontal bars represent scribed by the sum of a saturable term dis*SE. playing Michaelis-Menten kinetics with a Km value of 8.7 mM plus a second nonsaturable term. As the initial concentration of L-tryptoDISCUSSION phan in the perfusate rises, the proportion of L-tryptophan absorbed via the active transThe morphological changes in the intestiport system is thought to decrease. Therefore. nal mucosa after MMC pretreatment are the passive absorption component of L-trypshown in Fig. 1. At 48 hr, MMC caused se- tophan was more strongly depressed than the vere derangements in the mucosa which were active one by MMC pretreatment, because characterized by villous atrophy, mitotic ar- the ratio of percentage absorbed in treated to rest in the crypts, etc. However, villous epi- that in control animals decreased with an inthelium started to recover within 120 hr. In crease in the initial concentration of L-tryptorodents, the median transit time of columnar phan in the perfusate. Three mechanisms can be considered in alcells from the base of the crypts to the top of the villi is reported to be 2 to 3 days (Lipkin, tered absorption of drugs following treatment 198 1). Judging from this cell renewal cycle with antitumor drugs: (I) intraluminal interand the morphological changes of the intestiactions, (2) mucosal permeability, and (3) cirnal mucosa presented in Fig. 1, MMC culatory changes (Robinson et al., 1966; Koseemed to induce toxic effects on the intes- muro et uf., 1974; Venho, 1976; Jolly and tinal mucosa as a result of mitotic arrest in Fletcher, 1977; Cape1 et al., 1979: Siber et al., the dividing cells. Not only morphological 1980; Cobden et al., 1981; Senius et al., changes, but also changes in the absorption of 1981). sulfanilamide were shown to depend on the The pH of the intestinal perfusate and the timing of MMC pretreatment. lengths of the intestines used for the abAbsorption of the various drugs, which sorption experiment were not significantly have different physicochemical properties different between the control and MMC-
422
MIZUNO
treated animals. Intestinal blood flow in MMC-treated rats was reduced to about 85% in the controls. Influence of blood flow on drug absorption could be significant if the absorbability of the solutes is high (Winne and Remischovsky, 1970; Winne, 1979). The extent of decreased absorption of each drug was variable (cephalexin, -32%; sulfanilamide, -23%: salicylic acid - 10%; L-tryptophan ( 1 mM), -22%: D-tryptophan (1 mM), -2 I%), and there was no relationship between the decreased absorption of each drug and its inherent absorbability. Therefore, the reduction in intestinal blood flow by itself could not account for the decreased drug absorption. The increase in mucus release may interfere with the diffusion of solutes through the gut mucosa. However, Yamamoto et al. ( 1984a) indicated that even a 2-fold increase in mucus released during systemic anaphylaxis had no effect on the absorption of drugs. Thus, it is likely that the increase in mucus released observed in the present study (1.6fold increase) is not sufficient to modulate drug absorption, at least not in the present experimental system. An increase in intraluminal protein content might decrease drug absorption by increasing the fraction bound to macromolecules. But the percentage binding of salicylic acid, sulfanilamide, and cephalexin with intraluminal macromolecular substances was not more than about 6% in all cases. These results suggest that the majority of drugs in the luminal solution existed as free forms and, therefore, intraluminal protein would not be responsible for the decreased absorption of drugs. Various techniques have been used to determine the absorption surface area: the counting of epithelial cells (Roche et al., 1970; Senius et al., 1981) histometric techniques (Altmann, 1974) and measurement of intestinal wet weights (Komuro et al., 1974). Komuro et al. ( 1974) investigated the relation between the diminution of intestinal tissues brought about by treatment with an antitumor drug, N,N’.N”-triethylene triphos-
ET AL.
phoramide (“thio-TEPA”), and intestinal absorption of drugs. They reported that a decrease in the drug (thiopental, its absorption was considered to be controlled by surface area change) correlated well with a thioTEPA-induced decrease in intestinal weight. On the other hand, they also found a relationship between intestinal weight and villus height, which was measured by a histometric technique under a microscope as an index of effective absorption surface area. Therefore. we determined intestinal wet weights to assess the change in absorption surface area. From these observations, it is concluded that the decrease in absorptive surface area is primarily responsible for the decreased absorption ofdrugs in in situ systems in rats pretreated with MMC. Other alterations of intraluminal, membranous, and circulatory factors might take place, but these factors might be masked in the present system by the decreased absorptive surface area. REFERENCES AL-TMANN. G. G. (1974). Changes in the mucosa of the small intestine following methotrexate administration or abdominal X-irradiation. ilmer. J. Anat. 140, 263280.
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C EFFECT ON INTESTINAL
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R. J., AND LEVIN,
M. J. (1980).
Increased gastrointestinal absorption of large molecules in patients after 5-fluorouracil therapy for metastatic colon carcinoma. Cancer Res. 40,3430-3436. SPIES,J. R., AND CHAMBERS, D. C. (1948). Chemical determination of tryptophan: Study of color-forming reactions of tryptophan, pdimethylaminobenzaldehyde, and sodium nitrite in sulfuric acid solution. Anal. Chem. 20,30-39. VENHO, V. M. K. (1976). Effect of methotrexate on drug absorption from the rat small intestine in situ and in vitro. Acta Pharmacol. Toxicol. 38,450-464. WAKAKI, S.. MARUMO, H.. TOMIOICA, K., SHIMIZU. G., KATO, E.. KAMADA, H., KUDO. S., AND FUJIMOTO. Y. (1958). Isolation of new fractions of antitumor mitomycins. Antibiot. Chemother. 8,228-240. WINNE, D. (1979). Influence of blood flow on intestinal absorption ofdrugs and nutrients. Pharmacol. Ther. 6, 333-393.
WINNE, D.. AND REMISCHOVSKY (1970). Intestinal blood flow and absorption of non-dissociable substances. J. Pharm. Pharmacol. 22,640-64 1. YAMAMOTO, A., NAKAMURA, J., TAKADA, S.. TAKEDA, M..
HASHIDA.
M.,
KIMURA,
T.,
AND
SEZAKI,
H.
(1984a). Drug absorption from the gastrointestinal tract and immunity: The mechanism of the decreased absorption of salicylic acid during systemic anaphylaxis, I. J. Pharmacobio-Dyn. 7,728-736. YAMAMOTO. A., NAKAMURA, J., TAKADA. S., TAKEDA, M.. HASHIDA. M., KIMURA. T., AND SEZAKI, H.
(1984b). Drug absorption from the gastrointestinal tract and immunity: The mechanism of the decreased absorption of salicylic acid during systemic anaphylaxis, II. J. Pharmacobio-Dvn. 7.737-746.
AND
APPLIED
PHARMACOLOGY
Characterization
97,415-423
(1989)
of Mitomycin C-Induced
Gastrointestinal
Damage
I. In Situ Recirculation Experiment MASAYUKI
MIZUNO, MITSURU
SHIGEKI KAWABATA, TAKESHI HAMAURA, HASHIDA, AND HITOSHI SEZAKI’
Department Vj’Basic Pharmaceutics. Faculty ofPharmuceutical Sciences, Kyoto University. kjnto 606, Japun
Received February 5. 1%‘; accepted October 16. 1988 Characterization of Mitomycin C-Induced Gastrointestinal Damage. I. In Situ Recirculation Experiment. MIZUNO, M., KAWABATA, S., HAMAURA, T.. HASHIDA. M.. AND SEZAKI, H. ( 1989). Tosicol. Appl. Pharmucol. 97, 4 15-423. The effects of mitomycin C (MMC), an antitumor agent, on the intestinal absorption of various drugs in rats were investigated. Based on microscopic observations, preadministration of a single intravenous dose of MMC (3 mg/kg) caused serious degeneration of epithelial cells. villous atrophy, and mitotic arrest in crypts at 48 hr after pretreatment. At this time point, absorption of sulfanilamide, salicylic acid, cephalexin, and L-tryptophan was shown to be significantly decreased by means of an in situ recirculation technique. The histological changes and the decrease in absorption of sulfanilamide. a model for passively absorbed drugs, were shown to depend on the timing of MMC pretreatment. Maximal effects were observed 48 hr after dosing. The MMC-induced reduction in the absorption of drugs was not a result of differences between treated and control animals with respect to pH of the drug solution, binding of drugs with intraluminal macromolecules, or intestinal mucosal blood flow. The absorption of sulfanilamide from the small intestine in the in situ system correlated well with the wet weight of the small intestine regardless of pretreatment dose or route. This suggests that the change in absorptive surface area of the intestinal mucosa may play a major role in the MMC-induced decrease in absorption capacity ofthe intestine. IB 1989 Academic Press. Inc
In the treatment of neoplastic diseases, the use of antitumor drugs is often interrupted due to indiscriminate action on both tumor and normal cells (Lazo and Hacker, 1985). The gastrointestinal tract is often a target of chemotherapeutic agents because it is a rapidly proliferating tissue. Severe gastrointestinal effects such as malabsorption, anorexia, atrophy of the tracts, and diarrhea are often limiting factors in therapy with these agents. Mitomycin C (MMC) is widely used in cancer chemotherapy, but its utility is limited by the side effects of severe bone marrow suppression and gastrointestinal damage (Lown,
1979). In several mammalian species high doses or prolonged continuous MMC treatment have caused injury to the small intestines, characterized by villous atrophy and glandular distortion (Philips et al., 1960). It is not known if these morphological alterations in the gastrointestinal tract following MMC administration are associated with alterations in absorptive function. A number of studies have shown that changes in the absorption of nutrients and drugs following treatment with antitumor drugs are explained in terms of a decrease in the active transport capacity (Robinson ef al., 1966; Jolly and Fletcher, 1977; Cape1 et al., 1979; Chen, 1982), diminished water flux
’ To whom correspondence should be addressed. 415
004 1-008X/89 $3.00 Copyright Ci 1989 hy Academic Press, Inc. All rights of reproduction in any form reserved.
416
MIZUNO
through the intestinal membrane (Venho, 1976) loss of mucosal surface area (Komuro et al., 1974: Senius et al.. 198 l), and impaired mucosal integrity (Siber et al., 1980; Cobden et al., 198 1). However, little is known about the mechanisms of disordered absorption. Therefore, the present investigation was aimed at the elucidation of disorders in intestinal absorptive functions induced by MMC pretreatment. Several factors that could influence the drug absorption process, such as intraluminal interactions, mucosal permeability, and circulatory changes, were investigated. An in situ preparation was used to explore the possible mechanisms in reduced absorptive capacity. METHODS Materials. MMC and cephalexin were kindly supplied by Kyowa Hakko Kogyo Company, Tokyo, Japan, and Takeda Chemical Industries, Osaka, Japan, respectively. Fluorescein isothiocyanate-conjugated dextran (FITCD. average molecular weight 65.600) and bovine serum albumin were purchased from Sigma Chemical Company, St. Louis, Missouri. 6-Carboxyfluorescein (h-CF) was obtained from Eastman Kodak Company, Rochester. New York. All other reagents used in these experiments were of reagent grade and were used without further purification. ,4nimal.s. Age (6-7 weeks old)- and weight ( 180-220 g)-matched groups of male Wistar rats (Shiruoka Agricultural Cooperative Association for Laboratory Animals, Shizuoka. Japan) were used. Animals had free access to water and food for at least 3 days before the experiment, and were maintained on 12-hr light-dark cycles. After this period. rats were weighed and given a bolus intravenous injection of MMC (in physiological saline) into the left femoral vein under light ether anesthesia. Unless otherwise stated, the MMC dose was 3 mg/kg. In some experiments. the animals were submitted to an overnight fast to remove gastric contents. They were then lightly anesthetized with ether and administered 10 mg/ kg MMC by gastric intubation [dissolved in isotonic bicarbonate buffer. pH 8.3, to protect MMC against gastric acidity(Wakaki el al., 1958)]. Control rats received comparable volumes of physiological saline. All injections were given between 1200 and 1300 hr. Both groups were freely provided with food and water after MMC pretreatment. Preparation of‘dn~g solution. The isotonic phosphate buffer solution (pH 6.5) was prepared from 0.123 M Na2HP0, and 0.163 M NaHLPOJ. Drugs were dissolved
ET
AL.
in this buffer solution at the stated concentrations for the absorption experiments. Absorption esprriments. The procedure for the in situ absorption study on rat small intestine was the same as that described by Kakemi et ul. (I 967). Briefly, each animal was anesthetized with sodium pentobarbital(20 mg/ kg, ip) and the small intestine was cannulated for in situ recirculation. The entire length of the small intestine. from the pylorus to the ileocecal junction. was used for the absorption studies. The bile duct was ligated in all experiments. Rectal temperature was maintained at 37°C. Forty milliliters ofdrug solution (pH 6.5) was recirculated in the small intestine at the rate of 5 ml/min. At the end of the recirculation period, the perfusate was recovered and the intestine was washed with phosphate buffer, pH 6.5. The absorbed amount of drug was calculated from the difference in the amount of drug in the initial and final solutions. Afeusuremmt yfintestinul mucosal hloodflo~~. Intestinal mucosal blood flow was measured by the hydrogen gas clearance method (PHG 201. Unique Medical Co.. Ltd.. Tokyo, Japan) (Yamamoto et ~1.. 1984b) in rats under anesthesia with sodium pentobarbital(20 mg/kg. ip). The theoretical considerations were based largely on Kety’s approach to blood-tissue exchange of inert gases (Kety and Schmidt. 1945). Intestinal blood flow is expressed as milliliters per minute per 100 g tissue weight. of’ protein
DctcrFniFlUtiWI
and
mucin
in thr
intertinu/
The operation and recirculation techniques were the same as those used in the absorption experiments. After recirculation of pH 6.5 buffer solution for I hr in control and MMC-treated rats, the perfusate was collected as completely as possible. After centrifugation ofthe perfusate at 3000 rpm for IO min. the supernatant was assayed for protein and mucin. Protein was determined according to the method of Lowry el al. ( 195 I ) using bovine serum albumin as standard. Mucin was determined spectrophotometrically at 620 nm by the anthrone method (Roe, 1955) using D-ghCOSe as standard. /ztminul
I?indiqy thrperlksatr.
.rolution.
of drug.\
with
mucromokrdar
.xlrhstarwes
irl
Drugs tested for binding were salicylic acid. sulfanilamide. and cephalexin. To estimate the binding ofdrugs with macromolecular substances in intestinal luminal solution in vitro. two methods were adopted (Yamamoto (I/ ul.. 1984a). In the equilibrium dialysis method. each drug was dissolved in pH 6.5 buffer solution at a concentration of I mM. Visking cellulose tubing containing the intestinal perfusate was immersed in a drug solution and equilibrated for 72 hr at 4°C. After equilibration, the concentration of drugs in the external medium was determined as described below. In the ultrafiltration method, ultrafiltration was performed by using the “Micropartition System” (MPS-I. Amicon. Lexington. MA). This filtration system is purported to efficiently retain macromolecular substances with molecular weights greater than 10,000. The filtrate obtained was assayed to determine the free drug concentration.
MITOMYCIN
C EFFECT
ON
Histological ohwrvatiorz. Specimens were fixed in 10% (v/v) formaldehyde, sectioned, and stained with hematoxylin and eosin. Histological examination was performed by light microscopy. .Itzaiytical methods. Salicylic acid was extracted into chloroform after adding HCI to the test samples. It was then extracted with 0.1 N NaOH, and determined spectrophotometrically at 295 nm. Sulfanilamide was diazotized and coupled with 2-diethylaminoethyl-I-naphthylamine. The colored derivative was extracted into isoamyl alcohol by the addition of NaCI. The complex present in the organic phase was determined spectrophotometrically at 555 nm. D- and L-Tryptophan were determined colorimetritally (600 nm) according to the method of Spies and Chambers (1948). A reversed-phase high-pressure liquid chromatography (HPLC) method (Kimura et al., 1983) was used to determine cephalexin. FITC-D and 6-CF were determined spectrofluorimetrically. Excitation and emission wavelengths were 490 and 520 nm, respectively, for both drugs. Statistical ona~~.~ls. Results were analyzed statistically with Student’s t test, Differences with p < 0.05 were considered significant,
INTESTINAL
ABSORPTION
417
mg/kg), absorption of both 6-CF, which is poorly absorbed, and the high-molecular weight compound FITC-D, was not altered. Absorption of sulfanilamide, salicylic acid, and cephalexin, and of the actively transported L-tryptophan and its optical isomer Dtryptophan significantly decreased irrespective of the degree of ionic dissociation at pH 6.5. In the case of L-tryptophan, the ratio of percentage absorbed in the treated to that in the control animals decreased as the initial concentration of L-tryptophan in the perfusate rose: 1 mM, 0.783 + 0.067; 5 mM, 0.662 -t 0.053; 50 mM, 0.622 + 0.095. To ascertain that the observed changes were derived from pretreatment with MMC, the dose and time dependency of the decrease in drug absorption was characterized using sulfanilamide as a model for passiveIy absorbed drugs (Tables 2 and 3). Table 2 shows that the applied doses (1.2, 2.4, and 3.0 mg/ kg) of MMC caused significant decreases in sulfanilamide absorption. However. within RESULTS the dose ranges examined, no correlation was found between the magnitude of the decrease The general responses of animals to the in sulfanilamide absorption and the dose of MMC treatment were compared with those MMC. In Table 3, the effect of MMC treatreported by Philips et al. (1960). MMCment times on sulfanilamide absorption is treated rats decreased their food intake and presented. At 48 hr after MMC pretreatment. body weight within 48 hr of drug administrathe maximal decrease in sulfanilamide abtion, as previously reported (Mizuno et al., sorption was observed (p < 0.05). At 72 hr the 1986a; Mizuno et ul., 1986b). Light microsabsorption had already returned to the concopy of intestinal tissue taken from MMCtrol level. This time course of the change in treated rats (at 48 hr) showed serious degener- sulfanilamide absorption paralleled the patation of the epithelial cells, villous atrophy, tern of morphological changes in the intestiand mitotic arrest in the crypts, which were nal mucosa. coupled with some disorders in submucosal Table 4 summarizes the effect of MMC tissue (Fig. 1B). At 120 hr the villous epithepretreatment on the various physiological lium started to recover (Fig. 1C) and the gen- and experimental parameters. Wet weights of eral architecture and height of the villi whole small intestines were significantly reseemed to be restored to those of normal tis- duced in the MMC-treated rats (to about 65% of the control). The mean pH of the luminal sue (Fig. IA). The intestinal absorption of drugs possess- solution, measured 1 hr after recirculation of ing various physicochemical properties was the buffer solution (pH 6.5), was virtually measured in control and MMC-treated rats identical in both groups. The lengths of the by means of the in situ recirculation tech- intestines used for perfusion were not signifinique. The results are summarized in Table cantly different among the experimental 1. Forty-eight hours after MMC treatment (3 groups. Intestinal blood flow was significantly
418
MIZUNO
ET AL.
FIG.1, Intestinal muc :osal tissue (X 100) 48 hr after the intravenous administration of physiological saline (A) or a single dose of 3 mg/kg mitomycin C (B) and 120 hr after 3 mg/kg mitomycin C (C). Each section was stained with hematc xylin-eosin.
MITOMYCIN
C EFFECT ON INTESTINAL
419
ABSORPTION
TABLE I EFFECT OF MITOMYCIN
C ON INTESTINAL
Concentrationh (mW
Time’ (min)
Control
I 1 1 1 5 50 1 10 1 0.1 mg/ml
60 60 60 30 30 30 60 60 60 60
33.0 5 0.4 (5)’ 47.9 t 2.2 (6) 67.6 2 2.5 (5) 63.6 k 2.8 (4) 49.9 + 2.7 (5) ll.9+-0.7(6) 62.8 ri- 3.6 (6) 20.5 I 1.8 (7) 1.7 + 0.4 (6) I.1 +-0.4(6)
ABSORPTION
OF DRUGS”
Percentage of drug absorbed d DrUgS Cephalexin Sulfanilamide Salicylic acid L-Tryptophan D-Tryptophan 6-CF FITC-D
MMC-treated 22.3 f I .7 (6) 36.6 i 2.5 (6) 60.8 f I.1 (7) 49.4 f 5.1 (4) 33.0 f 2.7 (5) 7.3? 1.1 (6) 49.3 -+ 2.8 (6) 13.6 f 2.0 (5) 0.9 f 0.3 (6) 1.7 f 0.6 (8)
P
-co.01 -co.02 <0.02 <0.05
’ Mitomycin C (3 mg/kg) or physiological saline was given intravenously 48 hr before the absorption experiments. Intestinal absorption of each drug was determined by means of in situ recirculation technique. ’ Initial concentration ofdrugs in the perfusate. ’ Intestinal absorption of each drug was determined in 30 or 60 min. d Values are Xr& SE. ’ Number of animals per group in parentheses. ’ Not significantly different from control.
reduced 48 hr after dosing with MMC; mean values at this time were about 85% those from the control rats. The amount of intraluminal protein released during recirculation of the pH 6.5 buffer solution increased nearly twofold in MMC-treated rats. Intraluminal mu-
DOSAGE EFFECTOF INTRAVENOUS MITOMYCIN CON INTESTINAL ABSORPTION OF SULFANILAMIDE IN RATS”
Control I.2 2.4 3.0
Percentage of drug absorbed’ 47.9 39.5 36.1 36.6
?I 2.2c(6)d ?I 2.2 (5) i 2.7 (4) i 2.5 (6)
TABLE 3 TIME COURSE OF CHANGE IN INTESTINAL ABSORPTION OF SULFANILAMIDE AFTER ADMINISTRATION OF MITOMYCIN C”
TABLE 2
Dose of MMC (w/kg)
cus release, measured by employing the sugar content originating from mucin as an index, significantly increased in MMC-treated rats. Binding of salicylic acid, sulfanilamide and
P <0.05
0 Absorption of sulfanilamide was determined using the in situ recirculation technique at 48 hr after iv injection of 1.2,2.4. or 3 mg/kg mitomycin C. h Intestinal absorption of sulfanilamide is expressed as the percentage of drug absorbed within 60 min. ’ Values are .U+ SE. d Numbers ofanimals per group in parentheses.
Time after MMC (hr) Control 24 48 72 96
Percentage of drug absorbed b 47.9 49.3 39.5 41.8 46.4
+ f f f f
2.2’16)” 2.3 (4) 2.2 (5) 2.8 (4) 1.5 (5)
P NS’ to.05 NS NS
a Absorption of sulfanilamide was determined using the in situ recirculation technique at 24, 48, 72, and 96 hr after iv administration of mitomycin C ( 1.2 mg/kg). ’ Intestinal absorption of sulfanilamide is expressed as percentage of drug absorbed within 60 min. ’ Values are .Ir? SE. d Numbers of animals per group in parentheses. ‘Not significantly different from control.
420
MIZUNO
ET
TABLE
AL.
4
Control Intestinal wet weight(g) Intestinal length (cm) pH of the perfusate’ Intestinal blood flow (ml/mitt/100 g tissue) Mucus and protein content (fig/ml) Mucus Protein
MMC-treated
5.75 i- O.l4h(43)’ 101.6 * 3.0 (7) 6.4 + 0.02 (9) 102+7.7
P
3.76&0.18(37) 97.9 -t- 2.5 (4) 6.4 f 0.02 (6)
(II)
171 k 13.5(10)
<0.05
of perfusate 20.2 2 1.99 168.9 f 14.8
(8) (5)
31.7*4.48(j) 314.2 ir 47.9 (6)
r? These values were measured 48 hr after intravenous administration ’ Values are I+ SE. ’ Numbers ofanimals per group in parentheses. ‘Not significantly different from control. ” pH values were measured at the end ofthe absorption experiment:
cephalexin with macromolecular substances in the perfusate was unaffected by MMC treatment (Table 5). The mean value of the binding of these drugs was less than 6% in all cases. To correlate the reduction in absorptive surface area of the intestine with the decrease TABLE
of MMC
initial
(3 mg/kg)
pH was 6.5.
in drug absorption, the percentage absorption of sulfanilamide was plotted against intestinal wet weight. Figure 2 shows that intestinal absorption of sulfanilamide decreased with a reduction in intestinal wet weight (correlation coefficient 0.8846, p < 0.05) regardless of the route of exposure to MMC. 5
BINDINGOFDRUGSWITH MACROMOLECULARSUBSTANCESIN-WEPERFUSATEOEXAINED FROMCONTROLANDMITOMYCINC-TREATEDRATS' Percentage Drug Salicylic
acid
Sulfanilamide Cephalexin
Treatment
LJltrafiltration’
Control MMC’ Control MMC Control MMC
0.50 i 0.25 3.27 f 0.34 4.12?6.37(4) 5.77 * 4.37 6.67 t I .45 0.23 + 0.23
of bound
drug (%)” Equilibrium
(4)” (4)” (4) (4) (4)’
dialysis”
1.46 zlz 0.37 (5) 2.17-+0.55(5) 1.21 -+0.31(5) 1.16-tO.45(5) 0.54 -t 0.17 (5) 3.12 & 0.77 (5)X
’ Binding of drugs with macromolecular substances in intestinal solution was estimated ilz pi/n, by two methods. Drugs tested for binding were salicylic acid, sulfanilamide. and cephalexin. * Values are .iY? SE. ’ Ultrafiltration method: Ultrahltration was performed by using the “Micropartition System” (MPS- I. Amicon). d Equilibrium dialysis method: Visking cellulose tubing containing the intestinal perfusate was immersed in a drug solution and equilibrated for 73 hr at 4°C. ’ Numbers of experiments per group in parentheses. ’ MMC (3 mg/kg) was injected intravenously 48 hr before the experiments, x Significantly different from control (p < 0.02).
MITOMYCIN
C EFFECT ON INTESTINAL
ABSORPTION
421
and are absorbed via different transport systems, significantly decreased 48 hr after MMC pretreatment, although absorption of poorly absorbable (Mizuno et al., 1985) 6CF and high-molecular-weight FITC-D remained unchanged. Siber et al. (1980) reported that the gastrointestinal absorption in vivo of f’4C]polyvinylpyrrolidone (average molecular weight 11,000) and poorly absorbable tobramycin increased in patients receiving 5-fluorouracil therapy. Considering the postulation by Hughes ( 197 1) that fever and I infection during cancer therapy might be 6.0 L.0 5.0 3.0 caused by viable bacteria or their products Intestinal wet weight (g) leaking across damaged bowel epithelium, FIG. 2. Relationship between intestinal wet weight and the barrier permeability of intestinal epitheintestinal absorption of sulfanilamide under several exlium to macromolecules should be carefully perimental conditions. The percentages of sulfanilamide characterized. absorbed in 60 min are plotted against the wet weight of each intestine. (a) Control; (b) animals 48 hr after iv Kakemi el al. ( 1970) reported that the abadministration of I .2 mg/kg MMC: (c) 48 hr. iv. 2.4 mg/ sorption rate of L-tryptophan determined by kg MMC: (d) 48 hr. iv, 3 mg/kg MMC; (e) 48 hr. po, the in situ perfusion method could be de10 mg/kg MMC. Vertical and horizontal bars represent scribed by the sum of a saturable term dis*SE. playing Michaelis-Menten kinetics with a Km value of 8.7 mM plus a second nonsaturable term. As the initial concentration of L-tryptoDISCUSSION phan in the perfusate rises, the proportion of L-tryptophan absorbed via the active transThe morphological changes in the intestiport system is thought to decrease. Therefore. nal mucosa after MMC pretreatment are the passive absorption component of L-trypshown in Fig. 1. At 48 hr, MMC caused se- tophan was more strongly depressed than the vere derangements in the mucosa which were active one by MMC pretreatment, because characterized by villous atrophy, mitotic ar- the ratio of percentage absorbed in treated to rest in the crypts, etc. However, villous epi- that in control animals decreased with an inthelium started to recover within 120 hr. In crease in the initial concentration of L-tryptorodents, the median transit time of columnar phan in the perfusate. Three mechanisms can be considered in alcells from the base of the crypts to the top of the villi is reported to be 2 to 3 days (Lipkin, tered absorption of drugs following treatment 198 1). Judging from this cell renewal cycle with antitumor drugs: (I) intraluminal interand the morphological changes of the intestiactions, (2) mucosal permeability, and (3) cirnal mucosa presented in Fig. 1, MMC culatory changes (Robinson et al., 1966; Koseemed to induce toxic effects on the intes- muro et uf., 1974; Venho, 1976; Jolly and tinal mucosa as a result of mitotic arrest in Fletcher, 1977; Cape1 et al., 1979: Siber et al., the dividing cells. Not only morphological 1980; Cobden et al., 1981; Senius et al., changes, but also changes in the absorption of 1981). sulfanilamide were shown to depend on the The pH of the intestinal perfusate and the timing of MMC pretreatment. lengths of the intestines used for the abAbsorption of the various drugs, which sorption experiment were not significantly have different physicochemical properties different between the control and MMC-
422
MIZUNO
treated animals. Intestinal blood flow in MMC-treated rats was reduced to about 85% in the controls. Influence of blood flow on drug absorption could be significant if the absorbability of the solutes is high (Winne and Remischovsky, 1970; Winne, 1979). The extent of decreased absorption of each drug was variable (cephalexin, -32%; sulfanilamide, -23%: salicylic acid - 10%; L-tryptophan ( 1 mM), -22%: D-tryptophan (1 mM), -2 I%), and there was no relationship between the decreased absorption of each drug and its inherent absorbability. Therefore, the reduction in intestinal blood flow by itself could not account for the decreased drug absorption. The increase in mucus release may interfere with the diffusion of solutes through the gut mucosa. However, Yamamoto et al. ( 1984a) indicated that even a 2-fold increase in mucus released during systemic anaphylaxis had no effect on the absorption of drugs. Thus, it is likely that the increase in mucus released observed in the present study (1.6fold increase) is not sufficient to modulate drug absorption, at least not in the present experimental system. An increase in intraluminal protein content might decrease drug absorption by increasing the fraction bound to macromolecules. But the percentage binding of salicylic acid, sulfanilamide, and cephalexin with intraluminal macromolecular substances was not more than about 6% in all cases. These results suggest that the majority of drugs in the luminal solution existed as free forms and, therefore, intraluminal protein would not be responsible for the decreased absorption of drugs. Various techniques have been used to determine the absorption surface area: the counting of epithelial cells (Roche et al., 1970; Senius et al., 1981) histometric techniques (Altmann, 1974) and measurement of intestinal wet weights (Komuro et al., 1974). Komuro et al. ( 1974) investigated the relation between the diminution of intestinal tissues brought about by treatment with an antitumor drug, N,N’.N”-triethylene triphos-
ET AL.
phoramide (“thio-TEPA”), and intestinal absorption of drugs. They reported that a decrease in the drug (thiopental, its absorption was considered to be controlled by surface area change) correlated well with a thioTEPA-induced decrease in intestinal weight. On the other hand, they also found a relationship between intestinal weight and villus height, which was measured by a histometric technique under a microscope as an index of effective absorption surface area. Therefore. we determined intestinal wet weights to assess the change in absorption surface area. From these observations, it is concluded that the decrease in absorptive surface area is primarily responsible for the decreased absorption ofdrugs in in situ systems in rats pretreated with MMC. Other alterations of intraluminal, membranous, and circulatory factors might take place, but these factors might be masked in the present system by the decreased absorptive surface area. REFERENCES AL-TMANN. G. G. (1974). Changes in the mucosa of the small intestine following methotrexate administration or abdominal X-irradiation. ilmer. J. Anat. 140, 263280.
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C EFFECT ON INTESTINAL
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MIZUNO, M.. YOSHIDA, M., HASHIDA, M.. KIMURA. T.. AND SEZAKI, H. (1985). Effect of mucosal lipid components on the membrane permeation of water-soluble drugs. Chem. Pharm. Bull. (Tokyo) 33.2932-2940. PHILIPS,
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