WIHITWINWWHHIHIIMWW1HIHWIMHHII 115 237 THS LIBRARY “PR/“Y MIChigan State University University Michigan State This is to certify that the thesis entitled THE ABSORPTION AND GLUCURONIDATION 0F DIETHYLSTILBESTROL BY THE RAT SMALL INTESTINE presented by JEROME LASKER has been accepted towards fulfillment of the requirements for J‘LS- Jegree in _EHARMACQLQGY ///1,/ 6/1/ //////// ajor professor Date W an THE ABSORPTION AND GLUCURONIDATION OF DIETHYLSTILBESTROL BY THE RAT SMALL INTESTINE BY Jerome Lasker A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pharmacology 1977 ABSTRACT THE ABSORPTION AND GLUCURONIDATION OF DIETHYLSTILBESTROL BY THE RAT SMALL INTESTINE BY Jerome Lasker Absorption and glucuronidation of 14C-DES were studied in orally- and aborally-located segments of rat small intestine. Following place- ment of DES (50, 500, 1000, or 2500 nmoles) into the segment lumen, amounts of DES and diethylstilbestrol monoglucuronide (DESG) entering the portal circulation were quantitated in the venous blood draining the segment. DESG accounted for nearly 50% of the radioactivity appearing in the blood at each dose in both intestinal regions. Regional differences in total DESG formation were not observed through- out the dose range. Regional absorptive differences were observed only at 2500 nmoles applied DES (aboral > oral). Alterations in parameters of DES absorption and metabolism were produced by pretreat- ment with PB, 3-MC, PCN, or PBB and by concurrent application of SKF-SZSA; effects varied with the compound employed. These results indicate that intestinal glucuronidation of certain compounds during absorption may be significant throughout the small intestine over a wide range of concentrations. ACKNOWLEDGEMENTS I welcome this opportunity to express my sincere appreciation to my advisor, Dr. Douglas E. Rickert, whose unending inspiration and guidance have been invaluable during the course of these studies. I also wish to acknowledge the contributions of committee members Drs. Theodore M. Brody, Jay I. Goodman, and Steven D. Aust. Special thanks are offered to Mrs. Betty Lou Schoepke for her constructive criticism, technical assistance, and friendship. The studies described in this thesis were supported in part by grant numbers GM 22535 and lSOlRR 05772 from the National Institute of General Medical Sciences. 11 TABLE OF CONTENTS ACKNOWLEDGEMENTS TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES INTRODUCTI N Objectives Overview of Intestinal Drug Absorption and Metabolism-- Glucuronide Formation by the Small Intestine Consequences of Glucuronide Conjugation of Xenobiotics- Factors Affecting Glucuronide Formation by the Small Intestine Regional Distribution of Intestinal Glucuronide Forma- tion Enzyme Induction in the Small Intestine Inhibition of Intestinal Drug Metabolism Methods of Studying Intestinal Drug Absorption and Metabolism Purpose METHODS Animals Chemicals Pretreatments Preparation of Intestinal Segments Determination of DES and DESG in Venous Blood, Luminal Contents, and Intestinal Tissue Page ii iii vii \DU'INH 10 14 15 20 22 24 26 26 26 28 28 31 Determination of MODES in Venous Blood, Luminal Contents and Intestinal Tissue Statistics iii 32 32 TABLE OF CONTENTS (continued) Page RESULTS 33 Absorption and Glucuronidation of DES 33 Effects of Pretreatments on the Absorption and Glucu- ronidation of DES 41 Effects of Fasting and SKF-525A on the Absorption and Glucuronidation of DES 47 Intestinal Absorption of MODES 52 DISCUSSION 57 Regional Absorption and Metabolism by the Small Intestine 57 Absorption of MODES by the Small Intestine 61 Effect of Altered Nutritional State on the Intestinal Absorption and Metabolism 62 Alterations in Intestinal Absorption and Metabolism by Previous Exposure to Foreign Compounds 63 SUMMARY AND CONCLUSIONS 69 BIBLIOGRAPHY 73 iv Table LIST OF TABLES Segment Location, Length, and Blood Flow Total DES-Related Material, DES, and DESG in Venous Blood, Intestinal Wall, and Lumen Contents Sixty Minutes After Placing DES in the Intestinal Lumen Comparison of Parameters Associated with DES Ab- sorption and Glucuronidation in Oral and Aboral Regions of the Rat Small Intestine Effects of PB and 3-MC Pretreatment on Total DES- Related Material, DES, and DESG in Venous Blood, Intestinal Wall, and Lumen Contents Sixty Minutes After Placing 1 mM DES in the Intestinal Lumen---- Effects of PCN Pretreatment on Total DES-Related Materials, DES, and DESG in Venous Blood, Intesti— nal Wall, and Lumen Contents Sixty Minutes After Placing 1 mM DES in the Intestinal Lumen ---------- Effects of PBB Pretreatment on Total DES-Related Material, DES, and DESG in Venous Blood, Intesti- nal Wall, and Lumen Contents Sixty Minutes After Placing 1 mM DES in the Intestinal Lumen ---------- Effects of DES Pretreatment on Total l4C-DES— Related Materials, l4C-DES, and 14DESG in Venous Blood, Intestinal Wall, and Lumen Contents Sixty Minutes After Placing 1 mM DES in the Intestinal Lumen Effects of 48 hr Food Deprivation on Total DES- Related Material, DES, and DESG in Venous Blood, Intestinal Wall, and Lumen Contents 60 Minutes After Placing 1 mM DES in the Intestinal Lumen---- Effects of SKF-525A on the Total DES-Related Ma- terial, DES, and DES in Venous Blood, Intestinal Wall, and Lumen Contents 60 Minutes After Placing 5 mM DES in the Intestinal Lumen Page 34 38 40 44 46 48 49 50 51 LIST OF TABLES (continued) Table Page 10 Amounts of MODES, Total DES-Related Material and DES in the Venous Blood, Intestinal Wall, and Lumen Contents 60 Minutes After Placing 5 mM MODES or 5 mM DES in the Intestinal Lumen --------- 55 vi Figure LIST OF FIGURES Page Conjugation of DES with Glucuronic Acid to Form DESG 8 In_Vivo Intestinal Preparation with Intact Arterial Supply and Complete Collection of Venous Blood---- 30 Cumulative Amounts of Total DES-Related Material, DES and DESG Appearing in Venous Blood of Orally- and Aborally-Located Segments of Rat Small In- testine 36 Rate of DES Appearance as a Function of Segment Blood Flow in Orally- and Aborally-Located Seg- ments at 2 and 5 mM DES 43 Structure of MODES (3,4-di[p-methoxyphenyl]hex- 3-ene) 54 vii INTRODUCTION Objectives Many drugs and other foreign compounds gain entry into the body via ingestion. The onset, duration and intensity of action of these compounds are at least partially determined by the rate at which they are absorbed and enzymatically transformed in the body. Traditionally, the liver has been regarded as the main, if not the only, significant site of drug biotransformation. Evidence has been accumulated over the past two decades which suggests that the small intestine, as well as other extrahepatic tissues, may play an important role in this process. Since the small intestine is a major site for the absorption of xenobiotics, metabolism occurring during the absorption process may be an important determinant of xenobiotic bioavailability. Further- more, the ability of extrahepatic tissues to metabolize drugs and other foreign compounds may contribute significantly to the total body capacity for biotransformation and could be of major importance in determining the efficacy and/or toxicity of such compounds. At the present time the extent to which intestinal metabolism, and extra- hepatic metabolism in general, can affect the disposition of a foreign compound is poorly understood. The objectives of this research were three-fold, all of which relate to examination of the absorption and metabolism of a foreign 2 compound by the rat small intestine under conditions closely approxi- mating those found in 2122: The first objective was to determine whether metabolism of a foreign compound, diethylstilbestrol (DES), was significant during the absorption process. The second objective was to test for alterations in the intestinal absorption and meta- bolism of DES produced by exposure to various agents and altered nutritional states. The agents that were examined were those that offered some potential for interaction in an environmental or clinical situation or those which could yield insights into control of intesti— nal drug-metabolizing function. The final objective was to determine if changes in absorption could be produced by structural modification of DES so that the sites for its metabolism were blocked. Overview of Intestinal Drungbsorption and Metabolism The gastrointestinal tract receives a number of diverse chemical compounds ingested as nutrients, environmental contaminants, and the therapeutic agents. Drugs are commonly given by mouth since this is the most convenient route and it is the only one of practical impor- tance for self-administration. Absorption, in general, takes place along the entire length of the gastrointestinal tract, but the chemi- cal properties of each compound determine whether it will be absorbed in the strongly acidic stomach or nearly neutral intestine. The large surface area of the intestinal villi, the presence of bile, and the rich blood supply all favor intestinal absorption, however (1). The cell that performs the function of absorption is the columnar epithelial cell of the intestinal villi. These cells form the epi- thelial lining of the small intestinal lumen and constitute the main 3 barrier to absorption (2). The principles governing the absorption of drugs and other foreign compounds from the intestinal lumen are the same as for passage of these compounds across biological membranes elsewhere. Most drugs have been shown to be absorbed by simple passive diffusion through lipoid regions or aqueous channels of the epithelial cell membrane or by filtration through membrane pores (3). Low degree of ionization, high lipid/water partition coefficient of the non-ionized form, and small atomic or molecular radius of water- soluble substances all favor rapid absorption (3). There is evidence that a drug can be absorbed by a specialized carrier-mediated process (facilitated diffusion or active transport) if its chemical structure is similar enough to that of a substrate naturally transported (4,5). In addition, vesicular transport, such as pinocytosis, has been impli- cated in the absorption of some drugs from the small intestine (6). The first communication on the possible role of tissues other than the liver on the metabolism of foreign substances was presented by Herter and Wakeman in 1899, describing the conjugation of phenol with sulfuric acid by rabbit small intestinal homogenates (7). Despite their results and those of several other early investigators concerning intestinal sulfate conjugation, interest in the role of the gastrointestinal tract in drug metabolism was not greatly stimulated until some 50 years later. In 1954, Hartiala (9) and Shirai and Ohkubo (10) were able to demonstrate gastroinestinal glucuronide conjugation. Since then, different types of gastrointestinal meta- bolic reactions have been explored. It appears that most of the enzymatic pathways for drug metabolism found in hepatic tissue are found in intestinal tissue as well (8). 4 The columnar epithelial cells of the small intestinal mucosa are equipped with a wide variety of enzyme systems capable of metabolizing the various xenobiotics presented to them. In all Species studied, the activity of these enzyme systems per mg microsomal protein or per gram intestinal mucosa is generally well below that of the liver (11,12). These xenobiotic-metabolizing enzymes, which biochemically may be viewed as catalyzing both non-synthetic and synthetic reactions, are capable of oxidation, reduction, hydrolysis, and conjugation. Several examples of these metabolic functions include: the oxidation of ethanol by intestinal ADH (13); the hydroxylation of benzo(a)pyrene by the intestinal monooxygenase system (which is quite similar to that found in the liver and requires cytochrome P450 and NADPH cytochrome c reductase) (14); reduction of Z-acetylaminofluorene (15); hydrolysis of cardiac glycosides (l6); sulfate conjugation of p-nitrophenol (17); and glucuronidation of salicylamide, 5-hydroxytryptamine, and various steroids by intestinal glucuronyltransferase (18,19,20). In most cases, these biotransformation reactions appear to change lipid- soluble nonpolar compounds into less lipid soluble polar metabolites. This would then serve to decrease their transfer into the body in an active form and to facilitate their elimination from the body. Although these biochemical changes are often referred to as detoxifi- cations, they do not always result in the reduction or abolition of toxicity. In several cases, metabolism by the small intestine enhances toxicity (8). 5 Glucuronide Formation by the Small Intestine One of the potentially important pathways of foreign compound biotransformation in the small intestine is conjugation with glucuro- nic acid (glucuronidation). Quantitatively, glucuronidation appears to be the most common synthetic process by which drugs are chemically altered by the mammalian organism (21). The capacity for glucuronide formation is greatest in the liver, with small intestinal mucosa, kidney, spleen, brain, skin, lung, muscle and retroperitoneal fat, and placenta displaying somewhat lower capacities (22,23). Glucuronidation was first demonstrated in rat small intestinal mucosal homogenates by Hartiala in 1954 using o-aminophenol as sub- strate (9). Since then, numerous investigators have shown that a variety of substrates in various animal species undergo glucuronida- tion by the small intestine both in_gi££2_and in_vivg, Among the sub- strates for glucuronide formation are; bilirubin, 5-hydroxytrypta- mine, l-naphthol, p-nitrophenol, salicylate, salicylamide, thyroxine, triiodothyronine, retinoic acid, and morphine (8). Moreover, natural and synthetic steroids (estradiol, estriol, testosterone derivatives, dienestrol, and diethylstilbestrol) have been found to form glucuro- nides in the small intestinal mucosa (20,24,25). It appears that the distribution of the enzymes and nucleotides relevant to glucuronide synthesis in the columnar epithelial cell of the intestinal mucosa is quite similar to that in the hepatocyte. The enzyme directly responsible for glucuronide formation is uridine diphosphate (UDP) glucuronyltransferase (UDP glucuronate-B-glucu- ronosyltransferase [acceptor unspecific] EC 2.4.1.17, UDPGT). This membrane-bound enzyme, which has been localized in the microsomal 6 fraction of small intestinal mucosal homogenates, catalyzes the trans— fer of glucuronic acid from UDP glucuronic acid (UDPGA) to the accep- tor aglycone (26,27). UDPGA and UDP glucose dehydrogenase, the enzyme catalyzing synthesis of the oxidized sugar nucleotide, are found in the cytosol of the columnar epithelial cell (28,29). UDPGA is the only cofactor requirement of UDPGT (21). The conjugation of DES with glucuronide acid is illustrated in Figure 1. As shown, UDPGA has the a-configuration at the glucuronic acid—phosphate link. Therefore, this compound is not affected by B- glucuronidase, a group-specific enzyme present in both intestinal microorganisms and the intestinal mucosa. B-Glucuronidase catalyzes the hydrolysis of B-glucosiduronic acids to free glucuronic acid and aglycone. The glucuronides formed from the glucuronic acid moiety of UDPGA invariably have the B-configuration and are susceptible to B- glucuronidase-catalyzed hydrolysis. Since the glucuronides of a number of foreign compounds are excreted into the bile, intestinal hydrolysis in these cases may result in the recirculation of the compound via the bile, intestine, and liver. This enterohepatic circulation has important consequences, for it may greatly prolong biological half-life (30). Substrates for UDPGT are those that possess phenolic, alcoholic, carboxylic, amino, or sulhydryl groups or are converted to such com- pounds in the body by oxidation, reduction or some other metabolic process. The reaction proceeds via backside nucleophilic attack by an electron—rich atom (O, N, or S) of the acceptor molecule on carbon atom l of the glucuronic acid moiety of UDPGA (Figure l). The resul- tant glucuronides have either an ester or an ether linkage between the .AUmmnv mwficoudos wocoa HocummnfinumHASBmfle atom on saum oncoususfiw cuss mmo co coaummsmaou .H magmas H ouswwm m ammo \o I o: NIH—v 0/ as: + o 0 Q N _ :08 o :0 mo cowuomaaoo mumaaaou mam maaaom Hmfiumuum acoucfi sufi3 coaumumaoum Hmcfiumouaw o>H>.mW "N ouswflm 30 N wuswflm uncut a . . , oiupzmmu: . . . <4:22 <4:22_> z. 31 mixture. One-half ml of an isotonic saline—20% ethanol solution containing DES (0.1, 1.0, 2.0, or 5.0 mM) plus a tracer amount of 14C- DES was then instilled into the lumen by injection. A 200 uM SKF-SZSA + 5 mM DES hydroalcoholic solution was used in the inhibition studies. The final specific activity of injected DES ranged from 500- 1000 dpm/nmole. In the studies utilizing MODES, 5 mM l4C—MDDES (final specific activity, 1000 dpm/nmole) was injected in an equivalent volume of the above vehicle. All venous blood draining the segment was collected in heparinized, calibrated tubes for 6 ten-minute periods. The intestinal venous blood flow rates reported in Table l were calculated by dividing the total volume of venous blood collected during a given time period by the duration (10 min) of that period. The loop was kept moist during the procedure with surgical sponges moistened with saline. Body temperature was maintained at 37°C by use of a heat lamp and rectal temperature probe. At the conclusion of the collection period, the loop was excised, the contents removed and both were frozen for later analysis. Systemic blood and bladder urine samples were obtained prior to sacrifice of the animal. Determination of DES and DESG in Venous Blood, Luminal Contents, and Intestinal Tissue Whole blood, luminal contents and intestinal tissue were homo- genized in methanol. The homogenates were centrifuged and the super- nates were removed and evaporated to dryness. The methanol—insoluble material was dissolved in formic acid and decolorized with 30% H202. Residues remaining after evaporation of the supernates were either redissolved in methanol for determination of total radioactivity or subjected to florosil column chromatography to separate DES from DESG 32 (88). The column eluates, methanol solutions, and solubilized, decolorized methanol-insoluble materials were mixed with ScintiverseR (Fisher Scientific Co., Livonia, Mich.) for liquid scintillation counting. Radioactive compounds in the methanol-soluble portions of blood, luminal contents, and intestinal tissue were identified by thin—layer chromatography. Solvent systems used were n-propranol:30% NH (7:3 3 v/v) and butanol:acetic acid:water (4:1:1 v/v) (88). Determination of MODES in Venous Blood, Luminal Contents, and Intestinal Tissue Whole blood, luminal contents and intestinal tissue were homo- genized in benzene. The homogenates were centrifuged and the super- natant layers were removed. The benzene-insoluble material was suspended in methanol, recentrifuged and the supernate removed. The benzene and methanol supernates were evaporated to dryness. Residues remaining after evaporation were redissolved in a small volume of the original extraction solvent. Aliquots of each solution were subjected to thin layer chromatography to separate MODES from its metabolites. The solvent system employed was n-propranol:isopropanol:water (10:9:1 v/v). The remainder of the benzene and methanol solutions were mixed with ScintiverseR for liquid scintillation counting. Methanol-in- soluble materials were analyzed for radioactivity as in Section 5. Statistics Statistical evaluation of the data was by the Student's t-test or analysis of variance (factorial design). Regression lines were deter- mined by the method of least squares. The level of significance was chosen as p<.05 (89). RESULTS Absorption and Glucuronidation of DES Table 1 shows that the regions of the intestine selected for use varied little among experiments. Oral segments were in the first quarter and aboral segments were in the last quarter of the small intestine. Variations in segment length were small, and the data presented here is from segments accounting for approximately 6% of the rat small intestine. Blood flow rates varied little among animals and intestinal regions; the concentration of DES placed in the lumen had no significant effect on the rate of venous blood flow. When DES was instilled into the lumen of isolated intestinal segments, all methanol-soluble radioactivity in intestinal venous blood, luminal contents, or intestinal tissue co—chromatographed with authentic DES or authentic DESG. No significant amounts of radio- activity were found in the systemic circulation, the urine, or the bile. Amounts of aglycone plus glucuronide in the methanolic extracts of intestinal venous blood, luminal contents, and intestinal tissue closely approximated the total radioactivity is determined separately in each sample. In addition, no significant amounts of radioactivity were found in the methanol-insoluble portions of blood, luminal con- tents, or intestinal tissue. Figure 3 is a plot of the cumulative amounts of total DES-related material, DES, and DESG appearing in the segment venous blood. 33 34 .q.ma~.ooa mp3 woummu mamaacm Ham mo cowuocsn Hmomolooafi ou umuocfisam oHuoaha scum mafiummuafi Hamam mo .2.m.m H Aaov auwama mmmum>m mSHQ .mwaoaa comm mudomoumou :8 o.m paw .mmaoad coca monommuamu 2E o.m .mmHoac com mucommummu 2E o.H .moaoaa 0m mo mmn mo unsoam Hmuou m muammmuamu SE H.o .man .mmmo sumo ca soausaom oHHosoonouv>n m «o as m.o mmB mmov onHo so.owmm.o mo.onmn.o mo.o«om.o mo.onam.o Hmuon< Aaaa\aav «use 30am Ho.OHnm.o mo.o«os.o oa.onoq.o Ho.OHom.o Hugo wooam maoam> unmawmm HmafiummuaH ~.on.o m.HHm.o o.HHm.s o.oHo.m Hmuon< m.oHH.o a.OH~.o m.oao.o o.oam.m Hmuo AauV “cmammm HmafiumoucH mo auwamg o.mflm.mh N.~Hm.oa m.qflm.wa o.mam.am Hmuoa< Aaov umuucfiaam appease scum o.ofio.q~ m.qnn.- o.HHm.aH H.~Ha.o~ Hmuo u: swam HmaflumwuaH mo moamumfio o.m o.~ o.H H.o cowwmm umumamumm GAZEV defiumuuamocoo man vowanm¢ .mHmaHdm m mo .z.m.m H amoa mnu mH msam> comm Boam vooam tam .nuwamq .cOfiumooa unmame H mqmHampo mo wooan msoam> SH wcHummmam ummn was .mmn .HMHuouma vmumamuummm Hmuou mo muasoam m>Humasado .m mustm 2 a: E 55 3 \l \\\ E E E ‘\1 \ :=§§§;§=8*8° 1mM l IL . E 2 g . ._ 8 33 35 8% |.. a 2 ---8 - .° -° W.-§.-§.-°°838 “" 2 2 S: E .S' .5: '58 8:: . QE '3: \ 8: cl- N 2 _ --_ --O 51-...§§§§33° o 2 2 s E .5 .5 E 3 '- s 375‘ E 3;: a: I- a 2 O ? o . a «o ‘ to . - ~ v - N N 1- :- (“IOW u) SONVHVBJJV SAILV'IOWHO (swam u) SONVHVBJdV 3AI1V'IflWflO T10 09 6C in 37 Subsequent to the initial 10 minutes, the amount of each increased in a linear fashion. Increases in applied DES concentration produced increases in the amounts of total DES-related materials, DES, and DESG collected from aboral segments at each time point. This was also true for oral segments at DES concentrations up to 2 mM. Increasing the concentration of DES in oral segments from 2 to 5 mM produced no increase in the cumulative amounts of total DES-related material, DES, or DESG appearing in the venous blood. Table 2 summarizes the results obtained at the end of the 60 minute collection periods. Significant differences between regions of intestine in amounts of total DES—related material, DES, and DESG appearing in venous blood draining the absorption sites occurred only at a DES concentration of 5 mM. As noted earlier, this was due to a failure of the oral segment to increase the amounts of each appearing in venous blood when the DES concentration was raised from 2Vto 5 mM. The amounts of total DES-related material, DES, and DESG found in the intestinal wall at the end of 60 minutes all increased with in- creasing DES concentration in'both regions of the intestine. The increases seen were not, however, directly proportional to the in- creases in applied DES concentration. Increases in DES concentration produced less of an increase in total DES-related material in oral segments than in aboral segments. This was apparently due to a greater accumulation of DES in the aboral segments, since DESG levels were similar in both regions. Total DES-related material remaining in the lumen at the end of 60 minutes increased with dose in oral segments, but there was no increase in DES-related material remaining in the lumen of aboral 38 TABLE 2 Total DES-Related Material, DES, and DESG in Venous Blood, Intestinal Wall, and Lumen Contents Sixty Minutes After Placing DES in the Intestinal Lumen Each value is the mean : S.E.M. of 3 animals. Values are expressed as nmoles. Applied DES Concentration (mM)a Quantity In Compound Region 0.1 1.0 2.0 5.0 Venous Blood Total Oral l4: 1 139: 9 250:25 266:23b Aboral 21: 5 140: 2 203:35 431:41 DES Oral 8: O 77: 8 135:15 145:20b Aboral 12: 2 77: 2 93:19 226: 9 DESG Oral 6: l 62: 4 102: 5 121: 7 Aboral 10: 2 56: 5 109:20 187:36 Intestinal Wall Total Oral 6: l 40: 5 65: 4 151:49 b Aboral 10: 3 48: 1 122:41 681:143 DES Oral 5: l 27: 5 50: 4 122:29 b Aboral 9: 3 39: 4 108:39 588:125 DESG Oral 1: O 9: 1 15: 3 34:10 Aboral l: O 10: 2 16: 3 77:27 Lumen Total Oral l6: 2 126: 4 364:58b 515:81b Aboral 10: 2 130:11 243: 7 199:41 DES Oral l4: 1 91: 4 282:27 335:12b Aboral 8: l 107:11 203: 4 148:25 DESG Oral 3: 1 29: 4 53: 6 161:71b Aboral 2: l 28: 6 38+ 2 42:19 TOTAL RECOVERY 0 Oral 74: 8 61: 3 57: 6 34: 3b Aboral 81:10 63: 2 68: 8 52: 4 aThe dose was 0.5 ml of a hydroalcoholic solution in each case. Thus, 0.1 mM represents a total amount of DES of 50 nmoles, 1.0 mM represents 500 nmoles, 2.0 mM represents 1000 nmoles, and 5.0 mM represents 2500 nmoles. b Significantly different from oral region (p<0.05) by Student's 0 Values are expressed as percent. 39 segments when the DES concentration was raised from 2 to 5 mM. The amounts of both DES and DESG in aboral segments at these applied concentrations of DES were lower than in aboral segments. The total recovery of applied DES decreased with increasing DES concentration. This was especially marked in oral segments of the small intestine. In order to more fully understand the effects that dose and region of intestine may have on the quantity and identity of DES- related material entering the hepatic portal circulation, it was of interest to compare the parameters in Table 3. Each parameter was compared by a 2x4 factorial analysis of variance. The percentage of dose appearing in the segment venous blood appeared to decrease with increasing dose. Statistical analysis yielded a significant F ratio for concentration, but the F ratio for interaction between concentration and region was also significant. Thus, as might be anticipated from the data presented earlier, the effect of concentration of DES on the amounts of DES-related material entering the portal circulation depends on the region studied. An estimate of the total conjugative capacity in the oral and aboral regions of the intestine is also given in Table 3. Although each increase in DES concentration produced an increase in total DESG formation, those increases were not proportional to dose above 1 mM. The F ratio for DES concentration was significant, but the F ratios for comparison of regions and interaction were not. Thus, total conjugation capacity towards DES is the same in both areas of the intestine over the range of DES concentrations applied. 40 .mmHoaa comm muaommummu 2E o.m paw .mmaoad oooa muammonmmu 28 o.~ .moaoad oom mucmmmpmmp 2E o.H .moaoac on mo mma mo uadoam Hauou m mucommuawu 2E H.o .mdnH .mmmo zoom CH dOHusaom UHHonoonouu%£ m mo H8 m.o mm3 omov msy a .mHmmamcm HMUHumHumum How uxmu moms ammo mm H qu m Hmm MHoq Oqu Hmuon< vooan msonm> unmawmm SH wcHummamm H Hoq N HHH Hqu Mqu ammo mHMHHmumE vmumamulmmo Hmuou «0 N nHHmom wHHNoH qum mHMH ammon< Amoaoacv mousaHa co aH mucwawmm oHHHHm m Hes: mHOOH NHHH Hmuo HmcfiummuaH sn emauom Oman Hmuoa N HmH c HON HHwN mHHq Hmuon< mOusaHa oo cH vooan msoam> H HHH m “mu NHwN mHmN Hugo unmamom OH maHumOaam macs mo N o.m o.~ o.H H.o aOmem Houmamumm QAZEV GOHuwuucmoaoo man meHma« .mamaHaw m mo .z.m.m H some OSu mH osam> sowm doaHummuaH Hamam umm osu mo waOmem Hmuon< cam Hmuo aH coHumchouaosao mam aOHuaHomn< mmn nuH3 kumHoomm< muouwamumm mo :omHHmmaoo m mqmHw mH mma mo .AmamlmlxmnmHmcm£a%xocuoalaHleq.mv ammo: mo wusuosuum um oustm 54 m mhswfih mmo \o»: NIHW \QOI o/o Nxo IO \ mIo $5-03; caveatosmzdg s v.8 muses. \o»: «:0 8m: 0 /o N m :0 :oo ...:o\ 55 TABLE 10 Amounts of MODES, Total DES-Related Material and DES in the Venous Blood, Intestinal wall, and Lumen Contents 60 Minutes After Placing 5 mM MODES or 5 mM DES in the Intestinal Lumen Compound Venous Bloodb Intestinal Wallb Lumenb Total Recoveryc d,e e MODES 388:14 224:14 583:59 48: 2 Total DES- Related Material 266i23 151:49 515i81 34¢ 3 DES 145:20 122:29 335:12 ----- aEach value is the mean : SEM of 3 animals. Only oral segments were used in this series of experiments. Values are expressed as nmoles. Total dose of DES or MODES was 2500 nmoles. 0 Values are expressed as percent. dSignificantly different from total DES—related material (p<0.05) using Student's t-test. eSignificantly different from DES (p<0.05) using Student's t-test. 56 Differences between the two compounds in regard to the total recovery of radioactive material and amounts of each found in the intestinal wall were not observed. DISCUSSION Regional Absorption and Metabolism by the Small Intestine The metabolism of DES during its absorption from isolated intesti— nal segments was significant; about one-half of the drug was conju- gated with glucuronic acid. DESG was released into the blood and intestinal lumen. Since no accumulation of glucuronide in the intesti— nal wall was found (Table 2), the rate of release corresponded with the rate of DESG formation. Bock and White (37) found that in the perfused rat liver, the level of UDPGA did not decrease even during prolonged glucuronidation at maximal rates, indicating that the re- generation of this nucleotide could not be exhausted. In the intesti- nal mucosa, the formation of UDPGA must also be high since a rela- tively constant and high rate of DESG formation was maintained by this tissue (Figure 3). Other studies have suggested that the small intestine has the capability for drug metabolism but few of these have examined intesti- nal metabolism in sign over a range of drug concentrations. Barr and Riegelmann (87) have demonstrated capacity-limited appearance of salicylamide glucuronide in the intestinal venous blood of rabbits at concentrations of salicylamide greater than 1 mM. These results suggest that, in the case of salicylamide, the ratio of free drug to conjugated drug delivered to the liver is dependent upon the dosage 57 58 applied. Neither the appearance of DESG in the venous blood of aboral segments nor the total glucuronide formation (both regions) was clearly capacity-limited over the range of DES concentrations studied here. Both may be capacity-limited at DES concentrations higher than 5 mM, but low solubility of DES in aqueous solution prevented the use of higher concentrations. In the case of DES, the ratio of free drug to conjugated drug presented to the liver remained constant over the applied dosage range (Table 3). Using the isolated intestinal segment technique, it was found that exit of DESG from the mucosal cell is bidirectional, appearing in both the blood and the intestinal lumen. Occurrence of DESG in the lumen under the in;§i£2_conditions used in this study gives reasonable evidence that significant amounts of drug metabolites may be expected to be found in the intestinal lumen during a normal absorption process in the intact animal. This may be an important consideration in explaining the presence of drug metabolites in the lumen that were previously assumed to be due solely to enterohepatic circulation and biliary excretion. Release of drug metabolites into the lumen could also influence the rate and extent of drug absorption. In the case of DES, a cycling process may occur where the DESG that is released into the lumen is hydrolyzed to DES by bacterial or mucosal B-glucuroni- dase, reabsorbed, conjugated, and released again into the lumen where the cycle would be repeated until either the drug is eliminated in the feces or completely absorbed. Barr and Riegelmann (87) suggested that intestinal blood flow was an important determinant of drug absorption. They demonstrated that the rate of absorption of salicylamide was blood flow rate-limited 59 while the rate of absorption of the more polar glucuronide was diffu- sion rate-limited and independent of the rate of intestinal venous blood flow. In the case of DES absorption, the rate of appearance of both DES and DESG in the venous blood were independent of the rate of blood flow over the range of blood flows observed in this study. The differences between this study and that of Barr and Riegelmann can probably be explained on the basis of the differing chemical proper- ties of the two compounds studied and/or the different species used. Whether the rate of intestinal venous blood flows reported in this study or that of Barr and Riegelman approximate values in the intact animal is unknown. It should be mentioned here that several animals in this study exhibited very low rates of intestinal venous blood flow, resulting from either peripheral hemorrhaging due to surgical manipulation or faulty cannulation of the mesenteric vein (data ob- tained from these animals were not used in result calculations). The rates of appearance of DES and DESG in the venous blood of segments from these animals were significantly lower (50-75%) than animals that did not exhibit any overt preparational abnormalities. Therefore, it is conceivable that intestinal blood flow may limit absorption when the blood flow through the absorbing region is very low. Intestinal blood flow is governed by sympathetic tone, and thus may be influenced by a number of factors, such as emotional state, pathological condi— tions, the presence of chyme or by drugs (90). It is possible that any one or combination of these factors may significantly alter the rate and extent of absorption of a compound from the intestine by altering blood flow through the absorbing regions. 60 Some workers have reported a decrease in intestinal drug-meta— bolizing capability with increasing distance from the pyloric sphinc- ter (62-66). Aitio g£_al, (63) showed that the rate of synthesis of o-aminophenol glucuronide and the activity of UDPGT in whole wall slices of rat ileum was 15% of that in slices of duodenum. Neither total DESG formation nor appearance of DESG in the venous blood was found to be decreased in aboral segments of intestine in this study. Hanninen and co-workers (29) suggested that the rapid decrease ob- served from the pyloric sphincter downward in conjugative capacity and UDPGT levels in the rat were due to the rapid absorption of inducing compounds in the oral regions of the intestine and their lack in the chyme in aboral regions. In a later study, these same investigators were able to demonstrate an increase in o-aminophenol glucuronide synthesis and UDPGT levels in the rat intestine following 3-MC ad- ministration (72). Total DESG formation was not increased by any compound employed in this study, however, including 3-MC. These conflicting results may provide evidence for the existence of a multiplicity of intestinal glucuronyltransferases with differing degrees of inducibility. In that case, a nonuniform distribution of UDPGT activity towards some substrates but not others such as DES may exist in the small intestine. The regional differences in absorption of DES found in this study are of a rather complex nature. Few marked differences between regions were observed at applied DES concentrations less than 5 mM. At 5 mM DES, however, quantities of DES removed from the lumen and appearing in the intestinal wall were greater in aboral than oral segments. Aboral segments were capable of increasing the absorption 61 of DES and DESG into the venous blood as the applied concentration of DES was increased to 5 mM; oral segments were not. The total recovery of DES-related materials was also higher in aboral segments. These regional differences cannot be explained at present, due to compli- cating factors (to be discussed in Section 3) encountered in this study. Absorption of MODES by the Small Intestine The intestinal absorption of MODES was examined to determine whether changes in the parameters of DES absorption could be produced by modifying DES so that it was no longer a substrate for intestinal UDPGT. It was found that methylation of the phenolic hydroxy groups of DES prevented glucuronide conjugation of this drug by the rat intestine. The amount of MODES appearing in the venous blood of the intestinal segment was significantly greater than the amount of DES at the same applied concentration. While this increase in absorption can be explained in terms of the inhibition of glucuronidation, MODES also appears to be better absorbed than DES from the intestine. The amount of MODES appearing in the venous blood was also greater than the amount of total DES-related material. Addition of methyl groups to the phenolic hydroxy groups of DES would abolish the ability of the molecule to undergo hydrogen bonding in aqueous solution, thus increasing its lipid solubility. Since it is known that lipid solu- bility is a determinant of intestinal absorption (3), better absorp- tion of MODES from the intestine may be a result of the increased lipophilicity of this compound. 62 The concept of inhibition of intestinal drug metabolism by structural modification of the drug may have important clinical appli- cations. Many drugs have poor therapeutic effects when administered orally as compared to that obtained when they are given by other routes. While these differences have been explained as being due to poor absorption of these drugs from the intestine, results presented in this study demonstrate that the decreased therapeutic effects may be due to intestinal metabolism rather than poor absorption. In the case of DES, as much as 50% of the total drug-related material entering the portal circulation was in an inactive form due to in- testinal metabolism. If MODES were shown to have estrogenic activity approaching that of DES, then the structural modification could result in increased amounts (nearly 300% greater) of active drug entering the circulation. It seems possible that the decreased or erratic therapeutic effects of many drugs given orally could be improved by structural modification of these compounds. Drugs are given most commonly by mouth since this is the most convenient route, the most economical one, and the only one of practical importance for self-administration. Therefore, improving the therapeutic effects of orally-administered drugs would be beneficial to both the patient and the physician. Effect of Altered Nutritional State on Intestinal Absorption and Metabolism Mietanen and Leskinen suggested that the nutritional state of the animal was an important determinant of p-nitrophenol glucuronide synthesis in rat intestinal homogenates (36). This does not appear to be the case for DESG formation by isolated rat intestinal segments, 63 since a 48 hour fast had no significant effect on this process. However, the difference between this study and that of Mietanen and Leskinen may be due to the differing lengths of the fasting period used (48 versus 72 hours). Since the work of Dutton and Story (91), UDPGA is known to be formed from glucose via UDP glucose. Fasting animals have been shown to produce UDPGA in both in_vi£rg_and in_gigg_ experiments in smaller amounts than well-fed animals; the extent of the reduction in hepatic UDPGA formation is dependent upon the length of the fast (92). It is conceivable that 48 hours of food deprivation is not a long enough period to decrease intestinal intracellular production of UDPGA, and thus, to decrease the formation of DESG. On the other hand, the different procedures employed to examine intesti— nal glucuronide synthesis could possibly account for the differences between the two studies. Alterations in Intestinal Absorption and Metabolism bngrevious Exposure to Foreign Compounds As established enzyme inducers such as PB, 3-MC and polyhaloge- nated biphenyls can increase xenobiotic-metabolizing enzyme activity in the liver, the potential for increases in these same drug-metabo- lizing enzyme activities in the small intestine exists. However, various stimulators of hepatic drug metabolism have generally not been effective in the stimulation of glucuronide synthesis by the intestine (8). Since previous studies were performed utilizing ingitrg tech- niques, it was desirable to determine whether the lack of stimulatory effect of these compounds extended to a more physiological situation, the isolated intestinal segment. None of the various chemical pre- treatments employed in this study was found to increase total DESG 64 formation by the intestine. Only one, PBB, was found to increase the DESG/DES ratio in the venous blood draining the segment. PBB may have stimulated the intestinal conjugation of DES, but because of the large animal to animal variation and the small samples size used, the increase that was observed was not statistically significant. Although it has previously been demonstrated that 3-MC administered intra- gastrically was able to stimulate intestinal o-aminophenol synthesis 32;!l522 (72), preliminary experiments utilizing isolated intestinal segments indicate that DESG formation is not increased by intragastric administration of 3-MC. The hepatic metabolism of many compounds is enhanced by previous exposure. The effect of previous exposure on metabolism occurring during the absorption process has not been examined either in_vitrg_or in_vivg, In this study, prior exposure of the rat to DES did not increase the conjugation of this compound by the small intestine. These results, as well as those obtained in the other pretreatment studies, provide additional evidence that the capacity of a chemical to induce glucuronide synthesis in the liver does not necessarily correlate with the capacity of a chemical to induce this process in the small intestine. Glucuronide synthesis in the liver and intestine may be under different control mechanisms. On the other hand, it is also possible that the compounds employed in this study are not direct inducers but must first be metabolized to be active as stimulators of glucuronide synthesis. This metabolic activation may not take place in the intestinal mucosal cells. More work is needed to explain the apparent lack of effect of inducers on intestinal metabolism. 65 Pretreatment with PB decreased both the absorption and glucuroni- dation of DES in the oral and aboral regions of the intestine. This effect of PB pretreatment was not due to a decreased rate of dis- appearance of DES from the intestinal lumen. The amount of DES remaining in the lumen after 60 minutes was less in phenobarbital pretreated animals than in control animals. In contrast, a decrease in the rate of DES disappearance from the lumen apparently was respon- sible for the decreased amounts of aglycone and glucuronide appearing in the venous blood of PCN-pretreated animals. Intraperitoneal administration of PCN resulted in inflammation of the intestine and diarrhea; either of these conditions may have affected normal intesti- nal absorptive function. Administration of 3-MO also decreased the amount of DES appearing in the venous blood of oral segments. As mentioned previously, a complicating factor was encountered in this study of the absorption and metabolism of DES by the rat small intestine. In all_experiments, it was found that the total recovery of DES-related material was incomplete. This recovery decreased with increasing dose in both areas of the intestine; total recovery was higher in aboral segments. PB and DES pretreatment also decreased the recovery of radioactive material from intestinal segments. The total recovery of MODES was not significantly different from that of DES; experiments utilizing 5 mM dose solutions of these compounds resulted in less than 50% recovery of radioactive material. The reason(s) for the above observations are not known. Incomplete recovery of radio- activity could arise artifactually due to shortcomings in either the preparation or assay used. Several pieces of evidence suggest that the integrity of the preparation as a closed system was not compro— mised. 1) Radioactivity was not found in the systemic circulation, 66 bile, or urine of any of the animals used in this study. 2) Whole body digests of rats, prepared at the conclusion of sample collection, contained no radioactivity. 3) Finally, preliminary experiments utilizing the same preparation and other compounds salicylamide and p- nitrophenol) yielded complete recovery of all materials applied. That the assay for DES and DESG was sufficiently quantitative is supported by the good agreement between the sum of DES and DESG obtained in each experiment and the total radioactivity determined independently (see Table 2 and Methods). Furthermore, removal of the intestinal segment immediately following application of the dose yielded complete re- covery of radioactivity. It seems unlikely then that the incomplete recoveries obtained in this study were a result of flaws in the preparation and/or assay. An alternate explanation may be that the 14C-label is lost from DES and MODES via metabolism. Since the DES and MODES used in this study were radiolabelled at the ethyl side chain (monoethyl-l-lAC), it is possible that a volatile 14C-containing product was formed. Such a product would not be detected by the assay methods used or by the thin-layer chromatographic procedures employed to identify radioactive compounds in the blood, lumen, and segment wall (see Methods). It should be noted that several previous studies dealing with DES metabolism in the rat have used DES labelled in the same position as that used in this study, and often, total recoveries are not reported (93-95). There is some evidence in the literature to indicate that a portion of the monoethyl—l-IAC label may be lost after administration of radiolabelled DES to rats. Fischer and Weissinger have reported an age-related decrease in the recoveries of DES-related material in the 67 young rat (96). Aschbacher and co-workers have noted decomposition of l4C—DES in ruminant feces. Materials formed included acetic and proprionic acid. It is not known whether the decomposition was enzymatic or spontaneous or if it occurred in the gastrointestinal tract (97). Other workers have reported that, in addition to conju- gation with glucuronic acid, DES is extensively oxidized in the rat. Metzler (100) has recently shown that DES is metabolically oxidized and cleaved at the stilbene double bond to 4'-hydroxypropriophenone. This metabolic oxidation of DES is believed to proceed via the hepatic mixed-function oxidase system, and has been shown to be enhanced by pretreatment with various inducers of hepatic microsomal drug meta- bolism, such as phenobarbital and 3-MC (98,99). Mixed-function oxi- dase activity has also been demonstrated in small intestinal mucosa (11,12,64-66). This activity is highest in the upper small intestine, decreases with increasing distance from the pyloric valve, and can also be enhanced by pretreatment with certain compounds (11,64-66). The lower recoveries of DES-related materials from oral intestinal segments and in both phenobarbital and DES pretreated animals obtained in this study suggest that a pathway for DES metabolism in the rat small intestine which results in the formation of a volatile product is worth considering. Further evidence for the existence of this type of metabolic pathway is provided by the results of the SKF-525A experiments (Table 7). Concurrent administration of SKF-525A, an inhibitor of oxidative drug metabolism in the liver and intestine, resulted in 95% recovery of DES-related material from oral intestinal segments. The increase in total DESG formation observed in these segments was possibly due not to a stimulatory effect of SKF-SZSA on 68 intestinal glucuronide synthesis, but rather to an inhibitory effect of this compound on the oxidative metabolism of DES by the intestine. The decrease in the absorption of DES in 3-MC pretreated rats and the decrease in both DES absorption and glucuronidation in PB-pretreated rats could be explained if these compounds stimulated a pathway for DES metabolism which resulted in the formation of a volatile product. SUMMARY AND CONCLUSIONS The in sign isolated intestinal segment technique appears to be a suitable method with which to study small intestinal absorption and metabolism. By cannulating the mesenteric vein and leaving the arterial supply intact, the amounts of drug and its metabolites, if any, entering the portal circulation can be quantitated directly. Use of this technique allows the investigation of intestinal absorptive and metabolic function under conditions closely approximating those found in the intact animal; factors (hepatic metabolism, tissue distribu- tion, and urinary excretion) which complicate other techniques used to evaluate intestinal absorption and metabolism in_vivg_are eliminated using the isolated intestinal segment. Results obtained using this technique may eventually prove to be more indicative of in_vivg_ function than other techniques more commonly used. Utilizing the isolated rat intestinal segment, it was shown that metabolism of a model compound, DES, was significant during the absorption process; as much as 50% of the drug appearing in the venous blood draining the segment was conjugated with glucuronic acid. The capacity of the rat small intestine to glucuronidate DES was exten- sive, since this process could not be saturated at applied DES con— centrations up to 5 mM. Furthermore, this capacity appears to be uniformly distributed throughout the small intestine. In contrast, regional differences in absorptive capacity were determined; aborally- 1ocated segments displaying a somewhat greater capacity to absorb DES 69 70 and DESG than orally-located segments. Intestinal blood flow was not found to be a determinant of DES or DESG absorption in either region of the intestine over the range of blood flows reported. The significance of intestinal glucuronidation of ingested drugs and other xenobiotics occurring during the absorption process is poorly understood. The results presented in this study indicate that intestinal glucuronidation of certain compounds during absorption may be extensive over a wide range of drug concentrations and in all regions of the small intestine. This may have considerable pharma- cological implications in the absorption of a drug in its active state, for a significant amount of the drug may already be deactivated before presentation to the liver. Since the immediate consequence of intestinal glucuronidation of ingested compounds is to decrease their transfer into the body in an active form and to facilitate their elimination from the body, this process, as well as other intestinal drug-metabolizing functions, can be considered part of a physiological defense mechanism. From this point of view, it is of interest to find these detoxification mechanisms present in the organ that is the first and main route for the entrance of foreign compounds, the gastro- intestinal tract. It seems likely that intestinal metabolism has an effect on the hepatic disposition of ingested compounds. Although reports con- cerning the disposition of preformed glucuronides are conflicting, it is clear that the metabolic and excretory functions of the liver can be markedly altered, depending on whether a compound arrives at the liver as a glucuronide conjugate or as the aglycone. Because of this, alterations in intestinal glucuronidation of ingested xenobiotics 71 could lead to changes in the metabolism, distribution, and excretion of these compounds. Alterations in the aglycone/glucuronide ratio in the portal blood presented to the liver would play a major role in affecting these changes. It was therefore an objective of this study to determine whether alterations in intestinal metabolism of an ingested compound could be produced. Employing various treatments known to affect drug metabolism in the liver, it was shown that only one, previous exposure to PBB, altered the DESG/DES ratio in the venous blood draining the intestinal segment; this ratio was significantly increased. Pretreatment with 3- MC, PCN, or PB, all simulators of hepatic glucuronidation, resulted in decreased amounts of DES appearing in the portal circulation. None of these compounds, including PBB, were shown to increase total DESG formation by the intestine. In fact, PB decreased intestinal glucuro- nide synthesis. SKF-525A, an inhibitor of hepatic glucuronide syn- thesis, increased intestinal DESG formation as well as the amounts of both aglycone and glucuronide entering the portal circulation. Alteration of the nutritional state of the animal by fasting was shown to have no effect on parameters of DES absorption or metabolism. Methylation of the phenolic hydroxy groups of DES prevented glucuroni- dation of this compound during absorption. As a result, increased amounts of unaltered drug appeared in the circulation. The results of the above experiments would appear to indicate that the capacity of a treatment to alter glucuronide synthesis in the liver does not necessarily correlate with the capacity of a treatment to alter this process in the same way in the small intestine as well. It may be that factors that regulate drug metabolism in extrahepatic 72 tissues differ from regulatory factors in the liver. However, the various effects on parameters of DES absorption and metabolism ob— tained in the pretreatment experiments as well as those obtained with concomitant administration of SKF-525A are of a rather complex nature. These results are further complicated by the fact that recovery of applied materials in all experiments conducted in this study were consistently lower than that tolerated within limits of experimental error; recoveries varied with dose, region, and treatment. 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