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DRUG METABOLISM REVIEWS, 9(2), 259-279 (1979)
Influence of Gut Microflora on Bioavailability* H. G. BOXENBAUM, I. BEKERSKY, M. L. JACK, AND S. A. KAPLANt Department of Pharmacokinetics 'and Biopharmaceutics Hoffmann-LaRoche Inc. Nutley, New J e r s e y 07110 I. 11. 111.
IV.
.................................... ACCESS OF COMPOUNDS TO THE HUMAN INTESTINAL FLORA ................................ EXAMPLES OF BACTERIAL INFLUENCE ON BIOAVAILABILITY .............................. A. Isonicotinuric and Salicyluric Acids ............... B. Sulfasalazine (Salicylazosulfapyridine, SAS) and Other Azo Compounds ................... C. Cyclamate ...................................... CONCLUSIONS ...................................... Acknowledgments ................................... References ......................................... INTRODUCTION
260 261 264 264 270 273 277 278 278
*Presented at Symposium on Drug Disposition in Man held in Sarasota, Florida, November 6-11, 1977 under the auspices of the American Society for Pharmacology and Experimental Therapeutics. +To whom requests for reprints should be addressed.
259 Copyright 0 1979 hy Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage' and retrieval system, without permission in writing from the publisher.
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I. INTRODUCTION The relationship between the human intestinal flora and host is of great importance, It i s well established that intestinal bacteria play a role in normal physiological processes and can contribute to pathological states. Additionally, it has become increasingly apparent that microbial flora a r e capable of metabolizing a variety of foreign or drug substances, thus potentially influencing and/or altering drug activity and toxicity, Metabolism by microorganisms in the gastrointestinal tract may occur after drug administration, thereby affecting the bioavailability and physiological disposition profile of the drug. Such metabolic activity in the gastrointestinal tract is influenced by many factors. These factors include delayed absorption which may be due to the intrinsic properties of the drug or an effect of the dosage form, diffusion or secretion of drug and/or metabolite into the gastrointestinal tract, and/or drug action on the gastrointestinal microflora per s e with subsequent enhanced metabolism, i. e., metabolism adaptation. Therefore, gastrointestinal microbial metabolism studies must consider distribution of microorganisms within the human gastrointestinal tract, the rate and extent of drug absorption, and the gastrointestinal transit time. Pharmacological and toxicological implications of gastrointestinal microbial metabolism reflect alteration in metabolism following chronic drug administration, drug activation, increase in anerobes in the upper gastrointestinal tract due to disease states, and gastrointestinal disorders resulting from antimicrobial drug therapy, Whereas most tissue metabolic processes result in more hydrophilic derivatives which a r e generally eliminated from the body more rapidly than the parent drug, gastrointestinal microbial metabolism often results in the formation of more lipophilic derivatives due to hydrolysis o r reduction of glycosidic linkages, deconjugations, dehydroxylations, and decarboxylations. Such lipophilic metabolites tend to be absorbed into the systemic circulation and therefore can exert pharmacological and toxicological effects. This series of events may be the physicobiochemical basis of enterohepatic recycling of drug. Extensive reviews of drug metabolism via intestinal microorganisms a r e presented by Scheline [ 1, 21. Some of the metabolic conversions known to occur in the gastrointestinal tract a r e reported in Table 1. This presentation will focus on some of the biopharmaceutical and pharmacokinetic aspects of drug metabolism by the microorganisms of the gastrointestinal microflora.
INFLUENCE OF GUT MICROFLORA ON BIOAVAILABILITY
261
TABLE 1 Some Metabolic Conversions Known to Occur in the Gastrointestinal Tract. Adapted from Ref. 1
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Reaction
Specific groups metabolized
Hydrolysis
Glycosides, glucuronides, sulfate esters, amides, esters sulfamates, nitrates
Dehydroxy lation
C- Hy droxy1, N- hydroxy 1
Decarboxylation Dealkylation
0-Alkyl, N-alkyl
D ehalogenation Deamination Reduction
Double bonds, nitro groups, azo groups, aldehydes, ketones, alcohol, N-oxides
11. ACCESS O F COMPOUNDS TO THE HUMAN INTESTINAL FLORA Compounds may be exposed to intestinal microorganisms via diffusion o r secretion into the gastrointestinal tract. A factor generally not appreciated is that a "typical" human subject secretes 8.2 liters of fluids daily into the gastrointestinal tract, a s illustrated in Fig. 1. This includes 1 . 5 liters saliva, 0.5 liter bile, 2.5 liters gastric juice, 0.7 liter pancreatic juice, and 3.0 liters of intestinal secretions [ 31. One example of a compound which is exposed to the microflora by this general secretion mechanisms is the bile acid, glycocholic acid [ 41. This glycine conjugate is excreted via the bile into the small intestine, where some escapes reabsorption from the ileum and passes into the colon, wherein the glycine moiety is metabolically removed by bacteria. Compounds may also be exposed to gastrointestinal flora following oral ingestion. The most important factors that can influence whether o r not a compound undergoes microbial alteration is the distribution of microorganisms within the human gastrointestinal
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262
BOXENBAUM ET AL.
FIG, 1. Secretion of fluids into the gastrointestinal tract of a typical human subject. Adapted from Ref. 3.
tract. Scheline [ 13 reviewed much of the literature in this area and indicated that the stomach, duodenum, jejunum, and upper ileum a r e only sparsely populated. Increasing numbers of organisms exist in the distal ileum, and a significant increase is seen a t the ileocecal valve. The numbers and distribution of organisms a r e illustrated in Fig. 2. It should be noted that most of the organisms associated with the stool, and therefore presumably the large intestine, a r e obligate anerobes. In excess of 99% of the total cultivable fecal flora of normal adults are obligate, nonsporing anerobes. It has been determined that a least 20% of the mass of the feces consists The presence of anerobes in feces requires of live bacteria [6-83. that specimens for in vitro studies must be maintained under a 10% H,-90% N, mixture.
INFLUENCE O F GUT MICROFLORA ON BIOAVAILABILITY
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l0l2
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10 lo
10
8
10
10
10
O0
E?j
ANAEROBES BACTEROIDES 8 RELATED SPECIES, LACTOBACILLI CLOSTRlDlA
El COLIFORMS
M
GRAM
PosinvE
FLORA
STREPTOCOCCI, LACTOBACILLI, STAPHYLOCOCCI, YEAST
FIG. 2. Diagrammatic scheme of microbial populations of the human gastrointestinal tract. Taken from Ref. 5.
Another factor influencing accessibility of a foreign compound to microbes i s the extent and rate of absorption of the compound. Obviously, any compound which is rapidly and completely absorbed from the upper gastrointestinal tract will not be exposed to the microflora unless subsequently secreted back into the tract. Therefore, compounds exhibiting poor absorption characteristics will have the greatest exposure potential to the intestinal flora. Examples to be discussed include compounds having an azo bond o r a glycine conjugate, both of which tend to be poorly absorbed from the upper gastrointestinal tract. The time course of microbial transformation following oral administration will be partially dependent on gastrointestinal transit time. Eve [ 9 1 reviewed gastrointestinal transit time, including observations of mean passage times of food residues through the stomach, small intestine, upper large intestine, and lower large intestine. On an average, food residues reach the microflora of the upper large
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264
BOXENBAUM E T AL.
intestine after 5 hr. Thus one could generally expect some delay following drug ingestion before microbial metabolism would occur. It should be noted, however, that these transit times represent movement of unabsorbed food residues in an average man consuming an average diet. Transit of an unabsorbed drug taken after an overnight fast would probably occur much more rapidly. Drug development study designs, therefore, should consider gastrointestinal microbial metabolism. Such metabolism may influence the absorption, distribution, and excretion profiles of drugs. There may be differences in quantity of microorganisms in fasted vs fed animals. The observations based on large experimental doses in animal may not be predictable of small therapeutic doses in man. Variability in number, type, and location of microorganisms may exist, Variability in biliary excretion may result from regeneration of parent compound in the intestine o r liberation of various metabolites which may undergo further metabolism. Microbial metabolism may be responsible for the prolonged retention of drug o r metabolite in systemic circulation. In this regard, the influence of bacterial metabolism on the bioavailability and pharmacokinetic profiles of isonicotinuric acid, salicyluric acid, sulfasalazine, and cyclamate will be discussed.
111. EXAMPLES OF BACTERIAL INFLUENCE ON BIOAVAILABILITY A. Isonicotinuric and Salicvluric Acids The hydrolysis of glycine conjugates by gastrointestinal bacteria is well documented I:1, 2, lo]. Isonicotinuric, salicyluric, p-aminohippuric, and p-acetylaminohippuric acids appear to be hydrolyzed by gut bacteria following oral administration [ 10, 111. The structure of isonicotinuric acid (INU) and isonicotinic acid (INA) a r e presented in Fig. 3. Following intravenous administration of INU to human subjects, intact INU is recovered in the urine, indicating no systemic hydrolysis of INU to INA [ l o 1 However, when INU i s administered orally on an empty stomach, approximately 7% of the dose was absorbed intact from the stomach and/ o r small intestine, The remaining unabsorbed material then passed to the lower gastrointestinal tract where bacteria hydrolyzed it to INA. This latter compound was subsequently absorbed into the systemic circulation, where some was reconjugated with glycine, forming the originally administered compound, INU. Urinary excretion rate curves for one subject in two studies at oral solution doses of 129 and 476 mg a r e illustrated in Fig. 4.
INFLUENCE O F GUT MICROFLORA ON BIOAVAILABILITY
0
265
0
II
II
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C -OH
6 6 N
N
lsonicotinuric Acid (INU)
lsonicotinic Acid (INA)
FIG. 3. Structures of isonicotinuric and isonicotinic acids. Dose: 129 mg
100.0
RATIO:
Dose: 476 mg
m3,
RATIO:
100.0
INU
50.0
w, INU
50.0!
m-- -m INA
10.0:
z
0 W
K 0 X U
1.0:
4
0 W
I-
a K
0.5’
1
5
10
15
20 25
30
J
5
10
15
20 25
TIME ( HOURS 1 FIG. 4. Urinary excretion rate c u r v e s following o r a l administration of isonicotinuric acid (INU) to a healthy human subject. Isonicotinic acid (INA) rates e x p r e s s e d in t e r m s of m o l e equivalents INU. Adapted f r o m Ref. 10.
30
266
BOXENBAUM ET AL.
INA
RAT10: INU
r
1
..-..
~
'
r
INA
KANAMYCIN PRE TREATMENT
LlNOMYClN PRE -TREATMENT
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10.01
7 5.0 '
L
a K
.'
5
10
15
20
25
5
10
15
20
25
TIME ( HOURS 1
FIG. 5. Influence of antibiotic pretreatment on the urinary excretion rate curves following oral administration of approximately 125 mg isonicotinuric acid (INU). Isonicotinic acid (INA) rates expressed in terms of mole equivalents INU. Taken from Ref. 10.
Initially INU was observed in the 0 to 5 h r postadministration interval. Subsequently, the unabsorbed INU which continues down the gastrointestinal tract was converted to INA. The larger the dose of INU, the more pronounced the effect, a s indicated by the INA/INU ratios, equivalent to one in the first study and greater than one in the second study. The urinary excretion profiles indicate that once in systemic circulation, INA i s then reconjugated with glycine to form INU a s demonstrated by the increase in INU urine levels observed in the excretion curves. In order to confirm that microorganisms were responsible for the gastrointestinal hydrolysis of INU to INA, studies were undertaken in which the gastrointestinal flora were eliminated. Anerobic bacteria can be largely eliminated with little o r no change in the aerobes by administration of lincomycin, whereas anerobes a r e not destroyed with kanamycin [11. The urinary excretion profiles following the pretreatment of subjects with kanamycin and lincomycin a r e presented in Fig. 5. The microbial metabolism of INU was confirmed,
INFLUENCE O F GUT MICROFLORA ON BIOAVAILABILITY Dose:
Ratio:
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5.0[
267
49 rng (rectal)
:;,"
->
1
. 5
10
15
20
TIME (HOURS]
FIG. 6. Urinary excretion rate curves following rectal administration of isonicotinuric acid (INU). Isonicotinic acid (INA) expressed in terms of mole equivalents INU. Taken from Ref, 10. since pretreatment with lincomycin destroyed the anerobic microorganisms and thereby prevented the formation of INA. Kanamycin, which did not destroy anerobes, was ineffective in obliterating the metabolism to INA. The excretion rate profiles of INU and INA following rectal administration of INU a s a retention enema are presented in Fig. 6. The urinary excretion profiles indicate immediate and very extensive INA formation, the ratio of INA/INU being much greater than one for the small dose of 49 mg INU.
BOXENBAUM ET AL.
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5.0
ti 'I
1.0. D
-
0.5
0.1
i
0.05. 5
10
15
20
25
TIME (HOURS)
FIG. 7. Influence of propantheline administration on the urinary excretion rate curves following oral administration of isonicotinuric acid (INU). Isonicotinic acid (INA) rates expressed in terms of mole equivalents of INU. Taken from Ref. 10.
The same subject was given multiple doses of the antichloinergic agent, propantheline, and the resulting urinary excretion profile is reported in Fig. 7. This multiple dose pretreatment with propantheline resulted in prolonged gastrointestinal transit time of INU, allowing for more extensive INU absorption from the upper gastrointestinal tract and delayed onset of INA formation.
269
INFLUENCE O F GUT MICROFLORA ON BIOAVAILABILITY TABLE 2
Hydrolysis of INU Following 12 h r Anerobic Fecal Incubation Studies.a Taken from Ref. 1 0
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Percent recovery Sample contents Medium + INU Medium + INU + feces Medium + INU + feces + antibiotics Medium + INA + feces
INU
98.5 2.80 92.6 0
INA 0
91.8
4.41 96.3
a
Incubation medium consisted of phosphate buffer, glucose, yeast extract, and peptone a s described by Scheline [27].
The results of in vitro anerobic fecal incubations on the hydrolysis of INU a r e reported in Table 2. The INU was shown to be stable in the medium per se, but to be extensively hydrolyzed when incubated anerobically with feces. In the presence of antibiotics, the anerobic metabolism of INU was essentially eliminated. These results further confirm that the microorganisms responsible for the hydrolysis of INU a r e anerobic. Salicyluric acid ( M U ) , like INU, undergoes gastrointestinal microbial metabolism to salicylic acid (SA) prior to absorption, Following 3 mg/kg SAU i.v. administration, only SAU plasma levels a r e observed; hydrolysis of SAU to SA did not occur [12]. SAU was also administered orally at 3 mg/kg to a healthy human subject, and the plasma levels profile a r e presented in Fig. 8. It was estimated that approximately 80% of the SAU dose was absorbed intact from the upper gastrointestinal tract, and that the remaining SAU apparently passed down to the large intestine and was subjected to microbial hydrolysis to SA. It should be noted that formed SA, following absorption will be further metabolized to SAU in vivo. However, due to the rapid elimination rate of SAU and the sensitivity limitation of the assay, no prolonged SAU levels were observed a s with INU.
BOXENBAUM ET AL.
270 10 7
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5
-
SUBJECT A
.Salicyluric
3
Acid (SAU)
8-4
E
'
2
2
Y
c
0
4
4 J 1 a &
2
0.1
'5
0.5
1
0.3
a
0
8 '
ld
/
d
nl
\
/
\ \
/
0.2
\
/
b
/
/
\
I
\
\
6
0.1 0.01
I
I
10
I
20
1
30
40
1
50
Time (hours)
FIG. 8. Salicyluric and salicylic acid plasma concentration-time curves following oral administration of 3 mg/kg of salicyluric acid to a healthy subject.
B.
Sulfasalazine (Salicylazosulfapyridine, SAS) and Other Azo Compounds
Salicylazosulfapyridine (SAS) is used clinically in the treatment of ulcerative colitis [IS]. The structure of sulfasalazine and the bacterial metabolic products, 5-aminosalicylic acid and sulfapyridine, a r e presented in Fig. 9 [ 143. Following sulfasalazine administration, Das et al. (151 report some sulfasalazine is absorbed from the
1
60
INFLUENCE OF GUT MICROFLORA ON BIOAVAILABILITY
271
SULFASALAZINE
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HOOF
BACTERIAL A 2 0 REDUCTASE
S .AM I NOSALlCY LI C ACI D
SU LFAPY R I DIN E
FIG. 9. Sulfasalazine and bacterial metabolic products. Adapted from Ref. 14.
small intestine, but most of the drug reaches the cecum where splitting of the azo linkage occurs, yielding sulfapyridine and 5-aminosalicylic acid. Sulfapyridine is then absorbed from the colon and i s subsequently acetylated and conjugated to the glucuronide in the liver prior to excretion in the urine. The 5-aminosalicylic acid metabolite is also absorbed and is excreted in the urine a s both intact 5-aminosalicylic acid and as acetyl 5-aminosalicylic acid. Schrdder and Campbell [ 161 administered SAS at a dose of 5 g/ day to nine healthy subjects over a period of 1 0 days. SAS and sulfapyridine serum concentration-time curves in a typical subject following the first 4 g-dose are presented in Fig. 10. Approximately 85% of the administered dose was recovered in the urine and about 5% in the feces. The urinary excretion of sulfasalazine ranged from 1.7 to 10% of the administered dose in the nine subjects. About 40 to 50% of the dose was excreted in the urine a s sulfapyridine and its metabolites; approximately 30% of the dose was excreted a s 5-aminosalicylic acid and its metabolites. The feces consisted of sulfapyridine and metabolites of 5-aminoalicylic acid; no intact sulfasalazine was detected in the feces. These results demonstrate the efficiency of the bacteriological metabolism of sulfasalazine. Since sulfapyridine is present in the colon, it is conceivable that this agent is active by virtue of its antibacterial properties. In
BOXENBAUM ET AL.
272
100
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70
4
d
50
Sulfapyridine
\
\
I
!30 2E
\
w 20
E
0
u
5a
2
10 7
5
I
I
4
I
I
8
1
I
12
I
I
16
I
I
I
20
Time (hours)
FIG. 10. Sulfasalazine and sulfapyridine serum concentration-time curves following single dose oral administration of 4 g to a healthy volunteer. Data taken from Ref. 18.
reviewing the literature, however, Goldman and Peppercorn [ 171 were cautious in their appraisal of the possible beneficial effects of this sulfonamide. SAS therapy has only a minor influence on the bacterial constituents of the fecal flora. It was noted, however, that currently available methods a r e not capable of detecting changes in the host-microflora relationship a t the mucosal level. The influence of gastrointestinal flora on SAS has toxicological implications since observed adverse effects have been correlated with sulfapyridine serum levels. Toxicity did not correlate with either intact SAS or 5-aminosalicylic acid levels r15, 18, 191. Additional work has also been reported on other azo compounds. Walker [20] reviewed much of the literature on the fate of these compounds in man and other species. It was noted that compounds
1 24
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INFLUENCE O F GUT MICROFLORA ON BIOAVAILABILITY
273
in this group a r e the most common synthetic colorings used i n foods, pharmaceuticals, and cosmetics. Regarding metabolism, intestinal azo reduction systems a r e considerably more active and nonspecific than the hepatic azo reductase in animals. Twenty-one species of bacteria were screened as to their ability to reduce 14 azo compounds. Some of the bacteria were normal constituents of the mammalian gut, and overall, azo-reducing activity was fairly general and relatively nonspecific. C.
Cyclamate
Cyclamic acid (cyclohexylsulfamic acid) a s its sodium o r calcium salt had been widely used as an artificial sweetening agent. In 1969 it was removed from the United States market because its metabolite, cyclohexylamine, was suspected of being a bladder carcinogen in rats [ 211. Cyclamic acid is a strong acid with a pKa of 1.9 and is poorly absorbed, resulting in a prolonged residence time of the compound in the gastrointestinal tract. Figure 11 illustrates the metabolic fate of cyclamic acid in man [ 22-25]. Following single dose oral administration to three humans on a cyclamate-free diet, intact compound was primarily excreted unchanged in feces and urine. Approximately 30% of the dose was found in urine and 50977 in feces. Trace amounts of cyclohexylamine (< 0.002% of dose) were also detected in urine. In additional studies, the same three subjects were placed on a diet containing cyclamate for a period up to 30 days. The results reported in Table 3 indicate that on continued cyclamate exposure, cyclohexylamine urinary levels increased in the three subjects approximately 26-, 80-, and 8,650-fold. Thus there appeared to be variability, particularly in subject B. S.D. whose cyclohexylamine urinary levels increased dramatically. Removal of cyclamate from the diet resulted in a decreased conversion to cyclohexylamine. Thus there appears to be a reversible, self-inducible metabolic transformation of cyclamic acid to cyclohexylamine in man. Drasar [25] has shown that the metabolic conversion of cyclamic acid to cyclohexylamine in rats does not occur following either parenteral administration of cyclamate o r with incubations of cyclamate with the liver, spleen, kidney, o r blood preparations as shown in Table 4. These results indicate the cyclohexylamine formation occurs solely in the gut as the result of microorganism metabolism; that when cyclamate was incubated with the feces of a cyclamate pretreated %onverter, cyclohexylamine w a s formed; and when cyclamate was removed from the diet, the fecal organisms showed less ability to form cyclohexylamine.
BOXENBAUM ET AL.
27 4
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ORAL ADMINISTRATION
Cyclamic A c i d
Urinary Excretion (S30%)
Fecal Excretion
-Gut Flora
(
z: 50%)
6
Cyclohexylarnine
I
Systemic Absorption
Urinary k c r e t i o n (Major Pathway)
6
Cyclohexylamine
6"" OH
4-
Trans-cyclohexane-l,2-diol
(yoH Cyclohexanol
(Minor Metabolites)
FIG. 11. Metabolic degradation of cyclamic acid by bacterial flora. Adapted from Refs. 22 and 24.
INFLUENCE OF GUT MICROFLORA ON BIOAVAILABILITY
275
TABLE 3
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Cyclohexylamine Levels in the Urine of bbbjects Treated with Cyclamatea
Time on cyclamate (days)
Cyclohewlamine in urine (YJof dose) of three subjects B. S. D. P. C. H. A.G.R.
0
< 0.002
< 0.002
< 0.002
1
0.07
< 0.002
0.003
5
5.8
0.005
0.013
10
17.3
0.015
0.014
12
13.5
0.005
0.007
15
16.4
0.034
0.071
20
-
0.053
0.160
25
-
0.052
0.140
0.014
0.050
26 27 28
a
Three human subjects received 3 g of calcium cyclamate each day. Subject B. S.D. discontinued the daily dose of cyclamate on Day 18 and remained on a cyclamate-free diet. On Day 26, this subject received 3 g of calcium cyclamate. Taken from Ref. 22.
In summary, orally ingested cyclamate is primarily excreted in urine and feces intact. Moderate cyclamate exposure in the diet increases the ability of gastrointestinal microorganisms to convert cyclamate to cyclohexylamine. Microbially produced cyclohexylamine is then absorbed from the large intestine and is primarily excreted intact in the urine. Trace amounts of two metabolites,
3.25 2.40
-
-
-
1.94 0.62
-
0.78 0.43
0.3 33.6 34.0
-
-
0.3 61.5 69.6
-
-
-
0.4 54.0 14.9
-
-
-
-
16
14
13
-
< 0.05 < 0.05
< 0.05
< 0.05
< 0.01
Normal animals
a The r a t s were on a cyclamate diet for 4 to 4.5 months prior to receiving 100 mg of calcium cyclamate administered orally. The cyclamate diet consisted of allowing the r a t s free access to food but their water contained 0.5% (w/v) calcium cyclamate. Taken from Ref. 25.
Contents of: Duodenum-ileum (H2) Caecum (H2) Colon-rectum (H2)
Hind-gut contents: Anerobic (N2) Aerobic (air)
< 0.05
< 0.05
Liver, kidney, spleen, blood
< 0.05
< 0.05
< 0.05
< 0.05
Controls
43 21
33 30
22 60
Rat no.
Whole animal
Material tested
(o/o of administered o r added cyclamates)
Conversion into Cyclohexylamine
Metabolism of Cyclamate by Rat Tissues and Gut Contentsa
TABLE 4
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INFLUENCE OF GUT MICROFLORA ON BIOAVAILABILITY
277
TABLE 5
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Drugs in Which Iuminal and/or Mucosal Metabolism I s Suggested. Taken from Ref. 26 Conjugation: Estrogens Salicylamide L-Dopa 2- Methyldopa
Ester hydrolysis: Acetylsalicylic acid Organic nitrates Propoxyphene Meperidine Methadone Penta zoc ine Dexamethasone phosphate
Reduction: Hydrocortisone Cortisone A ldoste rone Progesterone Testerone
N-acetylation: p-Aminobenzoic acid Certain sulfonamides Amide hydrolysis: p-Aminohippuric acid Hippuric acid
trans-cyclohexane-l92-diol and cyclohexanol, have also been detected. When cyclamate is removed from the diet, the ability of the gastrointestinal flora to produce cyclohexylamine is considerably reduced. Many drugs a r e suspected of undergoing metabolism by microorganisms in the gastrointestinal tract and/or by gastrointestinal mucosa prior to absorption. A list of such drugs, compiled by Riegelman, is given in Table 5 .
IV.
CONCLUSIONS
In conclusion, it has been demonstrated by several investigators that gastrointestinal flora are capable of metabolizing foreign compounds via a variety of metabolic pathways. This process i s influenced by the accessibility of agents to the microbial flora of the large intestine due to physicochemical, physiological, o r pathological factors. This metabolism may result in the formation of pharmacologically and/or toxicologically active products.
BOXENBAUM E T AL.
278 Acknowledgments
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The clinical studies with salicyluric acid were conducted by Dr. M. M. Reidenberg and supported by grant NIH RR 47 from the National Institute of Health.
REFERENCES R. R. Scheline, Pharmacol. Rev., 25, 451 (1973). R. R. Scheline, J. Pharm. Sci., 57, 2021 (1968). J. S. Lockwood and H. T. Randall, Bull. N.Y. Acad. Med., 25, 228 (1949). J. B. Carey, Jr., in Diseases of the Liver, 3rd ed. (Schiff, ed.), Lippincott, Philadelphia, 1969, pp. 103-146. S. A. Broitman and R. A. Giannella, in Topics in Medicinal Chemistry: Absorption Phenomena, Vol. 4 (J. L. Rabinowitz and R. M. Myerson, eds.), Wiley-Interscience, New York, 1971, pp. 265-321. B. S. Drasar, M. Shiner, and G. M. McLeod, Gastroenterology, 56, 71 (1969). S. L. Gorbach and R. Levitan, in Progress in Gastroenterology, Vol. 2 (G. B. J. Glass, ed.), Grune and Stratton, New York, 1970, pp. 252-275. B. S. Drasar, M. J. Hill, and R. E. 0. Williams, in Metabolic Aspects of Food Safety (F. J C. Roe, ed.), Blackwell, Oxford, 1970, pp. 245-260. I. S. Eve, Health Phys., 12, 131 (1966). H. G. Boxenbaum, G. S. Jodhka, A. C. Ferguson, S. Riegelman, and T. R. MacGregor, J. Pharmacokinet. Biopharm., -2, 211 (1974). W. C. Hlilsmann and L. W. S. van Eps, Clin. Chim. Acta, 15, 233 (1967). I. Bekerslg, H. G. Boxenbaum, M. H. Whitson, C. V. Puglisi, R. Pocelinko, and S. A. Kaplan, Anal. Lett., I_o, 539 (1977). N. Svartz, Acta Med. Scand., 110, 577 (1942). AMA Drug Evaluations, 2nd ed., Publishing Sciences Group, Acton, Massachusetts, 1973, pp. 562-563. K. M. Das. M, A. Eastwood, J. P. A. McManus, and W. Sircus,-N. Engl. J. Med., 289, 491 (1973). H. Schroder and D. E. S. Campbell, Clin. Pharmacol. Ther., 13, 539 (1972).
.
INFLUENCE OF GUT MICROFLORA ON BIOAVAILABILITY
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[181 C191
C231
[241 1251 C261
279
P. Goldman and M. A. Peppercorn, Gastroenterology, 65, 166 (1973). H . Schroder and D. A. P. Evans, Gut, 1_3, 278 (1972). K. M. Das, M. A. Eastwood, J. P. A . McManus, and W. Sircus, Wd., 14, 637 (1973). R. Walker, Food Cosmet. Toxicol., S, 659 (1970). J. M. Price, C. G. Biava, B. L. Oser, E. E. Vogin, J. Steinfeld, and H. L. Ley, Science, 167, 1131 (1970). A. G. Renwick and R. T. Williams, Biochem. J., 129, 869 (1972). B. S. Drasar, A. G. Renwick, andR. T. Williams, E d . , 123, 26 (1971). C G . Renwick and R. T. Williams, E d . , 129, 857 (1972). B. S. Drasar, A. G. Renwick, and R. T. Williams, K d . , 129, 881 (1972). S. Riegelman and M. Rowland, J. Pharmacokinet, Biopharm., -1, 419 (1973). R. R . Scheline, Acta Pharmacol. Toxicol., 2, 275 (1966).
DRUG METABOLISM REVIEWS, 9(2), 259-279 (1979)
Influence of Gut Microflora on Bioavailability* H. G. BOXENBAUM, I. BEKERSKY, M. L. JACK, AND S. A. KAPLANt Department of Pharmacokinetics 'and Biopharmaceutics Hoffmann-LaRoche Inc. Nutley, New J e r s e y 07110 I. 11. 111.
IV.
.................................... ACCESS OF COMPOUNDS TO THE HUMAN INTESTINAL FLORA ................................ EXAMPLES OF BACTERIAL INFLUENCE ON BIOAVAILABILITY .............................. A. Isonicotinuric and Salicyluric Acids ............... B. Sulfasalazine (Salicylazosulfapyridine, SAS) and Other Azo Compounds ................... C. Cyclamate ...................................... CONCLUSIONS ...................................... Acknowledgments ................................... References ......................................... INTRODUCTION
260 261 264 264 270 273 277 278 278
*Presented at Symposium on Drug Disposition in Man held in Sarasota, Florida, November 6-11, 1977 under the auspices of the American Society for Pharmacology and Experimental Therapeutics. +To whom requests for reprints should be addressed.
259 Copyright 0 1979 hy Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage' and retrieval system, without permission in writing from the publisher.
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I. INTRODUCTION The relationship between the human intestinal flora and host is of great importance, It i s well established that intestinal bacteria play a role in normal physiological processes and can contribute to pathological states. Additionally, it has become increasingly apparent that microbial flora a r e capable of metabolizing a variety of foreign or drug substances, thus potentially influencing and/or altering drug activity and toxicity, Metabolism by microorganisms in the gastrointestinal tract may occur after drug administration, thereby affecting the bioavailability and physiological disposition profile of the drug. Such metabolic activity in the gastrointestinal tract is influenced by many factors. These factors include delayed absorption which may be due to the intrinsic properties of the drug or an effect of the dosage form, diffusion or secretion of drug and/or metabolite into the gastrointestinal tract, and/or drug action on the gastrointestinal microflora per s e with subsequent enhanced metabolism, i. e., metabolism adaptation. Therefore, gastrointestinal microbial metabolism studies must consider distribution of microorganisms within the human gastrointestinal tract, the rate and extent of drug absorption, and the gastrointestinal transit time. Pharmacological and toxicological implications of gastrointestinal microbial metabolism reflect alteration in metabolism following chronic drug administration, drug activation, increase in anerobes in the upper gastrointestinal tract due to disease states, and gastrointestinal disorders resulting from antimicrobial drug therapy, Whereas most tissue metabolic processes result in more hydrophilic derivatives which a r e generally eliminated from the body more rapidly than the parent drug, gastrointestinal microbial metabolism often results in the formation of more lipophilic derivatives due to hydrolysis o r reduction of glycosidic linkages, deconjugations, dehydroxylations, and decarboxylations. Such lipophilic metabolites tend to be absorbed into the systemic circulation and therefore can exert pharmacological and toxicological effects. This series of events may be the physicobiochemical basis of enterohepatic recycling of drug. Extensive reviews of drug metabolism via intestinal microorganisms a r e presented by Scheline [ 1, 21. Some of the metabolic conversions known to occur in the gastrointestinal tract a r e reported in Table 1. This presentation will focus on some of the biopharmaceutical and pharmacokinetic aspects of drug metabolism by the microorganisms of the gastrointestinal microflora.
INFLUENCE OF GUT MICROFLORA ON BIOAVAILABILITY
261
TABLE 1 Some Metabolic Conversions Known to Occur in the Gastrointestinal Tract. Adapted from Ref. 1
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Reaction
Specific groups metabolized
Hydrolysis
Glycosides, glucuronides, sulfate esters, amides, esters sulfamates, nitrates
Dehydroxy lation
C- Hy droxy1, N- hydroxy 1
Decarboxylation Dealkylation
0-Alkyl, N-alkyl
D ehalogenation Deamination Reduction
Double bonds, nitro groups, azo groups, aldehydes, ketones, alcohol, N-oxides
11. ACCESS O F COMPOUNDS TO THE HUMAN INTESTINAL FLORA Compounds may be exposed to intestinal microorganisms via diffusion o r secretion into the gastrointestinal tract. A factor generally not appreciated is that a "typical" human subject secretes 8.2 liters of fluids daily into the gastrointestinal tract, a s illustrated in Fig. 1. This includes 1 . 5 liters saliva, 0.5 liter bile, 2.5 liters gastric juice, 0.7 liter pancreatic juice, and 3.0 liters of intestinal secretions [ 31. One example of a compound which is exposed to the microflora by this general secretion mechanisms is the bile acid, glycocholic acid [ 41. This glycine conjugate is excreted via the bile into the small intestine, where some escapes reabsorption from the ileum and passes into the colon, wherein the glycine moiety is metabolically removed by bacteria. Compounds may also be exposed to gastrointestinal flora following oral ingestion. The most important factors that can influence whether o r not a compound undergoes microbial alteration is the distribution of microorganisms within the human gastrointestinal
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BOXENBAUM ET AL.
FIG, 1. Secretion of fluids into the gastrointestinal tract of a typical human subject. Adapted from Ref. 3.
tract. Scheline [ 13 reviewed much of the literature in this area and indicated that the stomach, duodenum, jejunum, and upper ileum a r e only sparsely populated. Increasing numbers of organisms exist in the distal ileum, and a significant increase is seen a t the ileocecal valve. The numbers and distribution of organisms a r e illustrated in Fig. 2. It should be noted that most of the organisms associated with the stool, and therefore presumably the large intestine, a r e obligate anerobes. In excess of 99% of the total cultivable fecal flora of normal adults are obligate, nonsporing anerobes. It has been determined that a least 20% of the mass of the feces consists The presence of anerobes in feces requires of live bacteria [6-83. that specimens for in vitro studies must be maintained under a 10% H,-90% N, mixture.
INFLUENCE O F GUT MICROFLORA ON BIOAVAILABILITY
263
l0l2
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10 lo
10
8
10
10
10
O0
E?j
ANAEROBES BACTEROIDES 8 RELATED SPECIES, LACTOBACILLI CLOSTRlDlA
El COLIFORMS
M
GRAM
PosinvE
FLORA
STREPTOCOCCI, LACTOBACILLI, STAPHYLOCOCCI, YEAST
FIG. 2. Diagrammatic scheme of microbial populations of the human gastrointestinal tract. Taken from Ref. 5.
Another factor influencing accessibility of a foreign compound to microbes i s the extent and rate of absorption of the compound. Obviously, any compound which is rapidly and completely absorbed from the upper gastrointestinal tract will not be exposed to the microflora unless subsequently secreted back into the tract. Therefore, compounds exhibiting poor absorption characteristics will have the greatest exposure potential to the intestinal flora. Examples to be discussed include compounds having an azo bond o r a glycine conjugate, both of which tend to be poorly absorbed from the upper gastrointestinal tract. The time course of microbial transformation following oral administration will be partially dependent on gastrointestinal transit time. Eve [ 9 1 reviewed gastrointestinal transit time, including observations of mean passage times of food residues through the stomach, small intestine, upper large intestine, and lower large intestine. On an average, food residues reach the microflora of the upper large
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BOXENBAUM E T AL.
intestine after 5 hr. Thus one could generally expect some delay following drug ingestion before microbial metabolism would occur. It should be noted, however, that these transit times represent movement of unabsorbed food residues in an average man consuming an average diet. Transit of an unabsorbed drug taken after an overnight fast would probably occur much more rapidly. Drug development study designs, therefore, should consider gastrointestinal microbial metabolism. Such metabolism may influence the absorption, distribution, and excretion profiles of drugs. There may be differences in quantity of microorganisms in fasted vs fed animals. The observations based on large experimental doses in animal may not be predictable of small therapeutic doses in man. Variability in number, type, and location of microorganisms may exist, Variability in biliary excretion may result from regeneration of parent compound in the intestine o r liberation of various metabolites which may undergo further metabolism. Microbial metabolism may be responsible for the prolonged retention of drug o r metabolite in systemic circulation. In this regard, the influence of bacterial metabolism on the bioavailability and pharmacokinetic profiles of isonicotinuric acid, salicyluric acid, sulfasalazine, and cyclamate will be discussed.
111. EXAMPLES OF BACTERIAL INFLUENCE ON BIOAVAILABILITY A. Isonicotinuric and Salicvluric Acids The hydrolysis of glycine conjugates by gastrointestinal bacteria is well documented I:1, 2, lo]. Isonicotinuric, salicyluric, p-aminohippuric, and p-acetylaminohippuric acids appear to be hydrolyzed by gut bacteria following oral administration [ 10, 111. The structure of isonicotinuric acid (INU) and isonicotinic acid (INA) a r e presented in Fig. 3. Following intravenous administration of INU to human subjects, intact INU is recovered in the urine, indicating no systemic hydrolysis of INU to INA [ l o 1 However, when INU i s administered orally on an empty stomach, approximately 7% of the dose was absorbed intact from the stomach and/ o r small intestine, The remaining unabsorbed material then passed to the lower gastrointestinal tract where bacteria hydrolyzed it to INA. This latter compound was subsequently absorbed into the systemic circulation, where some was reconjugated with glycine, forming the originally administered compound, INU. Urinary excretion rate curves for one subject in two studies at oral solution doses of 129 and 476 mg a r e illustrated in Fig. 4.
INFLUENCE O F GUT MICROFLORA ON BIOAVAILABILITY
0
265
0
II
II
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C -OH
6 6 N
N
lsonicotinuric Acid (INU)
lsonicotinic Acid (INA)
FIG. 3. Structures of isonicotinuric and isonicotinic acids. Dose: 129 mg
100.0
RATIO:
Dose: 476 mg
m3,
RATIO:
100.0
INU
50.0
w, INU
50.0!
m-- -m INA
10.0:
z
0 W
K 0 X U
1.0:
4
0 W
I-
a K
0.5’
1
5
10
15
20 25
30
J
5
10
15
20 25
TIME ( HOURS 1 FIG. 4. Urinary excretion rate c u r v e s following o r a l administration of isonicotinuric acid (INU) to a healthy human subject. Isonicotinic acid (INA) rates e x p r e s s e d in t e r m s of m o l e equivalents INU. Adapted f r o m Ref. 10.
30
266
BOXENBAUM ET AL.
INA
RAT10: INU
r
1
..-..
~
'
r
INA
KANAMYCIN PRE TREATMENT
LlNOMYClN PRE -TREATMENT
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10.01
7 5.0 '
L
a K
.'
5
10
15
20
25
5
10
15
20
25
TIME ( HOURS 1
FIG. 5. Influence of antibiotic pretreatment on the urinary excretion rate curves following oral administration of approximately 125 mg isonicotinuric acid (INU). Isonicotinic acid (INA) rates expressed in terms of mole equivalents INU. Taken from Ref. 10.
Initially INU was observed in the 0 to 5 h r postadministration interval. Subsequently, the unabsorbed INU which continues down the gastrointestinal tract was converted to INA. The larger the dose of INU, the more pronounced the effect, a s indicated by the INA/INU ratios, equivalent to one in the first study and greater than one in the second study. The urinary excretion profiles indicate that once in systemic circulation, INA i s then reconjugated with glycine to form INU a s demonstrated by the increase in INU urine levels observed in the excretion curves. In order to confirm that microorganisms were responsible for the gastrointestinal hydrolysis of INU to INA, studies were undertaken in which the gastrointestinal flora were eliminated. Anerobic bacteria can be largely eliminated with little o r no change in the aerobes by administration of lincomycin, whereas anerobes a r e not destroyed with kanamycin [11. The urinary excretion profiles following the pretreatment of subjects with kanamycin and lincomycin a r e presented in Fig. 5. The microbial metabolism of INU was confirmed,
INFLUENCE O F GUT MICROFLORA ON BIOAVAILABILITY Dose:
Ratio:
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5.0[
267
49 rng (rectal)
:;,"
->
1
. 5
10
15
20
TIME (HOURS]
FIG. 6. Urinary excretion rate curves following rectal administration of isonicotinuric acid (INU). Isonicotinic acid (INA) expressed in terms of mole equivalents INU. Taken from Ref, 10. since pretreatment with lincomycin destroyed the anerobic microorganisms and thereby prevented the formation of INA. Kanamycin, which did not destroy anerobes, was ineffective in obliterating the metabolism to INA. The excretion rate profiles of INU and INA following rectal administration of INU a s a retention enema are presented in Fig. 6. The urinary excretion profiles indicate immediate and very extensive INA formation, the ratio of INA/INU being much greater than one for the small dose of 49 mg INU.
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5.0
ti 'I
1.0. D
-
0.5
0.1
i
0.05. 5
10
15
20
25
TIME (HOURS)
FIG. 7. Influence of propantheline administration on the urinary excretion rate curves following oral administration of isonicotinuric acid (INU). Isonicotinic acid (INA) rates expressed in terms of mole equivalents of INU. Taken from Ref. 10.
The same subject was given multiple doses of the antichloinergic agent, propantheline, and the resulting urinary excretion profile is reported in Fig. 7. This multiple dose pretreatment with propantheline resulted in prolonged gastrointestinal transit time of INU, allowing for more extensive INU absorption from the upper gastrointestinal tract and delayed onset of INA formation.
269
INFLUENCE O F GUT MICROFLORA ON BIOAVAILABILITY TABLE 2
Hydrolysis of INU Following 12 h r Anerobic Fecal Incubation Studies.a Taken from Ref. 1 0
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Percent recovery Sample contents Medium + INU Medium + INU + feces Medium + INU + feces + antibiotics Medium + INA + feces
INU
98.5 2.80 92.6 0
INA 0
91.8
4.41 96.3
a
Incubation medium consisted of phosphate buffer, glucose, yeast extract, and peptone a s described by Scheline [27].
The results of in vitro anerobic fecal incubations on the hydrolysis of INU a r e reported in Table 2. The INU was shown to be stable in the medium per se, but to be extensively hydrolyzed when incubated anerobically with feces. In the presence of antibiotics, the anerobic metabolism of INU was essentially eliminated. These results further confirm that the microorganisms responsible for the hydrolysis of INU a r e anerobic. Salicyluric acid ( M U ) , like INU, undergoes gastrointestinal microbial metabolism to salicylic acid (SA) prior to absorption, Following 3 mg/kg SAU i.v. administration, only SAU plasma levels a r e observed; hydrolysis of SAU to SA did not occur [12]. SAU was also administered orally at 3 mg/kg to a healthy human subject, and the plasma levels profile a r e presented in Fig. 8. It was estimated that approximately 80% of the SAU dose was absorbed intact from the upper gastrointestinal tract, and that the remaining SAU apparently passed down to the large intestine and was subjected to microbial hydrolysis to SA. It should be noted that formed SA, following absorption will be further metabolized to SAU in vivo. However, due to the rapid elimination rate of SAU and the sensitivity limitation of the assay, no prolonged SAU levels were observed a s with INU.
BOXENBAUM ET AL.
270 10 7
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5
-
SUBJECT A
.Salicyluric
3
Acid (SAU)
8-4
E
'
2
2
Y
c
0
4
4 J 1 a &
2
0.1
'5
0.5
1
0.3
a
0
8 '
ld
/
d
nl
\
/
\ \
/
0.2
\
/
b
/
/
\
I
\
\
6
0.1 0.01
I
I
10
I
20
1
30
40
1
50
Time (hours)
FIG. 8. Salicyluric and salicylic acid plasma concentration-time curves following oral administration of 3 mg/kg of salicyluric acid to a healthy subject.
B.
Sulfasalazine (Salicylazosulfapyridine, SAS) and Other Azo Compounds
Salicylazosulfapyridine (SAS) is used clinically in the treatment of ulcerative colitis [IS]. The structure of sulfasalazine and the bacterial metabolic products, 5-aminosalicylic acid and sulfapyridine, a r e presented in Fig. 9 [ 143. Following sulfasalazine administration, Das et al. (151 report some sulfasalazine is absorbed from the
1
60
INFLUENCE OF GUT MICROFLORA ON BIOAVAILABILITY
271
SULFASALAZINE
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HOOF
BACTERIAL A 2 0 REDUCTASE
S .AM I NOSALlCY LI C ACI D
SU LFAPY R I DIN E
FIG. 9. Sulfasalazine and bacterial metabolic products. Adapted from Ref. 14.
small intestine, but most of the drug reaches the cecum where splitting of the azo linkage occurs, yielding sulfapyridine and 5-aminosalicylic acid. Sulfapyridine is then absorbed from the colon and i s subsequently acetylated and conjugated to the glucuronide in the liver prior to excretion in the urine. The 5-aminosalicylic acid metabolite is also absorbed and is excreted in the urine a s both intact 5-aminosalicylic acid and as acetyl 5-aminosalicylic acid. Schrdder and Campbell [ 161 administered SAS at a dose of 5 g/ day to nine healthy subjects over a period of 1 0 days. SAS and sulfapyridine serum concentration-time curves in a typical subject following the first 4 g-dose are presented in Fig. 10. Approximately 85% of the administered dose was recovered in the urine and about 5% in the feces. The urinary excretion of sulfasalazine ranged from 1.7 to 10% of the administered dose in the nine subjects. About 40 to 50% of the dose was excreted in the urine a s sulfapyridine and its metabolites; approximately 30% of the dose was excreted a s 5-aminosalicylic acid and its metabolites. The feces consisted of sulfapyridine and metabolites of 5-aminoalicylic acid; no intact sulfasalazine was detected in the feces. These results demonstrate the efficiency of the bacteriological metabolism of sulfasalazine. Since sulfapyridine is present in the colon, it is conceivable that this agent is active by virtue of its antibacterial properties. In
BOXENBAUM ET AL.
272
100
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70
4
d
50
Sulfapyridine
\
\
I
!30 2E
\
w 20
E
0
u
5a
2
10 7
5
I
I
4
I
I
8
1
I
12
I
I
16
I
I
I
20
Time (hours)
FIG. 10. Sulfasalazine and sulfapyridine serum concentration-time curves following single dose oral administration of 4 g to a healthy volunteer. Data taken from Ref. 18.
reviewing the literature, however, Goldman and Peppercorn [ 171 were cautious in their appraisal of the possible beneficial effects of this sulfonamide. SAS therapy has only a minor influence on the bacterial constituents of the fecal flora. It was noted, however, that currently available methods a r e not capable of detecting changes in the host-microflora relationship a t the mucosal level. The influence of gastrointestinal flora on SAS has toxicological implications since observed adverse effects have been correlated with sulfapyridine serum levels. Toxicity did not correlate with either intact SAS or 5-aminosalicylic acid levels r15, 18, 191. Additional work has also been reported on other azo compounds. Walker [20] reviewed much of the literature on the fate of these compounds in man and other species. It was noted that compounds
1 24
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INFLUENCE O F GUT MICROFLORA ON BIOAVAILABILITY
273
in this group a r e the most common synthetic colorings used i n foods, pharmaceuticals, and cosmetics. Regarding metabolism, intestinal azo reduction systems a r e considerably more active and nonspecific than the hepatic azo reductase in animals. Twenty-one species of bacteria were screened as to their ability to reduce 14 azo compounds. Some of the bacteria were normal constituents of the mammalian gut, and overall, azo-reducing activity was fairly general and relatively nonspecific. C.
Cyclamate
Cyclamic acid (cyclohexylsulfamic acid) a s its sodium o r calcium salt had been widely used as an artificial sweetening agent. In 1969 it was removed from the United States market because its metabolite, cyclohexylamine, was suspected of being a bladder carcinogen in rats [ 211. Cyclamic acid is a strong acid with a pKa of 1.9 and is poorly absorbed, resulting in a prolonged residence time of the compound in the gastrointestinal tract. Figure 11 illustrates the metabolic fate of cyclamic acid in man [ 22-25]. Following single dose oral administration to three humans on a cyclamate-free diet, intact compound was primarily excreted unchanged in feces and urine. Approximately 30% of the dose was found in urine and 50977 in feces. Trace amounts of cyclohexylamine (< 0.002% of dose) were also detected in urine. In additional studies, the same three subjects were placed on a diet containing cyclamate for a period up to 30 days. The results reported in Table 3 indicate that on continued cyclamate exposure, cyclohexylamine urinary levels increased in the three subjects approximately 26-, 80-, and 8,650-fold. Thus there appeared to be variability, particularly in subject B. S.D. whose cyclohexylamine urinary levels increased dramatically. Removal of cyclamate from the diet resulted in a decreased conversion to cyclohexylamine. Thus there appears to be a reversible, self-inducible metabolic transformation of cyclamic acid to cyclohexylamine in man. Drasar [25] has shown that the metabolic conversion of cyclamic acid to cyclohexylamine in rats does not occur following either parenteral administration of cyclamate o r with incubations of cyclamate with the liver, spleen, kidney, o r blood preparations as shown in Table 4. These results indicate the cyclohexylamine formation occurs solely in the gut as the result of microorganism metabolism; that when cyclamate was incubated with the feces of a cyclamate pretreated %onverter, cyclohexylamine w a s formed; and when cyclamate was removed from the diet, the fecal organisms showed less ability to form cyclohexylamine.
BOXENBAUM ET AL.
27 4
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ORAL ADMINISTRATION
Cyclamic A c i d
Urinary Excretion (S30%)
Fecal Excretion
-Gut Flora
(
z: 50%)
6
Cyclohexylarnine
I
Systemic Absorption
Urinary k c r e t i o n (Major Pathway)
6
Cyclohexylamine
6"" OH
4-
Trans-cyclohexane-l,2-diol
(yoH Cyclohexanol
(Minor Metabolites)
FIG. 11. Metabolic degradation of cyclamic acid by bacterial flora. Adapted from Refs. 22 and 24.
INFLUENCE OF GUT MICROFLORA ON BIOAVAILABILITY
275
TABLE 3
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Cyclohexylamine Levels in the Urine of bbbjects Treated with Cyclamatea
Time on cyclamate (days)
Cyclohewlamine in urine (YJof dose) of three subjects B. S. D. P. C. H. A.G.R.
0
< 0.002
< 0.002
< 0.002
1
0.07
< 0.002
0.003
5
5.8
0.005
0.013
10
17.3
0.015
0.014
12
13.5
0.005
0.007
15
16.4
0.034
0.071
20
-
0.053
0.160
25
-
0.052
0.140
0.014
0.050
26 27 28
a
Three human subjects received 3 g of calcium cyclamate each day. Subject B. S.D. discontinued the daily dose of cyclamate on Day 18 and remained on a cyclamate-free diet. On Day 26, this subject received 3 g of calcium cyclamate. Taken from Ref. 22.
In summary, orally ingested cyclamate is primarily excreted in urine and feces intact. Moderate cyclamate exposure in the diet increases the ability of gastrointestinal microorganisms to convert cyclamate to cyclohexylamine. Microbially produced cyclohexylamine is then absorbed from the large intestine and is primarily excreted intact in the urine. Trace amounts of two metabolites,
3.25 2.40
-
-
-
1.94 0.62
-
0.78 0.43
0.3 33.6 34.0
-
-
0.3 61.5 69.6
-
-
-
0.4 54.0 14.9
-
-
-
-
16
14
13
-
< 0.05 < 0.05
< 0.05
< 0.05
< 0.01
Normal animals
a The r a t s were on a cyclamate diet for 4 to 4.5 months prior to receiving 100 mg of calcium cyclamate administered orally. The cyclamate diet consisted of allowing the r a t s free access to food but their water contained 0.5% (w/v) calcium cyclamate. Taken from Ref. 25.
Contents of: Duodenum-ileum (H2) Caecum (H2) Colon-rectum (H2)
Hind-gut contents: Anerobic (N2) Aerobic (air)
< 0.05
< 0.05
Liver, kidney, spleen, blood
< 0.05
< 0.05
< 0.05
< 0.05
Controls
43 21
33 30
22 60
Rat no.
Whole animal
Material tested
(o/o of administered o r added cyclamates)
Conversion into Cyclohexylamine
Metabolism of Cyclamate by Rat Tissues and Gut Contentsa
TABLE 4
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INFLUENCE OF GUT MICROFLORA ON BIOAVAILABILITY
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TABLE 5
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Drugs in Which Iuminal and/or Mucosal Metabolism I s Suggested. Taken from Ref. 26 Conjugation: Estrogens Salicylamide L-Dopa 2- Methyldopa
Ester hydrolysis: Acetylsalicylic acid Organic nitrates Propoxyphene Meperidine Methadone Penta zoc ine Dexamethasone phosphate
Reduction: Hydrocortisone Cortisone A ldoste rone Progesterone Testerone
N-acetylation: p-Aminobenzoic acid Certain sulfonamides Amide hydrolysis: p-Aminohippuric acid Hippuric acid
trans-cyclohexane-l92-diol and cyclohexanol, have also been detected. When cyclamate is removed from the diet, the ability of the gastrointestinal flora to produce cyclohexylamine is considerably reduced. Many drugs a r e suspected of undergoing metabolism by microorganisms in the gastrointestinal tract and/or by gastrointestinal mucosa prior to absorption. A list of such drugs, compiled by Riegelman, is given in Table 5 .
IV.
CONCLUSIONS
In conclusion, it has been demonstrated by several investigators that gastrointestinal flora are capable of metabolizing foreign compounds via a variety of metabolic pathways. This process i s influenced by the accessibility of agents to the microbial flora of the large intestine due to physicochemical, physiological, o r pathological factors. This metabolism may result in the formation of pharmacologically and/or toxicologically active products.
BOXENBAUM E T AL.
278 Acknowledgments
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The clinical studies with salicyluric acid were conducted by Dr. M. M. Reidenberg and supported by grant NIH RR 47 from the National Institute of Health.
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INFLUENCE OF GUT MICROFLORA ON BIOAVAILABILITY
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