Clinical Science (1993) 84, 675-679 (Printed in Great Britain)
675
Localization of cyclosporin A absorption in rat small
bowel and the effect of bile
Yilmaz CAKALOGLU, George MARINOS, Joanne MARSDEN*, Timothy j. PETERS*,
Roger WILLIAMS and j. Michael TREDGER
Institute of Liver Studies and *Department of Clinical Biochemistry, King's College Hospital and
King's College School of Medicine and Dentistry, London, U.K.
(Received 17 November 1992/21 February 1993; accepted 23 February 1993)
1. Cyclosporin A absorption was examined after the
instillation of approximately 2 mg/kg into four segments (mean length 15cm) of rat small bowel,
isolated in situ in fed Wistar female rats: SI (duodenum and proximal jejunum distal to the common bile
duct); SII (distal jejunum); SIII (proximal ileum) and
SIV (distal ileum).
2. Cyclosporin A concentrations in whole blood were
assayed by an enzyme-mediated immunoassay for up
to 4 h in samples drawn from the femoral vein and
used to determine the following pharmacokinetic parameters: the area under the blood cyclosporin A
concentration versus time curve (AUC, 0-4 h), the
maximum blood concentration of cyclosporin A
(C,,), the time to reach Cmx.(t,,), the absorption
half-life (ti,), the elimination half-life (tii), and the
mean residence time (MRT).
3. Cyclosporin A absorption in SI (AUC, 991p g I-' h)
was nearly double that in more distal segments and
decreased progressively (SII, 533pg I-' h; SIII,
470pg l-'h; SIV, 419pg P'h). There were corresponding differences in Cm,: 327pg/l in SI and
201 &I, 169pg/l and 151pg/l in SII, SIII and SIV,
respectively. T
, was shorter in SIV (0.9h) than in
other segments (1.3-1.5h), but there were no significant differences between the segments for ti,, tii or
MRT.
4. In the presence of continuous bile flow (diverted
via a cannula for SIV), cyclosporin A absorption
significantly increased by 23% in SI and by 50% in
SIV, but the differential between absorption in SI and
SIV was maintained.
5. We conclude that cyclosporin A is absorbed
throughout the rat small intestine with the greatest
absorption rate in the proximal duodenum and jejunum, and that bile significantly augments cyclosporin
A absorption in both the proximal and particularly
the distal small bowel.
INTRODUCTION
The intestinal absorption of cyclosporin A (CyA)
is highly variable with a low mean bioavailability of
30% after oral administration in man [l, 21. Experimental [3, 41 and clinical [5,6] studies have shown
that bile flow and the length of small bowel are the
most important determinants of CyA absorption,
and studies in situ in rats and rabbits [3, 71 support
the hypothesis that CyA is mainly absorbed from
the upper small intestine [a]. Similar conclusions
were drawn by Drewe et al. [9], who reported
recently that CyA is predominantly absorbed in the
small bowel when administered locally to different
parts of human gastrointestinal tract. However, it is
unclear whether CyA is equally absorbed in all
parts of the small bowel and whether any differences
result from the amounts of bile present in the upper
and lower part of the small intestine. Because of
important clinical implications for patients with
intestinal disorders or after surgery, we have investigated the extent of CyA absorption in different
segments of small bowel and have studied the effect
of bile in a rat model of CyA absorption.
MATERIALS AND METHODS
Animals and protocol
Fed Wistar female rats weighing 275-325 g were
anaesthetized by intraperitoneal administration of
fentanyl/fluanisone (Hypnorm; Janssen Pharmaceuticals) and midazolam (Hypnovel; Roche) in
water (1:4). After laparotomy, the target segment of
small bowel was identified (see below) and both
ends were carefully ligated to preserve the vascular
supply. The femoral vein was cannulated with a
22 G Wallace cannula which was heparinized to
permit withdrawal of blood samples. Approximately
2mg/kg CyA, prepared by suspending 50pl of
100mg/ml Sandimmun oral CyA solution (Sandoz,
Key words: bile, cyclorporin A, intestinal absorption, pharmacokinetia.
Abbreviations: AUC, area under the blood cyclorporin A concentration versus time curve; CyA, cyclorporin A EMIT, enzymemediated immunoassay; MRT, mean residence
time.
Correspondence: Dr J.Michael Tredger, Institute of Liver Studies, King's College School of Medicine and Dentistry, Berremer Road, London SE5 9PJ, U.K.
676
Y. Cakaloglu et al.
Camberley, Surrey, U.K.) in lOml of Hepes buffer
(150mmol/l NaCl, 10mmol/l Hepes, pH 7.9, was
injected transmucosally into the target segment
using a 27 G hypodermic needle. Blood samples
(200pl) were drawn from the femoral vein at 15, 30,
45, 60, 90, 120, 150, 180, 210 and 240min into
EDTA. Immediately after taking the last blood
sample, rats were killed and the small bowel was
removed to measure the length of segment and the
complete small bowel. There were no macroscopic
changes in the appearance of the small bowel at the
end of the procedure.
-- o
-
0.
"1
A
OJ
0
30
60
90
I20
Ducdcnum +pmximd jejunum
Diiul jejunum
A P r o x i d ileum
150
180
210
240
Time (min)
Isolation of small bowel segments in sku
The length of the whole small bowel from the
pyloric sphincter to the end of the ileum measured
between 60 and 70cm. The duodenum, from the
pyloric sphincter to the ligament of Treitz, measured
approximately 10cm. No visual distinction could be
made between jejunum and ileum. From the ligament of Treitz, the proximal 25cm of the small
bowel was designated as jejunum (25 cm) and the
remainder (35-40 cm) as ileum. CyA absorption was
studied in four adjacent segments (length 13-17 cm,
mean 15cm): duodenum from distal to the common
bile duct plus proximal jejunum (SI, n = 10); distal
jejunum (SII, n=6); proximal ileum (SIII, n=6);
distal ileum (SIV, n=9).
Effect of bile flow
The same experimental procedure was repeated in
the presence of normal bile flow into SI ( n = 5 ) and
SIV (n=5). For SI the top ligature was placed just
above the common bile duct allowing bile to flow
freely into the segment during the entire procedure.
For SIV, bile flow was diverted from the common
bile duct into the distal ileum via a Portex PPlO
cannula. Bile flow for the 4 h period was relatively
constant at 750 pl/h (n= 3).
CyA assay and pharmacokinetic analysis
The concentration of CyA in whole blood was
measured by an enzyme-mediated immunoassay
(EMIT; SYVA U.K., Maidenhead, Berks, U.K.),
which determines parent CyA concentrations specifically. The area under the blood CyA concentration
versus time curve (AUC) for 4h, the absorption
half-life (ria), the elimination half-life (ti;,), the maximum blood concentration of CyA (C,,,), the time
to reach C,,, (rmax,) and the mean residence time
(MRT) were calculated with the Strip curve-fitting
program [lo]. Experimental groups showed homo-
Fig. 1. Whole blood concentrations of CyA after absorption from
different segments of rat small intestine. Each point shows the mean
for the number of determinations detailed in the Materials and methods
section.
geneous variances and statistical comparisons were
performed using an unpaired Student's t-test (SPSS,
Chertsey, Surrey, U.K.) with P values of <0.05
indicating significance.
RESULTS
Blood concentration profiles for the four segments, presented as mean values in Fig. 1, showed a
significantly higher AUC over 4 h in duodenum and
proximal jejunum (SI, AUC 991 pg l - ' h ) than in
the other segments (419-533pg l - ' h , Table 1).
AUC values in distal jejunum (SII), proximal ileum
(SIII) and distal ileum (SIV) decreased progressively
to 54%, 47% and 42% of that in the SI, respectively,
but were not significantly different from each other
(Table 1). The mean maximum CyA blood level
(C,,,,) was also higher in SI (327pg/l) than in SII
(201 pg/l, P ~ 0 . 0 5 )or SIII and SIV (169pg/l and
151 pg/l, respectively, P < O.OOOl), but t,,,. was significantly (P<O.O5) shorter in distal ileum (SIV, 0.9h)
than in other segments (1.3-1.5 h, Table 1). CyA was
rapidly absorbed from each segment of small bowel
with a negligible lag time and was detected in all
15 min blood samples. Mean values of the length of
the segments, cia, tij, and MRT were not significantly
different between segments I-IV, although values of
tij, and MRT were at least 30% greater in the distal
ileum than in more proximal segments.
Effect of bile
The diversion of endogenous bile flow into SI and
SIV considerably augmented CyA absorption
(Fig. 2) leading to a significant (P<0.05) increase in
,,,
mean AUC (23% in SI, 50% in SIV) and mean C
(27% in SI, 42% in SIV) (Table 2). Bile had no
significant effect on t,,,,, ti., rjj. or MRT in SI
(Table 2). In SIV, all four parameters increased in
Cyclosporin A absorption in rat small bowel
Table I. Pharmacokinetic parameters of CyA absorption in different segments of rat small bowel. All values are shown as
means fSD. SI, duodenum plus proximal jejunum; Sll, distal jejunum; SIII, proximal ileum; SIV. distal ileum. Statistical significance:
Y<O.OS versus SIV, bP<0.W5 versus Sll. cP<O.OW1 verus Slll and SIV, dP<O.OW1 SI versus other segments.
15.5fl.2
15.Of 1.3
14.5+ 1.5
15.4f1.3
SI
Sll
Slll
SIV
3.6 & 1.5
2.2f 1.1
2.9 f I.O
4.1 f 3.8
0.3f0.1
0.5f0.2
0.5f0.3
0.3f0.2
5.8 f 2.0
4.1 f 1.4
4.7 f I.4
7.0 f 5.3
1.3f0.3’
I .5 f 0.5’
1.5f0.5’
0.9 f0.3
327f4Sb*(
201 f 93
169f81
151 f22
991 f W d
533 & 203
470 f 21 6
419f48
Table 2 Effect of endogenous bile flow on CyA absorption in duodenum plus proximal jejunum (SI) and distal ileum
(SIV). All values are shown as means f SD. Statistical significance: ‘Pc0.05 versus without bile; bPc0.05 cPcO.O1 and dP<0.005
versus without bile; cP<0.05 SI versus SIV with bile; ‘P=O.O6 SI versus SIV with bile.
SI
Bile (-)
Bile (+)
SIV
Bile (-)
Bile (+)
0.3fO.l
0.5 f0.2
3.6f 1.5
3.6f 1.3
5.8 f 2.0
5.9 f I .7
1.3f0.3
1.5f0.6
327 f45
421 f 10ZZe
991 f 144
1289f2831.‘
0.3 f0.2
0.4 f 0.3
4.1 f 3.8
6.7 f 3.9
l.Of5.3
10.1 f5.6
0.9f0.3
I .5 f 0.6b
151 f22
254fW
419f48
841 f372d
the presence of bile, but only the greater t,,, (1.5
versus 0.9 h without bile) achieved significance
(P <0.05). Although bile enhanced CyA absorption
in SIV more than in SI, the absolute differences
between absorption in the two segments were
retained with higher values of C,,, (P<0.05)and
AUC (P=O.O6) in SI than in SIV in the presence of
bile (Table 2).
H loom
i;/
0
- . - - With bile flow
0-0
0
Without bile flow
..--.
0-0
0
30
60
90
iio
is0
With bile flow
Without bile flow
180
iio
240
Time (min)
Fig. 2 Effect of bile on CyA absorption in SI (duodenum and
proximal jejunum, a ) and SIV (distal ileum, b). Each point shows the
mean and SEM for the number of determinations detailed in the Materials
and methods section.
F
DISCUSSION
CyA absorption was shown to occur throughout
the small bowel in this experimental study performed using segments of rat small bowel isolated in
situ, but the greatest absorption occurred in the
proximal segments comprising duodenum and
proximal jejunum. The observed capacity for CyA
absorption throughout the small intestine extends
the recent findings of Drewe et al. [9], who showed
that CyA is predominantly absorbed from the small
intestine when it is introduced into different parts of
the gastrointestinal tract in human subjects. On the
basis of the observation that the absorption of drug
introduced into the duodenum was approximately
twice that when introduced into the ileum, Drewe et
al. [9] proposed that the length of functionally
intact small bowel was the most important determinant of CyA absorption. This interpretation conflicts with the evidence of Tarr and Yalkowsky [l 13,
who showed that doubling the length of the intestinal loop exposed to CyA, in rats, did not significantly increase the fraction of CyA absorbed. Our
results may explain these apparently discrepant
observations in the context of an absorption
678
Y. Cakaloglu et al.
window for CyA administered by the peroral route.
This concept envisages a localized region of gastrointestinal tract where CyA absorption occurs on the
basis of a relatively short duration of absorption
and a short mean lag time (0.4 h after oral administration in man [12] and <0.25 h after direct addition to gut loops in this study). What is clear from
our present results is that the absorption window
for CyA is not limited to the proximal small bowel,
although duodenum and proximal jejunum appear
to have a higher capacity for CyA absorption than
the distal segments. In addition to these anatomical
considerations, the site and extent of CyA absorption may also be determined by other factors, such
as transit time and drug solubility.
The distal parts of the small intestine, including
the distal jejunum and proximal and distal ileum,
were each shown to absorb approximately one-half
of that absorbed in the proximal small intestine.
The residence time in the gut and the degree of
intestinal permeability for a given drug are important elements of intestinal absorption. The significantly greater absorption of CyA from the most
proximal segment (SI) of small bowel in the
presence of a similar residence time for SI-SIII may
be explained by a higher permeability of duodenum
and proximal jejunum than of the other segments.
This is consistent with the report of Sawchuk and
Awni [7] suggesting that the permeability for CyA
absorption is greater in the proximal small bowel of
rat than in the distal portion. The capacity for CyA
absorption throughout the small intestine may also
clarify observations of two absorption maxima after
peroral administration of CyA [13, 141. It has been
proposed that the early peak represents absorption
from the upper small bowel with a short lag time
between dosing and the first appearance of CyA in
blood. The second peak may result from the absorption of CyA in the lower part of the small bowel
secondary to either enterohepatic recirculation or
resolubilization. Because human bile contained predominantly CyA metabolites with very little parent
CyA ( c1% of administered dose being excreted in
the bile) [IS], it seemed likely that late absorption
of residual CyA was responsible. Our present findings of a relatively high capacity for CyA absorption
in the distal small bowel also favour this resolubilization hypothesis. An additional implication is that
poor solubilization may normally limit the uptake
of cyclosporin to amounts below the total absorptive capacity of the complete small bowel. The
relevance of this to improving CyA therapy requires
confirmation in the intact small bowel and in the
presence of an abundance of bile to optimize
solubilization.
Bile is clearly important for CyA absorption [3-5,
16, 171. The extremely hydrophobic nature of CyA
requires the drug to form micelles with bile salts for
adequate absorption, and diverted bile flow, by
Roux-en-Y anastomosis excluding the upper small
bowel from CyA absorption or the presence of an
open biliary T-tube results in a decrease of approximately 50% in CyA bioavailability [3,18]. In the
second part of this study, bile was shown to significantly increase CyA absorption by similar amounts
in both the proximal (SI) and distal (SIV) segments
of the small bowel, but with a greater proportional
increase (50%) in SIV than in SI (23%). The greater
difference between absorption in SI and SIV in our
initial experiment (without bile flow) might be
explained by the presence of a higher concentration
of residual bile in the upper small intestine. .Bile
would accumulate before segments were isolated
and the common bile duct was ligated because rats
which have no gallbladder continuously excrete bile
into the bowel. However, it should be noted that
bile salt absorption occurs predominantly in the
terminal ileum [ 191, and the continued differences
between absorption in SI and SIV in the presence of
complete bile flow suggests that other factors may
be responsible. These may include absorptive surface area, which is known to be higher in the
proximal small bowel than in the distal small bowel
[20], intestinal permeability and blood flow, and
these may underlie the greater capacity of the
proximal small bowel for CyA absorption. Irrespective of the factors determining the site of maximal
CyA absorption, the effect of bile is not site-limited
and occurs throughout the small intestine.
ACKNOWLEDGMENTS
Dr Atholl Johnson, Analytical Unit, St George’s
Hospital Medical School, London, kindly provided
the Strip computer program. We are grateful to
Sandoz Pharmaceuticals for financial support.
REFERENCES
I. Ptachcinski RJ, Venkataramanan R. Burckart GJ. Clinical pharmacokinetics of
cyclosporin. Clin Pharmacokinet 1986; II: 107-32.
2. Kahan BD, Grevel J. Optimization of cyclosporine therapy in renal
transplantation by a pharmacokinetic strategy. Transplantation 1988: 46:
63 1-44,
3. Whitington PF. Kehrer BH, Whitington SH. Schneider B, Black DD. The
effect of biliary enterostomy on the pharmacokinetia of enterally
administered cyclosporine in rats. Hepatology 1989; 9 393-7.
4. Venkataramanan R, Perez HD, Schwinghammer T et al. Effect of bile on
cyclosporine absorption in dogs. Res Commun Chem Pathol Pharmacol 1986;
5 3 137-40.
5. Tredger JM, Naoumov NV. Steward CM et al. lnfluence of biliary T-tube
clamping on cyclosporine pharmacokinetia in liver transplant recipients.
Transplant Proc 1988; 10 512-5.
6. Whitington PF. Emond JC, Whitington SH, Broelsch CE. Baker AL.
Small-bowel length and the dose of cyclosporine in children after liver
transplantation. N Engl J Med 1990; 322: 733-8.
7. Sawchuk RJ, Awni WM. Absorption of cyclosporine from rabbit small
intestine in sifu. J Pharmacol Sci 1986; 7 5 1151-6.
8. Grevel J. Absorption of cyclosporine A after oral dosing. Transplant Proc
1986; 18: 9-15,
9. Drewe J, Beglinger C. Kissel T. The absorption of cyclosporine in the human
gastrointestinal tract. Br J Clin Pharmacol 1992; 3 3 39-43.
Cyclosporin A absorption in rat small bowel
10. Johnson A, Woolard RE. Strip: an interactive program for the analysis of drug
pharmacokinetia. J Pharmacol Methods 1983; 9 193-200.
II. Tarr BD, Yalkomky SH. Enhanced intestinal absorption of cyclosporine in
rats through the reduction of emulsion droplet size. Pharm Res 1989: 6 40-3.
12. Grevel J, Nuesch E, Abisch E, Kutz K. Pharmacokinetics of oral cyclosporin A
(Sandimmun) in healthy subjects. Eur J Clin Pharmacol 1986; 31: 21 1-6.
13. Reymond JP* Steimer JL. Niederberger W. On the dose dependency of
cyclosporin A absorption and disposition in healthy volunteers. J
Pharmacokinet Biopharm 1988; 16 331-53.
14. Gupta SK, Benet LZ. Absorption kinetics of cyclosporine in healthy
volunteers. Biopharm Drug Dispos 1989; 10: 591-6.
15. Venkataramanan R. Stan1 TE, Yang S et al. Biliary excretion of cyclosporine
A in liver transplant recipients. Transplant Proc 1985; 17: 286-9.
679
16. Mehta MU, Venkataramanan R, Burckan GJ et al. Effect of bile on
cyclosporine absorption in liver transplant recipients. Br J Clin Pharmacol
1988.25: 579-84.
17. Eriaon BG, Todo S, Kam I et al. Role of bile and bile salts on cyclosporine
absorption in dogs. Tnnplant Proc 1987; I 9 124B-9.
18. Naoumov NV, Tredger JM, Steward CM et al. Cyclosporin A
pharmacokinetia in liver transplant recipients in relation to biliary T-tube
clamping and liver dysfunction. Gut 1989; 30: 391-6.
19. Berkow R. The Merck manual of diagnosis and thenpy. Rahway, NJ: Merck
and Co, 1982: vol. I, 567.
20. Levin RJ. Fundamental concepts of structure and function of the intestinal
epithelium. In: Duthie HL, Wormsley KG, eds. Scientific basis of
gastroenterology. London: Churchill Livingstone, 1979 308-37.