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DRUG METABOLISM AND DISPOSITION
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
Vol. 27, No. 2
Printed in U.S.A.
ROLE OF THE LIVER AND GUT IN SYSTEMIC DIPHENHYDRAMINE CLEARANCE IN
ADULT NONPREGNANT SHEEP
SANJEEV KUMAR,1 K. WAYNE RIGGS,
AND
DAN W. RURAK
Division of Pharmaceutics and Biopharmaceutics, Faculty of Pharmaceutical Sciences (S.K. and K.W.R.) and
British Columbia Research Institute of Child and Family Health, Department of Obstetrics and Gynecology, Faculty of Medicine (D.W.R.),
The University of British Columbia, Vancouver, British Columbia, Canada
(Received July 13, 1998; accepted September 28, 1998)
This paper is available online at http://www.dmd.org
ABSTRACT:
liver and a significant extrahepatic systemic clearance component
is evident. Using the calculated total hepatic blood flow values, it
was found that 98.6 6 9.2% of the i.v. dose eventually was delivered to the “hepatoportal” system. Because the drug delivered to
the hepatoportal system is almost completely eliminated in a single pass (hepatic extraction ;94%), this indicates a lack of any
significant pulmonary drug uptake. Also, because only ;32.5% of
the i.v. dose is metabolized in liver, the gut is most likely responsible for the clearance of the remainder. This gut contribution to
systemic DPHM clearance was confirmed in a separate direct
study in four sheep where the steady-state DPHM gut extraction
ratio was 49.0 6 3.0%. Thus, gut accounts for a significant proportion (>50%) of DPHM systemic clearance in sheep in spite of a very
high hepatic drug extraction efficiency.
Diphenhydramine or 2-(diphenylmethoxy)-N,N-dimethylamine
(DPHM)2 is a potent classical histamine H1 receptor antagonist. It has
been widely used during human pregnancy for the treatment of nausea
and vomiting, insomnia, allergic rhinitis, and common coughs and colds.
For several years, our laboratory has been examining the comparative
maternal–fetal pharmacokinetics and metabolism of this drug in chronically instrumented pregnant sheep (Yoo et al., 1990; Kumar et al., 1997).
Our current focus is to elucidate the organs and metabolic pathways
involved in DPHM clearance in the mother and the fetus.
The major routes of DPHM metabolism in many species (e.g., dog,
rhesus monkey, human) include conversion to diphenylmethoxyacetic
acid (DPMA) and DPHM-N-oxide metabolites (Drach and Howell,
1968; Drach et al., 1970; Chang et al., 1974). The urinary excretion of
DPMA (including its amino acid conjugates) and DPHM-N-oxide
may account for ;40 to 60% and ;5 to 15% of the administered dose,
respectively, in these species (Drach and Howell, 1968; Drach et al.,
1970; Chang et al., 1974). However, we have found that these metabolic pathways collectively account for only ;1 to 2% of total
DPHM dose in maternal as well as fetal sheep (unpublished data).
This has prompted us to reexamine the role of the liver in DPHM
systemic clearance and to study other potential organs of drug clearance in sheep. Hence, in the current study, we have examined the
relative importance of the liver and gut in systemic elimination of
DPHM in adult sheep.
Materials and Methods
These studies were supported by funding from the Medical Research Council
of Canada. S.K. was supported by a University of British Columbia Graduate
Fellowship. D.W.R. is the recipient of an investigatorship award from the British
Columbia Children’s Hospital Foundation.
1
Present address: Department of Drug Metabolism, Merck Research Laboratories, Rahway, NJ 07065-0900.
2
Abbreviations used are: DPHM, diphenhydramine; [2H10]DPHM, deuteriumlabeled DPHM; DPMA, diphenylmethoxyacetic acid; [2H10]DPMA, deuteriumlabeled DPMA; AUC, area under the plasma concentration versus time profile;
Cmax, peak plasma concentration; EH, hepatic first-pass extraction ratio; p.v.,
portal venous; QH, total hepatic blood flow.
Animals and Surgical Preparation. A total of nine adult female nonpregnant sheep were used in these studies. All studies were approved by the
University of British Columbia Animal Care Committee, and the procedures
performed on sheep conformed to the guidelines of the Canadian Council on
Animal Care. Surgery was performed aseptically under halothane (1–2%) and
nitrous oxide (60%) in oxygen anesthesia, after induction with i.v. sodium
pentothal (1 g) and intubation of the ewe. Polyvinyl catheters (Dow Corning,
Midland, MI) were implanted in a femoral artery and a femoral vein in all nine
sheep. In the five sheep (average body weight, 70.1 6 10.5 kg) to be used for
hepatic DPHM first-pass extraction studies, a sterile catheter was implanted in
a branch of one of the mesenteric veins with the catheter tip advanced to the
main mesenteric vein in the direction of hepatic portal vein. In the four sheep
Send reprint requests to: Dr. K. Wayne Riggs, Associate Professor, Faculty of
(average body weight, 64.0 6 7.9 kg) to be used for DPHM gut uptake studies,
Pharmaceutical Sciences, The University of British Columbia, 2146 East Mall,
a catheter was implanted in the main hepatic portal venous trunk just before its
Vancouver, BC, Canada V6T 1Z3. E-mail: [email protected]
entry into the liver, as described below. A longitudinal abdominal incision was
297
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We investigated the contribution of the liver and gut to systemic
diphenhydramine (DPHM) clearance in adult nonpregnant sheep in
two separate studies. In the first study, a simultaneous 50-mg
bolus each of DPHM and its deuterium-labeled analog
([2H10]DPHM) was administered to five sheep via the femoral (i.v.)
and the portal venous (p.v.) routes in a randomized manner. Arterial
plasma concentrations of DPHM, [2H10]DPHM, and their deaminated metabolites, DPMA (diphenylmethoxyacetic acid) and
[2H10]DPMA, were measured using gas chromatography–mass
spectrometry. The hepatic first-pass extraction of DPHM after p.v.
administration was 94.2 6 3.7%. However, the area under the
plasma concentration versus time profile of the metabolite after i.v.
dosing was only 32.5 6 14.0% relative to that after p.v. administration. Thus, only ;32.5% of the i.v. dose is metabolized in the
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KUMAR ET AL.
equations. Subscripts “parent” and “metabolite” stand for parent drug and
metabolite, respectively.
The systemic clearance of drug was calculated as:
(CL)systemic 5
~Dose)i.v.
(AUCparent)i.v.
(1)
where, i.v. refers to i.v. administration of the parent drug.
The hepatic first-pass extraction ratio (EH) of drug is given by:
EH 5 1 2
~AUCparent)p.v.
(AUCparent)i.v.
(2)
The fraction of the i.v. administered drug metabolized in the liver was calculated from AUCs of the DPMA metabolite after p.v. and i.v. administration of
the parent drug, as:
fH 5
~AUCmetabolite)i.v.
(AUCmetabolite)p.v.
(3)
This equation assumes linear pharmacokinetics, sole hepatic formation of the
metabolite, and complete metabolism of the p.v. dose in the liver.
Total hepatic blood flow (p.v. 1 hepatic arterial) was estimated from the
relationships of the well stirred model of hepatic elimination (Wilkinson and
Shand, 1975; Pang and Gillette, 1978):
QH 5
F
1
AUCparent
Dose
G F
2
i.v.
AUCparent
Dose
G
(4)
p.v.
where, i.v. and p.v. refer to the doses and respective AUCs during i.v. or p.v.
administration of the parent drug.
The amount of i.v. administered drug eventually delivered to the hepatoportal system (gut 1 liver) was calculated from estimated hepatic blood flow
and its systemic arterial AUC (Wilkinson and Shand, 1975):
Amount delivered to the hepatoportal system 5 QH z ~AUCparent)i.v.
(5)
This subsequently was converted to the percentage of administered dose
delivered to the hepatoportal system.
DPHM gut uptake studies. Steady-state systemic clearance of the drug is
calculated as:
(CL)systemic 5
ko
(Css)MA
(6)
where ko is the DPHM infusion rate and (Css)MA is the steady-state femoral
arterial plasma DPHM concentration during gut uptake studies.
The steady-state gut extraction of the drug (Eg) is calculated as (du Souich
et al., 1995):
Eg 5 1 2
~Css)PV
(Css)MA
(7)
where (Css)MA is the steady-state DPHM concentration in femoral arterial
plasma (before the gut) and (Css)PV is the steady-state DPHM concentration in
the p.v. plasma (after the gut).
Statistical Analyses. All data are reported as the mean 6 S.D. The femoral
arterial plasma AUCs of the parent drug and metabolite after i.v. and p.v.
DPHM administration were compared using a paired t test. The achievement
of steady-state in plasma was established according to two criteria: 1) the slope
of plasma concentration versus time curve should not be significantly different
from zero and 2) the coefficient of variation of the measured concentrations
should be ,15%. The steady-state DPHM concentrations in femoral arterial
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made to gain access to various compartments of the ruminant sheep stomach.
A prominent branch of the main gastric vein was identified either on the
surface of the rumen or the omasum and a segment of the intact vessel was then
carefully isolated from the surface of the stomach compartment. An ;12- to
18-inch length of a sterile polyvinyl catheter was then advanced into this vessel
toward the direction of liver via the main gastric vein and into the hepatic
portal vein. The catheter was secured in place using sterile silk sutures and was
anchored to the surface of the rumen or the omasum. All catheters were
tunneled s.c. and exteriorized via a small incision on the flank of the ewe and
were stored in a denim pouch when not in use. Catheters were flushed daily
with approximately 2 ml of sterile 0.9% sodium chloride containing 12 U of
heparin/ml to maintain catheter patency. Intramuscular injections of 500 mg of
ampicillin were given to the ewe on the day of surgery and for 3 days
postoperatively. After surgery, animals were kept in holding pens with other
sheep and were given free access to food and water. The sheep were allowed
to recover for 3 to 8 days before experimentation. The position of the portal
venous catheter was verified in all animals by autopsy at the end of each
experiment.
Experimental Protocol. DPHM hepatic first-pass extraction studies. Equimolar
amounts of DPHM and its deuterium-labeled analog ([2H10]DPHM), equivalent to 50 mg DPHM, were administered simultaneously but separately via the
femoral (i.v. route) and mesenteric vein [portal venous (p.v.) route] catheters
in a randomized manner. Serial samples of femoral arterial plasma (;3 ml)
were collected at 5, 10, 20, 30, and 40 min and at 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8,
10, and 12 h after drug injection.
The deuterium-labeled parent drug ([2H10]-DPHM) used in hepatic DPHM
uptake studies above was synthesized and purified in our laboratory (Tonn et
al., 1993). The doses of [2H10]DPHM were corrected for the mass difference
(due to the presence of deuterium labels) compared with the unlabeled drug.
DPHM gut uptake studies. DPHM gut uptake was measured under steadystate conditions in four adult sheep with implanted p.v. catheters. For this
purpose, a 20-mg i.v. bolus-loading dose of DPHM was administered at the
beginning of the experiment via the femoral venous catheter to four sheep. This
was followed immediately by an infusion of unlabeled DPHM at a rate of 670
mg/min for 6 h. Simultaneous femoral arterial (before the gut) and p.v. (after
the gut) blood samples (;3 ml each) were collected every hour for the entire
duration of DPHM infusion (6 h) to estimate the steady-state gut extraction of
the drug.
All doses were prepared in sterile water for injection and were sterilized by
filtering through a 0.22-mm nylon syringe filter (MSI, Westboro, MA) into a
capped empty sterile injection vial.
Blood samples collected above during all experiments were placed into
heparinized Vacutainer tubes (Becton Dickinson, Rutherford, NJ) and gently
mixed. These samples were then centrifuged at 2000g for 10 min. The plasma
supernatant was removed and placed into clean borosilicate test tubes with
polytetrafluoroethylene (PTFE)-lined caps. All samples were stored frozen at
220°C until the time of analysis.
Drug and Metabolite Analysis. The concentrations of DPHM, [2H10]DPHM,
and their corresponding deaminated metabolites, DPMA and [2H10]DPMA, in
femoral arterial plasma samples collected during hepatic DPHM uptake studies
were measured using previously developed gas chromatographic–mass spectrometric (GC-MS) analytical methods (Tonn et al., 1993, 1995). The femoral
arterial and p.v. plasma samples collected during DPHM gut uptake studies
were analyzed only for concentrations of the parent drug, i.e., DPHM, using
the above GC-MS analytical method (Tonn et al., 1993) The linear calibration
range of the above assays is 2.0 to 200.0 ng/ml for DPHM and [2H10]DPHM
and 2.5 to 250.0 ng/ml for DPMA and [2H10]DPMA. The inter- and intraday
coefficients of variation are ,20% at the limit of quantitation and ,10% at all
other concentrations (Tonn et al., 1993, 1995).
Pharmacokinetic Analyses. DPHM hepatic first-pass uptake studies. Areas under the plasma drug or metabolite concentration versus time curves
(AUCs) from time 0 to the last sampling point were calculated using the linear
trapezoidal rule (Gibaldi and Perrier, 1982). This area under the curve was then
extrapolated to time infinity by adding the factor, Clast/K; Clast is the plasma
concentration of the drug or metabolite at the last sampling time, and K is the
terminal elimination rate constant (Gibaldi and Perrier, 1982). The total area
under the curve up to time infinity is referred to as AUC in the following
LIVER AND GUT DIPHENHYDRAMINE UPTAKE IN ADULT SHEEP
299
E1154 received 50 mg of DPHM via the p.v. route and an equimolar dose of [2H10]DPHM via the femoral venous route. The routes of administration of DPHM and
[2H10]DPHM were reversed in E102.
and p.v. plasma during gut uptake studies also were compared against each
other using a paired t test. The significance level was p , .05 in all cases.
Results
DPHM Hepatic First-Pass Uptake Studies. Figure 1 shows the
typical femoral arterial plasma concentration versus time profiles of
the parent drug and metabolite after simultaneous and randomized
administration of DPHM and [2H10]DPHM via i.v. and p.v. routes in
two sheep. In E1154, DPHM was given via the p.v. route and
[2H10]DPHM via the i.v. route. In E102, the order of administration
was reversed, i.e., DPHM was administered i.v. and [2H10] was
administered via the portal route. The average maximal plasma concentrations (Cmax) of the metabolite generated from the form of drug
administered via the p.v. route were significantly higher compared
with the Cmax of metabolite generated from the form of drug given i.v.
(447.8 6 175.9 versus 90.7 6 75.8 ng/ml; paired t test, p , .005). The
times of occurrence of plasma Cmax of the metabolite (Tmax) after p.v.
administration ranged from 5 to 20 min compared with 10 to 90 min
after i.v. administration. Table 1 presents the calculated femoral
arterial AUCs of the parent drug and metabolite, hepatic first-pass
extraction ratios, and the fraction of i.v. administered dose metabolized in liver. The AUC of the form of parent drug administered via
the p.v. route was smaller compared with that of the form administered i.v. (paired t test, p , .005). The AUC of the metabolite
generated from the form of parent drug administered via the p.v. route,
however, was significantly larger compared with that generated from
the form administered via the i.v. route (paired t test, p , .01). The
hepatic first-pass extraction of the drug was high and ranged from
90.4 to 99% (mean 94.2 6 3.7%; Table 1). The fraction of i.v. parent
drug dose metabolized in liver ranges from 18.2 to 50.4% (mean
32.5 6 14.0%; Table 1). Thus, the fraction of drug metabolized/
eliminated by extrahepatic tissues will be 49.6 to 81.8% (mean 67.5 6
14.0%).
Table 2 presents the estimates of systemic clearance, total hepatic
blood flow, and percentage of i.v. dose that is eventually delivered to
the hepatoportal system (gut and liver). The average percentage of the
i.v. dose delivered to the hepatoportal system was not significantly
different from 100% (unpaired t test, p . .3).
DPHM Gut Uptake Studies. Figure 2 shows the average concentration versus time profiles of DPHM in femoral arterial and p.v.
plasma in four sheep during the 6-h DPHM infusion period. DPHM
was at steady-state during the 2- to 6-h DPHM infusion period in both
femoral arterial as well as p.v. plasma according to the established
criteria. The steady-state femoral arterial and p.v. plasma concentrations, systemic clearance, and estimates of gut extraction of DPHM in
four sheep are presented in Table 3. The p.v. plasma concentrations of
DPHM were significantly lower compared with its femoral arterial
plasma concentrations throughout the experimental period in all animals (paired t test, p , .005 in all cases). The gut extraction of the
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FIG. 1. Representative femoral arterial plasma concentration versus time profiles of the parent drug (DPHM and [2H10]DPHM, A and C) and metabolite (DPMA and
[2H10]DPMA, B and D) in E1154 (A and B) and E102 (C and D).
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KUMAR ET AL.
TABLE 1
Femoral arterial AUCs of the parent drug (DPHM or [2H10]DPHM) and metabolite (DPMA or [2H10]DPMA), parent drug EH, and fraction of i.v. administered
parent drug dose metabolized in the liver during hepatic first-pass uptake studies
EH
Fraction of i.v.
Dose
Metabolized in
Liver (fH)
0.904
0.904
0.958
0.955
0.990
0.942 6 0.037
0.504
0.182
0.441
0.270
0.230
0.325 6 0.140
AUC
Ewe
Parent (i.v.)
Parent (p.v.)
Metabolite (i.v.)
20,437.5
14,728.5
14,140.9
9,812.6
7,311.1
13,286.1 6 5,042.8
1,952.8
1,411.5
595.3
437.8
76.6
894.8 6 767.2b
Metabolite (p.v.)
ng z min/ml
1158a
1154
139a
102a
989
Mean 6 S.D.
73,888.5
6,811.4
11,518.8
16,360.9
12,513.7
24,218.7 6 27,973.8
146,681.7
37,415.4
26,096.7
60,500.2
54,520.1
65,042.8 6 47,634.9c
a
In these ewes, [2H10]DPHM was given via the p.v. route and DPHM via the femoral venous route. In the other animals, the route of administration for the two forms of drug was reversed.
Significantly lower than the parent drug AUC after i.v. administration ( p , .005).
c
Significantly larger than metabolite AUC after i.v. administration of the drug ( p , .01).
b
TABLE 2
Ewe
1158a
1154
139a
102a
989
Mean 6 S.D.
Systemic
Clearance
Estimated Total
Hepatic Blood
Flow
ml/min/kg
ml/min/kg
45.1
54.1
56.8
85.1
78.5
63.9 6 17.0
41.4
59.5
59.0
74.0
78.9
62.6 6 14.7
% of i.v. Dose
Delivered to Gut
and Liver
91.8
109.9
103.8
86.9
100.5
98.6 6 9.2
a
In these ewes, [2H10]DPHM was given via the p.v. route and DPHM via the femoral venous
route. In the other animals, the route of administration for the two forms of drug was reversed.
FIG. 2. Average femoral arterial and p.v. plasma concentration versus time
profiles of DPHM in four sheep during 6-h DPHM infusion.
drug in individual animals ranged from 46.3 to 53.4% (mean 49.0 6
3.0%).
Discussion
DPHM was extracted extensively across the sheep liver with an
estimated mean hepatic first-pass extraction ratio of 94.2 6 3.7%
(Table 1).
The shape of the metabolite plasma profile is highly dependent on
the route of drug administration (Pang, 1981). Thus, a significantly
higher Cmax of the metabolite was observed after p.v. dosing as
compared with that after i.v. administration. Also, the Cmax values of
the former metabolite tended to occur at earlier times compared with
those of the latter. This is because p.v. administration results in an
almost instantaneous metabolism of ;94.2% of the administered dose
(see above) and an apparent “bolus” injection of large amounts of the
generated metabolite into the circulation. In contrast, the drug administered i.v. is more gradually metabolized and leads to a slower
increase in metabolite plasma concentrations.
It has been suggested that the arterial AUCs of the metabolite after
p.v. and i.v. administration of equal doses of the drug should be
relatively equal, provided the contribution of renal or peripheral
elimination to total clearance is ,10% and linear pharmacokinetics
exist (Pang, 1981; Houston and Taylor, 1984). This is true in spite of
the widely differing shapes of metabolite plasma profiles obtained
after these routes of administration (see above). However, if renal or
peripheral elimination of the drug is .10%, the metabolite AUC after
p.v. administration becomes larger compared with that after i.v. administration (Pang, 1981; Houston and Taylor, 1984). This is because
if renal or peripheral elimination of the drug is negligible (,10%), the
majority of the drug will undergo hepatic metabolism after all routes
of administration. Thus, similar amounts of the metabolite eventually
will be formed from equal doses of the drug given via the p.v. and i.v.
routes, leading to similar arterial metabolite AUCs. However, if renal
or peripheral elimination is .10%, a larger fraction of the i.v. dose
will be eliminated via this route and less will be available for hepatic
metabolism; this then will lead to the formation of smaller amounts of
the metabolite after i.v. dosing in comparison with that after p.v.
administration.
Earlier we observed that DPHM exhibits linear pharmacokinetics in
adult sheep with negligible renal and biliary clearances (,0.5% of
total body clearance) over the dose range used in this study (Yoo et
al., 1990; Tonn, 1995). Thus, in the absence of any peripheral elimination, the AUCs of the DPHM metabolites after i.v. and p.v. drug
administration should be equal. A lower metabolite AUC after i.v.
compared with that after p.v. administration in our DPHM hepatic
first-pass studies provides clear evidence for significant peripheral
drug elimination. Also, the data presented in Table 1 indicate that
almost the entire p.v. DPHM dose is metabolized in the liver. This
mainly results from the metabolism of ;94.2% of the dose during its
first pass through the liver; plus, at least some of the remaining drug
undergoes hepatic metabolism during subsequent passes. Thus, from
the relative metabolite AUCs after p.v. and i.v. administration, it
appears that only 32.5 6 14.0% of the i.v. administered drug is
metabolized in the liver and the rest likely is eliminated via peripheral
mechanisms. This analysis assumes sole hepatic formation of the
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DPHM systemic and intrinsic clearances, estimated hepatic blood flows, and
percentage of i.v. administered dose eventually delivered to the “hepatoportal
system” in five adult sheep
301
LIVER AND GUT DIPHENHYDRAMINE UPTAKE IN ADULT SHEEP
TABLE 3
Steady-state femoral arterial and p.v. plasma concentrations, systemic clearance, and gut extraction of DPHM in four sheep during gut uptake experiments
Steady-State Plasma Concentration
Ewe
Systemic Clearance
Femoral Arterial
p.v.
ng/ml
E0224
E4140
E1225A
E6216
Mean 6 S.D.
a
296.7
197.3
451.7
323.3
317.2 6 104.8
Gut Extraction
(Eg)
ml/min/kg
148.2
105.9
210.5
173.7
159.6 6 44.0a
40.3
50.0
20.3
35.1
36.4 6 12.4
0.501
0.463
0.534
0.463
0.490 6 0.030
Significantly lower compared with femoral arterial plasma concentrations.
mated contribution of the liver appears to be near the high end
(18.2–50.4%). Also, the systemic clearances of DPHM during the gut
uptake study appear to be somewhat lower compared with those
during the hepatic first-pass experiments. These may be related to
interanimal variability and the small number of animals used in these
two studies. The overall combined data indicate that gut uptake of the
drug may account for ;50 to 80% of DPHM systemic clearance. The
mechanism of this DPHM gut uptake remains unknown and may
involve simple binding of the drug to tissue components, its secretion
into the lumen via specific transporters (e.g., P-glycoprotein), or
metabolism via pathways other than the formation of DPMA.
Lately, there has been an increased interest in the detailed study of
the underlying mechanisms of gut uptake/metabolism of drugs and its
role in determining drug absorption and bioavailability. Oral bioavailability of midazolam, cyclosporine, verapamil, and nifedipine in humans appears to be highly dependent on their CYP3A-mediated
metabolism in the gut mucosa (Hebert et al., 1992; Paine et al., 1996;
Thummel et al., 1996; Fromm et al., 1996; Holtbecker et al., 1996;
Wandel et al., 1998). Polarized basolateral-to-apical drug transport/
secretion in the intestine, mediated via P-glycoprotein, also has been
demonstrated to be an important factor in determining the absorption
and bioavailability of cyclosporine and paclitaxel (Asperen et al.,
1997; Lown et al., 1997; Sparreboom et al., 1997). The majority of the
above studies have assessed the effect of gut uptake/metabolism/
secretion on the bioavailability of the drug after oral administration.
Apart from a few isolated attempts (du Souich et al., 1995), there
appear to be few systematic studies on the extent of gut uptake of
drugs from the systemic circulation and its role in systemic drug
clearance. For midazolam, gut drug uptake from the systemic circulation was negligible, presumably because of inaccessibility of the
intestinal CYP3A to the circulating drug (Paine et al., 1996). Other
investigators have assumed that the gut drug uptake from the systemic
circulation is insignificant (Hebert et al., 1992; Holtbecker et al.,
1996). Our data with DPHM show that this assumption may not
necessarily be true for all drugs. Thus, pharmacokinetic analyses of
hepatoportal drug disposition based on this assumption may not be
entirely accurate, as was previously argued by Lin et al. (1997).
In summary, our studies demonstrate that gut drug uptake from the
systemic circulation is responsible for ;50 to 80% of DPHM systemic clearance in adult sheep and the liver accounts for the remainder. These data also indicate that the assumption of negligible gut drug
uptake from the systemic circulation may not be universally true and
that it should be explicitly examined in pharmacokinetic studies
designed to assess the role of the gut in drug bioavailability and
clearance.
Acknowledgments. We thank Eddie Kwan for his help with the
animal surgeries.
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metabolite; if the metabolite is also formed in peripheral organs, the
percentage metabolized in the liver will be overestimated using this
approach (eq. 3). This is because peripheral metabolite formation will
contribute more toward the metabolite AUC after i.v. than after p.v.
administration because of the much higher parent drug concentrations
after i.v. dosing. It should be noted that the above conclusions are
valid in spite of the fact that the DPMA metabolite is not the only
contributor to DPHM clearance; it actually accounts for only a very
small fraction of DPHM elimination (;1%, unpublished data). Because the fraction of dose metabolized via a particular metabolic
pathway is constant in a linear pharmacokinetic system, the plasma
concentration-time profiles of all other metabolites also will exhibit a
similar behavior (Houston and Taylor, 1984).
The parent drug pharmacokinetic data after i.v. and p.v. administration can be used to estimate total hepatic blood flow (QH) using the
well stirred model of hepatic clearance (Wilkinson and Shand, 1975;
Pang and Gillette, 1978). The average estimate of QH obtained in
these studies (62.6 6 14.7 ml/min/kg; Table 2) is in excellent agreement with the reported values for adult nonpregnant sheep (56.6 6
18.0 ml/min/kg, Katz and Bergman, 1969; 48.6 ml/min/kg, Boxenbaum, 1980). It should be emphasized that the above estimation of QH
assumes a lack of significant DPHM uptake by the gut. However, the
hepatic extraction of DPHM is high (;94%), and, thus, the presence
of any gut drug uptake will not alter significantly the overall DPHM
extraction across the hepatoportal system (i.e., observed 94% versus
maximum possible 100%). Using these QH estimates and eq. 5, we
found that almost the entire i.v. dose (98.6 6 9.2%, Table 2) was
eventually delivered to the hepatoportal system. Only a very small
fraction of drug delivered to this system can escape uptake/metabolism because the extraction of DPHM across the liver alone is nearly
complete (;94%). Thus, the liver and/or gut are the major organs
responsible for elimination of the DPHM i.v. dose. Consequently, this
provides evidence for a lack of any significant first-pass DPHM
uptake by the lung. Moreover, because the liver metabolizes only
;32.5% of the i.v. dose, the gut is likely responsible for the elimination of the remaining ;67.5%.
This possible gut uptake of DPHM was confirmed in the second
direct study, where steady-state DPHM gut extraction was estimated
to be 49.0 6 3.0%. As discussed above, it appears that the gut and
liver are responsible for almost the entire DPHM systemic clearance.
Because the gut and liver are anatomically arranged in series and the
major contributor to hepatic blood flow is p.v. flow (;80%, Katz and
Bergman, 1969), roughly they will account for ;49.0 and ;51.0% of
DPHM systemic clearance, respectively. In fact, contribution of the
liver will be somewhat greater and that of the gut will be somewhat
lower because of the presence of hepatic arterial flow component that
bypasses the gut. This estimate of the gut contribution to DPHM
systemic clearance is near the low end of the range predicted from our
hepatic first-pass experiments (i.e., 49.6 – 81.8%), whereas the esti-
302
KUMAR ET AL.
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