drug enterohepatic circulation and disposition...
TRANSCRIPT
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REVIEWS Drug Discovery Today �Volume 19, Number 3 �March 2014
Drug enterohepatic circulation anddisposition: constituents of systemspharmacokinetics
Yu Gao1,4, Jingwei Shao1,4, Zhou Jiang1, Jianzhong Chen1,2, Songen Gu1,Suhong Yu1, Ke Zheng3 and Lee Jia1
1Cancer Metastasis Alert and Prevention Center, College of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou 350002, China2 School of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou 350108, China3 Research Institute of Functional Materials, College of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou 350002, China
Drug disposition information constitutes a part of systems pharmacokinetics, and becomes imperative
when a drug shows significant effects at its disproportionally low blood concentration. The situation
could result from outweighing the parent drug in tissues over in blood and/or from its active
metabolites. Fractions of certain drugs absorbed from the intestine to the systemic circulation via the
portal vein can return to the intestine via the bile duct and the sphincter of Oddi – a complementary
nonrenal elimination route termed the enterohepatic circulation (EHC). Here, we critically evaluate the
existing methods, techniques and animal models used for determining drug distribution, elimination
and EHC, and collectively portray characteristics of 43 drugs that undergo EHC. EHC could represent an
unexplored way to excrete unwanted substrates out of the body. The interdisciplinary analysis
galvanizes our efforts to overcome technical gaps in drug discovery and development.
IntroductionSystems pharmacokinetics is an emerging approach applied to
pharmaceutical ADME. It is a pharmacokinetics-based interdisci-
plinary field of study that focuses on complex interactions
between drugs and the patients who take the drugs. This subject
creates synergy at the interface between systems biology and
pharmacokinetics. One of the outreaching aims of systems phar-
macokinetics is to model and discover emergent properties of
enzymes, cells, tissues and the body as an integral system where
theoretical description is only possible using systems pharmaco-
kinetics techniques.
As part of ADME, and systems pharmacokinetics, drug distribu-
tion refers to the reversible transfer of drugs from one location to
another within the body. Distribution of drugs to and from blood
and other tissues occurs at various rates and to various extents.
Definitive information on the distribution of a drug requires mea-
surement of the drug in various tissues [1,2], about which we will
discuss here in detail. There are several factors that determine the
Corresponding author: Jia, L. ([email protected])4 These authors contributed equally to the work.
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distribution pattern of a drug with time, including delivery of the
drug to tissues via the blood, ability to cross tissue membranes,
binding to proteins within blood and tissues [3], and partitioning
into fat. Tissue uptake, commonly called extravasation, plays a part
in equilibrating the diffusion between a tissue and the blood perfus-
ing it. Distribution can be rate-limited by either perfusion or perme-
ability. A perfusion-rate limitation predominates when the tissue
membranes present basically no barrier to distribution. This con-
dition is likely to be met by small lipophilic drugs diffusing across
most membranes of the body, and by most drugs (except macro-
molecules) diffusing across capillary walls of muscle and subcuta-
neous tissues.
When a drug enters the bloodstream, rapid circulation of the
blood mixes the drug throughout the entire blood in minutes.
Permeation of the drug from the blood capillaries into the tissues
begins immediately and the process is called drug distribution. At
the same time, the drug is being removed from the bloodstream,
mainly by the liver and kidneys by a process called elimination.
Drugs are eliminated in the unchanged form and/or as metabolites
of the parent drugs. Some drugs are excreted via the bile. Others,
especially volatile drugs, are excreted in the breath.
er � 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.drudis.2013.11.020
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Some drugs eliminated via the bile could be reabsorbed from the
gastrointestinal tract into the systemic circulation and excreted
into bile again, resulting in multiple peaks in the plasma-concen-
tration–time profile, which prolongs the apparent elimination
half-life of the drugs. This process is termed the enterohepatic
circulation (EHC). No-one knows which physicochemical proper-
ties EHC drugs should possess to be excreted out of the circulation
into the gastrointestinal tract. This question has been interesting
us for many years, and here we compile almost all the information
we have about drugs undergoing EHC (Table 1), and try to under-
stand the unique but less utilized physiological process. Versatile
methods have been developed to help understand each step of the
pharmacological process of EHC. We provided the guidelines and
comprehensively and critically evaluated these methods used for
drug metabolism and plasma-protein-binding studies [3–5]. It is
0
2000
4000
6000
8000
10000
12000
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Kidney
Lu
14C
-car
ben
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im in
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(g/g
)
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50
100
150
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(a)
(b)
Small intesti
ne
Large in
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T
Acc
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FIGURE 1
Typical drug in vivo distribution and elimination study results expressed as a histogr
test drug in the gastrointestinal tract after a single oral administration. The test dru
same concentration levels in tissues after absorption. The carcass including fur, skinvisually shows a rat housed in a metabolism cage, and the feces and urine are c
equally important to provide a guideline for the experimental
design of drug tissue distribution, elimination and EHC studies
to improve the understanding of systems pharmacokinetics. Here,
we outline the comprehensive and rational approaches to deter-
mining the parameters of drug distribution and elimination, and
offer the largest data pool of those drugs that undergo EHC.
Drug distribution and eliminationThe purpose of distribution and elimination studies is to deter-
mine the target tissues, kinetic disposition and mass balance of the
drug along with its major excretion route, cumulative excretion (%
of administered dose) with time and excretion rate (ng/ml/h) at
different intervals after a single administration of the investiga-
tional drug (Fig. 1). The distributed drugs are removed from the
body by metabolism and excretion. The liver and kidneys are two
ngsHeart
SpleenLive
r
Muscle
Thyroid
Brain
Blood
Tumor
KidneyLungs
Heart
SpleenLive
r
Muscle
Thyroid
BrainBlood
Metabolism cage
Feces
Feces
Urine
ime (h)
Urine
4-8 8-24
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am. (a) Tissue distribution study visually illustrates high concentrations of the
g distributed to various blood well-perfused tissues and reached almost the
, bone and skull contains only 0.3% of the test drug. (b) The elimination studyollected at intervals after oral dosing [1].
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TABLE 1
Typical drugs that undergo enterohepatic circulation
Drug name (molecular weight) Chemical structure Mechanisms, clinical applications and references
ML970 (453.92 g/mol) A DNA minor-groove-binding agent for cancer
treatment [34]
Mifepristone (429.59 g/mol) Abortion, an emergency contraceptive [28,29]
Metapristone (415.25 g/mol) Cancer metastasis chemopreventive
a-Amanitin (918.97 g/mol) An inhibitor of RNA polymerase II [35]
Tesofensine (328.28 g/mol) Weight loss [36]
Meloxicam (351.40 g/mol) A nonsteroidal anti-inflammatory drug with analgesic
and fever reducer effects [37]
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TABLE 1 (Continued )
Drug name (molecular weight) Chemical structure Mechanisms, clinical applications and references
Lorazepam (321.2 g/mol) Anxiolytic, sedative, hypnotic, anticonvulsant muscle
relaxant [38]
Ciclosporin (1202.61 g/mol) An immunosuppressant drug, used for post-allogeneicorgan transplant [39]
3-Iodothyron-amine (355.17 g/mol) Modulates cardiac function, induces negative inotropiceffects and decreases cardiac output [40]
Glutathione (307.32 g/mol) The major endogenous antioxidant, regulation of thenitric oxide cycle. It is used for metabolic and
biochemical reactions [41]
Cysteinyl leukotrienes (625.77 g/mol) Involved in asthmatic and allergic reactions and acts tosustain inflammatory reactions [42]
Montirelin (408.47 g/mol) Thyrotropin-releasing hormone analog [43]
Chenodeoxy- cholic acid (392.57 g/mol) Reduce the saturation of cholesterol in the bile [44]
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TABLE 1 (Continued )
Drug name (molecular weight) Chemical structure Mechanisms, clinical applications and references
Folate (441.4 g/mol) It is necessary for the production and maintenance of
new cells, for DNA synthesis and RNA synthesis, and forpreventing changes to DNA and, thus, for preventing
cancer [45]
Mycophenolic acid (320.34 g/mol) Prevention of organ transplant rejection [46]
Colchicine (399.437 g/mol) The treatment of acute flares of gout and familial
Mediterranean fever. An anti-inflammatory agent forlong-term treatment of Behcet’s disease [47]
Norethisterone (298.419 g/mol) Oral contraceptive. Used for treatment of premenstrual
syndrome and help prevent uterine hemorrhage [48]
Methapyrilene (261.387 g/mol) An antihistamine and anticholinergic agent [49]
25-Hydroxy-vitamin D (400.64 g/mol) Increase fractional absorption of calcium from the gut
[50]
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TABLE 1 (Continued )
Drug name (molecular weight) Chemical structure Mechanisms, clinical applications and references
Vitamin B12 (1355.37 g/mol) Treatment of vitamin B12 deficiency, cyanide
poisoning and hereditary deficiency of transcobalaminII. Also given as part of the Schilling test for detecting
pernicious anemia [51]
Piroxicam (331.348 g/mol) Anti-inflammatory and analgesic drug, relieves the
symptoms of rheumatoid arthritis, osteoarthritis,
primary dysmenorrhoea and postoperative pain [52]
Ezetimibe (409.4 g/mol) Used for hypercholesterolaemia and homozygoussitosterolemia [53]
Baicalin (446.36 g/mol) A herbal supplement believed to enhance liver health
[32]
Progesterone (314.46 g/mol) Used for premenstrual tension syndrome, threatenedabortion and habitual abortion [54]
Chloramphenicol (323.132 g/mol) A bacteriostatic antimicrobial agent [55]
Digitoxin (764.939 g/mol) A cardiac glycoside used for the treatment of various
heart conditions [56]
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TABLE 1 (Continued )
Drug name (molecular weight) Chemical structure Mechanisms, clinical applications and references
Azithromycin (748.984 g/mol) The treatment of many different infections [57]
Isotretinoin (300.44 g/mol) Used mostly for cystic acne. Also employed for a
number of cancers and a few severe skin conditions
[58]
Rifampicin (822.94 g/mol) A bactericidal antibiotic drug of the rifamycin group[59]
Cholestyramine (776.87 g/mol) Acts to increase the basal metabolic rate, affect protein
synthesis, help regulate long bone growth and
neuronal maturation, and increase the body’ssensitivity to catecholamines by permissiveness [60]
Doxycycline (444.435 g/mol) Used to treat chronic prostatitis, sinusitis, syphilis,
chlamydia, pelvic inflammatory disease, acne, rosacea
and rickettsial infections [61]
Methotrexate (454.44 g/mol) Used for treatment of cancer, autoimmune diseases,
ectopic pregnancy and for the induction of medical
abortions [62]
a-Tocopherol (430.71 g/mol) Antioxidant, a regulatory effect on enzymatic activities,
gene expression and neurological functions, and
inhibition of platelet aggregation [63]
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TABLE 1 (Continued )
Drug name (molecular weight) Chemical structure Mechanisms, clinical applications and references
Irbesartan (428.53 g/mol) An angiotensin II receptor antagonist used mainly for
the treatment of hypertension [64]
Dexloxiglumide (461.379 g/mol) A cholecystokinin antagonist, inhibits gastrointestinal
motility and reduces gastric secretions [65]
Amiodarone (645.31 g/mol) Treatment of ventricular fibrillation and ventriculartachycardia atrial fibrillation [66]
Indomethacin (357.787 g/mol) A nonsteroidal anti-inflammatory drug [67]
Toremifene (405.959 g/mol) An oral selective estrogen receptor modulator (SERM)
that helps oppose the actions of estrogen in the body[68]
Genistein (270.24 g/mol) A biological active flavonoid found in high amounts in
soy that was reported to inhibit cancer progression
[69]
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TABLE 1 (Continued )
Drug name (molecular weight) Chemical structure Mechanisms, clinical applications and references
Morphine (285.34 g/mol) Treatment of severe pain or severe coughing [70]
Warfarin (308.33 g/mol) An anticoagulant normally used in the prevention of
thrombosis and thromboembolism [71]
Ceftriaxone (554.58 g/mol) A third-generation cephalosporin antibiotic [72]
Imipramine (280.407 g/mol) Also known as melipramine, is a tricyclic
antidepressant (TCA) of the dibenzazepine group. It ismainly used in the treatment of major depression and
enuresis [73]
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major organs that clear the drugs. Urine is one of the primary
elimination routes for drugs and metabolites to be excreted out-
side of the body. Excretion via the biliary and intestinal routes is
also important for elimination of metabolites, unchanged drugs
and unwanted substances.
The elimination phase typically follows first-order kinetics.
Most drugs are predominantly excreted via the kidneys as their
metabolized products [4–6], where the molecules under 5 nm in
diameter can be directly filtered through glomeruli [7]. If the drug
is rapidly eliminated, resulting in a rapid drop in concentration of
the free drug, the drug will permeate back from tissues into the
bloodstream.
To interpret the pharmacological and toxicological profiles of a
drug, it is important to have a comprehensive knowledge of the
ADME of the drug [1,8,9]. Tissue distribution is an essential
procedure in the preclinical drug discovery process. To conduct
the ADME study cost-effectively and efficiently, a tissue distribu-
tion study should be performed along with a drug elimination
study to provide the entire information about the mass balance of
a drug in individual tissues, biological fluids and the rest of the
body after administration (Fig. 1). Tissue distribution studies
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examine the distribution and accumulation of the investigational
drugs in potential sites of action and/or the targeted toxic tissues
(e.g. lungs and kidney) of the drugs. Tissue distribution and
elimination information is extremely important when the drug
showing significant pharmacological effects is demonstrated with
low oral bioavailability because concentrations of the drug in
tissues outweigh those in blood [2,10]. Data obtained after admin-
istration of radiolabeled drug to animals provide the most defini-
tive information on the routes of drug clearance. There has been a
global agreement on the need to perform a single-dose tissue-
distribution study as part of the preclinical program to provide
information about tissue distribution of the investigational drugs
[11].
For repeated doses, however, there is no consensus about
whether or not the tissue distribution study should be conducted.
It seems logical to perform the tissue distribution study under the
following circumstances: (i) the apparent half-life of the drug in
target tissues significantly exceeds its elimination half-life in
plasma; (ii) the drug has incomplete elimination; (iii) the
drug is being developed for site-specific targeted delivery and,
hence, concerns about the extensive tissue accumulation of the
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compound should be explicated [12]; and (iv) the drug is clinically
intended for a long period of administration in which the poten-
tial enzymatic up- or down-regulation of the drug is a major
concern. It is suggested that the period of preclinical repeated
administration does not exceed 28 days [13], and the tissue dis-
tribution and elimination monitoring should extend to a certain
period beyond the end of dosing.
Different drugs distribute to their corresponding habituated
tissues. Even if the drug analogs have similar functions, they
can distribute to their individual preferable tissues. For example,
tissue-type plasminogen activator (tPA) and urinary-type plasmi-
nogen activator (uPA) are two key components of the plasminogen
activator system, which converts plasminogen to plasmin by
proteolytic cleavage. Although tPA and uPA are synthesized and
released in urine by distinct portions of the urinary tubules, and
both have similar abilities to activate plasmin, they have different
tissue and cellular distributions. tPA is produced by glomerular
cells. It is the principal plasminogen activator in circulating
plasma and has a crucial role in the control of intravascular fibrin
degradation [14]. By contrast, uPA is produced by renal tubules,
and has no fibrin-binding capability. uPA is a tissue-localized
plasminogen activator that is regarded as the critical trigger for
cell-mediated proteolysis during macrophage invasion, tumor cell
invasion and metastasis, angiogenesis, would healing and tissue
remodeling [15].
Considerations of radiolabelingThere are many advantages of using non-radiolabeled drugs with
high (or ultra) performance liquid chromatography (HPLC or
UPLC1) tandem mass spectrometry (MS/MS) over using radiola-
beled drugs for tissue elimination and distribution studies. For
example, the former could propose the potential metabolites
produced in addition to the quantity of non-radiolabeled parent
drugs. Moreover, there is a significant cost in dealing with radi-
olabeled compounds in terms of synthesis, handling and waste
disposal of radiolabeled compounds. However, the radiolabeled
compounds are still used when a HPLC with a radiochemical
detector is conveniently available.14C and 3H are commonly used radioisotopes. 14C and 3H have
long decay half-lives of 5730 years and 12.3 years, respectively.
Hence, there is no need for decay correction for 14C and 3H. This
makes 14C and 3H the most appropriate radioactive labels for a
mass balance study. It seems that 14C is more suitable than 3H as a
typical radioisotope because: (i) 14C is about eightfold more ener-
getic than 3H (0.156 Mev versus 0.019 Mev), therefore 14C can be
detected more easily with better sensitivity (if accelerator mass
spectrometry is used, the amount of 14C needed for the study could
be drastically reduced); (ii) the kinetic isotope effect–the greater
the mass the stronger the chemical bond, and this can affect
reaction rates [because the relative mass difference between 12C
and its radioactive isotope 14C (i.e. 17%) is significantly less than
the relative mass difference between 1H and 3H (i.e. 200%),
replacement of 12C with its radioactive isotope 14C would result
in a smaller impact on bond strength and a minor kinetic isotope
effect than replacement of 1H with the 3H]; (iii) 3H can be
unstable and lost when it exchanges with normal hydrogen in
water, thus the use of 14C as the radioisotope is preferable to the
use of 3H [1,4,16].
The radioactive isotope should be labeled at the metabolically
stable position such as the aromatic or alicyclic ring systems of the
drugs. If the radioactive isotope is incorporated into the metabo-
lically labile site of the drug, the isotope can be rapidly detached
from the drug, making the metabolic products no longer traceable.
The administered radiolabeled formulation is usually prepared by
mixing the non-radioactively labeled drug with the radiolabeled
ones so that the total concentration of the drug and its metabolites
could be quantified by determining the radioactivity of the radio-
active tracer.
Management of animals, dosing and samplingFor tissue distribution studies, the animals can be group-housed in
microisolator cages. For elimination studies in mice, the mice
should be group-housed in metabolism cages (n � 4 per cage) to
obtain pooled urine sufficient for analysis. For elimination studies
in rats, one rat should individually reside in a single metabolism
cage, and the individual data should be statistically pooled and
analyzed to determine the mean and the standard deviation of the
distributed and eliminated drug.
The radioactivity expressed as counts per minute (cpm) of drug
formulation should be carefully checked and recorded immedi-
ately before dosing to determine the mass balance of the test drug.
It is very important to account the amount of radioactive drug
contaminated at the injection site (such as the radioactivity at
injection site of the mouse tail). To reduce the work load and
animal number to be used, terminal sampling should not be
scheduled as busy as the early phase of drug-blood concentration
time course does. It sounds pharmacologically reasonable to col-
lect blood and tissue samples as early as possible after intravenous
(i.v.) dosing because blood levels of the drug decline exponentially
at the early stages post-dosing. The following time points of
sampling seem reasonable: 2 min, 1 � t1/2 (elimination half-life),
2 � t1/2, 4 � t1/2, 6 � t1/2 after i.v. dosing, and even longer if the
drug adheres to tissue tightly. Theoretically, drugs disappear from
the blood after five t1/2 following administration. It also seems
reasonable to collect blood and tissue samples at tmax (the time to
achieve peak blood levels of a drug) and 1 � t1/2, 2 � t1/2, 4 � t1/2,
6 � t1/2 after oral dosing. The endpoint of sampling should be
prolonged if the drug adheres to tissue tightly.
Tissue preparation including tissue digestion and de-colorizationSoon after sampling, tissues should be individually weighed to
calculate drug amount based on the tissue weight (mg/g of tissue)
[1,16]. Figure 1a represents a typical histogram used to express
tissue distribution of a drug following its oral administration [1].
For non-radiolabeled drugs [8], the tissues should be dissected and
homogenized in phosphate-buffered saline (pH 7.4, 10 mM) at a
ratio of tissue:buffer at 1:2 (w/v) followed by liquid–liquid extrac-
tion of the drug from the mixture [17]. For radiolabeled drugs [1],
extensive tissue homogenization might not be needed. Instead,
the commercial solubilizer (e.g. BTS-450 tissue solubilizer com-
posed of 0.5 N of tetraethylammonium hydroxide in toluene) can
be used. Tissue samples can be digested with 1 ml of BTS-450 at
508C until tissues are dissolved. Heating can remarkably increase
digestion rate. Drops of 30% H2O2 can be added to the solution to
de-color the tissue solution. Glacial acetic acid (70 ml) helps to
eliminate chemiluminescence.
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Sample homogenates might need to be diluted accordingly with
an appropriate medium or solvent to reach the appropriate ana-
lytical reading scope of the drug. 20 mg of feces can be incubated
in the BTS-450 solution at 40 8C for two hours followed by addi-
tion of 0.5 ml of isopropanol and 0.2 ml of 30% H2O2 (for de-
colorizing), respectively, to the mixture. For blood radioactivity
counting, 30 ml aliquots of the whole-blood can be transferred
onto the Ready Cap, a crystalline scintillator used for replacement
of the cocktail. When samples are made this is done at room
temperature, the Ready Cap can be placed into a standard 20 ml
liquid scintillation vial for a direct radioactivity count. To deter-
mine the tested drug remained in the carcasses (e.g. total radio-
activity recovery), the mouse carcasses after rough dissection can
be completely dissolved in 10 ml of NaOH (10 N) after incubation
at 508C for about three hours.
Whole-body autoradiographyDrug tissue distribution can be determined by quantitative whole-
body autoradiography using a densitometer [18]. Briefly, eutha-
nized animal body is embedded in 5% sodium carboxymethycel-
lulose using a dry-ice–heptane bath. Sagittal sections (20 mm) of
animal body are collected and dried on Scotch tape at �208C. In a
darkroom, the sections are calibrated with a radioactive standard
and then exposed to a commercial imaging film such as Kodak SB5
Scientific for 1–8 weeks followed by film development. However,
the technique takes a long time to obtain the study results, and the
method cannot be used to determine the tested drug remaining in
the body, which is important information for the total radio-
activity recovery.
Urinary and fecal eliminationThe volume of mouse urine containing a typical radioactive drug
should be quantitatively measured after collection. The trace of
urine inside of the metabolism cage should be carefully collected
for counting, especially during the first 8-h period after dosing
when the majority of the drug is urinated out. Urine and feces can
be collected at different time points after dosing (Fig. 1b) [1]. A rat
can urinate more than 50 ml urine overnight. Hence, caution must
be paid to the volume of the urine container to avoid urine to fill to
the brim of the container so that the urine volume can be accu-
rately recorded. Cumulative excretion (% of administered dose) of
the drug in urine and feces with time, and the excretion rate (ng/
ml/h) of the drug at different intervals after a single administra-
tion, should be determined to provide a complete picture of the
amount and the rate of the investigational drug excreted [6].
Mass balanceMass balance employs a radioactive tracer to investigate the ADME
of a drug and its metabolites in the animal body after its admin-
istration [19]. After administration of the radiolabeled drug, the
residual radioactivity from the drug containers should be sub-
tracted from the calculated administered dose to obtain the actual
administered dose. The radioactive biological samples such as
blood, urine and feces should be collected at times across the
entire period of the drug ADME study to allow determination of
pharmacokinetic parameters. The exhaled air might need to be
collected if the drug is volatile. Blood sampling is usually per-
formed at baseline, during infusion and before the end of infusion
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if the test drug is administered by infusion. Blood sampling should
be more frequent during the distribution phase and less frequent
during the terminal elimination phase. Sample collection becomes
unnecessary if less than 1% of administered radioactivity is
excreted. The samples should be immediately stored at �208Cor less after collection. For sample analysis, the plasma and urine
samples should be quantified with scintillation liquid after dilu-
tion. If the samples are less colored, the results can be corrected for
variation by automatic quench correction with the same matrix
characteristics. If the samples are more colored, the samples
should be pretreated with tissue solubilizers and hydrogen per-
oxide for complete digestion and de-colorization. Chemilumines-
cence can be diminished by choosing the appropriate liquid
scintillation solvents that allow the chemiluminescence to decay
before counting. In theory, all administered radioactivity can be
detected in the excreta, and the mass balance can be achieved with
full recovery of radioactivity. In practice, the administered radio-
activity cannot be totally recovered. A lower recovery, which is not
uncommon, could be caused by biological factors such as acci-
dental binding of the radioactive materials to irrelevant containers
and tissue components, a short decay half-life of the radioactive
materials used or radioactive loss through expiration.
Enterohepatic circulationEHC refers to the circulation of bile acid, drugs or other substances
from liver to the bile, followed by reabsorption into the small
intestine and transport back to the liver for systemic circulation.
Figure 2b illustrates the anatomic route of the EHC: drug-contain-
ing blood from the gastrointestinal tract flows via the mesenteric
vein and portal vein sequentially to the liver and hepatic vein into
the systemic circulation. The liver synthesizes and secretes the bile.
A fraction of bile that contains the recycling drug flows into the
duodenum through the sphincter of Oddi. Generally, bile is stored
in the gallbladder, which sporadically empties a fraction of bile.
The emptying is stimulated by food intake. The average bile flow
rate in humans is 1.5–2.0 ml/min/kg bodyweight. Bile provides a
route for the excretion of endogenous and exogenous molecules.
Numerous drugs eliminated via the bile in the unchanged or
conjugated form into the small intestine are available for reabsorp-
tion into the portal vein and systemic circulation (Table 1). After
metabolism in the liver, the drug and/or its metabolites enter into
the bile. With the flow of bile, they enter into the gallbladder and
are emptied into the small intestine. A fraction of the drug can re-
enter into the small intestine by passive diffusion or active trans-
port. Meanwhile, the glucuronide metabolite of the drug could be
de-conjugated by the enzyme existing in the intestinal microflora
that can cleave glucuronide conjugates, leading to the availability
of parent drug for reabsorption. Some part of what is absorbed will
be secreted into the bile again, and the rest will enter the systemic
circulation. Therefore, the existence of the EHC process extends
the drug residence time in the body. A drug undergoing EHC
usually shows the multiple-peak phenomenon in its plasma-con-
centration–time profile and the prolonged elimination half-life.
The liver is composed of two kinds of epithelial cells: hepato-
cytes and cholangiocytes. Hepatocytes secrete drugs, metabolites
and hormones into the bile. They are the only cells in the
body that convert cholesterol to bile acids. Cholangiocytes line
intrahepatic bile ducts and account for 3–5% of the liver cell
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(b)
Right and left hepatic ducts of liver
Hepatic vein
Gallbladder
Mucosawith folds
Cystic duct
Duodenum
Oddi’s sphincter
Main pancreatic and sphincter
Mesenteric vein
Jejunum
Pancreas
Common hepatic duct
After firstpass
Systemic
circulation
Portal vein
Accessory pancreatic duct
Blie duct and sphincter
(a)
Drug Discovery Today
FIGURE 2
Two routes for eliminating drugs or unwanted substances from systemic circulation: renal route and enterohepatic circulation (EHC) intestine. (a) The renal route:
drugs with molecular weight less than 30–40 kDa and measuring 5–6 nm in diameter are primarily excreted in urine via glomerular filtration. (b) The EHC intestineroute: the orally administered drugs are absorbed by the digestive system and enter the blood circulation. The drug-containing blood flows via the mesenteric
vein and portal vein sequentially to the liver and hepatic vein into the systemic circulation. Some drugs are metabolized in the liver and excreted in the bile.
Generally, bile is stored in the gallbladder and is discharged through the bile duct upon eating. A fraction of discharged bile that contains the recycling drugs or
unwanted substances flows into the duodenum through the sphincter of Oddi. A portion of the excreted substances (i.e. drugs, HIV virus) can thus be eliminated infeces. Another portion of the excreted substances can be reabsorbed across the intestinal mucosa, transported back to the liver via the portal vein and returned to
the circulation system.
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population. Cholangiocytes provide a large surface area for trans-
port between blood and bile, and play a significant part in bile
formation.
Factors that interfere with EHCThere are several factors influencing EHC. The rate-limiting step of
drug absorption is determined by the physiology of the gastro-
intestinal tract and the physicochemical properties of the drug
(pKa, water/lipid solubility, formulation). The patient’s physiolo-
gical conditions such as lumen pH, gastric emptying time, intest-
inal transit time, surface area, gastrointestinal disease, the food in
gastrointestinal tract and the intestinal microflora will affect drug
absorption. The intestinal microflora, by hydrolyzing biliary drug
conjugates, affects drug EHC. For instance, cysteine S-conjugate
beta-lyase manipulates metabolism of cysteine conjugates and
thus changes the circulation rate of cysteine [20]. These factors
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REVIEWS Drug Discovery Today �Volume 19, Number 3 �March 2014
Liver
Liver
Donor rat Recipient rat
Tube 2
Tube 1Bile duct
Bile duct
Gallbladder
Gallbladder
Duodenum
Duodenum
Drug Discovery Today
FIGURE 3
Schematic diagram of the hepato-duodenal shunt model for studying theenterohepatic circulation. The common bile duct of the donor rat (receiving
the test drug) has a polyethylene (PE)-10 tube inserted to direct the bile into
the duodenum of the recipient rat (receiving the bile). To balance the fluid
losses and gains in the two paired rats, the bile of the recipient rat is drainedback to the donor rat through another PE cannula.
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define the disintegration and dissolution rate as well as the solu-
bility of drugs in the gastrointestinal tract [21].
Properties of drugs experiencing EHC can be exemplified as
follows. The threshold molecular weight of drugs undergoing
the biliary route of excretion was estimated to be 500–600 Da
[22]. After more broadly gathering the existing data and carefully
analyzing the data, we identified more than 45 molecules under-
going EHC (Table 1), and their molecular weights ranged from 290
to 1300 Da. Drugs with molecular weights less than 30–40 kDa,
and measuring 5–6 nm in diameter, are excreted primarily in urine
via glomerular filtration (Fig. 2a) [7]. Many drugs are secreted into
bile and undergo some extent of EHC. These include morphine,
warfarin, indomethacin, cardiac glycosides, rifampicin, baicalin,
ciclosporin, piroxicam, progesterone, ceftriaxone and doxycycline
(Table 1).
Table 1 illustrates some of typical drugs that have been shown to
go through EHC, the process that can lead to an increase in the
half-life of the drugs. EHC occurs particularly with small, less-polar
drugs. Besides, it is reported that some kinds of cells, biomacro-
molecules and nanomedicines can undergo EHC. T lymphocytes
derived from the small intestine during active inflammation can
circulate to the liver via EHC to cause hepatic disease [23,24].
Biomacromolecules such as immunoglobulin A (IgA) and insulin-
like growth factor-I (IGF-I) could be reabsorbed from the intestine
lumen in a receptor-activated form into the portal blood for
recirculation [25]. Although the nanomedicines have been widely
studied and some have advanced into the clinic, there are few
reports about EHC of nanomedicines. It was reported that encap-
sulation of indomethacin into poly(DL-lactide) nanocapsules
could increase uptake of drug by liver macrophages and biliary
excretion of the encapsulated drug, leading to enhanced EHC of
indomethacin [26]. In another work, phosphatidylcholine-choles-
terol liposomes were demonstrated partially to reconstitute
absorption of palmitic acid from the small intestine to the liver
[27]. Consideration of the significance of EHC urges us to assess the
current data and information about these EHC drugs more care-
fully to understand and redefine properties and characteristics of
these drugs undergoing EHC (Table 1).
Experimental methods for determining EHCChemical interruption of EHCIf EHC is interrupted, such as through oral administration of the
activated charcoal that binds the EHC drug to prevent it from
reabsorption into the intestine, the half-life of the EHC drug will
be decreased.
The example is RU486 that has been used as an abortion pill
worldwide. RU486 resides in human body for a long time, and its
elimination t1/2 ranges from 26 to 51 hours depending on the dose
given. The long t1/2 resulted from the drug’s high plasma-protein-
binding rate and its EHC. It has been demonstrated that the bound
fraction of [3H] RU486 in plasma as determined by equilibrium
dialysis was 94% [28]. In humans, it was found that RU486 is
mainly excreted via bile. This triggered an investigation to see if
RU486 goes through the EHC [29]: healthy volunteers, after fasting
overnight, took a single dose of RU486 (200 mg per person) and
fasted again for another three hours, and then took 5 g of the
activated charcoal five times daily for one week. The non-charcoal
group took the same dose of RU486 only. Serum concentrations of
338 www.drugdiscoverytoday.com
RU486 were then measured by the radioimmunoassay, preceded
by chromosorb column chromatography. The area of serum-con-
centrationt–time-course of RU486 in the charcoal group was sig-
nificantly lower than those in the non-charcoal group (P < 0.05).
The t1/2 of RU486 (17 hours) was obviously decreased for the
charcoal group in comparison with the non-charcoal group (30
hours). Drugs where EHC can be chemically interrupted often
show a similar phenomena and their EHC properties can be
investigated by using the same method as mentioned above.
Hepato-duodenal shunt modelColchicine is a drug used to treat gout (Table 1). It was found that
colchicine exhibited EHC. A microdialysis method using a hepato-
duodenal shunt model has been developed to investigate the
mechanism of colchicine EHC [30].
The hepato-duodenal shunt model was designed as follows
[31,32] (Fig. 3): a donor rat (receiving the test drug) and a recipient
rat (receiving the bile from the donor rat) with matched age and
weight are chosen as the paired rat model. They are anesthetized
and the body temperature is maintained at 378C throughout the
experiment period. The bile duct of the donor rat is surgically
exposed and a 20 cm section of polyethylene (PE)-10 cannula is
inserted proximal to the liver of the donor rat. The other end of the
cannula is inserted into the bile duct in the duodenum of the
recipient rat. To balance the fluid losses and gains in the paired
rats, the bile of the recipient rat is drained back to the donor rat
through a PE-10 cannula. The use of nasobiliary catheters or biliary
T-tubes to collect bile samples is also an effective way to measure
EHC. The method is often conducted using small numbers of
patients [22].
Following annulations, the donor rat is intravenously adminis-
tered with the test drug through the femoral vein. The blood
samples are collected from the jugular vein of the donor and
recipient rats and assayed by liquid chromatography. It can be
observed that the EHC drug concentration in the donor rat blood
Drug Discovery Today � Volume 19, Number 3 �March 2014 REVIEWS
Reviews�POSTSCREEN
declines owing to the blocking of EHC. The EHC test drug level
increases in the recipient rat, resulting from the absorbing test
drug from the bile of the donor rat [30].
Bile duct ligation coupled with LC/MS/MSNaringenin (NAR) is a drug that exhibits vasodilatory, antioxidant,
antiulcer and antitumor effects. Using a sensitive LC/MS/MS
method to establish the plasma drug concentration curve of
NAR, Ma et al. [33] identified double peaks in the plasma NAR
concentration curve of the rats receiving NAR. The result suggested
the existence of EHC in the drug’s disposition. Under ether
anesthesia, a laparotomy was performed in each rat. The common
bile duct of each rat was cannulated with a PE-10 tube. The other
end of the tube was inserted through the celiac muscle of the rats,
and fixed on the back of the rats to collect bile at different
intervals. Before NAR administration, the surgical exposure was
sewn up. Following a 3-day rest, the rats were orally administered
30 mg/kg NAR and the bile fluid was withdrawn at 0–2, 2–4, 4–6, 6–
8, 8–12, 12–24 and 24–48 hours. Meanwhile, blood samples were
collected at 5, 15, 30 and 45 min as well as 1, 2, 4, 6 and 8 hours.
Plasma and bile samples were diluted tenfold before analysis of the
samples by HPLC and MS.
After administration by gastric gavage, most NAR was excreted
from bile in the form of glucuronide conjugates. The total plasma
concentration versus time curve of NAR in cannulated rats exhib-
ited no double peaks and relatively lower drug plasma concentra-
tion compared with that in normal rats. This animal model and
the difference in blood concentration of the test drug obtained
before and after the bile drainage can be used to predict whether
the EHC pathway exists with the test drug or its glucuronide
conjugate.
Concluding remarksSystems pharmacokinetics is the quantitative analysis of the
dynamic interactions between drugs and a biological system
to understand the behavior of the body system as a whole in
absorbing, distributing, metabolizing and excreting drugs, as
opposed to the behavior of individual biological constituents.
Thus, it has become the interface between systems biology and
pharmacokinetics. Application of systems pharmacokinetics can
now impact across all stages of drug discovery and development as
well as large-scale clinical trials.
The data of drugs (i.e. metabolite information and the asso-
ciated pharmacokinetic parameters generated from the tissue dis-
tribution and elimination studies) constitute the basis for systems
pharmacokinetics and for clarifying the efficacy and toxicity of the
test drugs. They could also provide the information on specific
effects of the drugs for target tissues and organs. To obtain an
accurate comprehensive knowledge of the pharmacokinetic char-
acteristics of a drug, however, it is important to design the drug
tissue distribution and elimination experiments logically and
correctly. In our previous publications [1,3–5,16], we have pro-
vided hands-on experimental designs for in vitro and in vivo drug
metabolism and protein binding studies. This review further
outlines the requirements and guidelines in the experimental
designs for drug tissue distribution and elimination, covering
animal selection, material preparation, dosing and sampling,
tissue preparation and analysis, elimination, and mass balance.
EHC is the typical pharmacokinetic characteristic that some
drugs have. The clinical significance of EHC is just being under-
stood, and the potential of the pathway has not been fully
tapped. We will continue our exploitation for the clinical impli-
cations of EHC.
AcknowledgementsThis research was supported by the National Natural Science
Foundation of China (no. 81201709 and no. 81273548), the
National Science Foundation for Fostering Talents in Basic
Research of China (no. J1103303), the China Postdoctoral Science
Foundation (no. 2012M511441 and no. 2013T60638) and the
Science and Technology Development Foundation of Fuzhou
University (2013-XQ-8).
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