Drug enterohepatic circulation and disposition ...cmapc.fzu.edu.cn/attach/download/2015/09/23/166937.pdfenterohepatic circulation and disposition: constituents of systems pharmacokinetics

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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.

326 www.drugdiscoverytoday.com 1359-6446/06/$ - see front matt

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

mailto:[email protected]

http://dx.doi.org/10.1016/j.drudis.2013.11.020

Drug Discovery Today � Volume 19, Number 3 �March 2014 REVIEWS

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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

Stomach

Tumor

Kidney

Lu

14C

-car

ben

daz

im in

tis

sue

(g/g

)

1h4h8h24h

0

50

100

150

200

250

(a)

(b)

Small intesti

ne

Large in

testine

T

Acc

um

ula

ted

elim

inat

ion

%

10

5

0-4

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

Drug Discovery Today

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 )


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]






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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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]






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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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]






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


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



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


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


(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)


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.


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



Liver

Liver

Donor rat Recipient rat

Tube 2

Tube 1Bile duct

Bile duct

Gallbladder

Gallbladder

Duodenum

Duodenum


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.

Review

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OSTSCREEN

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


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



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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