Pharmacokinetics—Absorption, Distribution, and Elimination☆

Ivo P Nnane, Janssen Research & Development, LLC, Spring House, PA, United States
© 2018 Elsevier Inc. All rights reserved.

Introduction 1
Importance of ADME 1
Routes of Drug Administration 2
Fate of Drugs in the Body 2
Absorption 2
Distribution 5
Elimination 6
Drug Transporters 7
Blood Concentration–Time Profile 8
Pharmacokinetic Models 8
One-Compartment Model—Instantaneous Intravenous Input 8
Common Pharmacokinetic Parameters 9
Elimination Half-Life 9
Volume of Distribution 9
Clearance 10
Bioavailability 10
Pharmacokinetic Software 10
Opportunities and Challenges for Studying ADME 11
Further Reading 12

Introduction

Pharmacokinetics, often described as what the body does to a drug, is the quantitative study of the kinetics of drug absorption,
distribution, metabolism, and excretion (ADME). The related discipline of pharmacodynamics evaluates the effects of drugs on the
body including the mechanisms of their action and the relationship between the concentration of the drug at the site of action and
the pharmacological response. The safety and efficacy of a drug is influenced by several factors, including its pharmacokinetic and
pharmacodynamic profiles in animals and humans. After administration of a drug, it undergoes ADME in the body, and interacts
with a biological target such as a receptor or enzyme to produce a pharmacological effect. Pharmacokinetic and pharmacodynamic
principles are used to relate the concentration of a drug in a suitable biological sample such as plasma to the pharmacological
response, and to understand the time course of the drug effect. Pharmacokinetic and pharmacodynamic techniques are often
applied in the drug discovery and development process, and in the optimization of drug therapy in the clinical setting. During drug
discovery and development, the pharmacokinetics of new chemical entities (small-molecules) and biopharmaceuticals for example
peptides and monoclonal antibodies (large molecules) are often evaluated. A good understanding of the ADME of small molecules
or protein drugs is critical for drug discovery and development and drug utilization since these processes impact therapeutic
outcomes in patients. The same principles of ADME for small molecule drugs generally apply to peptide and protein drugs,
however, there are some important differences. In this article, the application of pharmacokinetics in drug discovery and develop-
ment and in clinical practice is discussed. Current in vitro and in vivo approaches used to study ADME of small molecules are
discussed and notable differences for large molecule ADME are highlighted. The drug and metabolite concentrations in blood or
other biological samples are routinely measured in pharmacokinetic studies, and appropriate models and software are used to
calculate PK parameters such as clearance, volume of distribution and terminal half-life. The simple one-compartment pharmaco-
kinetic model after IV administration is used to illustrate the plasma concentration–time profile and to simplify our understanding
of drug ADME processes.

Importance of ADME

During drug discovery and development, successful drug candidates must have the right ADME properties and desirable pharma-
cological and toxicological profiles. Unfavorable ADME and toxicological profiles contribute significantly to the failure rates of drug
candidates during drug discovery and development. The ADME studies provide supportive information that facilitates the
interpretation of pharmacology and toxicology findings during drug discovery and development. As a result, pharmaceutical
companies are allocating considerable resources for the evaluation of ADME properties of drug candidates in early stages of drug

☆
Change History: June 2018. IP Nnane updated text and references.

Encyclopedia of Analytical Science, 3rd Edition https://doi.org/10.1016/B978-0-12-409547-2.14439-7 1

2 Pharmacokinetics—Absorption, Distribution, and Elimination

evaluation in order to find the right ADME balance and reduce the failure rates of drug candidates in later stages of drug
development. ADME studies allow the identification of drug candidates that would be sufficiently absorbed after dosing and
eliminated at acceptable rates so that an efficacious steady-state plasma concentration is attained in the target population following
administration of a suitable dosage regimen. Furthermore, regulatory authorities are demanding information on ADME profiles and
pharmacodynamic activities before new drug applications are approved. ADME studies are routinely performed in vitro and in
animals, and increasingly in humans during the drug discovery and development process, and the information that is generated
constitutes an important part of the new drug applications (NDA) or biologics license application (BLA) that are submitted to the
regulatory authorities such as the FDA or EMA. During drug discovery, new drug candidates are synthesized using combinatorial
chemistry or biotechnological processes, and lead drug candidates are then selected based on desirable biological activities, ADME
properties and toxicity information obtained in relevant in vitro and in vivo systems. A small number of drug candidates with
desirable ADME properties that are determined to be well tolerated, safe and effective in appropriate in vitro and in vivo models
may enter into human clinical trials for further testing. Thus, a good understanding of the ADME profiles of the selected drug
candidates and a full consideration of the factors that influence the plasma concentration–time profiles of drugs in vivo is pivotal in
drug discovery and development, clinical trial design and use of drugs in the clinical setting.

Routes of Drug Administration

Several routes of drug administration, including extravascular (oral (po), intramuscular (im), subcutaneous (sc), transdermal,
inhalation, etc.) and intravascular routes (intravenous (iv) and intra-arterial), are used to deliver drugs into the body. For small
molecule drugs, oral administration is the most common route because it is relatively convenient and safe. For example, several
small molecules including disease modifying anti-rheumatic drugs (DMARDS), steroids and non-steroidal anti-inflammatory drugs
are given orally for the treatment of rheumatoid arthritis and pain relief (Table 1). Many factors including the physicochemical
properties of drugs, type of formulation, physiology of the gastrointestinal tract (GIT), other drugs, and food affect the way in which
drugs are absorbed after oral administration. Although drug absorption occurs in the mouth and stomach, it takes place mostly in
the small intestine due to its large surface area. After oral administration, the drug passes through the intestinal wall and liver to
reach the general circulation. The drug metabolism enzymes in the intestinal wall and liver metabolize many drugs, thereby
decreasing the amount of drug that is bioavailable. This phenomenon is often referred to as first-pass metabolism. The acidic
environment and digestive enzymes in the stomach may also chemically degrade some drugs, resulting in erratic absorption. In
contrast, drugs administered intravenously do not undergo absorption and presystemic metabolism, and therefore the entire dose
reaches the general circulation intact.
Biotherapuetics including monoclonal antibodies (mAbs), antibody fragments, fusion proteins and peptides are commonly
administered by parenteral routes, such as intravenous (iv), subcutaneous (sc) or intramuscular (im) injection. During the last few
decades several targeted biotherapeutics including infliximab and adalimumab have been developed and approved for the
treatment of immune mediated inflammatory diseases such as rheumatoid arthritis (Table 2). For biotherapeutics, several
important features, such as molecular size, hydrophilicity, target expression, manufacturing processes etc. influence their ADME
properties. For therapeutic proteins, sc administration is often the preferred route of delivery since molecular size, hydrophilicity,
and gastric degradation preclude oral administration and gastrointestinal (GI) absorption.

Fate of Drugs in the Body

After a drug is administered, it undergoes several processes including absorption, distribution, metabolism, and excretion (ADME)
in the body. At the site of administration, the drug is liberated from the dosage form and dissolved in tissue fluids. Several factors
influence the rate and extent of dissolution of the drug at the site of absorption, including the solubility of the drug, particle size, and
surface area of a solid dosage form. During absorption, the unchanged drug is transferred from the site of absorption into the
systemic circulation. The drug molecules that reach in the general circulation are reversibly distributed between the blood and other
tissues in the body. The drug is eliminated from the body by excretory organs such as the kidney or by metabolism in the liver to
metabolites that are readily excreted. At the site of action, drug molecules interact with receptors to initiate a cascade of events that
elicit a pharmacological or toxicological response. In essence, after entering the body, the drug acts on the body to produce a
biological response, and simultaneously the body acts on the drug to eliminate it from the body (Fig. 1). The processes of ADME
directly impact the plasma concentration–time profile of a drug and ultimately its pharmacodynamic effects. Additionally, the
factors that influence ADME of drugs have a significant impact on the overall pharmacokinetics of the drug.

Absorption

The movement of a drug from the site of administration into the general circulation is referred to as absorption. After extravascular
administration, the drug molecules are transferred from the site of absorption, through a cell membrane, into the systemic
circulation. The physicochemical properties of the drug and the structure and physiology of the cell membrane influence the
Table 1 Pharmacokinetic parameters of some small molecules drugs used to treat rheumatoid arthritis

Molecular Human
weight Route of bioavailability Clearance (CL or Volume of distribution
Drug Target/Mechanism (Da) administration (%) Tmax CL/F) (Vss or Vss/F) Half-life

Methotrexate Antimetabolite; inhibits dihydrofolic acid reductase 454.45 Oral, 60 (oral) 1–2 h 80 mL/min at 0.4–0.8 L/kg 3–10 h
(Trexall) intramuscular, small doses
intravenous,
intra-arterial,
intrathecal
Leflunomide Immunomodulatory agent which inhibits dihydroorotate 270.2 Oral 82–95 6–12 h (Active 31 mL/h 0.13 L/kg 14–18 days
(Arava) dehydrogenase metabolite)
Sulfasalazine Related to the anti-inflammatory and/or immunomodulatory 398.39 Oral <15 10 h 1 L/h 7.5
1.6 L 7.6
3.4 h
(Azulfidine) properties
Celecoxib Nonsteroidal anti-inflammatory drug (NSAID). Inhibition of 381.38 Oral – 3h 27.7 L/h 429 L 11.2 h
(Celebrex) prostaglandin synthesis primarily through inhibition of
cyclooxygenase-2 (COX-2)
Rofecoxib Nonsteroidal anti-inflammatory drug (NSAID). Inhibition of 314.36 Oral 93 2–3 h 141 and 120 mL/ 91 L 17 h
(Vioxx) prostaglandin synthesis primarily through inhibition of min at 12.5 mg
cyclooxygenase-2 (COX-2) and 25 mg,
respectively
Etodolac A member of the pyranocarboxylic acid group of nonsteroidal 287.37 Oral 80–100 80
30 min 49 (
16) mL/h/kg 390 mL/kg 6.4 h

Pharmacokinetics—Absorption, Distribution, and Elimination

(Lodine) anti-inflammatory drugs (NSAIDs)
Ibuprofen Nonsteroidal anti-inflammatory drug. 206.28 Oral – – – 200 mL/kg 1.8–2.0 h
(Motrin) Related to prostaglandin synthetase inhibition-involves
inhibition of cyclooxygenase (COX-1 and COX-2).
Prednisone A corticosteroid with potent anti-inflammatory effects 358.43 Oral – 1–2 h – – 2–3 h
(Deltasone)
Oxaprozin A nonsteroidal anti-inflammatory drug (NSAID). Related to 293 Oral 95 3h 0.15–0.3 L/h 11–17 L 41.4–54.9 h
(Daypro) prostaglandin synthetase inhibition
Ketorolac NSAID. Related to prostaglandin synthetase inhibition 255.27 IV, oral and IM 100 44
34 min 0.02–0.05 L/h/kg 13 L 4.0–7.9 h
(Toradol)
Tramadol Centrally acting synthetic opioid analgesic 299.8 Oral 75 2.3 h 5.9 mL/min/Kg 2.6 and 2.9 L/kg in male 6.3
1.4 h
(Ultram) and female subjects,
respectively
Codeine An opioid analgesic, related to morphine; selective for the mu 299.36 Oral 90 60 min 2280
840 mL/ 3–6 L/kg 3–4 h
receptor min
Morphine An opioid agonist. Relatively selective for the mu receptor 758 IV/oral 25–40 15 min 20–30 mL/min/kg 1–6 L/kg 2 h
Acetylsalicylic Interferes with the production of prostaglandins through 180.16 Oral – 1–2 h 13–19 min
acid (Aspirin) acetylation of cyclo’oxygenase. Analgesic, anti-
inflammatory, antipyretic and platelet aggregation inhibitor
Acetominophen Analgesic and antipyretic properties; primarily involve central 151.16 IV/Oral 79 30 min to 2 h 0.27 L/h/kg 1 L/kg 2.4 h
(Tylenol) actions
Tofacitinib Janus Kinase inhibitor 504.5 (or IV/Oral 74 0.5–1 h – 87 L 3 h
(Xeljanz) 312.4
as free
base)

3
Source: FDA or EMA label for each drug listed.
 4
Pharmacokinetics—Absorption, Distribution, and Elimination
Table 2 Pharmacokinetic parameters of large molecules drugs used to treat rheumatoid

Molecular Human
weight Route of bioavailability Volume of
Biotherapeutic Target/Mechanism (kDa) administration (%) Tmax Clearance distribution Half-life

Abatacept Inhibits T lymphocyte activation by binding to CD80 and 92 IV, SC 78.6 (SC) – 0.13–0.47 mL/day/kg 0.02–0.13 L/kg 8–25 days
(Orencia) CD86
Adalimumab Binds specifically to TNF-alpha and blocks its interaction 148 SC 64 131
56 h 12 mL/h 4.7–6.0 L 10–20 days
(Humira) with cell surface TNF receptors
Anakinra Blocks the biologic activity of interleukin-1 (IL-1) alpha 17.3 SC 95 3–7 h 105 (27) mL/min 18.5(11) L 4–6 h
(Kineret) and beta
Certolizumab PEG-Fab’ fragment. Binds to human TNF-alpha 91 IV/SC 76–88 54 and 21.0 mL/h 6–8 L 14 days
pegol 171 h
(Cimzia)
Etanercept Dimeric soluble form of the TNF receptor that can bind 150 SC 76 69
34 h 0.066 L/h 10.4 L 102
30 h
(Enbrel) TNF-alpha
Golimumab Human monoclonal antibody that binds to TNF-alpha 150 IV/SC 53 2–6 days 4.9–6.7 mL/day/kg 58–126 mL/kg 14 days
(Simponi)
Infliximab Binds with high affinity to TNFa and inhibits binding of 149.1 IV – – 0.41
0.01 L/day 3.0–4.0 L 7.7–9.5 days
(Remicade) TNF-alpha with its receptors
Rituximab Monoclonal antibody that targets the CD20 antigen 145 IV – – 0.335 L/day 3.1 L 18.0 days (5.17–77.5 days)
(Rituxan) expressed on the surface of pre-B and mature
B-lymphocytes
Tocilizumab Binds specifically to both soluble and membrane-bound 148 IV/SC 80 – 0.29
0.10 mL/h/kg 6.4 L Decreased with decreasing
(Actemra) IL-6 receptors (sIL-6R and mIL-6R) at the 10 mg/kg concentrations from 18 to
6 days)
Sarilumab Human recombinant monoclonal antibody of the IgG1 150 SC – 2–4 days Dose dependent: up to
(Kevzara) subclass that binds to the IL-6 receptor 8–10 days

Source: FDA or EMA label for each drug listed.

 Pharmacokinetics—Absorption, Distribution, and Elimination 5

Receptor Response

Distribution Metabolism
Liver

Absorption
Absorption Blood Distribution
site

Distribution Kidney Excretion

Other
tissues

Fig. 1 Schematic representation of the fate of a drug in the body.

passage of the drug from the site of administration into the general circulation. Several additional factors including intrinsic subject
characteristics (such as body weight, sex, age, etc.), catabolic capacity at injection site, blood flow, formulation and drug product
characteristics, charge and drug concentration influence drug absorption. The transport of drugs across cell membranes is mediated
by several mechanisms, including passive diffusion, facilitated transport, active transport, endocytosis, exocytosis, and ion-pair
transport. Accumulated evidence indicates that passive diffusion is the most common mechanism of drug absorption for small
molecules drugs. Passive diffusion is driven by lipid solubility and the magnitude of the drug concentration gradient across the cell
membrane and governed by Fick’s first law according to Eq. (1).
DC
Flux ¼  DA (1)
h

The flux refers to the flow of drug molecules across an area (A) per unit time; DC/h is the concentration gradient divided by the
membrane thickness (h), and D is the diffusion coefficient of the drug. The negative sign indicates that drug transfer occurs from an
area of high concentration to one of low concentration.
In general, the membrane lipid bilayer is highly permeable to lipid-soluble drugs and poorly permeable to water-soluble drugs.
However, some embedded proteins in membranes can form channels, carriers, or pumps that facilitate the movement of polar drugs
across membranes. The rate of diffusion of a drug also depends on its molecular size, although the variation with molecular weight
is modest for drugs with molecular weights of 200–1000 Da. The degree of ionization of a drug also plays a significant role in the
diffusion of the drug across membranes. Since most drugs are either weak acids or bases, their degree of ionization influences their
ability to cross biological membranes. The proportion of the drug present in either the ionized or nonionized form depends on the
ionization constant, pKa, and pH at the absorption site. Acidic drugs are predominantly in the nonionized form when the pH value
of the environment in which they are present is below their pKa values, while basic compounds are predominantly unionized when
the pH value of the environment in which they are present is above their pKa value. For many drugs, the ionized species exhibits low
lipid solubility and poor membrane permeability except when specific carrier mechanisms are in operation. Conversely, the
unionized species of most drugs are lipid soluble and readily cross biological membranes. For example, a weak acid such as aspirin
will be concentrated in plasma (pH 7.4) with respect to the gastric juice (pH 3.0) because the pH of the stomach favors its
absorption. However, it is important to note that the large surface area of the ileum compared to the stomach plays a far more
significant role than the pH partition in drug absorption. For most small molecule drugs that are currently used for the treatment of
rheumatoid arthritis the oral bioavailability (F) is in the range of approximately 15%–100%, and the time to reach peak
concentrations in plasma (Tmax) after oral administration in humans is in the order of hours (Table 1).
For large molecules, additional factors that influence drug absorption and overall pharmacokinetic profile include catabolic
capacity in the lymphatic system, interaction with neonatal Fc-receptor (FcRn) and target interactions. For example, the SC
bioavailability for monoclonal antibodies (mAbs) currently approved for the treatment of rheumatoid arthritis is in the range of
approximately 50%–80%, and the time to reach the maximum systemic concentration in humans is generally in days (Table 2).

Distribution

Distribution is the reversible movement of drugs between the blood and other tissues in the body. After a drug is absorbed into the
systemic circulation, it mixes rapidly in the blood and is transferred reversibly into the various tissues and site of action in the body.
The rate and extent of distribution of drugs into different tissues depends on their physicochemical properties such as ability to cross
cell membranes, affinity of drugs to certain proteins and tissues, pH partition, and fat/water partition. In each of the body
compartments, drugs exist in both free and bound form (Fig. 2). Many acidic drugs that are tightly bound to plasma proteins
6 Pharmacokinetics—Absorption, Distribution, and Elimination

Plasma
D+P D−P

Biophase

D+R D−R

Other
tissues

Fig. 2 Schematic representation of drug (D) binding to plasma proteins (P) and receptors (R) in body compartments.

such as albumin reside mainly in the bloodstream. For many drugs, it is the free form that is pharmacologically active, because it can
readily cross capillary membranes. Therefore, changes in drug binding due to drug–drug interactions or disease conditions may
cause significant changes in redistribution and pharmacological effect of a drug in the body. Several drugs do not exhibit a uniform
pattern of distribution in the body. For example, thiopental tends to accumulate in body fat, chloroquine concentrates in melanin-
containing tissues such as the liver, while small polar drugs are distributed in total body water. Physiologic factors such as blood
flow to the tissues also influence the distribution of drugs in the body. Since lipophilic drugs cross cell membranes more readily
than hydrophilic ones, their distribution in highly perfused tissues is often significant. Conversely, accumulation of drugs in adipose
tissue is limited by its blood flow, which is <2% of the cardiac output. In contrast to small molecules that readily penetrate tissues,
distribution of large molecules such as monoclonal antibodies (mAbs) in the body is often limited by several additional factors
including size, charge, binding properties, neonatal Fc receptor (FcRn) interaction, formulation, production process, post-
translational modifications such as glycosylation, receptor-mediated transport and target-mediated tissue distribution. Interaction
of large molecules with FcRn is considered one of the critical factors that determines the long circulating half-lives of therapeutic
mAbs (Table 2).
The volume and pattern of distribution of a drug in different body compartments may have important implications on its
pharmacokinetics and pharmacodynamics in humans and animals. For most small molecule drugs, the apparent volume of
distribution ranges from the plasma volume to values that are much larger than the body volume. In contrast, the volume of
distribution of most mAbs is similar to the blood volume, suggesting limited distribution. However, tissue distribution studies
indicate that mAbs are distributed to many tissues including the site of action although at lower concentrations than observed in
blood.

Elimination

Drug elimination refers to the irreversible removal of drugs from the body by excretory organs such as the kidney and/or the liver.
Small molecule drugs are eliminated from the body mainly by the processes of renal excretion and hepatic metabolism. Drugs may
also be excreted from the body via the bile, feces, lungs, sweat, and breast milk. Excretion refers to the processes by which the body
removes the unchanged drugs and their metabolites through the organs of excretion such as the kidneys. Through the processes of
filtration, tubular reabsorption, and secretion, the kidneys remove mostly water-soluble drugs and their metabolites from the blood
and excrete them into urine. Several factors including plasma protein binding, urinary pH, the dissociation constant (pKa) of the
drug, urine flow, and renal blood flow can affect the efficiency of the kidneys to excrete drugs and metabolites. For example, drugs
that are tightly bound to plasma proteins are not readily filtered by the glomerulus. At low glomerular filtrate pH, weak acidic drugs
are unionized and reabsorbed in the tubules of the nephron, whereas at high pH they are ionized in the filtrate and excreted into the
urine. The efficiency of the kidney in excreting drugs may be impaired by several factors, including age and diseases states, such as
high blood pressure and diabetes. In such cases, the drug dosage may be adjusted especially if the drug is eliminated primarily by the
kidneys. It is important to note that large molecules or protein drugs are not filtered in the kidneys because of their molecular size,
structure, and charge except low molecular weight (MW) protein drugs (MW < 30 kDa).
Drug metabolism or biotransformation is the process by which a drug is chemically altered by enzymes in the body to produce a
metabolite, which may be more hydrophilic and readily excreted by the kidneys. Although biotransformation can occur in several
organs, the liver is the principal organ of drug metabolism. The cytochrome P450 family of enzymes in the liver mediate phase
I biotransformation reactions such as oxidation, reduction, hydroxylation, and hydrolysis of drugs. The cytochrome P450 has broad
substrate specificity, and the structural features of a drug may help to predict potential metabolic pathways. Cytochrome P450 3A
(CYP3A) is estimated to metabolize 50%–70% of drugs that are in clinical use. For example, it has been demonstrated that
 Pharmacokinetics—Absorption, Distribution, and Elimination 7

lopinavir, a protease inhibitor, produces inadequate therapeutic concentrations, when administered alone, due to extensive
metabolism by CYP3A isoenzymes. Phase II biotransformation reactions such as glucuronidation, sulfation, acetylation, and
glycine conjugation are carried out by transferases that attach endogenous macromolecules to drugs or their metabolites to produce
highly polar metabolites. Ezetimibe, a lipid-lowering drug, is extensively conjugated to ezetimibe-glucuronide, a pharmacologically
active metabolite. In addition, some drugs and metabolites are excreted into the bile. For most drugs, phase II metabolism must
occur before excretion into the bile. The drugs and metabolites in the bile are dumped into the GIT and excreted in the feces.
Interestingly, drugs and metabolites in the bile that enter the GIT are available for reabsorption into the bloodstream if they escape
decomposition and excretion. This phenomenon is often referred to as enterohepatic cycling.
A number of factors such as age, sex, fasting, disease, genetics, and pregnancy influence the metabolism of drugs. For example,
drug metabolism capacity is lower in infants because metabolic enzyme systems are only partially developed at birth. Other factors
such as physicochemical properties and physiologic variables affect drug metabolism; for example, lipid-soluble compounds are
primarily metabolized in the liver. Moreover, the protein binding characteristics of drugs also affect their intrinsic metabolic
clearance. Extensive plasma protein binding tends to retard hepatic metabolism since bound drugs generally do not have access to
metabolic sites such as hepatocytes in the liver. Although drug metabolism is essentially a deactivation process, some metabolites
may possess similar or different degrees of pharmacological or toxicological activity as the parent drug. For example, codeine is
O-demethylated to morphine, the most active metabolite, which has significantly greater affinity for the mu opioid receptor and
greater potency compared to codeine (Table 1). Additionally, prodrugs produce the desired therapeutic effects after biotransfor-
mation of an inactive form to an active metabolites in the body; Prednisone, a synthetic corticosteroid is bioactivated in the liver
into the active steroid prednisolone for suppressing the immune system and inflammation.
Several in vitro systems and animal models are available to evaluate metabolic stability of drugs, identify the enzyme systems
that are involved in their metabolism, and predict drug–drug interactions. The Michaelis–Menten expression (Eq. 2) is commonly
used to describe the relationship between the rate of metabolism (V) and the concentration of the substrate (C). This equation is a
fundamental interpretation of how an enzyme interacts with a drug, where Km is defined as the substrate concentration at half the
maximum rate of metabolism (Vmax). Interestingly, the rates of metabolism obtained from human in vitro systems such as liver
microsomes may be used to predict drug hepatic clearance in humans when the drug concentrations are low relative to Km. The
in vitro intrinsic clearance (CLint), a measure of the innate enzyme activity to metabolize a drug, is determined from the ratio of Vmax
to Km of the major metabolic pathways and forms the basis for the prediction of in vivo hepatic clearance. Using this method, the
intrinsic clearance obtained from human liver microsomal experiments is scaled up to in vivo hepatic clearance using appropriate
scaling factors such as microsomal protein content, protein binding, liver weight, and blood flow. This approach has been
demonstrated to provide good predictions of hepatic clearance of some small molecule drugs in animals and humans.
Vmax C
V¼ (2)
Km þ C

The principles of ADME of small molecule drugs generally apply to large molecules or protein drugs. However, there are
significant differences in the mechanisms of metabolism between small and large molecules. Large molecules are removed from the
body by several pathways including degradation by proteolysis, receptor-mediated clearance, target-mediated clearance, nonspecific
endocytosis, and formation of immune-complexes. Nonspecific proteolysis or catabolism of biotherapeutics occurs widely in the
body; protein drugs may be metabolized to peptides or amino acids in tissues by various cells. The resulting small peptides or amino
acids are excreted from the body by the kidneys or recycled for protein synthesis. Additionally, the presence of antidrug antibodies
(ADAs) has been shown to increase the clearance of biotherapeutics. For example, anti-tumor necrosis factor (TNF) monoclonal
antibodies (mAbs) such as adalimumab are effective for the treatment of RA (Table 2), but some patients may show poor response
because of the formation of ADAs which may lead to reduced systemic drug concentration. Concomitant use of disease-modifying
antirheumatic drugs (DMARDS), especially methotrexate, is associated with reduced ADAs, increased mAb concentrations and good
clinical response. In these cases, clinicians may modulate the dose of the mAb depending on the clinical response.

Drug Transporters

It has become apparent that transport proteins play a major role in regulating the absorption, distribution, and excretion of several
drugs. Briefly, accumulated evidence has demonstrated that P-glycoprotein (P-gp), a transporter that is embedded in several
biological membranes including the blood–brain barrier (BBB) and the GIT, plays an important role in the ADME of many
drugs. A major function of P-gp is the energy-dependent cellular efflux of endogenous substrates and the excretion of drugs and
metabolites into urine, bile, and the intestinal lumen. Some studies have shown that digoxin, a P-gp substrate, has significantly
higher bioavailability in subjects with reduced P-gp function. At the BBB, P-gp limits the accumulation of many drugs including
digoxin, vinblastine, dexamethasone, and cyclosporine in the brain. Although P-gp transports a wide range of substances with
diverse chemical structures, lipophilic and amphipathic drugs appear to be particularly good P-gp substrates. Interestingly, P-gp and
cytochrome P450 3A4 (CYP3A4) are both localized in tissues with major function for drug absorption and disposition, such as the
small intestine and liver; it also appears that the substrate specificities of P-gp and CYP3A4 show significant overlap. This
observation is of particular importance because P-gp and CYP3A4 may work in a coordinate fashion to influence drug absorption
8 Pharmacokinetics—Absorption, Distribution, and Elimination

and disposition in the body. The role of drug transporters in modulating drug absorption, distribution, and excretion may
contribute to variability in drug pharmacokinetics and pharmacodynamics.

Blood Concentration–Time Profile

A plot of the concentration of a drug in plasma against time is often referred to as the blood concentration–time profile. The
processes of ADME influence the concentrations of drugs in body fluids with respect to time. The drug concentrations are normally
measured in whole blood, or in plasma or serum generated from blood since the site of drug action (e.g., the receptor) is often not
accessible. It is important to differentiate drug concentrations in blood, plasma, or serum since the composition of these matrices
differ in terms of white cells, red cells, platelets, and soluble proteins. As a result of these differences, the drug concentration in
plasma may be higher than in whole blood and this may impact the calculated pharmacokinetic parameters. Other body fluids such
as saliva, urine and cerebrospinal fluid (CSF) are sometimes used for measurement of drug concentrations. Generally, it is assumed
that drug concentrations in these fluids are in equilibrium with the drug concentration at the site of action. For drugs that are rapidly
distributed in the body, changes in the concentrations of the drug in blood reflect changes in the concentration of the drug in other
tissues. In order to achieve a desirable therapeutic effect, the drug concentrations in the blood are maintained within a desirable
concentration range (Fig. 3). This is especially true when the drug concentration in plasma is rapidly equilibrated with the site of
action and there is a direct relationship between drug concentration and effect. However, there is no direct relationship between
drug concentration in plasma and therapeutic effect for many drugs.

Pharmacokinetic Models

A variety of pharmacokinetic approaches including non-compartmental model, compartmental models and physiologically based
pharmacokinetic models are used for the quantitative description of the drug concentration–time profiles in blood and other body
fluids. The most common model is to represent the body as one or more compartments between which the drug is transferred and
from which the drug is removed. Compartmental pharmacokinetic models are virtual structures with defined volumes that are used
to describe the ADME processes in the body following drug administration. The transfer of drug between the compartments and
elimination of drug from the compartments are commonly represented by first order rate constants. In spite of the inherent
assumptions and limitations of compartmental models, they are useful in calculating pharmacokinetic parameters and predicting
drug concentration in plasma, urine, and other tissues at different times after drug administration. Pharmacokinetic models are also
used to predict human pharmacokinetics based on allometric scaling of animal data, design optimum dosage regimens, estimate
tissue accumulation of drugs and their metabolites, explain drug–drug interactions and assess effects of disease on the ADME of
drugs in the body. In this article, the open one-compartment model (instantaneous intravenous input) will be highlighted to
illustrate how the main pharmacokinetic parameters are determined from drug concentrations in plasma versus time data.

One-Compartment Model—Instantaneous Intravenous Input

The one-compartment model represents the body as a single kinetically homogeneous entity with a defined volume into which the
drug is administered and from which drug elimination occurs (Fig. 4). This model assumes that the drug achieves instantaneous
distribution and equilibration throughout the body following drug administration. In the case on the one-compartment model
(instantaneous intravenous input), a single bolus dose of a drug is administered directly into the systemic circulation thereby

Fig. 3 Blood concentration–time curve after intravenous (IV) and oral (PO) administration of a single dose of a hypothetical drug showing the therapeutic range,
minimum effective concentration (MEC), and minimum toxic concentration (MTC).
 Pharmacokinetics—Absorption, Distribution, and Elimination 9

Input Vd Output

Kel

Fig. 4 Schematic representation of the one-compartment model showing drug input, a single first-order elimination step, and associated pharmacokinetic
parameters.

bypassing the process of absorption. The administered drug is then rapidly distributed throughout the body, and is removed from
the body by a single first-order elimination pathway (Fig. 4). In this case, the equation that describes the rate of change of drug
concentrations in plasma (dCp/dt) after intravenous bolus administration of a drug that follows first-order elimination is shown in
Eq. (3). The integrated form of Eq. (3) that describes the concentration of the drug in plasma (Cp) at any time (t) following drug
administration is depicted in Eq. (4). The elimination rate constant (kel) is a first order rate constant that controls the amount of the
drug eliminated per unit time. The pharmacokinetic parameters such as kel, elimination half-life (t1/2), volume of distribution (Vd),
and clearance (CL) that are determined from the blood concentration–time profile of a drug are useful in drug discovery and
development, and for the design of suitable dosage regimens in the clinical setting.
dCp
¼ kel Cp (3)
dt
Cp ¼ C0 ekel t (4)

Common Pharmacokinetic Parameters

Elimination Half-Life
The elimination half-life (t1/2) is the time required for the amount of drug in the body to decrease by half (Eq. 5). For drugs that
exhibit linear PK, the half-life is independent of the dose of the drug administered and is a useful indicator of how fast a drug is
removed from the body. It takes five elimination half-lives for  97% of the bioavailable dose to be eliminated from the body. The
half-life is useful to estimate the dosing interval, the duration of action of a drug, and the time required to attain steady-state plasma
concentrations during a multiple dosage regimen. The volume of distribution and clearance of a drug influence the half-life (Eq. 6).
For example, the antimalarial drug chloroquine has a high clearance but a very long half-life due to its large volume of distribution.
In some renal and hepatic diseases, the half-life may remain unchanged although the clearance and volume of distribution may
change by the same proportion. In such situations, the half-life is not a good indicator of drug elimination from the body:
0:693
t1=2 ¼ (5)
kel
0:693Vd
t1=2 ¼ (6)
CL

Volume of Distribution
The apparent volume of distribution (Vd) is a virtual volume within which the drug is distributed; it is required to relate the plasma
or blood concentration (Cp) to the amount (Ap) of a drug in the body. In fact, Vd is the proportionality constant when the plasma or
blood concentration of a drug is proportional to the amount of drug in the body. The amount of drug in the body at immediately (at
approximately time ¼ 0) after administration of a single intravenous dose is equivalent to the dose (D). In this case, knowledge of
the initial drug concentration in plasma (C0) provides a simple approach for estimating the Vd of a drug that distributes rapidly in a
single homogeneous compartment:
D
Vd ¼ (7)
C0

Vd is an important pharmacokinetic parameter that is used to calculate the priming dose of a drug that will achieve a required
steady-state plasma concentration. In addition, it is a useful indicator of the extent of drug distribution from the blood to the tissues.
Drugs that are highly bound to plasma proteins reside mainly in blood, resulting in a high blood concentration and a Vd that is
approximately the volume of blood (5 L) in the average adult human. On the other hand, drugs that are extensively distributed in
the body exhibit a large apparent Vd. In this case, the apparent Vd is likely to be greater than the true body volume. In general, the
10 Pharmacokinetics—Absorption, Distribution, and Elimination

apparent Vd for drugs ranges from the plasma volume (e.g., heparin has a Vd of about 5 L in a 70 kg human subject) to values that
are much larger than the body volume (e.g., chloroquine has a Vd of about 12.95 L in a 70 kg human subject).

Clearance
The total body clearance (CL) measures the efficiency with which a drug is irreversibly removed from the body by all organs of
elimination. Thus, the sum of the clearance values of individual organs of elimination is equal to the total systemic clearance. For
most drugs, the systemic clearance of a drug is influenced by the capacity of the liver to metabolize the drug and the inherent ability
of the kidney to excrete the unchanged drug. The total clearance, defined as the volume of blood cleared of the drug per unit time,
can be calculated from the intravenous bolus dose (D) and the area under the plasma concentration–time profile extrapolated to
infinity (AUC(0!1)) as shown in Eq. (8).
D
CL ¼ (8)
AUCð0!1Þ

The clearance by an organ of elimination (CLorgan) is also a function of blood flow to the specific organ of elimination (Qorgan)
and the extraction ratio (ER) as shown in Eqs. (9) and (10). The extraction ratio is an empirical measure of the efficiency of an
eliminating organ, and can be defined as the ratio of the rate of drug elimination by an organ [Qorgan  (Ca-Cv)] to the rate of drug
presentation to an organ (Qorgan  Ca). Thus, disease states that affect the condition of organs of elimination may alter blood flow,
extraction ratio, and ultimately the systemic clearance of drugs.
 
Ca  Cv
CLorgan ¼ Qorgan (9)
Ca
 
Ca  Cv
ER ¼ (10)
Ca

where Ca is the concentration of drug in the arterial blood entering the organ and Cv is the concentration of the drug in the venous
blood leaving the organ.

Bioavailability

The absolute bioavailability (F) of a drug refers to the fraction of unchanged drug that reaches the systemic circulation following
administration by any extravascular route. Several factors including the route of administration, the physicochemical properties,
dosage form, and the physiological state of an individual can affect the amount of the administered dose of a drug that reaches the
systemic circulation after extravascular administration. The fraction of the administered dose that reaches the systemic circulation
after administration by any extravascular route is often calculated from the ratio of the area under the plasma concentration–time
profile extrapolated to infinity (AUC(ext)) after extravascular administration to the area under the plasma concentration–time profile
extrapolated to infinity (AUC(iv)) after intravenous administration of the same dose as shown in Eq. (11).
AUCð ext Þ
F¼ (11)
AUCðivÞ

The area under the plasma concentration–time curve is a useful indicator of the amount of drug that reaches the systemic
circulation intact and of the duration it resides in the body before it is removed. The processes of ADME influence the plasma
concentration–time profile of an administered drug and its bioavailability. For example, drugs administered orally may undergo
degradation in the gastrointestinal tract, be metabolized in the gastrointestinal membrane or may be cleared by the liver before
entering into the systemic circulation. Thus, the oral bioavailability of a drug is likely to be less than that of and equivalent
intravenous dose. The reported bioavailability values of some small and large molecule drugs approved for the treatment of
rheumatoid arthritis are shown in Tables 1 and 2.

Pharmacokinetic Software

Pharmacokinetic data analysis is performed by noncompartmental, compartmental, or physiological based methods. In addition,
population pharmacokinetic analysis is performed using nonlinear mixed effects models and maximum likelihood estimation
methods. The noncompartmental method estimates PK parameters based on the principle of statistical moments, has few
underlying assumptions, and apply the trapezoidal rule for measurement of the area under the plasma drug concentration–time
curve. The compartmental method estimates PK parameters by using kinetic models to evaluate the plasma drug
concentration–time curve. Thus, analysis of ADME and drug concentration–time data involves graph plotting and tedious
 Pharmacokinetics—Absorption, Distribution, and Elimination 11

mathematical calculations. As a result, a variety of software packages have been developed and validated to streamline pharmaco-
kinetic data analysis, perform noncompartmental analysis (NCA) calculation, nonlinear model fitting and complex pharmacoki-
netic/pharmacodynamic modeling.
Several commercially available packages for pharmacokinetic data analysis including Phoenix WinNonlin/NLME, Kinetica,
GastroPlus, SimCyp, and NONMEM are widely used for PK modeling and simulation. Other cost-effective alternatives packages for
PK data analysis are available including Microsoft Excel, ADAPT and SAAM II are openly available and widely used for pharma-
cokinetic data analysis. Increasingly, software such as MATLAB, S-Plus, SAS, and R that are extensively used in the field of Science
and Engineering are being applied in pharmacokinetic modeling and simulations. These software for pharmacokinetic data analysis
simplify tedious calculations and provide rapid solutions to complicated pharmacokinetic equations. In addition, the software for
pharmacokinetic data analysis are also useful for experimental study designs, statistical data analysis, data manipulation, graphical
representation of data, pharmacokinetic model simulation, and prediction of drug ADME and drug effects. As a result, software for
pharmacokinetic data analysis have contributed to a better understanding of the ADME of drugs during drug discovery and
development, and drug use in the clinical setting.

Opportunities and Challenges for Studying ADME

Considerable knowledge on drug metabolism enzymes and transport proteins has been accumulated during the last 2–3 decades;
these findings define how quickly a drug is absorbed, distributed, and eliminated from the body. In vitro tests conducted early in
drug discovery and preclinical development help to determine the important drug metabolism enzymes and transport proteins that
contribute to drug absorption and disposition in humans. The current technologies are increasing the throughput of ADME studies
for small molecule drugs during the drug discovery and development process. For example, cell culture techniques have facilitated
the assessment of intestinal permeability for many small molecule drugs. A notable example is the use of CaCO-2 cells, an
immortalized human colon adenocarcinoma cell line that contains microvilli, expresses functional transport proteins and meta-
bolic enzymes, and retains many characteristics of the intestinal brush border, to predict oral bioavailability of small molecule drugs
based on the capacity of the drugs to cross the CaCO-2 cell monolayer. Although it has been demonstrated that bioavailability of
some small molecule drugs can be predicted with reasonable accuracy using the CaCO-2 model, in vitro approaches are still inferior
to animal studies. Several in vitro and animal models are also available to evaluate the distribution of small molecule drugs; the
bovine brain micro-endothelial capillary model (BBMEC), cultured from blood vessels of the blood brain barrier (BBB has been
used to predict the transport of drugs across the BBB). Additionally, plasma protein binding studies augment our understanding of
the relationship between total plasma drug concentrations and the unbound drug concentration that is available to interact with
receptors. In vitro methods such as liver microsomes, cultured hepatocytes, liver slices and recombinant enzymes are routinely used
to evaluate the metabolism of small molecule drugs. The high volume of information gathered from in vitro drug metabolism
studies is useful in selecting drug candidates for future development and for predicting in vivo intrinsic clearance of drugs.
Increasingly, in silico approaches are also being utilized to predict ADME profiles of drugs based on chemical structures,
physicochemical properties, crystal structure of biotransformation enzymes, and ADME profiles of a library of known drugs.
Computer programs and databases have been utilized to analyze vast amounts of ADME data and create predictive models based
on the chemical structure of potential drugs. In addition, hypothetical livers are being created in an attempt to model and predict
ADME of drugs in a real human liver following drug administration Although some insight into the various aspects of ADME can be
obtained from in vitro and in silico methods, there are no robust alternatives to intact animals to accurately and reliably predict the
pharmacokinetic profiles of drug candidates after administration to humans at the present time. The common laboratory animals
that are used for ADME studies in drug research and development are mice, rats, guinea pigs, rabbits, dogs, and non-human
primates (e.g., cynomolgus monkeys). Traditional in vivo techniques such as imaging technologies and whole-body autoradiog-
raphy with radiolabeled drugs are also used to characterize biodistribution of drugs in animals. The results from these animal ADME
studies are ultimately used to prediction human pharmacokinetics and appropriate starting dose before first-in-human studies are
initiated.
The in vitro and in vivo systems for the study of ADME of therapeutic biologics are often different from those for small molecules
because of their size and structure. For example, there are no reliable in vitro systems to predict bioavailability of therapeutic
biologics after sc administration. Furthermore, the metabolic stability of large molecules such as human IgG proteins cannot be
readily assessed in vitro using liver microsomes or cultured hepatocytes. Thus, validated in vitro systems that can mimic activities of
biotherapeutics (e.g., receptor mediated uptake, target binding, endocytosis, internalization, lysosomal digestion, and FcRn/IgG
interactions) are needed for metabolism studies of large molecules. However, the metabolic stability of small peptides and
recombinant human proteins with low MW can be assessed in vitro using similar approached used for small molecules drugs.
For example, characterization of major degradation products may help to identify labile spots of biotherapeutics, provide
information to assist in the design of new constructs with improved in vivo ADME properties, and identify potential elimination
pathways. In addition, liquid chromatography–mass spectrometry (LC-MS) to detect and quantify biologics and degradation
products has been successfully applied in ADME studies of some small peptides and recombinant biologics despite the limited
sensitivity of this method compared to immunoassays.
The animals that are routinely used for ADME studies and prediction of human pharmacokinetics of large molecules are non-
human primates (e.g., cynomolgus monkeys) and/or other relevant animal species used in the toxicology studies. Relevant species
12 Pharmacokinetics—Absorption, Distribution, and Elimination

for the study of ADME properties of large molecules during drug discovery and development must exhibit similar target-binding
properties to the biologic drug candidate as humans. Transgenic animals that express human targets or receptors have also been
used to assess and predict ADME of biologics in humans. It should be noted that target-mediated clearance and antidrug–antibody-
mediated clearance influence elimination of large molecules, and should be taken into consideration when selecting relevant
species for ADME studies. Therefore, appropriate in vitro technologies for the study of ADME of biotherapeutics are needed to
increase our understanding of the absorption and disposition, and the potential impact of antigenic burden and the presence of
anti-drug antibodies on pharmacokinetics of biotherapeutics in vivo.
A clear understanding of human ADME is an expected component of new drug applications and biologics license applications to
drug regulatory agencies. Therefore, it is important to conduct human ADME studies early in the clinical development program
since the results obtained from such studies will address critical drug development questions and may help to determine if
additional toxicology studies are needed. Innovative approaches such as the use of microtracer doses of radiolabeled drug and
accelerator mass spectrometry (AMS) technology have emerged to study human ADME; a common method that is used to study the
fate of a new drug in humans involves administration of the new drug or a radiolabeled form of the new drug to a small cohort of
healthy participants. After drug administration, samples of blood, plasma, urine, and fecal material are collected and analyzed for
parent drug and any metabolites using validated assays. The metabolic profile can be delineated by careful chromatographic
isolation of radiolabeled metabolites followed by structural identification with mass spectrometry and NMR. It has also been
possible to characterize the metabolic profile, distribution and elimination properties of some drug candidates without using
radiolabeled drug because of recent advances in LC-MS/MS instruments with greater sensitivity.
Ultimately, the study of ADME facilitates how new drug candidates are selected and optimized for absorption and elimination in
the body. Furthermore, a good understanding of drug absorption and disposition from in vitro, animal and human studies during
drug discovery and clinical development helps to support new drug applications and approvals, and drug use in the clinical setting.

Further Reading

Birkett, D. J. Volume of Distribution. Aust. Prescr. 1991, 11, 36–37.

Ekins, S. In Silico Approaches to Predicting Drug Metabolism, Toxicology and Beyond. Biochem. Soc. Trans. 2003, 31, 611–614.
Gunaratna, C. Drug Metabolism and Pharmacokinetics in Drug Discovery: A Primer for Bioanalytical Chemists, Part II. Curr. Sep. 2001, 19, 87–92.
Guttendorf, R. J. The Emerging Role of ADME in Optimizing Drug Discovery and Design. Science 1996. http://www.netsci.org/Science/Special/feature06.html.
Medina, F.; Plasencia, C.; Goupille, P.; et al. Current Practice for Therapeutic Drug Monitoring of Biopharmaceuticals in Rheumatoid Arthritis. Ther. Drug Monit. 2017, 39 (4),
364–369.
Obach, R. S. The Prediction of Human Clearance From Hepatic Microsomal Metabolism Data. Curr. Opin. Drug Discov. Devel. 2001, 4, 36–44.
Shargel, L.; Yu, A. B. C. Applied Biopharmaceutics and Pharmacokinetics, 4th ed.; Appleton-Century-Crofts: East Norwalk, 1993.
Smith, D. A.; van de Waterbeemd, H.; Walker, D. K. Pharmacokinetics and Metabolism in Drug Design. Methods and Principles in Medicinal Chemistry, 1st ed.; Vol. 13; Wiley-VCH:
Weinheim, 2001.
Spruill, W. J.; Wade, W. E.; DiPiro, J. T.; Blouin, R. A.; Pruemer, J. M. Concepts in Clinical Pharmacokinetics, 6th ed.; American Society of Health-System Pharmacists: Bethesda,
2014.
Tanaka, Y. Current Concepts in the Management of Rheumatoid Arthritis. Korean J. Intern. Med. 2016, 31, 210–218.
Ternant, D.; Bejan-Angoulvant, T.; Passot, C.; Mulleman, D.; Paintaud, G. Clinical Pharmacokinetics and Pharmacodynamics of Monoclonal Antibodies Approved to Treat Rheumatoid
Arthritis. Clin. Pharmacokinet. 2015, 54, 1107–1123.
Tett, S. E. Clinical Pharmacokinetics of Slow-Acting Antirheumatic Drugs. Clin. Pharmacokinet. 1993, 25, 392–407.
Welling, P. G. Pharmacokinetics, Processes, Mathematics, and Applications, 2nd ed.; American Chemical Society, Washington: Washington, D.C., 1997.
Vugmeyster, Y.; Xu, X.; Theil, F. P.; Khawli, L. A.; Leach, M. W. Pharmacokinetics and Toxicology of Therapeutic Proteins: Advances and Challenges. World J. Biol. Chem. 2012, 3,
73–92.