ADME in Drug Discovery: J. Vrbanac, R. Slauter

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C H A P T E R

3
ADME in Drug Discovery
J. Vrbanac, R. Slauter

O U T L I N E

Introduction39 Plasma Stability 51


Overview of ADME Science 39 Microsomal Stability 51
ADME in Drug Discovery 42
Drug Distribution and Excretion 52
Drug Absorption 43 Kinetics of Metabolism in Microsomes, Hepatocyte S9
Physicochemical Properties and Permeability 43 Fraction, and Hepatocytes 52
Membrane-Bound Drug Transporters 45 Rate of Drug Disappearance in Liver Microsomes or
ATP Binding Cassette (ABC) Transport Proteins: Hepatocytes52
P-glycoprotein (P-gp, MDR1, ABCB1) 46 In Vivo eADME Disposition and Balance Studies 52
BCRP (MXR, ABCG2) 46 Drug Distribution Using Molecular Imaging 53
BSEP (SPGP, ABCB11) 47
Drug Metabolism 53
Solute Carrier (SLC) Transport Proteins: Organic
Biotransformation: Drug Metabolite Profile 53
Anion Transporting Proteins (OATPs) 47
Role of Mass Spectrometry in Drug Development  56
OTC147
Feces and Other “Problem” Tissues 58
SLC Transport Proteins 47
Drug Disposition Studies Using MS Without
Role of Membrane Transporters on ADME
Isotopic Labeling 58
Characteristics of Drugs 47
Bioactivation59
Transporter Mediated Drug–Drug Interactions:
Kinetics of Metabolism 60
P-glycoprotein48
Drug–Drug Interactions (DDIs) 60
Organic Anion (OATs) and Organic Cation
CYP Inhibition and Phenotyping 60
Transporters (OCTs) 48
CYP Induction Studies 62
Transporter-Mediated Drug Resistance 49
Methodologies for Evaluating Drug Interactions Summary and Trends 62
With Transporters 49 Use of Preclinical ADME Data 62
Metabolism in the GIT and Liver: Stability Testing 51 Technologies Impacting ADME in Drug Discovery 63
Stability Testing: Plasma and Microsomal Stability 51
References65

INTRODUCTION questions include by what mechanism(s) is a drug elimi-


nated from the body, what concentrations are neces-
sary to produce therapeutic and toxic effects, does the
Overview of ADME Science
drug have long-term actions related to changes in gene
The drug discovery and development process repre- expression, and what are the kinetics of movement of a
sents an overlapping continuum of research activities drug in the body. The scientific discipline of preclinical
designed to address specific scientific questions. These drug discovery and development can be described as a

A Comprehensive Guide to Toxicology in Nonclinical Drug Development, Second Edition 39


http://dx.doi.org/10.1016/B978-0-12-803620-4.00003-7 © 2017 Elsevier Inc. All rights reserved.
40 3.  ADME IN DRUG DISCOVERY

risk assessment process, whereby data are used to esti- the interface of drug discovery and drug development,
mate the usefulness of some agent in preventing, curing, which as stated above is commonly now referred to as
or slowing the progression of human disease. In a very early ADME (eADME). Not all eADME topics will be
real sense, all of the development of therapeutics repre- covered. For example, plasma protein binding (PPB)
sents a risk–benefit assessment. For drug therapies, the has been omitted, since it is arguably less important to
preclinical phase of research allows subsequent clinical eADME than critical concepts such as stability and clear-
studies to be initiated and proceed with some knowl- ance [8].
edge of risk–benefit. It is an iterative process that var- The characterization of ADME properties of com-
ies in form between different programs at any one time. pounds early in the drug discovery process has well-
This process is constantly evolving, as new knowledge characterized value for the selection of better drug
and technologies are introduced: The research plan of candidates, and has become more important as tech-
today has both many general similarities and significant nologies impacting this process have developed and
differences from 25 years ago. matured [9–11]. The cytochrome P450 (CYPs) enzymes
The constants in this process are drug efficacy and are intimately involved in ADME. The catalytic cycle
drug safety evaluation, which together represent the sci- of the P450-dependent monooxygenase system is
ence of pharmacology, which is the science of drugs. Toxi- shown in Fig. 3.1 (showing the second electron inser-
cokinetic data, pharmacokinetics in a toxicology study, or tion step from cytochrome b5). Over the past 20 years,
the study of the relationship of exposure to toxicity, are an understanding of the biochemistry of the cyto-
important for the design of definitive safety studies (gen- chrome P-450 system and the role that CYP inhibi-
eral toxicology, safety pharmacology, developmental and tion, CYP phenotype, and CYP induction play in the
reproductive toxicology, etc.). Toxicokinetic data allow for identification of better drug therapies has impacted
estimation (calculation) of a safety margin in preclinical how preclinical ADME research is conducted [12–14].
studies and ultimately the early estimation of a therapeutic Consider that 20 years ago approximately 40% of
index in humans. In parallel, the study of absorption, dis- clinical drug failures could be tied to PK and ADME
tribution, metabolism, and excretion are central to finding drug characteristics, and today this failure rate is 10%
new, safe, and effective drugs. The central message of this or less for companies with comprehensive, state-of-
chapter is that early characterization of PK (pharmacoki- the-art preclinical discovery/development programs
netics; ADME) properties is critical to the development of addressing these issues [15]. The drug discovery pro-
successful drug discovery programs [1–7]. cess continues to evolve and early ADME evaluation
ADME scientists have two “customers” in the preclin- has become a routine part of the “big picture” process
ical setting: The drug discovery scientists, who provide to examine the utility of drug templates in the discov-
new chemical entities for evaluation in various pharma- ery of novel therapeutics. The FDA has released the
cology and toxicology screens, and the preclinical drug “Industry, Drug Interaction Studies, Study Design,
development scientists who perform more refined evalu- Data Analysis, Implications for Dosing, and Labeling
ations of safety and efficacy for preparation of the IND Recommendations” guidance, which provides much
(Investigational New Drug Application). ADME stud- needed regulatory guidance for many of the ADME
ies supply the toxicologist with critical measurements investigations discussed in this chapter [16].
of drug exposure that can be correlated with observed Definitions. As already stated, the two constants in
toxicity, which in turn directly relates to the therapeutic the drug discovery process are an assessment of drug
index (TI). The clinical TI is calculated by dividing the efficacy and drug safety. Pharmacology is divided
dose that causes toxicity in 50% of subjects (eg, cardiac into two distinct domains, the separate but interactive
arrhythmia) by the dose that causes a desired therapeu- domains of dynamics and kinetics. Pharmacodynamics
tic effect in 50% of subjects (eg, increase in cardiac out- (toxicodynamics) or PD (TD) is the study of the effects
put). Early on in the drug discovery and development of xenobiotics (drugs; foreign substances; opposite of
process, ADME scientists are interested in estimating endobiotics) on the body. Pharmacokinetics (toxicokinetics)
clearance (CL), bioavailability (F), and pharmacoki- or PK (TK) is the study of the effects of the body on
netic/pharmacodynamic (PK/PD) data for entry into the xenobiotic, or the study of the journey of the drug
compound libraries. In addition, ADME scientists are molecules (the atoms) within the body and excretion
charged with providing to their toxicology colleagues an from the body. Pharmacokinetics, in the broad sense
understanding of exposure (dose) and toxicity. Consid- of the term as defined by Leslie Benet [17], includes
erations include the PK/PD (or TK/TD; toxicokinetic/ concentration-time (C-T) kinetic relationships, chemi-
toxicodynamic) relationship, the role of metabolism cal reaction kinetics, and the formation of new chemi-
and transporters, induction of specific drug-metabo- cal structures (biotransformation; formation of drug
lizing enzymes, and drug accumulation. This chapter metabolites). As stated in Goodman and Gilman’s The
will address ADME in discovery research, or ADME at Pharmacological Basis of Therapeutics (2006):

I.  DRUG DISCOVERY, METABOLISM, AND PHARMACOKINETICS


Introduction 41

FIGURE 3.1  The catalytic cycle of the P450-dependent monooxygenase system, with the second electron insertion step from cytochrome b5
(alternatively, NADPH may serve this function).

“When a drug enters the body, the body begins immedi- Compounds that are rapidly metabolized in the liver have
ately to work on the drug: absorption, distribution, metabolism poor oral bioavailability. The common barrier to drug dis-
(biotransformation), and elimination. These are the processes
of pharmacokinetics. The drug also acts on the body, an inter-
tribution is the cell membrane, which is why in the absence
action to which the concept of a drug receptor is central, since of other mechanisms such as active transport (transport of
the receptor is responsible for the selectivity of drug action and nutrients, for example), substances moving into and out of
for the quantitative relationship between drug and effect. The the cell can pass across the plasma membrane as a result
mechanisms of drug action are the processes of pharmacody- of their lipophilic properties. Other properties determin-
namics.” [18].
ing ADME are not as obvious. For example, redistribution
   is the mechanism responsible for termination of action
It is now common practice to segregate pharmacoki- of thiopental, a highly lipophilic drug that rapidly par-
netics into: (1) The study of the ADME of a drug, and in titions into the brain to act rapidly and briefly and then
particular the ADME determined by following the dis- redistributes into other tissues, eventually concentrating
tribution of radioactivity, from the narrower definition in adipose tissue [19]. In this example, a physicochemical
of (2) PK as the sojourn of the parent drug into, through property of a drug dramatically affects drug kinetics and
and out of the blood, and in particular C-T plasma/blood therefore dynamics.
data. For nonbiologics (small molecules) these C-T data Drugs are administered by various routes of
are determined by highly selective and sensitive quan- administration:
titative methods developed for the parent drug and at   
times metabolites, now almost exclusively performed 1. S
 tarting outside the body including oral and topical
using liquid chromatography-mass spectrometry analy- (skin, nasal mucosa, ocular topical), or having an
sis (LC-MS). Another popular acronym in common usage intermediate starting location, such as rectal, vaginal,
is DM&PK, ie, drug metabolism and pharmacokinetics, and inhalation
which encompasses the broad definition of PK. Pharma-
2. P
 arenteral routes: Intravenous (IV), intramuscular
cokinetics of the parent drug and active or toxic metabo-
(IM), intraperitoneal (IP), subcutaneous (SC), and
lites in plasma/blood is covered in a separate chapter.
depositions (DEPOT)
Absorption, distribution, metabolism, and excretion   
of a xenobiotic is related to the intrinsic properties of the There are also special parenteral routes, such as intraar-
chemical structure, including its molecular weight, the ticular and various ocular parenteral routes (intravitreal
shape of the molecule (“chemical space”), the ionization and retrobulbar, for example). The oral route is by far the
properties, the degree of lipophilicity and water solubility most important route when discussing ADME and drug
of the various forms (charged and uncharged sites), and discovery. We will focus on this route in this chapter,
associations with macromolecules (eg, a tissue protein- and will not specifically discuss any unique kinetics and
binding drug). Some properties are of obvious relevance: ADME associated with other routes of administration.

I.  DRUG DISCOVERY, METABOLISM, AND PHARMACOKINETICS


42 3.  ADME IN DRUG DISCOVERY

ADME in Drug Discovery or lead compounds. The process eventually transitions


into a drug development phase, in which a small group
The drug discovery process is complicated and inter- of “lead compounds” are evaluated in a more stringent
disciplinary. Scientists must work with drug discovery manner, including in vivo testing using preclinical spe-
teams for a significant period of time to gain the experi- cies including but not limited to mouse, rat, dog, and
ence and clarity of scientific vision to lead drug discov- monkey. When successful, this process leads to selec-
ery programs. The overall process is usually described tion of a few (1–2) compounds as successful IND can-
as consisting of drug discovery and drug development didates and entry into Phase I Clinical Trials [20–25].
“phases,” with considerable overlap between these The target ID stage has changed with the sequencing of
phases [17]. The process (Fig. 3.2) can also be described the human genome and the introduction of the “omics”
in terms of preclinical and clinical phases, where there technologies of genomics, proteomics, and metabolo-
is a clear demarcation of activities (the term commer- mics. Although the anticipated revolutionary impact
cialization phase for late stage activities has also been of the “omics” and combinatorial chemistry is greatly
used). In the modern setting, the pharmacological basis improving the drug discovery process has not come
of therapeutics is a highly interactive, dynamic pro- to fruition, continued technological advances have
cess that includes several iterations of the following improved the process of evaluating and testing drug
processes: The identification of a drug target that will targets. New, safer, and more effective drug therapies,
produce the desired effect (decreasing blood pressure, both small molecule and large molecule (predominately
for example); the development of some methodology biologics), will be a part of our future [26–28].
to evaluate the effect(s) of compounds on this target Technological advances impacting the ADME part of
(assay development); the use of this assay to evaluate pharmacology research include:
a large number of compounds (to screen a drug library);   
and more refined testing of the pharmacological and 1. T  he ability to follow drug-related material in fluids
toxicological properties of the chemical template and/ and tissues without radioactive studies
2. The early application of PET/SPECT imaging of
biologics for early drug disposition studies
3. The successful identification of “biomarkers” useful
in characterizing PK/PD (TK/TD) relationships
4. The increased role of in silico in making predictions
of certain ADME properties for chemical templates
and individual compounds
  

Technological advances will continue to dramatically


impact the eADME research process. It is indeed an excit-
ing time for scientists active in the field of drug discov-
ery and development.
One of the most important aspects in determining
the “what and when” for studying ADME properties is
cost effectiveness, since cost per compound and the cost
of each step increases exponentially at each stage of the
drug discovery/development process. The vast major-
ity of compounds do not have the necessary intrinsic
properties to constitute effective and safe therapeutics
in man, and thus the real job of the drug development
scientist is to identify compounds with “losing” proper-
ties, which is a process of elimination, or as drug discov-
ery/development scientists are fond of saying, “finding
and killing the losers.” Thus the actual job of the drug
development scientist is to “kill” compounds/programs
that can be shown to represent future clinical failures.
Compounds that survive this process will have a far bet-
ter chance of success in the clinic and become marketed
drugs. eADME is a critical part of this evaluation process.
FIGURE 3.2  The traditional drug discovery and development
process, ADME focus in bold. Different individuals will draw this
So where are “ADME data” first gathered in the
differently. This is a highly complex and constantly evolving research drug discovery process? The answer is that, with the
process. exception of the very earliest stages of new compound

I.  DRUG DISCOVERY, METABOLISM, AND PHARMACOKINETICS


Drug Absorption 43
characterization, research protocols designed in part to
assess ADME properties occur at all stages of the drug
discovery/development process, including very early
characterizations as part of the first chemical proper-
ties listed in “Drug Chemical Libraries.” For example,
an assessment of CYP3A4 inhibition liability (covered
below) may be determined along with water solubility
and plasma stability, which represents one of the early
data points determined for new compounds.
The interest in ADME is easy to understand since fail-
ure of drugs in the clinic is due to any of three distinct
reasons: (1) Efficacy, (2) Safety, and (3) ADME (PK).
Confusing, isn’t it? This is why the authors prefer the FIGURE 3.3  Scheme for movement of drugs through the body fol-
older, all-encompassing term “kinetics”/“pharmacoki­ lowing oral administration.
netics.” However, ADME is used in this chapter by
default to common usage. half of the small molecule drugs on the market are pri-
Effective ADME programs can greatly impact success in marily cleared by metabolism. It is not surprising that
the clinic and early assessment of ADME characteristics has experimental protocols designed to approximate the
real merit in improving the drug discovery and develop- oral absorption process use tissues and enzymes associ-
ment process [15]. This chapter has been divided up into: ated with this process. The important role of GIT trans-
  

1. A  bsorption porters and metabolic enzymes in drug absorption is


2. D  istribution and Elimination a subject of considerable past and present scientific
3. Metabolism interest.
  
The above points concerning movement of drug from
Distribution and elimination are considered together, the GIT to the blood are very important, since most drugs
since they are often characterized together (eg, MS anal- are administered orally (PO). Physicochemical proper-
ysis of tissues and excreta) and elimination can be con- ties (eg, solubility), cell membrane permeabilities, speci-
sidered to be distribution out of the body. Some subjects are ficities for transporters, and drug-metabolizing enzyme
considered in more than one section. For example, stabil- substrate specificities are important in oral absorption,
ity and metabolism rates are part of the characterization and thus also in the characterization of compounds
of absorption. Large molecules and biologics will not be under evaluation.
considered in this chapter. The chapter “Use of Imaging
for Preclinical Evaluation” (eg, PET and SPECT) dis-
Physicochemical Properties and Permeability
cusses large molecules.
Scientists experienced with the drug discovery and
development process have coined the phrase “does it
DRUG ABSORPTION look like a drug” by which they mean do the physico-
chemical properties of the drug candidate fit the typi-
In order for a xenobiotic (drug) to reach the blood, the cal drug profile (fall within some characteristic range of
“central compartment,” when ingested orally (Fig. 3.3), properties observed for small molecules)?
it must first pass out of the gastrointestinal tract and be One of the more useful observations concerning physi-
delivered to the liver via the hepatic portal vein (the por- cochemical properties of drugs is the “Lipinski rule of
tal vein conducts blood from the digestive system, spleen, 5” which states that poor absorption or permeation is
pancreas, and gallbladder to the liver). The drug and its more likely when there are more than 5 H-bond donors,
metabolites are then available to move into the liver and 10 H-bond acceptors, the molecular weight (MW) is
from the liver to the blood, where they are then distributed greater than 500, and the calculated log P (ClogP) is greater
throughout the body by the arterial circulation [29]. There than 5 [30,31]. Small-molecule compounds (drug candi-
are two major anatomical and biochemical barriers to dates) with atypically large molecular weights and a large
movement of drug from the intestinal lumen to the blood: number of heteroatoms do not “look” like orally avail-
  
able drugs. One good example of a drug that successfully
1. T  he tissues between the intestinal lumen and the
entered clinical development but that does not “look”
portal blood
like it would exhibit significant oral bioavailability (F) is
2. The liver tissues
   tirilazad (Freedox). Tirilazad has a molar mass of 624.9 g/
The liver is the most important site of the metabo- mole, a logP > 5.0, and 6 basic nitrogen atoms (Fig. 3.4).
lism of xenobiotics, and in this capacity serves as a pro- The alicyclic tertiary amines in tirilazad represent good
tection system for the body from chemical insults. Over candidates for CYP metabolism. It is not surprising that

I.  DRUG DISCOVERY, METABOLISM, AND PHARMACOKINETICS


44 3.  ADME IN DRUG DISCOVERY

the oral bioavailability (F) for tirilazad is zero to extremely the “drug-ability” of compounds (a slang term referring
low. This compound did enter clinical trials as an IV drug. to certain properties of a compound or template as rela-
Not surprisingly, these physicochemical properties com- tive to overall “ideal” drug properties), exhibiting poor
plicated preclinical development of this drug. Tirilazad physicochemical properties (pharmaceutical properties)
also exhibited a species-specific metabolic toxicity in the is not always a “show stopper,” but can make drug devel-
dog, with a predominant pathway being N-dealkylation opment very difficult. Water solubility and lipophilicity
between the two chemical domains, forming a metabolite influence the dissolution of drugs in the GIT and the ulti-
that was cardio-toxic in the dog. This metabolic character- mate free drug concentration, since they determine the
istic eliminated the dog from consideration as the nonro- ability of the drug to dissolve in and move through cell
dent toxicology species. membranes and distribute throughout the body. Since
In Silico. The use of software to predict chemical, water solubility, lipophilicity, and permeability are impor-
pharmaceutical, and biological properties of compounds tant parameters in estimating drug absorption properties
from chemical structures is an area of intense interest. in vivo, they are discussed in this section.
This subject matter lies outside the scope of this chap- Solubility. The solubility of a compound in water is
ter and will only be mentioned briefly. Several recent measured at thermodynamic equilibrium in a saturated
overviews have been published [32–36]. In silico pre- solution. The concentration at saturation is determined by
diction of physicochemical properties has developed to LC-UV (LC-ultraviolet) or another appropriate analytical
the point of being relatively useful for log P, log D, pKa, procedure. This is usually done in both water and/or in
and lipophilicity, but prediction of water solubilities has phosphate buffered saline, pH 7.4, and at physiological
proven to be far more difficult. One reason for this is osmolality. Water solubility is commonly estimated in a
that predicting the various forms a solid can take (such high-throughput screening (HTS) setup by adding the
as crystalline vs. amorphous solid) is difficult for novel compound dissolved in dimethyl sulfoxide (DMSO) into
compounds. Prediction of ADME properties by in silico buffer or water at a wide final concentration range and
methods is highly variable and is less effective for novel noting the turbidity of the solution (if cloudy, then the
compound templates. drug is assumed not to be completely in solution).
Physicochemical properties (water solubility, log D, CHI, Log D. A partition coefficient is the ratio of the amount
stability). Physicochemical properties of compounds, of compound existing in a nonionized state in two
such as molecular weight, charge state, water solubility, immiscible solvents, usually n-octanol and water. The
and lipophilicity, in part result in the observed in vivo pH is adjusted so that the predominant form is the non-
ADME properties. As for their influence on what is called ionized form. This is expressed as log P:

FIGURE 3.4  The structure of tirilazad, Freedox. This compound is a good example of a drug that does not follow the Lipinski Rule of 5. The
molecular weight of tirilazad is 624.9 g/mole, the logP is greater than five, and there are six basic nitrogen atoms. These physicochemical prop-
erties complicated preclinical development of this drug. In addition, tirilazad exhibited a species-specific metabolic toxicity in the dog, with a
predominant pathway being N-dealkylation between the two chemical domains, forming a metabolite that was cardio-toxic in the dog. This
metabolic characteristic led to the monkey being the nonrodent toxicology species in drug development.

I.  DRUG DISCOVERY, METABOLISM, AND PHARMACOKINETICS


Drug Absorption 45
acceptor compartments through an artificial membrane
[unionized solute]octanol
Log Poctanol/water = log . containing lipid is determined. Multiwell plate “sand-
[unionized solute]neutral water
wiches” have been devised for high-throughput opera-
tion. Data obtained in this manner correlates well with
A more physiologically relevant measure is log D, Caco2 (a cell line used to study drug transport) data,
which is the ratio of nonionized form in octanol (or all passive movement from the GIT, movement through the
forms) to the nonionized plus ionized forms in water: skin, and distribution into the brain. Caco2 and MDCK
permeability are discussed in the next section.
[unionized solute]octanol
Log D(octanol/water) = Log [unionized solute]water
Membrane-Bound Drug Transporters
+ [ionized solute]water
It has become clear that drug transporters play a key
For drug research, these values are typically mea- role in the absorption, distribution, and elimination of
sured at pH 7.4, with the aqueous phase being buffered drugs into and out of organisms, including man. Recog-
such that the drug does not alter the pH. nition of this fact is critical in the discovery and develop-
Chromatographic hydrophobicity index (CHI) [37,38]. As ment of new therapeutic agents. This section will focus
with log D, the chromatographic hydrophobicity index on those transporters that have been well characterized,
(CHI) is a measurement of the lipophilicity of a drug. The and for which in vitro methods exist that can be used
elution properties of compounds are evaluated using a as screening tools for the rank ordering of drug candi-
rapid gradient, reversed-phase, liquid chromatography dates in the lead optimization activities leading up to
(RP-LC), typically with UV or MS detection. The analysis selection of a lead candidate(s) for further development.
is carried out under acidic, neutral, and basic conditions Because this is an active area of research and an area
(pH = 2.0, 7.4, and 10.5). CHI was originally calculated where regulatory guidance is still being formulated, this
by determining the isocratic retention factor (log k″) at section is expanded somewhat relative to other topics.
various acetonitrile concentrations and plotting log k′ as Fig. 3.5 displays important transporters effecting ADME
a function of that concentration. From this relationship, and their intracellular locations in the most important
the slope (S) and the intercept (log k′(w)) values were cell types (quantitatively). Specific transporters are
obtained, and the hydrophobicity phi(0) calculated as discussed below. Fig. 3.5 was adapted from the recent
−log k′(w)/S. There is a linear correlation between the CDER (Center for Drug Evaluation and Research) guid-
gradient retention time values, t(R), and the isocratically ance [16]. The expression of transporters in the GIT, the
determined phi(0) values. In practice, a plot of CHI vs. liver, and in renal tubules is displayed. Drug transport-
retention times for standards is used to determine CHI ers are membrane-bound, or in most cases, trans-mem-
for the test compound. brane, proteins that are present in all organisms. These
Parallel artificial membrane permeability assay (PAMPA). proteins work to pump a myriad of nutrients, cofactors,
PAMPA is a screening technique to estimate passive dif- and ions into the cell and mediate the efflux of cellu-
fusion permeability (transcellular permeation). PAMPA lar waste, environmental toxins, and xenobiotics out of
estimates passive diffusion alone with no consideration the cell. Transport using these proteins is of two general
of active transport. It is desirable to consider a large types: active or passive. The actions of these membrane-
pH range when considering absorption from the GIT. transport proteins may be passive in nature, facilitating
The apparatus consists of a donor compartment and an the passage of molecules down concentration gradients
acceptor compartment. The movement from donor to into or out of the cell via a process that does not require

FIGURE 3.5  Location of efflux and uptake transporters in the GIT, liver, and kidney thought to be important in drug ADME. MRP, multidrug
resistance associated protein; PEPT1, peptide transporter one; OATP, organic anion transporting polypeptide; OAT, organic anion transporter;
OCT, organic cation transporter; BCRP, breast cancer resistance protein; MDR1, multidrug resistance 1(P-glycoprotein (P-gp)); MATE, multidrug
and toxic compound extrusion protein.

I.  DRUG DISCOVERY, METABOLISM, AND PHARMACOKINETICS


46 3.  ADME IN DRUG DISCOVERY

energy (ATP or reducing equivalents). Conversely, many expression and functionality of P-gp can be modulated
transporters actively pump molecules and ions against by inhibition and induction, which can affect the phar-
their concentration gradients in an active transport pro- macokinetics, efficacy, safety, or tissue levels of P-gp
cess that requires energy [39–41]. substrates [43–45]. Initially discovered as a result of
In considering the transport of drugs in the discovery its interaction with multiple anticancer drugs, P-gp is
and development process, the most attention has been responsible for the efflux across biological membranes of
focused on transporters from two major superfamilies a broad range of therapeutic drugs. P-gp substrates tend
due to their roles in the uptake into and elimination of to share a hydrophobic planar structure with positively
drugs out of the cell, respectively. By virtue of these activ- charged or neutral moieties. These include structurally
ities, these membrane-transport proteins can cause drug and pharmacologically unrelated compounds, many of
resistance and significant drug–drug interactions. As a which are also substrates for CYP3A4, a major drug-
comprehensive review of this area is beyond the scope metabolizing enzyme in the human liver and GI tract.
of this chapter, we focus on the most well-characterized Alteration of MDR1 activity by inhibitors (drug–drug
transporters from the two major genetic superfamilies: interactions) affects oral absorption and renal clearance.
the ABC (ATP binding cassette) transporter family and Drugs with narrow therapeutic windows (such as the
the SLC (solute carrier) transporter family. cardiac glycoside digoxin and the immunosuppressants
Most ABC proteins are active transporters that hydro- cyclosporine and tacrolimus) should be used with great
lyze ATP to actively pump their substrates across mem- care if MDR1-based drug–drug interactions are likely.
branes. There are 49 known genes for ABC proteins, Cell lines that express P-gp, as well as polarized,
which can be grouped into seven subclasses or families inside-out membrane vesicles prepared from these cell
(ABCA to ABCG) [39]. The most commonly studied lines, can be used to determine whether a drug is a P-gp
transporters in the ABC superfamily are P-glycoprotein substrate or inhibitor. In these polarized cell mono-
(P-gp, MDR1) and the cystic fibrosis transmembrane layer preparations, P-gp is located in the apical plasma
regulator (CFTR). membrane. When efflux across the cell membrane is
The SLC superfamily includes facilitated transporters measured in these cell monolayers, the ratio of basal-to-
and ion-coupled secondary active transporters that reside apical to apical-to-basal flux is used to evaluate whether
in various cell membranes. Forty-three SLC families with P-gp may play a significant role in transporting drugs
approximately 300 transporters have been identified in across these cell monolayers. Transport across cells is
the human genome [40–42]. In view of the fact that mem- not always related to excretion; P-gp may also have a
brane drug transporter activity can have a major influence role in drug penetration into the central nervous system
on the pharmacokinetic, safety, and efficacy profiles of [46–48]. Likewise, a high efflux ratio does not always
drugs, several key questions become critically important translate into poor oral absorption. The involvement
for drug development. These questions include which of P-gp in absorption of a drug is more pronounced in
transporters are of clinical importance in drug absorption cases in which there is an apparent balance between
and disposition, and what in vitro methods exist that rep- metabolism and efflux.
resent viable methods for screening development candi-
dates for interactions with these transporters. These and BCRP (MXR, ABCG2)
other important factors in the discovery and development The human membrane transport protein known as
process are discussed below. the breast cancer resistance protein (BCRP) has been
shown to be responsible for resistance to a number of
ATP Binding Cassette (ABC) Transport Proteins: therapeutics. The BCRP transporter is encoded by the
P-glycoprotein (P-gp, MDR1, ABCB1) ABCG2 gene. As with other members of the ABC super-
P-gp (MDR1, ABCB1) mediates the ATP-dependent family of transporters, BCRP uses energy derived from
export of drugs from cells. As with all ABC-transport ATP hydrolysis to pump drugs and xenobiotics across
proteins, the ABC region of P-gp binds and hydrolyzes the plasma membrane. It serves to limit the absorption
ATP, and the protein uses the energy for transport of its of substrates, prevent them from entering the brain, and
substrates across the membrane. It is expressed in the also to mediate their hepatic elimination. The drugs to
luminal membrane of the brush-border cells in the small which BCRP can confer resistance in tumor cell lines
intestine, in the epithelial and other cells that comprise include mitoxantrone, methotrexate, topotecan deriva-
the blood–brain barrier (BBB), in the apical membranes tives, bisantrene, etoposide, SN-38, and flavopiridol
of hepatocytes, and in kidney proximal tubular epithelia. [49–51].
P-gp plays an important role in the intestinal absorp- BCRP is present in many normal tissues, for instance,
tion and in the biliary and urinary excretion of drugs, in the apical membrane of placental cells, in the bile
while in the cells of the BBB it helps limit entry of vari- canalicular membrane of hepatocytes, in the luminal
ous drugs into the central nervous system. The level of membranes of brush border epithelial cells in the small

I.  DRUG DISCOVERY, METABOLISM, AND PHARMACOKINETICS


Drug Absorption 47
intestine and colon, and in the venous and capillary circulation. OATP1B1 and OATP1B3 are liver specific
endothelial cells of almost all tissues [52]. The localiza- and show broad substrate specificity (statins, rifampicin,
tion of BCRP in those tissues with barrier or elimination and telmisartan). Inhibition of OATP-mediated uptake
functions results in the BCRP transporter having a sig- of several statins by cyclosporin A and rifampicin causes
nificant pharmacological role in the disposition of drugs clinically significant DDIs [39,40,53–55].
and xenobiotics.
OTC1
BSEP (SPGP, ABCB11) For the elimination of environmental toxins and meta-
The ABC superfamily transport protein known as the bolic waste products, the body is equipped with a range
bile salt export pump (BSEP) is encoded by the ABCB11 of broad-specificity transporters that are present in the
gene. BSEP is expressed in hepatocytes on the apical side liver, kidney, and intestine. The polyspecific organic
of the bile canalicular membrane. It serves to pump bile cation transporters OCT1, 2, and 3 (SLC22A1–3) medi-
salts from the liver into bile and as such is the predomi- ate the facilitated transport of a variety of structurally
nant facilitator of bile acid efflux in hepatocytes. diverse organic cations, including many drugs, toxins,
BSEP activity in the liver canalicular membrane is and endogenous compounds. OCT1 and OCT2 are found
inhibited by a number of drugs or drug metabolites. This in the basolateral membrane of hepatocytes, entero-
is potentially a significant mechanism for drug-induced cytes, and renal proximal tubular cells. OCT3 has more
cholestasis. Dysfunction of individual bile salt trans- widespread tissue distribution and is considered to be
porters such as BSEP is an important cause of cholestatic the major component of the extra-neuronal monoamine
liver disease. This can occur due to genetic mutation, transport system (or uptake-2), which is responsible for
suppression of gene expression, disturbed signaling, or the peripheral elimination of monoamine neurotransmit-
steric inhibition. ters. Studies with knockout mouse models have directly
In addition to bile salts, BSEP mRNA has been shown demonstrated that these transporters can have a major
to be induced by classical liver enzyme inducers. There impact on the pharmacological behavior of various sub-
is, however, a limited amount of information on whether strate organic cations. The recent identification of poly-
atypical BSEP inducers such as 3-methylcholanthrene morphic genetic variants of human OCT1 and OCT2 that
(3 MC) are also substrates of the export pump. BSEP severely affect transport activity thus suggests that some
mediates the transport of taurocholic acid (TC) very effi- of the interpatient differences in response and sensitivity
ciently. The rate and amount of transport into polarized to cationic drugs may be caused by variable activity of
membrane vesicles can be quantified using methods such these transporters [39,40].
as LC/MS/MS, and also by labeling with fluorescent or
radioactive (3H-TC) tags. Compounds that interact with SLC Transport Proteins
the transporter can modulate the rate of TC transport. If Among the SLC superfamily, two families (SLC21
a substance is a transported substrate, it might compete and 22) with polyspecific members have been identi-
with TC, thus reducing the rate of TC transport. If a com- fied, which together mediate the transport of a vari-
pound is an inhibitor of the transporter, it will block the ety of structurally diverse organic anions, cations, and
transport of TC into polarized membrane vesicles. Some uncharged compounds. The SLC21 family of organic
compounds can be cotransported with TC, increasing its anion transporting polypeptides is currently known to
rate of transport compared to the control level [39,40]. consist of nine members in humans, transporting a range
of relatively large (usually >450 Da), mostly anionic
Solute Carrier (SLC) Transport Proteins: Organic amphipathic compounds (compounds with both hydro-
Anion Transporting Proteins (OATPs) phobic and hydrophilic domains), including bile salts,
The organic anion transporting proteins (OATPs) eicosanoids, steroid hormones, and their conjugates. The
belong to the SLC gene superfamily of transporters and SLC22 family currently consists of 12 members in humans
are 12 trans-membrane domain glycoproteins expressed and rats, encompassing organic cation transporters
in various epithelial cells. Some OATPs are expressed (OCTs), the carnitine transporter (OCTN2/SLC22A5),
in a single organ, while others occur ubiquitously. The the urate anion-exchanger (URAT1/SLC22A12), and
functionally characterized members of the OATPs several organic anion transporters [39,40].
mediate sodium-independent transport of a variety of
structurally independent, mainly amphipathic organic Role of Membrane Transporters on ADME
compounds, including bile salts, hormones and their Characteristics of Drugs
conjugates, toxins, and various drugs. Uptake trans- The body is continuously exposed to a variety of
porters (OATPs, NTCP, OCT1, and OAT2) are local- environmental toxins and metabolic waste products. To
ized in the basolateral membrane. These transporters rid itself of these compounds, it is equipped with vari-
mediate the uptake of substrates into the liver from the ous detoxification mechanisms such as metabolizing

I.  DRUG DISCOVERY, METABOLISM, AND PHARMACOKINETICS


48 3.  ADME IN DRUG DISCOVERY

enzymes and transport proteins mediating their inacti- for the transporter, and provide an indicator of their
vation and excretion. For excretion, a plethora of trans- behavior in vivo. The availability of this type of infor-
membrane-transport proteins is present in the major mation for a specific drug can be useful in anticipating
excretory organs (liver, kidney, and intestine). The solute potential problems with its use in a clinical setting.
carrier (SLC) superfamily is by far the largest superfam-
ily of transporters, consisting of about 225 members in Transporter Mediated Drug–Drug Interactions:
humans. P-glycoprotein
While most of these transporters are highly special- Drug–drug interactions involving membrane trans-
ized, mediating facilitated transport of essential nutrients port can be classified into two categories. One is caused
(eg, glucose, amino acids, nucleosides, and fatty acids), by competition for the substrate binding sites of the
some members are more generalized. Due to their broad transporters, and the other by a change in the expression
substrate specificity, the latter are also termed polyspe- level of the transporters. As mentioned previously, P-gp
cific transporters. They play a major role in the elimina- has a very broad range of substrate specificity; hence
tion of, and protection against, noxious compounds. drug–drug interactions involving it are very likely. P-gp
P-gp can export an astonishing variety (chemically inhibitors, such as quinidine and verapamil, are known
diverse) of amphipathic drugs, natural products, and to increase plasma concentrations of digoxin, a cardiac
peptides from mammalian cells, powered by the energy glycoside, because they block its biliary and/or urinary
of ATP hydrolysis. The transporter consists of two excretion via P-gp inhibition. Since the therapeutic range
homologous halves, each with six membrane-spanning of digoxin is small, changes in its plasma concentration
helices and a cytosolic nucleotide binding domain. P-gp are potentially very serious.
has been purified and studied extensively, but its mecha-
nism of action is still not well understood. X-ray crystal Organic Anion (OATs) and Organic Cation
structures of P-gp bound to two cyclic peptide substrates Transporters (OCTs)
has shown that the protein has a large, flexible, drug- OCTs transport a number of drugs including cimeti-
binding cavity located within the membrane-bound dine, metformin, procainamide, and triamterene from
domain. Drugs can bind to several subsites within this the plasma into hepatocytes and renal tubular cells. As
pocket, via different sets of interactions, helping to with the cytochromes P450, a variety of different OAT
explain the unusual polyspecificity of the transporter. and OCT transporters exist. It is well known that proben-
P-gp substrates are generally lipid-soluble and inter- ecid inhibits the renal secretion of many anionic drugs
act with the protein within the membrane before being via organic anion transport systems. The renal clear-
either expelled into the extracellular aqueous phase or ance of furosemide, ciprofloxacin, and benzylpenicillin
moved to the extracellular aspect of the membrane. is reduced by coadministration of probenecid. OAT1
P-gp substrates include many drugs that are used is a candidate for the transporter responsible for these
clinically, and the protein plays an important role in drug interactions on the renal basolateral membrane because
absorption and disposition in vivo. It is a key determi- probenecid has been found to be able to inhibit OAT1
nant in the pharmacokinetic profile of many drugs, and, [39,40,53–55].
ultimately, the clinical response. The protein is located at Metformin’s uptake into the liver, where it exerts its
the luminal surface of the intestine, and limits absorp- pharmacologic effect, is mediated by OCT1, while its
tion of drugs from the gut. Its presence in the luminal elimination via the kidney is primarily due to OCT2
membrane of brain capillary endothelial cells also makes activity. The capacity of OCT2 to transport metformin
a major contribution to the BBB and strongly reduces is at least 10 times greater than OCT1. Thus OTC2 in
accumulation of many different drugs in the brain. The combination with the renal elimination of metformin is
physiological role of P-gp is thought to involve protec- primarily responsible for its pharmacological properties.
tion against toxic xenobiotics and endogenous metabo- Cimetidine is also known to be a substrate for OCT and
lites by efflux or secretion of these compounds following can compete with metformin for both OCT1 and OCT2.
absorption by other mechanisms. The transporter also Because OCT2 is primarily responsible for metformin’s
plays an important role in the multidrug resistance elimination, competition from cimetidine will result
(MDR) displayed by many human tumors, and it is an in reduced renal clearance of metformin and elevated
important factor in predicting the outcome of chemo- plasma concentrations. Procainamide is another known
therapy treatment [39,40,43–45]. OCT substrate. Its renal clearance has been reduced fol-
If a drug interacts strongly with P-gp, the compound lowing coadministration with several drugs, including
will likely have reduced absorption in the gut, very lim- amiodarone, levofloxacin, and cimetidine [53–55].
ited entry into the brain, and be unable to enter drug- The clinical outcome of drug–drug interactions based
resistant tumors. Screening drugs for their ability to on OAT or OCT inhibition will depend on the pharmaco-
compete with P-gp-mediated transport of a probe com- logical properties of the drug in question. For example,
pound can give quantitative information on their affinity inhibiting the hepatic uptake of a drug may reduce its

I.  DRUG DISCOVERY, METABOLISM, AND PHARMACOKINETICS


Drug Absorption 49
metabolism, leading to higher plasma concentrations. If uptake and export transporters in pancreatic carcinoma
the site of action of the drug is intrahepatic, however, cells. In addition, cytotoxicity studies with MRP5-
a reduction in the desired pharmacological effect also overexpressing or MRP5-silenced cells additionally
may occur, despite increased plasma concentrations. demonstrated a contribution of MRP5 to gemcitabine
Nevertheless, the resulting increase in the drug’s plasma resistance [57].
concentration may lead to an increase in side effects Chemotherapy is a major form of treatment for can-
unrelated to the drug’s therapeutic effect. An example cers. Unfortunately, the majority of cancers are either
would be that patients taking statins might have an resistant to chemotherapy or acquire resistance during
increased risk of myopathy, whereas those on metformin treatment. One of the mechanisms by which human
could have a greater risk of developing lactic acidosis. cancers develop multidrug resistance is the overexpres-
The effect of inhibited renal clearance will depend on the sion of efflux transport proteins on the plasma mem-
percent of drug eliminated via the kidney and its thera- brane of cancer cells. P-gp and MRP1 have been shown
peutic window. In general, clinically significant effects to confer resistance to a broad spectrum of chemothera-
will occur with drugs having at least 50% of their elimi- peutic agents. Several other human ATP-binding cas-
nation via renal secretion and that also have a narrow sette (ABC) transporters with a potential role in drug
therapeutic window [39,40,53–55]. resistance have been described as having a role in mul-
tidrug resistance. Among them, a novel protein, now
Transporter-Mediated Drug Resistance known as the breast cancer resistance protein (BCRP),
The multidrug-resistance protein (MRP) has been rec- mitoxantrone-resistance protein (MXR) [58], or pla-
ognized as being correlated with drug resistance in cancer centa-specific ABC protein (ABCP), were shown to be
chemotherapy for some time. MRP is a trans-membrane present in the plasma membrane of the drug-resistant
protein that is, in part, responsible for the resistance of cells overexpressing the transporter [59]. Such studies
human tumor cells to cytotoxic drugs. Stably transfected, provide strong evidence that BCRP is a cause of drug
MRP-overexpressing cells have been shown to be resis- resistance for certain types of chemotherapeutic agents,
tant to doxorubicin, daunorubicin, vincristine, VP-16, including mitoxantrone and topotecan, in tissue cul-
colchicine, and rhodamine 123, but not to 4′-(9-acridinyl- ture models. BCRP is prominently expressed in organs
amino) methanesulfon-m-anisidide or taxol. Intracellular important for absorption (the small intestine), distribu-
accumulation of antineoplastic drugs (daunorubicin, vin- tion (the placenta and BBB), and elimination (the liver
cristine, and VP-16) is decreased and the efflux of drug and small intestine) of drugs, and an increasing amount
(daunorubicin) is increased in these cells [39,40]. Accu- of evidence is now emerging to support the conclusion
mulation of daunorubicin has been shown to be reversed that BCRP also plays an important role in drug disposi-
when the plasma membrane of these cells is permeabi- tion [39,40].
lized using nonionic detergent. This would seem to dem-
onstrate conclusively that MRP lowers the intracellular Methodologies for Evaluating Drug Interactions
daunorubicin level by pumping the drug out of the cells With Transporters
against a concentration gradient, thereby identifying it as Drug–drug interaction involving hepatic membrane
a transmembrane efflux pump [56]. transporters can occur as a result of competition for the
Pancreatic cancers are among the tumor types that same substrate-binding site of the transporter, very tight
have proven to be most chemoresistant to a variety of binding, by binding that interferes with the transporter
chemotherapy agents. Chemoresistance of this nature allosterically leading to inhibition of transporter activity,
can be mediated by various cellular mechanisms, or by a change in expression level of transporters. This
including reduced uptake of the drugs into the target has the potential to alter the blood concentration time
cells; alterations within the cells, such as changes in the profiles of drugs, leading to elevated levels of a coadmin-
metabolism of the drugs; changes in the cellular capac- istered compound. Evaluating the substrate potential
ity for DNA repair; and an increased efflux of the drugs of a drug candidate for the hepatic uptake transporters
from the cells. In studies of human pancreatic carci- in vitro is particularly beneficial when the liver is the
noma cells, Hagmann et al. showed that in cells stably drug target. For example, the hepatitis C drugs alpha-
transfected with human transporter cDNAs, or in cells interferon and S-acyl-2-thioethyl esters or the HMGCoA
in which a specific transporter was knocked down by inhibitors (statins) must achieve adequate concentra-
RNA interference, 5-fluorouracil treatment affects the tions in the liver for pharmacological activity.
expression profile of relevant cellular transporters In addition to drug–drug interactions, hepatic trans-
including MRPs, and that MRP5 (ABCC5) influences porters also play a role in toxicities including cholestasis
the chemoresistance of these tumor cells [57]. Similarly, and hyperbilirubinemia. Drug-induced hepatotoxicity is
cell treatment with the nucleoside drug gemcitabine or a major problem in drug development and there is grow-
a combination of chemotherapeutic drugs can variably ing evidence that inhibition of bile acid transporters is a
influence the expression pattern and relative amount of contributing mechanism.

I.  DRUG DISCOVERY, METABOLISM, AND PHARMACOKINETICS


50 3.  ADME IN DRUG DISCOVERY

Hepatocytes in suspension, attached to tissue cul- Drug-transport assays in polarized cell monolayers
ture dishes or in primary sandwich culture, are all good can be used to screen for P-gp involvement in transport.
models of hepatic transport. The contribution of trans- P-gp, encoded by MDR1, is expressed in the human
porter-mediated uptake to hepatic clearance (CLH) was intestine, liver, brain, and other tissues. Localized to the
recognized when CLH was consistently underpredicted cell membrane, P-gp functions as an ATP-dependent
for many series of chemotypes using just metabolic efflux pump capable of transporting many structurally
stability for the calculations. Factoring in transporter- unrelated xenobiotics out of cells. Intestinal expression
mediated hepatic uptake, along with metabolic clear- of P-gp may affect the oral bioavailability of drug mol-
ance using hepatocytes in suspension, improved these ecules that are substrates for this transporter. P-gp sub-
predictions [39,40]. strates can be identified by a direct measure of transport
ABC transporter assays including epithelial cell bar- across polarized cell monolayers. Bidirectional transport
rier systems using Caco-2 or LLC-PK1 cells are widely (apical to basolateral and basolateral to apical) is mea-
accepted in vitro models used to rank the absorption of sured in Caco-2 cells, or in LLC-PK1 cells expressing P-gp
drug candidates. In addition to these standard models, cDNA and corresponding control cells. Quantitation of
we may specifically measure human P-gp-mediated the rate of transport and total mass transported can be
drug transport using cDNA-transfected LLCPK porcine achieved by a variety of methods including LC/MS/
cell lines. These human P-gp expressing cell lines allow MS, fluorescence, or by using a radio-labeled substrate
the study of this important efflux transporter without [39,40,47]. Evaluation of drug candidates as a substrate
interference from other expressed transporters. and inhibitor of P-gp should be performed according to
Alternatively, a less specific but faster ATPase assay in FDA guidance [16].
membranes or membrane vesicles allows determination As a means of studying the transport of drugs and
of whether the compounds of interest interact with ABC xenobiotics into and out of the brain, capillaries can be
transporters. ATP hydrolysis is required for in vivo drug isolated from brain and digested to separate out brain
efflux by ABC transporters. The membrane ATPase assay capillary endothelial cells for growth in cell culture. The
measures the phosphate liberated from drug-stimulated endothelial monolayer can be grown on porous mem-
ATP hydrolysis in ABC transporter membranes [42–47]. branes, which can be placed in side-by-side diffusion
Caco-2 cells are the most popular cellular model in chambers for measurement of drug transport across the
studies on passage and transport. They were derived monolayer in vitro. The problem with this approach is
from a human colorectal adenocarcinoma. In culture, that BBB-specific gene expression is severely downregu-
they differentiate spontaneously into polarized intes- lated in vitro. For example, the expression of the GLUT1
tinal cells possessing an apical brush border and tight glucose transporter or the LAT1 large neutral amino
junctions between adjacent cells, and they express acid transporter is downregulated >100-fold in cultured
hydrolases and typical microvillar transporters. This cell endothelium compared to freshly isolated brain capil-
line was first used as a model for studying differentia- laries. For example, l-DOPA for Parkinson’s disease is
tion in the intestinal epithelium and later for estimating effective, because this drug crosses the BBB on the LAT1
the relative contributions of paracellular and transcellu- endogenous transporter [39,40].
lar passage in drug absorption. Alternatively, these transport systems can be studied
Caco-2 cells, despite their colonic origin, express in in vivo. Drug transport from blood to cerebrospinal fluid
culture the majority of the morphological and func- (CSF) is a function of drug transport across the choroid
tional characteristics of small intestinal absorptive cells, plexus epithelium, which forms the blood-CSF barrier
including phase I and phase II enzymes, which can in vivo. This epithelial barrier is anatomically separate
be detected either by measurement of their activities from the BBB, which limits drug transport from the
toward specific substrates, or by immunological tech- brain into brain interstitial fluid (ISF) across the capil-
niques. CYP3A, which is present in almost all intesti- lary endothelium. The capillary endothelium (the BBB)
nal cells, is very weakly expressed in Caco-2 cells, but and the choroid plexus (the blood-CSF barrier) have dif-
expression levels can be increased by treatment with ferent transporter gene expression profiles. Drugs may
1α,25-dihydroxyvitamin D3, an inducer of CYP3A4, readily enter CSF, due to rapid transport across the cho-
or transfection of CYP3A4 cDNA. But the resulting roid plexus, but do not undergo significant transport
expression levels do not reach the levels observed into brain tissue, due to limited BBB transport. This is
in vivo. With regard to phase II enzymes, Caco-2 cells illustrated with azidothymidine (AZT), a treatment for
do express N-acetyl transferase and glutathione trans- neuro-AIDS. AZT is readily transported into CSF, but is
ferase. In summary, the Caco-2 cell transport assay not transported across the BBB.
appears to be a good and predictive approach to under- BBB active efflux transporters (AET) such as p-
standing transport across the intestinal absorptive bar- glycoprotein actively transport drugs from brain to
rier [39,40]. blood. There are many other BBB efflux systems for

I.  DRUG DISCOVERY, METABOLISM, AND PHARMACOKINETICS


Drug Absorption 51
both small and large molecules. The efflux transport- stability in plasma, typically a compound is incubated
ers can be measured using the brain efflux index (BEI) in plasma (blood) at approximately 10 μM at 37°C for
method, which involves direct injection of the drug into 30–60 min and its stability determined by an appropri-
the brain under stereotaxic guidance. The kinetics of ate analytical method, such as LC-UV or LC-MS. Viable
drug loss from the brain compartment (which is a func- drug candidates should be “relatively” stable under
tion only of BBB efflux transport) can then be quanti- these conditions.
fied [39,40].
Microsomal Stability
Common in vitro metabolism systems include cells
Metabolism in the GIT and Liver:
(hepatocytes), the S9 fraction, microsomes, and the
Stability Testing soluble fraction. The preparation of S9 fraction, soluble
Clearly, metabolism is important in determining the fraction, and microsomes is shown in Fig. 3.6. Micro-
amount of drug absorbed from the GIT into the blood- somes are artificial structures derived from pieces
stream. The absorption and distribution of a drug fol- of endoplasmic reticulum (ER) formed during tissue
lowing PO administration leading to some desired homogenization. As shown in Fig. 3.6 microsomes are
pharmacological effect occurring at a target organ, such prepared by differential centrifugation at 10,000 and
as the brain, requires that the metabolism of the drug is 100,000 × g and contain cytochrome P450 enzymes
not extensive, either in the gut or the liver. Following (CYPs), but do not contain soluble enzymes. The family
oral administration, the metabolism of drugs in the gut of CYP enzymes contained in microsomes are responsi-
tissues and/or the liver upon absorption from the intes- ble for most of the Phase I biotransformations of xeno-
tinal lumen is termed “first-pass metabolism.” biotics, and incubation of test material with hepatic
microsomal preparations in various species is the pri-
Stability Testing: Plasma and Microsomal Stability mary means by which the Phase I biotransformations
Several simple stability tests allow the assessment of xenobiotics (drugs) are determined. For microsomal
of the “drug-ability” of a compound or chemical tem- stability determination, the compound is typically incu-
plate. For example, if a compound is highly unstable in bated in approximately 1.0 mg/ml microsomal protein,
a tissue (liver, for example) or fluid (blood, for exam- phosphate buffer, pH 7.4 at 37°C for 30 min. Compound
ple) that is likely to be encountered during the sojourn stability is then determined by an appropriate analyti-
from the intestinal lumen to interaction with the drug cal method such as LC-UV or LC-MS. Viable drug can-
receptor (brain, for example), then activity following didates should have a species-specific, predetermined
oral administration will by definition be low. For drug percentage remaining under these conditions. Early in
library screening stability testing, typically a one data- the drug discovery process, microsomal stability test-
point determination (with a zero time-point control) is ing is often undertaken in an HTS format using micro-
made, in which the time and other parameters (such as somes from rodent [60].
protein concentration) are such that a certain percentage
of loss of a compound can be used as a screening data
point (information about whether to keep or eliminate a
compound, or information to consider in the context of
additional compound data) [1,2,4,5].

Plasma Stability
Drugs must have sufficient stability in the body to
exert a pharmacological effect over a reasonable period
of time. A wide variety of compounds are unstable (are
degraded) when incubated in blood or plasma at rates
that are inconsistent with the PK properties necessary
for drug therapeutics. Plasma (blood) stability deter-
mination is a widely used, simple test that can elimi-
nate compounds in drug discovery screens. As already
stated above, the drug discovery/development process
involves not only the identification of drug targets and
demonstration of desirable activities in model systems,
but also the elimination of compounds that must, by def-
inition, fail in the clinic and poor plasma stability usually FIGURE 3.6  Preparation of microsomal, S9, and soluble fractions
represents one such characteristic. For determination of commonly used in drug metabolism studies.

I.  DRUG DISCOVERY, METABOLISM, AND PHARMACOKINETICS


52 3.  ADME IN DRUG DISCOVERY

DRUG DISTRIBUTION AND EXCRETION used as an estimate of what to expect in vivo. To some
extent, the popularity of in vitro systems has been driven
For simplicity, we have combined drug distribution and by their use by numerous research groups and the even-
drug excretion in this section. Modern eADME studies that tual commercialization of in vitro test products that
evaluate drug-related material distribution by MS usually are economical and effective. However, there is a lot of
analyze specific tissues, blood/plasma, and excreta. For “bang for the buck” in using in vitro systems in drug dis-
example, when a drug is administered by IV or PO, samples covery research, especially in identifying drugs that will
of blood (plasma), tissues (brain, for example), and excreta fail in the clinic, as previously noted. In vitro systems
(urine and feces) are taken at specific time intervals and are include intact cells such as perfused liver preparations,
analyzed for drug-related material by LC-MS, giving both liver slices, freshly prepared, or frozen hepatocytes and
distribution and excretion data. This has been made pos- other cell lines. Cell fractions include S9 fraction, soluble
sible by the revolutionary changes in MS technology over fraction (cytosol), and microsomes (Fig. 3.6).
the last 20–25 years, allowing for rapid development of sen-
sitive and specific methods for the quantitative analysis of Rate of Drug Disappearance in Liver Microsomes
drugs in tissues and fluids by LC-MS (expanded below). or Hepatocytes [64]
In addition, another tool available to the ADME scientist
is the direct determination of xenobiotics (and endogenous A typical experiment for stability screening pur-
compounds) in tissue slices by desorption of compounds poses using microsomes involves incubation of drug at
for analysis by MS imaging (MSI). In this technique, com- approximately 3 μM. Typical conditions would be incu-
pounds are desorbed into the gaseous phase for analysis bation of the drug at 37°C with 100 mM phosphate buf-
using either matrix-assisted laser desorption ionization fer pH = 7.4, 1.0 mM nicotinamide adenine dinucleotide
(MALDI) or secondary ion mass spectrometry (SIMS) [61]. phosphate (NADPH) and approximately 1.0  mg/ml
To summarize, MS allows for the qualitative and quanti- microsomal protein. Reaction starts with the addition of
tative analysis of small and large molecules in tissues and NADPH (time zero). Typically, 5 to 7 time points are taken
fluids without the use of radiotracers. The evolving tech- (0, 2, 5, 10, 20, 30, and 60 min, for example) and the reaction
nologies surrounding the use of radioactive tags to study is stopped by the addition of methanol or acetonitrile. The
the disposition of biologics early in drug discovery using drug is analyzed using LC-MS and the kinetics of disap-
PET and SPECT are discussed in a separate chapter. pearance data are entered into drug (chemical) libraries.
The study of the distribution of a xenobiotic is the Determination of the rate of disappearance of the drug
study of its movement into, through, and out of body using plated hepatocytes and hepatocytes in suspension
compartments. Data are expressed in terms of C-T for has become more popular recently. Although hepatocyte
the parent drug and its metabolites. Kinetic analysis can preparations represent a more complicated and more
afford insight into drug properties and PK/PD relation- expensive experiment, all hepatic enzymes are available
ships. A concept central to any discussion of distribution to act on parent compound and metabolites, including
is the volume of distribution. The (apparent) volume of dis- Phase II enzymes. This represents a significant advantage
tribution (VD) is a pharmacokinetic “parameter” used to over microsome and S9 preparations. However, a disad-
quantify the distribution of a medication between plasma vantage with using this approach to kinetic analysis is that
and the rest of the body. Certain changes in physiologi- the kinetics of movement of the drug across membranes
cal function(s) and certain disease states may alter VD. becomes a complicating factor, although it can be argued
This is discussed in detail in the chapter on PK (Chapter that this is a situation closer to that encountered in vivo.
4). The reader should consider Chapter 4 as part of the In summary, there are advantages and disadvantages of
ADME material and refer to this chapter to gain a more using whole cells, microsomes, or S9 fractions and often
in-depth understanding of blood-plasma PK. when choosing drugs to enter development, all these data
are available for consideration, or should be available.
Kinetics of Metabolism in Microsomes,
Hepatocyte S9 Fraction, and Hepatocytes In Vivo eADME Disposition and Balance
Over the past 25 years or so there has been an increase
Studies
in the use of in vitro systems as models used to estimate The impact of new analytical techniques and instru-
the in vivo ADME properties of drugs and chemical mentation, and improvements in existing technologies
templates under evaluation for drug-ability [62,63]. These on the way we conduct biochemical studies, cannot be
systems represent an important component of allome- overstated [65]. For example, the ability of MS to obtain
tric scaling for estimation of drug properties in man. For high-quality spectra or quantify molecules in biological
example, the rate of metabolism (and metabolic clear- matrices has increased by several orders of magnitude;
ance) observed in vitro for hepatic preparations can be 104–107 times, depending upon the application, between

I.  DRUG DISCOVERY, METABOLISM, AND PHARMACOKINETICS


Drug Metabolism 53
the late 1960s and today. The impact of electrospray (ESI) if available, and determine relative response factors for
and other MS techniques on the ability to detect, quan- drug metabolites by LC-MS-RAM (radioactivity monitor-
tify, and identify molecules in biological matrices has been ing) analysis. The RAM analysis is quantitative since the
profound (a Nobel Prize was awarded for the invention of specific activity is known and thus relative response fac-
ESI) [66]. The biochemists and pharmacologists of 50 years tors can be estimated and subsequent experiments can be
ago relied heavily on the use of spectroscopy and isotopes normalized in a quantitative manner [71].
such as 14C, or 13C in the elucidation of metabolic path- Cassette Dosing. As detailed above, modern MS instru-
ways. Multistep purifications, spectral absorbance, mass mentation is more sensitive and easier to use than older
spectral, and nuclear magnetic resonance spectroscopy techniques and also exhibits reasonable analyte selec-
(NMR) analysis were used, which involved a consider- tivity and sensitivity. Modern ± ESI and ± APCI coupled
able investment in time. Today, modern instrumentation with tandem quadrupole instruments has allowed PK
techniques have allowed the determination of xenobiotic studies in which drugs are administered concomitantly,
metabolites in a fraction of the time with online combina- a technique commonly known as “cassette dosing” or
tions of MS, NMR, and radioactivity detection. “n-in-1” dosing (5-in-1; a mixture of five compounds
Continued advances in analytical instrumentation have in the same formulation, for example). In these studies,
even allowed the rapid estimation of the disposition of drug mixtures are dosed IV and PO and LC-MS analy-
drug-related materials without the use of isotopes. The sis methods are used to quantitatively analyze samples
introduction of tandem quadrupole MS and advancements taken at appropriate time points. Either selected ion
in LC-MS interfaces (ESI; atmospheric pressure chemical monitoring (SIM) or selected reaction monitoring (SRM)
ionization, APCI) have greatly improved the practicabil- can be used for the MS analysis. Although caution must
ity of studying absorption in vivo [2,6,67–70]. In fact, mod- be applied in the use of this approach, the screening
ern MS technology has allowed for early PK data (in vivo) value of cassette dosing can be high when applied to
to be obtained in drug discovery programs for tens to a compounds within a specific chemical template [72,73].
few hundreds of candidate compounds. Generic LC-MS-
MS methods, or methods needing little development, can
be used to quantitatively analyze a drug in plasma with Drug Distribution Using Molecular Imaging
high sensitivity and selectivity. Provided that sufficient Over 80% of investigational new drugs fail during drug
amounts of compound are available (tens of mg), drugs development because of unsatisfactory absorption, distri-
can be dosed to mice or rats by PO and IV and blood sam- bution, metabolism, excretion, and toxicity (ADME and
ples taken at appropriate intervals to obtain early PK data. toxicity; ADME) characteristics. Tools that help to pre-
For a typical study, 10–12 samples are taken by each route dict the ADME responses early in development present
of administration with the last time point at 12 or 24 h post- an advantage as they avoid wasting valuable resources
dose. More abbreviated protocols are also useful if less on compounds that are bound to fail. A multitude of
accuracy is considered acceptable for ranking drug candi- noninvasive, high-resolution imaging technologies are
dates. Typically, tissue and fluid samples are homogenized now used regularly in ADME studies. Drug companies
and then extracted using a generic method for extraction of currently have hundreds of thousands of potential drug
drug-related material. This could be as simple as addition compounds; however, very few (<1:10,000) will eventu-
of one volume of water to urine and analysis by LC-MS or ally enter the market as an FDA-approved treatment.
as complicated as homogenization of feces in buffer using The techniques available today for real-time molecular
glass beads and sonication, followed by one or two extrac- imaging of the disposition of drugs in the body provide a
tions. For LC-MS-MS analysis using tandem quadrupole unique, early opportunity to identify which drugs will fail
MS, methods are developed by first evaluating the rela- in the later stages of drug development, thereby improv-
tive response factors using the four combinations of ± ESI ing the quality of the molecules ultimately selected to
and ± APCI (+/−; positive and negative ions detected) and move forward. These technologies have particular, funda-
then examining the appearance of the product-ion mass mental advantages in the study of large molecules (bio-
spectrum for determination of selected reaction monitor- logics). Again, this area is so important that a separate
ing (SRM) precursor-product ions that impart the greatest chapter is dedicated to it (see Chapter 35).
selectivity and specificity. LC conditions are quickly evalu-
ated and then quantitative analysis can be performed for
selected fluid/tissue samples. In addition, gradient LC-MS DRUG METABOLISM
analysis using repetitive scanning MS can be performed to
examine the drug metabolite profile in a semi-quantitative
Biotransformation: Drug Metabolite Profile
manner. Results of these analyses are used to determine
how additional samples are best analyzed. One addi- Mammals have evolved mechanisms to rapidly metab-
tional technique is to metabolize the radio-labeled drug, olize and eliminate potentially harmful substances from

I.  DRUG DISCOVERY, METABOLISM, AND PHARMACOKINETICS


54 3.  ADME IN DRUG DISCOVERY

biological sources (xenobiotics) that have been ingested. The metabolism of xenobiotics hence represents a dou-
Examples include the plethora of toxins associated bled-edged sword: It allows their elimination from the
with the kingdoms of fungi (mushrooms, for example) body, but may also convert the drugs to toxic substances,
and plants. Mammals must be able to metabolize and including highly reactive carcinogenic, mutagenic, or
eliminate toxins present in plants or the plant cannot be teratogenic molecules. Electrophilic metabolites can react
consumed as food. The same mechanisms for handling with nucleophilic macromolecules such as nucleic acids
plant toxins, both enzymatic and transport systems, are and proteins leading to cell damage [1,2,4,7].
involved in the metabolism of drugs. It is worth not- The most common enzyme types and biotransforma-
ing here that many of the compounds used as drugs are tion reactions are listed in Tables 3.1 and 3.2. In general,
found in plants or were derived from plant chemical tem- reactions that aid in the elimination of xenobiotics (drugs)
plates (eg, morphine from the opium poppy and digitalis involve oxidative, reductive, hydrolysis, and transferase
from foxglove [18,19]). These metabolism systems for reactions that increase the water solubility of the com-
dealing with xenobiotics are critical, since many ingested pound (decrease the lipophilicity). Xenobiotic-metabo-
xenobiotics are lipophilic and would accumulate in the lizing enzymes are grouped into two classes: Phase I and
body without metabolism with subsequent elimination. Phase II reactions. Phase I reactions include oxidation,
Drugs are also usually lipophilic, an intrinsic property reduction, and hydrolysis reactions, and Phase II reac-
that allows them to pass through cell and intracellular tions refer to conjugations of xenobiotics with glucuronic
membranes to reach drug receptors. The same property acid, sulfuric acid, specific amino acids, and other groups,
that allows drugs to reach receptors, however, also allows this conjugation occurring at functional groups contain-
them to accumulate in the absence of some clearance ing oxygen, sulfur and nitrogen (-OH, -NH-, -COOH, -SH,
mechanism involving metabolism or active transport. for example). Hydrolysis reactions, such as cleavage of an

TABLE 3.1  Table of Enzymes, Subcellular Fraction Localizations, and Reactions for Drug-Metabolizing Enzymes
Enzyme Types Subcellular Fraction; Reaction

PHASE I

Cytochrome P450s (CYP) ERa; C and O oxidation, dealkylation,


and other oxidations

Flavin-containing monooxygenases (FMO) ER; N, S, and P oxidation

Epoxide hydrolases (EH) ER, cytosol; hydration of epoxides

Reductases Cytosol, reduction reactions

Monoamine oxidase Oxidative deamination of monoamines


HYDROLASES

Esterases ER, cytosol; hydrolyzes ester bonds

Amidases ER, cytosol; hydrolyzes amide bonds


PHASE II TRANSFERASES (COFACTOR)

UDP-glucuronosyltransferases (UGT) (uridine diphosphate ER; addition of glucuronic acid


glucuronic acid)

Sulfotransferases (SULT) (3″-phosphoadenosine-5″-phosphosulfate) Cytosol: Addition of sulfate

Glutathione-S-transferases (GST) (glutathione) ER, cytosol; addition of glutathione

Miscellaneous transferasesb
MISCELLANEOUS ENZYMES

Alcohol dehydrogenase Cytosol; oxidation of alcohols

Aldehyde dehydrogenase Cytosol; oxidation of aldehydes

NADPH-quinone oxidoreductase (NQO) ER; reduction of quinones


aEndoplasmic reticulum (ER).
bAmino acid transferases, methyltransferases, and N-acetyltransferases (NACT) are found in the soluble fraction and the ER. Conjugation reactions vary across species. The cofactor
for methyltransferases is S-adenosyl methionine and the cofactor for N-acetyltransferases is acetyl CoA. Methylation and acetylation generally do not lead to more polar products
with increased water solubility.

I.  DRUG DISCOVERY, METABOLISM, AND PHARMACOKINETICS


Drug Metabolism 55
TABLE 3.2  Table of Schematics of General Reaction Types Involved in Drug Metabolism
Reaction Type Chemistry

OXIDATIVE REACTIONS

Aromatic Hydroxylation 5 5

2+

Aliphatic Hydroxylation 2+
5 5

N-Dealkyation
+
5 1 5 1
+ +

O-Dealkyation +
2 5 2 5

N-Oxidation 5 5

1 1
2
5 5

S-oxidation 5 5
2
6 6

5 5

Deamination 2+
5 + 5 +

1+ 2

HYDROLYSIS REACTIONS

Ester hydrolysis 5 2 5 2+
5  2+
5
2 2

Amide hydrolysis +
+
5 1 5 2+
5 1
+ 5
2 2

Epoxide hydrolysis 5 5
5 5
+
2 2+
+2 +

CONJUGATION REACTIONS

Glucuronidation 2
+2 2
2+ +2 2+
2 5
28'3  2 25
2+
+2 +2
2+
2+

Continued

I.  DRUG DISCOVERY, METABOLISM, AND PHARMACOKINETICS


56 3.  ADME IN DRUG DISCOVERY

TABLE 3.2  Table of Schematics of General Reaction Types Involved in Drug Metabolism—cont’d
Reaction Type Chemistry

Sulfation 2
5 2+  3$36 5 2 6 2+  3$3
2

Amino acid conjugation 2 2 &2+


& +
5 2+ 5 1
+ 5

Glutathione conjugation 5*6+ 5*6+

ester, amide, or phospho-amide bond can result in loss of noted that the liver is also the primary metabolic clearing
drug activity; however, in specific situations prodrugs are mechanism for a wide range of endogenous chemicals,
activated in this manner. Phase I oxidation reactions are including cholesterol, steroid hormones, and vitamins.
primarily carried out by cytochrome P450 enzymes (P450; Important sites of metabolism of drugs other than the
CYP) and flavin-monooxygenases (FMO) [74]. liver, ie, the extrahepatic sites of metabolism, include the
The importance of cytochrome P450 in metabolism GIT, lung, and kidney. For example, extrahepatic metab-
cannot be overstated. CYP enzymes play essential roles olism of propofol by glucuronidation has been observed
in the biosynthesis of important endogenous compounds, during liver transplantation [79].
endobiotics, such as steroids/sterols, the family of arachi- As a result of dramatic improvements in analytical
donic acid derived, locally acting hormones, the eico- instrumentation, the ADME properties of an NCE (new
sanoids, and other physiologically important compounds chemical entity) can be studied with some degree of con-
and intermediates. They also play important roles in the fidence by administering the unlabeled drug (without
metabolism of lipophilic compounds including fats, and a radioactive tag) to a preclinical species and collecting
their role in the elimination of xenobiotics and drugs is excreta, blood, and tissues and using modern LC-MS
critical. As already noted, the flip side of their beneficial instrumentation to determine the amount of parent drug
effects in eliminating toxins is that these same reactions (unchanged drug) present and the approximate amounts
can produce reactive compounds. CYP reactions include and structures of metabolites. (Note: A popular term is
oxidation of carbon and oxygen atoms, and N-dealkyl- “drug-related material” since this is an all-encompassing
ation and O-dealkylation. Epoxides are hydrolyzed by term that covers nonenzymatic processes.)
soluble and microsomal expoxide hydrolase and FMO is Traditional ADME studies require the synthesis of
involved in the oxidation of nitrogen, sulfur, and phos- a radio-labeled standard with the radio-isotope in a
phorous. Phase II reactions include UDP-glucuronosyl- position that is metabolically stable. The drug is then
transferases (UGTs) [75], which catalyze the addition of administered by PO or IV, or by the route under study,
glucuronic acid, forming glucuronides, sulfotransferases and the journey of the radio-labeled atoms through the
(SULTs) [76], which catalyze the addition of sulfate, and body is tracked by collecting samples and determining
glutathione-S-transferases (GST) [77], which add glutathi- the amount of radioactivity present, a quantitative exer-
one, a reaction that is a major mechanism for elimination cise since the specific activity is known and the quench
of electrophilic compounds and metabolites. This reac- can be determined. LC-RAM (radioactivity monitor-
tion is a critical detoxification mechanism. Other enzymes ing) is also usually performed, resulting in a quantita-
catalyze the addition of amino acids, acetyl groups, and tive metabolic profile. When protocols include collection
methyl groups. Additional important enzymes include of all excreta, these studies are usually termed balance
NADPH-quinone oxidoreductase (reduction of qui- studies or radioactive balance studies. The following dis-
nones), alcohol dehydrogenases, and aldehyde dehydro- cusses what can now be accomplished without the use
genases (shown in Table 3.1). of radio-isotopes [2,6].
The vast majority of drugs on the market are metab-
olized to some extent in vivo, primarily in the liver. In Role of Mass Spectrometry in Drug Development
fact, hepatic metabolism is the primary clearance mech- [61,67–71,80]
anism for many drugs. Drugs with high hepatic blood In the middle of the twentieth century, the use of both
clearance (the product of hepatic blood flow and hepatic stable and radio-isotopes in biochemical studies cou-
extraction ratio (QH · EH)) include desipramine, lidocaine, pled with analysis by MS, NMR, and photon spectros-
morphine, propranolol, and verapamil [78]. It should be copy made it possible to understand in great detail the

I.  DRUG DISCOVERY, METABOLISM, AND PHARMACOKINETICS


Drug Metabolism 57
important metabolism/catabolism reactions occurring for metabolites by computer, since only a limited num-
in living systems and in addition how living systems ber of biotransformations are possible. This means that
interact with xenobiotics biochemically. More recent a finite number of exact masses can be derived from
advances in MS have made it possible to quantitatively the elemental composition of the parent drug. Since the
and qualitatively follow the path of xenobiotics through elemental composition of a drug is often dissimilar to
the body in the absence of radio-labeled tags. Prior to that of endogenous compounds (number of heteroatoms
the widespread use of ESI and APCI, analysis of biologi- relative to carbon atoms, presence of halogens, etc.), this
cal samples by MS was limited to GC–MS with chemical approach can usually rule out endogenous compounds
derivatization, LC-MS techniques with serious limita- simply from their elemental composition. The use of
tions (thermospray, particle beam, moving belt, continu- proper control samples and the skillful interpretation
ous flow FAB (fast atom bombardment)), and off-line of product-ion mass spectra enhances the interpretative
desorption techniques (FAB, SIMS, and MALDI (matrix- value of these techniques.
assisted laser desorption/ionization)). ESI and APCI Currently, there is also significant interest in using
drastically changed this situation, since large or polar HR-MS in quantitative analysis. This has the distinct
small molecules can be analyzed online with LC elution advantage that MS–MS tuning is eliminated (collision
and without the need for chemical derivatization. cell gas pressure and collision energy) and all the mass
The pairing of ESI and APCI with another revolution- data can be collected all of the time, allowing for reversed
ary technique, the tandem quadrupole analyzer, changed searches of data for metabolites that are not part of the
how drug research has been conducted over the last original analysis scheme. Expect to see rapid advances in
25 years [67–71]. Quantitative methods, with or without quantitative analysis by LC-MS, as the technology con-
stable-isotope-labeled internal standards (termed stable- tinues to evolve rapidly [61,67–71,80,81].
isotope dilution mass spectrometry when stable isotopes are With modern MS instrumentation it is possible to detect
employed), that are sensitive, highly selective, and rug- drug-related material, usually at nM concentrations, and
ged could be developed rapidly using these combined to estimate the absorption, distribution, metabolism, and
technologies. Drug metabolites, especially Phase II con- excretion of a compound over the timespan of the in-life
jugates, could be detected with a high degree of confi- protocol, plus a day or two to process and then analyze
dence by sifting through LC-MS repetitive scanning data samples. However, when the analysis employs a nonradio-
and searching for logical mass additions and losses and labeled drug and MS, there is always the possibility that
then obtaining product-ion mass spectra of the pseudo- the molar relative response factor has decreased signifi-
molecular ions (MH+, [M−1]−, for example). cantly. The relative molar response factor, or RF, is the
Each product ion observed in a tandem mass spec- response observed for a known quantity of a molecule
trometer product-ion mass spectrum conveys two types in isolation and is an intrinsic property of the molecule.
of information: 1) the mass of the ion and 2) the neutral Another problem in conducting studies without the use
loss producing this ion, and the tandem mass spectrom- of radioactivity is the frequently encountered phenome-
eter can scan for product ions or neutral losses character- non of “suppression of ionization.” This can occur when
istic of a given structure or substructure [61,67–71,80–82]. coeluting material interferes with the ionization of drug-
Thus metabolites of xenobiotics could also be “screened” related material. However, this problem can be over-
for and specifically detected using parent and neutral come for the most part by analyzing one to two samples
loss scans [68–70]. As an example, glucuronide, glutathi- containing high concentrations of drug-related mate-
one, or sulfate conjugates of drugs exhibit characteristic rial by all four typically-used ionization modes ( ± ESI
neutral losses (positive ion) of 176, 129 (272 in negative and ± APCI), and by employing additional techniques
ion), and 80 Da in their product-ion mass spectra, and such as derivatization chemistry, an underutilized tech-
this information can be used to screen for these conju- nology in the opinion of the authors. Also, avoidance of
gates. Specific neutral losses and product ions can also short columns and attention to possible formation of par-
be used to screen for Phase I drug metabolites and this ent drug from N-oxides, N-glucuronides, and acyl gluc-
approach can at times be quite selective [69,70]. How- uronides are important considerations, with or without
ever, this approach could also miss metabolites where a radio-labeled drug standard.
these fundamental characteristics of the product-ion In the absence of a radio-labeled standard, knowl-
mass spectrum have changed (specific ions or neutral edgeable attention to detail greatly reduces the chances
losses not observed). of missing important metabolites due to a fundamen-
More recently, there has been an increased interest tal change in the properties of the molecule (altered
in the use of high-resolution analyzers for both quali- RF) or because of other issues. For example, repeating
tative and quantitative analysis of biological samples analyses using a fundamentally different stationary
[81,82]. This approach has the advantage that MS data phase or separation chemistry will expose most prob-
obtained at sufficiently high resolution can be searched lems with suppression of ionization. For situations in

I.  DRUG DISCOVERY, METABOLISM, AND PHARMACOKINETICS


58 3.  ADME IN DRUG DISCOVERY

which this is a known problem (eg, analysis of feces), counting and by LC-RAM. The data obtained indicates
a more comprehensive approach would be to fraction- the distribution and kinetics of movement of the drug-
ate the sample using normal phase LC and then to related material [2,6,61,67–70,80].
repeat the analysis (reversed phase-LC), analyzing spe- Disposition Without Radioactivity. As with plasma
cific fractions using a range of ionization techniques. PK and metabolism studies, and as already noted, one
This process can be automated in the modern laboratory way to estimate the results obtained in the disposition
setting. Finally, another pitfall of not using radioactive study just described is to use modern MS without radio-
labeling is that some compounds exhibit low recoveries labeled drug. The unlabeled drug is administered, and
from various fluids and tissues [2,6,61,67–70,80]. samples are taken in the same manner. Then each sam-
ple is analyzed by MS using both ESI and APCI ioniza-
Feces and Other “Problem” Tissues tion techniques and positive and negative ion detection.
As noted above, one situation where matrix effects Although standards are not available for drug metabo-
are always a problem is in the extraction and analysis lites, the metabolic profile of the species under study
of drug-related material from feces. If a radio-labeled can be estimated qualitatively using the combined data
compound has been administered, it is a simple exer- obtained from plated hepatocytes and microsomes and
cise to count the sample before and after extraction and LC-UV analysis.
thus determine recovery. Recoveries from this matrix In man, the vast majority of enzymes responsible for
are usually between 70 and 90% but can often be less the biotransformations important in drug clearance are
than 50%. There are techniques to address this problem. expressed in the liver. Other tissues may contribute to
For most tissues and fluids, using one to two extrac- the observed metabolite profile; however, the qualita-
tion techniques on selected samples is recommended, tive nature of the profile is contained in the combined
such as solid-phase extraction and dilute-and-shoot. hepatocyte and microsomal metabolism data set, which
For “problem” tissues, such as feces or certain tough tis- can be used to search for metabolites in the unknown
sues like ocular sclera, sonicating with glass beads such samples collected from the nonradioisotope disposi-
that nanoparticle-sized material remains at the time of tion in vivo study. Collection of product-ion mass spec-
extraction seems to cover most of the situations encoun- tra (MS–MS data) with spectral interpretation leads to
tered. The above techniques having been noted, and as structural information for metabolites generated in the
a cautionary note, there will always be some degree of hepatocyte and microsomal metabolism studies. As pre-
uncertainty when using nonradiolabeled compounds. viously mentioned, the power of repetitive scanning
A comprehensive review of the literature dealing with high-resolution MS applied to the analysis of complex
approaches that take full advantage of modern MS mixture has been discussed as a simpler and more gen-
instrumentation for this application is beyond the pres- eral method to qualitatively and quantitatively analyze
ent overview; however, a skilled analyst familiar with drugs in biological matrices in support of early ADME
these issues can devise an analytical scheme that will studies [81].
result in an ADME report that can make broad state- Metabolism can be a direct cause of toxicity. The
ments (eg, “low excretion of drug-related material was biotransformation (metabolism) of drugs is a major
observed in urine”) with a high degree of confidence determinant of the pharmacological and toxicologi-
without the use of radio-isotopes [83]. cal responses observed in vivo in mammalian sys-
tems [84]. The enzymes involved in detoxification of
Drug Disposition Studies Using MS Without xenobiotics are for the most part the same enzymes
Isotopic Labeling responsible for the metabolism of xenobiotics to
Disposition Using Radioactivity. A typical drug dispo- reactive (toxic) species. A fundamental knowledge
sition study using radioactive standards would employ of these detoxification systems is therefore required
a 14C-labeled drug standard. The position of the 14C-label for broad appreciation of toxicology, and the reader
must have been demonstrated to be metabolically stable should familiarize themselves with this subject, which
in vitro and in vivo (for 14C-labeled drugs, carbon diox- is described in detail in general toxicology and phar-
ide would be trapped and counted in this experiment). A macology texts [85–87]. An example of species-specific
radioactivity license from the NRC must be in place, and metabolic toxicity has already been given in Fig. 3.4.
all procedures for safe handling and disposal of radioac- During preclinical development, tirilazad exhibited
tive materials must be followed throughout the study. a species-specific metabolic toxicity in the dog. This
Drug administration is usually oral, but other routes can was the direct result of a predominant metabolic path-
be indicated by the discovery program (IV, DEPOT, topi- way being N-dealkylation between the two chemical
cal, etc.). Following dosing, samples of tissues, fluids, domains, forming a metabolite that was cardio-toxic
and excreta (urine and feces) are taken at various time in the dog. The dog was thus eliminated as the nonro-
points, and the samples are analyzed by scintillation dent toxicology species.

I.  DRUG DISCOVERY, METABOLISM, AND PHARMACOKINETICS


Drug Metabolism 59
Bioactivation that cause toxicity. Frequently, these species cause tox-
Metabolism and the effects of xenobiotics, both icity in the organ where they were produced, so it is not
wanted and unwanted, are closely linked. Most com- surprising that the liver is often affected. (Note: Reac-
monly, metabolism results in a decrease in the effects of tive species can circulate, so the expression of toxicity
a xenobiotic on the body, both desired and undesired. may occur separate from reactive metabolite formation
Bioactivation to more toxic metabolites may also hap- [2,3,88–90].)
pen. Often the more toxic metabolites are electrophiles, Drug-induced liver toxicity is a significant cause of
which bind covalently to cellular macromolecules, such morbidity and mortality. In fact, drug-induced liver
as proteins and nucleic acids, resulting in a disruption toxicity is the leading reason for drug-marketing with-
of normal cellular processes. Fig. 3.7 (top) displays a drawals [91]. In the example shown in Fig. 3.7 (bottom),
general scheme of the role metabolism plays in toxicody- acetaminophen is metabolized to the electrophile N-ace-
namics (toxic effects of the drug on the body), and Table tyl-benzoquinoneimine. Normally, this species is rapidly
3.2 illustrates the reactions undergone by typical bio- removed by detoxification with glutathione. However,
chemical compounds. Metabolism can be responsible glutathione can be depleted at high doses of acetamin-
for the bioactivation of prodrugs to the active moiety, ophen, in which case, N-acetyl-benzoquinoneimine
often via hydrolysis of an ester or amide bond. Drugs reacts with the electron-rich heteroatoms of proteins
may be metabolized in one or more steps to compounds and nucleic acids, resulting in toxicity. Similar examples

FIGURE 3.7  General scheme for the role metabolism plays in toxicodynamics (the study of the toxic effects a xenobiotic has on a living system).

I.  DRUG DISCOVERY, METABOLISM, AND PHARMACOKINETICS


60 3.  ADME IN DRUG DISCOVERY

abound in the literature [1–6]. A comprehensive review a. The drug (or a metabolite of the drug) may
is beyond the scope this chapter, but an excellent review interfere with the metabolism of another drug
of metabolism and toxicity can be found in Principles and by inhibiting the enzyme(s) responsible for the
Methods of Toxicology by A. Wallace Hayes, fourth or fifth clearance of this second drug (the drug being
editions. studied is termed a perpetrator);
b. The metabolic clearance of a drug may be
Kinetics of Metabolism significantly decreased (the drug being studied
Kinetic protocols measure the rate of disappear- is termed a victim) by the action of a second drug
ance of a parent drug in various systems, usually inhibiting the enzyme(s) responsible for the
by LC-MS. Besides the quantitative data, qualitative clearance of the first drug (the reverse of the first
analysis of the samples (hepatocytes, microsomes) situation). In the situation of irreversible enzyme
may also be performed allowing for estimation of the inhibition, days may pass before the enzyme
drug metabolite profile. Kinetic experiments differ is fully resynthesized (assuming nonchronic
from stability experiments, primarily in the number administration).
of C-T samples collected. Such determinations can 2. The drug (or a metabolite of the drug) may induce
be used in determining early “drug-able” properties production of more drug-metabolizing enzyme
for chemical libraries, since compounds that are very protein via induction at the transcription level, and
unstable in liver microsomal preparations will have thus influence its own clearance or the clearance of a
high first-pass metabolism, and hence are not suitable concomitantly administered drug(s); and finally
for oral administration. 3. Concomitantly administered drugs can interfere
Hepatic microsomes can be either prepared fresh or with transport proteins that are important in drug
purchased from various suppliers. Suppliers of micro- disposition, thus altering the disposition of one of
somes typically provide CYP isoform characteriza- or both of the coadministered drugs. However, the
tion and suggested protocols for their use. Typically, (quantitative) clinical relevance of this mechanism is
the drug is incubated at a range of concentrations in low compared to metabolism interactions.
  
microsomal protein (approximately 1.0 mg/ml) with
These three categories are the best characterized mecha-
NADPH and buffers. Samples would be taken at time
nisms for DDIs, and clear quantitative and qualitative data
zero, plus four to five other points up to 45–60 min (0,
exist in the clinical literature demonstrating some effect in
5, 10, 20, 30, and 45 min, for example). The data are
man. In order to characterize the likelihood of these inter-
useful in three general ways. The first is as a screening
actions in the clinic (the DDI liability), various in vitro and
data point early in the drug discovery process, since
in vivo tests have been validated in the preclinical setting.
compounds with very poor stability in microsomes
To summarize, a drug of type 1a above is referred to as
would be expected to be eliminated rapidly from the
a DDI perpetrator new chemical entity (NCE), and 1b is a
body by liver metabolism or have very high first-pass
DDI victim characterization of an NCE (assumes competi-
metabolism. This is analogous to the situation whereby
tive inhibition). CYP Inhibition studies (1a; perpetrator)
stability in plasma can be used as a compound library
attempt to address the question: Does the NCE inhibit
screening tool. Second, these kinetic data can be used
CYPs that are important to the elimination of other drugs
to estimate the drug’s PK, and third to make interspe-
that are likely to be coadministered in the course of effec-
cies comparisons [2,16].
tive therapeutics? CYP phenotyping studies (1b; victim)
attempt to address the question: Which CYP(s) are capa-
Drug–Drug Interactions (DDIs) ble of significantly metabolizing the NCE, and are thus
relevant to drug clearance, and what is the likelihood that
The deleterious effects of drugs can arise directly, or, coadministration of a drug that inhibits this particular
as already discussed, from metabolites produced by bio- CYP will result in a DDI [93]? The relevance of either situ-
transformation. A third way in which drugs can produce ation will depend on the target disease state and likely
unwanted side effects is via their interactions with other comedications. Finally, induction of drug-metabolizing
drugs through their direct actions on drug-metabolizing enzymes at the level of transcription can also be an effect
enzymes, ie, “inhibition” and “phenotype” drug–drug of a xenobiotic, which is discussed below (the reader is
interactions (DDIs) [1,92]. The most important DDIs fall also referred to CDER guidance for an extended discus-
into three general types: sion of the characterization of this phenomenon) [16].
  

1. A
 drug may interfere by inhibiting CYP enzymes,
either reversibly (competitive inhibition) or CYP Inhibition and Phenotyping
irreversibly (time- or mechanism-based inhibition). CYP inhibition studies using human liver enzymes are
This can happen in two general ways: important screens in drug development. The major human

I.  DRUG DISCOVERY, METABOLISM, AND PHARMACOKINETICS


Drug Metabolism 61
CYPs are available as cloned, expressed purified enzyme and specific substrates still represents an active area of
preparations, and a number of highly specific substrates inquiry. (Table 3.3 is representative of the most important
have been identified and characterized. It is therefore pos- CYPs but is still an incomplete list.)
sible to examine the test compound’s ability to inhibit a A typical inhibition study involves incubating the test
CYP enzyme in isolation, or in crude preparations such compound at selected screening concentrations (0.3–30
as microsomes or S9 fraction. The preferred method is to μM, for example) and determining its effect on the kinet-
use human liver microsomal preparations pooled from a ics of formation of the marker substrate metabolite. The
range of donors. Pools can be from mixed sex donors or resulting data are expressed as a percent inhibition at a
from males and females separately. Various marker sub- test concentration. If inhibition is noted at the lower test
strates for specific CYPs have been identified in the scien- concentrations, then a more exhaustive study is under-
tific literature [16,94–96]. Inhibition liability is determined taken to determine an IC50, which is the concentration of
by noting the effect of the test species on the metabolism drug required for 50% inhibition in vitro. Another term
of these CYP-selective marker substrates in human liver commonly encountered is the Ki.
microsomes. The appearance of the metabolite is noted
with and without the test compound. Analysis of these Ki = IC50 / (1 + [S] /Km )
metabolites is typically performed by stable-isotope dilu-
tion MS, which is rapid and cost-effective in volume [13]. A typical phenotype determination study would exam-
The phenotyping experiment asks the question “what ine the metabolism of the test species at two concentra-
CYP(s) are responsible for the metabolism of the test com- tions (1.0 and 10 μM, for example) that were representative
pound?” by determining the kinetics of disappearance of of observed or projected human plasma concentrations,
the test compound in human liver microsomes and deter- and incubate each CYP-specific inhibitor at these concen-
mining the effects of various marker inhibitors to specific trations. Care must be taken to use appropriate concen-
CYPs on the disappearance of test article. Table 3.3 lists trations of inhibitor to maintain the inhibition selectivity,
representative marker substrates and inhibitors, sourced eg, quinidine is a selective inhibitor of CYP2D6 at 1 μM
from the CDER website. The investigation of the accept- but will also inhibit CYP3A4 at higher concentrations.
ability of old and novel compounds as specific inhibitors The phenotyping experiment identifies which CYPs are

TABLE 3.3  Representative “CYP Chemical Probes”


CYP Representative Substratea Reaction Representative Inhibitora Representative Inducerc

1A2 Phenacetin Phenacetin O-deethylation Furafylline Omeprazoleβ-


Naphthoflavone(2)3-
Methylcholanthrene

2A6 Coumarin Coumarin 7-hydroxylation Tranylcypromine Dexamethasone

2B6 Bupropion Bupropion hydroxylation Phencyclidineb Phenobarbital

2C8 Paclitaxel Paclitaxel 6α- Montelukast Rifampin


hydroxylation

2C9 Diclofenac Diclofenac Sulfaphenazole Rifampin


4′-hydroxylation

2C19 S-Mephenytoin S-Mephenytoin Ticlopidineb Rifampin


4′-hydroxylation

2D6 Dextromethorphan Dextromethorphan O- Quinidine None identified


demethylation

2E1 Chlorzoxazone Chlorzoxazone Diethyldithiocarbamateb None identified


6-hydroxylation

3A4/5 Midazolam Midazolam Ketoconazole Rifampin


1′-hydroxylation

3A4/5 Testosterone Testosterone 6β-


hydroxylation
aRepresentative marker substrates and inhibitors from CDER guidelines (incomplete). http://www.fda.gov/Drugs/DevelopmentApprovalProcess/DevelopmentResources/DrugInter-
actionsLabeling/ucm093664.htm. Other substrates include nicotine (2A6), efavirenz (CYP2B6), amodiaquine (CYP2C8), tolbutamide and S-warfarin (2C9), bufuralol (CYP2D6),
chlorzoxazone (CYP2E1), nifedipine and atorvastatin (CYP3A4/5), and lauric acid (CYP4A11).
bAcceptable inhibitor (not listed as a preferred inhibitor).
cReference [16].

I.  DRUG DISCOVERY, METABOLISM, AND PHARMACOKINETICS


62 3.  ADME IN DRUG DISCOVERY

capable of metabolizing the NCE. This helps to work out CYP activities are determined using marker substrates
the likelihood that concomitant administration of another in an analogous manner to hepatic microsomes, and the
marketed drug with the test species would result in a DDI inducing activities of test drug and control drug(s), ie,
in which the metabolic clearance of the new drug could known inducers are compared. A test drug change in
decrease, leading to drug accumulation and increased CYP activity equal to or greater than 40% of the control
pharmacological effect. It should always be kept in mind inducer is considered enough to warrant further study.
that both inhibition and phenotyping studies are esti- In addition to changes in the expression of genes encod-
mates used to indicate whether specific clinical studies ing CYP enzymes, the effect on UGT enzymes such as
looking for DDIs are warranted. UGT1A1, UGT1A4, UGT1A6, UGT1A9, UGT1B7, and
It should also be noted that if the in vivo clearance transporters such as P-glycoprotein may be determined.
mechanism is excretion of an unchanged drug or signifi-
cant Phase II conjugation, we only need to be concerned
about the NCE being a perpetrator of DDIs, not a vic- SUMMARY AND TRENDS
tim. Also, clinical tests must eventually be performed
to more accurately determine the actual clinical liabil-
Use of Preclinical ADME Data
ity. In addition, the relevance of a DDI is also related to
the therapeutic area. Drugs with a narrow therapeutic The process of interpreting all of the data generated in
index represent one important situation. Treatment of a drug discovery program is highly complex. Assessment
life-threatening conditions, such as infectious diseases of eADME data is a critical component of this assessment.
or cancer, may represent situations where potential DDIs If a drug cannot be delivered to the drug target recep-
need to be clinically managed with drug plasma concen- tor at therapeutic concentrations for the needed time
tration monitoring and dose modification. Clearly, clini- period, then the drug will not be an effective therapeu-
cal management of potential DDIs is irrational for DDIs tic in humans. Drug candidates that will be very rapidly
that could occur for over-the-counter (OTC) drugs, such metabolized in the liver or are delivered poorly to the
as nonsedating antihistamines, and therefore the poten- target organ are eliminated by a robust eADME program
tial DDI liability for OCTs is kept very low. early in the evaluation process. The first job of the ADME
drug discovery scientist is to find and eliminate the “los-
CYP Induction Studies ers.” The second job of the ADME scientist is to evaluate
The first type of DDI we discussed was the inhibition of groups of compounds with regard to their likely perfor-
one or more CYPs by concomitantly administered drugs, mance relative to one another in the clinic. If a long half-
either a marketed drug (victim) or the test article (perpetra- life is very important, then preclinical studies are used to
tor). Another type can occur if the test agent induces one estimate this property. The process of estimating PK prop-
or more of these drug-metabolizing enzymes, and thus erties (ADME properties) in man from the data obtained
changes the metabolic clearance of a drug (either concomi- in preclinical species is termed allometric scaling. In this
tantly administered drugs or the test agent). The human exercise, in vitro human data are also taken into consid-
hepatocyte induction assay (HIAA) has become a standard eration. Allometric scaling is a valuable part of the drug
preclinical test to estimate this liability in the clinic [16]. discovery and development process [97–99]. A third job
Typically, human hepatocytes are plated and incubated of the ADME scientist is to help the toxicologist and clini-
with the test article, and as a control also incubated with cal scientist estimate clinical doses and derive a therapeutic
drugs known to induce the synthesis of CYP proteins, such index based solely on animal data. Finally, a fourth job of
as phenobarbital, rifampin, dexamethasone, or omeprazole the ADME scientist is to identify drug metabolites that
(Table 3.3). The test drug concentration should be based humans will be exposed to that preclinical toxicology spe-
upon clinical data, if available, or expected human plasma cies were not exposed to in the same relative amounts (dis-
concentrations. Three concentrations spanning the targeted cussed in Chapter 4, “Pharmacokinetics/Toxicokinetics”).
therapeutic concentrations should be employed. If clinical One very important situation occurs when a drug
estimates are not available, then concentrations covering a metabolite is absent or present at low concentrations in
range of at least two orders of magnitude should be used, the circulation of rodents and the nonrodent toxicology
with the maximum being 10 times greater than the high- species (dog, monkey), but represents significant expo-
est concentration anticipated in man. At least three donors sure in man. Significant exposure has been arbitrarily
should be used to account for intersubject variability, and defined as a plasma concentration (exposure as quanti-
the study should be performed in triplicate. fied by AUC or Cmax) that is greater than or equal 10% of
Three endpoints have been used as a determination the parent drug. CDER has issued guidance for address-
of induction: mRNA, enzyme protein concentration, ing this situation as Metabolites in Safety Testing, or MIST
and CYP enzyme activities. For IND-enabling stud- [100–104]. Modern NMR and MS technologies are critical
ies, phenotype measurements (activities) are preferred. tools in identifying problems early so that the appropriate

I.  DRUG DISCOVERY, METABOLISM, AND PHARMACOKINETICS


Summary and Trends 63
testing of metabolites can be done prior to introduction Mass spectrometry has always been an important
into humans [105]. One technique uses microdoses of the technology impacting drug research.
14C-labeled drug and analysis by accelerator mass spec- More recently, the power of repetitive scanning at
trometry (AMS). The drug is administered to humans in high resolution has been “rediscovered” as a powerful
very low absolute doses (weight, moles) and spiked with technique for the quantitative and qualitative analysis of
very low amounts of 14C-labeled drug, both of which are complex mixtures, a trend encouraged by the introduc-
low enough not to be of significant toxicological concern tion of high-resolution analyzers better adapted to ESI,
(abbreviated safety evaluations are performed, in any primarily time-of-flight (TOF) [106–108] and Orbitrap
case). LC separation is performed on plasma samples and [109,110] mass analyzers.
fractions are collected approximately every 30 s. The ratio Resolution is the ability of a mass analyzer to dis-
of 14C/13C is determined by AMS in each sample and is tinguish one distinct mass-to-charge ratio (m/z) from
used to quantify the amount of parent drug or metabo- another, and is usually defined as M/ΔM, where ΔM
lite present. Another approach involves the metabolism of is a defined resolving power such as peak width at ½
radiolabeled and stable-isotope labeled compounds, thus peak height for a mass vs. intensity peak. Figs. 3.8 and
providing standards for determination of the amounts 3.9 display mass spectral data obtained at R = 300 and
of metabolites produced in a biological system (from the at R = 30,000, respectively. The data in Fig. 3.8 were
radiolabeled standards) and stable-isotope labeled inter- obtained at unit resolution using a quadrupole mass
nal standards for performing stable-isotope dilution MS. analyzer, which operates at approximately unit resolu-
In this approach, high-resolution MS analysis is used tion across the mass range (the resolution at m/z 300 is
instead of MS–MS [71]. R = 300 and the resolution at m/z 700 is R = 700). Mass
m/z 300 can clearly be distinguished from mass m/z
301. However, the mass m/z 300.25 cannot be distin-
Technologies Impacting ADME in Drug
guished from m/z 300.27 at unit resolution, and thus their
Discovery signal intensities cannot be recorded separately. The unit
The evolution of molecular imaging technologies, resolution displayed in Fig. 3.8 stands in sharp contrast
especially PET and SPETC, are important enough to with Fig. 3.9 (R = 30,000). This is an important distinc-
have been covered in a separate chapter (see Chapter 35, tion (unit vs. high resolution) since many endogenous
“Use of Imaging for Preclinical Evaluation”). compounds represent interferences when using nominal

FIGURE 3.8  LC-MS spectra obtained using a quadrupole instrument (dashed line, nominal resolution) and an Orbitrap high-resolution
analyzer (solid line). Resolution (mass-resolving power) is M/ΔM, where ΔM is a defined resolving power. Nominal resolution data: Displayed is
MH+ (m/z 289; quadrupole instrument) and the isotopic cluster observed for testosterone, the so-called A + 1 (m/z 290) and A + 2 (m/z 291) ions.
Resolution that can distinguish one nominal mass from another is also called unit resolution. A simple way to define and calculate is to define ΔM
as the peak width at ½ height and calculate directly from a profile mass spectrum. High-resolution data: Displayed is MH+ (m/z 260) and the iso-
topic cluster observed for propranolol for the A + 1 (m/z 261) and A + 2 (m/z 262) ions. This mass spectrum was obtained at a resolution of 30,000.

I.  DRUG DISCOVERY, METABOLISM, AND PHARMACOKINETICS


64 3.  ADME IN DRUG DISCOVERY

Mass Spectrum
m/z 430.29591

Metabolite
m/z 433.19731

Intensity m/z 432.30956


Endogenous Compound
m/z 433.31316

m/z 431.29928

430 431 432 433 434

Mass m/z
FIGURE 3.9  Mass spectrum from LC-MS data obtained at high resolution (Orbitrap mass analyzer). These data were obtained for an
in vivo, radiolabeled ADME study for a biological sample. Both endogenous (naturally found in the body) and xenobiotic (drug) materials are
easily distinguished by the differences in mass defect for the different empirical formula. In this example, a drug metabolite at m/z 433.19,731 is
clearly differentiated from an endogenous compound (or possibly another xenobiotic) at m/z 433.31,316. This mass spectrum was obtained at a
resolution of 30,000. The determination of mass at high resolution allowing for the determination of empirical formulas greatly aids in the elucida-
tion of structures of drug metabolites and in selectivity of an assay for quantitative analysis studies. High-resolution MS coupled with LC will
continue to greatly impacting eADME research.

mass analysis. However, when operated at high resolu- In a typical low-resolution (nominal resolution)
tion, the mass defect (the mass difference from nominal mass analysis experiment, previous metabolism data
mass) differences between drugs, drug metabolites, and is considered, and various scanning experiments are
endogenous compounds can be resolved and observed performed, involving repetitive scanning of the data
(detected) separately. The appearance of more than one (say from m/z 100 to m/z 600 every 0.5 s for a drug of
species at a particular nominal m/z value does not inter- mass 300) and reanalysis with SIM, SRM, and MS–MS
fere with the detection and quantitation of the other spe- scans (neutral loss and parent ion scans). When the
cies since they represent distinct signals. data from m/z 250 to 350 are examined for metabo-
Limitations can occur, however, when the elemen- lites, 100 distinct pseudo-molecular ion m/z values
tal composition of the molecular ion is identical to the can be distinguished. When the mass analysis occurs
endogenous contaminants. For drugs that are either of at R = 30,000, in theory, up to 3 million distinct pseudo-
endogenous origin (a prostaglandin such as PGE2 or molecular ion m/z values can be distinguished. As
estradiol, for example), then this approach has limita- long as there is sufficient metabolite present to gener-
tions. However, most drugs have ratios of elements and ate a signal in the ion source, and the relative response
ring structures that are uncommon in endogenous com- factor has not changed dramatically, then the metab-
pounds, such as high number of hetero atoms relative olite will be detected by HRMS, provided that the
to carbon, or halogen atoms attached to carbons. Also, elemental composition is distinguishable from endog-
there are also a limited number of additions and subtrac- enous compounds.
tions of masses (exact masses) that are possible through The term relative response is important, since this is
biotransformaton reactions, hence there are a limited currently the major limitation to quantitative analysis.
number of m/z values (nominal and exact) that can be As stated above, relative response factors (RF) are arbitrary
assigned to metabolites of a given parent drug. All this ratios of the responses observed for two compounds
lends itself perfectly to using computers to search for under identical situations, say for LC-ESI-MS (posi-
distinct m/z values for metabolites of a starting mate- tive ion). As an example, if a drug undergoes aromatic
rial, thus automating the search for drug metabolite hydroxylation and the response for the same number of
masses in the spectra obtained when analyzing biologi- moles of parent drug are compared to the same number
cal materials [61,67–71,80,81]. (Note: When using HRMS of moles for the metabolite, this response will differ by
for structure ID, it is incorrect to use the term empirical some factor, RF, either an increase or a decrease relative to
formula determination; elemental composition should be the parent drug. If the response observed was 110% that
used instead.) of the parent drug, then the relative response factor is 1.1,

I.  DRUG DISCOVERY, METABOLISM, AND PHARMACOKINETICS


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