NLM Citation: Chung TDY, Terry DB, Smith LH.

In Vitro and In Vivo

Assessment of ADME and PK Properties During Lead Selection and
Lead Optimization – Guidelines, Benchmarks and Rules of Thumb. 2015
Sep 9. In: Markossian S, Grossman A, Arkin M, et al., editors. Assay
Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the
National Center for Advancing Translational Sciences; 2004-.
Bookshelf URL: https://www.ncbi.nlm.nih.gov/books/

In Vitro and In Vivo Assessment of ADME and PK

Properties During Lead Selection and Lead
Optimization – Guidelines, Benchmarks and Rules of
Thumb
Thomas D.Y. Chung,1 David B. Terry,2 and Layton H. Smith3
Created: September 9, 2015.

Abstract
Assessment of the pharmacological properties of small molecule chemical compounds is critical to the initial
selection or identification of a chemical lead, and during the further lead optimization to elucidate the Structure-
Activity Relationships (SAR) and Structure Property Relationships (SPR), and ultimately to select the
compound(s) that will enter Investigational New Drug (IND)-enabling studies. While extensive discussion of
how Absorption, Distribution, Metabolism, and Excretion (ADME) of compounds affects their ultimate
pharmacokinetics (PK) is beyond the scope of this chapter, herein, we provide guidelines for ADME and PK
assessments, benchmarks and practical “rules of thumb” for selecting compounds with sufficient PK to be viable
efficacious drugs.

Flow Chart of a Two-tier Approach for In Vitro and In Vivo

Analysis

Author Affiliations: 1 Conrad Prebys Center for Chemical Genomics, Sanford Burnham Prebys Medical Discovery
Institute, La Jolla, CA. 2 Conrad Prebys Center for Chemical Genomics, Sanford Burnham Prebys Medical Discovery
Institute at Lake Nona, Orlando, FL. 3 Conrad Prebys Center for Chemical Genomics, Sanford Burnham Prebys Medical
Discovery Institute at Lake Nona, Orlando, FL.
2 Assay Guidance Manual

Background
As well-reviewed in the Assay Guidance Manual (AGM) chapter on Early Drug Discovery and Development
Guidelines, there is an evolving paradigm for drug discovery and early development with the academic and non-
profit enterprises focused on delivering innovative, novel, new chemical entities (NCE) through collaboration,
partnering and licensing optimized leads for final clinical development to pharmaceutical companies. That
chapter outlined the overall process, critical steps and key decision points at each step. Each of these steps and
associated technologies, protocols, techniques, and case examples are covered elsewhere in the AGM. Ultimately,
an exemplary compound(s) emerges from systematic elucidation of the Structure Activity Relationship (SAR)
through examining the potency, specificity and selectivity of analogs around a chemical scaffold. This comprises
identification of a chemical lead, where the most potent, specific and selective compound(s) are chosen.
Assessments of the pharmacological properties of Absorption, Distribution, Metabolism, and Excretion (ADME)
of a candidate chemical lead(s) are critical to their initial selection, and establishes benchmarks against which
compounds synthesized during lead optimization can be evaluated. Further improvements in ADME properties
during lead optimization are sought, while preserving the potency and selectivity of the chemical lead(s), though
sometimes more efficacious compounds have lower in vitro potencies, but better ADME properties.
These activities often reside in an exploratory pharmacology group that provides in vitro and in vivo
pharmacologic and physicochemical property analysis of biologically active small molecules in support of small
molecule probe/drug discovery projects. Early pharmacological assessment has been adopted within the
pharmaceutical industry as a critical feature of a robust drug/probe discovery process. This is because the
development and optimization of useful molecules is a multi-parameter process. Simply designing new analogs
and developing a SAR for increased potency against the biological target is inadequate for the development of
small molecule probes or drugs suitable for cellular, tissue, or whole animal disease model(s). The assessment
and optimization of Structure-Pharmacologic/Property-Relationships (SPR) is a further critical step for efficacy
evaluation. In addition to assessing compound characteristics such as solubility, protein binding, and serum
stability, the data allows the chemistry team to prioritize different structural classes and rank order them not
only based on potency but also in relation to potential downstream absorption or metabolism liabilities. An
excellent additional overview of pharmacokinetics (PK) can be found on the online version of the Merck Manual
(http://www.merckmanuals.com/professional/clinical-pharmacology/pharmacokinetics/overview-of-
pharmacokinetics)
Prior to actual dosing in animals, a number of relatively rapid and cost effective in vitro assays can serve as
surrogates and indicators of the ADME fate of compounds in vivo. Improvements in ADME properties of
compounds translate to their improved PK properties. Simply stated, if a compound is rapidly absorbed, well
distributed, minimally metabolically degraded and not rapidly eliminated, while not being toxic, then it more
likely will rapidly achieve peak levels in the blood, maintain the desired levels (n-fold above the IC50) for a
longer duration, before falling to low trough levels, and ultimately being cleared by the body.
In the sections below, we describe the basic component and provide high level protocols for a two-tiered
approach for these key in vitro ADME assays (see Flow Chart). We provide an example of an ADME table, and
benchmarks for a series of probe compounds developed through the NIH Molecular Libraries Program (MLP)
by our group when we were part of the Molecular Libraries Probe Production Centers Network (MLPCN). We
then summarize a two-tiered approach to doing pharmacokinetic studies (see Flow Chart). First an abbreviated
“rapid” assessment for compound exposure (R.A.C.E.) developed by us (LHS), then the more typical
“comprehensive” pharmacokinetic analysis used during late lead optimization toward candidate selection for
final preclinical IND.
Ultimately, the in vivo efficacy of an optimized lead will be better served by having good pharmacological
properties, so that the compound administered at a given dose actually achieves the required concentration, for
In Vitro / In Vivo Assessment of ADME and PK Properties During Lead Selection / Optimization 3

sufficient duration in the target tissue to achieve the desired biological effect, while minimizing any undesired off
target effects. Improvement in these ADME properties is sought prior to actual dosing in animals to assess PK,
and certainly for larger compound efficacy studies, since animals are expensive and the ethics of sacrificing
animals in poorly designed studies uninformed by pharmacological guidance are indefensible.

In Vitro Analysis - Low Compound Requirements and Relative

Moderate Capacity
Lipophilicity
Pharmacologic question addressed: “Will my parent compound be stored in lipid compartments or how well will
my parent compound bind to a target protein?”
Lipophilicity is an important physicochemical property of a potential drug. It plays a role in solubility,
absorption, membrane penetration, plasma protein binding, distribution, CNS penetration and partitioning into
other tissues or organs such as the liver and has an impact on the routes of clearance. It is important in ligand
recognition, not only to the target protein but also CYP450 interactions, HERG binding, and PXR mediated
enzyme induction.
Lipophilicity is typically measured as the neutral (non-ionized) compound distribution between non-aqueous
(octanol) and aqueous (water) phase and the result is expressed as a 10-base logarithm of the concentration
ratios between these phases (partition coefficient), log P.
Another common measure for lipophilicity is the distribution coefficient, log D, which takes into account the
compound’s ionized and non-ionized forms, and therefore the measurement is done at different pH values.
Typically the most interesting is pH 7.4, since the majority of known drugs contain ionizable groups and are
likely to be charged at physiological pH.
Assay Design:
• Test articles are assayed in triplicate
• One concentration of test article (typically 10 μM)
• n-Octanol is the partition solvent
• Ratio of buffer: Octanol is 1:1 (other ratios available)
• Positive control: Testosterone (high log D7.4 value)
• Negative control: Tolbutamide (low log D7.4 value)
Analysis: LC/MS/MS measurement of parent compound
Report: Log D7.4 value
Quantity of test article required: 1.0 - 2.0 mg
Summary of Assay:
Lipophilicity of compounds is assessed using the golden standard “shake-flask” method. The compound is
dissolved in a solution with equal amounts of octanol and water, shaken for 3 hours, and then measured for the
amount of compound in each phase. Log D values are calculated by the log ([compound]octanol /
[compound]buffer).

Solubility
Pharmacologic question addressed: “What is the bioavailability of my compound?”
4 Assay Guidance Manual

Aqueous solubility, another common physicochemical parameter for drug discovery compounds, is an
important analysis as it reflects the bioavailability of the compound. The ability of a compound to dissolve in a
solvent to give a homogenous system is one of the important parameters to achieve a desired concentration of
drug in systemic circulation for the desired (anticipated) pharmacological response. Formulation and routes of
administration, especially oral dosing, are challenging for poorly soluble drugs, as it limits the absorption of
compound from the gastrointestinal tract. Also, poor solubility will affect other AMDE/DMPK analyses, if some
fraction of the compound precipitates and is unavailable (e.g. in assays for metabolite stability and various CYP
identification/inhibition/induction assays). Also, since the majority of known drugs contain ionizable groups,
the aqueous solubility is assessed over a range of pH values.
Assay Design:
• Test articles are assayed in duplicate
• One concentration of test article (typically 1 μM)
• Phosphate buffered solution (other buffers available)
• Three point pH range (5.0, 6.2, 7.4)
• Positive control: Diclofenac (high solubility)
• Negative control: Dipyridamole (low solubility)
• Background control: DMSO only
Analysis: UV spectrophotometry measurement of parent compound
Report: Amount of compound dissolved (μM)
Quantity of test article required: 1.0 - 2.0 mg
Summary of Assay:
The compound is dissolved in buffer solutions at the indicated pH values. The compound is allowed to reach
thermodynamic equilibrium by incubating for 18 hours. Compound UV absorption is compared to fully
saturated solution in 1-propanol.

Hepatic Microsome Stability

Pharmacologic question addressed: “How long will my parent compound remain circulating in plasma within the
body?”
The assay uses subcellular fractions of liver, microsomes, to investigate the metabolic fate of compounds. Liver
microsomes consist mainly of endoplasmatic reticulum and contain many drug-metabolizing enzymes,
including cytochrome P450s (CYPs), flavin monooxygenases, carboxylesterases, and epoxide hydrolase (1). Liver
microsomes are available commercially (example, Xenotech, LifeTechnologies and DB Biosciences) as frozen
preparations that are usually prepared in bulk with pooled livers from sacrificed mice, rat or human cadavers. As
a result, hepatic microsomal metabolic activity can vary significantly from batch to batch. Therefore, in critical
studies, it is recommended that planning to obtain the same lot of microsomes be considered in the
experimental plans. If lots of microsomes do run out, a few bridging comparisons to establish comparable values
for microsomal stability of reference compounds, should be done.
Assay Design:
• Test articles are assayed in triplicate
• Human liver microsomes (or other species as needed) (0.5 mg/mL)
• One concentration of test article (typically 10 μM)*
• Two time points t = 0 and t = 60 min*
• Positive control: Substrates with known activity
In Vitro / In Vivo Assessment of ADME and PK Properties During Lead Selection / Optimization 5

• Negative control: NADPH deficient

Analysis: LC/MS/MS measurement of parent compound at specific time points
Report: % metabolism of the test article (single time point); also, intrinsic clearance and half-life (multiple time
points)*
Quantity of test article required: 1.0 - 2.0 mg
Summary of Assay:
Metabolic stability of compounds are assessed at a single concentration (typically 10 μM) at t = 0 and at t = 60
min. Stability of compounds are tested in human (other species available) liver microsomes. Compounds are
tested in triplicate with or without NADP wells as a negative control for P450 metabolism. Each assay will
include a substrate with known activity (such as the CYP3A4 substrate testosterone) as a positive control (2).
*Note: the number of concentrations/time points in the assay can be expanded for drug development SAR
efforts.

Plasma Stability
Pharmacologic question addressed: “Is my compound degraded in plasma?”
In addition to hepatic metabolism, compounds are also subjected to degradation/modification by enzymes in
plasma, particularly hydrolysis and esterases. Thus, the stability of test compounds in plasma is an important
parameter, which not only affects in vivo results, but also the bioanalytical assay strategy and design.
Investigation of plasma stability should be performed early in the discovery process in order to assess potential
degradation and/or protein binding issues.
Assay Design:
• Test articles are assayed in triplicate
• Two concentrations (10 μM and 100 μM or if known Cmax and 10x Cmax)
• Two time points t = 0 and t = 180 min
• Positive control: Procaine (50 μM)
• Negative control: Procainamide (50 μM)
Analysis: LC/MS/MS detection of the remaining test article
Report: % parent compound remaining
Quantity of test article required: 1.0 - 2.0 mg
Summary of Assay:
A solution of test compound in plasma is prepared and incubated for a predetermined time period. Aliquots are
removed at pre-defined time points and analyzed by LC/MS/MS. The peak area for the parent compound is
compared to the time zero sample in order to assess the amount of compound still available.

Plasma Protein Binding

Pharmacologic question addressed: “What percent of the compound plasma protein is bound, to which component
(sub-fraction), and what is the free fraction available to cover the target?”
The binding of test compounds to plasma proteins is an important factor affecting drug efficacy, metabolism and
pharmacokinetic properties. In many cases, drug efficacy is determined by the concentration of free drug
(unbound), rather than the total concentration in plasma. If the drug is highly bound to plasma proteins, the
6 Assay Guidance Manual

amount of drug available to reach the target is reduced. Subsequently, the efficacy of that compound may be
significantly reduced. Therefore, information on the free drug fraction is essential for drug development and
may be helpful in correlating with in vivo efficacy.
Rapid equilibrium dialysis (RED) is an accurate and reliable method for determining the degree to which a
compound binds to plasma proteins. Plasma spiked with test compound is added to the center chamber of a
commercial plate based RED device. Blank, isotonic sodium phosphate buffer is added to the peripheral
chamber of the RED device and the plate is incubated at 37°C for 4 hours. Equilibrium of free compound is
achieved by the diffusion of the unbound compound across the dialysis membrane. Several manufacturers
provide RED devices (Thermo Scientific). Aliquots of the buffer and the plasma are taken at pre-determined
time points and the concentration of free and bound test compound is determined by LC/MS/MS analysis.
Assay Design:
• Test articles are assayed in duplicate
• Test articles are mixed with human plasma (other species available)
• One concentration of test article (10 μM, different concentrations available)
• One time point (t = 4 hours at 37°C)
• Positive control: Propranolol (high binding) and Metoprolol (low binding)
• Negative control: No plasma (PBS only)
Analysis: LC/MS/MS detection of the test compound in plasma and in buffer
Report: % compound bound
Quantity of test article required: 1.0 - 2.0 mg
Summary of Assay:
Human or specific species of interest plasma in the sample chamber are spiked with test compounds at 100x
dilution of stock solution (typically 10 mM in DMSO). The chamber is sealed, and the compound is dialyzed
against PBS, pH 7.4 at 37°C for 4 hours. Aliquots from each chamber (plasma and PBS) are collected and the
concentrations of compound in each sample are determined by LC/MS/MS. Adjustments are made for non-
specific binding.

Screening Cytotoxicity / Hepatotoxicity Test

Pharmacologic question addressed: “Is my compound too toxic to be therapeutic?”
Cytotoxicity is a well-established and easily accessible endpoint to gather early information about the general /
acute toxic potential of a test article. The in vitro cytotoxicity test with primary hepatocytes is used to identify the
cytotoxic potential of a test substance. The relative cell viability upon incubation with test article compared to
the solvent control is determined (single point).
The ATP-lite 1step Cytotoxicity Assay (PerkinElmer) is a single reagent addition, homogeneous, luminescence
ATP detection assay that measures the number of live cells in culture wells.
Assay Design:
• Primary hepatocytes (other cells available)
• 12-dose concentration response curve (CRC) of the test article (100x IC50 or 50 µM maximum
concentration)
• Two replicates of CRC
• One incubation time 24 hours
• Positive control: Compounds with known toxicity
In Vitro / In Vivo Assessment of ADME and PK Properties During Lead Selection / Optimization 7

• Negative control: Compound with known non-toxicity

• Background control: Vehicle only
Analysis: Luminescence is measured from 550 - 620 nm.
Report: IC50
Quantity of test article required: 1.0 - 2.0 mg
Summary of Assay:
Hepatocyte cells are incubated for 24 hours with known toxic and non-toxic compounds at a range of different
concentrations. At the end of the incubation period the cells are loaded with the ATP-liteTM 1step ATP
monitoring reagent and scanned using an automated plate reader with luminescence detection (Tecan Infinite
M200 reader) to determine the number of active cells.
CYP450 Inhibition Profiling
This assay extends the findings of the microsomal stability assay. Pharmacologic question addressed: “Does my
compound inhibit a key oxidative metabolic enzyme that would lead to subsequent drug-drug interactions?”
Cytochrome P450s (CYPs) are a superfamily of heme-containing enzymes that mediate the inactivation and
metabolism of many drugs as well as endogenous substances. Compounds that inhibit P450s may cause the toxic
accumulation of other substrates. CYP inhibition profiling examines the effects of a test compound on the
metabolism of other known enzyme substrates of the five primary drug human metabolizing CYP: 1A2, 2B6,
2C9, 2D6, 3A4. The levels of the CYP isoform marker substrate and metabolites are measured in the presence
and absence of a test compound by LC/MS/MS.
Assay Design:
• Five CYP isoenzymes: 1A2, 2B6, 2C9, 2D6, 3A4
• Test articles are run in triplicate
• One concentration of human liver microsomes (0.5 mg/mL)
• One concentration of test item (10 µM)
• One time point 0.5 hours
• Positive control: CYP marker reaction (Table 1)
• Negative control: NADPH deficient reaction control
Analysis: LC/MS/MS detection (appearance of metabolite)
Report: Data are expressed as % inhibition of selected metabolites formation for each CYP450 enzyme (1A2,
2B6, 2C9, 2D6, 3A4).
(See FDA guideline for CYP substrates:
http://www.fda.gov/Drugs/DevelopmentApprovalProcess/DevelopmentResources/DrugInteractionsLabeling/
ucm093664.htm)
Quantity of test article required: 1.0 - 2.0 mg
Summary of Assay:
In an assay similar to the metabolic stability assay, liver microsomes are used to determine the CYP450
inhibition profile of test compounds by measuring the % metabolism of a known substrate. Microsomes (and
NADPH regenerating system) are dispensed into a 96-well plate containing a substrate and test compound (10
µM), and the reaction is allowed to proceed for 0.5 hours at 37°C with shaking. The reaction is quenched by the
8 Assay Guidance Manual

addition of MeOH, centrifuged and the amount of product is measured by LC/MS/MS. Each plate will contain a
known inhibitor of each CYP450 profiled as positive control and NADP-/- negative controls.

Table 1: Inhibition profiling for the five primary drug human metabolizing cytochrome P450s.
CYP Enzyme Substrate Metabolite Known Inhibitor
1A2 Phenacetin Acetaminophen Furafylline
2B6 Bupropion Hydroxybupropion Ticlopidine
2C9 Diclofenac 4-hydroxydiclofenac Tienilic Acid
2D6 Dextromethorphan Dextrorphan Paroxetine
3A4 Testosterone 6β-hydroxytestosterone Azamulin

Permeability
Pharmacologic question addressed: “How well is my drug absorbed in the gastrointestinal tract?”
Evaluating compound permeability through a cell monolayer is a good indication of intestinal permeability and
oral bioavailability. The Parallel Artificial Membrane Permeability Assay (PAMPA) provides a high throughput,
non-cell based method for predicting passive, transcellular intestinal absorption, the process by which the
majority of small molecule drugs enter circulation. In the PAMPA method, an artificial membrane immobilized
on a filter is placed between a donor and acceptor compartment. The compound is introduced in the donor
compartment. Following the permeation period, the amount of compound in the donor and acceptor
compartments are quantified using scanning UV spectrophotometry.
The gastrointestinal tract (GT) has a pH range from pH 1 – 8. The pH of the blood is constant at pH 7.4;
therefore it is possible for a pH gradient to exist between the GT and the plasma that can affect the transport of
ionizable molecules. In an effort to mimic this pH gradient in vitro, alternative assays with pH 7.4 for the
acceptor compartment and pH values 5.0, 6.2, and 7.4 in the donor compartment are used.
PAMPA is a well-established and predictive assay that models the absorption of drugs in the gut. However,
PAMPA is an artificial system that may provide inaccurate and potentially misleading results. Despite these
limitations, PAMPA can be a useful tool to prioritize lead compounds in early stages of development. The colon
carcinoma (Caco-2) cell permeability assay is the industry standard for in vitro prediction of intestinal
absorption of drugs, but it too has limitations. Caco-2 cells require extensive culturing (>20 days), and often fail
to form the cohesive monolayer necessary for uniform transport of compounds across the cell layer. The assay
requires a significant amount of compound to perform the assay (typically ~20 mg). Together, the limitations of
time and compound consumption decrease the value of the results obtained by Caco-2 at the early stages of drug
discovery. One variant of PAMPA is the Blood Brain Barrier (BBB) PAMPA in where the artificial monolayer
contains brain specific membrane components, such as sphingolipids.
Assay Design:
• Test articles are run in triplicate
• One concentration (25 µM)
• One time point (18 hours)
• One pH (7.4) or three point pH range (5.0, 6.2, 7.4) for acceptor compartment
• Single polar membrane lipid (phosphatidylcholine in dodecane)
• Multiscreen PVDF membrane (0.45 µm)
• Positive control: Verapamil (high permeability)
• Negative control: Theophylline (low permeability)
In Vitro / In Vivo Assessment of ADME and PK Properties During Lead Selection / Optimization 9

Analysis: The concentration of the compound remaining in the donor well, diffused through the membrane and
into the acceptor well, and reference compounds are measured by UV spectrophotometry
Report: Bin the results as high, medium, or low predicted absorption and report direct permeability units (10-6
cm/s)
Quantity of test article required: 5.0 - 7.0 mg
Summary of Assay:
A lipid bilayer is established on a membrane filter and a test compound solution is added to the top of the
membrane-lipid interface. The ability of compounds to passively diffuse through the lipid treated membrane is
an indication of the overall compound permeability. This approach is helpful in compound profiling and
supporting the relative rank ordering of compounds.

Example of In Vitro ADME Profiling Assays

Table 2 below shows an example tabulation of the ADME properties assessed and evaluated as a requirement for
nomination of a chemical probe in the Molecular Libraries Program (MLP) for probes ML301, ML314 and a few
key related analogs, as described in the NCBI MLP probe report: http://www.ncbi.nlm.nih.gov/books/
NBK184496/ that describes a novel small molecule agonist for the Neurotensin 1 Receptor (NTR1).
ADME profile of ML301 (Probe 1) and related compounds: ML301, its intriguing naphthyl analog
(MLS-0446079), and the prior art, pyrazole, were evaluated in a detailed in vitro ADME screen as shown in Table
2. Despite its structural similarity (imidazole vs. pyrazole) to the prior art compound, ML301 exhibited
substantial advantages in this testing, especially with regard to plasma and microsomal stability. These three
compounds all exhibited good solubility due to the presence of the carboxylic acid moiety.
The PAMPA assay is used as an in vitro model of passive, transcellular permeability. The compounds exhibited
good overall permeability, inversely related to the pH of the donor compartment. Because these NTR1 agonists
are envisioned as predecessors of psychoactive drugs, a preliminary assessment of their potential to cross the
BBB was performed. When incubated with an artificial membrane that models the BBB, much lower
permeability was observed. These observations are also consistent with the carboxylic acid function in the
compounds, and may present an opportunity for future enhancements.
Plasma protein binding is a measure of a drug's efficiency of binding proteins within blood plasma. The less
bound a drug is, the more efficiently it can traverse cell membranes or diffuse. Drugs that are highly bound to
plasma proteins are confined to the vascular space, thereby having a relatively low volume of distribution. In
contrast, drugs that remain largely unbound in plasma are generally available for distribution to other organs
and tissues. The imidazole scaffold compounds (ML301 and its MLS-0446079) exhibited substantial protein
binding, but significantly lower than that of the prior art, pyrazole.
The stability of small molecules and peptides in plasma may strongly influence in vivo efficacy. Drug candidates
are susceptible to enzymatic processes, such as those mediated by proteases or esterases in plasma. They may
also undergo intramolecular rearrangement or bind irreversibly (covalently) to proteins. ML301 showed
excellent stability in plasma, significantly better than that of either analog.
The microsomal stability assay is commonly used to rank compounds according to their metabolic stability,
which influences how long the candidate may remain intact while circulating in plasma. ML301 showed
excellent stability in human and modest stability in mouse liver homogenates, which was much better than that
observed for the prior art analog, MLS-0437103. None of the compounds showed toxicity (>50 µM) toward
human hepatocytes.
10 Assay Guidance Manual

ADME profile of ML314 (Probe 2): As described above for ML301, in vitro ADME screening was also conducted
for ML314. Consistent with its aqueous solubility data, ML314 exhibited high permeability in the PAMPA assay
with increasing pH of the donor compartment. When incubated with an artificial membrane that models the
BBB, ML314 was found to be highly permeable. ML314 was highly bound to plasma protein and exhibited very
high plasma stability. ML314 was metabolized rapidly when incubated in vitro with human and mouse liver
microsomes. This result is not completely surprising because of the presence of several unsubstituted aryl and
alkyl positions and Ar-OMe ethers, which are prone to oxidation, hydrolysis, conjugation and other metabolic
reactions. ML314 showed a >15-fold window for toxicity (LC50 = 30 μM) towards human hepatocytes.
Improving the metabolic stability and toxicity profile of ML314 represents a challenge as well as an avenue for
further optimization studies in the future.

Table 2: Summary of in vitro ADME/T properties of NTR1 agonists ML301 (& analogs) and ML314.
ML314
MLS-0446079
MLS-0437103 ML301 (Probe 2)
ADME/T Assay Panel Component (ML301-napthyl
(prior art pyrazole) (Probe 1) β-arrestin
analog)
biased
>52 / >52 / >125 / 9.0 /
Aqueous Solubility in pION’s buffer (μg/mL) [μM]a 52.9 / >155 / >155 102.6 / >145 / >145 >52 0.52
pH 5.0/6.2/7.4 [113 / >296 / >296] [247 / >297 / >297] [>99 / >99 / [>297 / 21.4 /
>99] 1.2]
Aqueous Solubility in 1x PBS, pH 7.4 (μg/mL) [μM]a ND ND ND 0.45 [1.1]
PAMPA Permeability, Pe (x10-6 cm/s) 1163 / 2145 /
1267 / 725 / 70 953 / 145 / 12 363 / 17 / 6
Donor pH: 5.0 / 6.2 / 7.4 Acceptor pH: 7.4 2093
BBB-PAMPA Permeability, Pe (x10-6 cm/s)
4.8 1.1 1.2 399
Donor pH: 7.4 Acceptor pH: 7.4

Plasma Protein Binding Human 1 μM / 10 μM 99.60 / 99.71 97.93 / 97.96 98.69 / 98.70 99.45 / 99.22
(% Bound) Mouse 1 μM / 10 μM 96.63 / 97.10 96.43 / 95.41 92.15 / 91.36 99.67 / 98.84
Plasma Stability (%Remaining at 3 hrs) Human/
88.94 / 70.74 76.00 / 75.97 100 / 100 100 / 99.55
Mouse
Hepatic Microsome Stability (% Remaining at 1hr)
75.50 / 45.67 100 / 75.70 100 / 59.48 1.36 / 0.16
Human/Mouse
Toxicity Towards Fa2N-4 Immortalized Human
>50 >50 >50 29.6
Hepatocytes LC50 (µM)
a Solubility also expressed in molar units (μM) as indicated in italicized [bracketed values], in addition to more traditional μg/mL units.
ND = Not Determined

In Vivo Analysis - High Compound Requirements and Low

Capacity
Rapid Assessment of Compound Exposure (R.A.C.E.)
This experiment is a rapid and efficient compressed in vivo PK screening method to determine the
pharmacokinetic attributes of novel chemical probes (3). A small cohort of animals (4 mice or 2 rats/
experiment), is administered compound orally (p.o.) or by intraperitoneal injection (i.p.) at a single dose (5 - 50
mg/kg). Blood samples are collected at 20 and 120 minutes. The plasma samples are analyzed with a mini-
standard curve (Figure 1). This experiment provides a snapshot of compound exposure sufficient to estimate
In Vitro / In Vivo Assessment of ADME and PK Properties During Lead Selection / Optimization 11

total compound exposure as the area under the curve (AUC(20-120 min)), providing a rank order of estimated
AUC values to prioritize compounds for further investigation.
Another utilization for R.A.C.E. is to evaluate varying formulation excipients for improving solubility and
absorption. Generally, formulation excipients approved by the FDA are available for this assay. Please refer to
reference (4) for FDA approved excipients and amounts.
Assay Design:
• Test articles are formulated for p.o. or i.p. dosing
• One dose of test article (5 – 50 mg/kg)
• Two time points (t = 20 min and 120 min)
Analysis: LC/MS/MS detection of the test compound(s) in plasma samples
Report: Estimated AUC(20-120 min) and a rank order of compound exposure
Quantity of test article required: 5.0 - 50.0 mg

Comprehensive Pharmacokinetic Analysis

Compounds that show promising PK profiles or that are further developed, can be subjected to a comprehensive
pharmacokinetic analysis and metabolite identification study. An example set of graphs representing typical time
course drug plasma concentrations following oral dosing can be found in Wikipedia http://en.wikipedia.org/
wiki/Pharmacokinetics and are reproduced to illustrate the key related parameters: time to reach (tmax)
maximal concentration (Cmax) of compound, time to reduce concentration by half of the initial value (t1/2),
dosing interval (τ), and area-under-curve (AUC) or total compound exposure (Figure 2).
A minimum of six 300 gram rats are required, per compound tested, for appropriate sampling. For each drug,
the compound will be formulated in suitable vehicle to typically 1.0 mg/mL (final concentration) and
administered to 3 rats at 1 mg/kg i.v. into a femoral vein catheter. The same substance is administered to an
additional 3 rats at 2 mg/kg by oral gavage (direct dosing of compound into the stomach through a feeding tube).
Blood is drawn (0.25 mL) via the femoral artery catheter at 5, 15, and 30 minutes and at 1, 2, 4, 6, 8 and 24 hours
post dose for a total maximum volume of 2.25 mL. Plasma samples are analyzed by LC/MS/MS. A
comprehensive PK analysis requires 6-12 animals and blood collections at 8 time points.
Assay Design:
• Single test article is formulated for both p.o. and i.v. dosing
• I.v and p.o. doses of test article (e.g., 1.0 mg/kg and 2.0 mg/kg)
• Nine time points (5, 15, and 30 min and at 1, 2, 4, 6, 8 and 24 hours).
Analysis: LC/MS/MS detection of the test compound(s) in individual plasma samples
Report: Time course of plasma drug concentration versus time, PK parameters (for example, AUC, t1/2, oral
bioavailability) and metabolite identification
Quantity of test article required: 10 - 100 mg

Suggested Equipment and Resources

Automated workstation: A flexible versatile high-throughput/high-capacity workstation system for automated
liquid handling is a useful work horse of the pharmacology lab. The system should be capable of processing
compound solutions for analysis and executing the various in vitro profiling assays. A variable spanning 8
channel pipette arm, a 96-well multichannel pipetting head, and plate gripper arm are useful for reformatting
12 Assay Guidance Manual

Figure 1: Example of data from a previously performed RACE study. The purpose of this study was to select which compound from a
series exhibited the highest estimated exposure. (A) Representative exposure data from two compounds in a series obtained by
sequential RACE studies (n=3 mice/time point; vehicle mice showed no compound in the plasma, data not shown). (B) Results from
the GraphPad Prism5 AUC analysis. **eAUC (estimated exposure (AUC20-120 min) using a “trapezoidal” estimation of AUC
(Copyright © 2012 John Wiley & Sons, Inc., republished with permission from (3)).

Figure 2: Time course of drug plasma concentrations over 96 hours following oral administrations every 24 hours (τ). Absorption half-
life is 1 hour and elimination half-life is 12 hours. Note that in steady state and in linear pharmacokinetics AUCτ=AUC∞. Steady state
is reached after about 5 × 12 = 60 hours (courtesy of Helmut Schütz).
In Vitro / In Vivo Assessment of ADME and PK Properties During Lead Selection / Optimization 13

samples between vials, tubes, and 96-well plates. Specialized devices include vacuum filtration and magnetic
separation modules, as well as integrated devices for heating, cooling, and shaking samples.
Multimodal Plate Reader: A multi-mode plate reader capable of UV/Vis, top/bottom fluorescence, and flash/
glow luminescence read modes accessible to the plate gripper arm of a workstation is useful for unattended
reading for a batch of samples.
Liquid Chromatography/Mass Spectrometric/Mass Spectrometry (LC/MS/MS): This technology is the
workhorse of all ADME/T and PK analyses of samples from both the in vitro and in vivo assay. After extraction
of biomatrix from small molecules, the small molecule sample and any related compounds (metabolized,
hydrolyzed, or broken down compounds) are first separated through high-performance liquid chromatography
(HPLC). The effluent from the HLPC are monitored in real-time for ultraviolet absorbance at 254 and 280 nm,
and absorbing samples are diverted into an on-line electrospray unit to introduce samples into the mass
spectrometer, followed by sequential quadrapole selection and detection of mass/charge (m/z) distributions of
samples that are characteristic of parent and derived molecules. For example, we use a Shimadzu UPLC coupled
with an automated AB Sciex API 4000 MS/MS system with QTRAP detection. This is a high performance hybrid
triple quadrupole/linear ion trap system with excellent dynamic range and sensitivity. However, this is a very
mature sector of the MS market and several vendors with comparable instruments exist.
Microplate Scintillation and Luminescence Counter: For studies with radiolabeled compounds, scintillation
devices are still required. Currently a few manufacturers still make multi-wall, microtiter plate based scintillation
counters (e.g. PerkinElmer TopCount NXT™), these devices can also plate luminescence readers.

References
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