问题：【Humana Press】【Optimization in Drug Discovery-In Vitro Methods MPT】
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Recent analyses of drug attrition rates reveal that a significant number of
drug candidates fail in the later stage of clinical development owing to
absorption, distribution, metabolism, elimination (ADME), and toxicity
issues. Lead optimization in drug discovery, a process attempting to uncover
and correct these defects of drug candidates, is highly beneficial in lowering
the cost and time to develop therapeutic drugs by reducing drug candidate
failures in development.
At present, parallel synthesis combining with high-throughput screening
has made it easier to generate highly potent compounds (i.e., hits). However,
to be a potential drug, a hit must have drug-like characteristics in addition to
potency, which include optimal physicochemical properties, reasonable pharmacokinetic
parameters, and good safety profiles. Therefore, research tools
must be available in drug discovery to rapidly screen for compounds with
favorable drug-like properties, and thus adequate resources can be directed
to projects with high potential. Optimization in Drug Discovery: In Vitro
Methods is a compilation of detailed experimental protocols necessary for
setting up a variety of assays important in compound evaluation. A total of
25 chapters, contributed by many experts in their research areas, cover a
wide spectrum of subjects including physicochemical properties, absorption,
plasma binding, metabolism, drug interactions, and toxicity.
A good pharmacokinetic profile has long been recognized as an important
drug-like characteristic. Pharmacokinetic parameters are affected by
many properties of drug molecules such as physicochemical nature, absorption,
metabolic stability, and so on. Physicochemical properties influence
the transfer of a drug across cell membranes, and thus affect absorption and
distribution of the drug. Chapter 1 provides experimental methods measuring
pKa solubility and lipophilicity, the most fundamental physicochemical
properties of a drug candidate. These parameters are vital for preparing an
optimal drug formulation. A good absorption profile is another important
requirement for drug effectiveness. Drug absorption is primarily governed
by a passive transport mechanism, and many drug transporters play a very
important role in absorption and disposition. Several chapters address different
issues on this aspect. The Caco-2 model described in Chapter 2 is
most commonly used to investigate drug absorption mechanism. Parallel
artificial membrane permeability (PAMPA) is a rapid screening approach
for the earliest ADME primary screening of research compounds. Chapter 3
covers a wide background about PAMPA and also experimental procedures
to perform the assay. The perfused rat intestinal model outlined in Chapter 4
has long been considered the definitive or “gold standard” for evaluation of
drug absorption. Because CNS drugs need to penetrate the blood–brain barrier,
the brain microvessel endothelial cell model has recently been proposed
(Chapter 5) to screen compounds targeting CNS diseases. Because of important
roles played by the transporter P-glycoprotein (MDR1) in absorption and
resistance in anticancer chemotherapy, an enzymatic activity-based method is
described in Chapter 6 for rapidly screening MDR1 substrates; the following
chapter outlines a different approach to investigate the involvement of drug
transporters using oocytes injected with cRNAs (Chapter 7).
Plasma protein binding may also have significant effects on a wide variety
of phenomena such as pharmacokinetics, drug–drug interaction potential,
or interindividual variability. Chapters 8 and 9 present several different
methods evaluating plasma protein binding, which include equilibrium dialysis,
ultrafiltration and isothermal titration calorimetry. It is obvious that each
approach has unique advantages.
Optimal metabolic stability is essential for a drug to have lasting pharmacological
effects on the action site. With respect to optimizing pharmacokinetic
parameters such as bioavailability and clearance, the metabolic
stability of drug candidates can be determined from in vitro incubations with
either hepatocytes or microsomes as described in Chapter 10. As stated, each
metabolism system has clear advantages and bears different objectives.
Characterization of major metabolites in drug discovery has unique objectives:
(1) identifying potent metabolites with better “drug-like” properties;
(2) understanding the metabolism fate of drug candidates; and (3) using
metabolism information to guide new synthesis and generate more stable
compounds. Chapter 11 outlines methods for identifying oxidative metabolites
using microsomes or S9 fractions. Glucuronidation catalyzed by UDPglucuronosyltransferases
(UGTs) represents another important drug metabolism
pathway. Chapter 12 describes a general approach identifying UGTs responsible
for metabolizing a given drug candidate.
For safety reasons, drug–drug interaction has been of increasing concern
in drug discovery. Most drug interactions involve alternations in the metabolic
pathways within the cytochrome P450 (CYP) system. Induction of
CYP expressions by a given drug could lead to lower efficacy of the drug
and coadministered agents. CYP induction can be evaluated using human
hepatocytes as described in Chapter 13. Inhibition of CYPs is currently recognized
as the major mechanism for drug–drug interactions observed in
clinic. CYP inhibition can be assessed in different in vitro systems. Chapter
14 describes a high throughput approach screening for 13 individual CYPs
by using fluorescent substrates and cDNA-expressed enzymes, and Chapter
15 presents a traditional method assessing the inhibition of those major
CYPs in human liver microsomes. Each system has its own advantages and
limitations, and the decision to use a particular approach depends on the
goal of the drug evaluation. CYP inhibition can be further classified into
reversible and irreversible, and understanding the inhibition mechanism is
critical for compound selection in drug discovery. A systemic approach is
given in Chapter 16 to identify mechanism-based CYP inhibitors. Drug
interaction related to the phase II reaction of glucuronidation is not covered
in this book.
Toxicity is a major cause of drug candidate failures in both clinical development
and after market launch. One aspect of toxicity results from the interaction
of a drug or its metabolites with either nucleic acids or specific
proteins important in normal cellular function. The interaction of xenobiotics
with DNAs potentially results in DNA damage or covalent modifications,
leading to genotoxicity. As assessment of genotoxicity remains an important
aspect ingenotoxicity assessment. In Chapter 17, detection of DNA adducts is
described using 32P-postlabeling combining with PAGE or HPLC radioactive
analysis; analysis of CYP-mediated covalent DNA adducts is presented
in Chapter 18. Two methods detecting DNA damage at the level of individual
eukaryotes induced by xenobiotics are provided, including a traditional
Comet assay (Chapter 19) and a rapid cell-based reporter system
(Chapter 20). Although the Ames test has long been used to detect mutagens
and possible carcinogens, an improved version assay given in Chapter 21
significantly reduces background resulting from contamination in S9 fractions.
Also, a modified mouse lymphoma assay (MLA) is outlined in Chapter 22,
because this assay has been recommended as one of core toxicology tests.
Toxicity caused by interaction of a drug or its metabolites with cellular
proteins is more difficult to detect, simply because both targeting proteins
and interaction mechanism (covalent or noncovalent) are largely unknown
at the drug discovery stage. Because of the complexity of this aspect, this
book only treats several topics of more general interest. As QT prolongation
caused by interaction of drug molecules with HERG channels remains to be
a common concern in drug discovery, a high throughput in vitro assay is
devised in Chapter 23 to screen compounds for interaction with HERG.
Recently, it has been recognized that covalent modification of cellular proteins
by reactive drug metabolites is potentially associated with idiosyn-
Preface vii
cratic toxicity. Reactive metabolites generated by CYPs can be trapped by
the addition of glutathione to in vitro incubations and structurally characterized
using mass spectrometry (Chapter 24). Another well-known class of
reactive metabolites is acyl glucuronides formed by the Phase II conjugation.
Acyl glucuronides are electrophilic intermediates that are unstable and
can interact with amino acid residues to form covalent protein adducts. The
last chapter presents a new in vitro assay assessing the reactivity of acyl
glucuronides (Chapter 25).
Each chapter contains introduction, materials, methods, and notes sections.
The introduction contains important background information. The
materials section lists all the equipment and reagents necessary to carry out
the assay, while step-by-step protocols are outlined in the methods section.
Finally, information dealing with common and unexpected experimental
problems is detailed in the notes section.
We want to express our tremendous gratitude to all contributors who were
so receptive to contributing drug discovery and development, several chapters are devoted
to chapters to this book. Without their time and
energy, this book would not have been possible

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回复人：smileashes,▲ () 时间：2011-07-15 20:55:38 编辑	1楼
good

回复人：baoyouzhao,▲ () 时间：2011-07-15 09:27:08 编辑	2楼

回复人：jianghai,★★★★★ (学到老，活到老！) 时间：2011-07-13 11:50:38 编辑	3楼

回复人：jimuwei,★★★ () 时间：2011-07-13 21:42:41 编辑	4楼

回复人：shennt,▲▲ (从事有机合成工作) 时间：2011-07-14 11:14:33 编辑	5楼
看看，谢谢

回复人：ky3000t,▲▲ () 时间：2011-07-16 16:27:20 编辑	6楼

回复人：枫叶子,▲ (选择了机遇就选择了风险，选择了求索就选择了磨难.) 时间：2011-07-16 17:52:32 编辑	7楼
谢谢分享！

回复人：ypwang,▲ (接受论坛规则和论坛协议) 时间：2011-08-05 06:34:08 编辑	8楼
谢谢分享！

回复人：samilk,▲ (做药物化学的。) 时间：2011-08-06 01:44:30 编辑	9楼

回复人：oyqc888,▲ (青山绿水，蓝天白云) 时间：2011-11-18 12:29:34 编辑	10楼

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