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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5849_Библиотеки_им_академика_М_И_Перельмана
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8.2.3 Bile Flow, Drug-Induced Cholestasis, and Inhibition
of Biliary Efflux Transporters
The flow of bile from the liver into the small intestine serves several important
physiologic roles. Bile contains high levels of bile acids which serve to aid in the
digestion of fat and nutrients from dietary intake. In the liver, these bile acids help in
the solubilization and subsequent elimination of cholesterol across the canalicular
membrane and prevent the formation of gallstones in the biliary ducts and gall
bladder. Bile flow also represe nts the predominant pathway for elimination of
bilirubin and bilirubin glucuronide, the breakdown product of hemoglobin. The
proper maintenance of biliary flow relies upon mechanisms of uptake of biliary
constituents from the blood across the sinusoidal membrane, fluid flow through the
hepatocyte, and subsequent active and passive processes of excretion across the
canalicular membrane into the bile duct. Conjugated and unconjugated bile acids are
actively transported into the bile predominantly via the bile salt export pump or BSEP,
localized at the canalicular membrane of the hepatocyte. In addition to BSEP, other
transport mechanisms are important for the proper excretion of components into the
bile. These include ABCG5/ABCG8, the heterodimer protein responsible for the
excretion of cholesterol directly from the liver, MDR3, the flippase responsible for the
movement of phosphatidyl choline from the inner to the outer leaflet of the canalicular
membrane, and ATP8B1, the flippase responsible for the translocation of phosphatidyl serine from the outer to the inner leaflet of the bilayer [37–39]. Genetic disorders
that result in the lack of function of BSEP, ATP8B1, or MDR3 all result in the clinical
manifestation of cholestasis in some form (reviewed in Refs. 40 and 41).
In addition to its intrinsic physiologic functions, bile flow and biliary excretion can
play a key role in the elimination of xenobiotics. Many drugs and metabolites of drugs
have been shown to be eliminated into the bile via active transport mechanisms. For
example, the muscle relaxant vecuronium [42] and the cardiac glycoside digoxin are
extensively excreted into the bile by P-glycoprotein (MDR1) [43]. The canalicular
transporter MRP2 plays a role in the excretion of several HMG-CoA reductase
inhibitors including pravastatin [44] and the breast cancer resistance protein (BCRP)
has been shown to be important for the biliary excretion of several fluoroquinolones [45]. Although MDR1, MRP2, and BCRP are thought to be responsi ble for the
biliary excretion of most drugs and their metabolites, some recent results have
implicated BSEP in the direct excretion of pravastatin into the bile [46]. Several
studies also highlight the overlapping role of these transporters to facilitate the
excretion of xenobiotics and their metabolites [47–49].
Potential interactions of therapeutic agents with the endogenous mechanisms of
bile flow and biliary excretion include decreased drug clearance as a result of reduced
biliary function due to underlying cholestatic disease as well as direct interaction of
a drug with hepatobiliary function, resulting in bland cholestasis. Several reports have
described the impact of hepatic disease on the disposition of drugs and mechanisms of
hepatobiliary excretion [50, 51], and should be considered in the drug development
process to understand the potential that these patient populations will be targeted in
routine clinical treatment. The ability of drugs to inhibit the normal excretion of bile
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from the liver can lead to the accumulation of bile acids within the cell [52, 53].
Although compensatory mechanisms (e.g., multidrug resistance-associated protein
3, MRP3) at the sinusoidal membrane of the liver may help transport bile acids from
the hepatocyte back into the blood, some drugs and their metabolites have been
shown to inhibit multiple mechanisms of transport within the liver, at sinusoidal and
canalicular membranes [54, 55]. Due to their detergent like properties, cellular
accumulation of bile acids and their conjugates can lead to hepatic necrosis [56]. As
mentioned above, genetic mutations in BSEP result in intrahepatic cholestasis.
Analogous to genetic disruption, inhibition of BSEP by drugs and their metabolites
has been shown to result in cholestasis in vivo. BSEP inhibition by compounds such
as cyclosporine, nefazodone, bosentan, glibenclamide, and troglitazone (described
in Section 8.4) has been suggested to contribute to the underlying mechanism of
hepatotoxicity observed clinically [56–60].
8.3 ASSAYS AND TEST SYSTEMS TO MEASURE VARIOUS
TYPES OF DILI
Assays and test systems to measure various types of DILI are discussed with attention
to in vitro assay systems with sufficient predictivity and throughput for lead
optimization programs of the drug discovery process.
Since the normal human liver has an enormous capacity to adapt, repair and
regenerate itself, any in vitro system that lacks the ability to adapt to injury can only be
viewed as restricted to investigating the initial chemical insult(s) to the liver. Whole
cell-based models typically have more capability to cellular defense and repair
than cell-free systems, and are increasing utilized to study DILI mechanisms and
processes. Human liver cell-based systems, due to the presence of human-specific
liver transporters and drug-metabolizing enzymes, are becoming more mainstream as
well as cells isolated from animal species (e.g., rat, dogs, monkeys) [61, 62].
Human liver cell lines (exemplified by the HepG2 hepatocarcinoma cell line),
despite their limitations in drug-metabolizing capacity (low) and cell cycle status
(proliferating cells), are still frequently used to study the general cytotoxic potential
of the parent drug molecule. Cellular necrosis can be measured by the amount of
intracellular ATP levels left after a period of drug treatment, as only healthy cells can
produce ATP. Since the mitochondrion is the major source of cellular ATP,this simple
assay can be an initial surrogate assay (although not a definitive test) for drug-induced
mitochondrial injury. Since the parental HepG2 cell line has low levels of CYP450
enzymatic activity, engineered HepG2 cell lines have been used to express various
CYP450 enzymes (although not engineered in the same cell line). In addition to the
HepG2 line, other cell lines include the HepaRG (a clonal selection of the HepG2 cell
line) [63, 64], the THLE (an SV40 tumor antigen immortalized human liver cells)
[65], and their CYP450-expressing daughter cell lines [65]. These CYP450-expressing cell lines may be used to investigate CYP450 metabolite-specific toxicities to
these cells. Most of these cell lines can only be used to study drug- or metaboliteinduced necrosis or apoptosis, but not cholestasis.
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Primary cells, in particular, primary hepatocytes cultured in defined media, can
sustain differentiated liver morphology and function for extended culture time
(typically at 1 week or more). These differentiated liver-specific traits include:
nondividing and polynuclear phenotypes, expression of phase-I and -II drug-metabolizing enzymes that are inducible, and expression of drug uptake and efflux
transporters that are responsive to perturbations. Primary human hepatocytes cultured
in the sandwiched configuration (either Collagen/Collagen, or Collagen/Matrigel
sandwich, with hepatocytes between two layers of matrix), reesta blish cellular
polarity over time in culture where functional sinusoidal and canalicular transporters
are localized to the proper membrane surfaces of the hepatocytes. These transporters
are capable of facilitating vectorial transport of compounds across the hepatocyte and
compound accumulation within the cellular compartment and the bile canaliculi
compartment can be quantified. Therefore, the primary hepatocyte sandwiched model
is an optimal in vitro model to study both hepatobiliary drug transport [49, 66] and
drug-induced cholestasis [67, 68].
Other in vitro methodologies have been developed to complement the whole cell
sandwich-cultured hepatocyte model. BSEP-transfected Sf9 membrane vesicles are
a useful tool to understand the intrinsic potential for a compound to directly inhibit
BSEP-mediated bile acid transport across the canalicular membrane [69, 70]. This
system does however have the potential to incorrectly predict the true impact of BSEP
inhibition in vivo since in the intact liver, compounds must first cross the sinusoidal
membrane of the hepatocyte to gain access to the proposed site of inhibition.
Compounds such as estradiol-17b-
D-glucuronide do not appear to directly inhibit
BSEP, rather, may cause endocytic retrieval of the transporter in intact cells,
suggesting that the entire complement of cellular machinery is required to elicit the
true impact of bile acid transport inhibition [71]. Some data support that compound
excretion into the canali cular space is required to inhibit BSEP, so-called transinhibition that has been observed by metabolites of progesterone as well as estradiol17 b-
D-glucuronide [72, 73]. Membrane vesicles derived from the canalicular
membranes of intact hepatocytes have also been utilized to demonstrate the potential
for xenobiotics to inhibit mechanisms of bile acid transport [58]. Although these
systems integrate the function of other transport proteins present at the apical
membrane of the hepatocyte, technical challenges regarding purity and the absence
of a sinusoidal membrane may limit the ultimate translation of these data to the in vivo
setting.
While the simple cellular ATP measurement provides a rapid screen without much
mechanistic insight, other more informative assays can measure more subtle insult
to cellular health, and provide more insights toward mechanistic understandings of
DILI. Cellular imaging aimed at measuring prelethal events (as opposed to simple cell
loss due to cell detachment from the cell culture surface), can provide both a direct
correlation to histopathology and information on intracellular organelles affected
by such drug insults. In cellular imaging, cells are typically treated with drug or
metabolite with or without imaging dyes. When drug is treated without dyes, the
imaging reagents can be added post drug treatment to highlight organelle morphology. These imaging reagents can highlight specific cellular phenotypes relevant to
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toxicology such as apoptosis (programmed cell death), steatosis (neutral lipid
accumulation), phospholipidosis (phospholipid accumulation), oxidative stress
(production of reactive oxygen species), mitochondria stress (mitochondria membrane potential), and cholestasis (decreased bile acid flux) [74]. These cellular
morphologies are either inspected manually under an appropriate microscope, or
more commonly, rapidly captured and digitized by an automated microscope
equipped with a semiautomated image analysis algorithm. The image analysis
algorithms can continuously capture and measure any objects of interest, and can
be programmed to alert the researcher on any “abnormal” or “findings of interest” at
the cellular-, field-, well-, treatment-, or compound-level. This whole field of efficient
cellular imaging analysis, termed high-content screening (HCS, as opposed to
HTS for high-throughput screening), have practically exploded in the past decade
to a highly sophisticated and organized scale to discover both novel pharmacology,
toxicology, systems biology, and systems medicine [74–79]. In the spirit of “see ing is
believing,” high-content screens provide or enable:
(a) visual proof for a given biological conclusion,
(b) multiple cellular phenotypes that can be measured at the same time from
within the same cell, thus lending the technology endless possibilities to
investigate cellular dynamics and processes in pharmacology and toxicology,
(c) drug precipitate, fluorescent or colored compound, that can be easily deci-
phered, minimizing both false positives and false negatives in traditional HTS,
(d) accurate cell count from a treated sample, thus any “findings of interest” can be
normalized by cell count, minimizing false conclusions derived due to the loss
of cell attachment, and
(e) probably the most significant, in vitro correlation to in vivo histopathology.
Detailed primary hepatocyte cell culture, staining, imaging, and image analysis
protocols have recently been published [80–82]. The hepatocyte imaging assay
technology (HIAT), identified over half of the hepatotoxic drugs tested including
many idiosyncratic hepatotoxicants, while maintaining a low false-positive rate of
0–5%, in a validation study using over 300 drugs [80]. The HIAT utilized a
combination of epifluorescent molecules to simultaneously probe the intensity and
location of several intracellular organelles and species. Nuclei count , shape, and
intensity were stained by the DNA-specific anthraquinone dyes Draq5 [83]
(Figure 8.1a). Intrahepatocyte levels and distributions of reduced glutathione were
imaged by monochlorobimane [84] (Figure 8.1b); monochlorobimane is a nonfluorescent hydrophobic probe that readily permeates into cells and is conjugated with
glutathione to form the fluorescent glutathione bimane, an organic anion [85]. This
fluorescent glutathione conjugate displayed a strong cytosolic fluorescence in healthy
hepatocytes. It is also a substrate for the canalicular multispecific organic anion
transporter MRP2, which mediates the ATP-dependent secretion of a wide range of
organic anions over the canalicular membrane into bile. The primary human
hepatocytes were cultured in a confluent sandwiched culture that facilitates the
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formation of bile canaliculi (BC) and the function of a family of multispecific
transporters including MRP2, MDR1, and BSEP in between two adjacent hepatocytes
(Figure 8.1b) [86]. The maintenance of normal hepatobiliary efflux function of such
transporter can also be measured by the signal intensity of the bimane fluorescence in
the BC region (Figure 8.1b). In addition, HIATutilizes a fluoresceine-based probe for
reactive oxygen species, and a potential-sensitive dye to measure mitochondrial
membrane potential [87]. Healthy hepat ocytes exhibit normal nuclear morphology
(Figure 8.1a), strong intracellular glutathione fluorescence with even stronger signals
in the BC regions (Figure 8.1b), very little reactive oxygen species (Figure 8.1c), and
strong mitochondrial membrane potential (Figure 8.1d). Deviations from such normal
morphology upon drug treatment can signal one or more potential mechanisms of
drug-induced liver injury [80]. Some examples of such morphological deviations
are shown in Figure 8.2a–d. Drug-induced inhibition of BSEP can be quantitatively
measured by the efflux of a BSEP-selective fluorescent probe, cholyl–lysyl fluoresceine (CLF), into the bile canaliculi space of similarly sandwi ched cultured human
hepatocytes [81].
For any test system to be useful for lead optimization, it is important that the system
is robust enough for assay optimization. The field of HTS has benefited from both
laboratory automation and development of sensitive imaging instruments [88]. Image
analysis algorithms have been sufficiently automated to allow meaningful extraction
of data from a large number of cellular features over many cellular samples [79, 88,
89]. The intelligent combination of these technologies enabled automated statistical
mining of cellular images for novel cellular phenotype combinations relevant for both
drug efficacy [79, 90] and drug toxicity [80, 89]. A new era of phenotype-based drug
screening has been born, but now it is fully dependent on novel combinations of
Figure 8.1 Representative epifluorescence images of healthy hepatocytes from the HIAT.
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molecular probes and systems biology, as opposed to the heritage of phenotypic
descriptions.
It is important to maintain a low false-positive rate for any toxicological assay
system to be useful for lead optimization. Since a false-positive finding or false alarm
on an otherwise quite safe drug in the lead optimization stage, could potentially lead to
disinterest or even abandonment of viable drug candidate, it is imperative that any
assay systems be subject to thorough evaluation and validation using a sufficient
number of “safe” drugs. It is quite common that there exists an imbalance in the
number of toxic versus safe drugs in many scholarly publications on potential
assay and test systems for DILI [91–94]. Since the number of toxic drugs outnumbers
the safe ones by a very wide margin in these studies, a potential side effect of
optimizing the assay sensitivity as much as possible is decreased specificity (i.e.,
increasing false-positive rate without truly knowingly). In a few recent studies with
more balanced number of safe (i.e., negative) drugs, the findings are quite illuminating [25, 26, 80, 95, 96].
There is no shortage of clinically beneficial drugs that can cause a transient
increase in serum ALT activity, no increase in total serum bilirubin (TBL) concentration, and do not cause serious hepatotoxicity in the clinic. Well-known examples
of such drugs include the Alzheimer’s medication tacrine, HMG-CoA reductase
inhibitors or statins such as simvastatin, widely used pain killers such as aspirin,
effective antidepressants such as fluoxetine, paroxetine, and buspirone, the antihypertensive drug propranolol, and the selective estrogen receptor modulator (SERM)
raloxifene [97–99]. These drugs should be classified as DILI negatives, and should be
included in the drug validation list of any test systems to better assess their falsepositive rate, before such test systems can be used for routine testing or prospective
prediction of DILI. All of these drugs were classified as DILI negatives by the
Figure 8.2 Representative epifluorescence images of abnormal hepatocytes from the HIAT.
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HIAT testing paradigm [80]. Hence the final conclusions from HIATare distinct from
what have been published about these compounds in the past [100–108].
The reason for the high specificity of the HIATmay be a combination of: (1) the drug
concentrations used have reasonabl e relevance to the in vivo situation (e.g., using
a common scaling factor of 100 to account for higher liver drug exposure to the liver
for an orally dosed drug and other patient-related idiosyncratic variables), (2) primary
hepatocytes in culture are nondividing cells and compared to hepatoma cell lines are
less sensitive to agents that may perturb the cell cycle [109]; and (3) primary hepatocyte
culture in the sandwiched configuration maintains more normal and balanced drug
metabolizing and transporter functions [86, 110–112]. HIAT was evaluated against a
large number of drugs (>300 in total) with over a hundred DILI negative drugs,
including those that cause a transient increase in ALT but not TBL (as discussed above).
As a result the low false-positive rate of the HIAT system is more likely than other
testing systems to be translated to a “real world” scenario. The high positive predictive
value toward the ultimate in vivo outcome (in this case serious DILI) is a striking
contrast to the current in vitro tests for drug-induced genotoxicity [96] or developmental toxicity [95], and should be acontinued emphasis in developing,evaluating,and
implementing in vitro test systems for the prediction of other drug-induced toxicity.
8.4 MEDICINAL CHEMISTRY STRATEGIES TO MINIMIZE DILI
Hepatotoxic drugs can sometimes cause DILI by a multitude of mechanisms. Because
of their role in insulin resistance, the family of PPARs—specifically PPARg, have
been leveraged as therapeutic targets for type-II diabetes mellitus. Several thiazolidinediones have been developed that are effective in controlling insulin levels in
diabetic patients. First reported in 1982, ciglitazone was the first molecule in this class
to show promise in preclinical models of diabetes [113] and subsequent optimization
of this series of molecules resulted in compounds such as troglitazone (Rezulin
),
pioglitazone (Actos
), and rosiglitazone (Avandia) (Figure 8.3).
Figure 8.3 Chemical structures of thiazolidinediones developed for the treatment of type-II
diabetes mellitus. Troglitazone and ciglitazone exhibited more hepatotoxicity than rosiglitazone or pioglitazone in the clinic.
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Troglitazone was removed from the United States market in 2000 due to several
reports of hepatotoxicity, whereas the latter two are still prescribed to patients in the
United States. Following these reports of adverse events with troglitazone, several
mechanistic studies probed the underlying reasons behind the toxicity observed.
Several hypotheses have since been proposed to explain the incidence of clinical
hepatotoxicity. Troglitazone, but not the other three glitazones, was shown to produce
a reactive metabolite in the presence of human liver microsomes [114]. In addition,
troglitazone and ciglitazone, but not rosiglitazone or pioglitazone, can induce
mitochondrial permeability transition (MPT) and cause mitochondrial dysfunction [115]. Recently, in a murine model of silent mitochondrial stress, troglitazone
treatment induces first an initial adaptive response, followed by a toxic response
involving oxidant injury to mitochondrial proteins [116]. Troglitazone and its
metabolite troglitazone sulfate were shown to be potent inhibitors of the bile salt
export pump in vitro in canalicular liver plasma membrane vesicles and in vivo in
rats [58]. Snow et al. demonstrated the potential for pioglitazone and rosiglitazone to
also inhibit BSEP-mediated transport of bile salts in a dose-depe ndent manner,
suggesting that the core thiazolidinedione moiety may represent a class effect on bile
acid homeostasis in vivo [117]. However, the earlier studies clearly indicated that the
sulfate metabolite of troglitazone was 10-fold more potent inhibitor of transport
than troglitazone itself, with an apparent IC
50
value of 0.4 mM for inhibition of BSEP-
mediated taurocholate transport in membrane vesicles. In vivo, troglitazone is
predominantly metabolized to this sulfate conjugate, whereas 62% of the dose of
rosiglitazone is excreted unchanged into the urine [118].
Target-based SAR of the thiazolidinediones has been well described (see Ref. 119
for extensive reviews). Structurally, all three of the PPARg activators contain the core
thiazolidinedione moiety with a single carbon linking a phenyl group. All three
structures also contain an ether functionality linking a lipophilic tail. This lipophilic
tail differentiates troglitazone and ciglitazone from the others, and by deduction could
therefore be at least in part, responsible for the observed toxicity. Troglitazone has
differentiated itself in a variety of in vitro experimental systems from pioglitazone
and rosiglitazone, as summarized above. Recent data from Huh-7 cells showed
that troglitazone, not pioglitazone or rosiglitazone, was capable of modulating the
farnesoid X receptor, a key transcription factor responsible for the regulation of target
genes responsible for bile acid homeostasis (e.g., BSEP) [21]. Although not directly
related to mechanisms of bile acid transport, troglitazone has been shown to be a more
potent inducer of CYP3A4 and CYP2B6, and is a more potent inhibitor of CYP3A4-,
CYP2C8-, and CYP2C9-mediated metabolism than pioglitazone or rosiglitazone [120]. Given that clinical doses of troglitazone result in plasma C
max
values
two- to fourfold higher than pioglitazone or rosiglitazone, the increased potency to
modulate several P450 mechanisms coupled with these higher systemic concentrations could further contribute to the hepatotoxicity observed. Overall, the absolute
mechanisms responsible for the toxicity of the thiazolidinediones still remain
somewhat debated [121]. It is clear that emerging data do support the differentiation
of troglitazone from other analogs that have shown limited signals of hepatic
dysfunction clinically.
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The case of four glitazones highlights the need for medicinal chemists to improve
the potency of the molecule towardits intended target (i.e., from ci- and tro- to rosi-, and
pioglitazone), while atthe sam e time maintain thesimplicity ofthe molecu le’s structure
(e.g., the more lipophilic tail in troglitazone may be a reason why it is associated with
the more promiscuity toward a varietyof “off-targets”by the troglitazone molecule). In
general, medicinal chemists will be well-advised to consider these strategies to
minimize a molecule’s propensity to induce hepatotoxicity:
(a) Increasing drug potency without introducing excessive molecular size, lipo-
philicity, and ionization state:
Despite the now well-known publication of the concept of drug-likeness,
such as the “rule of five” [122], it has been recognized that both the calculated
1-octanol–water partition coefficient (i.e., clog P, which is a measure of
a drug’s lipophilicity) and the median molecular weight (i.e., MW, which is
a general measure of a drug’s molecular size) have been increasing since
around 1985 [123]. Lipophilicity of a drug molecule reflects its likelihood
to transfer from aqueous phase to cell membranes and to protein binding
sites, which are mostly hydrophobic in nature. Some degree of lipophilicity
is obviously needed for a drug molecule to engage its intended target. If
lipophilicity is too high, there is an increased likelihood of binding to
multiple targets and resultant pharmacological promiscuity, or toxicity, as
well as poor solubility and metabolic clearance. Pharmacological promiscuity is predominantly controlled by lipophilicity and ionization state, with
bases being more promiscuous than acids, neutral compounds, or zwitterions [123].
It is proposed that the essence of lead optimization is to increase potency
without increasing lipophilicity or excessive molecular mass at the same
time [123]. In this regard, two usef ul metrics to guide the progress of lead
optimization are “ligand efficiency” (LE, Equation 8.1) and “ligand-lipophilicity efficiency” (LLE, Equation 8.2):
LE ¼ pIC
50
ðor pKiÞnumber of heavy atoms ð8:1Þ
LLE ¼ pIC
50
ðor pKiÞc log Pðor log DÞð8:2Þ
The average oral drug with a clog P 2.5 and potency in the range
1–10 nM suggests an LLE target of 5–7 or greater [123]. Increased LE
and LLE during the lead optimization stage are expected to increase the
overall therapeutic index of the lead series.
(b) Leverage noncompetitive binding, especially, for targets with a high concen-
tration of endogenous ligands, to lower drug exposure required for in vivo
efficacy.
Although the importance of good drug-like physicochemical properties
cannot be underestimated, these properties alone may not always be respon-
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sible for the lack of success in a target class. It is recognized that biochemical
mechanisms of drug action is as important as, if not more so, for the translation
into a better therapeutic index [124]. If IC
50
is the concentration required to
inhibit the functional response by one half, and K
i
is the binding affinity of the
drug for the target, the biochemical efficiency or BE can be defined by
Equation 8.3. Equation 8.4 is the equivalent of Equation 8.3, for the case of
agonism mechanism as opposed to antagonism. The in vivo therapeutic
effective dose, ED
50
, or the dose required to inhibit the functional response
by one half, is related to IC
50
or EC50, and specific pharmacokinetic (PK)
factors, as in Equations 8.5 and 8.6 [124].
IC
50
¼ Ki=BE ðð8:3Þ for antagonistsÞ
EC
50
¼ Kd=BE ðð8:4Þ for agonistsÞ
ED
50
¼ PKðIC50Þðð8:5Þ for antagonistsÞ
ED
50
¼ PKðEC50Þðð8:6Þ for agonistsÞ
In essence, BE is dependent upon the biochemical mechanism of action
(e.g., competitive, noncompetitive, irreversible, quasiirreversible, etc.). BE is
inversely related to the potency, consistent with concept that the less efficient
a drug’s mechanism of action, the greater the concentrations of drug (thus IC
50
or EC50) required to achieve the required potency. High drug concentrations
may compromise the safety of the drug and decrease the therapeutic window.
Therefore, another metric of successful lead optimization is to increase a drug
candidate’s BE. One way to achieve this goal is to leverage noncompetitive or
nonequilibrium mode of biochemical modulation [124]. This is especially
important when the concentration of the endogenous ligand of the intended
target is high or highly fluctuating in an open biological system (e.g.,
concentration of ATP for kinase inhibitors, concentration of other endogenous
substrates, and cofactors for other enzymes).
(c) Take therapeutic C
max
into consideration when predicting DILI.
Since the liver is potentially exposed to a higher effective C
max
for an orally
administered drug, pharmaceutical scientists should not be misled by the
apparent low IC
50
, Ki,orKdin a cell-free and plasma protein-free test system
in vitro. As demonstrated by recently published studies of over 300 drugs, the
toxicological response of the liver is largely driven by in vivo liver C
max
exposure, which is higher than measured systemic C
max
values for an orally
administered drug [80]. In the preclinical and drug discovery phase, the
“systemic C
max
needed” to ensure continuous efficacy of a drug candidate
in the simplest case can be estimated from the minimal efficacious in vivo
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