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Current
Drug Metabolism
ISSN: 1389-2002
Current Drug Metabolism
Volume 7, Number 2, February 2006
Contents
Induction of Cytochrome P450 3A4 and P-Glycoprotein by the
Isoxazolyl-Penicillin Antibiotic Flucloxacillin Pp. 119-126
J. Huwyler, M.B. Wright, H. Gutmann and J. Drewe
[Abstract]
Fluoxetine Metabolism and Pharmacological Interactions:
The Role of Cytochrome P450 Pp. 127-133
R. Mandrioli, G.C. Forti and M.A. Raggi
[Abstract]
Prediction of Drug-Drug Interactions for AUCoral
of High Clearance Drug from In Vitro Data: Utilization
of a Microtiter Plate Assay and a Dispersion Model
Pp. 135-146
T. Yamamoto, A. Suzuki, Y. Kohno, K. Nagata and Y. Yamazoe
[Abstract]
Predicting the Pharmacokinetics of Acyl Glucuronides and
Their Parent Compounds in Disease States Pp. 147-163
J.H. Liu and P.C. Smith
[Abstract]
Preclinical Pharmacokinetics: An Approach Towards Safer and
Efficacious Drugs Pp. 165-182
S.S. Singh
[Abstract]
The Role of Blood-Brain Barrier Studies in the
Pharmaceutical Industry Pp. 183-203
A. Reichel
[Abstract]
Recent Advances in Molecular Modeling and Medicinal
Chemistry Aspects of Phospho-Glycoprotein Pp. 205-217
E. Srinivas, J.N. Murthy, A.R.R. Rao and G.N. Sastry
[Abstract]
Abstracts
[Back to top]
Induction of Cytochrome P450 3A4 and P-Glycoprotein by
the Isoxazolyl-Penicillin Antibiotic Flucloxacillin
J. Huwyler, M.B. Wright, H. Gutmann and J. Drewe
Clinical findings indicate that co-administration of the
isoxazolyl-penicillin flucloxacillin with cyclosporine may
reduce the plasma concentrations of cyclosporine. We have
explored in the present study if induction of cytochrome P450
3A4 or P-glycoprotein may offer a mechanistic explanation
of the observed effects. Flucloxacillin is neither an inhibitor
nor a substrate of drug metabolizing cytochrome P450 isoenzymes
(CYP3A4, 1A2, 2C9, 2C19 and 2D6) or P-glycoprotein as shown
by an in vitro assay for CYP inhibition, a fluorescent
indicator assay for P-glycoprotein inhibition and a functional
P-glycoprotein ATPase assay. However, incubation of human
LS 180 colorectal adenocarcinoma cells with flucloxacillin
led to a dose-dependent induction of MDR1 as well as of CYP3A4
mRNA, which was also confirmed in primary human hepatocytes.
At high concentrations, flucloxacillin activated the human
Pregnane-X-Receptor, PXR, a ligand-dependent transcription
factor that is the target of many drugs that induce CYP3A4,
with consequences for the metabolism of other drugs. Liver
microsomes from control rats or rats, which received for 3
consecutive days 100 mg/kg of oral flucloxacillin, were used
to study the metabolism and metabolite pattern of midazolam,
a model substrate of CYP 3A4. There was a trend towards a
higher intrinsic microsomal clearance of midazolam using microsomes
from fluclox-acillin treated rats. In addition, there was
a significant increase in the formation of the principal midazolam
metabolites 1-hydroxy midazolam, 4-hydroxy midazolam and 1,4-dihydroxy
midazolam as compared to controls. These findings in-dicate
that flucloxacillin has the potential to induce expression
of both CYP3A4 as well as P-glycoprotein, most likely through
activation of the nuclear hormone receptor PXR. This would
offer an explanation for the observed clinical drug-drug interactions
between the antibiotic and cyclosporine.
[Back to top]
Fluoxetine Metabolism and Pharmacological Interactions:
The Role of Cytochrome P450
R. Mandrioli, G.C. Forti and M.A. Raggi
A review with 103 references. Fluoxetine is the parent drug
of the SSRI (selective serotonin reuptake inhibitor) antidepressant
class, and is still one of the most highly used drugs of this
class world-wide. Fluoxetine now has largely (albeit not completely)
substituted older and less safe drugs such as tricyclic antidepressants.
Different cytochrome P450 isoforms are involved in the metabolism
of fluoxetine, however, the main active metabolite, norfluoxetine,
is produced by the CYP2D6 action in the human liver. In this
paper, the main metabolic characteristics of fluoxetine will
be reviewed, with particular attention paid to the role of
cytochrome isozymes. The pharmacological interactions of the
drug will be overviewed, especially those concerning other
drugs used in psychiatric clinics, such as antipsychotics
and antidepressants and the relationships between pharmacological
interactions and cytochrome activity will be discussed. Recently,
much attention has been drawn to the therapeutic drug monitoring
(TDM) of fluoxetine, and in particular to the analysis of
fluoxetine enantiomers for which enantiomeric separations
and enantioselective metabolism will also briefly be mentioned.
[Back to top]
Prediction of Drug-Drug Interactions for AUCoral
of High Clearance Drug from In Vitro Data: Utilization
of a Microtiter Plate Assay and a Dispersion Model
T. Yamamoto, A. Suzuki, Y. Kohno, K. Nagata and Y. Yamazoe
The purpose of this study was to propose a new method to
predict in vivo drug-drug interactions (DDIs) for
a high clearance drug from in vitro data. As the
high clearance drug, NE-100 (N, N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine
monohydrochloride) was used. First, approach based on Iu/Ki
value was used for the prediction of DDIs between NE-100 and
concomitant drugs. When the Ki values
(Ki-cal) obtained from the microtiter
plate (MTP) assay and the reported Ki
values (Ki-rep) for these drugs were
used to predict increases at levels of NE-100 AUCoral
(AUCoral ratio), the AUCoral
ratios from the Iu /Ki-cal
correlated with those from the Iu/Ki-rep. This result suggests
that the Ki-cal from the MTP assay can
be used for prediction of DDIs instead of the Ki-rep
value. Second, a new approach combining the inhibition rate
(R) calculated from the MTP assay and two physiological models
was used to predict DDIs. When the AUCoral
ratios of NE-100 by various drugs were predicted using the
R value and the well-stirred model, the ratios were similar
to those predicted using the Iu/Ki.
However, after co-administration of drugs such as quinidine,
propafenone and thioridazine (potent inhibitors of CYP2D6),
the NE-100 AUCoral ratios predicted from
the dispersion model was much greater than those from well-stirred
model. This result shows that application of the dispersion
model to the prediction method using the R value might sensitively
and precisely predict the increased levels of AUCoral
by DDIs for high clearance drug, compared with the prediction
method using IuKi value.
[Back to top]
Predicting the Pharmacokinetics of Acyl Glucuronides and
Their Parent Compounds in Disease States
J.H. Liu and P.C. Smith
Acyl glucuronides are potentially reactive intermediates,
which not only undergo hydrolysis and intramolecular acyl
migration, but also bind irreversibly to plasma protein
in vitro and in vivo. To evaluate the impact
of renal failure, liver dysfunction and other disease states
on the pharmacokinetics of acyl glucuronides and their parent
compounds, a pharmacokinetic model has been established. The
model has been successfully utilized to predict the pharmacokinetics
of six compounds, diflunisal (DF), valproic acid (VPA), zomepirac
(Z), suprofen (S), R-etodolac (R-ET), S-etodolac (S-ET), and
their acyl glucuronides in various simulated disease states
in experimental animals. Modeling studies revealed that altering
the metabolic pathways of these compounds had significant
impact on exposure and clearance of acyl glu-cuoninde. The
simulation results also indicated that disease states that
affect irreversible metabolic pathways other than glucuronidation
may have major impacts on the apparent plasma clearance of
the parent compound or exposure to the reactive acyl glucuronide
as well. The study concluded that the model is sufficiently
robust and applicable for pharma-cokinetic studies of acyl
glucuronides and their parent compounds in various disease
states that may modulate drug clearance. The model is also
applicable to understanding the complex disposition of other
drugs subject to conjugation, especially those that can be
reversible and undergo enterohepatic recycling, such as sulfation
and glycine conjugation.
[Back to top]
Preclinical Pharmacokinetics: An Approach Towards Safer
and Efficacious Drugs
S.S. Singh
Lack of efficacy and toxicity are considered to be major
reasons for drug failures and pharmacokinetics governs them
to a large extent. Compound with favorable pharmacokinetics
is more likely to be efficacious and safe. Therefore, the
preclinical pharmacokinetic evaluation should be comprehensive
enough to ensure that compounds do not fail in the clinic.
Preclinical ADME screening facilitates early elimination of
weak candidates and directs the entire focus of the drug development
program towards fewer potential lead candidates. Hence, it
is mandatory that the pre-clinical candidates are subjected
to as many possible reality checks. Reliance on in-vitro
tests should be minimized because they do not represent the
real physiological environment but rather slow down the pace
of a drug discovery program. Com-pounds can be straight away
subjected to in-vivo high throughput screens such
as cassette dosing, cassette analysis or rapid rat screen
etc. Candidates with the desired in-vivo pharmacokinetic
profile may be further profiled in-vitro, using assays
such as metabolic stability, reaction phenotyping, CYP-450
inhibition and induction, plasma protein binding etc. in human
microsomes, human recombinant CYP-450 enzymes and human plasma.
This also provides an early indication of whether the compound
which worked in animals would work in human as well.
In-vitro metabolic stability profile is a qualitative
as well as quantitative comparison of metabolism of a compound
in human and animal models. It helps in identifying the right
model for toxicity studies. Extensive metabolism is generally
considered a liability as it limits the systemic exposure
and shortens the half-life of a compound. Several strategies
such as reduction of lipophilicity, modification and / or
blocking of metabolically soft spots and use of enzyme inhibitors;
have been developed to combat metabolism. In spite of several
concerns, the fact that active metabolites of several mar-keted
drugs have been developed as drugs with better efficacy, safety
and pharmacokinetics profile; cannot be denied. Therefore,
instead of considering metabolic instability a liability it
can be exploited as a tool for discovering better drugs. It
is equally important to identify the metabolic pathways of
the drug candidates by conducting in-vitro CYP450
reaction phenotyping assays. The identification of drug metabolizing
enzymes involved in the major metabolic pathways of a compound
helps in predicting the probable drug-drug interactions in
human. Compounds with more than one meta-bolic pathway have
less likelihood of clinically significant drug interactions.
In-vitro CYP450 inhibition and induction screens
are used to evaluate the potential of compound towards drug
– drug interactions and the most prone candidates may
either be discarded or taken ahead with a caution. It is known
that only unbound drug is pharmacologically active and therefore
the assessment of bound fraction by the estimation of plasma
protein binding of a compound is another important parameter
to be explored in-vitro. In addition to the process
of ‘weeding out’ weak candidates early in the
drug discovery process, it is equally important to identify
the probable causes of poor ADME exhibited by some com-pounds
as this information is useful to medicinal chemists for improving
upon backbones that exhibit un favorable pharmacokinetic profile.
Toxicity study is the foundation of an INDA (Investigational
new drug application) and there-fore, the final selection
of a compound can be performed only after proper toxicological
evaluation in animal models. Toxicokinetics forms an integral
part of toxicity study and is used to assess the exposure
of candidates in toxicity models and correlate the drug levels
in blood and various tissues with the toxicological findings.
Although in-vivo screening of compounds in animal
models and in-vitro assays in human recombinant CYP-450
enzymes help in drug candidate selec-tion, both approaches
have their own limitations. There is no certainty that the
selected candidates will exhibit the desired target PK profile
in human and real human PK remains suspense until the compound
enters Phase-1 clinical trial. The recognition of human micro
dosing, (HMD) by medicines and healthcare products regulatory
agency (MHRA) and European agency for evaluation
of medicinal products [EMEA] is a stepping stone in the
direction of obtaining human PK data early in the preclinical
stage. This would gradually shift the focus of early drug
development away from animal studies directly towards safe
and ethical studies in human yielding more relevant and reliable
pharmacokinetic data. HMD would provide an answer to the growing
public demand for a reduction in the use of animals for pharmaceutical
development.
[Back to top]
The Role of Blood-Brain Barrier Studies in the Pharmaceutical
Industry
A. Reichel
The blood-brain barrier (BBB) remains one of the greatest
challenges for the discovery and development of treatments
for CNS disorders, which to this day remains one of the riskiest
disease areas in terms of clinical success rates. Although
the BBB is currently seen predominantly as a permeability
obstacle for CNS drug delivery, it is becoming increasingly
clear that the BBB has many more implications for the pharmaceutical
industry impacting on CNS pharmacology and pathology, CNS
pharmacokinetics and pharmacodynamics, and adverse CNS effects,
to name but a few areas.
The present review does not intend to summarize the activities
in the field of BBB research per se, which has been ac-complished
by a number of excellent recent reviews, but instead to provide
an overview of the role of BBB studies from a pharmaceutical
industry perspective.
This review will elaborate on the specific needs in terms
of BBB-related issues across the different drug discovery
and development phases, i.e. target identification and validation,
lead generation and optimization, candidate selection and
profiling, preclinical development and clinical studies. The
specific approaches taken will be discussed in terms of spe-cific
requirements, questions to be asked, feasibility, interpretability,
and impact. It becomes clear that few of the exist-ing BBB
models fully meet the requirements of the industrialized drug
discovery process, highlighting the need for an array of new
or modified tools and approaches that are more effective in
helping make decisions which are more specifi-cally tailored
to the various stages of the lengthy process from target to
the clinic. In looking at the numerous ongoing activities
in the area of BBB research from the drug discovery and development
point of view, an attempt has been made to place a stronger
emphasis on the applicability of particular techniques and
approaches, to identify gaps and areas for future activities.
In order to materialize the considerable knowledge gained
in recent years, the review is intended to fos-ter an increased
awareness of the need to better integrate basic academic research
with the specific requirements of the pharmaceutical industry
for the search of effective and safe new CNS medicines.
[Back to top]
Recent Advances in Molecular Modeling and Medicinal
Chemistry Aspects of Phospho-Glycoprotein
E. Srinivas, J.N. Murthy, A.R.R. Rao and G.N. Sastry
Phospho-glycoprotein (P-gp) is an efflux transporter expressed
in many organs (ex: kidney, lung, liver and spleen) and in
hormone producing or responsive tissues (ex: adrenal cortex,
testis and placenta). It is involved in many important physiological
functions. Among them the major one is extrusion of xenobiotics
in order to detoxify the cells. This property of P-gp is associated
with multidrug resistance (MDR) for many pathological conditions.
While the ex-perimental determination of three-dimensional
structure is not yet successful, the transmembrane (TM) 5,
6, 11 and 12 are sensitive to mutations and contain substrate
binding sites. Designing of potential and selective inhibitors
of P-gp is still hampered by a lack of information upon the
three dimensional structure of P-gp. The design of P-gp inhibitors
was traditionally driven by quantitative structure activity
relationship studies, which is complicated by factors such
as differ-ent types of assays, multiple drug binding sites
and diverse chemical structures. Clearly a conclusive and
predictive SAR does not seem to be practical, despite progress
in the last few years towards more specific SAR suggesting
well defined structural features responsible for activity.
Advances made recently in solving the crystal structure of
prokaryotic ATP binding cassette proteins (ABC) transporters,
Ec-MsbA, Vc-MsbA and BtuCD yielded suitable templates for
construc-tion of homology models of P-gp. Few molecular dynamics
(MD) simulations aimed at elucidating the functional dynam-ics
of ABC transporters have provided useful insights to their
mechanism and structure. The present review aims at the general
overview of importance, expression, structure, organization
and drug binding sites of P-gp. This review also highlights
recent developments in the homology modeling, molecular dynamics
simulations of P-gp and progress in QSAR, pharmacophore modeling
of P-gp modulators.
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