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Current
Drug Metabolism
ISSN: 1389-2002
Current Drug Metabolism
Volume 8, Number 5, June 2007
Contents
Editorial Pp. 405
Mechanism-Based Inactivation of Cytochrome P450 Enzymes: Chemical
Mechanisms, Structure-Activity Relationships and Relationship
to Clinical Drug-Drug Interactions and Idiosyncratic Adverse
Drug Reactions Pp. 407-447
A.S. Kalgutkar, R.S. Obach and T.S. Maurer
[Abstract]
Drug-Drug Interactions via Mechanism-Based Cytochrome
P450 Inactivation: Points to Consider for Risk Assessment
from In Vitro Data and Clinical Pharmacologic Evaluation
Pp. 449-462
K. Venkatakrishnan and R.S. Obach
[Abstract]
Evaluation of Human Pharmacokinetics, Therapeutic
Dose and Exposure Predictions Using Marketed Oral Drugs
Pp. 463-479
D.F. McGinnity, J. Collington, R.P. Austin and R.J. Riley
[Abstract]
GSTP1 and MTHFR Polymorphisms Are Related with Toxicity
in Breast Cancer Adjuvant Anthracycline-Based Treatment
Pp. 481-486
R. Zárate, S. González-Santigo, J. de la
Haba, E. Bandres, R. Morales, J. Salgado, A. Gómez,
E. Aranda and J. García-Foncillas
[Abstract]
Genetic Polymorphism of the Flavin-Containing
Monooxygenase 3 (FMO3) Associated with Trimethylaminuria
(Fish Odor Syndrome): Observations from Japanese Patients
Pp. 487-491
H. Yamazaki and M. Shimizu
[Abstract]
Phenotyping of Cytochrome P450 2E1 In Vitro
and In Vivo Pp. 493-498
L. Ernstgård, G. Johanson, A.-S. Karlsson and M.
Warholm
[Abstract]
Metabolism of Dietary Polyphenols and Possible Interactions
with Drugs Pp. 499-507
J.D. Lambert, S. Sang, A.Y. H. Lu and C.S. Yang
[Abstract]
Presystemic Metabolism of Orally Administered Peptide
Drugs and Strategies to Overcome It Pp. 509-517
A. Bernkop-Schnürch and T. Schmitz
[Abstract]
Calcium Metabolism and Oxidative Stress in Bone Fractures:
Role of Antioxidants Pp. 519-525
S.A. Sheweita and K.I. Khoshhal
[Abstract]
Abstracts
[Back to top]
Editorial
The topic of mechanism-based inactivation (MBI) of
cytochrome P450 enzymes has been recently emerging as one
of ever increasing importance. While our knowledge of some
of the biochemistry and chemistry of the phenomenon of MBI
of P450 enzymes has been with us for over three decades, the
ramifications of MBI in drug discovery and clinical pharmacokinetics
have been appreciated only more recently. While drug label
warnings and contraindications due to P450 based drug-drug
interactions (DDI) are numerous, the only drug known to be
removed from the market due to an unfavorable drug-drug interaction
profile (as a perpetrator of DDIs) was in fact a mechanism-based
inactivator (mibefradil). Work done by Hall and co-workers
over the past several years (Mayhew, et al., 2000;
Wang, et al., 2004), as well as that of other eminent
investigators has elevated the science such that we can now
relate in vitro MBI to in vivo DDI. The
importance of MBI in the development of new drugs has not
gone unnoticed by government regulatory authorities. The FDA
has included an assessment of MBI as an essential component
to drug development in its recently released draft guidance
document (FDA, 2006).
Because of the importance of this topic, Current Drug Metabolism
is publishing two back-to-back comprehensive reviews on MBI
that were written by colleagues of mine at the research laboratories
of Pfizer, Inc. In the first, the types of structures that
can cause MBI of cytochrome P450 enzymes are described, as
a useful reference for those involved in the chemical design
of new drugs. The implications of MBI regarding DDI and pharmacokinetics
are alluded to as a form of an introduction to the topic.
In the second, the details are laid out regarding the mathematics
behind how in vitro MBI data can be used in the development
of drugs, for the prediction of DDI, and the design of clinical
study strategies. We hope that the readers of Current Drug
Metabolism find these two articles to be of value to their
research efforts aimed toward the discovery and development
of safe and effective new agents that will lack the property
of causing DDI via mechanism-based inactivation of
P450 enzymes.
FDA (2006) Guidance for Industry. Drug Interaction Studies
- Study Design, Data Analysis, and Implications for Dosing
and Labeling http://www.fda.gov/cber/gdlns/interactstud.htm
(accessed, March 27, 2007).
Mayhew BS, Jones DR, Hall SD. (2000) An in vitro
model for predicting in vivo inhibition of cytochrome
P450 3A4 by metabolic intermediate complex formation. Drug
Metab Dispos 28: 1031-1037.
Wang YH, Jones DR, Hall SD. (2004) Prediction of cytochrome
P450 3A inhibition by verapamil enantiomers and their metabolites.
Drug Metab Dispos 32: 259-266.
Chandra Prakash
Editor-in-Chief
Current Drug Metabolism
[Back to top]
Mechanism-Based Inactivation of Cytochrome
P450 Enzymes: Chemical Mechanisms, Structure-Activity Relationships
and Relationship to Clinical Drug-Drug Interactions and Idiosyncratic
Adverse Drug Reactions
A.S. Kalgutkar, R.S. Obach and T.S. Maurer
Cytochrome P450 constitute a superfamily of heme-containing
enzymes that catalyze the oxidative biotransformation of structurally
diverse xenobiotics including drugs. Inhibition of P450 enzymes
is by far the most common mechanism which can lead to DDIs.
P450 inhibition can be categorized as reversible (competitive
or non-competitive) or irreversible (mechanism-based inactivation).
Mechanism-based P450 inactivation usually involves bioactivation
of the xenobiotic to a reactive intermediate, which covalently
modifies an active site amino acid residue and/or coordinates
to the heme prosthetic group. Covalent modification of P450
enzymes can also lead to hapten formation and can in some
cases trigger an autoimmune response resulting in toxicological
consequences. Compared to reversible inhibition, irreversible
inhibition more frequently results in unfavorable DDIs as
the inactivated P450 enzyme has to be replaced by newly synthesized
protein. For these reasons, most drug metabolism groups within
pharmaceutical companies have well-established screening paradigms
to assess mechanism-based inactivation of major human P450
enzymes by new chemical entities followed by in-depth mechanistic
studies to elucidate the mechanism of P450 inactivation when
a positive finding is discerned. A deeper understanding of
the process leading to enzyme inactivation by drug candidates
can lead to rational chemical intervention strategies to circumvent
the P450 inactivation/bioactivation liability. Apart from
structure-activity relationship studies, methodology to predict
the magnitude of in vivo metabolic DDIs using in
vitro P450 inactivation data and predicted human pharmacokinetics
of the candidate drug also exists and can be utilized to project
the extent of clinical DDIs against P450 enzyme-specific substrates.
In this review, a comprehensive analysis of the biochemical
basis and known structure-activity relationships for P450
inactivation by xenobiotics is described. In addition, the
current state-of-the-art of the methodology used in predicting
the magnitude of DDIs using in vitro P450 inactivation
data and human pharma-cokinetic parameters is discussed in
detail.
[Back to top]
Drug-Drug Interactions via Mechanism-Based Cytochrome
P450 Inactivation: Points to Consider for Risk Assessment
from In Vitro Data and Clinical Pharmacologic Evaluation
K. Venkatakrishnan and R.S. Obach
This commentary discusses the approaches to, and key considerations
in the in vitro-in vivo extrapolation of drug-drug
interactions (DDI) resulting from mechanism-based inactivation
(MBI) of cytochrome P450 (CYP) enzymes and clinical pharmacologic
implications. In vitro kinetic assessment and prediction
of DDI produced via reversible inhibition and MBI
rely on operationally and conceptually distinct approaches.
DDI risk assessment for inactivators requires estimation of
maximal inactivation rate (kinact)
and inactivator potency (KI)
in vitro, that need to be considered in context of
the biological turnover rate of the enzyme (kdeg)
and clinical exposures of the inactivator (I), respectively,
to predict interaction magnitude. Risk assessment cannot be
performed by a simple comparison of inactivator potency against
in vivo exposure since inactivation is both concentration
and time-dependent. MBI contour plots tracking combinations
of I:KI and kinact:kdeg
resulting in identical fold-reductions in intrinsic clearance
are proposed as a useful framework for DDI risk assessment.
Additionally, substrate-specific factors like fraction of
the total clearance of the object drug via the enzyme
being inactivated (fm(CYP)) and the bioavailability fraction
across the intestine for CYP3A substrates (FG)
are important determinants of interaction magnitude. Sensitivity
analysis of predicted DDI magnitude to uncertainty in input
parameters is recommended to inform confidence in predictions.
The time course of reversal of DDI resulting from CYP inactivation
is determined by the half-life of the enzyme which is an important
consideration in the design and interpretation of clinical
DDI studies with inactivators.
[Back to top]
Evaluation of Human Pharmacokinetics, Therapeutic
Dose and Exposure Predictions Using Marketed Oral Drugs
D.F. McGinnity, J. Collington, R.P. Austin and R.J. Riley
In this article approaches to predict human pharmacokinetics
(PK) are discussed and the capability of the exemplified methodologies
to estimate individual PK parameters and therapeutic dose
for a set of marketed oral drugs has been assessed. For a
set of 63 drugs where the minimum efficacious concentration
(MEC) and human PK were known, the clinical dose was shown
to be well predicted or in some cases over-estimated using
a simple one-compartment oral PK model. For a subset of these
drugs, in vitro potency against the primary human
targets was gathered, and compared to the observed MEC. When
corrected for plasma protein binding, the MEC of the majority
of compounds was ≤ 3 fold over the respective in
vitro target potency value. A series of in vitro
and in vivo experiments were conducted to predict
the human PK parameters. Metabolic clearance was generally
predicted well from human hepatocytes. Interestingly, for
this compound set, allometry or glomerular filtration rate
(GFR) ratio methods appeared to be applicable for renal CL
even where CLrenal> GFR.
For ~90% of compounds studied, the predicted CL using in
vitro-in vivo (IVIV) extrapolation together with a CLrenal
estimate, where appropriate, was within 2-fold of that observed
clinically. Encouragingly volume of distribution at steady
state (Vss) estimated in
preclinical species (rat and dog) when corrected for plasma
protein binding, predicted human Vss
successfully on the majority of occasions – 73% of compounds
within 2-fold. In this laboratory, absorption estimated from
oral rat PK studies was lower than the observed human absorption
for most drugs, even when solubility and permeability appeared
not to be limiting. Preliminary data indicate absorption in
the dog may be more representative of human for compounds
absorbed via the transcellular pathway. Using predicted
PK and MEC values estimated from in vitro potency
assays there was a good correlation between predicted and
observed dose. This analysis suggests that for oral therapies,
human PK parameters and clinical dose can be estimated from
a consideration of data obtained from in vitro screens
using human derived material and in vivo animal studies.
The benefits and limitations of this holistic approach to
PK and dose prediction within the drug discovery process are
exemplified and discussed.
[Back to top]
GSTP1 and MTHFR Polymorphisms Are Related with Toxicity
in Breast Cancer Adjuvant Anthracycline-Based Treatment
R. Zárate, S. González-Santigo, J. de la
Haba, E. Bandres, R. Morales, J. Salgado, A. Gómez,
E. Aranda and J. García-Foncillas
We have analyzed several members of drug-metabolizing enzymes
(DMEs) and other polymorphisms in genes implicated in tumor
aggressivity regarding possible links between specific genetic
variability in systemic drug bioavailability and toxicity
in breast cancer patients treated with adjuvant anthracycline-based
treatment.
PCR-RFLP and sequencing analyses technique were used for evaluating
fourteen previously identified polymorphisms in 94 patients.
GSTP1A>G and MTHFR 1298A>C genotypes
remained as significant predictors in a multivariate logistic
regression analysis. GSTP1 polymorphism was linked
to haematological GIII-IV toxicity (P = 0.044, HR=
6.4, 95% CI = 1.05 to 39. Increased and significant HR was
obtained for MTHFR-1298 AC+CC group when
non-haematological toxicities GIII-IV toxicities were evaluated
(HR = 24; 95% CI = 2.3 to 254), P = 0.008.
Our results suggest that GSTP1 and MTHFR
genotypes may be consider relevant and independent factors
of toxicity in adjuvant anthra-cycline-based treatment of
breast cancer.
[Back to top]
Genetic Polymorphism of the Flavin-Containing Monooxygenase
3 (FMO3) Associated with Trimethylaminuria (Fish Odor Syndrome):
Observations from Japanese Patients
H. Yamazaki and M. Shimizu
Trimethylaminuria (fish odor syndrome) is a metabolic disorder
characterized by the inability to convert malodorous dietary-derived
trimethylamine (TMA) to odorless TMA N-oxide by the
flavin-containing monooxygenase 3 (FMO3). Mutations of the
FMO3 gene were investigated in Japanese trimethylaminuria
that showed low FMO3 metabolic capacity. Novel polymorphisms
in the FMO3 gene causing stop codons at Cys197, Trp388,
Gln470 or Arg500 of FMO3 were discovered in self-reported
trimethylaminuria Japanese volunteers. Different metabolic
capacities of FMO3 were observed for Asn114Ser, Thr201Lys,
Arg205Cys or Met260Val FMO3 variants in addition to common
Glu158Lys, Val257Met, and Glu308Gly FMO3. Estimated allelic
frequencies for these novel mutated FMO3 genes for
the Japanese population examined was ~1-4 % in this Japanese
cohort. Recombinant Arg500stop (94% of the whole FMO3 structure)
and several missense FMO3 variants showed no detectable activity
and different effects on N- and S-oxygenation
activities, respectively. The family members of Japanese probands
who were heterozygous for these nonsense mutants generally
showed moderate TMA N-oxygenation metabolic capacity,
suggesting that heterozygotes for the nonsense mutations will
exhibit trimethylaminuria symptoms only if they have, on the
other chromosome, a mutation that substantially impairs enzyme
activity. In addition, other causal factors for decreased
FMO3 metabolic capacity such as liver damage or menstruation
and treatment with copper chlorophyllin are also included
in this minireview. The present article provides fundamental
information for the importance of future investigations of
the human FMO3 gene associated with trimethylaminuria
(fish odor syndrome).
[Back to top]
Phenotyping of Cytochrome P450 2E1 In Vitro and In
Vivo
L. Ernstgård, G. Johanson, A.-S. Karlsson and M.
Warholm
The aim of the present study was to develop and improve methods
for phenotyping of CYP2E1, an important enzyme in the biotransformation
of many industrial chemicals, therapeutic drugs and endogenous
substances. The possibility to measure CYP2E1 activity in
lymphocytes by using p-nitrophenol as a substrate
and CYP2E1 protein levels by flow cytometry were studied in
vitro. Further, the conventional chlorzoxazone method
for in vivo phenotyping was studied by adjusting
the dose to body weight in 10 healthy volunteers. Finally,
the possibility to obtain the chlorzoxazone metabolic ratio
in saliva samples was investigated.
No CYP2E1 protein in lymphocytes was detected by using flow
cytometry. Some enzyme activity was found in the experiments
with p-nitrophenol, however, it could not be verified
that it was catalyzed by CYP2E1. Chlorzoxazone and 6-hydroxychlorzoxazone
were not detectable in saliva samples.
The present in vivo experiments, combined with our
previous data (in total 356 experiments in 50 subjects) show
that the metabolic ratio increases with decreasing absorbed
dose, expressed as the sum of chlorzoxazone and 6-hydroxychlorzoxazone
in plasma at 2 h. The increase becomes pronounced at sum concentrations
below 100 μM.
In conclusion, chlorzoxazone metabolism in vivo remains
the only available method for CYP2E1 phenotyping. The administered
dose as well as the absorption of the probe influences the
chlorzoxazone ratio. We suggest that a dose of 10 mg chlorzoxazone
per kg body weight is used to estimate the CYP2E1 phenotype.
Further, metabolic ratios should be disregarded if the sum
of plasma chlorzoxazone and 6-hydroxychlorzoxazone is below
100 μM
(blood sampled after 2 h).
[Back to top]
Metabolism of Dietary Polyphenols and Possible Interactions
with Drugs
J.D. Lambert, S. Sang, A.Y. H. Lu and C.S. Yang
Polyphenolic compounds are abundant in the human diet and
gram quantities are ingested daily. The consumption of polyphenols
is expected to rise due to the use of dietary supplements
and public health initiatives promoting the consumption of
more fruits and vegetables. It is known that these dietary
polyphenols are extensively metabolized. Many of these compounds
are therefore are expected to compete with other substrates
of Phases I, II, III enzymes and transporters. In addition,
some dietary polyphenols may induce certain drug metabolizing
enzymes and affect the metabolism of important therapeutic
agents. This review will discuss 1) the metabolism of dietary
polyphenols using green tea polyphenols (catechins) as an
example, 2) inhibition of drug metabolism by polyphenols,
and 3) induction of drug metabolizing enzymes by dietary polyphenols.
The potential consequences of these effects on drug metabolism
will also be discussed.
[Back to top]
Presystemic Metabolism of Orally Administered Peptide
Drugs and Strategies to Overcome It
A. Bernkop-Schnürch and T. Schmitz
To date, the majority of therapeutic peptides and proteins
have to be administered via parenteral routes, which are painful
and inconvenient. Consequently, “injectable-to-oral-conversions”
are highly on demand. Apart from a poor membrane uptake, however,
an extensive presystemic metabolism of orally given peptide
drugs is responsible for a comparatively very poor oral bioavailability.
This presystemic metabolism in the gastrointestinal tract
is based on luminally secreted enzymes (I) including pepsins,
trypsin, chymotrypsin, elastase and carboxypeptidase A/B,
on brush border membrane bound enzymes (II) including various
carboxypeptidases and aminopepti-dases and on cytosolic enzymes
(III). In addition, thiol-disulphide exchange reactions between
orally administered peptide drugs and sulfhydryl bearing components
of the gastrointestinal juice are responsible for a presystemic
metabolism.
Strategies to avoid a presystemic metabolism in the gastrointestinal
tract are on the one hand based on chemical modifications
of peptide drugs in order to make them more stable towards
an enzymatic attack. On the other hand various formulation
techniques are applied in order to protect therapeutic peptides,
being incorporated in appropriate carrier systems. They include
liposomes, nano-/microparticles and matrix tablets comprising
various auxiliary agents such as enzyme inhibitors and multifunctional
polymers.
Within this review an overview about “the enemy’s
strength” and the current strategies to avoid a presystemic
metabolism of orally administered peptides is provided.
[Back to top]
Calcium Metabolism and Oxidative Stress in Bone Fractures:
Role of Antioxidants
S.A. Sheweita and K.I. Khoshhal
Calcium ion is an essential structural component of the skeleton.
There is growing evidence for the importance of nutrition
in the maintenance of bones and joints health. Nutritional
imbalance combined with endocrine abnormalities may be involved
in osteoporosis. For example, essential fatty acids and their
metabolites were reported to have beneficial action in osteoporosis.
The mechanism by which fatty acids prevent osteoporosis may
involve inhibition of pro-inflammatory cytokines, which are
known to have a major role in osteoporosis through induction
of oxidative stress which had adverse effects on the skeleton.
Other risk factors for osteoporosis, such as smoking, hypertension
and diabetes mellitus are also associated with increased oxidative
stress and free radicals levels.
When bone fracture occurs, a remarkable yield of free radicals
is generated by the damaged tissues. However, controlled production
of free radicals by normally functioning osteoclasts could
accelerate destruction of calcified tissues and assist bone
remodeling. Enhanced osteoclastic activity observed in bone
disorders may have been responsible for increased production
of reactive oxygen species [ROS] in the form of superoxide,
which is evident by increased levels of serum malondialdehyde
[MDA] levels. One of the most damaging effects of ROS is lipid
peroxidation, the end product of which is MDA which also served
as a measure of osteoclastic activity. Inhibition of the antioxidant
enzymes activities, such as superoxide dismutase and glutathione
peroxidase, was found to increase superoxide production by
the osteoclasts which represented by increased levels of MDA.
Therefore, oxidative stress is an important mediator of bone
loss since deficiency of antioxidant vitamins has been found
to be more common in the elderly osteoporotic patients. It
is concluded from this review that increased free radical
production overwhelms the natural antioxidants defense mechanisms,
subjecting individuals to hyperoxidant stress and thus leading
to osteoporosis. In addition, administration of antioxidants
might protect bones from osteoporosis and also might help
in the acceleration of healing of fractured bones.
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