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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5195_Библиотеки_им_академика_М_И_Перельмана.pdf
quantitative approaches to blood samples is also being explored, such as the
quantification of exosomes, secreted circulating vesicles that are derived from
the cellular endosomal compartment, that contain drug-metabolizing enzyme and
transporters. Exosomes of hepatic origin may offer a snapshot of expression of
these drug-metabolizing enzymes in the liver and potentially serve as a measure
of individual drug biotransformation capacity in vivo.
62
Finally, it is important to remember there are key limitations to proteomic
analyses, even when tissue samples are available. Protein quantitation is not a
perfect surrogate for function. Concomitant medications and post-translational
conformational modifications that may go unnoticed in a quantitative proteomic
analysis have the potential to impact enzyme activity. Furthermore, trafficking of
proteins to sites of action may not be completely efficient or may exist in the
form of a dynamic equilibrium. This is particularly relevant for drug
transporters, many of which have endogenous ligands and participate in
homeostatic processes regulating their localization on cellular membranes. To
quantify the amount of drug transporters on cellular surfaces (i.e., those that may
be capable of regulating intracellular drug concentration), additional
approaches are currently being developed to supplement targeted proteomic
analysis. Application of purification procedures allows transporters to be
selectively measured in the purified plasma membrane fraction, thereby
excluding any transporters present in internalized membrane vesicles from the
analysis. Use of reference proteins, such as aquaporin, can further confirm
transporter regional localization in tissues like the kidney, leading to higher
confidence in the robustness of the data.63 The potential importance of
quantitative proteomics to link in vitro studies with in vivo outcomes in
translational pharmacology research and the subsequent clinical applications
has been recognized, and efforts have been initiated to establish best practices
for this important new tool.
58
METABOLOMIC TOOLS
Several analytical approaches can be utilized for metabolomic and
metabonomic investigations, depending on the analytes of interest, but the most
used platforms are nuclear magnetic resonance (NMR) spectroscopy and LC or
gas chromatography coupled with mass spectral detection.64 Analogous to
proteomics, one popular strategy for conducting these metabolomic studies is to
use a global profiling approach (i.e., global metabolomics) to measure the
concentrations of all detectable small molecules in a biologic sample.
Evaluated holistically, the relative levels of these compounds from one
individual to the next constitute what is often referred to as an individual unique
“metabolomic fingerprint.” Using a statistical approach known as principle
component analysis (PCA), the differentness of groups of individual
metabolomic fingerprints can be evaluated and related to drug responsiveness
or the functional activity of the biologic pathways relevant to drug disposition.
If associations are discovered, a patient’s unique metabolomic fingerprint could
then be compared for similarities to these predetermined patterns to identify the
optimal drug to use and to guide dose selection. Global metabolomics, when
coupled with small molecule libraries, can also be used to identify a promising
subset of small molecules within the biologic matrix that may reflect an outcome
of interest. Targeted metabolomics, the assessment of these discrete small
molecule groups, can be subsequently applied and may even result in the
discovery of singular endogenous biomarkers that reflect the activity of drugmetabolizing enzymes and transporters. The identification of endogenous
biomarkers to replace exogenously administered probes for phenotyping drug
disposition pathways would be of great utility for pediatric medicine where
nontherapeutic pharmacologic effects of probes drugs should be minimized. A
metabolomic approach was applied to identify an endogenous compound with a
molecular mass of 444.3 Da as a potential biomarker of CYP2D6 activity from
pediatric urine samples.
65
Combining metabolomic and genome-wide genotyping data has revealed
common genetic variations that are associated with variability in metabolic
phenotypes involving the corresponding biochemical pathways.66 Metabolomics
in conjunction with simultaneous gene expression analysis of the transcriptome
provides additional mechanistic insights, leading to a more “systems-based”
understanding of cellular processes, especially in the context of drug-related
perturbations.67 A particularly exciting application of pharmacometabolomics is
the identification of metabolic signatures and corresponding cellular pathways
to predict drug response
68,69
; the approach has not been applied to drug response
in pediatrics to any appreciable extent so far.
APPLICATIONS OF PEDIATRIC
PHARMACOGENETICS AND
PHARMACOGENOMICS
DRUG BIOTRANSFORMATION AND CONCENTRATIONDEPENDENT TOXICITY
Clinical observation of patients with high drug concentrations/excessive or
prolonged drug responses together with the realization that the biochemical
traits (subsequently identified as proteins involved in drug biotransformation)
were inherited provided the origins of the concept of pharmacogenetics. Indeed,
with few exceptions, the major consequence of pharmacogenetic
polymorphisms in drug-metabolizing enzymes is concentration-dependent
toxicity due to impaired drug clearance and, to a lesser extent, reduced
conversion of prodrugs to therapeutically active compounds. For most
cytochromes P450, genotype–phenotype relationships are influenced by
development in that fetal expression is limited (with the exception of CYP3A7)
and functional activity is acquired postnatally in isoform-specific patterns.
Furthermore, clearance of some compounds appears to be greater in children
relative to adults, obscuring the correlation between genotype and phenotype in
neonatal life through adolescence.30 The ontogeny of several phase I and phase
II drug biotransformation pathways has been exhaustively reviewed.
70
Comprehensive reviews of cytochromes P450 pharmacogenetics in general,
71
and for individual drug biotransformation enzymes, such as CYP2B6,
72
CYP2C9,73 CYP2C19,74 CYP2D6,
75–77
the CYP3A subfamily,78 glucuronosyl
transferases (UGT),
79,80
sulfotransferases (SULTs),81 N-acetyltransferases
(NATs),82 and thiopurine S-methyltransferase (TPMT)83 are also available.
Salient features of the more common polymorphisms of clinically relevant drugmetabolizing enzymes are discussed briefly below.
CYP2C9
Although several clinically useful compounds are substrates for CYP2C9, the
effects of allelic variation are most profound for drugs with narrow therapeutic
indices, such as phenytoin,
84,85
warfarin,86 and tolbutamide.
87,88
Several allelic
variants of CYP2C9 have been observed in population studies, and an up-todate listing is maintained by the Pharmacogene Variation (PharmVar)
Consortium (https://www.pharmvar.org/gene/CYP2C9). Individual alleles are
designated by the gene name (CYP2C9) followed by an asterisk and an Arabic
number; CYP2C9*1 designates, by convention, the fully functional wild-type
allele. The CYP2C9*2 allele results in an amino acid substitution at position
144 of the CYP2C9 protein and is associated with an approximately 5.5-fold
decreased intrinsic clearance for (S)-warfarin relative to the wild-type
enzyme.89 The conservative isoleucine to leucine change at position 359
characteristic of the CYP2C9*3 allele occurs within a region of the protein that
affects substrate orientation in the active site and, as a result, produces a
considerable (27-fold) decrease in intrinsic (S)-warfarin clearance.90 The risk
of bleeding complications in patients treated with warfarin and concentrationdependent phenytoin toxicity is most pronounced for individuals with a
CYP2C9*3/*3 genotype. A decrease in activity has been observed for the
relatively rarer CYP2C9*5 allele due to an aspartate to glutamate change at
position 360, also within the active site of the enzyme.91 Interestingly, the
CYP2C9*8 allele, resulting in a substitution of arginine with histidine at
position 150, demonstrated increased activity for metabolizing tolbutamide in
vitro.92 However, the allele was associated with reduced in vivo phenytoin and
warfarin clearance in subsequent clinical studies. Additional uncommon
CYP2C9 alleles with evidence suggesting reduced activity in vivo are
CYP2C9*6, CYP2C9*11, and CYP2C9*12.
73
Approximately one-third of the Caucasian population carries a variant
CYP2C9 allele (*2 and *3 alleles, most commonly), whereas the *2 and *3
alleles are less common in African Americans, Chinese, Japanese, or Korean
populations. In contrast, the *5 and *6 alleles have been detected in African
Americans but are each found in less than 0.01% of Caucasians. The *8 and *11
alleles are also more common in individuals of African ancestry, but the
differences in allele frequency compared to other populations are less
pronounced than for the *5 and *6 alleles.
73
A pediatric case illustrating the clinical relevance of CYP2C9
pharmacogenetics involved the administration of phenytoin to a 2-year-old child
who was homozygous for the CYP2C9*2 allele.93 During standard-of-care
administration of phenytoin to treat status epilepticus, the patient developed
acute toxicity. Measurement of phenytoin plasma levels revealed phenytoin
concentrations in excess of threefold the upper therapeutic limit. Symptoms of
phenytoin intoxication took over 5 days to abate, and monitoring of drug levels
in the patient revealed that it took almost a week for phenytoin concentrations to
fall within the normal range, thus illustrating the fact that in addition to the
potential for genetic polymorphisms to result in dramatic elevations in drug
concentrations, they can also extend the duration of time a patient is exposed to
potentially toxic drug concentrations relative to patients with an extensive
metabolizer phenotype who may for other reasons (e.g., greater dose) achieve
the same elevated concentrations. Another important contributor to the marked
increases in phenytoin levels experienced by this patient was the presence of a
nonfunctional allele for CYP2C19. Whereas CYP2C19 only accounts for about
10% of phenytoin clearance in CYP2C9 extensive metabolizers,94 it can play a
much more dominant role in CYP2C9 poor metabolizers. Cases like this
highlight the importance of considering polymorphisms in secondary pathways
of drug metabolism, especially when a patient is genetically deficient in or
taking an inhibitor of the major pathways of drug metabolism.
CYP2C19
Originally reported as “mephenytoin hydroxylase” deficiency, the CYP2C19
poor-metabolizer phenotype is present in 3% to 5% of the Caucasian population
and 20% to 25% of Asians. Although several defective alleles have been
identified, the two most common variant alleles, CYP2C19*2 and CYP2C19*3,
result from single-base substitutions that introduce premature stop codons and,
consequently, truncated polypeptide chains that possess no functional activity.
95
In Japanese adults treated with lansoprazole, amoxicillin, and clarithromycin
for Helicobacter pylori infection, the eradication rate for CYP2C19 poor
metabolizers (97.8%) and heterozygous extensive metabolizers (one functional
CYP2C19 allele; 92.1%) was significantly greater than that observed in
homozygous extensive metabolizers (72.7%; p < 0.001). Of the 35 patients in
whom initial treatment failed to eradicate H. pylori, 34 had at least one
functional CYP2C19 allele, and eradication could be achieved with higher
lansoprazole doses in almost all cases.96 Given that the frequency of the
functional CYP2C19*1 allele is considerably greater in Caucasians (~84%)
compared to Japanese (~55%),95 eradication failure can be expected to occur
more frequently in Caucasians.
The CYP2C19*17 allele, characterized by two variants in the 5′-upstream
region of the CYP2C19 gene, occurs at a frequency of 18% in Swedish and
Ethiopian populations and approximately 4% in a Chinese population.
97
CYP2C19*17 is associated with “ultrarapid” activity as measured by
omeprazole metabolite ratio97 and decreased serum concentrations of substrates,
such as escitalopram.98 Both CYP2C19*2 and CYP2C19*4, an uncommon allele
resulting in loss of function via impaired gene transcription, have been
observed to be in linkage disequilibrium with CYP2C19*17 in some
populations. The *17 allele is believed to be unable to impact phenotype when
present on the same chromosome as the *2 or *4 allele. Additional uncommon
alleles resulting in loss of function in CYP2C19 are *5, *6, *7, and *8. A
detailed summary of CYP2C19 allelic variants reported to date can be found at
https://www.pharmvar.org/gene/CYP2C19.
CYP2C19 substrates, such as proton pump inhibitors, voriconazole, and
escitalopram, are used clinically in pediatric patient populations, and therefore,
it is reasonable to expect that pharmacogenetic considerations should guide
dosing strategies in children as well as in adults. The 2018 CPIC recommends
that voriconazole not be administered to children who are either CYP2C19
poor or ultrarapid metabolizers whenever an appropriate alternative drug (e.g.,
amphotericin B) is available.99 Among children receiving escitalopram or
citalopram, CYP2C19 poor-metabolizer status has been associated with greater
incidence of adverse drug reactions and drug discontinuation. Conversely, faster
metabolizer status was associated with more rapid treatment response.
100
Simulations leveraging CYP2C19 pharmacogenetics and clinical
pharmacokinetic data suggest poor metabolizers require, on average, only 50%
of the dose administered to extensive metabolizers to achieve comparable
systemic exposure, and ultrarapid metabolizers were estimated to require an
approximately 50% greater dose.
101
Although CPIC has provided dosing
recommendations for CYP2C19 antidepressant substrates, actual experience to
support recommendations in children is limited.
102
CYP2D6
The CYP2D6 gene locus is highly polymorphic with more than 130 allelic
variants identified to date (https://www.pharmvar.org/gene/CYP2D6; Table
5.1).77 For CYP2D6, allelic variants are the consequence of point mutations,
single-base pair deletions or additions, and gene rearrangements or deletion of
the entire gene that result in a reduction or complete loss of activity. Inheritance
of two recessive loss-of-function alleles results in the “poor-metabolizer
phenotype,” which is found in about 5% to 10% of Caucasians and about 1% to
2% of Asian subjects. In Caucasians, the *3, *4, *5, and *6 alleles are the most
common loss of functional alleles and account for approximately 98% of poormetabolizer phenotypes.
103
CYP2D6 activity is lower, on a population basis, in
Asian and African American populations due to a lower frequency of
TABLE 5.1
nonfunctional alleles (*3, *4, *5, and *6) and a relatively high frequency of
alleles that are associated with decreased activity relative to the wild-type
CYP2D6*1 allele. In Asians, CYP2D6*10 has an allele frequency of
approximately 50%, whereas CYP2D6*17 and CYP2D6*29 occur at relatively
high frequencies in subjects of black African origin.
104
A schematic
representation of some of the more common allelic variants is presented in
Figure 5.3. At the other end of the spectrum, the presence of CYP2D6 gene
duplication/multiplication events, which occur at a frequency of 1% to 2% in
Caucasians,
75–77
most often is associated with enhanced clearance of CYP2D6
substrates, although cases of increased toxicity due to increased formation of
pharmacologically active metabolites have also been reported.
105
In addition to
gene duplication/multiplication events, the CYP2D6 gene locus is also subject
to gene rearrangements, hybrid structures, and tandem arrangements involving
two or more nonidentical gene copies.77 Examples of relatively frequent
structural variants are presented in Figure 5.4.
Internet Re sources for Pharmacoge netics and Pharmacogenomics
Topic URL
General
pharmacogenetic
sites
Pharmacogenetics
Knowledge Base
https://www.pharmgkb.org/
Pharmacogene
Variation
Consortium
https://www.pharmvar.org/
Pharmacogenetics:
allelic variants of
drug-metabolizing
enzymes
CYP2C9 https://www.pharmvar.org/gene/CYP2C9
CYP2C19 https://www.pharmvar.org/gene/CYP2C19
CYP2D6 https://www.pharmvar.org/gene/CYP2D6
CYP3A4 https://www.pharmvar.org/gene/CYP3A4
CYP3A5 https://www.pharmvar.org/gene/CYP3A5
CYP3A7 https://www.pharmvar.org/gene/CYP3A7
UGTs https://www.pharmacogenomics.pha.ulaval.ca/ugt-alleles-
nomenclature/
NAT1 http://nat.mbg.duth.gr/Human%20NAT1%20alleles_2013.htm
NAT2 http://nat.mbg.duth.gr/Human%20NAT2%20alleles_2013.htm
NUDT15 https://www.pharmvar.org/gene/NUDT15
All sites were accessible on September 5, 2019.
Figure 5.3 Common CYP2D6 allelic variants. The CYP2D6 gene consists of nine exons, and individual
alleles are defined by the presence of key single-nucleotide variants, such as single-nucleotide
polymorphisms, insertions, and deletions, throughout the gene relative to the reference CYP2D6*1 allele
(top of figure). The different types of dashed lines refers to the functional consequence of allelic variants of
the activity of the protein, with solid line representing fully functional activity (CYP2D6*1, CYP2D6*2),
dashed line representing complete loss of activity (CYP2D6*3, CYP2D6*4, CYP2D6*6), and dotted line
representing partial activity (CYP2D6*10, CYP2D6*17, CYP2D6*29).
Figure 5.4 Structural variation in the CYP2D6 locus. A: The reference gene locus containing a single
copy of the CYP2D6 gene downstream of the CYP2D8 and CYP2D7 pseudogenes. A gene deletion event
results in the complete loss of CYP2D6, resulting in the CYP2D6*5 allele. B: Gene duplication and
multiplication events. Two or more (multiple) copies of a CYP2D6 allelic variant may be present in tandem
on a single chromosome. Duplication/multiplication events may involve fully functional alleles
(CYP2D6*2xN, CYP2D6*35xN, where N represents the number copies present, e.g., CYP2D6*2x2 for a
duplication), decreased function (CYP2D6*9xN, CYP2D6*41xN), or nonfunctional (CYP2D6*4xN) gene
copies. C: Hybrid and tandem events. Rearrangements involving two or more nonidentical gene copies can
also occur within the CYP2D locus. The top two lines represent examples of hybrid genes. CYP2D6*13 is
considered a CYP2D7–CYP2D6 hybrid as it is composed of DNA sequence from the CYP2D7
pseudogene and DNA derived from the downstream CYP2D6 genes (top line). The CYP2D6*36 allele is
an example of a CYP2D6–CYP2D7 hybrid and consists primarily of CYP2D6 sequence with a 3′-region
derived from CYP2D7 (second line). These hybrid structures generally do not code for functional protein.
The bottom two lines represent examples of two commonly observed tandem events: CYP2D6*68+*4
(inactive) and CYP2D6*36+*10, a partial function allele with an activity score of 0.25 due to the presence
of the partial function *10 allele. REP6, REP7, REPdup, and REPdel refer to repeat elements within the
locus. (Adapted from Nofziger C, Turner AJ, Sangkuhl K, et al. PharmVar GeneReview: CYP2D6. Clin
Pharmacol Ther 2020; 107:154-170.)
CYP2D6 is involved in the biotransformation of more than 40 therapeutic
entities, including several β-receptor antagonists, antiarrhythmics,
antidepressants, antipsychotics and morphine derivatives. Of these,
atomoxetine, codeine, dextromethorphan, diphenhydramine, fluoxetine,
imipramine, risperidone, and tramadol are commonly encountered in pediatrics.
In vitro studies indicate that fetal liver microsomes have very limited CYP2D6
activity (~1% of adult values), but CYP2D6 protein is detectable in all samples
from newborns.
106
A second study utilizing a relatively large number of
pediatric liver samples revealed that CYP2D6 protein and activity were similar
between fetal liver samples obtained during the third trimester of pregnancy and
liver samples obtained from infants in the first week of life, and both protein
and activity remained relatively constant after 1 week of age up to 18 years. The
data further imply that genetic variability, rather than ontogeny, is primarily
responsible for the observed variability in catalytic activity.
107
Similar results
have been observed in an in vivo longitudinal phenotyping study involving more
than 100 infants over the first year of life. This study utilized dextromethorphan
as a probe compound and the urinary ratio of dextromethorphan to dextrorphan
as a measure of CYP2D6 activity. Although considerable interindividual
variability in CYP2D6 activity was observed, no relationship between
CYP2D6 activity and postnatal age was apparent between the ages of 2 weeks
and 12 months.
108
Similarly, a cross-sectional study involving 586 children (480
Caucasians and 106 African Americans) indicated that the distribution of
CYP2D6 phenotypes in children was comparable to that observed in adults by
at least 10 years of age, and probably much earlier.40 Cumulatively, these in
vitro and in vivo indicate that developmental factors are less important than
genetic variation as determinants of CYP2D6 variability in children.
Drug accumulation with an increased risk of concentration-dependent
toxicity is of particular concern in CYP2D6 poor metabolizers. Indeed, a
fluoxetine-related death has been reported in a 9-year-old child with multiple
neuropsychiatric disorders who was subsequently determined to be a CYP2D6
poor metabolizer by genotype analysis. Measurements of blood and liver
concentrations of fluoxetine at autopsy were several-fold higher than expected,
consistent with CYP2D6 genotype.
109
On the other hand, poor metabolizers may
experience decreased efficacy or therapeutic failure when prescribed drugs are