Biomarkers of CYP2D6 and CYP3A4 Variability in Pediatrics

This proposal represents a continuation of previous and ongoing longitudinal studies to
characterize the relative contribution of pharmacogenetics and ontogeny to variability in
CYP2D6 and CYP3A4 activity between birth and adolescence. Specifically, the overall goal of
the research program is to test the hypothesis that genetic variability, rather than
ontogeny, is the primary source of variability in CYP2D6 and CYP3A4 activity during
adolescence. This study will extend our current series of longitudinal phenotyping studies
and will be a cross-sectional study in children aged 7-15 years, studied on seven occasions,
6 months apart.

Children will be recruited from Children's Mercy Hospitals and Clinics, The Children's Mercy
Hospital Northland Campus, and The Children's Mercy South Campus.

Subjects participating in this study will consist of a total of 220 children and adolescents
from 7 to 15 years of age with a gender and ethnic distribution representative of the
patient population served by Children's Mercy Hospital (CMH; from a representative 12 month
period) as follows: White, 59.6%; African-American, 31.9%; Hispanic, 4.9%; Asian,
0.6%;Native American, 0.1%; Other, 2.9%. The gender distribution is 44.4% female and 55.6%
male. An equal number of pediatric patients who meet the DSM-IV criteria for a primary
diagnosis of ADHD or ADD (~33%) and age- and sex-matched controls (~67%) will be recruited.
The patients represent a population of children who can reasonably expect to be treated with
a medication that is primarily dependent upon CYP2D6 for its elimination from the body. An
example is atomoxetine, also known as Strattera®. Each patient will be asked to invite a
friend to participate in the phenotyping study with them, providing a non-ADHD population as
a reference. Additional phenotyping visits will occur every six months (totaling 7 visits).
Justification: Assuming a 10% attrition rate for the second visit, the sample size is
expected to provide approximately 25 patients and 25 controls (50 subjects total) in each
initial 2-year age bin: 7.0 to 9.0 y, 9.1 to 11.0 y, 11.1 to 13.0 y, and 13.1 to 15.0 y.

Subjects will be enrolled via informed permission/assent and will be studied as outpatients
utilizing resources provided by the Pediatric Clinical Research Unit located in the Division
of Clinical Pharmacology and Medical Toxicology at Children's Mercy Hospital. For the ADHD
group, patients being treated with atomoxetine at the time of study enrollment will be
allowed to participate. Although in vitro studies indicate that atomoxetine inhibits CYP2D6
activity by ~50%, there does not appear to be any effect in vivo as single dose
pharmacokinetics of the CYP2D6 substrate, desipramine, were unaffected by four days of
atomoxetine 60 mg b.i.d. Thus, we do not expect any substantial effect on DM
biotransformation. Furthermore, if during the six month interval between phenotyping visits
ADHD patients are prescribed medications that are known CYP2D6 inhibitors, they will be
allowed to continue in the study; the dose and start date of these medications will be
recorded and the effect of the inhibitor on CYP2D6 phenotype (urinary metabolite ratio and
endogenous biomarker) will be determined. These data points will be excluded from the
ontogeny and genotype-phenotype correlation analysis

Pre-study visit. (about 1 hour long) On a screening visit, prospective subjects (and their
parents) will be provided with a description of the proposed study. After all their
questions have been answered, they will be given a copy of the permission/assent form to
review and sign Subjects will then have their medical history and use of medications
reviewed, including the use of non-prescription and herbal remedies. All subjects will be
given a complete physical examination by a physician, including vital signs (blood pressure,
heart rate, breathing rate, temperature, height and weight) and an assessment of pubertal
development by Tanner stage. Breast development will be assessed by visual inspection. Pubic
hair will be assessed by visual inspection. Testicular volume will be measured by direct
comparison to orchidometer beads. Tanner Staging will be assessed by a gender appropriate
physician. Blood will be drawn by needle stick for serum chemistries, liver function tests
and a hematology panel; a portion will also be retained for DNA testing (approximately 2
teaspoons of blood total). Urine will be collected for a Urinalysis with Micro. Numbing
cream may be applied to the site to minimize discomfort. A urine pregnancy test will be
performed for females of child bearing age.

The DNA testing, referred to "genotyping" will be conducted by a combination of technologies
designed to detect variations from single nucleotide level (including polymorphisms,
insertions and deletions), to larger gene deletions, rearrangements, hybrid structures and
copy number variations using established methods in the Developmental Pharmacology and
Experimental Therapeutics Laboratory. As next-generation sequencing technology becomes
established, existing technologies will be replaced by next-generation sequencing for its
ability to assess the entire gene region rather than at individual locations.

Study Day 1 (about 5 hours long) Within approximately one week of the pre-study visit,
subjects will be admitted to the study unit. The child must refrain from eating 2 hours
prior to the scheduled visit. Prior to dosing, the subjects will completely empty their
bladders and provide a blank urine sample. Each subject will then be given a single 0.5
mg/kg dose of DM as Robitussin Cough (7.5 mg/5 ml; alcohol-free, fruit punch flavor). All
urine produced over the 4-hour study period will be collected. Urine will be retained for
analysis of DM and its three metabolites, and for metabolomic analyses. One hour after
dosing, the subjects will be provided with a small meal that they will select from the
standard hospital menu. A pregnancy test will be performed for females of child bearing age.
Subjects will be asked to provide breaths (inspiratory and expiratory) for ten minutes with
a handheld indirect calorimeter (ie. MedGen/Body Gem: also known as "GEM") with a clip on
their nose. The measurements obtained for resting metabolic rates will occur prior to dosing
and 4 hours post dose.

Subjects will be asked how they are feeling during the study and before going home.

Study Day 2-7 (about 5 hours long) The remaining visits will occur every 6 months following
Study Day 1; the study sessions will be delayed for 1-2 weeks if the subjects are suffering
from flu-like symptoms. The child must refrain from eating 2 hours prior to the scheduled
visit. For females, a pregnancy test will be conducted using the pre-dose urine sample prior
to DM dosing.

Subjects will then have their medical history and use of medications reviewed, including the
use of non-prescription and herbal remedies. Subjects will be given a complete physical
examination by a physician, including vital signs (blood pressure, heart rate, breathing
rate, temperature, height and weight) and an assessment of pubertal development by Tanner
stage. Breast development will be assessed by visual inspection. Pubic hair will be assessed
by visual inspection. Testicular volume will be measured by direct comparison to
orchidometer beads. Tanner Staging will be assessed by a gender appropriate physician.

Subjects will be admitted to the study unit. Prior to dosing, the subjects will completely
empty their bladders and provide a blank urine sample. Each subject will then be given a
single 0.5 mg/kg dose of DM as Robitussin Cough (7.5 mg/5 ml; alcohol-free, fruit punch
flavor). All urine produced over the 4-hour study period will be collected. Urine will be
retained for analysis of DM and its three metabolites, and for metabolomic analyses. One
hour after dosing, the subjects will be provided with a small meal that they will select
from the standard hospital menu. A pregnancy test will be performed for females of child
bearing age. Subjects will be asked to provide breaths (inspiratory and expiratory) for ten
minutes with a handheld indirect calorimeter (ie. MedGen/Body Gem: also known as "GEM" with
a clip on their nose. The measurements obtained for resting metabolic rates will occur prior
to dosing. Subjects will be asked how they are feeling during the study and before going
home.

Inclusion Criteria:

- Males and females between 7 and 15 years of age.

Exclusion Criteria:

- Inability to have blood drawn for the screening lab tests

- Current therapy with medications known to inhibit CYP2D6: *fluoxetine (Prozac®)

- sertraline (Zoloft®)

- paroxetine (Paxil®)

- venlafaxine (Effexor®)

- imipramine

- nortriptyline

- desipramine

- amitriptyline

- fenfluramine

- terbinafine

- cyclobenzaprine

- haloperidol (Haldol®)

- metoprolol

- quinidine

- propafenone (Rythmol®)

- cimetidine (Tagamet®)

- tamoxifen

- over-the-counter diphenhydramine-containing drugs

- including Benadryl and generics and the cough and cold preparations

- Dytuss®

- Tusstat®

- Robitussin®

- pro-drugs codeine

- tramadol

- hydrocodone

- oxycodone (Percodan®, Percocet®) that are converted by 2D6 into their active
forms.

- Illicit drug use, treatment within the past 2 months with paroxetine or fluoxetine,
or the past six months with terbinafine are also exclusion criteria.

- Inability or unwillingness to fast 2 hours prior to the study session

- Existence of diagnosis which may influence absorption and gastric emptying; such as
reflux , inflammatory bowel disease, or Crohn's disease.

- A demonstrated adverse reaction to previous dextromethorphan exposure

- Impaired hepatic or renal activity, or physical examination as determined by the Sub
Investigator's discretion.

- Pregnancy

- Body-mass index (BMI) <5th percentile