The ultimate goal of this proposed investigation is to establish the role of genetic
variation and development in key transporters on the dose-exposure relationship of two
commonly used statin drugs in children. This study is the first step in a series of
investigations aimed at determining the mechanisms behind variations in physiologic response,
clinical efficacy and significant adverse effect risk that surround the statin drugs in
children and adolescents.
Research Design and Methods
1. Trial Design 1.1. Investigational Agents Pravastatin 20 mg tablet (ages 8-13 years), 40 mg
tablet (>14 years) Simvastatin 10 mg tablet (ages 8-17), 20mg (>17 years) Commercial supplies
of pravastatin and simvastatin that are FDA approved for use in pediatric dyslipidemia will
be used. Pravastatin and simvastatin from the same source and lot will be used for all
subjects. The doses designated above are chosen according to previous adult and pediatric
data 26, 28-32, 41 and are consistent with current labeling for pravastatin and simvastatin.
Although fixed doses within a pre-specified age range will be used, dose data will be
analyzed corrected for weight (mg/kg) based on the patient's weight at time of dosing.
1.2. Risk/Benefits All subjects with a LDL >130mg/dl (95% percentile) will be eligible for
this study, consistent with current clinical criteria for statin therapy. The risks
associated with participation in this single-dose pharmacokinetic study are expected to be
minimal given the known adverse event profile for the study articles and also, the limited
exposure. The most common mild adverse effects of statins are headache, myalgia, and
gastrointestinal symptoms (abdominal pain, dyspepsia, diarrhea, constipation). However, in
children these adverse effects occur with the same frequency as placebo 14, 15, 18. Rare
adverse effects include elevation of hepatic transaminases and myopathy, but these are
generally observed with chronic treatment. Furthermore, no cases of hepatic failure with
statins have been reported to date 52. There is also a small risk associated with placement
of the intravenous (IV) catheter that will be used to draw serial blood samples for
pharmacokinetic analysis and screening/safety labs. Finally, there is the risk of loss of
confidentiality for the participating subjects. Methods to protect PHI and data handling are
specifically outlined in the CMH CPR protocol Sections 4 & 5 (CMH IRB# 12040220). All of the
above risks associated with this non-therapeutic clinical trial are minimal.
There is no direct benefit to participating subjects, although there may be benefit for
children in the future as it is expected that the information to be gained from the study can
be generalized to the larger population of pediatric patients who may require statin
treatment. The purpose of the study is to determine how genetic variation impacts pravastatin
and simvastatin plasma concentrations following recommended doses of the drug in a patient
population that has not previously been studied. Thus, with this knowledge, future protocols
could subsequently be developed to effectively "personalize" dosing for pediatric subjects
with the aforementioned SNPs who are taking pravastatin or simvastatin, thereby improving
efficacy and safety for the individual patient.
1.3. Study Design/Type This will be a single center, open label, randomized, cross-over study
in patients with dyslipidemia comparing the pharmacokinetics of pravastatin and simvastatin,
in patients with greater than or equal to one variant allele in the SLCO1B1 gene (-11187
and/or c.521) to patients with the wild-type/wild-type genotype.
1.4. Population Sample The CMH Cardiology Pharmacogenomics Repository (CPR) (CMH IRB#
12040220) database will be accessed to determine subjects meeting inclusion criteria. Once
the target population and an equal set of age-, sex- and Tanner Stage-matched controls are
identified, the patients will be invited to participate in a single center, open label,
randomized, cross-over, pharmacokinetic study of pravastatin and simvastatin.
1.5. Subject Recruitment Once a target population and an equal set of age- and sex-matched
controls are identified from the CPR, prospective participants will be informed about the
study. Initial contact will occur by way of a prepared telephone script. Those that agree to
participate will be scheduled for a visit to the CMH Clinical Research Unit for a screening
visit. At this visit, the study will be explained to the patient/patient's family,
permission/assent/consent will be obtained, a physical examination (including Tanner
staging), and screening laboratory testing will be performed. Participants for whom
permission/assent/consent has been obtained and inclusion criteria are met, will be scheduled
to come back to the CMH Clinical Research Unit within 6 to 14 days for the first study drug
1.6. Subject Withdrawal Participation in the pharmacokinetic trial of pravastatin and
simvastatin is completely voluntary. As part of the informed permission/assent/consent
process; subjects are informed that they can withdraw their permission/assent/consent at any
time. If at any time, study subjects have social, philosophical, religious or family concerns
related to the study, they may decide to withdraw permission/assent/consent.
A study subject or an authorized third party (PCP) may revoke permission/assent/consent at
anytime. The request to revoke permission/assent/consent may be made verbally or in writing
and addressed to study personnel (as indicated in the permission/assent/consent form). Study
personnel will take appropriate steps to respect the will of the study subject and ensure
that the study subject is able to withdraw without having a compromise in personal integrity
or medical evaluation/treatment. In the event permission/assent/consent to continue is
withdrawn after partial collection of samples or data, only existing data will be analyzed
and tabulated. The subject can be withdrawn from the study by the investigators, if at any
point, in the judgment of the investigators, that the subject's continued participation may
be associated with the production of unanticipated risks (e.g., a late onset hypersensitivity
reaction following administration of the study article).
1.7. Subject Study Visits The study will involve three total visits. One screening visit and
2 drug study day visits.
Screening Visit (Approximately 1 hour) -Upon arrival for the screening visit, the study will
be reviewed in depth with the participating subject and subject's parents/legal guardian when
applicable. They will be provided sufficient time to review the permission/assent/consent
form, have all questions answered, and concerns addressed. If they agree to participate, they
will sign the permission/assent/consent form.
-Following permission/assent/consent, screening labs will be drawn. If the patient is unable
to complete the study labs at this time, a time will be arranged to have these performed at
least 1 day prior to Study Drug Day #1. The laboratory evaluation will consist of the
1. Research chemistry panel (Sodium, Potassium, Chloride, Carbon Dioxide, Anion Gap,
Calcium, Phosphorus, Magnesium, Glucose, BUN, Creatinine, Total Protein, Albumin,
Bilirubin, ALT, AST, Alkaline Phosphatase, GGT, LDH, Cholesterol, Uric Acid and
Triglyceride) (3 mL blood in mint green or red top with yellow ring gel tube)
2. Complete blood count with differential (1 mL in a 3 mL EDTA lavender top tube or 250 uL
in a microtainer)
3. Creatine phosphokinase (0.2ml in gel mint green or red top w/ gold ring)
4. C-reactive protein (1 ml of blood in a mint green or red top tube w/ yellow ring gel; or
-If the patient is currently taking a statin, they will be asked to discontinue this
agent until after the completion of the entire study.
-Finally, scheduling of the subsequent study days will take place at this visit. Study
Drug Day #1 will be scheduled 6 days and up to 14 days following the screening visit.
Study Drug Day #2 will be scheduled 6 days and up to 14 days following Study Drug Day
Drug Study Day #1 (Approximately 10 hours) -Upon arrival to the Children's Mercy
Hospital Clinical Research Unit, the subject will have a screening physical examination
including Tanner staging. For females, a urine specimen will be obtained for beta-human
chorionic gonadotropin pregnancy test.
-Once cleared to participate, the subject will have an indwelling, silastic cannula (21
or 24 Ga) placed in a peripheral vein on the hand or forearm for the purpose of repeated
blood sampling to support the pharmacokinetic aims of the study. Patency of this cannula
will be maintained throughout the study period using a sterile solution of heparinized
-A single oral dose of either simvastatin or pravastatin will be ingested with 150ml of
tap water after an overnight fast. The sequence of statin administered will be
randomized between the two study visits. For two hours following study article
administration, subjects will remain fasted with only small sips of water permitted. A
standard, age-appropriate heart healthy meal will be served 2 hours after statin
administration and a snack will be served in the afternoon of the study day. Water will
be permitted ad libitum.
- In the event that the patient vomits within 2 hours of ingestion of Study Drug Day
#1, they will be withdrawn from the study for that day and rescheduled. The subject
will be rescheduled up to 14 days after the screening visit. If they are unable to
be rescheduled within 14 days of the original screening labs, the labs will be
redrawn that day. In the event that the patient vomits within 2 hours of ingestion
of Study Drug Day #2, they will be withdrawn from the study for that day and
rescheduled. The subject can be rescheduled 6 days and up until 14 days after Study
Drug Day #1. If a more than 14 days, but no more than 21 days have transpired since
Study Drug Day#1, the subject can be rescheduled, but must have repeated screening
- Serial venous blood samples (1.5 ml each) will be drawn from the indwelling venous
cannula to measure plasma drug and drug metabolite levels of the ingested drug
before ingestion (time 0), and at 0.5, 1, 1.5, 2, 3, 4, 6, and 8 hours post
ingestion. Should the patency of the indwelling cannula be lost, it will either be
placed or alternatively, remaining blood samples will be obtained by sterile
venipuncture at the desire of the study subject. After the samples are drawn, they
will be placed into glass tubes containing potassium EDTA and gently mixed by
repeated inversion. They will then be immediately centrifuged at 4ºC for a total of
10 minutes at 600g (~2000RPM) and will immediately be placed in an ice bath to be
transported to the Clinical Pharmacology Laboratory where they will be processed.
The cellular component of the time 0 samples will be retained for future isolation
of leukocyte DNA. The plasma component of each of the repeated blood sample
obtained to support the pharmacokinetic objectives of the study will be removed by
manual aspiration and transferred to a labeled, polypropylene cryovial which will
be placed at -80ºC where it will be stored until analysis.
- Once the 8 hour post-dose sample is taken, blood (5.2 ml) for study safety labs
will be drawn, the IV cannula will be removed, and the patient will be discharged
from the Clinical Pharmacology unit after the Drug Study Day #2 is confirmed with
the study subject and/or subject's parent(s).
Drug Study Day #2 (Approximately 10 hours) -Following a minimum of 6 days and up to 14
days after the first drug ingestion day (wash out period), the subject will brought back
to the CMH Clinical Research Unit for another single dose pharmacokinetic study.
Following a physical examination and insertion of a peripheral IV cannula, the alternate
statin that was not administered on the first drug study day will be given with 150 ml
tap water after an overnight fast. The same blood sampling scheme will be used as
described for study day #1. Blood for study safety labs will be obtained after the last
drug level sample is drawn and the patient will then be discharged from the CMH Clinical
Research Unit. The total amount of blood required for the entire study is summarized in
the following table.
Study Day Amount of Blood Screening Visit 5.2ml (Screening Labs) Drug Study Day #1
13.5ml (PK study labs) 5.2ml (Safety labs) Drug Study Day #2 13.5ml (PK study labs)
5.2ml (Safety labs)
Total 42.6ml (Over a 1 month period of time)
One week after completion of Drug Study Day #2 or in the event of early withdrawal, the
family will be contacted via telephone to screen for post-study adverse events as
defined section 5.
1.10. Randomization The proposed study is a single-dose pharmacokinetic study comparing
two statin drugs, simvastatin and pravastatin. The sequence of administration of the two
statins will be randomized for each study participant.
The Children's Mercy Hospital Investigational Drug Service (IDS) Pharmacy will be
responsible for the randomization procedure and will maintain control of the sequence
randomization. This is an open label study, so neither the investigators nor subjects
will be blinded to the sequence randomization.
2. Laboratory Evaluation 2.1. Drug and Metabolite Analysis After ingestion of a single statin
dose, the corresponding drug and its metabolites will be quantified. Pravastatin is
administered in its active, open acid form and does not have any significantly active
metabolites 27. The most common inactive metabolite isolated in plasma is 3'α-isopravastatin
and it is formed non-enzymatically primary in the acidic conditions of the stomach 53.
Therefore, after pravastatin ingestion, serial plasma levels of pravastatin acid and
3'alpha-isopravastatin will be assessed. (Additional details of plasma sampling intervals are
outlined in Section 1.9). Simvastatin is administered in its inactive, lactone form and
undergoes hydrolysis to the major active metabolite which is beta-hydroxy simvastatin acid.
Minor metabolites are 6'-hydroxy-simvastatin, 3'hydroxy-simvastatin, 3''-hydroxy-simvastatin,
6'exomethylene-simvastatin, 6'-hydroxymethyl-simvastatin, and 6'-hydroxycarbonyl-simvastatin
25. Several of these metabolites, when converted to their acid forms, have reported
activities in dogs but seem to accumulate in the bile with little detectable in plasma 54.
Therefore, in this study, only serial plasma levels of simvastatin and simvastatin acid will
be assessed. (Additional details of plasma sampling intervals are outlined in Section 5.0).
Analytical strategies for the determination of simvastatin and simvastatin acid:
Simvastatin and simvastatin acid will be determined using a high-throughput salting-out
assisted liquid/liquid extraction (SALLE) method with acetonitrile and mass spectrometry
compatible salts for simultaneous LC-MS/MS analysis as previously described 55. This method
has been extensively validated according to FDA guidelines, and more importantly,
demonstrates minimal interconversion of simvastatin and simvastatin acid during sample
preparation. A brief outline of the method is as follows. Plasma samples will be thawed at
4ºC in a temperature-controlled water bath, and kept at this temperature during the procedure
to minimize analyte interconversion. To an aliquot of sample, a stable deuterated
isotope-labeled internal standard of simvastatin and simvastatin acid (Toronto Research
Chemicals) are added to allow stable isotope dilution mass spectrometry. The sample is then
extracted by the SALLE procedure at pH 4.5 to minimize the interconversion of simvastatin
between the lactone and acid form. The extract is stable in the autosampler for 33 hours at
4ºC, and will be analyzed by LC-MS/MS within this timeframe. Sample analysis will occur on a
Waters TQ-S triple quadupole mass spectrometer coupled to a Waters Acquity Ultra Performance
Liquid Chromatograph (UPLC-MS-MS), utilizing the chromatographic conditions (including
column) as previously described 55. Simvastatin and its internal standard will be monitored
in positive ion-mode utilizing the 450-285 and 453-285 transitions of the methyl ammonium
adducts respectively. Simvastatin acid and its internal standard will be measured in negative
ion-mode due to poor ionization in positive mode. Transitions for these analytes will be
435-319 and 438-319. All plasma samples will be run in duplicate for each sampling time
interval to assure validity. The remaining plasma not used in the analysis will be stored
within the Clinical Pharmacology laboratory in dedicated freezers/refrigerators as outlined
in the CPR Repository. Use of these samples for further study will be at the discretion of
the CPR director as outlined in the CPR protocol (CMH IRB# 12040220).
Analytical strategies for the determination of pravastatin and pravastatin lactone:
A large number of analytical strategies for the determination of pravastatin and pravastatin
lactone have been described in the literature 56-59, however many of these are labor
intensive and not as high throughput as the methodology presented for simvastatin. Due to the
structural similarities between simvastatin and pravastatin it is highly likely that the
analytical method for simvastatin can be adapted for the analysis of pravastatin and
pravastatin lactone with minimal change. In the unlikely event that the simvastatin method
cannot be adapted to pravastatin and pravastatin lactone analysis, we will utilize the method
presented by Vlckova et al 60.
3 Assessment of Pharmacokinetics 3.1 Pharmacokinetic Parameters Pharmacokinetic analyses will
be conducted using Kinetica version 5.0 (Thermo Electron, Philadelphia, PA). Pravastatin,
simvastatin, and metabolite plasma concentration vs. time data will be curve fit using a
peeling algorithm to generate initial monoexponential parameter estimates. Final estimates of
the terminal elimination rate constant (delta z) will be determined from an iterative, linear
least squares regression algorithm. A model-independent approach will be used and parameters
of interest determined as follows. Individual Cmax and Tmax will be obtained by direct
examination of the plasma concentration versus time profile. The area under the plasma
concentration versus time curve during the sampling period (AUC0-n) will be calculated using
the mixed log-linear method where n refers to the final sampling time with quantifiable drug
or metabolite concentrations. Extrapolation of the AUC to infinity (AUC0-infinity) will be
achieved by the summation of AUC0-n + Cpn/delta z, where Cpn is the last observable plasma
concentration calculated from the curve fit of the terminal slope of the plasma concentration
vs. time curve and delta z is the apparent terminal elimination rate constant. Absorption and
elimination half-life (t½) will be calculated from the initial and terminal portions of the
plasma concentration vs. time curve for each analyte of interest. In the case of metabolites,
the corresponding half life term will be that used to denote metabolite formation.
4 Statistical Plan 4.1 Statistical Methods Pravastatin and simvastatin pharmacokinetic data
for the study cohort will be examined initially using standard descriptive statistics (i.e.,
arithmetic mean, geometric mean, standard deviation, coefficient of variation, 95% confidence
limits). A repeated measures analysis of variance model will be employed to determine
treatment, period, and sequence effects for the resultant primary and secondary
pharmacokinetic parameter estimates. Pharmacokinetic parameters reflective of exposure (Cmax,
AUC0-n, AUC0-infinity) will be log-transformed using the natural logarithm, the 90%
confidence intervals for the difference in the means calculated and the antilog of the
confidence limits evaluated against the pre-established bioequivalence criteria as defined in
our power calculations. The significance limit accepted for all statistical analyses will be
alpha = 0.05.
The contribution of different alleles to variation in the pharmacokinetic parameters of
pravastatin and simvastatin will be investigated with multiple regression analysis. The
genotype class (non-carriers and carriers) will be treated as independent variables. For Aim
#1, the pharmacokinetic variables for each drug will be compared between non-carriers and
carriers of variant alleles using independent t-test. Similarly, for Aim #2, the ratio of
each pharmacokinetic parameter in carriers to non-carriers (e.g. AUCcarrier/AUCnon-carrier)
for pravastatin will be compared to the corresponding ratios following simvastatin
administration. All analyses will be performed in SPSS version 18.0 (SPSS, Chicago, IL).
4.2 Subject Population(s) for Analysis Previously studies have shown that highly hydrophilic
statins, such as pravastatin, are more dependent on the OATP1B1 transporter compared to more
lipophilic statins, such as simvastatin 41, 45. It is theorized that the upstream promoter
region (-11187) might have a more pronounced impact on the genotype-phenotype relationship as
seen in the previous pediatric study 49. Therefore, our study will be powered to observe a
dose-exposure difference in -11187 allelic variants and wild-type/wild-type with pravastatin.
There are anticipated to be 150 subjects enrolled in the CPR database meeting inclusion
criteria. The frequency of SLCO1B1 SNPs (-11187G>A, 7-8%) from previous adult studies 61,
suggests approximately ~19-22 heterozygous subjects (according to the Hardy Weinberg
Equation) in the anticipated number of participants from the CPR database. With a 90%
participation/10% attrition rate, there would be ~17-20 subjects.
On the basis of previous data on the adult pharmacokinetics of pravastatin in the variant
alleles described above 45, a sample size of 18 in each group (-11187G>A genotype and
wild-type control) will have 80% power to detect a difference in means of -101.0 in AUC (0-∞)
(the difference between a Group 1 mean, m1, of 102.7 and a Group 2 mean, m2, of 203.7, which
is nearly a 2-fold difference) assuming that the Group 1 standard deviation, s1, is 69.1 and
the Group 2 standard deviation, s2, is 126.4 using a two group Satterthwaite t-test with a
0.050 two-sided significance level.
Therefore, our target sample size is 18 subjects with SLCO1B1 -11187G>A allele. With age- and
sex-matched controls, the total sample size is 36 subjects.
5. Adverse Events An adverse event is defined as any unintended change in the body structure
(signs) or body function (symptoms), whether or not considered drug-related. During the
entire duration of the study, subjects will be instructed to report all adverse events. All
adverse events, whether volunteered, elicited, or noted on physical examination, will be
recorded throughout the study, (i.e., from the time of the first dose administration on Day 1
up to 1 week after the entire study completion or subject withdrawal from the study). The
severity of adverse events will be categorized as mild, moderate, or severe according to FDA
The Investigator will make a determination of the relationship of the adverse event to the
study drug using a four-category system (not related, unlikely, suspected, and probable)
according to FDA guidelines. A serious adverse drug experience is any adverse test material
event occurring at any dose that results in any of the outcomes defined by the FDA.
An unexpected adverse experience is any adverse experience that is not identified in nature,
severity, or frequency in the current approved drug labeling or elsewhere in the general
investigational plan. Any serious and unexpected adverse clinical event, whether or not
related to the study drug, will be reported verbally to the primary investigators within 24
hours of notification.
Subsequently, a Serious Adverse Event (SAE) Report Form will be completed and Children's
Mercy Hospital and Clinics Institutional Review Board (IRB) will be notified. A statement
from the Principal Investigator outlining the details of the adverse reaction will accompany
this form. Laboratory reports and other safety reports will be forwarded as they become
NOTE: The term "severe" is often used to describe the intensity (severity) of a specific
event (as in mild, moderate, or severe myocardial infarction); the event itself, however, may
be of relatively minor medical significance (such as a severe headache). This is not the same
as "serious," which is based on participant/event outcome or action criteria usually
associated with events that pose a threat to the participant's life or functioning.
Seriousness (not intensity) serves as a guide for defining regulatory reporting obligations.
Ethical aspects This study will be conducted according to US and international standards of
Good Clinical Practice (FDA regulations 21 CFR 312 for IND studies and FDA guidance E6) for
all studies. Applicable government regulations and University of Missouri-Kansas
City/Children's Mercy Hospital and Clinics research policies and procedures will also be
This protocol and any amendments will be submitted to the Children's Mercy Hospital and
Clinics Institutional Review Board (IRB) for formal approval to conduct the study. The
decision of the IRB concerning the conduct of the study will be made in writing to the
All subjects and parent/legal guardians, when applicable, will be provided a
permission/assent/consent form describing this study and providing sufficient information for
subjects to make an informed decision about their participation in this study. This consent
form will be submitted with the protocol for review and approval by the IRB. Formal
consent/permission/assent will be obtained, using the IRB-approved forms, before a subject is
submitted to any study procedure. The consent/permission/assent form must be signed by the
subject or legally acceptable surrogate, and the investigator-designated research
professional obtaining the consent.