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The pharmacokinetics and pharmacodynamics of vincristine in the adolescent and young adult population compared to younger patients
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The pharmacokinetics and pharmacodynamics of vincristine in the adolescent and young adult population compared to younger patients
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Content
THE PHARMACOKINETICS AND PHARMACODYNAMICS OF VINCRISTINE IN
THE ADOLESCENT AND YOUNG ADULT POPULATION COMPARED TO
YOUNGER PATIENTS
by
Leidy L. Isenalumhe
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(CLINICAL AND BIOMEDICAL INVESTIGATIONS)
AUGUST 2015
Copyright 2015 Leidy L. Isenalumhe
TABLE OF CONTENTS
Abbreviations iii
List of Tables iv
List of Figures v
Abstract vi
Chapter 1: Introduction 1
Chapter 2: Methods 10
Chapter 3: Data Analysis 22
Chapter 4: Preliminary Results 26
Chapter 5: Preliminary Discussion 33
References 34
Appendix A: Pediatric Modified Total Neuropathy Scale 36
Appendix B: USC RedCap Data Collection 39
ii
ABBREVIATIONS
AAPC Averaged annual percentage change
AIC Akaike Information Criterion
ALL Acute lymphoblastic leukemia
AMC Amino-4-methylcoumarin
AUC Area under the curve
AYA Adolescent and Young adult
CCI Cancer Center internal review board
CEP72
CMT1A Charcot-Marie Tooth Disease
CNS Central Nervous system
COG Children’s Oncology group
CTCAE
4.0
National Cancer Institute Common Terminology Criteria for Adverse
Events, version 4.0
CYP Cytochrome p450
CYP3A Cytochrome p450 3A
CYP3A5 Cytochrome p450 3A5
HR-ALL High Risk Acute Lymphoblastic Leukemia
InsP3R inositol 1,4,5-triphophate receptor
M1 Vincristine metabolite 1
M2 Vincristine metabolite-2
NCS-1 Neuronal calcium sensor-1
PBS Phosphate buffered solution
PD Pharmacodynamics
PK Pharmacokinetics
PMP22 Peripheral myelin protein 22
PRG Progress review group
SEER Surveillance Epidemiology and End Results
SR-ALL Standard Risk Acute Lymphoblastic Leukemia
TCEP Tris (2-carboxyethyl) phosphine
VCR Vincristine
VRNT Vincristine related neuropathy
iii
LIST OF TABLES
Table 1: Genetic Variations of Cytochrome P450 3A5
Table 2: Specimen Time Point
Table 3: Mass spectrometer setting for each of the analytes
Table 4: Patient demographics in categorized in Tanner Stage
Table 5: Race/ethnicity of patient enrolled in this study
Table 6: Vincristine pharmacokinetics by Tanner Stage
Table 7: Future analysis
iv
LIST OF FIGURES
Figure 1: Study Schema
Figure 2: Distribution of patients by age of enrollment, Tanner Stage and Sex
Figure 3: Effect of Tanner Stage on Vincristine AUC
Figure 4: Effect of age on Vincristine AUC
Figure 5: Effect of sex on Vincristine exposure (AUC)
Figure 6: Effect of BMI on Vincristine AUC
Figure 7: Vincristine AUC and phase of therapy
Figure 8: Vincristine AUC versus antifungal use
v
ABSTRACT
Improvement in survival from many forms of cancer among the adolescent and young
adult (AYA) population has lagged behind that of younger patients. One contributing
factor may be differences in tolerance of cancer treatments that lead to reduced adherence
and efficacy. It has been shown that the AYA population has an increased prevalence of
vincristine related neuropathy (VRNT), where dosage attenuation and/or increasing the
dosing intervals which may decrease their cancer survival and reduced quality of life.
Previous studies evaluated the relationship of age and vincristine pharmacokinetics (PK)
have resulted in conflicting conclusions. These studies demarcated the AYA population
as >10 years of age, which not only included adolescents and young adults, but also
young children and pre-adolescent children. The proper characterization of this age group
is critical, since developmental changes, as characterized by the Tanner staging, are
associated with physiological variation that could significantly affect drug distribution
and metabolism. Additionally, there are currently no means for predicting VRNT in any
patient, regardless of age.
We hypothesize that the pharmacokinetics/pharmacodynamics of vincristine
differs between AYA and younger children, which may contribute to the excess VRNT
found in the AYA population. One of our aims is to show that the PK of vincristine and
its metabolite M1 will differ between young children and AYA as defined by Tanner
stage. This is a novel way to define AYA compared with previously published VCR PK
studies. Additionally, we hypothesize that calpain, a calcium-dependent protease that is
believed to be associated with calcium mediated cell injury in the nervous system can
vi
serve as a biomarker predicting for VRNT in this age group. Hence, compared to
baseline, serum calpain will increase after administration of vincristine. Changes in
serum calpain, particularly if correlated with vincristine and/or M1 PK, would support
further study of calpain as a biomarker of VRNT.
vii
CHAPTER 1
INTRODUCTION
The Adolescent and Young Adult (AYA) Progress Review Group (PRG) define
the AYA population as ages 15 to 39. In this age group, about 68,000 people were
diagnosed with cancer in 2002, an incidence rate approximately eight times more than
children under age 15
1
. Historically, improvement in survival for many forms of cancer
among the adolescent and young adult (AYA) population has lagged behind that of
younger patients. According to the Surveillance Epidemiology and End Results (SEER)
Cancer Statistics Review 1975-1997, improvement in 5-year survival, as reflected in the
average annual percent change (AAPC) in US cancer mortality rates was significantly
lower among patients ages 15 to 39 compared with ages 1 to 14 years
1
.
Reasons for this disparity remain uncertain and largely unexplored. One
contributing factor to this disparity may be differences in tolerance of cancer treatments
that lead to delays in the administration of chemotherapy or increase in the length of
dosing interval, where one or both of these strategies may reduce efficacy and adherence.
Recent studies involving a variety of pediatric cancers have documented increased
treatment-related toxicity amongst AYA vs. younger children, suggesting the problem of
age-dependent differences in chemotherapy tolerance is both pervasive and clinically
important.
Vinca-alkaloids, such as vincristine, are widely use chemotherapy agent against
various pediatric malignancies. By binding to tubulin, vincristine inhibits microtubule
1
assembly leading to abortion of mitosis and subsequent apoptosis
2
. Notably, microtubules
are essential in axonal transport. Among vinca-alkaloids, vincristine has the highest
binding affinity onto tubulin; hence it is the most neurotoxic
3
. Accordingly, the dose-
limiting effect of vincristine is neurotoxicity, which limits general maximum dosage to
2mg. However, its vast utility, and pervasiveness in various chemotherapeutic pediatric
regimens, make it an exemplary component for examination to better understand side
effect profiles and the effect on prognosis for chemotherapeutic agents at large.
Vincristine is eliminated by hepatic-mediated metabolism, via the cytochrome
p450 3A (CYP3A) family of enzymes. It is metabolized to primary inactive metabolite
M1, with minor metabolites M2 and M4
4,5
. It is preferentially metabolized by CYP3A5
compared to CYP3A4. Vincristine clearance is higher in CYP3A5 expressers than non-
expressers
4,5
. Reduced vincristine exposure as measured by area under the concentration-
time curve (AUC) has correlated with lower incidence neurotoxicity. In a recent study it
was shown that CYP3A5 expressers experience less vincristine related neurotoxicity
(88% vs 100%, P=0.035) and shorter length of their treatment course with neurotoxicity
6
.
The chart below summarizes the most important genotype/phenotype relationships for
CYP3A5 Alleles and CYP3A5 expression
5
.
6
Table 1: Genetic variation of Cytochrome P450 3A5.
Allele Phenotype
CYP3A5∗1/∗1 (homozygous) High CYP3A5 expression
CYP3A5∗1 (heterozygous) High CYP3A5 expression
2
This may explain the difference in vincristine-related neurotoxicity (VRNT) by
race/ethnicity, with Caucasians having higher rates of VRNT compared to African
American
7
. African Americans have a higher percentage of CYP3A5 expression when
compared to other ethnicities
5
.
There is a greater incidence and severity of vincristine-related neurotoxicity
(VRNT) reported in AYA patients. Recently published results from the Fourth Intergroup
Rhabdomyosarcoma Study (IRS-IV) showed a significantly higher incidence of VRNT in
patients ages 10 to 14 years compared with those ages 5 to 9 years (OR 2.8, p < 0.001),
and even higher incidence in those ages 15 to 20 years (OR 4.18, p < 0.001). This led to
dose reductions for patients >10 years of age
8
. In another study describing VRNT in
children treated for rhabdomyosarcoma or Wilms Tumor, Cox Regression analysis was
used to evaluate the relationship between the probability of developing Stage 3 or 4
VRNT as a function of cumulative dose (mg/kg). With increasing age, there was a steady
increase in the probability of developing VRNT for a given cumulative dose/kg
(P<0.001)
9
. This difference was only significant in the younger age cohorts ≥2 years of
age.
CYP3A5∗3 /*3 (homozygous) Low CYP3A5 Expression
CYP3A5∗3 (heterozygous) Low CYP3A5 Expression
CYP3A5∗5 Low CYP3A5 Expression
CYP3A5∗6 Very low activity of CYP3A5
CYP3A5∗7 No activity of CYP3A5
3
In a current Children’s Oncology Group (COG) clinical trial comparing standard
vs. dose-intensive vincristine for treatment of relapsed precursor-B acute lymphoblastic
leukemia, AALL0433, patients >13 years experienced excess dose-limiting peripheral
neurotoxicity compared with younger children. In patients >13 years, Grade 3
neuropathy was seen in 1/21 (5%) with standard doses of vincristine (1.5mg/m
2
,
maximum dose 2 mg) vs. 9/26 (35%) on intensified therapy (2mg/m
2
, maximum dose
2.5mg) (p=0.015), while no dose-related difference was observed in patients <13 years
(p=0.245)
10
.
Additionally, genetic variations may lead to higher rates of VRNT in certain
patient populations. A recent publication showed that inherited variant of promoter region
of CEP72 gene was associated with a higher incidence of vincristine-related peripheral
neuropathy
11
. It is well established that patients with the most common type of Charcot-
Marie Tooth Disease (CMT1A), associated with duplication of the peripheral myelin
protein 22 (PMP22) have exacerbation of their neuropathies or unmask their disease after
exposure to vincristine
12,13
.
The exact mechanism for the increased VRNT incidence in this population is not
well delineated, where increase side effects/decrease treatment tolerance may contribute
to dose attenuation/alterations, treatment delays and decreased adherence, which may
then impair overall survival. A better understanding of this difference could lead to more
rationale vincristine dosing and more informed development of therapeutic strategies for
AYA patients. Given the prominent role vincristine currently plays in treatment of AYA
cancers including ALL, lymphomas, soft tissue sarcomas and Ewing sarcoma, its
4
anticipated impact could be substantial. A better understanding of this difference could
lead to more rationale vincristine dosing and more informed development of therapeutic
strategies for AYA patients.
The pharmacokinetics studies of vincristine in adults and children have shown
large interpatient and intrapatient variability
14
. Only two published studies have
compared vincristine pharmacokinetics (PK) in young children and AYA, and the two
studies had conflicting outcomes. The first study included 54 patients, divided into 2
groups based on age. They evaluated vincristine PK in 45 children <10 years of age and 9
patients ≥10 years of age and found that when vincristine clearance was normalized to
body weight, there was a statistically significant difference between the two groups (20.4
ml/min/kg vs. 9.4 ml/min/kg, p=0.024), and that the clearance in both groups was faster
than the published rate in adult patients
15
. The second study included 98 patients and
again used 10 years of age as the cut-off between young children (n=72) and adolescents
(n=26); no association between age and vincristine clearance was detected
16
. In both of
the above studies, adolescence was defined as >10 years of age. However, the mean age
for initiation of gonadarche is 11 and 11.5 to 12 years in girls and boys, respectively
17
.
This is critical because the previous studies likely included patients 10 years or older
whose developmental physiology was more consistent with childhood.
In order to further understand this observation, for which we may be able to
develop preventive strategies to reduce the development of neuropathy, we will employ
Tanner Staging rather than an arbitrary age breakpoint to stratify patients because AYA
physiology differs dramatically from that of younger children. Developmental changes,
5
as characterized by the Tanner staging system rather than age, are associated with
changes in body composition, increased liver size, and increased levels of growth
hormone, all which may significantly affect drug distribution and metabolism
18,19
.
Several studies have shown that increased levels of hormones such as estrogen
and androgen are associated with reduction in phase I enzyme function. One particular
study demonstrated a Tanner stage-dependent decrease in caffeine clearance by the phase
I enzyme CYP1A2
20
. This suggests that puberty may lead to alter expression of these
types of enzymes.
Although VRNT is common and potentially debilitating side effect and has higher
rates among the AYA population, there is currently no established biomarker for VRNT.
The mechanism of action of vinca alkaloids is the inhibition of polymerization of
microtubules. For some time it was thought that this inhibition, which also occurs in
axons, was the major mechanism of VRNT. Recent research has been conducted to better
characterize the pathways involved in VRNT as well as the neuropathy induced by
paclitaxel, a vinca alkaloid derivative with a mechanism of action similar to vincristine.
One of the major pathways involves neuronal calcium sensor-1 (NCS-1), a calcium
binding protein mainly expressed in neurons and neuroendocrine cells, and the calcium-
dependent protease, calpain. Binding of vincristine or paclitaxel to NCS-1 enhances
intracellular calcium signaling through the interaction of NCS-1 with the inositol 1,4,5-
triphosphate receptor (InsP3R) and modulates the function of the InsP3R in a calcium-
dependent manner. Treatment with paclitaxel or vincristine also activates calpain which
cleaves a number of substrates, including NCS-1
21–25
. This cleavage of NCS-1 results in a
6
decrease in the activity of the InsP3R, resulting in decreased generation of intracellular
calcium signals, which induces axonal injury thereby causing peripheral neuropathy
21–25
.
It has been shown that the addition of a calpain inhibitor to neuroblastoma cells treated
with paclitaxel normalizes NCS-1 and intracellular calcium levels. The same study
examined the effects of inducing a mutation in the calpain binding site on the NCS-1
protein. By inhibiting calpain inactivation of NCS-1, normal calcium levels were
maintained
23
. In a murine model, the calpain inhibitor AK295 reduced histologic
evidence of paclitaxel dose-dependent axonal degeneration in dorsal root ganglia by
approximately 50%. AK295 also protected against neuropathy in the mice as measured
by behavioral and electrophysiologic testing
22
.
Other researchers used vinblastine to promote axonal injury in retinal ganglion
cells in mice, and showed that the axonal damage was prevented by inhibiting calpain
activity
25
. Calpains are ubiquitous cytosolic proteolytic enzymes involved in both
physiological and pathological cellular functions. Pathological cellular insults lead to
more generalized calpain activation, which leads to cell death. It is believed to be
associated with calcium mediated cell injury in ischemic strokes
21–23
. In summary, there
are in vitro and animal data to suggest that calpain is a potential biomarker for
neurotoxicity. Changes in serum calpain, particularly if correlated with vincristine and/or
M1 PK, would support further study of calpain as a biomarker of VRNT.
We summarized how VRNT is a pervasive issue among the AYA cancer
population. Finding a way to minimize these side effects may have positive impact on the
7
overall morbidity and outcome on this high risk population. Stratifying the AYA
population by Tanner stage may lead to a more appropriate physiological classification.
Hypothesis/Specific Aims
We hypothesize that the pharmacokinetics (PK) and/or the pharmacodynamics
(PD) of vincristine differ between AYA and younger children and contribute to the
observed excess VRNT in the AYA population.
Specific Aim 1: Develop a non-parametric population model of vincristine and M1
PK in children and adolescents.
Hypothesis: The PK of vincristine and its metabolite M1 will differ between young
children and AYA as defined by Tanner stage. We hypothesize that the vincristine
clearance will be lower in AYA population compared to younger children. Additionally,
AUC will be higher in the AYA population when compared to younger children.
The goal of this study is to evaluate the concentration of vincristine in relations to
the production of primary metabolite M1 and M2 over time and to determine if there is a
difference in pharmacokinetics (PK) among Tanner Stages. This study also examined the
PK of vincristine and M1 and M2. For the first time, patients will be classified based on
their physiologic maturity (Tanner Stage)—rather than age—to determine whether
having a more adult phenotype is associated with prolonged vincristine exposure. This
will increase our likelihood of detecting a developmentally-driven difference in
vincristine metabolism and, if successful, provide evidence that Tanner staging is a more
reliable method for classifying adolescents for purposes of chemotherapy dosing. Further,
8
we will not only characterize vincristine PK differences between the two groups, but will
for the first time examine differences in the PK of M1 and M2.
Specific Aim 2: Measure repeated serum calpain with initiation of vincristine and
again after four weeks of therapy.
Hypothesis: Compared to baseline, serum calpain, which is associated with
neurodegeneration will increase after administration of vincristine. We hypothesize that
there is an association between calpain and vincristine administration, as well as
vincristine induced peripheral neuropathy. An association between Vincristine/M1 PK
and calpain would substantiate further investigation of calpain as a biomarker of VRNT
and development of risk models of VRNT.
9
CHAPTER 2
METHODS
The study was set up as a cross-sectional pilot study. The length of the study for
each patient was 4 weeks. The list of evaluation at each time point is displayed in Table
2. Patients were enrolled while they were either in the Induction or Maintenance Phase of
therapy and stratified based on phase. Week 1 of the study included a baseline
neurological assessment, PK lab processing, Calpain lab assessment and Tanner Stage
assessment. Week 4 of the assessment included neurological examination, Calpain level
and genetic testing (Refer to Table 2 and Figure 1).
Due to the small sample size of this pilot study we wanted to ensure we recruited
patients with specific characteristics in order to be able to analyze our hypothesis and
specific aims. We employed purposeful sample selection for patients with the following
characteristics: History of VRNT, No history of VRNT, equal number of male, female
and Tanner Stage categories.
Patients
Recruitment took place from September 2014 to March 2015. Patients were
approached for participation in to the study if they met the following inclusion criteria;
diagnosis of Standard Risk Acute Lymphoblastic Leukemia (SR-ALL) or High Risk
Acute Lymphoblastic Leukemia (HR-ALL) actively treated at Children’s Hospital of Los
Angeles, 1-24 years of age, Induction (newly diagnosed) or Maintenance CR1 phase of
10
therapy, scheduled to receive vincristine as part of their treatment regimen. The
exclusion criterion was lack of central line in induction phase.
Eligible patients were identified from the hospital inpatient service, investigators
clinical practice and clinic schedules of the oncology staff. The inpatient and outpatient
patient list were reviewed daily for potential patients. The study and consent forms were
approved by the hospital and Cancer Center internal review board and designated IRB
number CCI-14-0092 and CCI 14-005, respectively. The clinical trial was registered at
ClinicalTrials.gov and designated the following identifier, NCT02360930. Written
informed consent was obtained from each patient and assent was obtained from patients
when deemed necessary. A total of 40 patients were approached for enrollment of which
30 agreed to enroll in the study.
Patients with SR-ALL were treated per Children’s Oncology group protocol
AALL0932 and for HR-ALL via AALL1131. Patients were treated as either on study or
off study based on family and primary oncologist joint preference. Vincristine was
administered at standard dose of 1.5 mg/m
2
, with max dose of 2mg via intravenous push.
Each patient received vincristine weekly x 4 if they were currently in Induction phase or
monthly if currently in Maintenance phase of therapy. Additional chemotherapy agents in
the induction phase and around the time of the scheduled specimen collection, included
intrathecal cytarabine and intrathecal methotrexate, prednisone or dexamethasone,
pegasparginase and daunorubicin if stratified as HR-ALL. In patients in the maintenance
phase, additional chemotherapy agents included mercaptopurine, methotrexate and
intrathecal methotrexate.
11
Figure 1: Study Schema
Table 2: Specimen collection time point
Week 1 Week 4
Timed Vincristine PK blood samples
Timed Calpain levels
Baseline Neurological assessment
Tanner stage examination
Timed Vincristine PK blood sample
Timed Calpain Levels
Genetic blood testing
Neurological assessment
12
Tanner Staging
Permission was obtained from the parent and the subject to perform Tanner
Staging assessment. To avoid inter-observer variability, a single investigator performed
the Tanner Staging examination at week-1 of the trial. Patients were classified into
Tanner Stage ≤2 and Tanner Stage ≥4 stage using standard Tanner Stage criteria
26
. Since
the Tanner Staging examination took place at time of vincristine PK blood collection, the
investigator was blinded to the PK results while conducting the examination and
recording results of Tanner Stage.
Vincristine Pharmacokinetics Measurements
The pharmacokinetic (PK) measurements were obtained around a single
vincristine dose. For patients in the Induction phase of therapy, PK measurements were
obtained around the first vincristine dose. Blood samples were collected prior to infusion
(0 min), 10 minutes, 30 minutes, 1 hour, 12 hours and 24 hours following the first dose of
vincristine.
For patients in Maintenance phase, the PK measurements were obtained around
one of their scheduled monthly vincristine infusions. We collected timed blood samples
prior to infusion (0 min), 10 minutes, 30 minutes, 1 hour and 24 hours following the dose
of vincristine. In ALL patients in Maintenance, a 12 hour blood sample was not feasible
since these patients were treated in the outpatient setting and were not able to return for
that specified time point.
13
Each specimen was labeled with appropriate patient information (name, MRN,
study ID, collection date and time). We collected 1ml per each time point and placed it in
1.8 mL sodium citrate tube. Each sample was inverted 8-10 times to mix contents
thoroughly and it was then placed on ice slurry immediately.
The specimens were then centrifuge at 1500 x G (~3000 RPM) for 15 minutes at
4°C. The plasma was carefully transferred to a 2mL cryovial. We insured that there was
a minimum plasma volume of 0.5mL. Then it was stored at -70°C. The light blue sodium
citrate tube was stored at 4°C, which was later used for processing of calpain levels.
Synthesis of M1:
Vincristine (12 µL) at 30 µM was preincubated with recombinant CYP3A5 (100 pmol)
and 100 mM Na
2
HPO
4
with 5 mM MgCl
2
, pH 7.4 (total volume 1 mL). The reaction was
initiated with the addition of NADPH (50 mM stock and then dilute to 10 mM, where 10
µL) (0.5 mM final concentration). After 40 min, the incubation was quenched with an
equal volume of acetonitrile, chilled, and centrifuged. The supernatant (100 µL) was
diluted with 0.2 % formic acid and separated by HPLC with UV detection (254 nm),
where the M1 retention was identified at 31 minutes.
Synthesis of M2:
Vincristine (VCR) M2 was synthesized by the oxidizing VCR using horseradish
peroxidase and hydrogen peroxide. For reaction vincristine sulfate 100 µM (200 µL),
was mixed with horseradish peroxidase (0.2 mg Invitrogen), in 100 mM Na
2
HPO
4
pH 7.2
14
buffer (350 µl), and hydrogen peroxide (125 µL), was added to the solution and incubated
at 37°C in a shaking for 2 h. The solution is then extracted with 1 mL methylene chloride
for three times. The organic layer (upper layer) was removed and reextacted for a total of
three separate extraction. The organic extract was consolidated and then dried using a
steady stream of dried nitrogen gas at room temperature. The residue resolved in 100 µL
of methanol, where each injection was 50 µl to separate the M2 using HPLC using a
DAD detector set at 254 nm. The eluent fraction with a retention time of 48 minutes was
collected and the entire collection was dried to yield M2. Standard curve of used
vincristine to correlate the concentration of M2.
Isolation of Vincristine Metabolites:
After biosynthesis of either M1 or M2, the metabolites were isolated using a gradient
HPLC separation method. An Agilent1100 Series (Agilent Technologies) HPLC linked
onto a diode array detector was set to 254 nm. M1, M2, vincristine, and vinorelbine
(internal standard) were achieved using a C18 column (Phenomenex, Torrance, CA) at a
flow rate of 0.4 ml/min.
The mobile phase consisted of 0.2% formic acid (mobile phase A) and methanol (mobile
phase B). Analytes were eluted using a series of linear gradients: 0 min/20% B, 7
min/20% B, 42 min/56% B, 42.1 min/80% B, 52 min/80% B, 52.1 min/20% B. The
analytes were monitor at a wavelength of 254 nm. =
15
Sample preparation for Mass spectrometry:
To 50 µL of plasma, an aliquot of 50 µL of the internal standard (vinblastine) was added
and vortex thoroughly. The sample was extracted by adding 350 µl of methanol and
vortexed thoroughly. The samples spin at 13,000 rpm for 10 min and the supernatant was
transferred into a fresh tube. The supernatant was evaporated to dryness by stream of
filtered dry air, and reconstituted in 50 µl of 80% methanol with 0.1% formic acid.
Plasma Quantification of Vincristine and Metabolites:
Extract plasma samples were quantified for vincristine, M1, and M2 using a
metabolomics assay where all of the analytes are quantified using a validated LC-MS
assay. The reconstituted sample (50 µL) was injected into a Shimadzu Prominence LC
system coupled onto a Sciex API 4000 mass spectrometer (Applied Biosystems) with an
electrospray Turboionisation (ESI) interface. The analytes were separated using a C18
column (Hypersil, Thermo Scientific) (50 x 2.1 mm, 5µm particle size). The MS was
operating in the positive mode using electron turbospray. Mass spectrometer settings are
summarized in Table 3.
Table 3: Mass spectrometer setting for each of the analytes.
Compound DP CE CXP
VCT 55.00 32.00 9.00
VBT 55.00 25.00 10.00
M1 46.00 31.00 21.00
M2 76.00 55.00 13.00
16
The mobile phase consisted of 0.1% formic acid in water (mobile phase A) and
0.1% formic acid in methanol (mobile phase B). The flow rate was set to 0.33 ml/min and
the injection volume was 30 µL.) The mobile phase gradient was started with 30% of
Buffer B and run for 1.9 min, which linearly increased to 90% acetonitrile over 2 min,
and the mobile phase was in 90% B over 4 min. Multiple reaction monitor was used to
quantify the analytes using the following M
+
→T
+
413.3 → 353.6 (m/z) for vincristine
and 408.4 → 376.3 (m/z) for vinblastine (Internal standard), 397.4→ 337.6 (m/z) for M1
and 839.5→ 779.8 (m/z) for M2. Analyst 1.6.2 (Applied Bioscience) was used for data
acquisition and processing. The source temperature was set at 500°C. The curtain gas was
set at 25 U* and the collision gas 12 U*. The ion spray voltage was set to 4.5 kV. The
accumulation time was 200 ms (refer to Table 3)
Calpain Measurements
Calpain was collected at week 1 and week 4 of the study period. For the week 1
collection, the specimen was collected in 2 ways. We collected 3ml of whole blood prior
to infusion. It was placed in sodium citrate light blue tube and gently inverted 8-10 times
and placed on ice immediately. The specimen was then centrifuged at 1500 X G (~3000
RPM) for 15 minutes at 4ºC. The plasma was then transferred to a 2ml cryovial and
stored at -70º. We ensured that there was a minimum plasma volume of 0.5mL. The
sodium citrate tube was stored at 4ºC.
17
Additionally, all the sodium citrate tubes from each vincristine PK blood draw
and week 1 calpain blood draw were stored, collected and used to obtain PBMC in order
to determine calpain levels. The original blood volume was marked directly on the
sodium citrate tube. Each sodium citrate tube was processed for peripheral blood
mononuclear cells (PBMC) isolation. We then replaced plasma volume in the sodium
citrate tube by filling the tube to the previously marked volume with phosphate buffered
solution (PBS) and gently re-suspended the blood back into a homogenous mixture. All
the contents from the Sodium Citrate tube were transferred to a 15ml conical tube.
Calpain activity will be measured using the Calbiochem® Calpain Activity Kit,
Fluorogenic. This assay measures the calpain activity of both isoforms, calpain-1 and
calpain-2 utilizing a synthetic calpain substrate, Suc-LLVY-7-Amino-4-methylcoumarin
(AMC), where the AMC portion of the substrate is fluorogenic. In the presence of
calpain, calcium, and the reducing agent tris (2-carboxyethyl) phosphine (TCEP), the
substrate is cleaved releasing AMC. AMC is then measured fluorometrically at an
excitation wavelength of ~360-380 nm and an emission wavelength of ~440-460 nm. The
calpain activity is determined by subtracting the activity obtained using the inhibition
buffer from the activity detected with the activation buffer. The inhibition buffer contains
the calcium chelator BAPTA, which completely blocks calpain activity. Calpain activity
can be quantitated using an AMC calibration curve or can be displayed in relative
fluorescence units (RFU). The kit includes highly purified native human calpain-1, which
will be included in every assay as a positive control.
18
Genetics Processing
At the 4-week mark of the study, we obtained one whole blood sample from each
patient. We collected 2 mL whole blood per time point and place in EDTA purple top
tube. The specimen was then gently inverted 8-10 times to mix contents thoroughly. The
specimen was stored at 4°C. Each specimen will be sent for the following genetic testing:
race/ethnicity, CYP3A5 allele, CEP720, and for genetic mutation of CMT1A, PMP22
gene.
PBMC Isolation (calpain and genetic sample)
We noted the volume of blood and recorded the volume on the PBMC Calculation
worksheet as “Volume of useable whole blood.” We then diluted blood in the 50mL (or
15mL if performing 4WK Calpain PBMC Isolation) conical tube with PBS in a 1:1 ratio.
The calculated Ficoll was dispensed into 50ml (or 15mL – 4WK Calpain) Conical tubes
then overlay diluted blood on the Ficoll Layer. We centrifuged the tubes at 400 G for 30
minutes at 20°C and we ensure that the brakes on the centrifuge were turned off. We
then carefully removed the white cells and transferred them into new 15mL conical tube
and filled it with PBS up to the 14ml line. The specimen was then centrifuge at 400 G for
10 minutes and PBS was removed. The cells were then washed a second time @ 400 G
for 10 minutes. The CPS was transferred into the 15ml conical tube with the cell pellet
and re-suspended to uniformity. The PBMCs were then frozen overnight at -80°C then
transferred to liquid nitrogen for shipment.
19
Neurological examination
Neurological assessment of each participant was conducted at the week 1-time point
of the study and again at the week 4-time point of the study. This assessment was
structured to document baseline neurological deficits of each patient and/or to determine
if and when they developed vincristine related neuropathy (VRNT).
We used a three-part approach to enhance the screening for the presence of VRNT.
To avoid inter-observer variability, a single investigator performed the neurological
assessment of each subject. The investigator practiced the assessment on regular subjects
prior to starting the trial.
For the first part of this neurological assessment we employed the Pediatric-Modified
Total Neuropathy Score
27
, which is a validated measure of chemotherapy induced
peripheral neuropathy in children with cancers outside of the central nervous system, who
are aged 6-18 years of age.
For the second part of the assessment we used the National Cancer Institute Common
Terminology Criteria for Adverse Events, version 4.0 (CTCAE 4.0) for the following
adverse events: peripheral motor neuropathy, peripheral sensory neuropath and
paresthesia. This assessment was performed at week 1 and week 4 of neurological
examination and recorded in real time.
Additionally, a thorough chart review was conducted at the following time points:
a) Week 1: To determine if the subject had a previous history of central or peripheral
neuropathy as well as its etiology.
b) Week 4: To determine if the subject had developed VRNT.
20
c) At time of data analysis: To determine if the participants had developed VRNT
The medical record was reviewed for diagnosis of VRNT, peripheral neuropathy, dose
adjustment of vincristine secondary for peripheral neuropathy, physical or occupational
therapy referral secondary to VRNT, inpatient and outpatient prescription for gabapentin
or neurontin. Since baseline and 4 week neurological examination were performed and
recorded prior to obtaining the result of the VCR PK, calpain and genetic testing, the
investigator was blinded to these results when performing this exam. The investigator
was not blinded to the Tanner Stage. The same investigator performed all the physical
examination on each patient.
Data collection
The following data were collected for each patient: Diagnosis, Risk stratification of ALL,
Sub-type of ALL (B or T cell), leukemia CNS status at time of diagnosis, date of birth,
age at diagnosis, weight, height, and race/ethnicity. Additionally, each patient’s
medication list was reviewed at Week1 and Week 4 and recorded in real time.
21
Chapter 3
Data Analysis
All of the patient’s data points, lab results and neurological examination results were
stored and saved using RED-CAP software. All the data were imported by one
investigator. We have conducted a preliminary analysis on the data that has been obtained
thus far. We are currently in the process of reviewing each participant data point to
ensure accuracy.
Population Vincristine PK model:
One and two-compartment structural models have been previously reported. Using the
Pmetrics non-parametric populations modeling and simulation package for R
28
, we will
fit all vincristine concentrations measured from the study subjects to both structural
models with direct input into a central compartment. Models will be discriminated on the
basis of the Akaike Information Criterion (AIC), observation vs. prediction bias and
imprecision, and parsimony, with the fewest parameters possible. We will also test for the
effect on AIC of inclusion of covariates such as allometric body size and Tanner stage.
After selecting the optimal vincristine model, we will add measured M1 and fit models,
which add one or two compartments for M1 kinetics to the base vincristine model,
discriminating models as before. If Tanner stage is included in the final model because of
improvement in the AIC, we will reject the null hypothesis for this aim. From the model
we will be able to calculate each participant's plasma vincristine and metabolite areas
under the curve (AUCs) as well as each participant’s peripheral compartment vincristine
22
and metabolite AUCs as measures of the amount of vincristine and metabolites
kinetically distributing to tissues outside of the vascular space. Because the model will
allow calculation of vincristine and metabolite concentrations at any time point, AUC by
this method can be calculated with much higher resolution than by the non-
compartmental method. We will use these AUCs as alternatives to the non-
compartmental estimates in analyses of the relationship of physical maturation to overall
disposition of vincristine in children and AYA, as well as the relationship between
vincristine pharmacokinetics and VRNT.
Assay Development
Production of M1:
M1 was produced by adding vincristine into recombinant human CYP3A5, where the M1
is isolated using HPLC. In Figure 2, the M1 metabolite isolated and collected. The
structure of M1 was confirmed using MS, where the molecular weight and the fragments
are consistent with M1.
Production of M2
M2 was produced by reacting vincristine with peroxidase, which will oxidize vincristine
to produce M2. Similarly, the reaction mixture was separated using HPLC to isolate and
purify M2, where the structure was confirmed using MS.
23
Metabolic Assay:
The concentration of vincristine, M1 and M2 were measured using a validated plasma
assay. This assay was validated with a lower level detection (LLD) of 0.05 ng/mL. An
exemplary chromatogram summarizes the separation of vincristine, M1 and M2.
Neurological Assessment
We are currently in the process of analyzing the neurological data that was
obtained. The modified pediatric neuropathy scale is only validated for patients ages 6-
18years of age. The age range of our study patients was from 2 years of age to 22 years of
age. Unfortunately, our study population was composed of patients from ages of 2 to 22
years of age; hence we included a population for which this neuropathy scale has not
been validated.
Additionally, a few patients, regardless of age were unable to complete the full
examination. Our most reliable neurological assessment of VRNT is the National Cancer
Institute Common Terminology Criteria for Adverse Events, version 4.0 (CTCAE 4.0)
assessment conducted at week 1 and week 4 (peripheral motor neuropathy, peripheral
sensory neuropathy and paresthesia) and the extensive chart review that was conducted
for diagnosis of VRNT.
For each adverse event we will summarize the toxicity grade. We will use
multivariate analysis to determine if there is an association between toxicity score and
VCR AUC, VCR peripheral AUC, VCR Clearance, CEP720 mutation. Additionally, we
24
have calculated each patient’s cumulative VCR dose and cumulative VCR exposure to
determine if there is also a correlation with the grade of toxicity.
Genetic Analysis
We obtained genetic specimen on 29/30 patients. Previous research has shown that there
is a difference in CYP3A5 allele and how quickly or efficient vincristine is metabolized.
We believe that this may be a confounding factor that may have an influence on our
hypothesis. We plan on analyzing each patient for CYP3A5 allele, CEP70 and genetic
analysis for Charcot Marie tooth disease.
25
CHAPTER 4
PRELIMINARY RESULTS
Patients/Demographics
Patient recruitment was conducted from September 2014 to March 2015. We
approached 40 patients and recruited 30. Of the 30 patients, 46.7% of the patients were
determined to be in Tanner stage ≤ 2 and 53% were Tanner Stage ≥ 4. The female to
male ratio was 1:1 (Figure 2). The majority of patients were diagnosed with HR-ALL.
For the patients with Tanner stage ≤ 2, there a large percentage of them were in the
Induction phase of therapy (71.4%). For the patients with Tanner Stage ≥ 4, the majority
of them were in the Maintenance phase of therapy (75%).
Tanner ≤2 Tanner ≥4
Tanner Stage N (%) 14 (46.7%) 16 (53%)
Age (Median years) (range) 5.44 (2-8 years) 17.8 (13-21)
Male (%) 50% 50%
Phase of Therapy (N: %)
• Induction
• Maintenance
10/14 (71.4%)
4/14 (28.5%)
4/16 (25%)
12/16 (75%)
Disease Risk N (%)
• SR-ALL
• HR-ALL
9/14 (64.2%)
5/14 (35.7%)
0/16 (0%)
16/16 (100%)
Table 4: Patient demographics categorized in Tanner Stage
The majority of patients were of Hispanic/Latino origin (26/30 participants). Only
1/30 patient were from Southeast Asian/Pacific Islander and 3/30 patients were
Caucasians (Refer to Table 4).
26
Race/Ethnicity Tanner ≤2
N (%)
Tanner ≥4
N (%)
American Indian 0 (0%) 0 (0%)
Asian American 0 (0%) 0 (0%)
Southeast Asian/Pacific Islander (N=1) 1 (3.3%) 0 (0%)
Hispanic/Latino (N=26) 10 (38.5) 16 (61.5%)
White (non-hispanic/latino) (N=3) 3 (10%) 0 (0%)
African American (N=0) 0 (0%) 0 0%)
Table 5: Race/ethnicity of patient enrolled in this study
Figure 2: Distribution of patients by Age of enrollment, Tanner Stage and sex
27
VCR PK Analysis
The plasma concentrations of all of the patients were calculated using trapezoidal
rule. Pharmacokinetic parameters that were calculated include Tmax, Cmax, half-life
(t1/2), clearance (Cl) and AUC.
There was no statistical significant difference vincristine clearance between the
lower and higher Tanner Stages (P-value= 0.577). The mean vincristine clearance of
Tanner Stage ≤2 is 0.049 ± 0.048 and the vincristine clearance of Tanner Stage ≥ 4 is and
.058 ± 0.035 (Table 6). The mean AUC for patients in Tanner Stage ≤2 was less than
Tanner Stage ≥ 4, but not statistically significant (Table 6). Hence, in our preliminary
analysis of the data, this pilot study failed to show a difference in vincristine AUC and
clearance between the Tanner Stages. In a univariate analysis, only age and phase of
therapy were statistically significant (refer to Table 6 and Figure 3-8).
Tanner ≤ 2 Tanner ≥ 4 P-Values
Cmax (ng/ml) 55.57 ± 59.13 31.07 ± 26.32 0.170
Tmax(hr) 0.17 ± 0.01 0.24 ± 0.22 0.215
Half-life(hr) 5.45 ± 2.51 3.46 ± 3.16 0.070
AUC (ng*hr/ml) 50.26 ± 66.42 147.22 ± 20.95 0.319
Clearance (ml/hr) 0.049 ± 0.048 0.058 ± 0.035 0.577
Table 6: Difference of vincristine pharmacokinetics between the Tanner staging
Vincristine AUC between female and male were compared, where Figure 5 summarizes
the data. No differences between the two sexes were found in this study, where the p-
value was 0.407. Additional analysis evaluated the impact of weigh will have on
vincristine disposition. In Figure 6, summarizes the difference between normal weight
28
and obesity, where no statistical difference were detected between normal weight and
overweight/obese patients.
Figure 3: Effect of Tanner Stage on Vincristine AUC
29
Figure 4: Effect of age on vincristine AUC
Figure 5: Effect of sex on vincristine exposure (AUC)
30
Figure 6: Effect of BMI on vincristine AUC
Figure 7: Vincristine AUC and phase of therapy
31
Figure 8: Vincristine AUC versus antifungal use
32
CHAPTER 5
PRELIMINARY DISCUSSION
The results presented are from a preliminary analysis. A more extensive analysis of the
data obtained will be performed. This is the first time in which Tanner Stage has
evaluated the pharmacology of vincristine. In our preliminary univariate analysis of this
pilot study we were unable to identify a difference between vincristine PK and level of
Tanner Stage. One of the major potential confounding factors may be the CYP3A5 allele
for each patient. It has been established that CYP3A5 expressers have a higher clearance
of vincristine compared to patients who do not express this protein
6
.
We are currently in the process of analyzing each patient’s genetic variations of
CYP3A5 to determine if this variation contribute to the results we obtained. Table 8
summarizes our future analysis for completion of the trial.
Table 8: Future analysis
Assessment Association
Model AUC
VCR AUC
M1 AUC
M2 AUC
Peripheral VCR AUC
Peripheral M1 AUC
Peripheral M2 AUC
VCR Clearance
Tanner Stage
Age
Phase of Therapy
Sex
Fluconazole use
Fluconazole exposure
CYP3A5 Allele
CTCAE 4.0
33
REFERENCES
1. Closing the gap: research and care imperatives for adolescents and young
adults with cancer. A report of the Adolescent and Young Adult Oncology
Program Review Group. Natl Cancer Inst 2006.
2. Himes RH, Kersey RN, Heller-Bettinger I, Samson FE. Action of the vinca
alkaloids vincristine, vinblastine, and desacetyl vinblastine amide on
microtubules in vitro. Cancer Res 1976;36(10):3798–802.
3. Miltenburg NC, Boogerd W. Chemotherapy-induced neuropathy: A
comprehensive survey. Cancer Treat Rev 2014;40(7):872–82.
4. Dennison JB. Selective Metabolism of Vincristine in Vitro by CYP3A5. Drug
Metab Dispos 2006;34(8):1317–27.
5. Dennison JB, Jones DR, Renbarger JL, Hall SD. Effect of CYP3A5
Expression on Vincristine Metabolism with Human Liver Microsomes. J
Pharmacol Exp Ther 2007;321(2):553–63.
6. Egbelakin A, Ferguson MJ, MacGill EA, et al. Increased risk of vincristine
neurotoxicity associated with low CYP3A5 expression genotype in children
with acute lymphoblastic leukemia. Pediatr Blood Cancer 2011;56(3):361–7.
7. Dennison JB, Mohutsky MA, Barbuch RJ, Wrighton SA, Hall SD. Apparent
High CYP3A5 Expression Is Required for Significant Metabolism of
Vincristine by Human Cryopreserved Hepatocytes. J Pharmacol Exp Ther
2008;327(1):248–57.
8. Gupta AA, Anderson JR, Pappo AS, et al. Patterns of chemotherapy-induced
toxicities in younger children and adolescents with rhabdomyosarcoma: A
report from the Children’s Oncology Group Soft Tissue Sarcoma Committee.
Cancer 2012;118(4):1130–7.
9. Langholz B, Skolnik JM, Barrett JS, et al. Dactinomycin and vincristine
toxicity in the treatment of childhood cancer: A retrospective study from the
Children’s Oncology Group. Pediatr Blood Cancer 2011;57(2):252–7.
10. 2011 ASPHO Abstracts. Pediatr Blood Cancer 2011;56(6):897–973.
11. Diouf B, Crews KR, Lew G, et al. Association of an Inherited Genetic Variant
With Vincristine-Related Peripheral Neuropathy in Children With Acute
Lymphoblastic Leukemia. JAMA 2015;313(8):815.
12. Nakamura T, Hashiguchi A, Suzuki S, Uozumi K, Tokunaga S, Takashima H.
Vincristine exacerbates asymptomatic Charcot–Marie–Tooth disease with a
novel EGR2 mutation. neurogenetics 2012;13(1):77–82.
13. Weimer LH, Podwall D. Medication-induced exacerbation of neuropathy in
Charcot Marie Tooth Disease. J Neurol Sci 2006;242(1-2):47–54.
34
14. Van den Berg HW, Desai ZR, Wilson R, Kennedy G, Bridges JM, Shanks
RG. The pharmacokinetics of vincristine in man. Cancer Chemother
Pharmacol 1982;8(2):215–9.
15. Crom WR, de Graaf SS, Synold T, et al. Pharmacokinetics of vincristine in
children and adolescents with acute lymphocytic leukemia. J Pediatr
1994;125(4):642–9.
16. Frost B-M, Lönnerholm G, Koopmans P, et al. Vincristine in childhood
leukaemia: no pharmacokinetic rationale for dose reduction in adolescents.
Acta Paediatr 2003;92(5):551–7.
17. Rogol AD, Roemmich JN, Clark PA. Growth at puberty. J Adolesc Health
2002;31(6):192–200.
18. Veal GJ, Hartford CM, Stewart CF. Clinical Pharmacology in the Adolescent
Oncology Patient. J Clin Oncol 2010;28(32):4790–9.
19. Kennedy M. Hormonal Regulation of Hepatic Drug-Metabolizing Enzyme
Activity During Adolescence. Clin Pharmacol 38 Ther 2008;84(6):662–73.
20. Lambert G, Schoeller D, Kotake A, Flores C. The effect of age, gender, and
sexual maturation on the caffeine breath test. Dev Pharmacol Ther
1986;9(6):375–8.
21. Boehmerle W, Zhang K, Sivula M, et al. Chronic exposure to paclitaxel
diminishes phosphoinositide signaling by calpain-mediated neuronal calcium
sensor-1 degradation. Proc Natl Acad Sci 2007;104(26):11103–8.
22. Benbow JH, Mann T, Keeler C, et al. Inhibition of Paclitaxel-induced
Decreases in Calcium Signaling. J Biol Chem 2012;287(45):37907–16.
23. Benbow JH, DeGray B, Ehrlich BE. Protection of Neuronal Calcium Sensor 1
Protein in Cells Treated with Paclitaxel. J Biol Chem 2011;286(40):34575–82.
24. Wang MS. Calpain inhibition protects against Taxol-induced sensory
neuropathy. Brain 2003;127(3):671–9.
25. Ryu M, Yasuda M, Shi D, et al. Critical role of calpain in axonal damage-
induced retinal ganglion cell death. J Neurosci Res 2012;90(4):802–15.
26. Kliegman R, Stanton B. Nelson Textbook of Pediatrics. 19th ed. Elsevier
Saunders; 2011.
27. Gilchrist LS, Tanner L. The pediatric-modified total neuropathy score: a
reliable and valid measure of chemotherapy-induced peripheral neuropathy in
children with non-CNS cancers. Support Care Cancer 2012;21(3):847–56.
28. Neely MN, van Guilder MG, Yamada WM, Schumitzky A, Jelliffe RW.
Accurate Detection of Outliers and Subpopulations With Pmetrics, a
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Package for R: Ther Drug Monit 2012;34(4):467–76.
35
APPENDIX A:Pediatric-Modified Total Neuropathy Scale
Sensory Symptoms: _____ (record worst score for the three sensations)
“Do you have any parts of your body that are tingly, numb (can hardly feel), or hurt?”
____Tingly _____Numb _____ Hurt (record number for each
If yes, : Where you have those feelings?”
0 None
1 Symptoms limited to fingers or toes
2 Symptoms extend to ankles and wrists
3 Symptoms extend to knee or elbow
4 Symptoms above knee or elbow
Functional Symptoms: _____ (record worst score of the three questions)
“ Do you have trouble buttoning shirts or zipping zippers? “ _____
“ Do you have trouble Walking such as tripping frequently?” _____
“Do you have trouble going up or down stairs?” _____
If yes to any, “ Is it …..(read choices)”and record after each question
0 Not difficult
1 A little difficult
2 Somewhat difficulty
3 I need help
4 I can’t do that at all
Autonomic Symptoms: _____(record worst score of the three questions)
“Do you feel dizzy or light-headed when you get up out of bed?” _____
“Do your hands or feet feel hotter or colder than normal?” _____
0 Never
1 A little bit
2 Sometimes
3 Very much
4 Almost always
Semmes Semmes
Toes Finger
R R
L L
36
Clinical testing:
Light Touch Sensation: _____
0 Normal
1 Reduced in fingers/toes
2 Reduced up to wrist/ankle
3 Reduced up to elbow/knee
4 Reduced to above elbow/knee
Pin Sensibility: _____
0 Normal
1 Reduced in fingers/toes
2 Reduced up to wrist/ankle
3 Reduced up to elbow/knee
4 Reduced to above elbow/knee
Vibration Sensibility: _____
0 Normal
1 Reduced in fingers/toes
2 Reduced up to wrist/ankle
3 Reduced up to elbow/knee
4 Reduced to above elbow/knee
Strength: _____ (worst Score) (MRS Score R/L)
MRC level: Great Toe ____/____ ankle DF ____/____ Finger abd ____/____ Wrist Ext
____/____
0 Normal
1 Mild Weaknes (MRC 4)
2 Moderate Weakness (MRC 3)
3 Severe Weakness (MRC2)
4 Paralysis (MRC 1-0)
DTR: _____ (Achilles, Patellar)
0 Normal
1 Ankle reflex reduced (Achilles +1)
2 Ankle reflex absent (Achilles 0, Patellar +2)
3 Ankle reflex absent, other reduced (Achilles 0, Patellar +1)
4 All reflexes absent (all 0)
Med Mal Wrist
R R
L L
Knee Elb
R R
L L
Bioesth Bioesth
Toes Finger
R / R /
L / L /
Med Mal Wrist
R / R /
L / L /
Knee Elb
R / R /
L / L /
37
Total Score: ______/__32____
38
APPENDIX B: USC RedCap Data Collection
39
40
41
42
43
44
45
46
47
48
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Isenalumhe, Leidy L.
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Core Title
The pharmacokinetics and pharmacodynamics of vincristine in the adolescent and young adult population compared to younger patients
School
Keck School of Medicine
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Master of Science
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Clinical and Biomedical Investigations
Publication Date
07/31/2016
Defense Date
07/31/2015
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