Amie DavéNovember 16, 2007

Columbia College of Physicians and SurgeonsMorgan Stanley Children’s Hospital of New York- Presbyterian

New York University School of Medicine

Vincristine in Cancer Therapy Vinca-alkaloid derived from the periwinkle plantIntegral part of many pediatric protocols

ALL, Hodgkin’s disease, non-Hodgkins lymphoma, rhabdomyosarcoma, neuroblastoma, Ewing’s sarcoma, Wilms tumor

Mechanism of vincristine (VCR)Binds to tubulin disrupts microtubule function

mitotic arrest & cell deathInterfere with many biochemical processes: amino acid

uptake, inhibition of protein, DNA, & RNA synthesis, disrupts lipid & glucose metabolism

Vincristine-Induced NeuropathyNeurotoxicity is the dose-limiting toxicity of VCR therapyManifests as a sensory and/or motor neuropathyPresentation of neuropathy in children

Paresthesias, numbness, loss of proprioception, gait disturbances

More difficult to detect May be subtle

Believed to be related to cumulative dose in adults

Glutamine Neutral non-essential amino acid, frequent use as a

nutritional supplementGlutamine depletion over time in cancer patients May have a promising role in prophylaxis against VCR-

induced neuropathyProposed mechanisms of neuroprotectant effect

Related to circulating nerve growth factor (NGF) levels; glutamine’s ability to upregulate NGF mRNA

Glutamine enhances microtubule formation and/or stability

Low toxicity

Study Objectives Eligibility Criteria Evaluate effect of glutamine on

VCR-mediated antitumor efficacy in vitro

Describe the incidence of VCR-induced peripheral neuropathy in pediatric oncology patients

Investigate the efficacy of glutamine in ameliorating VCR-induced peripheral neuropathy

To investigate the effect of glutamine on serum nerve growth factor and glutamine levels

InclusionAges 5 to 21 yearsAbility to complete baseline

assessmentDiagnosis of leukemia or solid

tumors and expected to receive ≥ 15 mg/m2 of VCR over 30 weeks

Exclusion Primary CNS tumors or CNS

metastasesRecurrent disease Grade II, III or IV neurological

status by the NCI CTC 3.0 on clinical exam

Already received > 8mg/m2 of VCR during therapy at the time of consent

Study Instruments

Purdue Pegboard Test

Symbol Digits Test

Hand DynamometerGrip Strength

Study Design Double-blind, randomized, placebo-controlled trial

Baseline

Neurological Statusevaluated every 3 weeks

Meet criteria Randomization

Glutamine Placebo

Day 0

Day 42

Day 21

Day 0

Day 42

Day 21

Neurological Assessments• Clinical exam• Purdue Pegboard Test• Symbol Digits Test• Grip Strength Test

Blood Collection• Serum NGF• Serum glutamine • Baseline, Day 0, 21, & 42

Day 21 - discontinuesupplement

Randomization Criteria• Clinically progressive neurological findings

• Increase in one toxicity grade by the NCI CTC 3.0 on clinical exam

or• Clinically progressive neuropsychological findings

• ≥ 1/2 SD from patient’s own baseline score on any of the instruments that measure fine and gross motor function

Glutamine dose 6 g/m2 (max of 10 g/dose) BID

In vitro testingDavid J. Kroll, Ph.D. Senior Research PharmacologistResearch Triangle Park, NC

Physiologic levels of glutamine: 500 – 600 µMCCRF-CEM human T-cell leukemia cellsThe effects of 0.5, 2, 5, and 10 mM of glutamine on

cell cycle distributioncellular growth ratesensitivity to vincristine cytotoxicitysensitivity to L-asparaginase cytotoxicity

Results – In vitro testing Effect of glutamine culture conditions on growth of

CCRF-CEM human T-cell leukemia cells

Days

0 1 2 3 4 5

cel

l nu

mb

er/m

L X

10,

000

0

50

100

150

200

250

0.5 mM Gln2 mM Gln5 mM Gln10 mM Gln

Results – In vitro testing No effect of glutamine on cell cycle distribution

Sub G0 G0/G1 S G2/M

0.5 mM Gln 2.70 ± 0.85 46.06 ± 1.31 21.05 ± 0.45 30.13 ± 1.59

2 mM Gln 3.81 ± 0.70 46.55 ± 2.06 20.69 ± 0.40 28.81 ± 1.85

5 mM Gln 3.78 ± 1.76 47.01 ± 2.53 20.81 ± 0.28 28.17 ± 2.17

10 mM Gln 2.25 ± 0.19 45.45 ± 0.67 21.36 ± 0.16 30.90 ± 0.59

Results – In vitro testing No effect of glutamine on vincristine sensitivity in CCRF-CEM

cells

Vincristine IC50 (nM)

0.5 mM Gln 0.695

2 mM Gln 0.740

5 mM Gln 0.772

10 mM Gln 0.791

Results – In vitro testing Effect of glutamine on

CCRF-CEM response tovincristine

-10 -9 -8 -7 -60

25

50

75

1000.5 mM glutamine2 mM glutamine5 mM glutamine10 mM glutamine

log [vincristine] (M)

% c

ellu

lar

surv

ival

Clinical Trial Demographics 23 patients, 3 removed, 2 on observationAges 5 – 19 years

Mean 9.9, Median 8.510 males, 8 femalesDiagnosis

ALL – 16 Rhabdomyosarcoma – 2Ewings sarcoma – 2Wilms tumor – 2 Hodgkin’s Disease – 2

Results – RandomizationCriteria for randomization met in first 18 of 18 patientsCriteria met by

Clinical exam – 5 patientsNeuropsych. Testing – 6 patientsBoth – 7 patients

VCR-related neuropathy is prevalent in children with overt progression by clinical exam in 67% May be greater if in fact neuropsych. testing is picking up

signs at an earlier stage

Results – Neuropsych. TestingPotential practice effect with Symbol Digits test      

Neuropsychologic Test Mean change in z-

score* Range

Purdue Pegboard - Dominant -0.1 -2.5 to 1.72Purdue Pegboard - Non-dominant -0.54 -2.68 to 1.83Purdue Pegboard - Both -0.7 -2.34 to 1.11

Hand Dynamometer - Right -0.02 -1.76 to 1.71Hand Dynamometer - Left -0.46 -2.38 to 0.77

Symbol Digits† 0.89 .-0.54 to 2.84*Negative score indicates decline (z-score at randomization minus baseline z-score) from baseline to randomization test

†Ten patients administers symbols digits test, no norms for patients age five

Results – Cumulative VCR Cumulative dose of approximately 6 mg/m2 required for

detection of signs by clinical exam or neuropsych. testing in all but one patient

No difference in number of days to randomization between patients with ALL compared to other diseases

It is the cumulative dose as opposed to dose-intensity (mg/m2/28-day interval) that determines randomizationAmong six patients that received 6 mg/m2 ± 0.5, dose

intensity varied from 2.5 to 5.8 mg/m2/28-day interval

Results – Serum glutamine levelsNo significant difference found between glutamine levels at baseline and

at time of randomization (Z=-0.31, p=0.76)Significant correlation between baseline and the change in glutamine

(rho=-0.531, p=0.034)

Results – Serum glutamine levelsNo significant differences between ALL and other

diseases

Results – Glutamine & time to randomization Significant difference in the number of days to

randomization between patients who had a decrease and increase in glutamine (Z= -2.119, p=0.034) Decrease – mean 80.9 days, SD=36.96 Increase – mean 49.0 days, SD= 19.71

Findings No current evidence to suggest any adverse effect of glutamine on CCRF-

CEM leukemia cell growth, cell cycle distribution, or sensitivity to vincristine even at 20 times normal circulating concentrations

First 18 of 18 patients met criteria for randomization 12 patients randomized due to progression of score on clinical exam

Cumulative dose of approximately 6 mg/m2 required for detection of signs by clinical exam or neuropsych. testing

No differences in in glutamine levels between baseline and randomization No difference between patients with ALL and other diseases

Trends in glutamine levels may help predict who develops neuropathy

AcknowledgmentsStudy group –

E. Ladas RD MS, D. Hughes BA, S. Sands PsyD, D. Kroll PhD,

L. Vahdat MD, O. Bessmertny PharmD, M. Weiner MD,

K. Kelly MD, J. Glade Bender MDGCRC, Irving Center for Clinical Research at Columbia

UniversitySociety for Integrative Oncology