Clinical trial NCT01128218
Conditions: Brain Neoplasms
Keywords: Brain Neoplasms 5-ALA Aminolevulinic acid Fluorescence Gliomas Glioblastoma Surgery
Sponsor: Southern Illinois University
Start date: 2011-03-01; Completion date: 2021-02-01
Enrollment: 33 participants; Allocation: NON_RANDOMIZED; Intervention model: SEQUENTIAL

Brief summary:
Extent of resection is a very important prognostic factor affecting survival in individuals diagnosed with a malignant glioma. However, the infiltrative nature of the malignant glioma tumor cells produces indistinct borders between normal and malignant tissues, and the lack of easily identifiable tumor margins confounds attempts at total resection. The investigators propose to identify the borders of malignant gliomas intraoperatively using oral 5-aminolevulinic Acid (5-ALA) which results in fluorescence of the malignant cells and thereby provide an opportunity for more complete tumor resection.

When exogenous 5-ALA is provided at increased concentration the tumor cells will become fluorescent under ultraviolet light. This feature identifies the tumor cells intraoperatively and facilitates complete resection.

Data collection will include measurement of dose-limiting toxicity, tumor fluorescence, and tumor density. Data analysis will evaluate toxicity, sensitivity, and specificity of 5-ALA.

Following completion of the phase 1 portion of this trial, an additional 14 subjects will be entered at the recommended phase 2 dose level in order to further define the above parameters at the recommended phase 2 dose level.

Detailed description:
Specific Aims:

This study is intended to investigate the utility, safety and efficacy of 5-aminolevulinic acid (5-ALA) induced brain tumor fluorescence during malignant brain tumor resection. Specifically this study is intended to:

Establish a safe dose for oral 5-ALA administration. Determine the sensitivity and specificity of 5-ALA mediated fluorescence for malignant glioma tissue in the brain.

Background and Significance:

There is a considerable body of literature that suggests that completeness of resection is a positive factor for longer term survival in individuals with malignant glioma. Unfortunately, it is often difficult to completely remove a malignant brain tumor because during surgery it is sometimes very difficult to distinguish tumor from normal brain. It would be very helpful if there would be some way to help the surgeon make this distinction. Malignant glioma tumor cells (more so than normal cells) contain the biosynthetic pathways to produce protoporphyrin from a naturally occurring amino acid, 5-aminolevulinic acid (5-ALA). Protoporphyrin is the immediate precursor to hemoglobin (it is hemoglobin without the iron atom) and is fluorescent under blue light. When exogenous 5-ALA is provided at increased concentration, protoporphyrin concentration in the malignant cell increases at a rate far greater than normal brain cells and renders the malignant cell fluorescent red under blue light. This feature distinguishes the tumor cells from normal cells intraoperatively and facilitates complete resection.

Recent studies in Germany have confirmed the utility of pre-operative oral 5-ALA and intraoperative brain tumor fluorescence in aiding the resection of brain tumors in individuals with malignant brain tumors. These studies have led to oral 5-ALA to be approved for this indication by the European Medicines Agency, but oral 5-ALA has not been approved for this indication by the United States FDA. This proposal is a phase 1 and phase 2 trial that will hopefully lead to FDA approval of oral 5-ALA for intra-operative visualization of malignant brain tumors.

Experimental Plan and Methods:

In the phase 1 part of this proposed study, a minimum of 3 to a maximum of 19 patients will be administered oral 5-ALA 4 hours prior to surgery in cohorts of 3 at five escalating doses of 5-ALA (10, 20, 30, 40, or 50 mg/kg).

The following data will be collected:

* Dose-limiting toxicity data; i.e., nausea, vomiting, liver function, photo-sensitivity
* Tumor fluorescence assessed by neurosurgeon
* Tumor density from biopsies obtained by the neurosurgeon in will be assessed by neuropathology (Solid tumor, Tumor mixed infiltrating normal brain, No tumor)

This trial will evaluate:

* single dose toxicity of oral 5-ALA given pre-operatively;
* sensitivity and specificity of 5-ALA - Protoporphyrin IX (Pp IX) as an intraoperative fluorescent detection agent and aid for resection of tumor tissue remaining in the walls of the resection cavity of primary and recurrent malignant brain tumors;

Following completion of the phase 1 portion of this trial, an additional 14 subjects will be entered at the recommended phase 2 dose level in order to further define the above parameters at the recommended phase 2 dose level.

Arms: arm 1: Dose Level 1: Participants were given a one-time, single-dose administration of oral 10mg/kg Aminolevulinic Acid (5-ALA) arm 2: Dose Level 2: Participants were given a one-time, single-dose administration of oral 20mg/kg Aminolevulinic Acid (5-ALA) arm 3: Dose Level 3: Participants were given a one-time, single-dose administration of oral 30mg/kg Aminolevulinic Acid (5-ALA) arm 4: Dose Level 4: Participants were given a one-time, single-dose administration of oral 40mg/kg Aminolevulinic Acid (5-ALA) arm 5: Dose Level 5: Participants were given a one-time, single-dose administration of 50mg/kg Aminolevulinic Acid (5-ALA) arm 6: Phase 2: Participants were given a one-time, single-dose administration of 40mg/kg Aminolevulinic Acid (5-ALA)
Interventions: intervention 1: Tumor fluorescence
Intervention descriptions: intervention 1: Oral doses in phase 1 study of 10mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg and 50 mg/kg.

Recommended oral dose of phase 1 will be used in phase 2

Protocol documents:
Prot_SAP_000.pdf: 
1
Protocols [21 CFR 312.23(a) (6)]
IND #
105, 807
SCRIHS Protocol #
12-135
TITLE
A Phase 1 and 2 Study of 5-Aminolevulinic Acid (5-ALA) to 
Enhance Visualization and Resection of Malignant Glial Tumors 
of the Brain*
*
Nervous system neoplasms malignant:  Anaplastic astrocytoma (10002224),  Astrocytoma 
malignant NOS (10003572), Brain stem glioma (10006143), Ependymoma (10014967), 
Ependymoma malignant (10014968), Glioblastoma (10018336), Glioblastoma multiforme 
(10018337),  Gliosarcoma (10018340), Anaplastic  oligodendroglioma (10026659), 
Oligodendroglioma (10030286), Medulloblastoma (10027107), Mixed astrocytoma-ependymoma 
(10027743), Miscellaneous CNS primary tumor (10007959), Supratentorial primitive 
neuroectodermal tumor (10056672).
Coordinating Center
Southern Illinois University School of Medicine
and Statistician
Center for Clinical Research
Principal Investigator
Jeffrey W. Cozzens, MD
and Data Manager
Southern Illinois University School of Medicine
Department of Surgery, Division of Neurosurgery
PO Box 19680, Springfield, IL 62794-9680
217.545.8863
217.545.9752 fax, jcozzens@siumed.edu
Co - Investigators
José A. Espinosa, MD
Southern Illinois University School of Medicine
Department of Surgery, Division of Neurosurgery
PO Box 19680, Springfield, IL 62794-9680
217.545.8172
217.545.9752 fax, jespinosa@siumed.edu
Devin V. Amin, MD, PhD
Southern Illinois University School of Medicine
Department of Surgery, Division of Neurosurgery
PO Box 19680, Springfield, IL 62794-9680
217.545.8172
217.545.9752 fax, damin@siumed.edu
Study Coordinator
Barbara Cray Lokaitis, BA, CCRP
Southern Illinois University School of Medicine
751 North Rutledge Street, Suite 3100, Springfield, IL 62702
217.545.9737
217.545.4282 fax, blokaitis@siumed.edu
Protocol Type/Version #/Version Date
Original/Version 10.0/ 18 Jan 2017
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2
SCHEMA
There is a considerable body of literature that suggests that completeness of resection is a 
positive factor for longer term survival in individuals with malignant glioma.  Unfortunately, it is 
often difficult to completely remove a malignant brain tumor because during surgery it is 
sometimes very difficult to distinguish tumor from normal brain.  It would be very helpful if there 
would be some way to help the neurosurgeon make this distinction.  Malignant glioma tumor 
cells (more so than normal cells) contain the biosynthetic pathways to produce protoporphorin 
from a naturally occurring amino acid, 5-aminolevulinic acid (5-ALA).  Protoporphorin is the 
immediate precursor to hemoglobin and is fluorescent under deep blue light.  When exogenous 
5-ALA is provided at increased concentration, protoporphorin concentration in the malignant cell 
increases at a rate far greater than normal brain cells and renders the malignant cell fluorescent 
red under blue light.  This feature distinguishes the tumor cells from normal cells intraoperatively 
and facilitates complete resection.
Recent studies in Germany have confirmed the utility of pre-operative oral 5-ALA and 
intraoperative brain tumor fluorescence in aiding the resection of malignant brain tumors.  These 
studies have led to the approval of oral 5-ALA for this indication by the European Medicines 
Agency, but it has not been approved for this indication by the United States FDA.  This proposal 
is a phase 1 and phase 2 trial that will hopefully lead to FDA approval of oral 5-ALA for intra-
operative visualization of malignant brain tumors.
In the phase 1 part of this proposed study, a minimum of three to a maximum of *18 patients will 
be administered oral 5-ALA two-three hours prior to surgery in cohorts of three at five escalating 
doses of 5-ALA (10, 20, 30, 40, or 50 mg/kg).  
Dose-Escalation Schedule
Dose Level
Dose of 5-ALA
Level 1
10 mg/kg
Level 2
20 mg/kg
Level 3
30 mg/kg
Level 4
40 mg/kg
Level 5
50 mg/kg
(*Due to four patients receiving 40mg/kg rather than three, 19 total patients will be studied in 
phase 1. Protocol deviation approved by IRB on 17 Oct 2013.)
The following data will be collected:

Dose-limiting toxicity data.

Tumor fluorescence assessed by neurosurgeon (0 to +++) in three distinct areas of 
fluorescence (Strong fluorescence, Weak fluorescence, No fluorescence).

Tumor density from biopsies obtained by the neurosurgeon in the same three distinct 
areas of fluorescence and assessed by neuropathology (Solid tumor, Tumor mixed 
infiltrating normal brain, No tumor).

Neurosurgeon’s intra-operative estimate of residual tumor.

Neuroradiologist’s estimate of post-operative residual tumor on MRI.
This trial will evaluate:

The toxicity of a single dose of oral 5-ALA given pre-operatively.
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
The sensitivity and specificity of 5-ALA - Protoporphyrin IX (Pp IX) as an intraoperative 
fluorescent detection agent and aid for resection of tumor tissue remaining in the walls of 
the resection cavity of primary and recurrent malignant brain tumors.

The relationship of the neurosurgeon’s estimate of the extent of malignant glioma 
resection (as guided by tumor fluorescence) to the actual extent of resection determined 
by post-operative imaging.
Following completion of the phase 1 portion of this trial, an additional 16 subjects will be entered 
at the recommended phase 2 dose level in order to further define the above parameters at the 
recommended phase 2 dose level.
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TABLE OF CONTENTS
Page
SCHEMA .......................................................................................................................................2
1. OBJECTIVES
...................................................................................................................6
1.1
Primary Objectives ....................................................................................................6
2. BACKGROUND
...................................................................................................................6
2.1
5-ALA
...................................................................................................................6
2.2
Brain Tumors............................................................................................................11
2.3
Rationale .................................................................................................................13
2.4
Correlative Studies Background ............................................................................14
3. PATIENT SELECTION..........................................................................................................15
3.1
Eligibility Criteria .....................................................................................................15
3.2
Exclusion Criteria ....................................................................................................15
3.3
Inclusion of Women and Minorities .......................................................................16
4. TREATMENT PLAN..............................................................................................................16
4.1
5-ALA Administration..............................................................................................16
4.2
Definition of Dose-Limiting Toxicity ......................................................................19
4.3
Supportive Care Guidelines....................................................................................20
4.4
Duration of Therapy.................................................................................................20
4.5 
Duration of Follow-up..............................................................................................20
4.6
Criteria for Removal from Study.............................................................................21
5. DOSING DELAYS/DOSE MODIFICATIONS ........................................................................21
6. ADVERSE EVENTS: LIST AND REPORTING REQUIREMENTS.......................................21
6.1
Comprehensive Adverse Events and Potential Risks List (CAEPR) ..................21
6.2
Adverse Events Characteristics.............................................................................21
6.3
Expedited Adverse Event Reporting......................................................................21
6.4
Routine Adverse Event Reporting..........................................................................22
7. PHARMACEUTICAL INFORMATION ..................................................................................22
7.1
Drug (5-ALA).............................................................................................................22
7.2
Drug Availability.......................................................................................................22
7.3
Drug Ordering ..........................................................................................................22
7.4
Drug Accountability.................................................................................................22
8. CORRELATIVE/SPECIAL STUDIES....................................................................................22
8.1
Assessment of Visual Tumor Discrimination........................................................22
9. STUDY CALENDAR .............................................................................................................23
10. MEASUREMENT OF EFFECT..............................................................................................24
10.1
Definitions. ...............................................................................................................24
10.2
Guidelines for Evaluation of Measurable Disease................................................24
10.3
Confirmatory Measurement/Duration of Response..............................................24
11. REGULATORY AND REPORTING REQUIREMENTS ........................................................24
11.1
Expedited Adverse Event Reporting......................................................................24
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11.2
Data Reporting .........................................................................................................27
11.3
CTEP Multicenter Guidelines..................................................................................27
11.4
Cooperative Research and Development Agreement (CRADA)/
Clinical Trials Agreement (CTA) .............................................................................27
11.5
Data Safety Monitoring Committee ........................................................................27
11.6
Investigator Responsibilities..................................................................................29
12. STATISTICAL CONSIDERATIONS......................................................................................30
12.1
Study Design/Primary Endpoints...........................................................................30
12.2
Sample Size/Accrual Rate.......................................................................................31
12.3
Stratification Factors...............................................................................................32
REFERENCES ............................................................................................................................33
APPENDICES
APPENDIX A
Performance Status Criteria...........................................................................................A-1
APPENDIX B
UV Illuminated Operating Microscope............................................................................B-1
APPENDIX C
Eligibility Checklist .........................................................................................................C-1
Treatment Day Checklist/Data Sheet.............................................................................C-2
Post-operative Data Sheet.............................................................................................C-3
Pathologist Report Data Sheet ......................................................................................C-4
Biopsy/Fluorescence Correlations Data Sheet ..............................................................C-5
Intra-Operative Data Sheet ............................................................................................C-6
APPENDIX D
Letter from CTEP ...........................................................................................................D-1
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1.
OBJECTIVES
1.1 Primary Objectives:
1.1.1
Establish a safe dose for oral 5-ALA administration.
1.1.2
Determine the sensitivity and specificity of 5-ALA mediated fluorescence for 
malignant glioma tissue in the brain.
1.1.3 Compare the neurosurgeon’s intra-operative estimate of the extent of 
malignant glioma resection (as guided by tumor fluorescence) with the actual 
extent of resection determined by post-operative imaging.
2
BACKGROUND
2.1 Aminolevulinic Acid (5-ALA)
Aminolevulinic acid (also known as ALA, 5-ALA, 5-Aminolevulinic acid, D-Aminolevulinic 
acid, dALA or δALA) is a naturally occurring substance that is found in all organisms, 
including humans, and is necessary for part of the basic metabolic life processes (Figure 
1).  It is the first compound in the porphyrin synthesis pathway, the pathway that leads to 
hemoglobin in mammals and chlorophyll in plants.  In non-photosynthetic eukaryotes 
such as animals, insects, fungi, and protozoa, as well as the α-proteobacteria group of 
bacteria, it is produced by the enzyme 5-ALA synthase, from glycine and succinyl CoA. 
This reaction is known as the Shemin pathway.23 
Figure 1. 5-Aminolevulinic acid (C5H9NO3, MW 167.6)
Approximately 350 mg of 5-ALA is synthesized in humans each day for endogenous 
heme production.  Administration of exogenous 5-ALA results in the production of high 
intracellular (mitochondrial) concentrations of Protoporphyrin IX (Pp IX).  5-ALA (and 
other porphyrins) and Pp IX are excreted in the urine and the stool (via bile), respectively.  
PpIX has properties of strong fluorescence and can also act as a photosensitizing agent 
for photodynamic therapy - resulting in cell death.
2.1.1
Pharmacology
The metabolism of 5-ALA is the first step in the biochemical pathway resulting 
in heme synthesis.  5-ALA is not fluorescent or a photosensitizer, but rather a 
metabolic precursor of PpIX, which is fluorescent and a photosensitizer. The 
synthesis of 5-ALA is normally tightly controlled by feedback inhibition of the 
enzyme, 5-ALA synthetase, presumably by intracellular heme levels.  5-ALA, 
when provided to the cell, bypasses this control point and results in the 
accumulation of PpIX, which is converted into heme by ferrochelatase through 
the addition of iron to the PpIX nucleus (see Figure 2).
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Figure 2.  ALA synthase, the first enzyme of the heme biosynthetic pathway, catalyzes 
the condensation of glycine and succinyl coenzyme A to form ALA.  This enzyme is 
localized in the inner membrane of mitochondria. Separate genes encode erythroid and 
nonerythroid ALA synthases.  ALA dehydratase, a cytosolic enzyme, converts two 
molecules of ALA into a monopyrrole, porphobilinogen (PBG), with the removal of two 
molecules of water.  PBG deaminase catalyzes the condensation of four molecules of 
PBG to yield a linear tetrapyrrole, hydroxymethylbilane (HMB).  Uroporphyrinogen III 
cosynthase catalyzes the formation of uroporphyrinogen III from HMB.  
Uroporphyrinogen decarboxylase, a cytosolic enzyme, catalyzes four sequential 
decarboxylations of the carboxymethyl side chains in uroporphyrinogen III (an 
octacarboxyl porphyrin) to yield heptacarboxyl porphyrin, hexacarboxyl porphyrin, 
pentacarboxyl porphyrin, and, finally, coproporphyrinogen III (a tetracarboxyl porphyrin).  
This enzyme can also metabolize uroporphyrinogen I to coproporphyrinogen I.  
Coproporphyrinogen oxidase, a mitochondrial enzyme in mammalian cells, catalyzes the 
removal of the carboxyl group and two hydrogens from the proprionic groups of 
pyrrolerings A and B of coproporphyrinogen III to form vinyl groups at these positions, 
forming protoporphyrinogen.  This enzyme is unable to metabolize coproporphyrinogen I.  
Protoporphyrinogen oxidase mediates the oxidation of protoporphyrinogen IX to 
protoporphyrin IX, catalyzing the removal of six hydrogen atoms from the porphyrinogen 
nucleus.  Protoporphyrin, the final intermediate in the pathway, is the only intermediate 
that is an oxidized porphyrin.  Porphyrins in the oxidized state are reddish and fluoresce 
when exposed to long-wave ultraviolet light.  Porphyrinogens that leak into extracellular 
fluid undergo auto-oxidation and are excreted primarily as porphyrins.
2.1.2
Pharmacodynamics of 5-ALA
When exogenous 5-ALA is administered either orally, intravenously or 
topically, it is taken up by the cells and metabolized in the pathway described, 
resulting in a large temporary accumulation of PpIX in the mitochondria.  
Exogenous 5-ALA induces elevated PpIX levels in most (but not all) tissues 
that line body surfaces or body cavities.  It can be shown that PpIX 
preferentially accumulates in the mucosa of the rat colon, but to a much lesser 
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extent in the submucosa and muscle layers.3 Mucosal selectivity occurs for 
other organs (stomach, bladder) in vivo when 5-ALA is administered either 
intravenously or orally.17  Tissues that show no 5-ALA-induced PpIX include 
muscle (striated, smooth and cardiac), dermis, blood vessels, mature 
erythrocytes and most (but not all) other tissues of mesodermal origin.19  
However, since most of these mesodermal tissues have multiple components, 
a more detailed examination might reveal that a minority component in some 
tissues does in fact show a measurable degree of 5-ALA induced PpIX.
Malignant tissues usually accumulate more PpIX than do corresponding 
normal tissues from which they are derived.  Increased PpIX accumulation 
can be found in many malignant cell lines, as compared to their non-malignant 
counterparts.4  A number of studies have indicated that exogenous 5-ALA 
results in a selective and preferential accumulation of PpIX in diseased or 
neoplastic cells more than in normal cells.  In terms of tumor-to-normal tissue 
(T/N) ratio of formed PpIX, 5-ALA-mediated accumulation of PpIX contrasts of 
10:1 up to 90:1 have been reported depending on the pathologic condition, 5-
ALA drug dose, and time post-administration.1  
There are also certain non-malignant but altered tissues that show enhanced 
PpIX production in comparison to cells in their normal state.  Examples of 
these tissues are psoriatic, actinic keratotic and pre-keratotic lesions.  Thus, 
while an enhancement of 5-ALA induced PpIX accumulation is not specific for 
malignant tissues, in general it does indicate the presence of some 
abnormality.
It is unclear what the exact mechanism is for the selective accumulation of 
PpIX in neoplastic tissue over normal tissue when presented with exogenous 
5-ALA.  It is clear, however, that this selective accumulation is not due to 
differences in the cellular uptake of 5-ALA.13  Whatever the mechanism, it is 
obvious from a kinetic point of view that the slowest step between the uptake 
of 5-ALA and formation of PpIX must be considerably faster than the 
transformation of PpIX into heme to attain a larger accumulation of PpIX and 
this occurs more robustly in neoplastic cells than in normal cells.14  
PpIX is strongly fluorescent when activated by light at the far blue end of the 
visible spectrum in the Soret band (400nm).  It emits a characteristic 
fluorescence spectrum with two prominent peaks in the red at 630nm and 
700nm.  This property has been used by various surgical specialties to help 
identify tumor for resection and to evaluate the extent of resection.  
Specifically for neurosurgery an oral form of 5-ALA (Gliolan, Medac GmbH, 
Wedel, Germany) has been approved by the European Medicines Agency in 
September 2007 for for visualisation of malignant tissue during surgery for 
malignant glioma (WHO grade III and IV) 
(http://www.emea.europa.eu/pdfs/human/press/pr/42748007en.pdf).  The 
results of MC-ALS.8-I/GLI Phase I/II and subsequent Phase III trials in Europe 
have been summarized in a number of recent papers by Walter Stummer 
M.D. and his colleagues.28,29,31,33  The European Medicines Agency comments 
can be found at: 
http://www.emea.europa.eu/humandocs/PDFs/EPAR/gliolan/H-744-en6.pdf 
(also see section 2.3) and the package insert for Gliolan can be found at 
http://www.emea.europa.eu/humandocs/PDFs/EPAR/gliolan/H-744-PI-en.pdf
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The accumulation and fluorescence of PpIX in tumor cells is not the only 
biological property of this 5-ALA metabolite.  Photodynamic therapy of tumors 
is also possible with the accumulation of PpIX in tumor cells.  Photodynamic 
therapy involves the photoactivation of a photosensitizer which when 
activated will kill tumor cells.  The fundamental building block of most red-
absorbing, naturally occurring photosensitizers is 5-ALA.  These sensitizers 
including PpIX are can cause cell death when exposed to light of certain 
frequencies.  PpIX has the ability to absorb a photon of visible light and then 
transfer most of this absorbed energy to a molecule of oxygen.  This causes a 
transient increase in the chemical reactivity of the oxygen molecule, and 
converts it into a relatively strong oxidizing agent, singlet oxygen.  It is the 
singlet oxygen that kills cells by causing lethal oxidative damage to 
biologically important structures.
A topical form of 5-ALA has been approved by the FDA for the photodynamic 
therapy (PDT) treatment of actinic keratoses 5-ALA (Levulan Kerastick 
Topical Solution, DUSA Pharmaceuticals New York, Inc. Valhalla NY 10595).  
According to the presumed mechanism of action, photosensitization following 
application of Levulan Topical Solution occurs through the metabolic 
conversion of 5-ALA to PpIX, which accumulates in the skin to which Levulan 
Topical Solution has been applied.  When exposed to light of appropriate 
wavelength and energy, the accumulated PpIX produces a photodynamic 
reaction, a cytotoxic process dependent upon the simultaneous presence of 
light and oxygen. The absorption of light results in an excited state of the 
porphyrin molecule, and subsequent spin transfer from PpIX to molecular 
oxygen generating singlet oxygen, which can further react to form superoxide 
and hydroxyl radicals.  Photosensitization of actinic (solar) keratosis lesions 
using the Levulan Kerastick, plus illumination with the BLU-UTM Blue Light 
Photodynamic Therapy Illuminator (BLU-U), is the basis for Levulan 
photodynamic therapy of skin lesions.  It is presumed that the same 
mechanism could be utilized for photodynamic therapy of brain neoplasms 
using oral administration of 5-ALA, and this is the basis for future studies (but 
not this study).
It is the properties of the intracellular metabolism of 5-ALA, the subsequent 
intracellular accumulation of PpIX without extracellular spread, the selective 
and preferential accumulation of PpIX in neoplastic cells compared to normal 
cells and the fluorescent properties of the accumulated PpIX that form the 
basis for this proposal concerning the gross total resection of malignant brain 
tumors.  The photodynamic therapy benefits of 5-ALA/PpIX in the treatment of 
malignant gliomas will likely be addressed in a subsequent proposal.
2.1.3
Pharmacokinetics of 5-ALA
The pharmacokinetics of 5-ALA have been well studied.  5-ALA is supplied as 
the HCl salt which yields an acidic solution.  Intravenous administration of an 
unbuffered solution to animals can cause bradycardia and hypotension, 
whereas buffered 5-ALA does not.  Unfortunately, aqueous solutions of 5-ALA 
are unstable when buffered to pH 7.4.  This makes intravenous formulation 
difficult and sometimes inconsistent.36  For this reason there have been a 
number of studies to determine the characteristics of intravenous vs. oral 
formulations.  In dogs, the concentration of 5-ALA decreased rapidly after 
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intravenous bolus (terminal half-life of 19.5 ± 2.5 min after dosing) and was 
not significantly different from drug delivered orally (terminal half-life of 40.7 ± 
22.9 min after dosing).5  In a human pharmacokinetic study (N= 4) using a 
128 mg dose of sterile intravenous 5-ALA HCl and oral 5-ALA HCl (equivalent 
to 100 mg 5-ALA) in which plasma 5-ALA and PpIX were measured, the mean 
half-life of 5-ALA was 0.70 ± 0.18 h after the oral dose and 0.83 ± 0.05h after 
the intravenous dose.36  The oral bioavailability of 5-ALA was 50-60% with a 
mean Cmax of 4.65 ± 0.94 µg/mL.  PpIX concentrations were low and were 
detectable only in 42% of the plasma samples.  PpIX concentrations in 
plasma were quite low relative to 5-ALA plasma concentrations, and were 
below the level of detection (10 ng/mL) after 10 to 12 hours.  In another 
human study (N=6), the terminal half-life of an intravenous dose of 5-ALA was 
about 50 minutes for an intravenous dose and 45 minutes for an oral dose.6  
In this same study it was determined that peak plasma concentrations of 5-
ALA were achieved at 0.83 ± 0.20 h after administration of the oral dose. And 
that about 60% of 5-ALA was absorbed after oral administration.  The drug 
was either metabolized in the liver or other organs or rapidly excreted in the 
urine.6
5-ALA does not exhibit fluorescence, while PpIX has a high fluorescence 
yield. Time-dependent changes in surface fluorescence have been used to 
determine PpIX accumulation and clearance in actinic keratosis lesions and 
perilesional skin after application of Levulan Topical Solution in 12 patients.  
Peak fluorescence intensity was reached in 11 ± 1 h in actinic keratoses and 
12 ± 1 h in perilesional skin.  The mean clearance half-life of fluorescence for 
lesions was 30 ± 10 h and 28 ± 6 h for perilesional skin.  The fluorescence in 
perilesional skin was similar to that in actinic keratoses.  Therefore, the FDA 
has recommended that Levulan Topical Solution should only be applied to the 
affected skin. (Data from DUSA Pharmaceuticals New York, Inc., Valhalla, NY 
10595.)
The normal brain synthesizes very little PpIX,8,15,34 which provides good 
contrast to gliomas.24,31  It is interesting that considerably more PpIX is found 
in the brain when the hexyl derivative of 5-ALA (hexaminolevulinate) is given.  
Clinical measurements of PpIX pharmacokinetics have been made in blood 
plasma and from some easily accessible sites by fluorescence spectroscopy 
after different routes of 5-ALA delivery.6,9,20,21,35  One of the most important 
result is that PpIX has returned to baseline levels within 48 hours after dosage 
with 5-ALA, wherever measured and irrespective of the route of 
administration.9,21,36
2.1.4
Toxicity
In vivo pharmacological and toxicological effects that may be associated with 
elevated systemic levels of 5-ALA, Pp IX, and other intermediates of porphyrin 
biosynthesis in humans are well known, and characterize a group of metabolic 
diseases known as the porphyrias.  An association between high systemic 
concentrations of 5-ALA and neurological abnormalities is observed in Acute 
Intermittent Porphyria (AIP).  However, while there have been considerable 
interest in the association between elevated 5-ALA levels and the neurological 
symptoms of AIP, numerous attempts at clarifying the relationship have 
yielded inconsistent results and contradictory conclusions.  Most studies have 
shown that 5-ALA and Porphobilinogen (PBG = condensation of 2 molecules 
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of 5-ALA) are not neurotoxic in vivo when administered in large amounts to 
animals and humans.  Furthermore, the concentration of these precursors in 
the brain or cerebrospinal fluid of porphyric patients is substantially below that 
required for demonstrable toxicity in vitro.  Litman and Correia hypothesize 
that depletion of hepatic heme, a hallmark of AIP, leads to elevated 
tryptophan content and 5-hydroxytryptamine turnover in the brain, and is 
responsible for the neurological symptoms of AIP.16  It should be noted that on 
this basis, administration of exogenous 5-ALA would not lead to a depletion of 
hepatic heme and, therefore would not be expected to elicit the neurological 
symptoms of AIP.  Photosensitivity, associated with high systemic levels of 
PpIX, is a typical finding in Erythropoietic Protoporphyria (EPP) which is 
associated with decreased activity of ferrochelatase, the enzyme that converts 
PpIX to heme.  EPP is entirely cutaneous, with none of the hemolytic or 
neurological problems of the other porphorias.
Mild transient nausea and occasional vomiting have been observed following 
oral administration of high doses of 5-ALA.10,21,37  In one study, the incidence 
of nausea following 30 mg/kg and 60 mg/kg (oral) was 7% and 19%, 
respectively.37  No vomiting was observed with the lower dose, while 8% of 
the patients vomited following the higher dose.  In all instances the nausea 
was mild and short lived (less than 15 min.).  When vomiting occurred, it was 
mild and occurred only once in each patient.  The nausea or vomiting 
occurred within 2.5 to 3 hours after receiving the drug.
Abnormalities in liver function have been observed in patients following 
ingestion of 5-ALA.  Webber et al. observed that almost one-quarter of all 
patients who retained 5-ALA had at least one abnormality in liver function 
tests.37  This was unrelated to 5-ALA dose in that more patients receiving low 
doses (< 30 mg/kg) developed abnormalities than those receiving high doses.  
Significant variations in the time of appearance of the abnormalities were also 
noted (12 to 120 hr. postoperatively).  In all cases, liver function tests returned 
to normal 2 months after receiving 5-ALA.
The most common complaint, due to Pp IX photosensitization, is a very 
unpleasant pricking, itching or burning sensation under the skin after 
exposure to the sun.  A systemic load above 10 mg/kg is required to develop 
skin photosensitization (DUSA Pharmaceuticals) which lasts from 
approximately 24 to 48 hours following 5-ALA administration.  It is for this 
reason that patients will be kept in subdued light conditions for 48 hours 
following surgery. 
2.2 Brain Tumors
2.2.1
Background
Malignant gliomas are brain neoplasms that originate from the parenchymal 
elements of the brain. Malignant gliomas account for approximately 70% of the 
22,500 new cases of malignant primary brain tumors that are discovered in 
United States adults each year.38  Although these tumors are relatively 
uncommon, the prognosis is generally very poor with a median survival of 12-15 
months for individuals diagnosed with glioblastoma multiforme (GBM) and 2 to 5 
years for those with anaplastic astrocytoma (AA).  The median survival of a 
patient undergoing biopsy of GBM without surgical resection or further treatment 
is about 8 weeks.  For those patients who undergo surgery alone, the median 
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survival increases to 17 weeks.  The addition of radiation therapy post-surgical 
resection raises median survival to approximately 7 to 12 months; chemotherapy 
adds an additional few weeks to this median survival.  Overall, the 5-year relative 
survival rate for a primary malignant glioma is age dependent.  Survival rates are 
63.1% for age 0-19, 50.4% for age 20-44, 14.2% for age 45-64, and 4.9% for age 
>65.  Recurrence of the tumor at the site of surgical resection is the rule.  The 
recurrence is usually within 2 cm of the margin of the resection cavity in 80% of 
cases.  For patients with recurrent tumors undergoing repeat surgical resection, 
the re-operation may add as much as 14 to 30 weeks of additional survival.  
Nevertheless, this response is met with fatal growth in virtually all patients.  The 
5-year survival rate is less than 5%.2,22,38
2.2.2
Current Treatment of Malignant Brain Tumors
The current treatment of patients with malignant brain gliomas is traditionally 
divided into surgical, radiotherapeutic, chemotherapeutic, and experimental 
treatments.38  Surgery is usually performed to either fully resect or debulk the 
contrast-enhancing portion of the tumor.  Although surgery alone rarely cures a 
malignant brain tumor, reduced tumor burden allows the body’s own immune 
system or adjuvant therapies a greater chance of success.  Biopsies are usually 
reserved for tumors in areas of the brain that are considered eloquent (harboring 
critical cerebral functions; i.e. language, motor, vision, functions, etc.) or when the 
patient’s medical condition does not predict a safe surgical outcome.  
Radiotherapy plays a central role in the management of brain tumors in 
individuals older than 4 years of age.  Radiotherapy is generally regional, with a 
boost to the tumor bed, for a total of 6,000 cGy over 30 treatments in 6 weeks.  
Chemotherapy continues to be offered to brain tumor patients as adjuvant 
treatment.  It is usually delivered intravenously but oral agents such as Temodar 
have recently shown some efficacy and are now the standard of care.  
Chemotherapy impregnated wafers (BCNU) are sometimes implanted at the time 
of surgery.  Experimental protocols, including immunotherapy and gene therapy, 
round out the efforts attempting to better treat these deadly neoplasms.
There is general agreement amongst neurosurgeons and neuro-oncologists that 
the extent of resection is a major factor in the prognosis of individuals with 
malignant gliomas.2,22,30,38  Usually the extent of resection refers to resection of 
the contrast-enhancing portion of the tumor.  The ultimate goal in surgery is to 
achieve a “gross total” resection of the tumor which is defined as complete 
absence of the contrast-enhancing portion of the tumor on the immediate post-
operative scan.
There are a number of methods that the neurosurgeon uses to achieve the goal 
of gross total resection of a malignant glioma.  The first method is to dissect 
around the tumor and to develop the plane between the obvious tumor tissue and 
the surrounding normal brain.  In many instances this can be done – usually with 
the help of the operating microscope.  At times, however this plane can be very 
difficult to establish – especially when the dissection is close to the intracerebral 
ventricle where tumor is often difficult to distinguish from the normal surrounding 
brain.  It is very important in these instances to use other methods to help to 
identify residual tumor.  Some neurosurgeons have used intra-operative 
ultrasound with limited success.  Other neurosurgeons have employed the use of 
an intra-operative MRI scanner which is very expensive and cumbersome.  
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In the past decade a group of neurosurgeons in Europe led by Walter Stummer, 
M.D. have explored the use of intra-operative tumor fluorescence from 5-ALA-
PpIX to identify residual malignant glioma.24-33  These studies have established 
that this technique can be very successful in helping to achieve gross-total 
resection in malignant gliomas with great safety and effectiveness.  These studies 
have generated interest in this technique amongst neurosurgeons in the United 
States.  Independently, Dr. John Ruge from the Lutheran General Hospital in 
Park Ridge, Illinois and also the Principal Investigator of this proposal, Dr. Jeffrey 
Cozzens has traveled to Dusseldorf, Germany to observe brain tumor surgery 
with 5-ALA-PpIX fluorescence by Dr. Stummer.  Dr. Ruge has, in conjuction with 
DUSA Pharmaceuticals, initiated a Phase I/II study with the FDA for studying the 
use of 5-ALA in brain tumor surgery (see 
http://www.clinicaltrials.gov/ct2/show/NCT00671710?term=5-ALA&rank=12).   
(ClinicalTrails.gov identifier NCT00671710).  There is also a similar trial underway 
in Cleveland, Ohio under the direction of the National Cancer Institute and Dr. 
Robert Maciunas (see 
http://www.clinicaltrials.gov/ct2/show/NCT00752323?term=5-ALA&rank=8) 
(ClinicalTrials.gov identifier NCT00752323)       
2.3 Rationale for the Use of 5-ALA in the Surgery of Brain Tumors
Aminolevulinic acid (5-ALA) is a naturally occurring amino acid and a precursor in heme 
biosynthesis.  5-ALA is produced at the cytosolic surface of the mitochondrial membrane 
and then transported to the cellular cytosol for heme biosynthesis.  The availability of 5-
ALA in the cell cytoplasm is the rate limiting factor in heme biosynthesis in all but 
erythropoetic cells.  The heme biosynthetic enzymes are more active in glial tumors of 
the brain than in normal brain tissue.  As a result, addition of 5-ALA (the rate-limiting 
precursor) to malignant glial cells leads to an increase in the intracellular accumulation of 
protoporphorin IX (PpIX), a fluorescent intermediary in heme biosynthesis.  As a result, 
malignant glial cells show red fluorescence relative to dark normal brain tissue when 
viewed under deep blue light (440 nm). This effect allows the neurosurgeon to distinguish 
tumor from normal brain and therefore enables more complete surgical removal of 
malignant glial tumor from within normal brain tissue.
In a study in Europe for approval of 5-ALA by the European Medicines Agency, a similar 
study to this proposed study was performed (also see 
http://www.emea.europa.eu/humandocs/PDFs/EPAR/gliolan/H-744-en6.pdf
Study MC-ALS.28/GLI
The aim of this prospective, single-arm, uncontrolled multicentre (n = 4) phase II study 
was to determine the positive predictive value of tissue fluorescence, defined as the 
percentage of patients showing positive tumour cell identification in all biopsies taken 
from areas of weak and strong fluorescence.
The primary efficacy parameter was the positive predictive value of tissue fluorescence, 
defined as the percentage of patients with positive tumour cell identification in all biopsies 
taken from areas with weak and strong fluorescence.  A biopsy was termed "positive 
tumour cell identification" if the reference neuropathological institute observed a tumour 
cell content greater than 0%.
The mean tumour cellularity per patient (i.e. mean area on a section occupied by tumour 
cells) in strongly fluorescing biopsies was 79.1% ± 20.1%, whereas mean tumour 
cellularity in weakly fluorescing biopsies was 30.78% ± 27.88% per patient. A minimum 
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tumour cellularity of 4.5% was detectable by virtue of its (weak) fluorescence by the 
neurosurgeon.  Taking all biopsies together, mean tumour cellularity with strong or weak 
fluorescence was 79.1% ± 19.8%.
The number of truly-positive specimens (biopsies) was higher if the quality of 
fluorescence was strong (n=32) rather than weak (n=25).  Consequently, the positive 
predictive value of strong fluorescence was higher (100.0%; 90% CI: 91.1% - 100.0%) 
than that of weak fluorescence (83.3%; 90% CI: 68.1% - 93.2%).  In total, there were 28 
patients where all fluorescing biopsies showed tumor in all biopsies taken from areas of 
any fluorescence (strong or weak fluorescence) resulting in a positive predictive value of 
84.8% (90% CI: 70.7% - 93.8%).
Among 185 evaluated biopsies, seven sections did not reveal any tumour cells (false-
positive specimens). In all false-positive specimens, the quality of fluorescence in was 
weak.  Strongly fluorescing false-positive biopsies did not occur in this study.
The positive predictive value of tissue fluorescence at the biopsy level, defined as the 
number of tumour positive biopsies among all biopsies taken from areas of any 
fluorescence (weak and strong fluorescence), was 96.2% (90% CI: 93.0% - 98.2%). The 
positive predictive value was higher, if only strong fluorescent biopsies were taken into 
account (100.0%; 90% CI: 96.9% - 100.0%) compared to weak fluorescent biopsies only 
(92.2%; 90% CI: 85.9% - 96.3%).
This study only provides data for the positive predictive value.  Nothing was obtained for 
the negative predictive value.  The results showed that the strong fluorescent area has a 
much higher cellularity than the comparators areas and this makes the positive predictive 
value of strong fluorescence 100%.  It satisfactorily shows that the fluorescence is highly 
specific for tumour.  Since the major problem is to remove healthy tissue taken as tumour 
(false positives) the high specificity is reassuring.  The issue of real sensitivity remains 
but it less critical for the clinical use.
2.4 Correlative Studies Background
This study will run in conjunction with another study (currently being developed) that will 
look at the feasibility and effectiveness of digital subtraction magnetic resonance imaging 
(dsMRI) of the brain.  Currently the radiological assessment of the extent of brain tumor 
resection is performed utilizing the neuroradiologist’s subjective interpretation of the 
differences between a post-operative gadolinium enhanced image and a post-operative 
non-enhanced image.  dsMRI has the potential to digitally and objectively define and 
indentify the differences between these two images.  It also has the potential to measure 
the volume of any residual tumor.
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3
PATIENT SELECTION
3.1 Eligibility Criteria
Patients must have clinically documented primary brain tumor for which resection is 
clinically indicated.  The anticipated histology at resection should include:  Anaplastic 
astrocytoma (10002224), Astrocytoma malignant NOS (10003572), Brain stem glioma 
(10006143), Ependymoma (10014967), Ependymoma malignant (10014968), 
Glioblastoma (10018336), Glioblastoma multiforme (10018337), Gliosarcoma 
(10018340), Anaplastic oligodendroglioma (10026659), Oligodendroglioma (10030286), 
Medulloblastoma (10027107), Mixed astrocytoma-ependymoma (10027743), 
Miscellaneous CNS primary tumor (10007959), Supratentorial primitive neuroectodermal 
tumor (10056672)
3.1.1
Prior therapy is not a consideration in protocol entry.
3.1.2
Age ≥ 18 years.  Because no dosing or adverse event data are currently available 
on the use of 5-ALA in patients <18 years of age, children are excluded from this 
study but will be eligible for future pediatric phase 1 single-agent trials.
3.1.3
ECOG performance status <2 (Karnofsky >60%, see Appendix A).
3.1.4
Life expectancy is not a consideration for protocol entry. 
3.1.5
Patients must have normal organ and marrow function as defined below:
Leukocytes
>3,000/mcL
Absolute neutrophil count
>1,500/mcL
Platelets
>100,000/mcL
Total bilirubin
within normal institutional limits
AST (SGOT)/ALT (SGPT)
<2.5 X institutional upper limit of normal
Creatinine
within normal institutional limits
OR
Creatinine clearance
>60 mL/min/1.73 m2 for patients with creatinine 
levels above institutional normal.
3.1.6
The effects of 5-ALA on the developing human fetus are unknown.  5-ALA has 
unknown teratogenic or abortifacient effects.  For this reason, women of child-
bearing potential and men must agree to use adequate contraception (hormonal 
or barrier method of birth control; abstinence) prior to study entry and for the 
duration of study participation.  Should a woman become pregnant or suspect she 
is pregnant while participating in this study, she should inform her treating 
physician immediately.
3.1.7
Ability to understand and the willingness to sign a written informed consent 
document.
3.2 Exclusion Criteria
3.2.1
Prior therapy is not an exclusion criterion.
3.2.2
Patients may not be receiving any other investigational agents at the time of entry 
into the study.
3.2.3
History of allergic reactions attributed to compounds of similar chemical or 
biologic composition to 5-ALA.
3.2.4
Personal or family history of porphyrias.
3.2.5
Uncontrolled intercurrent illness including, but not limited to ongoing or active 
infection, symptomatic congestive heart failure, unstable angina pectoris, cardiac 
arrhythmia, or psychiatric illness/social situations that would limit compliance with 
study requirements.
3.2.6
Pregnant women are excluded from this study because 5-ALA is of unknown 
teratogenic or abortifacient effects.  Because there is an unknown but potential 
risk for adverse events in nursing infants secondary to treatment of the mother 
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with 5-ALA, breastfeeding should be discontinued if the mother is treated with 5-
ALA.
3.2.7
Patients who are cancer survivors or those who are HIV positive are not excluded 
from this study.
3.2.8
Patients with significant cognitive impairment (which in the judgement of the 
principal investigator leaves them incapable of giving informed consent) are 
excluded from this study. 
3.3 Inclusion of Women and Minorities
Both men and women and members of all ethnic groups are eligible for this trial.  The 
proposed study population is illustrated in the table below.
Race/Ethnicity (%)
Gender
White, not of 
Hispanic 
Origin
Black, not 
of Hispanic 
Origin
Hispanic
Asian or 
Pacific 
Islander
Unknown
Total
Male
25
5
5
5
10
50
Female
25
10
5
5
5
50
Total
50
15
10
10
15
100
4
TREATMENT PLAN
4.1 Aminolevulinic Acid (5-ALA) Administration
Treatment will be administered on an inpatient basis.  Reported adverse events and 
potential risks are described in Section 6.  One-time, single-dose administration of 5-ALA 
is planned, which precludes the need for dose modification.  No investigational or 
commercial agents or therapies other than those described below may be administered 
with the intent to treat the patient's malignancy in the peri-operative period.
4.1.1
Treatment Protocol
A maximum of thirty-three subjects with malignant brain tumors will participate in 
this trial.  Each patient will have been evaluated and found to have a malignant 
brain tumor by history and recent imaging studies (MRI) and deemed a surgical 
candidate based on current neurosurgical standards of care.  Consent will be 
obtained by the principal investigator or co-investigator.  Three subjects will be 
enrolled in each of the five phase 1 dose levels seen below according to the rules 
seen in the table in section 4.2.1.  After establishment of the safest effective dose, 
an additional 16 subjects will be enrolled at that dose level for the phase 2 study.  
The protocol is divided into two parts; the surgical procedure part and the post 
operative treatment part.
Dose-Escalation Schedule
Dose Level
Dose of 5-ALA
Level 1
10 mg/kg
Level 2
20 mg/kg
Level 3
30 mg/kg
Level 4
40 mg/kg
Level 5
50 mg/kg
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4.1.2
Surgical Phase
5-ALA will be mixed in the minimum volume of sterile water or juice immediately 
before use and given as a single bolus orally approximately three hours prior to 
induction of anesthesia for surgical resection of a malignant brain tumor.  Image-
guided microsurgical resection of the tumor will be undertaken.  The intensity of 
the white and blue light from the microscope will be measured at the shortest 
focal length of the microscope with a digital light meter before resection of the 
tumor is performed.  During tumor resection under the surgical microscope, the 
tumor bed will be illuminated with deep blue light via a high pass optical filter in 
the microscopes light chain.  The neurosurgeon will examine the tumor bed with a 
barrier filter in the optical chain of the surgical microscope (see Appendix B).
Under white light the neurosurgeon will take small biopsies from areas of obvious 
tumor and areas in the wall of the resection cavity that are judged to be normal 
(but possibly edematous), non-eloquent brain.
Under blue light, small biopsies will be taken from areas of strong fluorescence, 
weak fluorescence and non-fluorescing regions in the tumor resection cavity.  
At all times, the neurosurgeon will use their best judgment to remove or biopsy 
only that tissue that would normally be removed or biopsied as part of any brain 
tumor procedure that was not involved or part of an investigational study.
Specimens will be collected regardless of fluorescence.  All biopsy specimens, 
including those taken from the solid tumor, will be reviewed by a neuro-
pathologist. Residual fluorescent tissue in the tumor cavity will be surgically 
removed only if in the judgment of the neurosurgeon these areas would have 
been resected using white light alone and that this would lead to a more complete 
resection of tumor.  Pathologic confirmation of tumor type will be made by 
neuropathology.  
After surgical resection of the tumor, the neurosurgeon will judge the extent of 
resection by estimating the volume of remaining obvious tumor seen under white 
light or fluorescent tissue seen under blue light.
Data Collection in Operating Room
Under White Light
Obvious Tumor
Obvious Normal Brain (possibly edematous)
Biopsy for Histology
A
B
Tumor Content of Sample
X
X
Specimen Labeled
X
X
Under Blue Light
Strongly Fluorescent Tissue
Weakly 
Fluorescent 
Tissue
Non-Fluorescent Tissue
Biopsy for Histology
A (This can be the same 
biopsy as A under white light 
or it can be a new biopsy D)
C
B (This can be the same 
biopsy as B under white 
light or it can be a new 
biopsy E)
Fluorescence Intensity by 
Neurosurgeon 
X
X
X
Tumor Content of Sample
X
X
X
Specimen Labeled
X
X
X
Estimate made of extent 
X
X
X
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of Fluorescent 
Tissue/Tumor Resected
Specimens will be collected regardless of fluorescence.
Areas where biopsies have been made of fluorescent and non-fluorescent tissue 
within the tumor cavity will be graded by the neurosurgeon at the time of biopsy 
as 0 to +++ (3+) fluorescence.  This will be recorded in the operating room each 
specimen identified with a code.  The pathologist who reviews each specimen will 
be blinded as to the extent of fluorescence in the area where the biopsy was 
obtained. 
Grade
Definition
0
No fluorescence.  No difference 
between any of the tissues 
visualized.
No fluorescence
+
Low fluorescence.  Red glow is 
barely distigushed from surrounding 
non-fluorescent tissue.
++
Moderate fluorescence.  Dull red 
glow of fluorescent tissue.
Weak fluorescence
+++
High fluorescence.  Bright red glow 
of fluorescent tissue
Strong fluorescence
The neurosurgeon is responsible for: 

An overall assessment of the detection of tumor within the cavity provided by 
the fluorescence imaging. 

Judging the extent of resection of the tumor or fluorescent tissue.

Under white light:
o
Identifying at least one area of obvious tumor and biopsying that site.  
Then switch to blue light and grading the fluorescence at the site.
o
Identifying at least one area of obviously normal (but possibly edematous) 
brain in the wall of the tumor resection cavity in an area of non-eloquent 
brain and biopsying that site.  Then switch to blue light and grading the 
fluorescence at the site.

Under blue light:
o
Identifying at least one fluorescent area within the tumor cavity in an area 
of non-eloquent brain and biopsying and grading that site’s fluorescence.  
It may not be necessary to biopsy this area if this same area has already 
been biopsied under white light.
o
Identifying at least one non-fluorescent area within the tumor cavity in an 
area of non-eloquent brain and biopsying and grading that site’s 
fluorescence.  It may not be necessary to biopsy this area if this same 
area has already been biopsied under white light.
o
Identifying one area of weak fluorescence in an area of non-eloquent brain 
and biopsying and biopsying and grading that site’s fluorescence.  It may 
not be necessary to biopsy this area if this same area has already been 
biopsied under white light.
o
Intra-operative photographs of tumor fluorescence will be made during 
each surgery to help compare the degree of fluorescence from one patient 
to the other. 
The neuro-pathologist is responsible for:
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
Determining the extent of tumor cellularity for each specimen in categories of
o
Solid tumor
o
Infiltrating tumor/normal brain
o
No tumor (edematous peritumoral normal brain)
4.1.3
Post-Operative Follow-up
Patients will be assessed for completeness of tumor resection within 24 hours 
following surgery using MRI both with and without gadolinium enhancement.  A 
24 post-surgical MRI within 24 hours of surgery is the standard of care for anyone 
undergoing surgical resection of a brain tumor.  Subsequently, patients will be re-
imaged periodically to assess tumor progression according to the specific 
standard of care tumor treatment protocol determined by the Neuro-Oncologist.  
At these same intervals, when appropriate to the normal standard of care, routine 
liver function tests and physical examination will be performed.
4.1.4
Survival
Survival will be determined over two years from entry into the study by either 
periodic medical record review for patients seen in the SIU Neuro-Oncology 
Clinic, or by search of the Social Security Death Index (SSDI). 
Volumetric analysis will be performed with Tera-recon software on gadolinium 
contrast-enhanced T1W MRI to assess extent of resection (EOR) and residual 
tumor volume (RTV). These findings will then be correlated with progression free 
survival (PFS) and overall-survival (OS).
4.2 Definition of Dose-Limiting Toxicity
Dose-Limiting Toxicity (DLT) is determined by Adverse Events which are defined and 
graded following the CTEP, NCI Guidelines: Adverse Event Reporting Requirements of 
January 1, 2005 
(http://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/newadverse_2
006.pdf) and the CTC/CTCAE Criteria.  Expected Adverse Events (AE) have not been 
identified for 5-ALA by the NCI Comprehensive Adverse Event and Potential Risk list 
(CAEPR) but are identified in section 11.1.2 and 11.1.3 of this protocol.  Those AEs 
which are determined to be Grade 2 or higher and directly attributed to 5-ALA or its 
metabolites will be considered to be dose-limiting for the purposes of determining dose 
escalation.  At present, CTEP does not have a CAEPR or ASAEL for this agent (see 
Appendix D).
Since this study involves the one-time dose of the study drug, further dose modifications 
associated with the above adverse events will not be required.
4.2.1
Dose-Escalation Scheme
Dose escalation will proceed within each cohort according to the following 
scheme.  Three subjects will be entered sequentially at each of the five dose 
levels (10, 20, 30, 40, and 50 mg/kg).  This dose escalation scheme is more 
aggressive than standard accelerated dose escalation schemas published 
because we are already aware of safety data from Europe demonstrating no 
toxicity with oral doses of 50 mg/kg.  An additional three subjects will be added to 
the recommended phase 2 dose level according to the following scheme.  Three 
subjects are planned at each dose level in order to ensure that sufficient samples 
are available to determine fluorescent intensity differences between normal and 
malignant tissue at each dose level.
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Each dose escalation will be approved by a Data Safety Monitoring Board 
determined by SCRIHS (see Section 11.5).
Dose Escalation and Modification Rule Table
4.2.2
Phase 2 accrual of 16 additional subjects at the recommended phase 2 dose will 
commence once the phase 1 portion of this trial is completed.  All correlative 
studies will be continued in the phase 2 portion of the study in order to define the 
differential fluorescence between normal and malignant brain tissue, 
enhancement of tumor resection, and survival impact of preoperative 5-ALA.
4.3 Supportive Care Guidelines
Patients will be managed by standard of care procedures for post-operative brain 
resection patients.  In addition, patients will be sequestered in “low light” environment for 
48 hours following the administration of 5-ALA.  
Low light exposure conditions will start in the holding area immediately after ingestion of 
the study drug and will continue in all patient environments for at least 48 hours.  These 
conditions include the following:

Covering as much of the patient’s skin, mucous membranes and eyes as possible 
except for those areas that need to be exposed for clinical care and evaluation (ie. 
placement of intravenous access, preparation for general anesthesia, preparation for 
surgery, observation during and after surgery, etc.).  This will include the patient 
wearing dark glasses when awake.

Windows in patient care areas will be covered and doors closed.

Overhead room lights will be turned off in patient care areas. Lights that are 
necessary for patient care may be used at the minimal intensity required.  

Patient care areas do not need to be completely dark.  Minimal light for patient, 
caregiver and visitor safety is allowed.  Televisions, monitors and medical equipment 
may be used as necessary.  
# of Patients with DLT at 
a Given Dose Level
Escalation Decision Rule
0 out of 3
Enter 3 patients at the next dose level.
> 2 out of 3
Dose escalation will be stopped.  This dose level will be declared the 
maximally administered dose (highest dose administered).  Three (3) 
additional patients will be entered at the next lowest dose level if only 3 
patients were treated previously at that dose.
1 out of 3
Enter at least 3 more patients at this dose level.
- If 0 of these 3 patients experience DLT, proceed to the next dose level.
- If 1 or more of this group suffer DLT, then dose escalation is stopped, 
and this dose is declared the maximally administered dose.  Three (3) 
additional patients will be entered at the next lowest dose level if only 3 
patients were treated previously at that dose.
<1 out of 6 at highest dose 
level below the maximally 
administered dose
This is generally the recommended phase 2 dose.  At least 6 patients 
must be entered at the recommended phase 2 dose.
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4.4 Duration of Therapy
A one-time, single-dose administration of oral 5-ALA 3 hours prior to induction of 
anesthesia for surgical resection of a malignant glioma is planned.
4.5 Duration of Follow-up
Patients will be followed for 2 years after surgery or until death, whichever comes first.
4.6 Criteria for Removal from Study
Because this study involves the one-time dose of the study drug followed by surgery, the 
only criteria for removal from the study would be if the patient either could not ingest the 
drug or could not undergo surgery. 
5
DOSING DELAYS/DOSE MODIFICATIONS
One-time, single-dose administration of 5-ALA is planned, which precludes the need for 
intra-patient dose modification or delay. 
6. ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS 
The following list of AEs (Section 6.1) and the characteristics of an observed AE (Section 
6.2) will determine whether the event requires expedited (via Medwatch) reporting in addition 
to routine reporting.
6.1 Comprehensive Adverse Events and Potential Risks List (CAEPR)
The Comprehensive Adverse Event and Potential Risks list (CAEPR) provides a single, 
complete list of reported and/or potential adverse events (AE) associated with an agent 
using a uniform presentation of events by body system. 
We have contacted CTEP and we have been informed that CTEP does not have a 
CAEPR list for 5-ALA (see Appendix D).
6.2 Adverse Event Characteristics 
CTCAE term (AE description) and Grade
The descriptions and grading scales found in the revised NCI Common Terminology 
Criteria for Adverse Events (CTCAE) version 3.0 will be utilized for AE reporting.  All 
appropriate treatment areas will have access to a copy of the CTCAE version 3.0.  A 
copy of the CTCAE version 3.0 can be downloaded from the CTEP web site 
(http://ctep.cancer.gov).
Expectedness - AEs can be ‘Unexpected’ or ‘Expected’ for expedited reporting purposes 
only.  ‘Expected’ AEs are listed in sections 11.1.2 and 11.1.3 of this protocol.
Attribution of the AE
Definite – The AE is clearly related to the study treatment.
Probable – The AE is likely related to the study treatment.
Possible – The AE may be related to the study treatment.
Unlikely – The AE is doubtfully related to the study treatment.
Unrelated – The AE is clearly NOT related to the study treatment.
6.3 Expedited Adverse Event Reporting
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22
6.3.1
Phase 1 and phase 2 studies with investigational agents (Including hospitalization 
defined below):
- For hospitalization only – Any medical event equivalent to CTC grade 3, 4, or 5 
which precipitated hospitalization (or prolongation of existing hospitalization) must 
be reported regardless of designation as expected or unexpected and attribution.
- The FDA safety information and adverse event reporting system, Medwatch, 
FDA form 3500A will be used for reporting within 24 hours.
6.3.2
Expedited Reporting Guidelines – Medwatch Reporting Requirements for Adverse 
Events That Occur Within 30 Days1 of the Last Dose of the Investigational Agent 
on Phase 1 Trials
Phase 1 Trials
Grade 1
Grade 2
Grade 2
Grade 3
Grade 3
Grades
4 & 52
Unexpected
Expected
Unexpected 
and 
Expected
Unex-
pected
Expected
with 
Hospitali-
zation
without 
Hospitali- 
zation
with 
Hospitali-
zation
without 
Hospitali-
zation
Unexpected 
and
Expected
Unrelated
Unlikely
Not
Required
Not
Required
Not
Required
10 
Calendar
Days
Not
Required
10 
Calendar
Days
Not
Required
24-Hour;
5 Calendar
Days
Possible
Probable
Definite
Not
Required
10 
Calendar
Days
Not
Required
24-Hour;
5 Calendar
Days
24-Hour;
5 Calendar
Days
10 
Calendar
Days
Not
Required
24-Hour;
5 Calendar
Days
1 Adverse events with attribution of possible, probable, or definite that occur greater than 30 days after 
the last dose of treatment with an agent under a CTEP IND require reporting as follows:
AdEERS 24-hour notification followed by complete report within 5 calendar days for:

Grade 3 unexpected events with hospitalization or prolongation of hospitalization

Grade 4 unexpected events 

Grade 5 expected events and unexpected events
2 Although an AdEERS 24-hour notification is not required for death clearly related to progressive disease, a full 
report is required as outlined in the table.                                                                               December 15, 
2004
Note:  All deaths on study require both routine and expedited reporting regardless 
of causality.  Attribution to treatment or other cause must be provided.
Expedited AE reporting timelines defined:
“24 hours; 5 calendar days” – The investigator must initially report the AE via 
Medwatch within 24 hours of learning of the event followed by a complete 
Medwatch report within 5 calendar days of the initial 24-hour report.
“10 calendar days” - A complete Medwatch report on the AE must be submitted 
within 10 calendar days of the investigator learning of the event. 
Any medical event equivalent to CTCAE grade 3, 4, or 5 that precipitates 
hospitalization (or prolongation of existing hospitalization) must be reported 
regardless of attribution and designation as expected or unexpected with the 
exception of any events identified as protocol-specific expedited adverse event 
reporting exclusions.  
6.4 Routine Adverse Event Reporting
All Adverse Events must be reported in routine study data submissions.  AEs reported 
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23
through Medwatch must also be reported in routine study data submissions.
7.
PHARMACEUTICAL INFORMATION
7.1 Drug (5-ALA) - 5-ALA for oral administration will be provided by DUSA Pharmaceuticals 
New York, Inc., Valhalla, NY 10595.
7.2 Drug Availability - The study drug 5-aminolevulinic acid will be provided by DUSA 
Pharmaceuticals New York, Inc., Valhalla, NY 10595.
7.3 Drug Ordering - The study drug 5-aminolevulinic acid will be provided by DUSA 
Phamaceuticals, Inc. to the Memorial Medical Center or St. John’s Hospital Pharmacy.
7.4 Drug Accountability - The Investigator, or a responsible party designated by the 
Investigator, in cooperation with the Memorial Medical Center or St. John’s Hospital 
Pharmacy will maintain a careful record of the inventory and disposition of all agents 
received from DUSA Pharmaceuticals, Inc.
8.
CORRELATIVE/SPECIAL STUDIES
8.1 Assessment of Visual Tumor Discrimination
8.1.1
Overall assessment of Tumor Discrimination - With the tumor and tumor cavity 
exposed, the neurosurgeon will visualize the tumor and tumor cavity under white 
light and again under deep blue light illumination and assess the ability of 
fluorescent illumination to distinguish  tumor from normal brain tissue.  This 
subjective assessment will be graded by the neurosurgeon at the time of 
visualization from 0 to +++ (3+).
Grade
Definition
0
No fluorescence.  No difference 
between any of the tissues visualized.
No fluorescence
+
Low fluorescence.  Red glow is barely 
distigushed from surrounding non-
fluorescent tissue.
++
Moderate fluorescence.  Dull red glow 
of fluorescent tissue.
Weak fluorescence
+++
High fluorescence.  Bright red glow of 
fluorescent tissue
Strong fluorescence
8.1.2
Surgical Assessment of Fluorescence in Biopsy Specimens: 
Each biopsy specimen taken for this trial under blue light will be graded for 
fluorescence intensity at the time of biopsy by the neurosurgeon on a scale of 0 to 
+++ (3+).
8.1.3
Pathologic Assessment of Biopsy Specimens:  
All biopsy specimens, including those taken from the bulk tumor, will be reviewed 
by a neuropathologist.
9.
STUDY CALENDAR
Baseline evaluations are to be conducted within two weeks prior to start of protocol therapy.  
Scans and x-rays must be done within one week prior to the start of therapy.  Adverse 
events will be recorded up to 30 days following surgery.
Pre-
Day 1 
Day 2
Week 5
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24
Study
(Surgery)
5-ALA a 
X
Informed consent
X
Demographics
X
Medical history
X
Physical exam
X
X
X
Vital signs
X
X
X
X
Concurrent Meds
X
X
X
Height
X
Weight
X
X
X
Performance Status
X
X
X
Adverse Event 
Evaluation
X
X
X
X
CBC w/diff, platelets
X
X
X
X
Serum chemistry
X
X
X
X
EKG (as indicated)
X
MRI Brain gadolinium
X
X
B-HCG
X
Other correlative 
studies
X
10. MEASUREMENT OF EFFECT
In this study, the study drug 5-ALA is not expected to have any direct effect on tumor cells.  
Future studies may be designed to investigate the potential of 5-ALA and its photosensitizing 
metabolite PpIX in a study of photodynamic therapy, but in this study no attempt will be 
made to photo-activate the tumor cells.
The study will look at the specificity and sensitivity of 5-ALA/PpIX for malignant glioma cells. 
10.1 Definitions
Response and progression will be primarily evaluated in this study using MRI 
volumetric techniques which measure the volume of residual enhancing tumor.  
10.1.1 Extent of Resection
Volumetric MRI techniques measure difference in the areas of bright MRI signal 
when a post-operative T1 non-enhanced image is compared to a post-operative 
T1 gadolinium enhanced image.  These differences may be measured by post-
processing digital subtraction techniques (dsMRI – see section 2.4) or by outlining 
areas of new bright signal as judged by the neuroradiologist.  Extent of resection 
is divided into three categories which are defined as follows:
- Gross-total tumor resection (GTR) of tumor is defined as no measureable 
residual contrast enhancement in the immediate post-operative MRI.  
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25
- Near-total resection (NTR) is defined as measureable post-operative 
residual contrast enhancement greater than or equal to the volume of one 
MRI voxel (0.175 cm3) and less than or equal to 2.5 cm3. 
- Incomplete resection (IR) is defined as measureable post-operative residual 
contrast enhancement greater than 2.5 cm3.
10.2
Guidelines for Evaluation of Measurable Disease
All neuroradiological measurements should be taken and recorded in metric notation 
using standardized volumetric techniques.  These techniques have been discussed 
previously.7,11,12
10.3
Confirmatory Measurement/Duration of Response
10.3.1
Confirmation - N/A
10.3.2
Duration of Overall Response - N/A
10.3.3
Duration of Stable Disease - N/A
11.
REGULATORY AND REPORTING REQUIREMENTS
Adverse event (AE) reporting for this study is via the FDA safety information and adverse 
event reporting system, Medwatch, FDA form 3500A.  The descriptions and grading scales 
found in the revised NCI Common Toxicity Criteria (CTC) version 3.0 will be utilized for 
adverse event reporting and are available in the Clinical Research Department and 
available on line at http://ctep.cancer.gov/reporting/ctc.html.  Expected adverse events 
specific for 5-ALA are found below (Section 11.1.2).
11.1
Expedited Adverse Event Reporting (AE; formerly known as Adverse Drug 
Reaction)
11.1.1 Expedited Reporting Guidelines – Phase 1 studies with investigational agents 
(Including hospitalization defined in bullet 1 below)

For Hospitalization only – Any medical event equivalent to CTC grade 3, 
4, or 5 which precipitated hospitalization (or prolongation of existing 
hospitalization) must be reported regardless of designation as expected or 
unexpected and attribution.

The FDA safety information and adverse event reporting system, Medwatch, 
FDA form 3500A will be used for reporting within 24 hours.
11.1.2 Agent-Specific Expected Adverse Events List 
The list below guides the investigator in determining which AEs require expedited 
reporting.  Those AEs that do not require expedited reporting are reported in 
routine study data submissions.

Skin reaction - Grades 1 and 2.

AST/ALT elevation - Grades 1, 2, or 3.

Nausea - Grades 1 and 2. 
11.1.3 Other Expected Adverse Events
For this protocol, the following more serious expected adverse events are risks 
associated with surgical removal of a malignant brain tumor and do not require 
expedited reporting: 

Adverse reaction to anesthesia - Both local and general anesthesia involve 
risk.  There is a possibility of complication or injury from all forms of 
anesthesia and sedation.

Balance problems - Difficulty with balance or vertigo may occur as a result of 
the tumor itself or from the surgery to correct it.  Nausea and vomiting may 
also occur after surgery.

Bleeding - It is possible, though unusual, to experience an episode of 
bleeding, which may be extensive during or after surgery.  Bleeding may 
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26
require additional treatment or transfusion.  Certain medications, such as anti-
inflammatory drugs, may increase the risk of bleeding.

Blood clot development - Blood clots may occur with any type of surgery.  
Blood clots at the site of the surgery can block blood flow and cause 
complications, including pain, swelling, inflammation and tissue damage 
(stroke).

Brain injury - There is a risk that the procedure will cause injury of the 
surrounding brain.  The symptoms that result from the injury depend on the 
location of the tumor.

Cardiac complications - There is a chance that having the procedure could 
cause an irregular heartbeat or a heart attack.

Functional loss - It is possible to experience problems such as difficulty 
opening the mouth or chewing after surgery.  Also, speech, language and 
memory difficulties may occur after surgery.

Hydrocephalus - It is-possible that the normal flow of spinal fluid around the 
brain may be altered.  Additional treatment, including surgery, may be 
necessary to correct this.

Infection - Infection may occur not only at the incision site, but also within the 
bone flap.  Infection-related risks also include the development of meningitis 
(an infection which causes inflammation of the membranes covering the brain 
and spinal cord) or a brain abscess (localized collection of pus).

Loss of nerve function - Some loss of function may occur in the facial nerves 
as a result of the procedure.

Paralysis - it is possible that some paralysis or numbness may occur as a 
result of the surgery.

Post-operative neurologic decline - There is a small risk that neurologic 
function will decline following surgery and may produce such symptoms as 
personality changes or loss of cognitive function.  These problems are 
sometimes caused by post-operative hemorrhage (bleeding into or on the 
surface of the brain) or cerebral edema (accumulation of fluid that results in 
swelling and pressure on the brain).

Post-operative pain - It is possible, though unlikely, that pain or other 
symptoms will increase following the procedure.  A headache may linger 
anywhere from one week to one month following craniotomy and occasionally 
for a longer period of time.

Recurrence - There is a chance that the tumor may return to the same site.  
However, the risk of recurrence depends on the type of tumor and how much 
of it can be removed in the initial operation.

Respiratory difficulties - Breathing difficulties, which are usually temporary or 
post-operative pneumonia may occur as a result of surgery.  Pulmonary 
embolus could occur from blood clotting in the veins.

Seizure activity - Abnormal electrical activity in the brain, which can trigger 
seizures, may develop in response to the tumor itself or its removal.

Visual disturbances - There may be some changes in visual function caused 
by the surgery or by the tumor itself.
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27
11.1.4 Forms - None additional
11.1.5 Secondary AML/MDS – N/A
11.2
Data Reporting - In addition to adverse event reporting, all reports will be provided to 
the FDA as required by the Code of Federal Regulations.
11.3
CTEP Multicenter Guidelines - N/A
11.4
Cooperative Research and Development Agreement (CRADA)/Clinical Trials 
Agreement (CTA) N/A The agent, aminolevulinic acid (5-ALA), used in this protocol is 
provided by Collaborator, DUSA Pharmaceuticals New York, Inc., Valhalla, NY 
10595.
11.4.1 Aminolevulinic acid (5-ALA) may not be used for any purpose outside the scope 
of this protocol, nor can aminolevulinic acid (5-ALA) be transferred or licensed to 
any party not participating in the clinical study.  Collaborator data for 
aminolevulinic acid (5-ALA) are confidential and proprietary to Collaborator and 
shall be maintained as such by the investigators.
11.4.2 Any data provided to Collaborator(s) for Phase 3 studies must be in accordance 
with the guidelines and policies of the responsible Data Monitoring Committee 
(DMC), if there is a DMC for this clinical trial.
11.5
Data Safety Monitoring Plan
A minimum of 3 to a maximum of *18 subjects will be enrolled in this study.  Oral 5-
ALA will be administered 2-3 hours prior to surgery in cohorts of three at five 
escalating doses (10, 20, 30, 40, or 50 mg/kg).  (*Due to four patients receiving 
40mg/kg rather than three, 19 total patients will be studied in phase 1. Protocol 
deviation approved by IRB on 17 Oct 2013.)
Study investigators and study personnel will carefully screen each patient for side 
effects and adverse events.  Expected adverse events specific for 5-ALA are 
described in the Protocol (Section 11.1.2) and other expected adverse events are 
UNEXPECTED EVENT
EXPECTED EVENT
GRADES 2 – 3
Attribution of Possible, 
Probable or Definite
GRADES 4 and 5
Regardless of Attribution
GRADES 1 - 3
GRADES 4 and 5
Regardless of Attribution
Grade 2 - Expedited report 
within 10 working days.
Grade 3 - Report by phone 
to IDB within 24 hrs. 
Expedited report to follow 
within 10 working days.
(Grade 1 – Adverse Event 
Expedited Reporting NOT 
required.)
Report by phone to IDB within 
24 hrs. Expedited report to 
follow within 5 calendar days.
This includes all deaths within 
30 days of the last dose of 
treatment with an 
investigational agent 
regardless of attribution.
Any late death attributed to 
the agent (possible, probable, 
or definite) should be reported 
within 5 calendar days .
Adverse Event Expedited 
Reporting NOT required.
Report by phone to IDB 
within 24 hrs.  Expedited 
report to follow within 5 
calendar days.
This includes all deaths 
within 30 days of the last 
dose of treatment with an 
investigational agent 
regardless of attribution.
Any late death attributed to 
the agent (possible, 
probable, or definite) should 
be reported within 5 
calendar days.
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28
listed in Section 11.1.3. Subjects will be assessed for any unexpected serious 
adverse events such as significant changes in vital signs, nausea, vomiting, rashes, 
skin eruptions, and any long term changes in liver function.  Unauthorized disclosure 
of protected health information is also a risk of this study.
It is the responsibility of the Principal Investigator (PI), Dr. Jeffrey Cozzens to oversee 
the safety of this Phase I study.  However, since Dr. Cozzens holds the IND for the 
investigational drug, an independent data safety monitoring committee (DSMC) has 
been assigned to this study.  The DSMC will consist of Dr. Krishna Rao 
(hematology/oncology physician), Dr. Kathy Robinson (oncology research study 
specialist), and Dr. Chad Noggle (neuropsychologist).
The DSMC will meet after each cohort (every third subject) has received the study 
drug to assess safety data including side effects and fluorescence ratings.  The 
DSMC will discuss each adverse event and determine causality in consultation with 
the Principal Investigator.  In addition, the DSMC will review study progress (including 
data quality and timeliness, recruitment, accrual and retention); study procedures 
(including subject privacy and data confidentiality protection); outcomes of adverse 
events and any new literature to assess change to the risk/benefit ratio and to 
determine if modifications need to be made or the study terminated.  If any serious 
adverse events or unanticipated problems are identified and determined to be 
associated with the study protocol, the study will be stopped and immediately 
reported to SCRIHS and the FDA. The DSMC has the authority to stop the study and 
take whatever steps are necessary to protect the safety and well-being of research 
subjects until the IRB can evaluate the DSMC’s report.
Study personnel will be expected to immediately alert the DSMC to any unanticipated 
problems or serious adverse events. Unanticipated problems and adverse events will 
also be reported to SCRIHS in accordance with institutional policy 
(http://www.siumed.edu/adrfa/SCRIHS/AdverseEvent.pdf) and to the FDA as 
required.  Reports generated by the DSMC will be provided to SCRIHS at the time of 
continuing review.
To ensure the integrity of the data collected from study participants several 
procedures will be implemented. All study personnel involved in data collection will be 
thoroughly trained in the assessment methods thus ensuring consistent applications 
of procedures and measurement consistency across study subjects.  All data will be 
automatically saved to a computer hard drive and stored on a secure server (i.e., 
password protected) which is backed up daily.  All informed consent documents will 
be kept at the study site in a separate locked file cabinet with restricted access. 
Issues related to data integrity will be discussed as a recurring agenda item in the bi-
monthly study team meetings. 
The study coordinator is responsible for data security.  Data management activities 
include: using double data entry and monitoring outliers.  Data security measures 
include keeping hard copies of data double-locked at all times with access limited to 
investigators and approved study personnel using password protection and a secure 
server.
Reporting Adverse Events
We will follow 1996 and 2000 International Conferences on Harmonization, Sections 
E2 and E6 Good Clinical Practice and FDA regulations.  An adverse event (AE) is 
IRB NUMBER: 12-135
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29
defined as any untoward medical occurrence that does not necessarily have a causal 
relationship with this treatment.  A Serious Adverse Event (SAE) is defined as any 
untoward medical occurrence that results in death, is life-threatening, requires 
hospitalization or prolongation of hospitalization, results in persistent or significant 
disability/incapacity, or is a congenital anomaly or birth defect.
We will use the descriptions and grading scales found in the revised NCI Common 
Terminology Criteria for Adverse Events (CTCAE) Version 3.0 for AE reporting.  All 
participating sites will have access to the CTCAE guide and it can be downloaded at 
http://ctep.cancer.gov
Expectedness - Adverse Events can be ‘Unexpected’ or ‘Expected’ for expedited 
reporting purposes only.
Attribution of the Adverse Event
1
Definite
AE is clearly related to the study treatment
2
Probable
AE is likely related to the study treatment
3
Possible
AE may be related to the study treatment
4
Unlikely
AE is doubtfully related to the study treatment
5
Unrelated
AE is clearly not related to the study treatment
 
Expedited Adverse Event Reporting – Phase 1 studies with investigational agents 
(Including hospitalization below):

For Hospitalization only – Any medical event equivalent to CTC grade 3, 4, or 5 
which precipitated hospitalization (or prolongation of existing hospitalization) must 
be reported regardless of designation as expected or unexpected and attribution.

The FDA safety information and adverse event reporting system, Medwatch, FDA 
form 3500A will be used for reporting within 24 hours.
Expedited Reporting Guidelines

Medwatch Reporting Requirements for Adverse Events that Occur within 30 Days 
of the Last Dose of the Investigational Agent on Phase 1 Trials.
Phase 1 Trials
Grade 1
Grade 2
Grade 2
Grade 3
Grade 3
Grades
4 & 52
Unexpected
Expected
Unexpected 
and 
Expected
Unex-
pected
Expected
with 
Hospitali-
zation
without 
Hospitali- 
zation
with 
Hospitali-
zation
without 
Hospitali-
zation
Unexpected 
and
Expected
Unrelated
Unlikely
Not
Required
Not
Required
Not
Required
10 
Calendar
Days
Not
Required
10 
Calendar
Days
Not
Required
24-Hour;
5 Calendar
Days
Possible
Probable
Definite
Not
Required
10 
Calendar
Days
Not
Required
24-Hour;
5 Calendar
Days
24-Hour;
5 Calendar
Days
10 
Calendar
Days
Not
Required
24-Hour;
5 Calendar
Days
1 Adverse events with attribution of possible, probable, or definite that occur greater than 30 days after the last 
dose of treatment with an agent under a CTEP IND require reporting as follows:
AdEERS 24-hour notification followed by complete report within 5 calendar days for:

Grade 3 unexpected events with hospitalization or prolongation of hospitalization

Grade 4 unexpected events 

Grade 5 expected events and unexpected events
2 Although an AdEERS 24-hour notification is not required for death clearly related to progressive disease, a full report 
IRB NUMBER: 12-135
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30
is required as outlined in the table.                                                                                                      December 15, 2004
Note:  All deaths on study require both routine and expedited reporting regardless of 
causality.  Attribution to treatment or other cause must be provided.
Expedited Adverse Event Reporting Timelines (Definitions)

24 hours; 5 calendar days – The investigator must initially report the AE via 
Medwatch within 24 hours of learning of the event followed by a complete 
Medwatch report within 5 calendar days of the initial 24-hour report.

10 calendar days - A complete Medwatch report on the AE must be submitted 
within 10 calendar days of the investigator learning of the event. 

Any medical event equivalent to CTCAE grade 3, 4, or 5 that precipitates 
hospitalization (or prolongation of existing hospitalization) must be reported 
regardless of attribution and designation as expected or unexpected with the 
exception of any events identified as protocol-specific expedited adverse event 
reporting exclusions.  
Routine Adverse Event Reporting
All Adverse Events must be reported in routine study data submissions.  AEs 
reported through Medwatch must also be reported in routine study data 
submissions.
11.6
Investigator Responsibilities (21CFR312.50 & 21CFR312.53)
The investigator is responsible for ensuring that the investigation is conducted in 
accordance with the general investigational plan and protocols, maintaining an 
effective IND with respect to the investigation, and ensuring that FDA and all 
participating investigators are promptly informed of significant new adverse effects or 
risks with respect to the drug.
The investigator will maintain a current signed investigator statement (FDA 1572), a 
current Curriculum Vitae (CV), and a current clinical protocol.
12. STATISTICAL CONSIDERATIONS
12.1
Study Design/Primary Endpoints
12.1.1 Establish a safe dose for oral 5-ALA administration
NCI Common Toxicity Criteria will be used to quantify toxicity following 5-ALA 
administration.  Special attention will be paid to rash, pruritis, hepatic 
transaminitis, nausea and vomiting.  Based on previously reported German 
trial, a 30 mg/kg dose is anticipated to be as well-tolerated as 10 and 20 
mg/kg doses.
12.1.2 Determine the sensitivity and specificity of the Phase 1 recommended dose in 
discriminating between normal and malignant tissue intraoperatively.
Four methods are being employed to assess discrimination of tumor from 
normal brain tissue.

The neurosurgeon will assess the ability to distinguish tumor from normal 
brain tissue under ultraviolet illumination and grade the discrimination from 
0 to +++ (3+) (See Section 8.1.1).  Although arguably the most relevant 
measure, this is also the most biased and difficult to corroborate measure.

Under white light the neurosurgeon will assess and biopsy an area of 
obvious tumor and an area of obviously normal (but possibly edematous) 
non-eloquent brain.  The neurosurgeon will then assess whether these 
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31
areas correspond to areas of strong, weak or no fluorescence seen under 
blue light.

Under blue light, the neurosurgeon will assess the areas of fluorescence 
in the tumor cavity and will biopsy areas of strong fluorescence, weak 
fluorescence and no fluorescence.  It may not be necessary to repeat 
some of these biopsies if the area has already been biopsied under white 
light.  The pathologist will score tumor involvement in each biopsy 
specimen.
These data will be placed in the following grids – first a 3x3 grid as follows for 
an analysis of correlation, specifically a Cohen’s kappa will be calculated to 
assess the strength of agreement between fluorescence and tumor density:
Fluorescence vs. 
Tumor Density
Solid Tumor
Infiltrating 
tumor/normal 
brain
No tumor 
(edematous 
peritumoral 
normal brain)
High Fluorescence
(Number of 
areas 
identified)
(Number of areas 
identified)
(Number of 
areas 
identified)
Weak Fluorescence
(Number of 
areas 
identified)
(Number of areas 
identified)
(Number of 
areas 
identified)
No Fluorescence
(Number of 
areas 
identified)
(Number of areas 
identified)
(Number of 
areas 
identified)
Second, these data will also be placed in a simpler 2x2 grid for fluorescence 
sensitivity/specificity analysis:
Fluorescence vs. 
Tumor Density
Tumor
No Tumor
Fluorescence
a (Number of areas 
identified)
b (Number of areas 
identified)
No Fluorescence
c (Number of areas 
identified)
d (Number of areas 
identified)
Sensitivity=a/(a+c); specificity=d/(b+d)
Third, the following grid will be used to examine the sensitivity/specificity of 
white light examination of the tumor bed and tumor density:
White Light 
Examination vs. Tumor 
Density
Tumor
No Tumor
White Light Obvious 
Tumor
a (Number of areas 
identified)
b (Number of areas 
identified)
White Light Obvious No 
Tumor
c (Number of areas 
identified)
d (Number of areas 
identified)
Sensitivity=a/(a+c); specificity=d/(b+d)
Fourth, the following grid will be used to examine the relationship of the 
neurosurgeon’s intraoperative fluoroscopic aided estimate of the extent of 
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32
resection vs. the actual extent of resection as measured by post-operative MR 
imaging techniques (See definitions in section 10.1.1), specifically a Cohen’s 
kappa will be calculated to assess the strength of agreement between 
operative EOR and MRI EOR:
Operative EOR vs. 
MRI EOR
MRI GTR
MRI NTR
MRI IR
Operative GTR
(Number of 
areas 
identified)
(Number of 
areas 
identified)
(Number of 
areas 
identified)
Operative NTR
(Number of 
areas 
identified)
(Number of 
areas 
identified)
(Number of 
areas 
identified)
Operative IR
(Number of 
areas 
identified)
(Number of 
areas 
identified)
(Number of 
areas 
identified)
12.2 Sample Size/Accrual Rate
It is anticipated that this trial will accrue a mimimum of 3 patients and a maximum of 
*18 patients in the phase 1 portion of this protocol and 16 patients in the phase 2 
portion of the trial.  (*Due to four patients receiving 40mg/kg rather than three, 19 total 
patients will be studied in phase 1. Protocol deviation approved by IRB on 17 Oct 
2013.)
Total accrual of a maximum of 34 patients in 48 months is expected. 
Assuming that the estimated success rate of resection is 0.70, with a sample size of 34, 
we will have 80% power to detect a moderate kappa of 0.490 from the null hypothesis 
of zero intraclass kappa, using a large sample 0.050 level two-sided test.
Descriptive statistics will be presented as mean and standard deviation for continuous 
variables, as frequency and percentage for categorical variables. SAS 9.1 will be used 
to perform the statistical analyses, type I error (significance level) will be set as 0.05.
12.3 Stratification Factors
No stratification will be performed in this protocol.
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34
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APPENDIX A
Performance Status Criteria
ECOG Performance Status Scale
Karnofsky Performance Scale
Grade
Descriptions
Percent
Description
100
Normal, no complaints, no evidence 
of disease.
0
Normal activity.  Fully active, able 
to carry on all pre-disease 
performance without restriction.
90
Able to carry on normal activity; 
minor signs or symptoms of 
disease.
80
Normal activity with effort; some 
signs or symptoms of disease.
1
Symptoms, but ambulatory.  
Restricted in physically strenuous 
activity, but ambulatory and able 
to carry out work of a light or 
sedentary nature (e.g., light 
housework, office work).
70
Cares for self, unable to carry on 
normal activity or to do active work.
60
Requires occasional assistance, but 
is able to care for most of his/her 
needs.
2
In bed <50% of the time.  
Ambulatory and capable of all self-
care, but unable to carry out any 
work activities.  Up and about 
more than 50% of waking hours.
50
Requires considerable assistance 
and frequent medical care.
40
Disabled, requires special care and 
assistance.
3
In bed >50% of the time.  Capable 
of only limited self-care, confined 
to bed or chair more than 50% of 
waking hours.
30
Severely disabled, hospitalization 
indicated.  Death not imminent.
20
Very sick, hospitalization indicated. 
Death not imminent.
4
100% bedridden.  Completely 
disabled.  Cannot carry on any 
self-care.  Totally confined to bed 
or chair.
10
Moribund, fatal processes 
progressing rapidly.
5
Dead.
0
Dead.
A-1
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APPENDIX B
Ultraviolet-Illuminated Operating Room Microscope
Tumor Dye Project – Technical Aspects
5-aminolevulinic acid (5-ALA) – a naturally occurring non-fluorescing metabolic precursor, 
induces synthesis of protoporphyin IX (PpIX).  PpIX fluoresces when exposed to the excitation 
light of blue-violet (wavelength 375-400nm.) It gives off a light with characteristic peaks 635 and 
704nm.  This is best viewed by filtering (440 long pass filter) out most other wavelengths except 
a small amount of excitation light.
The excitation light source is a 400 W short-arc Xe-lamp and a short pass filter (440nm.) 
integrated into a standard Leica surgical microscope.  This filter can be switched on or off by the 
surgeon when needed.  Light is delivered through a commercially available liquid light guide.  
The Leica OH series microscope is modified so that these filters can be switched on and off 
whenever the surgeon wishes to observe the fluorescence.
Two special filters for the 5-ALA fluorescence are used in a standard Leica OH microscope.  The 
filters are a short pass (<440nm) filter which rotates in front of the microscope’s standard 400-
watt Xe light source.  In addition, a special long pass (>440nm) barrier filter in the optical path 
allows clear viewing of the red fluorescence.  Both filters are inserted and removed with one 
button on the microscope handle.  With the push of that one button, the standard Leica OH 
microscope can then go back to being the neurosurgical and spine microscope it was designed 
for.
Using these modifications intraoperative detection of tumor fluorescence is possible without 
disturbing the routine of the surgical procedure.  Switching between the two illumination modes 
does not require significant surgeon optical accommodation and does not interrupt the operation.  
All of these modifications are readily made to the standard neurosurgical microscope and 
requires no additional personnel in the operating room.
B-1
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39
Appendix C
Eligibility Checklist
Clinical/Radiological evidence of a primary malignant brain tumor
Age ≥ 18 years old
ECOG Performance Status ≤ 2 
Karnofsky ≥ 60%
Leukocytes ≥ 3,000/mcL
Absolute neutrophil count ≥ 1,500/mcL
Total bilirubin within normal institutional limits
AST (SGOT)/ALT(SGPT) ≤ 2.5 X institutional upper limit of normal
Creatinine within normal institutional limits
or Creatinine clearance ≥60mL/min/1.73 m2 
Not pregnant
Able to understand and sign informed consent
Not currently treated with other investigational agents
Not allergic to 5-ALA or similar drugs
No personal or family history of porphyria
No uncontrolled intercurrent illness including, but not limited to ongoing or 
active infection, symptomatic congestive heart failure, unstable angina 
pectoris, cardiac arrhythmia, or psychiatric illness/social situations that would 
limit compliance with study requirements
C-1
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40
Appendix C
Treatment Day Data Sheet
C-2
 
Date
Time
Initials
Result
 Pre-op
 
 
 
 
5-ALA Administration (dose)
 
 
 
 
Intra-op
Neurosurgeon JC   JE   DA    (circle one)
 
 
 
 
Tumor Visualization with UV Illumination
 
 
 
 
Photos Done?
 
 
 
 
 
 
 
 
 
Pathologist
 
 
 
 
 
 
 
 
 
White Light / Intensity by light meter
 
 
 
 
Area of obvious tumor identified
 
 
 
 
  - Biopsy obtained?
 
 
 
 
         Biopsy code label
 
 
 
 
  - Fluorescence grade
 
 
 
 
 Area of  normal brain in cavity identified
 
 
 
 
  -Biopsy obtained?
 
 
 
 
         Biopsy code label
 
 
 
 
  -Fluorescence grade
 
 
 
 
 
 
 
 
Blue Light
 
 
 
 
 
 
 
 
 
Area of strong fluorescence identified
 
 
 
 
  - Biopsy obtained?
 
 
 
 
         Biopsy code label
 
 
 
 
  - Fluorescence grade
 
 
 
 
Area of weak fluorescence identified
 
 
 
 
  - Biopsy obtained?
 
 
 
 
         Biopsy code label
  -Fluorescence grade
Area of no fluorescence identified
  - Biopsy obtained?
         Biopsy code label
  - Fluorescence grade
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41
Appendix C
Post-operative Data
Post-op MRI
Date
   Evaluation Method (Circle one)
dsMRI            Manual
   Extent of Resection:
Gross total       Near Total        Incomplete
Liver Function Tests
    AST (SGOT)/ALT(SGPT)
    Bilirubin
C-3
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42
Appendix C
Pathologist Report Data
Surgical Pathology Number ____________
Code
Pathology Summary
 Solid tumor
 Infiltrating tumor/normal brain
 No tumor (edematous peritumoral normal brain)
 Other________________________________________
 Solid tumor
 Infiltrating tumor/normal brain
 No tumor (edematous peritumoral normal brain)
 Other________________________________________
 Solid tumor
 Infiltrating tumor/normal brain
 No tumor (edematous peritumoral normal brain)
 Other________________________________________
 Solid tumor
 Infiltrating tumor/normal brain
 No tumor (edematous peritumoral normal brain)
 Other________________________________________
 Solid tumor
 Infiltrating tumor/normal brain
 No tumor (edematous peritumoral normal brain)
 Other________________________________________
 Solid tumor
 Infiltrating tumor/normal brain
 No tumor (edematous peritumoral normal brain)
 Other________________________________________
C-4
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43
Appendix C
Biopsy/Fluorescence
Correlations
Biopsy Results
Code/Type
Strongly Fluorescent
Weakly Fluorescent
No Fluorescence
__________
 WL/T
 WL/N
 BL
 Solid tumor
 Infiltrating tumor/normal 
brain
 No tumor (edematous 
peritumoral normal brain)
 Solid tumor
 Infiltrating 
tumor/normal brain
 No tumor (edematous 
peritumoral normal 
brain)
 Solid tumor
 Infiltrating 
tumor/normal brain
 No tumor (edematous 
peritumoral normal brain)
__________
 WL/T
 WL/N
 BL
 Solid tumor
 Infiltrating tumor/normal 
brain
 No tumor (edematous 
peritumoral normal brain)
 Solid tumor
 Infiltrating 
tumor/normal brain
 No tumor (edematous 
peritumoral normal 
brain)
 Solid tumor
 Infiltrating 
tumor/normal brain
 No tumor (edematous 
peritumoral normal brain)
__________
 WL/T
 WL/N
 BL
 Solid tumor
 Infiltrating tumor/normal 
brain
 No tumor (edematous 
peritumoral normal brain)
 Solid tumor
 Infiltrating 
tumor/normal brain
 No tumor (edematous 
peritumoral normal 
brain)
 Solid tumor
 Infiltrating 
tumor/normal brain
 No tumor (edematous 
peritumoral normal brain)
__________
 WL/T
 WL/N
 BL
 Solid tumor
 Infiltrating tumor/normal 
brain
 No tumor (edematous 
peritumoral normal brain)
 Solid tumor
 Infiltrating 
tumor/normal brain
 No tumor (edematous 
peritumoral normal 
brain)
 Solid tumor
 Infiltrating 
tumor/normal brain
 No tumor (edematous 
peritumoral normal brain)
__________
 WL/T
 WL/N
 BL
 Solid tumor
 Infiltrating tumor/normal 
brain
 No tumor (edematous 
peritumoral normal brain)
 Solid tumor
 Infiltrating 
tumor/normal brain
 No tumor (edematous 
peritumoral normal 
brain)
 Solid tumor
 Infiltrating 
tumor/normal brain
 No tumor (edematous 
peritumoral normal brain)
__________
 WL/T
 WL/N
 BL
 Solid tumor
 Infiltrating tumor/normal 
brain
 No tumor (edematous 
peritumoral normal brain)
 Solid tumor
 Infiltrating 
tumor/normal brain
 No tumor (edematous 
peritumoral normal 
brain)
 Solid tumor
 Infiltrating 
tumor/normal brain
 No tumor (edematous 
peritumoral normal brain)
C-5
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44
SCRIHS 10-035  5-ALA
Intra-Operative Data Sheet
Sample Code
Light
Area Biopsied
Fluorescence
(0 to +++)
 White
 Obvious Tumor
 Obvious Not Tumor
_________________
Time:
 Blue
 Strongly Fluorescent
 Weakly Fluorescent
 No Fluorescence
 White
 Obvious Tumor
 Obvious Not Tumor
_________________
Time:
 Blue
 Strongly Fluorescent
 Weakly Fluorescent
 No Fluorescence
 White
 Obvious Tumor
 Obvious Not Tumor
_________________
Time:
 Blue
 Strongly Fluorescent
 Weakly Fluorescent
 No Fluorescence
 White
 Obvious Tumor
 Obvious Not Tumor
_________________
Time:
 Blue
 Strongly Fluorescent
 Weakly Fluorescent
 No Fluorescence
 White
 Obvious Tumor
 Obvious Not Tumor
_________________
Time:
 Blue
 Strongly Fluorescent
 Weakly Fluorescent
 No Fluorescence
 White
 Obvious Tumor
 Obvious Not Tumor
_________________
Time:
 Blue
 Strongly Fluorescent
 Weakly Fluorescent
 No Fluorescence
Grade
Definition
0
No fluorescence.  No difference between any of the tissues 
visualized.
No 
fluorescence
+
Low fluorescence.  Red glow is barely distinguished from 
surrounding non-fluorescent tissue.
++
Moderate fluorescence.  Dull red glow of fluorescent tissue.
Weak 
fluorescence
+++
High fluorescence.  Bright red glow of fluorescent tissue
Strong 
fluorescence
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45
Estimate of extent of fluorescent tissue 
resected (%):
Estimate of extent of enhancing tumor resected (%):
C-6
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46
APPENDIX D
Dear Dr. Cozzens,
CTEP does not have a CAEPR for this agent at this time.  
For any future CAEPR requests, please submit additional details such as the protocol number, who 
the proposed trial’s sponsor will be, where the trial will be held, and who is expected to supply trial 
agent(s).  
Regards,
Yael
Yael R. Vinciguerra
Deputy Program Manager
Contractor - Technical Resources International
Protocol & Information Office
Operations & Informatics Branch
Cancer Therapy Evaluation Program
National Cancer Institute
301-402-7369 (Direct Line)
301-496-1367 (Main PIO Number)
For all PIO submissions, please e-mail: pio@ctep.nci.nih.gov
From: Jeffrey W. Cozzens, M.D. [mailto:jeff.cozzens@gmail.com] 
Sent: Monday, March 30, 2009 6:39 PM
To: NCI CTEP PIO with Contractors
Subject: 5-ALA
I am currently writing a proposal utilizing 5-aminolevulinic acid (5-ALA).  Please send me the current 
CAEPR or ASAEL for this drug.
_______________________________________
Jeffrey W, Cozzens, M.D., FACS
Neurosurgery
e-mail: jeff.cozzens@gmail.com
Phone: 847-570-1440
D-1
IRB NUMBER: 12-135
IRB APPROVAL DATE: 01/22/2017