untitled h1
untitled h2

Pr ot oc ol Nif urti m o x P h ase II  
V 0 7 0 6 A P h ase II Tri al of Nif urti m o x f or Refr act or y or Rel a pse d Ne ur o bl ast o m a or Me d ull o bl ast o m a.  
N C T 0 0 6 0 1 0 0 3 Versi o n 9 . 0 Dece m ber 0 4, 2 0 15
untitled h1
s t u d y

St u d y C hair:  
Giselle S h oller, M D  
N M T R C C hair 1 0 0 Mic hi ga n Street N E M C 2 7 2 Gra n d Ra pi ds, MI 4 9 5 0 3 Tel: ( 6 1 6) 2 6 7 - 0 3 3 2 Fa x: ( 6 1 6) 3 9 1 - 2 7 8 5 giselle.s h oller @ hele n de v osc hil dre ns. or g S T U D Y VI C E C H AI R :  
Willia m Fer g us o n, M D  
Pe diatric He mat ol o g y - O nc ol o g y Car di nal Gle n n o n C hil dre n's Me dical Ce nter/ Sai nt L o uis U ni versit y Sc h o ol of Me dici ne 1 4 6 5 S. Gra n d Bl v d.  
Sai nt L o uis, M O 6 3 1 0 4 P h o ne: ( 3 1 4)- 5 7 7- 5 6 3 8 Fa x: ( 3 1 4) 2 6 8- 4 0 8 1 E mail: fer g us ws @sl u.e d u
untitled h1
s a f e t

Gi na K. Ha n na, P har m D.  
P har macist 8 0 5 Ma g n olia Cree k Circle Orla n d o, F L 3 2 8 2 8 P h: 3 5 2- 2 5 6- 2 4 6 7 E- mail: gi na. ha n na @ g mail.c o m S E NI O R S T A TI S TI CI A N:  
Ta ka As hi ka ga, P h. D.  
Me dical Bi ostatistics  
& Bi oi nf or matics Facilit y U V M C olle ge of Me dici ne 2 7 Hills B uil di n g 1 0 5 Carri ga n Dri ve B urli n gt o n, V T 0 5 4 0 5 P h o ne ( 8 0 2) 6 5 6- 2 5 2 6 Fa x ( 8 0 2) 6 5 6- 0 6 3 2 E mail Ta ka mar u. As hi ka ga @ u v m.e d u Ja mes Wallace, M D  
De pt. of Ra di ol o g y U V M C olle ge of Me dici ne/ F A H C  
1 1 1 C olc hester A ve B urli n gt o n, V T 0 5 4 0 1 P h o ne: 8 0 2- 8 4 7- 3 5 0 6 E mail: ja mes. wallace @ vt me d net. or g Willia m R o berts, M D  
Pe diatric He mat ol o g y/ O nc ol o g y Ra d y C hil dre n's H os pital Sa n Die g o 3 0 2 0 C hil dre ns Wa y M C 5 0 3 5 Sa n Die g o, C A 9 2 1 2 3- 4 2 8 2 P h o ne: 8 5 8- 9 6 6- 5 8 1 1 Fa x: 8 5 8- 9 6 6 - 8 0 3 5 E mail: wr o berts @c hs d. or g Terrill F. Sa x o n, P h D  
C hair, Ass ociate Pr ofess or De pt. of E d ucati o nal Ps yc h ol o g y Office: B urles o n 2 1 8 A  
Ba yl or U ni versit y Wac o, Te xas 7 6 7 9 8 P h o ne: ( 2 5 4) 7 1 0- 6 1 0 1 E mail: Terrill _ Sa x o n @ ba yl or.e d u Ge ne vie ve Ber ge n da hl, R N  
Cli nical Pr o gra m Direct or N M T R C  
1 0 0 Mic hi ga n Street N E M C 2 7 2 Gra n d Ra pi ds, MI 4 9 5 0 3 Tel: ( 6 1 6) 2 6 7- 0 3 3 5 E- Mail: Ge ne vie ve. ber ge n da hl @ hele n de v osc hil dre ns. or g Al yssa Va n der Werff Cli nical Pr o gra m C o or di nat or N M T R C  
1 0 0 Mic hi ga n Street N E M C 2 7 2 Gra n d Ra pi ds, MI 4 9 5 0 3 Tel: ( 6 1 6) 2 6 7 -0 3 2 7 Fa x: ( 6 1 6) 3 9 1 -2 7 8 5 E- Mail: Al yssa. Va n der Werff @ hele n de v osc hil dre ns. or g Ti m ot h y Hi g gi ns, M D  
Ra di ol o gist Fletc her Alle n Healt h Care Mai n Ca m p us - Patric k 1 1 1 1 C olc hester A ve n ue B urli n gt o n, V T 0 5 4 0 1 P h: 8 0 2- 8 4 7- 3 5 9 2
untitled h1
data safet y a n

Gi na K. Ha n na, P har m D.  
P har macist 8 0 5 Ma g n olia Cree k Circle Orla n d o, F L 3 2 8 2 8 P h: 3 5 2- 2 5 6- 2 4 6 7 E- mail: gi na. ha n na @ g mail.c o m Wi nst o n H u h, M D  
Assista nt Pr ofess or, De part me nt of Pe diatrics Patie nt Care, Di visi o n of Pe diatrics, T he U ni versit y of Te xas M D A n ders o n Ca ncer Ce nter, H o ust o n, T X 7 7 0 3 0 T o m Geller, M D  
Pe diatric He mat ol o g y - O nc ol o g y Car di nal Gle n n o n C hil dre n's Me dical Ce nter/  
Sai nt L o uis U ni versit y Sc h o ol of Me dici ne 1 4 6 5 S. Gra n d Bl v d.  
Sai nt L o uis, M O 6 3 1 0 4 P h o ne: ( 3 1 4)- 5 7 7- 5 6 3 8 Fa x: ( 3 1 4) 2 6 8- 4 0 8 1 SECTION
PAGE  
| TABLE OF CONTENTS                                                                                                                                                |                                                                                                                                                                       |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| STUDY COMMITTEE                                                                                                                                                  |                                                                                                                                                                       |
| 1.0                                                                                                                                                              | PROTOCOL CONCEPT .                                                                                                                                                    |
| 2.0                                                                                                                                                              | OBJECTIVES                                                                                                                                                            |
| 2.1 Primary Objective: .                                                                                                                                         |                                                                                                                                                                       |
| 2 Secondary Objectives:                                                                                                                                          |                                                                                                                                                                       |
| BACKGROUND AND PRELIMINARY DATA.                                                                                                                                 |                                                                                                                                                                       |
| 3.0                                                                                                                                                              | 3.1 Clinical background ........................................................................................................................................... 9 |
| 3.2 Preclinical background                                                                                                                                       |                                                                                                                                                                       |
| 3.3 Nifurtimox Induces Apoptosis of Neuroblastoma Cells in vitro and in vivo. ............................ 11                                                    |                                                                                                                                                                       |
| 3.4 Nifurtimox is Cytotoxic to Medulloblastoma Cells in vitro and in vivo ...................................... 12                                              |                                                                                                                                                                       |
| 3.5 Pharmacokinetic Studies of Nifurtimox                                                                                                                        |                                                                                                                                                                       |
| 3.6 Assay Procedure:                                                                                                                                             |                                                                                                                                                                       |
| 3.7 HPLC separation: .                                                                                                                                           |                                                                                                                                                                       |
| 4.0                                                                                                                                                              | SUBJECT ELIGIBILITY AND STUDY DESIGN .                                                                                                                                |
| 4.1 Eligibility: .                                                                                                                                               |                                                                                                                                                                       |
| 4.2 Stratification                                                                                                                                               |                                                                                                                                                                       |
| 4.3 Subject Registration .                                                                                                                                       |                                                                                                                                                                       |
| 5.0                                                                                                                                                              | T TREATMENT PLAN                                                                                                                                                      |
| 5.1 Nifurtimox Dosing- Cycles 1-6                                                                                                                                |                                                                                                                                                                       |
| 5.2 Combination Therapy:                                                                                                                                         |                                                                                                                                                                       |
| STATISTICAL CONSIDERATIONS:                                                                                                                                      |                                                                                                                                                                       |
| 6.0                                                                                                                                                              | 6.1 Sample Size Discussion .                                                                                                                                          |
| 6.2 Statistical Analysis Approaches .                                                                                                                            |                                                                                                                                                                       |
| 7.0                                                                                                                                                              | DOSE ADJUSTMENTS                                                                                                                                                      |
| 7.1 Nifurtimox Dose Modifications ........................................................................................................................... 19 |                                                                                                                                                                       |
| 7.2 Topotecan/Cyclophosphamide Dose Modifications:                                                                                                               |                                                                                                                                                                       |
| 8.0                                                                                                                                                              | SUPPORTIVE CARE AND CONCOMITANT THERAPY                                                                                                                               |
| 9.0                                                                                                                                                              | REQUIRED OBSERVATIONS                                                                                                                                                 |
| 10.0 PHARMACOKINETIC STUDIES .                                                                                                                                   |                                                                                                                                                                       |
| 1.0 MONITORING OF POTENTIAL ADVERSE NEUROLOGICAL EFFECTS                                                                                                         |                                                                                                                                                                       |
| 12.0                                                                                                                                                             | OFF STUDY CRITERIA                                                                                                                                                    |
| EVALUATION/RESPONSE CRITERIA. ................................................................................................................ 29                |                                                                                                                                                                       |
| 13.0                                                                                                                                                             | SAFETY MONITORING                                                                                                                                                     |
| 14.0                                                                                                                                                             |                                                                                                                                                                       |
| 15.0                                                                                                                                                             | A ADVERSE EVENT REPORTING                                                                                                                                             |
| 16.0                                                                                                                                                             | SERIOUS ADVERSE EVENT REPORTING                                                                                                                                       |
| Nifurtimox Phase II: Nifurtimox for Neuroblastoma and Medulloblastoma V0706                                                                                      | Page 5 of 55                                                                                                                                                          |  
Amendment \#9.0 Version: December 04, 2015  
| 17.0                                                                                                                      | AGENT INFORMATION             |
|---------------------------------------------------------------------------------------------------------------------------|-------------------------------|
| 17.1 Nifurtimox                                                                                                           |                               |
| 17.2 Cyclophosphamide                                                                                                     |                               |
| 17.3 Topotecan                                                                                                            |                               |
| 17.4  Filgrastim .                                                                                                        |                               |
| 17.5 Pegfilgrastim .                                                                                                      |                               |
| 18.0                                                                                                                      | BIOLOGICAL STUDIES (Optional) |
| 18.2 Bone Marrow Sample Instructions (for all participating sites): .................................................. 42 |                               |
| 19.0                                                                                                                      | REFERENCES                    |
| Appendix I:  Structured Interview for Adverse Neurologic Events                                                           |                               |  
Appendix II: REQUIRED OBSERVATIONS .  
Appendix III: QOL Questionnaires .....................................................................................................................48
untitled h1
i n v e s

I ha ve rea d t he attac he d pr ot oc ol a n d a gree t hat it c o ntai ns all t he necessar y details f or perf or mi n g t he st u d y.  
I will pr o vi de c o pies of t he pr ot oc ol a n d of t he precli nical a n d cli nical i nf or mati o n  
(I n vesti gat or's Br oc h ure) o n t he test article, w hic h was f ur nis he d t o me b y t he Ne ur o blast o ma a n d Me d ull o blast o ma Tra nslati o nal Researc h C o ns orti u m ( N M T R C), t o all me m bers of t he st u d y tea m res p o nsi ble t o me w h o partici pate i n t he st u d y. I will disc uss t his material wit h t he m t o ass ure t hat t he y are f ull y i nf or me d re gar di n g t he test article a n d t he c o n d uct of t he st u d y.  
O nce t he pr ot oc ol has bee n a p pr o ve d b y t he I nstit uti o nal Re vie w B oar d  
(I R B)/I n de pe n de nt Et hics C o m mittee (I E C), I will n ot m o dif y t his pr ot oc ol wit h o ut o btai ni n g t he pri or a p pr o val of t he N M T R C a n d of t he I R B/I E C. I will s u b mit t he pr ot oc ol m o dificati o ns a n d/ or a n y i nf or me d c o nse nt f or m (I C F) m o dificati o ns t o t he N M T R C a n d t he I R B/ I E C, a n d a p pr o val will be o btai ne d before a n y m o dificati o ns are i m ple me nte d.  
I u n dersta n d t he pr ot oc ol a n d will w or k acc or di n g t o it, t he pri nci ples of G o o d Cli nical Practice ( G C P) [c urre nt I nter nati o nal C o nfere nce of Har m o nizati o n (I C H) g ui deli nes],  
a n d t he Declarati o n of Helsi n ki ( 1 9 6 4) i ncl u di n g all a me n d me nts u p t o a n d i ncl u di n g t he Sc otla n d re visi o n ( 2 0 0 0) a n d n otes of clarificati o n a d de d i n 2 0 0 2 a n d 2 0 0 4.  
I n vesti gat or's Si g nat ure Date  
  
I n vesti gat or's Pri nte d Na me I n vesti gati o nal Site Na me
untitled h1
a p h ase ii

T here is c urre ntl y n o c urati ve treat me nt f or c hil dre n wit h rela pse d/refract or y ne ur o blast o ma, a n d f or t hese c hil dre n t he 5- year s ur vi val rate is < 1 0 %. Alt h o u g h t here ha ve bee n re p orts t hat t he s ur vi val rate of c hil dre n wit h c he m ose nsiti ve rela pse d me d ull o blast o ma ca n a p pr oac h 4 0 % f oll o wi n g i nte nsi ve c he m ot hera p y c o m bi ne d wit h a ut ol o g o us ste m cell s u p p ort, m ore rece nt data l o o ki n g at s ur vi val of all patie nts rela psi n g after m o der n c o m bi nati o n c he m ot hera p y a n d ra diati o n is als o o n t he or der of 1 0- 1 5 %. As s uc h, ne w t hera pe utic a p pr oac hes are nee de d t o treat t hese c hil dre n.  
T his p hase II cli nical trial is s pecificall y desi g ne d t o assess t he a nti ne o plastic effect of t he a nti parasitic dr u g nif urti m o x w he n gi ve n i n c o m bi nati o n wit h c ycl o p h os p ha mi de a n d t o p oteca n t o c hil dre n wit h rela pse d or refract or y ne ur o blast o ma or me d ull o blast o ma.  
A rece ntl y c o m plete d p hase I trial of nif urti m o x i n s u bjects wit h rela pse d/refract or y ne ur o blast o ma esta blis he d a ma xi m u m t olerate d d ose of 3 0 m g/ k g/ da y. Precli nical w or k has s h o w n t hat si n gle-a ge nt nif urti m o x has b ot h i n vitr o a n d i n viv o c yt ot o xicit y a gai nst b ot h ne ur o blast o ma a n d me d ull o blast o ma cell li nes a n d xe n o graft m o dels. I n t he p hase I st u d y, nif urti m o x was i nitiall y gi ve n al o ne, a n d t he n c o m bi ne d wit h t o p oteca n a n d c ycl o p h os p ha mi de; res p o nses were see n i n b ot h p hases of t hera p y .  
T he prese nt st u d y is a p hase II trial of nif urti m o x wit h t he a d diti o n of sta n da r d d oses of c ycl o p h os p ha mi de, a n d t o p oteca n, i n s u bjects wit h eit her ne ur o blast o ma or me d ull o blast o ma t hat is i n first or s u bse q ue nt rela pse, or refract or y t o sta n dar d i nitial t hera p y. O ur pri mar y o bjecti ve is t o e val uate t he efficac y of nif urti m o x i n c o m bi nati o n wit h t hese c he m ot hera p y a ge nts. O ur sec o n dar y ai ms will be t o:  
- e val uate t he pea k ser u m le vels of nif urti m o x i n t hese s u bjects a n d c orrelate pea k le vel wit h res p o nse;  
- c orrelate t u m or ge n o mi c a nal ysis wit h res p o nse;  
- e val uate q ualit y of life of s u bj ects recei vi n g t his dr u g c o m bi nati o n;
untitled h1
2. 1. pri mar y

2. 1. 1. Test t he efficac y ( best res p o nse) a n d safet y of nif urti m o x i n c hil dre n wit h rela pse d or refract or y ne ur o blast o ma or me d ull o blast o ma, i n c o m bi nati o n wit h c ycl o p h os p ha mi de/t o p oteca n 2. 1. 2. E val uate Mea n Ti me t o Pr o gressi o n
untitled h1
2. 2. sec o n

2. 2. 1. 7 B E val uate t he correlati o n bet wee n t he p har macol o gic ser u m le vels of n if urti m o x wit h t u m or res p o nse.  
2. 2. 2. Q ualit y of life a n d ne ur oc o g niti ve e val uati o n/ q uesti o n naire.  
2. 2. 3. Bi ol o g y st u dies t o i ncl u de: ge n o mic a nal ysis of cells pre - a n d p osttreat me nt, c orrelati o n of i n vitr o res p o nse t o i n vi v o res p o nse, fl o w c yt o metr y of t u m or b ur de n i n b o ne marr o w a n d bi o mar ker de vel o p me nt.
untitled h1
3. 0 b a c

T his P hase II trial is desi g ne d t o e val uate t he a nti -t u m or efficac y of nif urti m o x i n c hil dre n wit h ne ur o blast o ma a n d me d ull o blast o ma.  
N if urti m o x is a s y nt hetic nitr of ura n c o m p o u n d t hat has bee n use d safel y si nce t he 1 9 7 0's f or t he treat me nt of C ha gas' Disease i n b ot h a d ults a n d c hil dre n. T he efficac y a n d t o xicit y of nif urti m o x i n a c o h ort of 6 2 c hil dre n wit h C ha gas' Disease has rece ntl y bee n re vie we d . I n 2 0 0 2, a patie nt wit h pr o gressi ve refract or y ne ur o blast o ma w h o was bei n g treate d wit h c ycl o p h os p ha mi de a n d t o p oteca n ac q uire d C ha gas ' Disease fr o m a bl o o d tra nsf usi o n, a n d was starte d o n t he rec o m me n de d d ose of nif urti m o x  
( 1 5- 2 0 m g/ k g/ da y); her t u m or s u bse q ue ntl y re gresse d . S u bse q ue ntl y 6 m ulti pl y rela pse d ne ur o blast o ma s u bjects were treate d wit h nif urti m o x ( 2 0 m g/ k g/ da y) c o m bi ne d wit h c ycl o p h os p ha mi de a n d t o p oteca n t hr o u g h c o m passi o nate release. Fi ve of t he si x s u bjects s h o we d a res p o nse t o treat me nt a n d t w o of t hese s u bjects attai ne d a c o m plete re missi o n o n t his t hera p y a n d re mai n wit ho ut e vi de nce of disease f or m ore t ha n o ne year after be gi n ni n g nif urti m o x.  
A P hase I trial of nif urti m o x i n c hil dre n wit h m ulti pl y rela pse d/refract or y ne ur o blast o ma has bee n c o m plete d. T he pri mar y o bjecti ve was t o deter mi ne t he ma xi m u m t olerate d d ose of t his me dicati o n , w hic h was f o u n d t o be 3 0 m g/ k g/ da y di vi de d i n 3 d oses. T he n o n- d ose-li miti n g t o xicities were mai nl y na usea a n d ne ur o pat h y. T he d ose li miti n g t o xicities of t w o patie nts at 4 0 m g/ k g/ da y were a gra de 3 p ul m o nar y he m orr ha ge a n d a gra de 3 ne ur o pat h y (re versi ble). T he P hase I trial als o e val uate d t u m or res p o nse.  
F o urtee n patie nts were e val ua ble f or efficac y f oll o wi n g nif urti m o x treat me nt al o ne. Acc or di n g t o R E CI S T criteria, 6 patie nts dis pla ye d sta ble disease, a n d 8 ha d pr o gressi ve disease. I n a d diti o n t o R E CI S T cr iteria, patie nts were als o e val uate d f or a cli nical res p o nse as defi ne d b y ra di ol o gical sta ble disease wit h eit her a 5 0 % decrease i n uri nar y catec h ola mi nes, cleara nce of b o ne marr o w disease, or res ol uti o n of MI B G  
acti vit y. A cli nical res p o nse was see n i n 2 ( 3 3 %) of t he 6 patie nts wit h sta ble disease. Of t hese patie nts, o ne ha d res ol uti o n of b o ne marr o w disease, w hic h was c o nfir me d o n s u bse q ue nt b o ne marr o w bi o psies, a n d o ne ha d greater t ha n 5 0 % decrease i n uri nar y catec h ola mi nes.  
Of t he te n patie nts t hat we re e val ua ble f or efficac y after 3 c ycles wit h nif urti m o x, c ycl o p h os p ha mi de a n d t o p oteca n, 3 s h o we d a partial res p o nse, 5 ha d sta ble disease, a n d 2 ha d pr o gressi ve disease acc or di n g t o R E CI S T
Nif urt i m o x P hase II: Nif urti m o x f or Ne ur o blast o ma a n d Me d ull o blast o ma V 0 7 0 6 Pa ge 9 of 5 5  
criteria. Of t he 5 patie nts wit h sta ble disease, a cli nical res p o nse was see n i n 3 ( 6 0 %) of t he m. O ne patie nt c o nti n ue d t o ha ve cleara nce of b o ne marr o w disease a n d a greater t ha n 5 0 % decrease i n uri nar y catec h ola mi nes. T he ot her t w o patie nts ha d a decrease i n uri nar y catec h ola mi nes b y o ver 5 0 %. T w o patie nts were n o n- e val ua ble f or t heir res p o nse t o c o m bi nati o n t hera p y. O ne patie nt die d fr o m pr o gressi ve disease pri or t o recei vi n g c o m bi nati o n t hera p y, a n d o ne patie nt ref use d f urt her treat me nt. T he mea n pr o gressi o n- free s ur vi val f or all 1 4 patie nts w h o be ga n treat me nt was 9 . 1 m o nt hs ( me dia n 4. 5 m o nt hs). T he mea n pr o gressi o n-free s ur vi val varie d de pe n di n g u p o n t he n u m ber of pre vi o us c he m ot hera p y re gi me ns. I n patie nts t hat ha d recei ve d < 3 pre vi o us c he m ot hera p y re gi me ns, t he mea n pr o gressi o n-free s ur vi val was 1 2. 5 m o nt hs ( me dia n 1 4. 0 m o nt hs), w hile i n patie nts t hat ha d u n der g o ne > 3 pri or c he m ot hera p y re gi me ns, t he mea n pr o gressi o n- free s ur vi val was 7. 6 m o nt hs ( me dia n 7. 6 m o nt hs). T he Ma ntel p val ue f or t his a nal ysis was 0. 3 9. Gi ve n t he e xte nsi ve pri or treat me nt t hese s u bjects ha d recei ve d, t hese res ults are e nc o ura gi n g a n d f or m t he basis of t he pr o p ose d st u d y utilizi n g a nif urti m o x d ose of 3 0 m g/ k g/ da y.  
T he c o m bi nati o n of c ycl o p h os p ha mi de a n d t o p oteca n was c h ose n f or c o m bi nati o n t hera p y beca use it has s h o w n efficac y i n t he treat me nt of rela pse d ne ur o blast o ma a n d me d ull o blast o ma; in a n earlier p hase II st u d y e val uati n g t his c o m bi nati o n c o n d ucte d b y t he Pe diatric O nc ol o g y Gr o u p, t here was a 3 4 % res p o nse rate a m o n g patie nts wit h rela pse d ne ur o blast o ma. We h y p ot hesize t hat t her e will be greater res p o nse i n c o m bi nati o n wit h nif urti m o x.  
M ulti ple palliati ve ra diati o n t hera p y re gi me ns ha ve bee n utilize d i n c hil dre n wit h pai nf ul b o n y metastases fr o m ne ur o blast o ma. N o s yste matic re vie w of t hese sc he d ules has bee n d o ne, b ut it has bee n o ur o bser vati o n t hat s h ort c o urses of ra diati o n ( o ne t o t hree da y s) ma y w or k as well as m ore pr ol o n ge d sc he d ules ( u p t o t hree wee ks) a n d will be sta n dar dize d as s u p p orti ve care f or b o ne pai n.
untitled h1
3. 2. precli nical bac

Nif urti m o x is a nitr o heter oc yclic c o m p o u n d c o ntai ni n g a nitr o gr o u p t hat is esse ntial f or its efficac y. Nif urti m o x has bee n s h o w n t o e xert its c yt ot o xic effect s pecificall y b y ge nerati n g free ra dicals. T he nitr o gr o u p ca n be re d uce d t o t he nitr o a ni o n ra dical i n cell -free s yste ms b y i nteracti n g wit h c yt oc hr o me P- 4 5 0 re d uctase, xa nt hi ne o xi dase, asc or bate, a n d catec h ola mi nes. Nitr o a ni o ns ca n t he n re d uce o x y ge n t o t he s u per o xi de a ni o n ra dical a n d h y dr o ge n per o xi de. I n C ha gas' disease, t he nitr o a ni o n free ra dicals a n d o x yra dicals ha ve been s h o w n t o be c yt ot o xic f or t he parasite T. cr uzi. T he re d ucti o n of t he nitr o gr o u p n ot o nl y ge nerates a ni o n ra dicals, b ut i nteracti o n wit h catec h ola mi nes a p pears als o t o ge nerate se mi q ui n o ne free ra dicals t hat e xacer bate da ma ge t o f u ncti o nall y i m p ortant bi o m olec ules, lea di n g t o a p o pt osis of ne ur o blast o ma cell li nes.  
Ne ur o blast o ma cells are k n o w n t o c o ntai n hi g h le vels of catec h ola mi nes, t here b y p ote ntiall y lea di n g t o relati vel y s pecific tar geti n g of t hese cells. T he i m p orta nce of t he i nteracti o n bet wee n nif urti m o x a n d catec h ola mi nes i n ne ur o blast o ma cell li nes was c o nfir me d b y t he re d ucti o n of c yt ot o xicit y b y pretreat me nt wit h A M P T, a t yr osi ne h y dr o x ylase i n hi bit or t hat re d uces t he t otal a m o u nt of catec h ola mi ne st ore d i n cells, als o re d uces nif urti m o xi n d uce d c yt ot o xicit y. I n a d diti o n, t he e n ha nce d se nsiti vit y of s y m pat hetic ne ur o ns - b ut n ot par asy m pat hetic ne ur o ns or n o n- ne ur o nal cells—t o nif urti m o x s u p p orts t his c o ncl usi o n ( S h oller, i n s u b missi o n).
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3. 3. nif urti m

We teste d t he effect of nif urti m o x o n gr o wt h of C H L A- 9 0, L A 1- 5 5 n, L AN 2, S M S K C N R, a n d S Y 5 Y ne ur o blast o ma cells i n c ult ure. Nif urti m o x i n hi bite d t he gr o wt h of all fi ve cell li nes i n a c o nce ntrati o n- de pe n de nt ma n ner. After 2 da ys of e x p os ure t o 2 0 mc g/ ml of nif urti m o x, t he n u m ber of cells is decrease d t o 5. 9 % of ve hicle -treate d c o ntr ols i n C H L A- 9 0  
( p < 0. 0 0 1), 3 1. 3 % i n L A 1- 5 5 n ( p < 0. 0 0 1), 3 2. 9 % i n L A- N 2 ( p < 0. 0 0 1),  
3. 6 % i n S M S- K C N R ( p < 0. 0 0 1), a n d 1. 8 % i n S Y 5 Y ( p < 0. 0 0 1). T he c o nce ntrati o n re q uire d t o reac h 5 0 % i n hi biti o n of gr o wt h (I C 5 0 ) f or t hese cell li nes was esti mate d t o be 1 2. 1 8 mc g/ ml f or C H L A- 9 0, 1 3. 6 2 mc g/ ml f or L A 1- 5 5 n, 1 7. 1 7 mc g / ml f or L A-N 2 , 1 0. 9 8 mc g / ml f or S M S-K C N R  
a n d 9. 1 5 mc g/ ml f or S Y 5 Y. I n c o ntrast, c yt ot o xic effects were n ot o bser ve d o n n or mal e pit helial cells i n c ult ure e x p ose d t o nif urti m o x.  
C o nc urre nt wit h t he decrease i n cell via bilit y, nif urti m o x treat me nt pr o d uces m or p h ol o gi cal c ha n ges c o nsiste nt wit h a p o pt osis a n d t hese m or p h ol o gic c ha n ges occ ur i n a d ose- de pe n de nt ma n ner. E x p os ure t o 1 µ g/ ml of nif urti m o x ca use d a decrease i n a x o n le n gt h c o m pare d t o c o ntr ol u ntreate d cells, a n d at 1 0 µ g/ ml nif urti m o x t his effect was i ncre ase d si g nifica ntl y wit h r o u n di n g of cells. At 2 0 µ g/ ml nif urti m o x, ne ur o blast o ma cells were r o u n de d u p a n d fl oati n g wit h o nl y a fe w cells re mai ni n g a d here nt t o t he plate.  
I n a d diti o n t o its effects i n vitr o, nif urti m o x was als o a ble t o re d uce t he gr o wt h of ne ur o blast o ma t u m ors i n a xe n o graft m o del. N u de mice  
(n u/ n u ) were i njecte d i n t he left fla n k wit h S M S K C N R cells a n d all o we d t o f or m pal pa ble t u m ors f or 1 0- 1 4 da ys. T he mice were t he n fe d 5 g f o o d pellets dail y wit h or wit h o ut 1 5 0 m g/ k g nif urti m o x. T here were n o si g ns of dr u g- relate d t o xicit y at t his d ose sc he d ule, w hic h res ults i n ser u m le vels a p pr o xi matel y e q ui vale nt t o a d ose of 1 2 m g/ k g i n h u ma ns. After 2 8 da ys of treat me nt ( w he n c o ntr ol mice reac he d ma xi m u m t u m or size of 3. 0c m )  
t he mice were sacrifice d a n d t heir t u m ors were e xcise d a n d wei g he d.  
Treat me nt wit h nif urti m o x res ulte d i n i n hi biti o n of t u m or gr o wt h: t he a vera ge t u m or wei g ht i n c o ntr ol mice was 1. 1 5 gra ms c o m par e d t o a n a vera ge wei g ht of 0. 3 gra ms i n mice recei vi n g nif urti m o x ( p = 0. 0 3 ).  
I m m u n o hist oc he mical stai ni n g s h o we d t hat t u m ors treate d wit h nif urti m o x ha d decrease d cell pr oliferati o n as e vi de nce d b y decrease i n Ki 6 7 p ositi ve stai ni n g ( 7 5. 3 % vers us 9 3. 2 %, p < 0. 0 0 1) a n d decrease i n mit otic i n de x  
( 3 3. 5 vers us 5 0. 2, p = 0. 0 1 2) as well as a n i ncrease i n a p o pt osis as e vi de nce d b y a n i ncrease i n t he p y k n otic i n de x ( 6 3. 7 vers us 3 9. 5, p = 0. 0 2 2).  
3. 4. Nif urti m o x is C yt ot o xic t o Me d ull o blast o ma C ells i n vitr o a n d i n vi v o We teste d t he effect of nif urti m o x o n gr o wt h of D 2 8 3 a n d D A O Y  
me d ull o blast o ma cells i n c ult ure. Nif urti m o x i n hi bit e d t he gr o wt h of t hese li nes i n a c o nce ntrati o n- de pe n de nt ma n ner. After 2 da ys of e x p os ure t o 2 0 mc g/ ml of nif urti m o x, t he n u m ber of cells is decrease d t o 7. 0 % of ve hicle-treate d c o ntr ols i n D 2 8 3 ( p < 0. 0 0 1) a n d 4 0 % i n D A O Y ( p < 0. 0 0 1).  
A m o use xe n o graft m o del was use d t o m ore f ull y e val uate t he effect of n if urti m o x. N u de mice were i njecte d stere ota xicall y i n t he cere bell u m wit h 1 0 D 2 8 3 cells a n d fe d f o o d wit h or wit h o ut nif urti m o x  
( 1 5 0 m g/ k g/ da y) f or 7 wee ks p ost-s ur ger y. T he a ni mals were t he n sacrifice d, a n d t he t u m ors re m o ve d a n d e val uate d b y i m m u n o hist oc he mistr y. I n viv o t u m or gr o wt h was i n hi bite d b y nif urti m o x, a n d hist ol o gic e val uati o n of t he t u m ors s h o we d t hat nif urti m o x treat me nt res ulte d i n a n i ncrease i n t he a p o pt otic rate.
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3. 5. p har mac

Des pite meta b olis m st u dies i n rats a n d d o gs dati n g t o 1 9 7 2, t here a p pears t o be little p u blis he d data re gar di n g p har mac o ki netics of nif urti m o x i n h u ma ns. T he pr o d uct br oc h ure refers t o li mite d P K st u dies i n healt h y a d ult v ol u nteers a n d patie nts wit h re nal fail ure w h o recei ve d si n gle d oses i n t he ra n ge of 1 0- 1 5 m g/ k g. After a n oral d ose t he ti me t o pea k plas ma c o nce ntrati o n was a b o ut 2. 5 h o urs, t he C ma x was 0. 7 5 1 µ g/ ml ( 7 5 1 n g/ ml) a n d t he T1/ 2 was a p pr o xi matel y 3. 5 h o urs. T here was lar ge i nter- patie nt varia bilit y wit h pea k c o nce ntrati o ns ra n gi n g bet wee n 3 5 6- 1 0 9 3 n g/ ml.  
A n H P L C assa y f or ser u m has bee n p u blis he d ( Pa ul os et al, J o ur nal of C hr o mat o gra p h y 4 3 3: 3 5 9- 3 6 2, 1 9 8 8) a n d t h e s pecifics are o utli ne d bel o w.  
T he se nsiti vit y usi n g detecti o n at 4 0 0 n m a n d se parati o n o n a C 1 8 c ol u m n was re p orte d t o be 0. 0 7 7 µ g/ ml ( 7 7 n g/ ml) ser u m , w hic h is a p pr o xi matel y 2 0 % of t he l o west pea k le vel re p orte d i n t he st u dies n ote d a b o ve.  
F urt her m ore, wit h a T 1/ 2 of ∼ 3. 5 h o urs a n d TI D d osi n g, t he assa y a p pears se nsiti ve e n o u g h t o d o at least li mite d P K st u dies o n c hil dre n as s mall as 1 0 k g: 1 t o 2 ml bl o o d sa m ples o btai ne d at 2 ti me p oi nts ( baseli ne a n d 1 8 0 mi n utes p ost- a d mi nistrati o n) w o ul d re q uire a t otal v ol u me of n o m ore t ha n 4 ml .
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3. 6. assa y pr

A 5 0 µ l ali q u ot of nitr of uraz o ne s ol uti o n ( 3 0 µ g/ ml) is a d de d t o 1. 0 ser u m sa m ple as a n i nter nal sta n dar d. Ser u m pr otei ns are preci pitate d wit h 0. 6 ml 1 M perc hl oric aci d , a n d the preci pitate re m o ve d b y ce ntrif u gati o n f or 1 0 mi n utes at 1 0 0 0 x g. T he li q ui d p hase i s as pirate d a n d t he aci d ne utralize d wit h 2 0 0 µ l of 1 M s o di u m h y dr o xi de. T he nif urti m o x  
(t o get her wit h t he nitr of uraz o ne sta n dar d) is e xtracte d i nt o a n or ga nic p hase of 3. 5 ml dic hl or o met ha ne f or 1 h o ur. A 3 ml ali q u ot of t he or ga nic p hase is e va p orate d t o dr y ness at 4 0 C, t he resi d ue diss ol ve d i n 2 0 0 µ l H P L C m o bile p hase b uffer, of w hic h 2 0 µ l ali q u ots are use d f or eac h H P L C se parati o n.
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3. 7. h p l

C hr o mat o gra p h y is d o ne o n a 1 5 c m, 3. 9 m m I D u B o n da pa k C 1 8 c ol u m n.  
T he m o bile p hase is met ha n ol- S ore nse n p h os p hate b uffer, p H 7. 0 ( 5 0: 5 0, v/ v). T he c hr o mat o gra p h y was d o ne at a rate of 1 ml/ mi n. T he sta n dar d el utes at a p pr o xi matel y 2 mi n utes a n d t he dr u g at 3 mi n utes.  
T h is assa y was re p orte d t o be li near o ver t he ra n ge of 0. 0 7 7- 2. 3 µ g/ ml  
( 7 7- 2 3 0 0 n g/ ml) a n d t he re pr o d uci bilit y base d u p o n a p o ole d ser u m sa m ple assa ye d re petiti vel y o ver a 2- wee k peri o d s h o we d a n i nter-da y c oefficie nt of variati o n of o nl y 4 %.
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4. 0 s u b

4. 1. 1. A ge: 0 -2 1 years at t he ti me of dia g n osis.  
4. 1. 2. Dia g n osis: Hist ol o gic verificati o n at eit her t he ti me of ori gi nal dia g n osis or rela pse of ne ur o blast o ma or me d ull o blast o ma .  
4. 1. 3. Disease Stat us: Refract or y or first or m ulti ple rela pse d ne ur o blast o ma, or me d ull o blast o ma t hat has rela pse d after, or is refract or y t o, a c he m ot hera p y- c o ntai ni n g treat me nt re gi me n.  
4. 1. 4. Meas ura ble disease, i ncl u di n g at least o ne of t he f oll o wi n g:  
Meas ura ble t u m or > 1 0 m m b y C T or M RI  
P ositi ve b o ne marr o w bi o ps y/as pirate.  
F or ne ur o blast o ma s u bjects o nl y, a p ositi ve MI B G  
F or me d ull o blast o ma s u bjects o nl y, p ositi ve C S F c yt ol o g y 4. 1. 5. C urre nt disease state m ust be o ne f or w hic h t here is c urre ntl y n o k n o w n c urati ve t hera p y.  
4. 1. 6. A ne gati ve uri ne pre g na nc y test is re q uire d f or fe male s u bject s of c hil d beari n g p ote ntial ( o nset of me nses or ≥ 1 3 years of a ge).  
4. 1. 7. B ot h male a n d fe male st u d y s u bjects nee d t o a gree t o use o ne of t he m or e effecti ve birt h c o ntr ol met h o ds d uri n g treat me nt a n d f or si x m o nt hs after treat me nt is st o p pe d. T hese met h o ds i ncl u de t otal a bsti ne nce ( n o se x), oral c o ntrace pti ves ("t he pill"), a n i ntra uteri ne de vice (I U D), le v o n or gestr ol i m pla nts ( N or pla nt), or me dr o x y pr o gester o ne acetate i njecti o ns ( De p opr o vera s h ots). If o ne of t hese ca n n ot be use d, c o ntrace pti ve f oa m wit h a c o n d o m is rec o m me n de d.  
4. 1. 8. Or ga n F u ncti o n Re q uire me nts S u bjects m ust h a ve a de q uate li ver f u ncti o n as defi ne d b y A S T or A L T < 1 0 x u p per li mit of n or mal S u bjects wit h o ut b o ne marr o w met astases m ust ha ve a de q uate b o ne marr o w f u ncti o n, defi ne d as A N C > 5 0 0/ µ l 4. 1. 9. I nf or me d C o nse nt: All s u bjects a n d/ or le gal g uar dia ns m ust si g n i nf or me d writte n c o nse nt. Asse nt, w he n a p pr o priate, will be o btai ne d acc or di n g t o i nstit uti o nal g ui deli nes E xcl usi o n Criteri a :  
4. 1. 1 0. Life e x pecta nc y < 2 m o nt hs or La ns k y sc ore < 5 0 %  
4. 1. 1 1. S u bjects t hat wei g h Less t ha n 3. 5 k g 4. 1. 1 2. I n vesti gati o nal Dr u gs: S u bjects w h o are c urre ntl y recei vi n g a n ot her i n vesti gati o nal dr u g are e xcl u de d fr o m partici pati o n.  
4. 1. 1 3. A nti -ca ncer A ge nts: S u bjects w h o are c urre ntl y recei vi n g ot her a ntica ncer a ge nts are n ot eli gi ble. S u bjects m ust ha ve f ull y rec o vere d fr o m t he effects of pri or c he m ot hera p y ( he mat ol o gical a n d b o ne marr o w s u p pressi o n effects), ge nerall y at least 3 wee ks fr o m t he m ost rece nt a d mi nistrati o n ( 6 wee ks f or nitr os o ureas).  
4. 1. 1 4. I nfecti o n: S u bjects w h o ha ve a n u nc o ntr olle d i nfecti o n are n ot eli gi ble u ntil t he i nfecti o n is j u d ge d t o be well c o ntr olle d i n t he o pi ni o n of t he i n vesti gat or.  
4. 1. 1 5. S u bjects w h o , i n t he o pi ni o n of t he i n vesti gat or, ma y n ot be a ble t o c o m pl y wit h t he safet y m o nit ori n g re q uire me nts of t he st u d y, or i n w h o m c o m plia nce is li kel y t o be s u b o pti mal, s h o ul d b e e xcl u de d .  
4. 2. Stratificati o n Str at u m I - First rela pse d a n d refract or y ne ur o blast o ma Str at u m II - M ulti pl y rela pse d ne ur o blast o ma Str at u m III - Rela psed /refract or y me d ull o blast o ma
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4. 3. s u bject

4. 3. 1. All sites will c o m plete a " S u bject Re gistrati o n F or m." T h is f or m will t he n be sca n ne d or fa xe d t o t he Researc h C o or di nat or at t he Ne ur o blast o ma a n d Me d ull o blast o ma Tra nslati o nal Researc h C o ns orti u m ( N M T R C) .  
4. 3. 2. Eac h s u bject will be gi ve n a u ni q ue i de ntifier n u m ber. T his u ni q ue i de ntifier will be ke pt o n a " S u bject I de ntificati o n L o g" at eac h site.
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5. 1. nif urti m

5. 1. 1. Ne ur o blast o ma ( Strat u m 1 a n d II s u bjects) - Nif urti m o x will be a d mi nistere d at 3 0 m g/ k g/ da y i n t hree di vi de d d oses f or e ver y da y of t he st u d y.  
5. 1. 2. Me d ull o blast o ma ( Strat u m III s u bjects) - Nif urti m o x will be a d mi nistere d at 2 0 m g/ k g/ da y i n t hree di vi de d d oses f or e ver y da y of t he st u d y.  
5. 1. 3.  
5. 1. 4. Nif urti m o x d oses will be r o u n de d t o t he nearest w h ole ta blet ( eac h ta blet is pr o vi de d as 1 2 0 m g/ta blet) . T here will be n o d ose i nterr u pti o ns u nless t o xicit y s u per ve nes (see Secti o n 7 . 0).  
5. 1. 4. 1. F or s u bjects ≤ 1 0 k g or < 1 year of a ge Nif urti m o x c o ntact t he N M T R C f or d osi n g.  
5. 1. 4. 2. Nif urti m o x ma y be gi ve n b y G t u be or N G t u be.  
5. 1. 4. 3. Nif urti m o x s h o ul d be gi ve n wit h f o o d 5. 1. 4. 4. Nif urti m o x ma y be cr us he d or c ut a n d gi ve n i n f o o d if s u bject is u na ble t o s wall o w 5. 1. 4. 5. If s u bjects v o mits after gi vi n g Nif urti m o x d ose:  
−If less t ha n or e q ual t o 3 0 mi n utes si nce d ose- re peat d ose  
−If o ver 3 0 mi n utes si nce d ose- d o N O T re peat d ose
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5. 1. 5. nif urti

Patie nt wei g ht ( k g) x 3 0 = _ _ _ _ _ _ ÷ 1 2 0 = _ _ _ _ _ _ \# of pills per da y. R o u n d n u m ber of pills per da y t o nearest w h ole ta blet t he n di vi de i nt o 3 e ve n ( w he n p ossi ble) or u ne ve n d oses.  
E xa m ple: S u bject wei g hs 2 7 k g. 2 7 k g x 3 0 m g/ k g/ da y =  
8 1 0 m g/ da y. Di vi de 8 1 0 m g/ da y b y 1 2 0 m g/ta blet = 6. 7 5 ta blets per da y. R o u n d u p t o 7 ta blets per da y, t he n di vi de i nt o 3 d oses.  
S u bject s h o ul d get 3 ta bs i n a m, 2 ta bs i n after n o o n, 2 ta bs at ni g ht t o e q ual 7 ta bs per da y.
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5. 1. 6. nif urti

or s o o ner at t he be gi n ni n g of eac h c ycle if a n oticea ble c ha n ge i n wei g ht occ urs.  
5. 2. C o m bi nati o n T hera p y :  
5. 2. 1. T he c o m bi nati o n of c ycl o p h os p ha mi de, a n d t o p oteca n, al o n g wit h t he nif urti m o x, will be use d i n eac h c ycle.  
5. 2. 2. Nif urti m o x will be gi ve n orall y t hree ti mes per da y per secti o n 5. 1.  
5. 2. 3. Eac h c ycle of c o m bi nati o n c he m ot hera p y will be gi ve n o ver 5 c o nsec uti ve da ys as f oll o ws:  
5. 2. 3. 1. Pre h y drati o n wit h 5 0 0 ml/ m D 5 W ½ N S I V o ver 3 0- 6 0 mi n utes, al o n g wit h a ntie metic t hera p y as per treati n g p h ysicia n prefere nce; 5. 2. 3. 2. C ycl o p h os p ha mi de, 2 5 0 m g/ m / d ose pre pare d per i nstit uti o nal sta n dar ds i n n or mal sali ne, I V, i nf use d o ver 3 0 mi n utes; 5. 2. 3. 3. T o p oteca n, 0. 7 5 m g/ m / d ose, pre pare d per i nstit uti o nal sta n dar ds i n n or mal sali ne, I V, i nf use d o ver 3 0 mi n utes; 5. 2. 4. T his 2 1 da y c ycle ( +/- 3 da ys) of nif urti m o x, c ycl o p h os p ha mi de, a n d t o p oteca n will be re peate d f or a t otal of 6 ti mes.  
5. 2. 5. S u bjects wit h o ut b o ne marr o w metastases m ust ha ve a de q uate b o ne marr o w f u ncti o n, defi ne d as A N C > 5 0 0/ µ l bef ore starti n g a n y c he m ot hera p y. S u bjects wit h d oc u me nte d b o ne marr o w i n v ol ve me nt w h o ha ve ot her wise rec o vere d fr o m pri or c yt ot o xic t hera p y ar e eli gi ble t o c o nti n ue t hera p y. If s u bjects ha ve bee n ne gati ve f or b o ne marr o w metastases, b ut d o n ot ha ve a de q uate b o ne marr o w f u ncti o n (as descri be d a b o ve), t he n t he b o ne marr o w ma y be rec hec ke d. If t here is n o b o ne marr o w i n v ol ve me nt t he n wait t o see if A N C rec o vers; fil grasti m or pe gfil grasti m ca n be use d t o sti m ulate marr o w. If t he y ha ve n ot rec o vere d wit hi n 2 wee ks s u bject will be ta ke n off pr ot oc ol t hera p y, b ut ma y re mai n o n nif urti m o x u n der t his I N D a n d be m o nit ore d per secti o n 9. 8  
5. 3. S u bjects wit h a pri or hist or y of seiz ures will be rec o m me n de d t o start o n a n a ntiseiz ure me dicati o n pri or t o starti n g o n Nif urti m o x.  
5. 4. At c o m pleti o n of c ycle 6 (last c ycle), s u bjects t hat are be nefiti n g fr o m t he st u d y dr u g ma y c o nti n ue t o recei ve nif urti m o x u n der t his I N D, as si n gle a ge nt or i n  
c o m bi nati o n t hera p y at treati n g p h ysicia n's discreti o n. If s u bjects e x perie nce pr o gressi o n or i nt olera nce at a n y p oi nt t hereafter t he y will be c o ns i dere d resista nt t o nif urti m o x a n d g o off st u d y. Treati n g p h ysicia ns will c o nti n ue t o re p ort t o xicities as descri be d bel o w a n d will re p ort res p o nse t o treat me nt as s u bjects are e val uate d.  
5. 5. If s u bjects are t olerati n g nif urti m o x, b ut are u na ble t o c o nti n ue o n st u d y f or reas o ns ot her t ha n pr o gressi o n or d ose li miti n g t o xicities (e. g. platelet or A N C  
c o u nts n ot rec o vere d wit hi n 2 wee ks of ne xt c ycle start), s u bjects ma y re mai n o n nif urti m o x u n der t he c urre nt I N D a n d c o nti n ue t o be m o nit ore d f or dr u g safet y a nd ma xi mal res p o nse t o nif urti m o x.
untitled h1
6. 0 s t a

T he sa m ple size disc ussi o n f oc uses o n t he pri mar y efficac y o utc o me meas ure d b y t he pr o p orti o n of s u bjects w h o, d uri n g t he 6 c ycles of treat me nt, ac hie ve a best t u m or res p o nse of eit her c o m plete res p o nse ( C R)  
or partial res p o nse ( P R) as defi ne d bel o w. A n a bs ol ute differe nce i n res p o nse rates c o m pare d t o c he m ot hera p y al o ne of ~ 1 5 % t o 2 0 % w o ul d be c o nsi dere d cli nicall y si g nifica nt. Eac h strat u m of t his trial will c o m pare a n ull h y p ot hesis pr o p orti o n wit h a n alter nati ve h y p ot hesis pr o p orti o n a n d will use a directi o nal 5 % T y pe I err or le vel. Eac h strata will use a n 8 0 %  
p o wer le vel. Strat u m I c o nsists of ne ur o blast o ma s u bjects i n first rela pse, a n d t he n ull res p o nse rate ( C R + P R) is ta ke n t o be 3 0 % w hile t he alter nati ve res p o nse rate ( C R + P R) is ta ke n t o be 5 0 % as defi ne d a b o ve. Strat u m II c o nsists of m ulti pl y rela pse d or refract or y ne ur o blast o ma s u bjects a n d t he n ull res p o nse rate is ta ke n t o be 2 0% w hile t he alter nati ve res p o nse rate is ta ke n t o be 4 0 %. Strat u m III c o nsists of m ulti pl y rela pse d or refract or y me d ull o blast o ma s u bjects, f or w h o m t he n ull h y p ot hesis res p o nse rates are als o 5 % a n d s o t he alter nati ve res p o nse rate is 2 0%. Pre vi o us sa m ple size j ustificati o ns f oc use d u p o n a si n gle sa m ple size deter mi nati o n f or all t hree strata. Strat u m I a n d II will be m o difie d t o i m ple me nt a t w o -sta ge mi ni ma x desi g n t o acc o m m o date a n i nteri m l o o k a n d st o p pi n g f or f utilit y d ue t o t he larger sa m ple size nee d w hile Strat u m III will retai n t he ori gi nal si n gle sta ge desi g n gi ve n t hat t he c urre nt sa m ple size accr ual is alrea d y at 2 0 s u bjects per strat u m.
untitled h1
strat u m i

A sa m ple size of n = 3 9 s u bjects will be re q uire d t o test t he 30 % vs. 5 0 %  
res p o nse rate h y p ot heses f or t he Strat u m I s u bject gr o u p usi n g a t w o-sta ge mi ni ma x desi g n wit h a n i nteri m l o o k w he n t he first 1 9 e val ua ble s u bjects are accr ue d a n d t he fi nal l o o k w he n 3 9 e val ua ble s u bjects are accr ue d. T he Nif urt i m o x P hase II: Nif urti m o x f or Ne ur o blast o ma a n d Me d ull o blast o ma V 0 7 0 6 Pa ge 1 7 of 5 5 Sta ge 1: Acce pt t he n ull h y p ot hesis if t he res p o nse rate is less t ha n or e q ual t o 6/ 1 9 f or t he 1st 1 9 s u bjects. Ot her wise c o nti n ue t o sta ge 2.  
T he pr o ba bilit y of st o p pi n g f or f utilit y at Sta ge 1 is 0. 6 7 w he n t he n ull h y p ot hesis is tr ue a n d 0. 1 0 5 w he n t he alter nati ve is tr ue.  
Sta ge 2: Acce pt t he n ull h y p ot hesis if t he res p o nse rate is less t ha n or e q ual t o 1 6 / 3 9. Ot her wise reject t he n ull h y p ot hesis.  
T he size of t he T y pe I err or rate is 4. 5 % w hile t he act ual p o wer is 8 0. 4 %.
untitled h1
strat u m i i

A sa m ple size of n = 3 3 s u bjects will be re q uire d t o test t he 2 0 % vs. 4 0 %  
res p o nse rate h y p ot heses f or t he Strat u m II s u bject gr o u p usi n g a t w osta ge mi ni ma x desi g n wit h a n i nteri m l o o k w he n t he first 1 8 e val ua ble s u bjects are accr ue d a n d t he fi nal l o o k w he n 3 3 e val ua ble s u bjects are accr ue d. T he t w o -sta ge decisi o n r ule f or t his desi g n bec o mes:  
Sta ge 1: Acce pt t he n ull h y p ot hesis if t he res p o nse rate is less t ha n or e q ual t o 4/ 1 8 f or t he 1st 1 8 s u bjects. Ot her wise c o nti n ue t o sta ge 2.  
T he pr o ba bilit y of st o p pi n g f or f utilit y at Sta ge 1 is 0. 7 2 w he n t he n ull h y p ot hesis is tr ue Sta ge 2: Acce pt t he n ull h y p ot hesis if t he res p o nse rate is less t ha n or e q ual t o 1 0/ 3 3. Ot her wise reject t he n ull h y p ot hesis.  
T he siz e of t he T y pe I err or rat e is 4. 6 % w hile t he act ual p o wer is 8 0. 1%.
untitled h1
strat u m iii

A sa m ple size of n = 2 1 will be re q uire d t o test t he 5 % vs. 2 0 % res p o nse rate h y p ot heses f or t he Strat u m III s u bject gr o u ps usi n g a si n gle sta ge desi g n.
untitled h1
t otal patie nts

Ass u mi n g t hat a p pr o xi mat el y 5 % of Strat u m I s u bjects will be n o n -  
e val ua ble, t he act ual sa m ple size nee de d will be n = 4 1. Ass u mi n g t hat a p pr o xi matel y 1 0 % of Strat u m II s u bjects will be n o n -e val ua ble, t he n t he act ual sa m ple size nee de d will be n = 3 6. Ass u mi n g t hat a p pr o xi matel y 1 0 % of Strat u m III s u bjects will be n o n -e val ua ble, t he n a sa m ple size of n  
= 2 3 eac h will be re q uire d f or Strata III.  
Nif urt i m o x P hase II: Nif urti m o x f or Ne ur o blast o ma a n d Me d ull o blast o ma V 0 7 0 6 Pa ge 1 8 of 5 5
untitled h1
6. 2. statistical a nal

Statistical a nal ysis of t he pri mar y o utc o me will be c o n d ucte d f or eac h of t he t hree s u bject strata se paratel y Strat u m I a n d II data will utilize a t w o -  
sta ge mi ni ma x a nal ysis as o utli ne d i n t he sa m ple size j ustificati o n.  
Strat u m III data will be a nal yze d o nl y w he n t he f ull sa m ple size f or t hat strat u m has bee n ac hie ve d. T he n u m ber of s u bjects wit h c o m plete ( C R)  
a n d partial ( P R) res p o nses, sta ble disease ( S D), a n d pr o gressi ve disease  
( P D), will be s u m marize d relati ve t o t he t otal n u m ber of e val ua ble s u bjects. A n e xact bi n o mial m o del will be use d t o test t he n ull a n d alter nati ve h y p ot heses as s pecifie d i n t he sa m ple size disc ussi o n a b o ve usi n g t he state d directi o nal 5 % si g nifica nce le vel. I n a d diti o n, e xact 9 5 %  
c o nfi de nce i nter vals f or t he pri mar y efficac y o utc o me usi n g t he bi n o mial m o del will be o btai ne d t o s u p ple me nt t he p- val ue calc ulati o ns a n d t o assist i n t he i nter pretati o n of t he o bser ve d res ults at t he c o m pleti o n of t he f ull 6 c ycles of treat me nt. F urt her e xa mi nati o n of baseli ne a n d treat me nt -relate d meas ures ass ociate d wit h t he pri mar y efficac y o utc o me ( C R + P R) rate will utilize a l o gistic r e gressi o n m o del w here t he o utc o me of i nterest will be t he gr o u p of s u bjects ac hie vi n g a C R or P R vs. ot her s u bjects.  
Statistical a nal ysis of t he sec o n dar y o utc o me meas ures , s uc h as q ualit y of life data, will utilize a wit hi n -s u bjects a p pr oac h gi ve n t he re peate d meas ures str uct ure of t he data as c ollecte d o ver t he m ulti ple o bser vati o nal ti me p oi nts. Ti me t o e ve nt a nal ysis usi n g Ka pla n- Meier pl ots a n d C o x re gressi o n m o dels will be use d t o e xa mi ne t he l o n ger ter m occ urre nce of cli nical o utc o mes (s uc h as s ur vi val) a n d t he i nfl ue nce of p ote ntial c o variates. T he p ote ntial f or missi n g data i n a n y l o n git u di nal st u d y is rec o g nize d. T h us, a se nsiti vit y a nal ysis of t he i m pact of missi n g data will be c o n d ucte d usi n g a m ulti ple i m p utati o n a p pr oac h if data ca n be ass u me d t o be missi n g at ra n d o m or c o m pletel y at ra n d o m. T hese res ults will be c o m pare d t o m o deli n g res ults base d u p o n li near mi xe d m o del res ults wit h o ut use of i m p utati o ns.
untitled h1
7. 1. nif urti m

7. 1. 1. A n y Gra de 5 t o xicit y attri b uta ble t o Nif urti m o x t hat occ urs wit hi n 3 0 da ys of ta ki n g st u d y dr u g, will re q uire a D S M B meeti n g t o be hel d wit hi n 7 da ys of re p orti n g t he t o xicit y. U ntil t he ti me t hat t he D S M B meets, n o ne w s u bjects ma y be e nr olle d o n st u d y. T he c o m mittee will e val uate t he relati o ns hi p of t he nif urti m o x t o t he t o xicit y.  
7. 1. 2. A n y s u bjects w h o de m o nstrate Gra de 3 or 4 C N S/ P N S t o xicit y attri b uta ble t o Nif urti m o x will ha ve nif urti m o x hel d. If s y m pt o ms res ol ve wit hi n 1 4 da ys, s u bjects ma y res u me nif urti m o x at a re d uce d dose of 2 0 m g/ k g/ da y  
( 1 5 m g/ k g/ da y f or me d ull o blast o ma). F or s u bjects t hat restart at t he re d uce d d ose: if t he y de m o nstrate a sec o n d Gra de 3 or 4 C N S/ P N S  
t o xicit y attri b uta ble t o Nif urti m o x at t his p oi nt t he y will ha ve nif urti m o x hel d a gai n. A gai n, if s y m pt o ms res ol ve wit hi n 1 4 da ys, s u bjects ma y res u me nif urti m o x at a re d uce d d ose of 1 5 m g/ k g/ da y ( 1 0 m g/ k g/ da y f or me d ull o blast o ma). If t he s y m pt o ms d o n ot i m pr o ve t o a Gra de 2 or less wit hi n 1 4 da ys t he y will be re m o ve d fr o m st u d y. F or s u bjects t hat resta rt at t he 1 5 m g/ k g/ da y ( 1 0 m g/ k g/ da y f or me d ull o blast o ma) re d uce d d ose: if t he y de m o nstrate a t hir d Gra de 3 or 4 C N S/ P N S t o xicit y attri b uta ble t o Nif urti m o x a gai n at t his p oi nt t he y will be re m o ve d fr o m st u d y.  
7. 1. 3. A n y s u bject w h o de m o nstrates Gra de 2 C N S/ P N S t o x icit y attri b uta ble t o Nif urti m o x ma y ha ve nif urti m o x hel d, at t he discreti o n of t heir treati n g p h ysicia n or t he PI, d uri n g a n e val uati o n of t he s y m pt o ms f or a peri o d u p t o 7 da ys ma xi m u m.  
7. 1. 4. S u bjects w h o de vel o p a n y ot her Gra de 4 t o xicit y attri b uta ble t o nif urti m o x  
( ot her t ha n he mat ol o gical t o xicit y or fe brile ne utr o pe nia) will ha ve t he dr u g hel d a n d will be f oll o we d u ntil res ol uti o n of t o xicit y t o ≤ Gra de 2. If s y m pt o ms r es ol ve t o ≤ Gra de 2 wit hi n 7 da ys, s u bjects ma y res u me nif urti m o x at a re d uce d d ose of 2 0 m g/ k g/ da y ( 1 5 m g/ k g/ da y f or me d ull o blast o ma). If s y m pt o ms d o n ot res ol ve t o ≤ Gra de 2 wit hi n 7 da ys, s u bjects will be re m o ve d fr o m st u d y a n d m o nit ore d u ntil t o xicit y is  
≤ Gra de 2. If s u bject e x perie nces a sec o n d Gra de 4 t o xicit y attri b uta ble t o nif urti m o x t he y will ha ve t he dr u g hel d a n d will be f oll o we d u ntil res ol uti o n of t o xicit y t o ≤ Gra de 2. If s y m pt o ms res ol ve t o ≤ Gra de 2 wit hi n 7 da ys, s u bjects ma y res u me nif urti m o x at a re d uce d d ose of 1 5 m g/ k g/ da y ( 1 0 m g/ k g/ da y f or me d ull o blast o ma). If s y m pt o ms d o n ot res ol ve t o ≤ Gra de 2 wit hi n 7 da ys, s u bjects will be re m o ve d fr o m st u d y a n d m o nit ore d u ntil t o xicit y is ≤ Gra de 2. If s u bject e x perie nces a t hir d Gra de 4 t o xicit y attri b uta ble t o nif urti m o x ( ot her t ha n he mat ol o gical t o xicit y or fe brile ne utr o pe nia) at t he re d uce d d ose le vel of 1 5 m g/ k g/ da y  
( 1 0 m g/ k g/ da y f or me d ull o blast o ma), t he y will be off st u d y. T here will be n o d ose re- escalati o ns all o we d.
untitled h1
7. 2. t o p

7. 2. 1. S u bjects w h o de vel o p a n y Gra de 4 t o xicit y t hat is attri b uta ble t o c ycl o p h os p ha mi de or t o p oteca n ot her t ha n t he c o m m o nl y a ntici pate d he mat ol o gic or i nfecti o us t o xicities will be re m o ve d fr o m st u d y b ut ma y c o nti n ue o n nif urti m o x u n der t he c urre nt I N D as l o n g as n o t u m or pr o gressi o n is d oc u me nte d. T hese s u bjects will c o nti n ue t o be e val ua ble f or t o xicit y.  
7. 2. 2. S u bjects e x perie nci n g pr ol o n ge d ( greater t ha n 7 da ys) Gra de 4 he mat ol o gic t o xicities attri b uta ble t o c ycl o p h os p ha mi de or t o p oteca n ma y ha ve t heir c ycl o p h os p ha mi de a n d t o p oteca n d ose re d uce fr o m recei vi n g dr u g o n Da ys 1 t hr o u g h 5 of eac h c ycle t o o nl y recei vi n g dr u g o n da ys 1 t hr o u g h 3 of eac h c ycle.  
7. 2. 3. Pri or t o starti n g eac h c ycle, t he A N C s h o ul d be > 5 0 0/ µ l (e xce pt i n s u bjects wit h d oc u me nt e d b o ne marr o w i n v ol ve me nt); see Sec. 5. 2. 5, 8. 3, 8. 4 a n d 8. 5 f or g ui deli nes if c o u nts are i na de q uate.  
7. 2. 4. S u bjects e x perie nci n g he mat uria i n e xcess of s p ora dic as y m pt o matic micr osc o pic he mat uria ma y ha ve M E S N A a d de d t o t heir c he m ot hera p y/ h y drati o n fl ui ds (s u g geste d t otal M E S N A d ose is 6 0 % of c ycl o p h os p ha mi de d ose). S u bjects wit h gr oss he mat uria, cl ots, or d ys uria des pite h y drati o n/ M E S N A will ha ve t o p oteca n/c ycl o p h os p ha mi de disc o nti n ue d.
untitled h1
8. 0 s u p

8. 1. C o nc urre nt a ntica ncer t hera p y ot her t ha n c ycl o p h os p ha mi de or t o p oteca n ma y n ot be a d mi nistere d t o s u bjects o n st u d y E X C E P T f or ra diati o n t o relie ve metastatic b o ne pai n as l o n g as t here are ot her meas ura ble areas of disease t o f oll o w f or sec o n dar y e n d p oi nt.  
8. 1. 1. Ra diati o n t hera p y ma y be gi ve n t o sites of pai nf ul b o ne metastases. Pri or t otal b o d y irra diati o n t o d oses < 2 0 G y will be per missi ble, h o we ver a n y pri or f ocal ra diati o n t hera p y t o t he s y m pt o matic site will e xcl u de t he s u bject fr o m t his sta n dar dize d ra diati o n t hera p y pla n. S u bjects will be treate d wit h t hree fracti o ns of 3 G y eac h. T he ra diati o n d ose ma y be prescri be d t o a p oi nt or v ol u me at t he discreti o n of t he treati n g ra diati o n o nc ol o gist. M ulti ple sites ma y be i ncl u de d a n d treate d per t he trial.  
8. 1. 2. Re -treatme nt ra diati o n t hera p y tec h ni q ues a n d d ose sc he d ules will be d o ne at t he discreti o n of t he treati n g ra diati o n o nc ol o gist.  
8. 2. Pr o p of ol: I n a past st u d y wit h nif urti m o x, a p ossi ble i nteracti o n bet wee n pr o p of ol  
a n d nif urti m o x was o bser ve d i n t w o s u bjects. T here is ver y li mite d i nf or mati o n b ut a te m p oral relati o ns hi p was n ote d wit h ti mi n g of me dicati o ns a n d a d verse e ve nt occ urre nce ( ne ur ot o xicit y), t he i nf or mati o n is n ot c o ncl usi ve at t his ti me.  
D ue t o t his p ossi ble i nteracti o n, t he f oll o wi n g g ui deli nes f or pr o p of ol use w hile ta ki n g nif urti m o x s h o ul d be f oll o we d:  
H ol d nif urti m o x f or 2 4 h o urs pri or t o pr ol o n ge d a nest hesia wit h pr o p of ol of > 1 h o ur. If Nif urti m o x ca n n ot be hel d pri or t o a pr oce d ure (f or e xa m ple e mer ge nc y sit uati o ns), a n d alter nati ve a ge nt s h o ul d be c o nsi dere d. S h ort d urati o n pr o p of ol use ( < 1 h o ur) has n ot elicite d a n y u nt o war d res p o nses i n past st u d y s u bjects , a n d t heref ore d oes n ot re q uire a n y c ha n ge i n nif urti m o x use pri or t o pr oce d ure.  
8. 3. M etr o ni daz ole : D uri n g t he c o urse of t his st u d y; a p ossi ble i nteracti o n bet wee n metr o ni daz ole a n d nif urti m o x has bee n o bser ve d i n t w o s u bjects. T here is ver y  
li mite d i nf or mati o n b ut a te m p oral relati o ns hi p was n ote d wit h ti mi n g of me dicati o ns a n d a d verse e ve nt occ urre nce ( se vere ne ur ot o xicit y). D ue t o t his p ossi ble i nteracti o n, t he f oll o wi n g g ui deli nes f or metr o ni daz ole use w hile ta ki n g nif urti m o x s h o ul d be f oll o we d:  
M etr o ni daz ole s h o ul d n ot be use d w hile ta ki n g Nif urti m o x. If a n ot her alter nati ve a ge nt ca n n ot be use d t he n h ol d nif urti m o x d uri n g metr o ni daz ole use. Nif urti m o x ma y be restarte d 4 8 h o urs after c o m pleti o n of last d ose of metr o ni daz ole.  
8. 4. S u p p orti ve c are: A p pr o priate a nti bi otics, bl o o d pr o d ucts, a ntie metics, i ntra ve n o us fl ui ds, a n d ge neral s u p p orti ve care are t o be use d as dee me d cli nicall y necessar y b y treati n g p h ysicia n.  
8. 5. F or Me d ull o bl ast o m a S u bjects - If, at start of c he m ot hera p y, platelet c o u nt is ≤  
5 0, 0 0 0/ µ l, platelet tra nsf usi o n rec o m me n de d t o re d uce t he ris k of C N S blee di n g.  
8. 6. If t he start of t he ne xt c ycle of c he m ot hera p y is dela ye d b y > 1 wee k after t he a ntici pate d start date , t he n fil grasti m or pe gfil grasti m s h o ul d be gi n 2 4- 4 8 h o urs after t he c o m pleti o n of da y 5 c he m ot hera p y a n d c o nti n ue u ntil ne utr o p hil rec o ver y (a d mi nistere d per treati n g i nstit uti o n's g ui deli nes) .  
8. 7. S u bject s w h o ha ve a seiz ure t hat is li kel y t o be sec o n dar y t o nif urti m o x t o xicit y will be starte d o n t he a nti -seiz ure me dicati o n o xcar baze pi ne . If t his me dicati o n is n ot t olerate d a n ot her a nti-seiz ure me dicati o n ma y be prescri be d i n c o ns ultati o n wit h ne ur ol o g y.  
8. 8. S u bjects w h o de vel o p a Gra de 2 or hi g her na usea or a n a versi o n t o s wall o wi n g t he nif urti m o x ta blets ma y s plit t he ta blets i n 2 a n d place t he m i n a Size 0 Gelca p, i n or der t o ma ke t he m easier t o t olerate.  
8. 9. F or pai n sec o n dar y t o t u m or s welli n g, de xa met has o ne at a d ose of 5 m g/ m BI D  
ma y be a d de d t o re gi me n.
untitled h1
9. 0 r e q

9. 1. Pri or t o st u d y e ntr y ( wit hi n 7 da ys of start of t hera p y):  
− P h ysical e xa m i ncl u di n g ne ur ol o gical e xa m a n d Cli nical T otal Ne ur o pat h y Scale ( T N Sc)  
− Uri ne catec h ola mi nes (f or ne ur o blast o ma o nl y)  
− Uri ne pre g na nc y test f or fe male s u bject s of c hil d beari n g p ote ntial  
( o nset of me nses or ≥ 1 3 years of a ge)  
− C B C wit h differe ntial  
− E lectr ol ytes, B U N, C reati ni ne,  
− L D H, A S T, A L T, B ilir u bi n, Ferriti n  
− Ne ur ol o gic str uct ure d i nter vie w (see a p pe n di x I) a n d baseli ne meas ures o n t he B RI E F a n d P C S Q o L: D T q uesti o n naire as well as t he P C S Q o L: M T D q uesti o n naire (as o utli ne d i n secti o n 1 1. 0).  
9. 2. Pri or t o st u d y e ntr y ( wit hi n 3 0 da ys of start of t hera p y)- m ust be d o ne after last pre vi o us c he m o d ose:  
− C T or M RI of meas ura ble disease sites  
− MI B G sca n ( n ot re q uire d if s u bject's disease is pre vi o usl y deter mi ne d t o be n ot MI B G a vi d or f or s u bjects wit h me d ull o blast o ma)  
− B o ne marr o w as pirate a n d bi o ps y (re q uire d f or ne ur o blast o ma s u bjects, o pti o nal f or me d ull o blast o ma. ( S h o ul d be str o n gl y c o nsi dere d f or me d ull o blast o ma s u bjects t hat ha ve l o w platelet a n d A N C c o u nts, a n d sl o w rec o veries of c o u nts). E xtra b o ne marr o w sa m ples t o be se nt (if s u bject a grees t o bi ol o g y p orti o n of st u d y) per secti o n 1 8. 2.  
− C S F cell c o u nt a n d c yt ol o g y ( me d ull o blast o ma s u bjects o nl y)
9. 3. Da y s 1 a n d 4 of C ycle 1: P har mac o ki netic st u dies are t o be dra w n pri or t o t he s u bject ta ki n g t heir 1st d ose of Nif urti m o x d ose a n d a gai n 3 h o urs f oll o wi n g t he o bser ve d m or ni n g d ose. See secti o n 1 0. 0 f or s pecific P K c ollecti o n a n d ha n dli n g i nf or mati o n.  
9. 4. Eac h wee k ( +/ - 3 da y wi n d o w) f or first 2 1 da ys:  
− P h ysical e xa m i ncl u di n g ne ur ol o gical e xa m  
− C B C wit h diffe re ntial  
− Electr ol ytes, B U N, creati ni ne  
− L D H, A S T, A L T, bilir u bi n 9. 5. Pri or t o e ver y 2 1 da y treat me nt c ycle ( +/- 3 da y wi n d o w f or c ycle start) wit hi n 7 da ys of start of ne xt c ycle:  
− P h ysical E xa m i ncl u di n g ne ur ol o gical e xa m  
− C B C wit h differe ntial  
− Electr ol ytes, B U N, creati ni ne  
− L D H, A S T, A L T, bilir u bi n  
− Uri ne catec h ola mi nes (f or ne ur o blast o ma s u bject s).  
Treati n g p h ysicia n ma y o btai n a d diti o nal st u dies at t heir discreti o n.
untitled h1
9. 6. a d diti

− M RI/ C T re -e val uati o n of disease sites  
− MI B G sca n (f or MI B G a vi d ne ur o blast o ma s u bjects o nl y)  
− B o ne marr o w bi o ps y a n d as pirate (if p ositi ve at st u d y e ntr y)  
− C S F f or cell c o u nt a n d c yt ol o g y ( me d ull o blast o ma o nl y a n d if p ositi ve at st u d y e ntr y)  
− Ne ur ol o gical str uct ure d i nter vie w (see A p pe n di x 1) , Cli nical T otal Ne ur o pat h y Scale ( T N Sc), a n d meas ures o n t he B RI E F a n d P C S Q o L: D T q uesti o n naire (as o utli ne d i n secti o n 1 1. 0 of pr ot oc ol).  
− A d diti o nal i ma gi n g or st u dies ma y be d o ne if cli nicall y i n dicate d b y s y m pt o ms, e xa m, or t u m or mar kers 9. 7. At t he E n d of Pr ot oc ol T hera p y ( C ycle 6) or at Earl y Wit h dra wal:  
− P h ysical E xa m i ncl u di n g ne ur ol o gical e xa m a n d Cli nical T otal Ne ur o pat h y Scale ( T N Sc)  
− C B C wit h differe ntial  
− Electr ol ytes, B U N, creati ni ne  
− L D H, A S T, A L T, Bilir u bi n  
− Uri ne catec h ola mi nes (f or ne ur o blast o ma s u bjects) Ne ur ol o gical str uct ure d i nter vie w (see A p pe n di x 1)  
− Meas ures o n t he B RI E F a n d P C S Q o L : D T q uesti o n naire (as o utli ne d i n secti o n 1 1 . 0 of pr ot oc ol).  
− M RI/ C T re -e val uati o n of disease sites  
− MI B G sca n (f or MI B G a vi d ne ur o blast o ma s u bjects o nl y)  
− B o ne marr o w bi o ps y a n d as pirate (if p ositi ve at st u d y e ntr y)  
− C S F f or cell c o u nt a n d c yt ol o g y ( me d ull o blast o ma o nl y a n d if p ositi ve at st u d y e ntr y)
Nif urt i m o x P hase II: Nif urti m o x f or Ne ur o blast o ma a n d Me d ull o blast o ma V 0 7 0 6 Pa ge 2 3 of 5 5  
− If t he s u bject wit h dra ws earl y, sca ns nee d n ot be re peate d if rece ntl y perf or me d u nless cli nicall y i n dicate d. A d diti o nal i ma gi n g or st u dies ma y be d o ne if cli nicall y i n dicate d b y s y m pt o ms, e xa m, or t u m or mar kers. Treati n g p h ysicia n ma y o btai n a d diti o nal st u dies at t hei r discreti o n.
untitled h1
9. 8. f oll o

9. 8. 1. Wit hi n 7 da ys of start of eac h c ycle ( +/- 3 da y wi n d o w f or c ycle start):  
− P h ysical E xa m i ncl u di n g ne ur ol o gical e xa m  
− C B C wit h differe ntial  
− Electr ol ytes, B U N, creati ni ne  
− L D H, A S T, A L T, bilir u bi n  
− Uri ne catec h ola mi nes (f or ne ur o blast o ma s u bjects).  
− Treati n g p h ysicia n ma y o btai n a d diti o nal st u dies at t heir discreti o n.  
9. 8. 2. Mi ni m u m of e ver y t hree c ycles ( or s o o ner if i n dicate d) :  
− M RI/ C T re -e val uati o n of disease sites  
− MI B G sca n (f or MI B G a vi d ne ur o blast o ma s u bjects o nl y)  
− B o ne marr o w bi o ps y a n d as pirate (if p ositi ve at st u d y e ntr y)  
− C S F f or cell c o u nt a n d c yt ol o g y ( me d ull o blast o ma o nl y a n d if p ositi ve at st u d y e ntr y)  
− Treati n g p h ysicia n ma y o btai n a d diti o nal st u dies at t h eir discreti o n.
untitled h1
9. 9. l o n

9. 9. 1. S u bject s will be f oll o we d at 3 0 da ys ( + 7 da ys) past last d ose of st u d y dr u g as f oll o ws:  
− P h ysical e xa m i ncl u di n g ne ur ol o gical e xa m  
− C B C wit h differe ntial  
− E lectr ol ytes, B U N, creati ni ne  
− L D H, A S T, A L T, bilir u bi n  
− Uri ne catec h ola mi nes (f or ne ur o blast o ma s u bjects)  
− Ne ur ol o gic str uct ure d i nter vie w (see a p pe n di x I)  
9. 9. 2. S u bjects will be f oll o we d f or l o n g ter m s urvi val b y c o ntact wit h pare nt or treati n g i nstit uti o n t o c o nfir m s ur vi val at t he f oll o wi n g ti me p oi nts (ti me fr o m last d ose of st u d y dr u g):  
− 2 m o nt hs  
− 3 m o nt hs  
− 5 m o nt hs  
− 7 m o nt hs  
− 9 m o nt hs  
− 1 year Nif urt i m o x P hase II: Nif urti m o x f or Ne ur o blast o ma a n d Me d ull o blast o ma V 0 7 0 6 Pa ge 2 4 of 5 5  
will be re vie we d b y ce ntral ra di ol o g y at t he U ni versit y of Ver m o nt a n d Fletc her Alle n Hea lt h Care acc or di n g t o secti o n 1 3. 0. All st u d y re q uire d
untitled h1
sca ns s h o

Al yssa Va n der Werff Cli nical Pr o gra m C o or di nat or N M T R C  
1 0 0 Mic hi ga n A ve n ue N E M C 2 7 2 Gra n d Ra pi ds, MI 4 9 5 0 3 T el: ( 6 1 6) 2 6 7 -0 3 2 7 Fa x: ( 6 1 6) 3 9 1 -2 7 8 5 E- Mail: Al yssa. Va n der Werff @ hele n de v osc hil dre ns. or g
untitled h1
1 0. 0 p h

1 0. 1. O n Da ys 1 a n d 4 of c ycle 1 P K s peci me ns will be c ollecte d.  
1 0. 1. 1. T he s u bject will h ol d ( n ot ta ke) t heir 1st d ose of nif urti m o x o n da ys o ne a n d f o ur u ntil arri vi n g t o t he cli nic a n d pre- d ose bl o o d s peci me n is c ollecte d.  
1 0. 1. 2. Pre -d osi n g P K sa m ple will be c ollecte d a n d pr ocesse d as per Secti o n 1 0. 3 a n d 1 0. 4 1 0. 1. 3. O nce t he pre- n if urti m o x s peci me n is c ollecte d t he s u bject will ta ke t heir prescri be d m or ni n g d ose of n if urti m o x. If gel ca ps ules are bei n g use d, t he nif urti m o x s h o ul d N O T be place d i n t he Gel Ca ps ules f or t his d ose.  
1 0. 1. 4. T hree h o urs f oll o wi n g ( +/- 1 5 mi n utes) t he s u bject's m or ni n g d ose of nif urti m o x, t he ne xt P K sa m ple will be c ollecte d.  
1 0. 1. 5. T he date a n d ti me of all s peci me n c ollecti o ns a n d of t he d ose of nif urti m o x will be n ote d i n t he s u bject's c hart.  
1 0. 2. P K S a m ple re q uire me nts:  
O ne 2 cc w h ole bl o o d s peci me n will be c ollecte d i n a ser u m se parat or t u be  
( S S T).  
1 0. 3. S peci me n H a n dli n g:  
1 0. 3. 1. S peci me n will be c ollecte d wit h al u mi n u m f oil wra p pe d ar o u n d t he t u be i n or der t o pr otect t he sa m ple fr o m li g ht deteri orati o n.  
1 0. 3. 2. F oll o wi n g c ollecti o n t he s peci me n will t o be all o we d t o cl ot at r o o m te m perat ure for n o less t ha n t we nt y t o t hirt y mi n utes.  
1 0. 3. 3. S peci me n will be s p u n i n a ce ntrif u ge at 1, 0 0 0- 1, 3 0 0 g f or 1 0- 1 5 mi n utes 1 0. 3. 4. F oll o wi n g ce ntrif u gati o n t he ser u m fr o m t he S S T will be ali q u ote d i nt o a plastic scre w ca p pe d cr y o vial.  
1 0. 3. 5. S peci me ns m ust be la bele d wit h t he s u bject's i nitials, u ni q ue st u d y i de ntifier, a n d date a n d ti me of s peci me n c ollecti o n.  
1 0. 3. 6. S peci me n will t he n be st ore d i n a - 7 0° C. freezer u ntil rea d y t o be s hi p pe d.  
1 0. 4. S peci me n s hi p pi n g :  
1 0. 4. 1. All s peci me ns will be s hi p pe d o n dr y ice f oll o wi n g t he fe deral I A T A  
hazar d o us S peci me n S hi p pi n g G ui deli nes 1 0. 4. 2. S peci me ns s h o ul d be batc h s hi p pe d (after a p pr o xi matel y 5 s u bjects) o n a M o n da y- T h urs da y (la b d oes n ot acce pt Sat ur da y deli veries) .  
1 0. 4. 3. S peci me ns will be s hi p pe d t o t he f oll o wi n g a d dress :
untitled h1
pi n g z h

Pe diatric O nc ol o g y Tra nslati o nal Researc h La b orat or y C o o pers La n di n g 1 3 4 5 M o nr oe A ve, S uite 1 2 1 Gra n d Ra pi ds, MI 4 9 5 0 5 Tel: 6 1 6- 4 8 6 - 8 6 4 5 Pi n g.z ha o @ hele n de v osc hil dre ns. or g
untitled h1
1 1. 0 m o

nif urti m o x ma n date cl ose m o nit ori n g f or p ote ntial a d verse ne ur ol o gic effects.  
Nif urti m o x is k n o w n t o cr oss t he bl o o d- brai n barrier, raisi n g t he p ossi bilit y of a d verse C N S effects a s well as peri p heral ne ur ot o xicit y. T he s y m pt o ms t o be m o nit ore d are g ui de d b ot h b y pri or re p orte d a d verse be ha vi oral/s u bjecti ve effects  
( w hic h mi g ht be ne ur ol o gicall y me diate d), as well as p ote ntial si de effects res ulti n g fr o m t he catec h ola mi ner gic ne ur oto xicit y of t he st u d y dr u g. P ote ntial catec h ola mi ner gic tar gets i ncl u de s y m pat hetic a dre ner gic ne ur o ns of t he a dre nal me d ulla, ne ur o ns of t he l oc us cer ule us i n t he me d ulla, a n d d o pa mi ner gic ne ur o ns of t he basal ga n glia. C ollecti vel y, t hese ne ur o ns m o d ulate a ut o n o mic  
(s y m pat hetic) car di o vasc ular f u ncti o n, m ot or c o ntr ol, atte nti o n, re war d s yste ms of t he brai n, a n d ne ur o-e n d ocri ne f u ncti o n (e. g., pr olacti n release).  
Alt h o u g h s o me dr u g i nf or mati o n s u m maries me nti o n peri p heral ne ur o pat h y as a p ossi ble si de eff ect of nif urti m o x, t hese refere nces a p pear t o reflect pl a usi ble ne ur o pat hic s y m pt o ms ( n u m b or wea k ha n ds, feet) rat her t ha n s pecific cli nical or la b orat or y e vi de nce of peri p heral ne ur o pat h y ( de presse d te n d o n jer ks, a b n or mal electr o p h ysi ol o gical fi n di n gs). A re vie w of t he literat ure di d n ot re veal a n y pri or defi niti ve e vi de nce of nif urti m o x -i n d uce d ne ur o pat h y. H o we ver, t o xicit y ma y bec o me e vi de nt at t he hi g her dr u g d oses t o be st u die d i n t his pr ot oc ol; t heref ore, i nter val ne ur ol o gical e xa mi nati o ns a n d q uesti o ns re gar di n g t o xicit y will be directe d t o detecti n g nif urti m o x -relate d ne ur o pat h y. Beca use o ne p ote ntial site of C N S ne ur ot o xicit y is t he l oc us cer ule us - a brai nste m n ucle us ass ociate d wit h re g ulati o n of atte nti o n a n d ar o usal —e xec uti ve f u ncti o n will be assesse d (e. g.,  
" be ha vi or re g ulati o n" a n d " metac o g niti o n" ) wit h t he Be h avi or R ati n g I n dex of Exec utive F u ncti o n ( B RI E F).  
T he Be h avi or R ati n g I nve nt ory of Exec utive F u ncti o n ( B RI E F) is desi g ne d t o assess e xec uti ve f u ncti o ni n g i n c hil dre n a n d a d olesce nts a ges 5- 1 8. It c o nsists of t w o p ossi ble rati n g f or ms - a pare nt a n d a teac her q uesti o n naire - w hic h re q uire 1 0- 1 5 mi n utes t o a d mi nister a n d 1 5- 2 0 mi n utes t o sc ore. We will o nl y be usi n g t he pare nt c o m plete d q uesti o n naire i n t his st u d y. T he B RI E F is s uite d f or b ot h h o me a n d sc h o ol setti n gs. T he B RI E F is usef ul i n e val uati n g c hil dre n wit h a wi de s pectr u m of de vel o p me ntal a n d ac q uire d ne ur ol o gical c o n diti o ns s uc h as lear ni n g disa bilities, Atte nti o n -Deficit/ H y peracti vit y Dis or der ( A D H D), Tra u matic Brai n I nj ur y ( T BI), L o w Birt h Wei g ht, T o urette's S y n dr o me, a n d Per vasi ve De vel o p me ntal Dis or ders/ A utis m.  
Eac h B RI E F q uesti o n naire c o ntai ns 8 6 ite ms i n 8 n o n o verla p p pi n g cli nical scales a n d 2 vali dit y scales. T hese t he oreticall y a n d statisticall y deri ve d scales f or m t w o br oa de r i n de xes:  
1) Be ha vi oral Re g ulati o n: I n hi bit, S hift, E m oti o nal C o ntr ol 2) Metac o g niti o n: I nitiate, W or ki n g Me m or y, Pl a n/ Or ga nize, Or ga nizati o n of Materials, M o nit or Assessi n g Q u ality of Life ( Q o L)  
T he q ua lit y of life ( Q o L) of c hil dre n/fa milies i n treat me nt is a n i m p orta nt c o nsi derati o n i n cli nical dr u g trials. F or t he c hil d, s ocial-e d ucati o nal be ha vi or ca n be affecte d b y c o g niti ve a bilities a n d ne ur ot o xicities a n d t he ot her si de- effects of treat me nt dr u gs. H o w Q o L is affecte d b y a s pecific st u d y dr u g is als o of i nterest gi ve n t hat Q o L is use d b y pare nts, p h ysicia ns a n d s u bject s as a meas ure of dr u g efficac y. T w o q uesti o n naires will be use d t o meas ure t he Q o L effects of t he st u d y dr u g(s) o n s u bjects a n d t hei r fa milies.  
T w o se parate q uesti o n naires will be use d t o e xa mi ne t w o as pects of Q o L: ( 1)  
H o w s u bjects' Q o L is affecte d b y nif urti m o x (a n d wit h c o m bi nati o ns of a d diti o nal a ge nts/t hera pies), a n d ( 2), h o w t he pare nts of pe diatric ca ncer patie nts percei ve t he i m p orta nce of vari o us Q o L effects w hile ma ki n g treat me nt decisi o ns f or t heir c hil d. F or t he f or mer, t he Pe di atric C a ncer S urvey: P are nt Assess me nt of C hil dre n's Q u ality of Life D uri n g Tre at me nt ( P C S Q o L: D T). T he sec o n d as pect of Q o L will be assesse d wit h Pe di atric C a ncer S urvey: F or P are nts M aki n g Decisi o ns ( P C S Q o L: M T D).  
T he pr o p ose d pla n a n d sc he d ule f or m o nit ori n g of a d verse ne ur ot o xic effects i ncl u des ( 1) serial ne ur ol o gic e xa mi nati o n, ( 2) iterati ve str uct ure d i nter vie ws ( See A p pe n di x 1 f or " Str uct ure d I nter vie w f or A d verse Ne ur ol o gic E ve nts" f or m) a n d  
( 3) B RI E F assess me nts a n d t w o Q o L q uesti o n naires ( A p pe n di x 3).  
| Ne ur ol o gic                                                                                                                                                                                                                         | Str uct ure d   | B RI E F   | P C S Q o L: D T   | P C S Q o L: M T D   |     |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------|------------|--------------------|----------------------|-----|
| E xa m                                                                                                                                                                                                                                 | I nter vie w    |            |                    |                      |     |
| Baseli ne                                                                                                                                                                                                                              | √               | √          | √ ^                | √ ^                  | √ ^ |
| E n d of                                                                                                                                                                                                                               | Wee k 1         | √          |                    |                      |     |
| E n d of                                                                                                                                                                                                                               | Wee k 2         | √          |                    |                      |     |
| E n d of e ver y C ycle                                                                                                                                                                                                                | √               |            |                    |                      |     |
| E n d of C ycles 2 & 4                                                                                                                                                                                                                 | √               | √ ^        | √ ^                |                      |     |
| E n d of Pr ot oc ol  T hera p y/ Earl y  Wit h dra wal                                                                                                                                                                                | √               | √          | √ ^                | √ ^                  |     |
| 3 m o nt h f oll o w u p  6 m o nt h f oll o w u p  9 m o nt h f oll o w u p  1 Year f oll o w u p  N ote . B RI E F: Be h avi or R ati n g I n dex of Exec utive F u ncti o n ( B RI E F); P C S Q o L: D T: P are nt Assess me nt of |                 |            |                    |                      |     |  
T he ne ur ol o gic e xa m a n d Cli nical T otal Ne ur o p at h y Scale ( T N Sc) will be perf or me d b y a pe diatric ne ur ol o gist or o nc ol o gist w h o is e x perie nce d at perf or mi n g ne ur ol o gical e xa ms o n c hil dre n i n t he a ntici pate d a ge gr o u p. S o me s pecific feat ures of t he e xa m t o be rec or de d i ncl u de dee p te n d o n refle xes, p u pil size, le n gt h- de pe n de nt se ns or y c ha n ges, a n d tre m or/ ot her a b n or mal s p o nta ne o us m o ve me nts.  
A n o utli ne of t he str uct ure d i nter vie w t o be c o n d ucte d is i ncl u de d i n A p pe n di x I of t his pr ot oc ol.  
Nif urt i m o x P hase II: Nif urti m o x f or Ne ur o blast o ma a n d Me d ull o blast o ma V 0 7 0 6 Pa ge 2 8 of 5 5 1 2. 1. Criteri a f or S u bject Off -T her a p y S u bjects will be re m o ve d fr o m t he st u d y t hera p y f or t he f oll o wi n g reas o ns:  
- Pr o gressi ve ne o plastic disease  
- S u bject or g uar dia n wit h dra ws c o nse nt t o c o nti n ue i n t he trial  
- S u bject de vel o ps a n i nterc urre nt ill ness t hat precl u des f urt her p artici pati o n, or re q uires a pr o hi bite d c o nc o mita nt treat me nt  
- T he I n vesti gat or wit h dra ws t he s u bject i n t he s u bject's best i nterests  
- S u bject is l ost t o f oll o w -u p ( defi ne d as t he i na bilit y t o c o ntact t he s u bject o n 3 se parate occasi o ns o ver a peri o d of 2 wee ks)  
- A d mi nistrati ve reas o ns (e. g., t he s u bject is tra nsferre d t o h os pice care)  
- A n a d verse e ve nt, w hic h i n t he o pi ni o n of t he I n vesti gat or, precl u des f urt her trial partici pati o n or f ulfills t he pr ot oc ol re q uire me nts f or wit h dra wal  
- Deat h 1 2. 2. Criteri a f or S u bject Off -St u d y S u bjects ma y be wit h dra w n fr o m t he st u d y c o m pletel y w hic h i ncl u des wit h dra wal fr o m s u r vi val f oll o w-u p f or t he f oll o wi n g reas o ns:  
- C o m pleti o n of all st u d y re q uire me nts  
- S u bject or g uar dia n wit h dra ws c o nse nt t o c o nti n ue i n t he trial (if t his occ urs, n o f urt her st u d y visits or data ma y be c ollecte d)  
- S u bject is l ost t o f oll o w -u p ( defi ne d as t he i na bilit y t o c o ntact t he s u bject o n 3 se parate occasi o ns o ver a peri o d of 2 wee ks)  
- Deat h
untitled h1
1 3. 1. res p

res p o nse" f or t he p ur p oses of a nal ysis of treat me nt effect. Best res p o nse is deter mi ne d fr o m t he se q ue nce of t he o bjecti ve stat uses descri be d bel o w.  
1 3. 2. Res p o nse Criteri a F or P atie nts wit h S oli d T u m ors - T his st u d y will use t he  
( R E CI S T) Res p o nse E val uati o n Criteria i n S oli d T u m or fr o m t he N CI.  
1 3. 3. Me as ur a ble dise ase: The prese nce of at least o ne lesi o n t hat ca n be acc uratel y meas ure d i n at least o ne di me nsi o n wit h t he l o n gest dia meter at least 1 0 m m.  
1 3. 3. 1. Meas ura ble Disease Res p o nse will be assesse d at e n d of C ycle 2 e n d of c ycle 4, a n d e n d of pr ot oc ol t hera p y.  
1 3. 3. 2. F or patie nts wit h Meas ura ble Disease: T his st u d y will use t he  
( R E CI S T) Res p o nse E val uati o n Criteria i n S oli d T u m or fr o m t he N CI.  
1 3. 3. 3. Serial meas ure me nt s of lesi o ns are t o be d o ne wit h C T or M RI.  
T he sa me met h o d of assess me nt is t o be use d t o c haracterize eac h i de ntifie d a n d re p orte d lesi o n at baseli ne a n d d uri n g f oll o w- u p.  
1 3. 3. 4. Q ua ntificati o n of Disease B ur de n T he s u m of t he l o n gest dia meter  
( L D) f or all tar get lesi o ns will be calc ulate d a n d re p orte d as t he Nif urt i m o x P hase II: Nif urti m o x f or Ne ur o blast o ma a n d Me d ull o blast o ma V 0 7 0 6 Pa ge 2 9 of 5 5  
disease meas ure me nt. F or t his e val uati o n, all sca ns will u n der g o ce ntral re vie w at t he U ni versit y of Ver m o nt.  
C o m plete Res p o nse ( C R): Disa p peara nce of all tar get lesi o ns.  
P arti al Res p o nse ( P R): At least a 3 0 % decrease i n t he disease meas ure me nt, ta ki n g as refere nce t he disease meas ure me nt d o ne t o c o nfir m meas ura ble disease at st u d y e ntr y .  
St a ble Dise ase ( S D): Neit her s ufficie nt decrease t o q ualif y f or P R or s ufficie nt i ncrease t o q ualif y f or P D fr o m st u d y e ntr y.  
Pr o gressi ve Dise ase ( P D): At least a 2 0 % i ncrease i n t he s u m of t he l o n gest dia meters of all tar get lesi o ns c o m pare d t o st u d y e ntr y, t he a p peara nce of u ne q ui v ocal ne w lesi o ns, or la b orat or y e vi de nce of cli nical pr o gressi o n ( e. g., s prea d t o b o ne marr o w or i ncreasi n g catec h ola mi nes).
untitled h1
1 3. 4. res p

1 3. 4. 1. T h ose patie nts wit h m or p h ol o gic e vi de nce of ne ur o blast o ma b y r o uti ne H  
a n d E stai ni n g ( N S E stai ni n g o nl y is n ot e val ua ble) will be e val ua ble t o assess b o ne marr o w res p o nse.  
C o m plete res p o nse: N o t u m or cells detecta ble b y r o uti ne m or p h ol o g y o n t w o s u bse q ue nt bilateral b o ne marr o w as pirates a n d bi o psies d o ne at least t hree wee ks a part after st u d y e ntr y.  
Pr o gressi ve dise ase : T u m or see n o n m or p h ol o g y o n t w o c o nsec uti ve b o ne marr o ws d o ne at least t hree wee ks a part i n patie nts w h o ha d N O t u m or i n b o ne marr o w at st u d y e ntr y. ( N ote:  
Patie nt ma y be declare d as pr o gressi ve disease i n b o ne marr o w after o nl y o ne dia g n ostic b o ne marr o w at t he discreti o n of t he treati n g p h ysicia n after disc ussi o n wit h t he st u d y c hair.)  
St a ble dise ase : Persiste nce of a n a m o u nt of t u m or i n t he b o ne marr o w b y m or p h ol o g y t hat d oes n ot meet criteria f or eit her c o m plete res p o nse or pr o gressi ve disease.
untitled h1
1 3. 5. res p

1 3. 5. 1. Patie nts w h o ha ve a p ositi ve MI B G sca n at t he sta rt of t hera p y will be e val ua ble f or MI B G res p o nse. T he use of 1 2 3I f or MI B G i ma gi n g is rec o m me n de d f or all sca ns. If t his ra di ois ot o pe is u na vaila ble at t he treati n g i nstit uti o n, t he use of t he sa me ra di ois ot o pe f or all MI B G sca ns f or a n i n di vi d ual patie nt is str o n gl y e nc o ura ge d. All MI B G's will be perf or me d at t he researc h i nstit uti o n a n d ce ntrall y re vie we d at t he U ni versit y of Ver m o nt.  
T he f oll o wi n g criteria will be use d t o re p ort MI B G res p o nse b y t he researc h i nstit uti o n o n t he e n d of c o urse re p ort f or ms: C o m plete res p o nse = c o m plete res ol uti o n of all MI B G p ositi ve lesi o ns P arti al res p o nse = res ol uti o n of at least o ne MI B G p ositi ve lesi o n, wit h persiste nce of ot her MI B G p ositi ve lesi o ns.  
St a ble dise ase = n o c ha n ge i n MI B G sca n i n n u m ber of p ositi ve lesi o ns (i ncl u des patie nts w h o ha ve sa me n u m ber of p ositi ve lesi o ns b ut decrease d i nte nsit y)  
Pr o gressi ve dise ase = De vel o p me nt of ne w MI B G p ositi ve lesi o ns
untitled h1
1 3. 6. ce ntr

1 3. 6. 1. T he res p o nse of MI B G lesi o ns will be assesse d o n ce ntral re vie w usi n g t he C urie scale as o utli ne d bel o w. Ce ntral re vie w res p o nses will be use d t o assess efficac y f or st u d y e n d p oi nt. N O T E: T his sc ori n g is N O T re q uire d t o be d o ne b y t he researc h i nstit uti o n f or e n d of c o urse res p o nse assess me nts.  
1 3. 6. 2. T he b o d y is di vi de d i nt o 9 a nat o mic sect ors f or oste o me d ullar y lesi o ns, wit h a 1 0t h ge neral sect or all ocate d f or a n y e xtra- osse o us lesi o ns. I n eac h re gi o n, t he lesi o ns are sc ore d as f oll o ws. T he a bs ol ute e xte nsi o n s c ore is gra de d as: 0 = n o site per se g me nt, 1 = 1 site per se g me nt, 2 = m ore t ha n o ne site per se g me nt, 3 = massi ve i n v ol ve me nt ( > 5 0 % of t he se g me nt).  
T he a bs ol ute sc ore is o btai ne d b y a d di n g t he sc ore of all t he se g me nts. See  
dia gra m of sect ors bel o w:  
  
1 3. 6. 3. T he rel ati ve sc ore is calc ulate d b y di vi di n g t he a bs ol ute sc ore at eac h ti me b y t he c orres p o n di n g pretreat me nt o verall sc ore. T he relati ve sc ore of eac h patie nt is calc ulate d at eac h res p o nse assess me nt a n d classifie d as bel o w :  
C o m plete res p o nse: all areas of u pta ke o n MI B G sca n c o m pletel y res ol ve d.  
P arti al res p o nse : Relati ve sc ore < 0. 2 (lesi o ns al m ost disa p peare d) t o < 0. 5 (lesi o ns str o n gl y re d uce d).  
St a ble dise ase : Relati ve sc ore > 0. 5 (lesi o ns wea kl y b ut si g nifica ntl y re d uce d) t o > 0. 9 (lesi o ns n ot re d uce d).  
P r o gressi ve Dise ase : Ne w Lesi o ns o n MI B G sca n.
untitled h1
1 3. 7. defi niti

1 3. 7. 1. T his will be utilize d as a basis t o i nte grate res p o nse at all sites defi ne d as meas ura ble i n t his st u d y, i ncl u di n g C T/ M RI lesi o ns w hic h meet R E CI S T  
criteria, MI B G p ositi ve lesi o ns, a n d b o ne marr o w disease. T hese criteria will be use d t o defi ne t he o verall res p o nse f or t he patie nt i n b ot h strata i n t he statistical a nal ysis.
untitled h1
1 3. 7. 2. c

Disa p peara nce of all tar get lesi o ns. N o e vi de nce of t u m or at a n y site (c hest, a b d o me n, li ver, b o ne, b o ne marr o w, n o des, etc.), a n d H V A/ V M A n or mal.
untitled h1
1 3. 7. 3. p

At least a 3 0 % decrease i n t he disease meas ure me nt f or C T/ M RI  
tar get lesi o ns, ta ki n g as refere nce t he disease meas ure me nt d o ne t o c o nfir m meas ura ble disease i n tar get lesi o ns at st u d y e ntr y. B o ne marr o w wit h C R. MI B G wit h eit her P R/ C R i n b o ne lesi o ns; MI B G  
ma y be S D or C R i n s oft tiss ue lesi o ns c orres p o n di n g t o lesi o ns o n C T/ M RI. H V A/ V M A ma y still be ele vate d.
untitled h1
1 3. 7. 4. pr

A n y o ne of t he f oll o wi n g:  
a) At least a 2 0 % i ncrease i n t he disease meas ure me nt f or C T/ M RI  
tar get lesi o ns, ta ki n g as refere nce t he s mallest disease meas ure me nt rec or de d si nce t he start of treat me nt.  
b) A p peara nce of o ne or m ore ne w lesi o ns or ne w sites of t u m or.  
c) P D as defi ne d a b o ve f or eit her b o ne marr o w or MI B G lesi o ns.
untitled h1
1 3. 7. 5. st

T he patie nt will be classifie d as sta ble disease if t here are n o ne w lesi o ns; n o ne w sites of disease , a n d t he y d o n ot fit t he criteria f or P D/ P R/ C R as a b o ve.
untitled h1
1 4. 0 s a

1 4. 1. T o xicit y data m ust be s u b mitte d t o t he St u d y C hair at t he e n d of eac h c ycle of t hera p y.  
1 4. 2. Gi na Ha n na, P har m D will ser ve as t he Safet y Officer f or t his pr ot oc ol . T he Safet y Officer will re vie w all t o xicit y data at eac h D S M B meeti n g or s o o ner if t here is a safet y c o ncer n.  
1 4. 3. T he N M T R C V 0 7 0 6 m o nit ori n g Pla n will be f oll o we d f or t his pr ot oc ol.
untitled h1
1 5. 0 a d

1 5. 1. Nif urti m o x is s u p plie d t hr o u g h a n I N D a n d t heref ore res p o nsi bilit y t o t he  
F D A i ncl u des:  
1 5. 1. 1. Re p orti n g a n y u ne x pecte d fatal or life- t hreate ni n g a d verse e x perie nce ass ociate d wit h use of t he dr u g b y tele p h o ne, fa x, or o ver ni g ht mail, n o later t ha n 7 cale n dar da ys after i nitial recei pt of i nf or mati o n.  
1 5. 1. 2. Re p orti n g a n y a d verse e x perie nce ass ociate d wit h use of t he dr u g t hat is b ot h seri o us a n d u ne x pecte d i n writi n g n o later t ha n 1 5 cale n dar da ys after i nitial recei pt of i nf or mati o n.  
1 5. 1. 3. S u b mitti n g a n n ual pr o gress re p orts.  
1 5. 1. 4. Fe brile ne utr o pe nia is a n e x pecte d res ult of t o p oteca n/c ycl o p h os p ha mi de a n d s o d o es n ot nee d t o be re p orte d.  
1 5. 2. A " Data F or m f or Rec or di n g A d verse E ve nts" will be pr o vi de d t o rec or d n o nseri o us A d verse E ve nts. All Gra de 3 ( C T C A E Versi o n 3. 0) or hi g her A E's  
(e xce pt he mat ol o gic) will nee d t o be ca pt ure d .  
1 5. 3. T he D S M B c haire d b y t he safet y officer will re g ularl y meet per N M T R C  
p olic y a n d D S M B c harter t o re vie w all safet y re p orts a n d deter mi ne w het her pr ot oc ol m o dificati o ns are warra nte d .
untitled h1
1 6. 0 s e

1 6. 1. All Seri o us A d verse E ve nt s (S A E's ) t hat occ ur at partici pati n g sites m ust be re p orte d t o t he c o or di nati n g site wit hi n 2 4 h o urs of disc o ver y a n d c o or di nati n g ce nters m ust disse mi nate t his i nf or mati o n t o t he ot her sites as s o o n as p ossi ble.  
All seri o us, u ne x pecte d e ve nts a n d Gra de 4 a n d Gra de 5 t o xicities, b ot h e x pecte d a n d u ne x pecte d , m ust be re p orte d t o t he St u d y C hair ( w h o will t he n re p ort it t o t he Safet y Officer) wit hi n 2 4 h o urs of disc o ver y. T he partici pati n g site will t he n be res p o nsi ble f or s u b mitti n g all re q uire d Me d watc hes t hat occ ur at t heir sites t o t he F D A (a n d pr o vi di n g a c o p y t o t he c o or di nati n g site st u d y c hair), a n d s u b mitti n g pa per w or k t o all t heir o w n l ocal I R B re q uire me nts.  
1 6. 2. Rel ate d ness t o St u d y Dr u g T he I n vesti gat or m ust atte m pt t o deter mi ne if a n a d verse e ve nt is i n s o me wa y relate d t o t he use of t he st u d y dr u g a n d defi ne a n attri b uti o n cate g or y. T his relati o ns hi p s h o ul d be descri be d as f oll o ws:  
| R E L A TI O N S HI P                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | A T T RI B U TI O N                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | D E S C RI P TI O N                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               |
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| T h e A E i s cl e arl y N O T r el at e d t o t h e  i nt er v e nti o n. T he e ve nt is clearl y d ue t o ca uses disti nct fr o m t he use of  t he st u d y dr u g, s uc h as a d oc u me nte d pre-e xisti n g  c o n diti o n, t he effect of a c o nc o mita nt me dicati o n, or a ne w  c o n diti o n w hic h, base d o n t he pat h o p h ysi ol o g y of t he  c o n diti o n, a n d t he p har mac ol o g y of t he st u d y dr u g, w o ul d  be u nrelate d t o t he use of t he st u d y dr u g.                                                                                            |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |
| U nr el at e d                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |
| U nr el at e d t o  i n v e sti g ati o n al                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |
| a g e nt/i nt er v e nti o n                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 | T h e A E i s d o u btf ull y r el at e d t o t h e i nt er v e nti o n.  A d verse e ve nt d oes n ot ha ve te m p oral relati o ns hi p t o  i nter ve nti o n, c o ul d rea dil y ha ve bee n pr o d uce d b y t he  s u bject's cli nical state, c o ul d ha ve bee n d ue t o  e n vir o n me ntal or ot her i nter ve nti o ns, d oes n ot f oll o w  k n o w n patter n of res p o nse t o i nter ve nti o n, d oes n ot  rea p pear or w orse n wit h rei ntr o d ucti o n of i nter ve nti o n. |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |
| U nli k el y                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 | T h e A E m a y b e r el at e d                                                                                                                                                                                                                                                                                                                                                                                                                                                                          | t o t h e i nt er v e nti o n.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |
| T he e ve nt f oll o ws a reas o na ble te m p oral se q ue nce fr o m  a d mi nistrati o n of t he st u d y dr u g a n d t he e ve nt f oll o ws a  k n o w n res p o nse patter n t o t he st u d y dr u g B U T t he e ve nt  c o ul d ha ve bee n pr o d uce d b y a n i nterc urre nt me dical  c o n diti o n w hic h, base d o n t he pat h o p h ysi ol o g y of t he  c o n diti o n, a n d t he p har mac ol o g y of t he st u d y dr u g, w o ul d  be u nli kel y relate d t o t he use of t he st u d y dr u g O R t he  e ve nt c o ul d be t he effect of a c o nc o mi ta nt me dicati o n. |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |
| P o s si bl e                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                | T h e A E i s li k el y r el at e d t o t h e i nt er v e nti o n.  T he e ve nt f oll o ws a reas o na ble te m p oral se q ue nce fr o m  a d mi nistrati o n of t he st u d y dr u g, t he e ve nt f oll o ws a  k n o w n res p o nse patter n t o t he st u d y dr u g A N D t he e ve nt  ca n n ot ha ve bee n reas o na bl y e x plai ne d b y a n i nterc urre nt  me dical c o n diti o n O R t he e ve nt ca n n ot be t he effect of a  c o nc o mita nt me dicati o n.                      |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |
| R el at e d t o                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |
| i n v e sti g ati o n al                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |
| a g e nt/i nt er v e nti o n                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 | Pr o b a bl e                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | T h e A E i s cl e arl y r el at e d t o t h e i nt er v e nti o n.  T he e ve nt f oll o ws a reas o na ble te m p oral se q ue nce fr o m  a d mi nistrati o n of t he st u d y dr u g, t he e ve nt f oll o ws a  k n o w n res p o nse patter n t o t he st u d y dr u g a n d base d o n t he  k n o w n p har mac ol o g y of t he st u d y dr u g, t he e ve nt is  clearl y relate d t o t he effect of t he st u d y dr u g. T he a d verse  e ve nt i m pr o ves u p o n disc o nti n uati o n of t he st u d y dr u g a n d  rea p pears u p o n re peat e x p os ure. |
| D efi nit e                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |
untitled h1
1 7. 1. nif urti

1 7. 1. 1. F or m ul ati o n a n d St a bilit y : T he dr u g is st a ble at r o o m te m per at ure, a n d is li g ht se nsiti ve.  
1 7. 1. 2. G ui deli nes f or A d mi nistr ati o n : See secti o ns u n der 5. 1.  
1 7. 1. 3. S u p plie r: T he dr u g will be pr o vi de d t o e ac h site b y B a yer i n pill f or m.  
| Ris ks a n d si de effects rel ate d t o nif urti m o x:  T h ose i n dic ati n g nee d f or me dic al atte nti o n:  C o m m o n                    | Occ asi o n al                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             | R are                                                    |    |                          |
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| H a p pe ns t o 2 1 -1 0 0                                                                                                                           | H a p pe ns t o 5 -2 0                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |                                                          |    |                          |
| c hil dre n o ut of e ver y 1 0 0                                                                                                                    | c hil dre n o ut of e ver y  1 0 0                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | H a p pe ns t o < 5 c hil dre n o ut of  e ver y 1 0 0   |    |                          |
| W hile t a ki n g                                                                                                                                    |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | S ki n R as h, he a d ac he or  verti g o ( dizzi n ess) | Ce | tral ner v o us s yste m |
| nif urti m o x:                                                                                                                                      | t o xicit y i ncl u di n g dis orie ntati o n  (c o nf usi o n ), dist ur ba nces of  e q uili bri u m s uc h as ata xia  (cl umsi ness or u nstea di ness),  a n d n ysta g m us ( u nc o ntr olle d  bac k -a n d-f ort h a n d/ or r olli n g  e ye m o ve me nts ); e xcitati o n,  f or getf ul ness, i ns o m nia (tr o u ble  i n slee pi n g ), irrita bilit y,  ps yc h osis ( m o o d or me ntal  c ha n ges ), seiz ures  (c o n v ulsi o ns), m uscle  wea k ness, per i p heral  ne ur o pat h y ( n u m b ness,  ti n gli n g, pai n, or wea k ness i n  ha n ds or feet) a n d tre m ors; a  hi g h e osi n o p hil c o u nt, fe ver,  i m p ote nce ( decrease d se x ual  dri ve or a bilit y), or a l o w w hite  bl o o d cell c o u nt wit h a ris k of  i nfecti o n ca n occ ur wit h hi g h  d oses |                                                          |    |                          |
| Us uall y disa p pear  s p o nta ne o usl y after  wit h dra wal of t he  me dicati o n.  L ate:  A n y ti me after  c o m pleti o n of  treat me nt | T here are n o k n o w n late si de effects of Nif urti m o x at t his ti me.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              |                                                          |    |                          |  
| T h ose i n dic ati n g nee d f or me dic al atte nti o n o nl y if t he y c o nti n ue or are b ot hers o me:  C o m m o n Occasi o nal   | Rare                                                                                                                                                                                               |                                                                             |
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| Ha p pe ns t o 2 1 -1 0 0 c hil dre n                                                                                                      | Ha p pe ns t o 5 -2 0 c hil dre n                                                                                                                                                                  | Ha p pe ns t o < 5 c hil dre n o ut of                                      |
| o ut of e ver y 1 0 0                                                                                                                      | o ut of e ver y 1 0 0                                                                                                                                                                              | e ver y 1 0 0                                                               |
| W hile ta ki n g                                                                                                                           | Gastr oi ntesti nal dist ur ba nces,                                                                                                                                                               |                                                                             |
| n if urti m o x:                                                                                                                           | s uc h as a n ore xia wit h wei g ht  l oss (l oss of a p petite),  e pi gastric pai n (a b d o mi nal or  st o mac h pai n), na usea a n d/ or  v o miti n g occ ur i n 2 0 -8 0 % of  patie nts. | Hea dac he or verti g o  ( dizzi ness) occ ur i n 1 0-  1 2 % of patie nts. |
untitled h1
1 7. 2. c ycl

1 7. 2. 1. S o urce a n d P h ar m ac ol o g y : A n al k ylati n g a ge nt, relate d t o nitr o ge n m ustar d, w hic h is bi oc he micall y i nert u ntil it is meta b olize d t o its acti ve c o m p o ne nts b y t he li ver p h os p ha mi dases. It is n o n- p hase- s pecific. T he dr u g a n d its meta b olites are e xcrete d e xcl usi vel y b y t he ki d ne y after pare nteral a d mi nistrati o n. T he plas ma half -life ra n ges fr o m 4 t o 6. 5 h o urs.  
1 7. 2. 2. F or m ul ati o n a n d St a bilit y : See pac ka ge insert.  
1 7. 2. 3. G ui deli nes f or A d mi nistr ati o n : T he cycl o p h os p ha mi de will be 2 5 0 m g/ m 2/ d ose pre pare d per i nstit uti o nal sta n dar ds i n n or mal sali ne a n d a d mi nistere d i ntra ve n o usl y o ver 3 0 mi n utes. See secti o ns u n der 5. 2.  
17 2 4 T o xicit y :  
C o m m o n Ha p pe ns t o 2 1 - 1 0 0 c hil dre n o ut of e ver y 1 0 0  
| 17 2 4                                                                                                                                                                  | T o xicit y :                                                                                              | C o m                            | m o n   |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------|----------------------------------|---------|
| Ha p pe ns t o 2 1 - 1 0 0  c hil dre n o ut of e ver y  1 0 0                                                                                                          | R are                                                                                                      |                                  |         |
| Ha p pe ns t o < 5  c hil dre n o ut of  e ver y 1 0 0                                                                                                                  |                                                                                                            |                                  |         |
| I                                                                                                                                                                       | m                                                                                                          | me di ate:                       |         |
| Wit hi n 1                                                                                                                                                              | - 2 da ys of                                                                                               |                                  |         |
| recei vi n g dr u g                                                                                                                                                     | Occ asi o n al                                                                                             |                                  |         |
| Ha p pe ns t o 5 - 2 0  c hil dre n o ut of  e ver y 1 0 0                                                                                                              | Tra nsie nt bl urre d  visi o n car diac  t o xicit y wit h  arr h yt h mias m y ocar dial  necr osis ( L) |                                  |         |
| Pr o                                                                                                                                                                    | m pt:                                                                                                      |                                  |         |
| Wit hi n 2                                                                                                                                                              | - 3 wee ks,                                                                                                |                                  |         |
| pri or t o t he ne xt  c o urse                                                                                                                                         | A n ore xia ( L),  na usea ( L),  v o miti n g( L)                                                         | Metallic taste ( L),             |         |
| I na p pr o priate  A D H 1                                                                                                                                             |                                                                                                            |                                  |         |
| M yel os u p pressi o n  ( L),  al o pecia ( L)                                                                                                                         | He m orr ha gic  c ystitis ( L)                                                                            |                                  |         |
| Del a ye d:  A n y ti me later  d uri n g t hera p y,  e xcl u di n g t he  a b o ve c o n diti o ns                                                                    | I m m u n os u p pressi o n,  g o na dal  d ysf u ncti o n /sterilit y  ( L)                               | P ul m o nar y  fi br osis 3( L) |         |
| L ate:  A n y ti me aft er  c o m pleti o n of  treat me nt                                                                                                             | Sec o n dar y  mali g na nc y,  bla d der fi br osis                                                       |                                  |         |
| 1 Less c o m m o n wit h l o wer d oses. 3 Ris k i ncre ase d wit h c hest r a di ati o n.  2 O nly wit h very hi g h d oses. ( L) T o xicity m ay als o occ ur l ater. |                                                                                                            |                                  |         |  
Nif urt i m o x P hase II: Nif urti m o x f or Ne ur o blast o ma a n d Me d ull o blast o ma V 0 7 0 6 Pa ge 3 7 of 5 5  
i nf or mati o n.
untitled h1
1 7. 3. t o

1 7. 3. 1. S o urce a n d P h ar m ac ol o g y : T o p oteca n A S is a se mis y nt hetic a nal o g ue of ca m pt ot heci n, a n al kal oi d deri ve d fr o m t he ca m pt ot heci n tree w hic h gr o ws wi del y t hr o u g h o ut Asia. T he dr u g is a s pecific T o p ois o merase -I i n hi bit or.  
T o p oteca n i nterferes wit h t he re pair acti vit y of t o p ois o merase I b y sta bilizi n g t he f or mati o n of a c o vale ntl y b o n de d D N A-T o p ois o merase I  
c o m ple x. T h us, t he 5'- p h os p h or yl ter mi n us of t he e nz y me catal yze d si n gle stra n d D N A  
brea k re mai ns b o u n d t o t he t yr osi ne of t he e nz y me t here b y i nterferi n g wit h re plicati o n. T he dr u g e xists as t w o s pecies i n e q uili bri u m i n a q ue o us s ol uti o ns. O ne is a m ore acti ve cl ose d-ri n g lact o ne a n d t he ot her a less acti ve o pe n ri n g f or m. Aci dic c o n diti o ns fa v or t he cl ose d ri n g lact o ne f or m w hile basic a n d p h ysi ol o gic p H fa v ors t he o pe n ri n g f or m. I n h u ma n plas ma t he lact o ne f or m is a b o ut 2 1 % b o u n d t o plas ma pr otei n. T he dr u g is e xcrete d 3 9 % i n uri ne a n d t he re mai n der i n t he st o ol, t h e latter is pres u ma bl y via biliar ye xcreti o n. I n rats, o ver 9 0 % of t he ra di oacti vit y fr o m [ 1 4 C]t o p oteca n was rec o vere d i n st o ol a n d uri ne i n t he first 9 6 h o urs. T he half life f or t he lact o ne f or m is 1 8 0 mi n utes.  
1 7. 3. 2. F or m ul ati o n a n d St a bilit y: See pac ka ge insert .  
1 7. 3. 3. G ui deli nes f or A d mi nistr ati o n : T o p oteca n at a d ose of 0. 7 5 m g/ m will be pre pare d per i nstit uti o nal sta n dar ds i n n or mal sali ne a n d a d mi nistere d i ntra ve n o usl y, i nf use d o ver 3 0 mi n utes. See secti o ns u n der 5. 2.  
1 7. 3. 4. T o xicit y :  
| R are                                                                                                                                                                                                                 |                                 |                             |       |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------|-----------------------------|-------|
| Ha p pe ns t o < 5  c hil dre n o ut of  e ver y 1 0 0                                                                                                                                                                |                                 |                             |       |
| I                                                                                                                                                                                                                     | m                               | me di ate:                  |       |
| Wit hi n 1                                                                                                                                                                                                            | - 2 da ys of                    |                             |       |
| recei vi n g dr u g                                                                                                                                                                                                   | C o                             | m                           | m o n |
| Ha p pe ns t o 2 1 - 1 0 0  c hil dre n o ut of  e ver y 1 0 0                                                                                                                                                        | Occ asi o n al                  |                             |       |
| Ha p pe ns t o 5 - 2 0  c hil dre n o ut of  e ver y 1 0 0                                                                                                                                                            |                                 |                             |       |
| Na usea, v o miti n g,  diarr hea ( L),  m uc ositis ( L), fl u - li ke s y m pt o ms ( L),  hea dac he, ras h ( L),  ele vate d  tra nsa mi nases,  ele vate d al kali ne  p h os p hatase,  ele vate d bilir u bi n | A b d o mi nal pai n,  ri g ors |                             |       |
| Pr o                                                                                                                                                                                                                  | m pt:                           |                             |       |
| Wit hi n 2                                                                                                                                                                                                            | - 3 wee ks,                     |                             |       |
| pri or t o ne xt c o urse                                                                                                                                                                                             | M yel os u p pressi o n,        |                             |       |
| al o pecia                                                                                                                                                                                                            |                                 |                             |       |
| Del a ye d:  A n yti me later                                                                                                                                                                                         | Ast he nia                      | Micr osc o pic  he mat uria |       |
| Nif urt i m o x P hase II: Nif urti m o x f or Ne ur o blast o ma a n d Me d ull o blast o ma V 0 7 0 6                                                                                                               | Pa ge 3 8 of 5 5                |                             |       |  
| d uri n g t hera p y,  e xcl u di n g t he a b o ve  c o n diti o ns  L ate:  A n yti me aft er  c o m pleti o n of  t hera p y   |
|-----------------------------------------------------------------------------------------------------------------------------------|  
( L) T o xicity m ay als o occ ur l ater.  
1 7. 3. 5. S u p plier : C o m merciall y a vaila ble. See pac ka ge i nsert f or f urt her i nf or mati o n.
untitled h1
1 7. 4. fil gr

1 7. 4. 1. S o urce a n d P h ar m ac ol o g y : Fil grasti m is a h u ma n gra ul oc yte c ol o n y sti m ulati n g fact or ( G- C S F), pr o d uce d b y rec o m bi na nt D N A tec h n ol o g y.  
Fil grastri m is a 1 7 5 a mi n o aci d pr otei n wit h a m olec ular wei g ht of 1 8, 8 0 0 dalt o ns ma n ufact ure d b y rec o m bi na nt D N A tec h n ol o g y utilizi n g E. c oli bacte ria i nt o w hic h has bee n i nserte d t he h u ma n gra n ul oc yte c ol o n y sti m ulati n g fact or ge ne. It differs fr om t he nat ural pr otei n i n t hat t he Na mi n o aci d i n met hi o ni ne a n d t he pr otei n is n ot gl yc os ylate d. G- C S F is a li nea ge s pecific c ol o n y-sti m ulati n g fact or whic h re g ulates t he pr o d ucti o n of ne utr o p hils wit hi n t he b o ne marr o w a n d affects ne u tro p hil pr o ge nit or pr oliferati o n, differe ntiati o n a n d selecte d e n d- cell f u ncti o nal acti vati o n( i ncl u di n g e n ha nce d p ha g oc ytic a bilit y, pri mi n g of t he cell ular meta b olis m ass ociate d wit h res pirat or y b urst, a nti b o d y de pe n de nt killi n g, a n d t he i ncrease d e x pressi o n of s o me f u ncti o ns ass ociate d wit h cell s urface a nti ge n). T he eli mi nati o n half- life is si milar f or s u bc uta ne o us a n d i ntra ve n o us a d mi nistrati o n, a p pr o xi matel y 3. 5 h o urs. T he ti me t o pea k c o nce ntrati o n wit h a d mi nistere d s u bc uta ne o usl y is 2 t o 8 h o urs.  
1 7. 4. 2. F or m ul ati o n a n d St a bilit y : See p ac ka ge insert.  
1 7. 4. 3. G ui deli nes f or A d mi nistr ati o n : Fil grasti m or pe gfil grasti m s h o ul d be gi n 2 4- 4 8 h o urs after t he c o m pleti o n of da y 5 c he m ot hera p y if nee de d, a n d c o nti n ue u ntil ne utr o p hil rec o ver y. Pe g fil grasti m ma y be s u bstit ute d at t he discreti o n of t he treati n g p h ysicia n.  
| Ris ks a n d si de effects rel ate d t o fil gr asti                                                                                                                                                                                                                                                                                    | m i ncl u de t h ose w hic h are:    |                                                                                                                                                                                                                                                                              |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| C o                                                                                                                                                                                                                                                                                                                                     | m                                    | m o n                                                                                                                                                                                                                                                                        |
| Ha p pe ns t o 2 1 - 1 0 0                                                                                                                                                                                                                                                                                                              |                                      |                                                                                                                                                                                                                                                                              |
| c hil dre n o ut of e ver y 1 0 0                                                                                                                                                                                                                                                                                                       | R are                                |                                                                                                                                                                                                                                                                              |
| Ha p pe ns t o < 5 c hil dre n o ut of  e ver y 1 0 0                                                                                                                                                                                                                                                                                   |                                      |                                                                                                                                                                                                                                                                              |
| -                                                                                                                                                                                                                                                                                                                                       | Ac hi n g or pai n i n t he  b o nes | Occ asi o n al                                                                                                                                                                                                                                                               |
| Ha p pe ns t o 5 - 2 0 c hil dre n o ut of  e ver y 1 0 0 - L ocal irritati o n at t he site of  t he i njecti o n  - Hea dac he  - Hi g her t ha n n or mal le vels of  li ver e nz y mes a n d uric aci d  i n t he bl o o d  - A l o w n u m ber of platelets i n  t he bl o o d w hic h ma y ca use  y o u t o br uise a n d blee d | -                                    | Aller gic reacti o ns w hic h  ca n be life t hreate ni n g  wit h s h ort ness of breat h,  l o w bl o o d press ure, ra pi d  heart rate, hi ves a n d facial  s welli n g. T his reacti o n is  ver y rare a n d has bee n  ass ociate d mai nl y wit h  i ntra ve n o us |
| Nif urt i m o x P hase II: Nif urti m o x f or Ne ur o blast o ma a n d Me d ull o blast o ma V 0 7 0 6                                                                                                                                                                                                                                 | Pa ge 3 9 of 5 5                     |                                                                                                                                                                                                                                                                              |  
| m ore easil y   |                                                                                                                                                                                                                                |                      |
|-----------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------|
| -               | L o w fe ver                                                                                                                                                                                                                   |                      |
| -               | E nlar ge me nt of t he s plee n  w hic h ma y ca use pai n i n t he  a b d o me n or left s h o ul der                                                                                                                        |                      |
| -               | W orse ni n g of s ki n ras hes                                                                                                                                                                                                |                      |
| -               | Hi g her t ha n n or mal w hite  bl o o d c o u nt                                                                                                                                                                             | a d mi nistrati o n. |
| -               | If y o u are k n o w n t o ha ve  sic kle cell disease,  fil grasti m ma y ca use a  sic kle cell crisis                                                                                                                       |                      |
| -               | R u pt ure of t he s plee n                                                                                                                                                                                                    |                      |
| -               | Diffic ult y breat hi n g a n d  l u n g da ma ge t hat ma y be  d ue t o t he w hite bl o o d  cells t hat are sti m ula te d b y  fil grasti m tra veli n g t o t he  l u n gs w he n t he y are  i nfla me d or i nfecte d. |                      |  
1 7. 4. 5. S u p plier: C o m merciall y a vaila ble. See pac ka ge i nsert f or f urt her i nf or mati o n.  
1 7. 5. Pe gfil gr asti m ( pe g yl ate d fil gr asti m, S D/ 0 1, Ne ul ast a ®) ( 0 7 2 0 0 6 1 7. 5. 1. S o urce a n d P h ar m ac ol o g y :. Pe gfil grasti m is t he pe g ylate d f or m of rec o m bi na nt met hi o n yl h u ma n G-C S F(fil grasti m). Pe gfil grasti m is pr o d uce d b y c o vale ntl y bi n di n g a 2 0- kil o dalt o n ( k D)  
m o n o met h o x y p ol yet h yle ne gl yc ol m olec ule t o t he N- ter mi nal met hi o n yl resi d ue of fil grasti m. T he m olec ular wei g ht of pe gfil grasti m is 3 9 k D. GC S F is a li nea ge s pecific c ol o n y -sti m ulati n g fact or w hic h re g ulates t he pr o d ucti o n of ne utr o p hils wit hi n t he b o ne marr o w a n d affects ne utr o p hil pr o ge nit or pr oliferati o n, differe ntiati o n, a n d selecte d e n d- cell f u ncti o nal acti vati o n (i ncl u di n g e n ha nce d p ha g oc ytic a bilit y, pri mi n g of t he cell ular meta b olis m ass ociate d wit h res pirat or y b urst, a nti b o d y de pe n de nt killi n g, a n d t he i ncrease d e x pressi o n of s o me f u ncti o ns ass ociate d wit h cell s urface a nti ge ns). After s u bc uta ne o us i njecti o n t he eli mi nati o n half- life of pe gfil grasti m ra n ges fr o m 1 5 t o 8 0 h o urs a n d t he ti me t o pea k c o nce ntrati o n ra n ges fr o m 2 4 t o 7 2 h o urs. Ser u m le vels are s ustai ne d i n m ost patie nts d uri n g t he ne utr o pe nic peri o d p ostc he m ot hera p y, a n d be gi n t o decli ne after t he start of ne utr o p hil rec o ver y, c o nsiste nt wit h ne utr o p hilde pe n de nt eli mi nati o n.  
1 7. 5. 2. F or m ul ati o n a n d St a bilit y : See pac ka ge i nsert.  
1 7. 5. 3. G ui deli nes f or A d mi nistr ati o n: Fil grasti m or pe gfil grasti m s h o ul d be gi n 2 4- 4 8 h o urs after t he c o m pleti o n of da y 5 c he m ot hera p y if nee de d, a n d c o nti n ue u ntil ne utr o p hil rec o ver y. Pe gfil grasti m ma y be s u bstit ute d at t he discreti o n of t he treati n g p h ysicia n.  
| Ris ks a n d si de effects rel ate d t o pe gfil gr asti   | m i ncl u de t h ose w hic h:                                                                                                                                                                                                | R are                                                                                                                                                                                                                                                                                              |                                                        |
|------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------|
| Ha p pe ns t o < 5 c hil dre n o ut of  e ver y 1 0 0      |                                                                                                                                                                                                                              |                                                                                                                                                                                                                                                                                                    |                                                        |
| -                                                          | Ac hi n g or pai n i n t he  b o nes  C o m m o n  Ha p pe ns t o 2 1- 1 0 0                                                                                                                                                 |                                                                                                                                                                                                                                                                                                    |                                                        |
| c hil dre n o ut of e ver y 1 0 0                          | Occ asi o n al                                                                                                                                                                                                               |                                                                                                                                                                                                                                                                                                    |                                                        |
| Ha p pe ns t o 5 - 2 0 c hil dre n o ut of  e ver y 1 0 0  | -                                                                                                                                                                                                                            | Aller gic reacti o ns w hic h  ca n be life t hreate ni n g  wit h s h ort ness of breat h,  l o w bl o o d press ure, ra pi d  heart rate, hi ves a n d facial  s welli n g. T his reacti o n is  ver y rare a n d has bee n  ass ociate d mai nl y wit h  i ntra ve n o us  a d mi nistrati o n. |                                                        |
| -                                                          | If yo u are k n o w n t o ha ve  sic kle cell disease,  fil grasti m ma y ca use a  sic kle cell crisis                                                                                                                      |                                                                                                                                                                                                                                                                                                    |                                                        |
| -                                                          | R u pt ure of t he s plee n                                                                                                                                                                                                  |                                                                                                                                                                                                                                                                                                    |                                                        |
| -                                                          | Diffic ult y breat hi n g a n d  l u n g da ma ge t hat ma y be  d ue t o t he w hite bl o o d  cells t hat are sti m ulate d b y  fil grasti m tra veli n g t o t he  l u n gs w he n t he y are  infla me d or i nfecte d. |                                                                                                                                                                                                                                                                                                    |                                                        |
| 1 7. 5. 5. S u p plier: C o                                | m                                                                                                                                                                                                                            | merci all y a v ail a ble. See p ac k a ge i nsert f or f urt her                                                                                                                                                                                                                                  |                                                        |
| i nf or                                                    | m ati o n.                                                                                                                                                                                                                   | -                                                                                                                                                                                                                                                                                                  | L ocal irritati o n at t he site of  t he i njecti o n |
| -                                                          | Hea dac he                                                                                                                                                                                                                   |                                                                                                                                                                                                                                                                                                    |                                                        |
| -                                                          | Hi g her t ha n n or mal le vels of  li ver e nz y mes a n d uric aci d  i n t he bl o o d                                                                                                                                   |                                                                                                                                                                                                                                                                                                    |                                                        |
| -                                                          | A l o w n u m ber of platelets i n  t he bl o o d w hic h ma y ca use  y o u t o br uise a n d blee d  m ore easil y                                                                                                         |                                                                                                                                                                                                                                                                                                    |                                                        |
| -                                                          | L o w fe ver                                                                                                                                                                                                                 |                                                                                                                                                                                                                                                                                                    |                                                        |
| -                                                          | E nlar ge me nt of t he s plee n  w hic h ma y ca use pai n i n t he  a b d o me n or left s h o ul der                                                                                                                      |                                                                                                                                                                                                                                                                                                    |                                                        |
| -                                                          | W orse ni n g of s ki n ras hes                                                                                                                                                                                              |                                                                                                                                                                                                                                                                                                    |                                                        |
| -                                                          | Hi g her t ha n n or m al w hite  bl o o d c o u nt                                                                                                                                                                          |                                                                                                                                                                                                                                                                                                    |                                                        |  
1 8. 0 BI O L O GI C A L S T U DI E S ( O pti o n al)  
1 8. 1. T u m or c ollecti o n a n d esta blis h me nt of pri mar y ne ur o blast o ma a n d  
me d ull o blast o ma cell li nes i n c ult ure (t o be perf or me d at t he t he  
Ne ur o blast o ma Tra nslati o nal Researc h La b ).  
1 8. 1. 1. Via ble, fres h t u m or > 0. 2 gra ms fr o m t u m or bi o ps y or 2 cc fr o m b o ne  
marr o w as pirate s h o ul d be place d i n R P MI tiss ue c ult ure me dia usi n g  
sterile tec h ni q ue f or cell li ne ge nerati o n. Cells s h o ul d be la bele d wit h  
s u bjects i nitials a n d st u d y n u m ber.  
*If t here is n ot a de q uate tiss ue a vaila ble t he n cells will be o btai ne d  
fr o m t he c yt o ge netics la b orat or y after c o m pleti o n of t heir testi n g.  
1 8. 1. 2. If e xcess tiss ue is a vaila ble, t he n s na p fr oze n t u m or tiss ue a p pr o xi matel y  
5 m m i n size s h o ul d be wra p pe d i n f oil, s na p fr oze n i n li q ui d nitr o ge n a n d  
st ore d at - 2 0º C. Tiss ue sa m ples will be c o de d. Fi xe d tiss ue re mai ni n g  
fr o m dia g n ostic e val uati o n ma y be use d.  
1 8. 1. 3. T u m or cells will be gr o w n t o 7 0 % c o nfl ue nc y a n d tra nsferre d t o  
Ne ur o basal me dia wit h E G F a n d F G F.  
1 8. 1. 4. Cell li nes will be mai ntai ne d i n c ult ure f or bi ol o g y st u dies. T hese will  
i ncl u de deter mi ni n g t he gr o wt h c ur ves a n d res p o nsi ve ness of cells deri ve d  
Nif urt i m o x P hase II: Nif urti m o x f or Ne ur o blast o ma a n d Me d ull o blast o ma V 0 7 0 6 Pa ge 4 1 of 5 5  
fr o m t hese t u m ors t o a variet y of gr o wt h fact ors, c yt o ki nes, ne ur otr o p hic fact ors, a n d c yt ot o xic a ge nts (s uc h as nif urti m o x). We will st u d y t he ge nes e x presse d b y t hese cells usi n g re verse -tra nscri ptase p ol y merase c hai n reacti o n ( R T - P C R), N ort her n bl ot a nal ysis, real ti me P C R, a n d D N A  
a n d m R N A micr o arra ys bef ore a n d after treat me nt wit h nif urti m o x a n d ot her a ge nts. F urt her st u d y of t he pr otei ns e x presse d b y t hese cells will be d o ne usi n g i m m u n oc yt oc he mistr y a n d Wester n bl otti n g. Fl o w c yt o metr y will be use d t o detect resi d ual ne ur o blast o ma i n b o ne marr o w sa m ples.
untitled h1
1 8. 2. b o

sa m ples s h o ul d be se nt at t he f oll o wi n g ti me p oi nts:  
All patie nts - E nr oll me nt F or B o ne Marr o w P ositi ve patie nts o nl y ; als o se n d sa m ples at: E n d of C ycle 2, E n d of C ycle 4, a n d E n d of Pr ot oc ol T hera p y (e n d of c ycle 6) .
untitled h1
1 8. 2. 2. s

Se n d gree n t o p (s o di u m he pari n) t u be (s) t o eac h of t he f oll o wi n g sites wit h a mi ni m u m of 2cc a n d prefera bl y 5cc of b o ne marr o w as pirate i n t he m. B ot h ca n be s hi p pe d r o o m te m perat ure- b ut nee d t o be se nt o ut sa me da y pri orit y o ver ni g ht ( m ust get t here wit hi n 2 4 h o urs of t he dra w ). S hi p me nts are o nl y acce pte d M o n da y t hr o u g h Fri da y, s o B o ne Marr o w dra w nee ds t o be d o ne a n d s hi p pe d o ut o n M o n da y t hr o u g h T h urs da y o nl y please. Please n otif y eac h site (at e -mail a d dress liste d bel o w of pe n di n g s hi p me nt arri vals). Please e ncl ose " B o ne Marr o w S hi p me nt Tra ns missi o n F or m" wit h eac h s hi p me nt - o ne f or eac h site.
untitled h1
pi n g z h

S pectr u m Healt h, C o o per's La n di n g 1 3 4 5 M o nr oe A ve ; S uite 1 2 1 Gra n d Ra pi ds, MI 4 9 5 0 5 Pi n g.z ha o @ hele n de v osc hil dre ns. or g T he ot her sa m ple will g o t o:  
S pectr u m Healt h Fl o w C yt o metr y La b Le m me n -H olt o n Ca ncer Pa vili o n 1 4 5 Mic hi ga n Street N E S uite 6 2 0 1 Gra n d Ra pi ds, MI 4 9 5 0 3 p h o ne: 6 1 6- 4 8 6- 6 2 3 3 1 8. 3. Bi o m ar ker De vel o p me nt Bi o mar kers are o bjecti vel y meas ure d a n d e val uate d i n dicat ors of n or mal bi ol o gical pr ocesses, pat h o ge nic pr ocesses, or p har mac ol o gic res p o nses t o Nif urt i m o x P hase II: Nif urti m o x f or Ne ur o blast o ma a n d Me d ull o blast o ma V 0 7 0 6 Pa ge 4 2 of 5 5  
a t hera pe utic i nter ve nti o n. I n o nc ol o g y, t here is partic ular i nterest i n t he m olec ular c ha n ges u n derl yi n g t he o nc o ge nic pr ocesses t hat ma y i de ntif y ca ncer s u bt y pes a n d t he sta ge of disease, assess t he e xte nt of t u m or gr o wt h, pr o vi de s urr o gate meas ures of disease pr o gressi o n or metastasis, or pre dict res p o nses t o i n vesti gati o nal pr o d ucts. T hese i n vesti gati o ns ma y be usef ul i n de vel o pi n g mar kers t o i de ntif y disease s u bt y pes, g ui de t hera p y a n d/ or pre dict disease pr o gressi o n. I n a d diti o n t o testi n g t he safet y a n d effecti ve ness of t he i n vesti gati o nal pr o d uct i n t his st u d y, we ma y atte m pt t o de vel o p bl o o d a n d/ or t u m or test(s) desi g ne d t o i de ntif y s u bjects m ost li kel y t o be nefit fr o m t he i n vesti gati o nal pr o d uct. A 5cc bl o o d sa m ple will be dra w n f or bi o mar ker de vel o p me nt at scree ni n g, c ycle 2, c ycle 4, a n d at e n d of st u d y, a n d at t he safet y f oll o w- u p visits.  
T o st u d y t he effects of h u ma n ge n o mic ( p he n ot y pic) variati o n o n dr u g res p o nse, we pla n t o c o n d uct e x pl orat or y p har mac o ge n o mic st u dies as part of t his st u d y. M olec ular a nal yses o n bi ol o gical sa m ples o btai ne d fr o m s u bjects o n t his st u d y ma y be use d t o i de ntif y res p o nse patter ns t o nif urti m o x, i de ntif y ca ncer s u bt y pes, sta ge disease, assess t he a m o u nt of t u m or gr o wt h or pre dict disease pr o gressi o n or metastasis. T hese m olec ular a nal yses will n ot i ncl u de e x pl orat or y p har mac o ge netic a nal ysis.  
All s u bjects will be as ke d t o p r o vi de paraffi n t u m or bl oc ks ( or a mi ni m u m of 2 0 u nstai ne d sli des, if a vaila ble) fr o m t he pri mar y a n d/ or metastatic t u m or t hat are arc hi ve d. I n a d diti o n, all s u bjects will be as ke d t o pr o vi de e xisti n g s na p fr oze n t u m or sa m ples.  
C orres p o n di n g pat h ol o g y re p orts s h o ul d als o be pr o vi de d. As a n o pti o nal part of t his st u d y, s u bjects ma y be as ke d t o u n der g o tiss ue sa m pli n g b y c ore bi o ps y of a l ocall y a d va nce d or metastatic lesi o n f or e x pl orat or y bi o mar ker a nal yses pri or t o t he start of st u d y treat me nt. T he t u m or tiss ue bi o psies m ust be accessi ble wit h mi ni mal m or bi dit y t o t he s u bject b y a perc uta ne o us c ore nee dle bi o ps y eit her wit h or wit h o ut t he ai d of a C T  
sca n or ultras o u n d g ui da nce. Bi o ps y s peci me ns will be o btai ne d usi n g sta n dar d sterile s ur gical tec h ni q ues. All t u m or bi o ps y sa m ples s h o ul d ha ve a p orti o n of t he bi o ps y stai ne d wit h he mat o x yli n a n d e osi n a n d re vie we d b y a pat h ol o gist t o c o nfir m t he prese nce a n d esti mate t he perce nta ge of t u m or tiss ue. T he p ur p ose of t his a nal ysis is t o de vel o p a n a p pr oac h f or t he i de ntificati o n a n d vali dati o n of tra nscri pts t hat ma y pre dict w hic h s u bjects are li kel y t o res p o n d t o nif urti m o x.  
1 8. 4. P h ar m ac o ge netic St u dies D N A will be c ollecte d a n d e xtracte d fr o m bl o o d a n d t u m or sa m ples t o be utilize d f or e x pl orat or y p har mac o ge netic a nal ysis. T hese p har mac o ge netics a nal yses are differe nt fr o m t he bl o o d a n d t u m or a nal ysis f or bi o mar ker de vel o p me nt as descri be d a b o ve beca use t he y are f oc use d o n e val uati n g differe nt i n herite d or disease-i n d uce d ge ne f or ms i n t u m or D N A t hat ma y i nfl ue nce t he differe nt res p o nses s u bjects ha ve t o t he sa me dr u g. T he g oals of t hese e x pl orat or y st u dies are t o i de ntif y p ote ntial ge netic mar kers t hat ma y hel p i n t he i n vesti gati o n of ca ncer a n d/ or s u bjects w h o ma y ha ve t he best p ossi ble res p o nse t o nif urti m o x. T he c urre nt pla n is t o c o n d uct e x pl orat or y p har mac o ge netic st u dies o n t u m or tiss ue t o e val uate f or m utati o ns of ge nes (e. g. M Y C N) i n a n atte m pt t o deter mi ne if t he prese nce of a n y of t hese m utati o ns/rearra n ge me nts c orrelates wit h res p o nse.  
A d diti o nal ge ne cate g ories f or a nal yses ma y i ncl u de t u m or s u p press or ge nes (e. g. p 5 3, P T E N), o nc o ge nes (e. g., N- m yc, M E T), cell a d hesi o n, i n vasi o n a n d metastasis ge nes (e. g., α v β 3, C D H 1, M M P's), ge nes i n v ol ve d i n re g ulati n g cell gr o wt h a n d t he cell c ycle (e. g., c ycli n D 1, G H R H ), i m m u ne f u ncti o n-relate d a n d t u m or a nti ge n ge nes (e. g., T N F,  
S C Y A 2, K L K 3), dr u g meta b olis m a n d tra ns p ort ge nes (e. g., C Y P 3 A 4/ 5, P X R, G S T M 1, A B C B 1), a n d ge nes i n v ol ve d i n a n gi o ge nesis (e. g., HI F 1 α V E G F, F G F R 3). T h ose rele va nt t o st u d y dr u g acti o n i ncl u de a p o pt osis ge nes (e. g., B C L 2, I A Ps, T N F S F 6) a n d t he p utati ve dr u g tar gets/ pat h wa ys.
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1 8. 5. s a

Bl o o d a n d t u m or sa m pl es c ollecte d f or bi o mar ker de vel o p me nt a n d p har mac o ge netic st u dies a n d a n y ot her c o m p o ne nts fr o m t he cells ma y be st ored f or u p t o 2 0 years t o researc h scie ntific q uesti o ns relate d t o ca ncer a n d/ or nif urti m o x. T he s u bject retai ns t he ri g ht t o ha ve t he sa m ple material destr o ye d at a n y ti me b y c o ntacti n g t he pri nci pal i n vesti gat or. T he PI/s p o ns or will be t he e xcl usi ve o w ner of a n y data, disc o veries, or deri vati ve materials fr o m t he sa m ple materials a n d is res p o nsi ble f or t he destr ucti o n of t he sa m pl e(s) at t he re q uest of t he researc h s u bject t hr o u g h t he pri nci pal i n vesti gat or or at t he e n d of t he st ora ge peri o d. If a c o m merci al pr o d uct is de vel o pe d fr o m t his researc h pr oject, t he PI/s p o ns or will o w n t he c o m mercial pr o d uct. T he s u bject will ha ve n o c o m mercial ri g hts t o s uc h pr o d uct a n d will ha ve n o c o m mercial ri g hts t o t he data, i nf or mati o n, disc o veries, or deri vati ve materials gai ne d or pr o d uce d fr o m t he sa m ple.
untitled h1
1 9. 0 r e

1. S h oller, G S, Kal k u nte, S, Gree nla w, C, Mc Carte n, K, F or ma n, E, A nti- T u m or acti vit y of Nif urti m o x o bser ve d i n a patie nt wit h Ne ur o blast o ma. J Pe diatr He mat ol O nc ol. 2 0 0 6 Oct; 2 8( 1 0): 6 9 3- 6 9 5 2. S olari A, Ortiz S, S ot o A, Ara nci bia C, Ca m pilla y R, C o ntreras M, Sali nas P,  
R ojas A, Sc he n o ne H, Treat me nt of Tr y pa n os o ma cr uzi- i nfecte d c hil dre n wit h Nif urti m o x: a 3 year f oll o w- u p b y P C R. J o ur nal of A nti micr o bial C he m ot hera p y.  
2 0 0 1; 4 8: 5 1 5- 5 1 9 3. Ra ma kris h na Ra o, D N, Mas o n, R, Ge nerati o n of Nitr o A ni o ns of S o me 5-  
Nitr of ura ns, 2 - a n d 5- Nitr oi mi daz oles b y N ore pi ne p hri ne, D o pa mi ne, a n d Ser ot o ni n. J. Bi ol. C he m. 1 9 8 7; 2 6 2( 2 4): 1 1 7 3 1- 1 1 7 3 6
St u d y I D \#:: _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ I nitials: _ _ _ _ _ _ _ _ _  
  
  
  
Date: _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ C ycle #: _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _  
  
  
  
  
Pr o vi der c o m pleti n g f or m Si g nat ure: _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _  
Please c o m plete wit h t he fa mil y.  
| T o xicit y                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                | Gra de   |    |    |    |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------|----|----|----|
| N o ne                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     | 1        | 2  | 3  | 4  |
| Hea dac he?  Li g ht Se nsiti vit y?  St o mac h u pset?  V o miti n g?  I m bala nce?  Dizzi ness/ Li g ht hea de d ness?  Pai n i n li m bs?  C ol d ha n ds/feet?  S peec h har d t o u n dersta n d?  Dis orie ntati o n/c o nf usi o n?  E xcessi ve slee pi ness?  Diffic ult y or c ha n ge i n slee p r o uti ne?  U n us ual m o ve me nts?  Tre m or?  U n us ual cr yi n g, irrita bilit y?  C ha n ge i n his/ her i nterest i n t o ys, pla y?  C ha n ge i n a p petite?  C ha n ge i n h o w his/ her e yes m o ve?  C ha n ge i n a bilit y t o c he w or s wall o w?  Seiz ure or ot her s u d de n c ha n ge i n le vel of  a ware ness? |          |    |    |    |  
I n eac h case of a p ositi ve, pare nts will be as ke d t o gra de t he se verit y of t he si g n or s y m pt o m i n a ma n ner t hat will all o w us t o cate g orize t he si g n/s y m pt o m as class 1- 5 *  
acc or di n g t o t he classificati o n s yste m as f oll o ws:  
| Gra de                                                                                                                                                                    | Pare nt's Q ualitati ve Descri pti o n of t he a d verse si g n/s y m pt o m                                   |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------|
| Gra de 1                                                                                                                                                                  | Prese nt o nl y as a si g n, n ot a pr o ble m i n a n y se nse                                                |
| Gra de 2                                                                                                                                                                  | C hil d c o m plai ns, b ut still t he s y m pt o m is n ot i nterferi n g wit h  c hil d's us ual acti vities |
| Gra de 3                                                                                                                                                                  | I nterferi n g wit h c hil d's us ual acti vities                                                              |
| Gra de 4                                                                                                                                                                  | Si g nifica ntl y a n d per vasi vel y effecti n g t he c hil d's dail y life                                  |
| [ * A gra de of 5 i n t he classificati o n c orres p o n ds t o Deat h a n d s o will clearl y n ot be t he  classificati o n res ulti n g fr o m pare nt i nter vie w.] |                                                                                                                |
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a p pe n di

| E n d of Pr ot oc ol  T hera p y  or Earl y  Wit h dra wal                                                                                                              |             |                  |                  |                               |                          |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------|------------------|------------------|-------------------------------|--------------------------|
| Pri or t o  eac h  c ycle‡                                                                                                                                              |             |                  |                  |                               |                          |
| Ti me P oi nt:                                                                                                                                                          | Prest u d y | C ycle 1, w k  2 | C ycle 1, w k  3 | E n d of c ycles  2 a n d 4 ¶ | 3 0 Da y  F oll o w- u p |
| P h ysical E xa m                                                                                                                                                       | X           | X                |                  |                               |                          |
| Ne ur o e xa m                                                                                                                                                          | X           | X                |                  |                               |                          |
| Ne ur o i nter vie w / T N Sc                                                                                                                                           | X           | X                | X                | X                             |                          |
| B RI E F assess me nt                                                                                                                                                   | X           | X                | X                |                               |                          |
| P C S Q o L: M T D                                                                                                                                                      | X           |                  |                  |                               |                          |
| P C S Q o L: D T                                                                                                                                                        | X           | X                | X                |                               |                          |
| Uri ne pre g na nc y test †                                                                                                                                             | X           |                  |                  |                               |                          |
| C bc/ diff  L ytes, B U N, Creat                                                                                                                                        | X           |                  |                  |                               |                          |
| L D H, A S T, A L T, Bili                                                                                                                                               | X           |                  |                  |                               |                          |
| Ferriti n                                                                                                                                                               | X           |                  |                  |                               |                          |
| H V A/ V M A ( N B o nl y)                                                                                                                                              | X           |                  |                  |                               |                          |
| C T or M RI                                                                                                                                                             | X           | X                | X                |                               |                          |
| MI B G *                                                                                                                                                                | X           | X                | X                |                               |                          |
| B o ne marr o w                                                                                                                                                         | X           | X #              | X #              |                               |                          |
| C S F cell  c o u nt/c yt ol o g y                                                                                                                                      | X §         | X § #            | X § #            |                               |                          |
| * = MI B G n ot re q uire d if s u bject's ne ur o blast o ma was pre vi o usl y deter mi ne d t o be n ot MI B G a vi d or f or s u bjects wit h me d ull o blast o ma |             |                  |                  |                               |                          |  
* = MI B G n ot re q uire d if s u bject's ne ur o blast o ma was pre vi o usl y deter mi ne d t o be n ot MI B G a vi d or f or s u bjects wit h me d ull o blast o ma  
\# = Re peat o nl y if p ositi ve at st u d y e ntr y  
† = O nl y f or fe male s u bjects of c hil d beari n g p ote ntial ( ≥ 1 3 years of a ge)  
‡ = testi n g t o occ ur wit hi n 7 da ys of t he start of eac h c ycle ( +/- 3 da ys)  
¶ = testi n g t o occ ur wit hi n 7 da ys of c o m pleti n g c ycle ( +/- 3 da ys)  
§ = Me d ull o blast o ma s u bjects o nl y